SHUGART_H.pdf

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PROBING BEHAVIOR AND HOST PREFERENCE IN THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) By HOLLY SHUGART A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019

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© 2019 Holly Shugart

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To Eugene Max Shugart

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4 ACKNOWLEDGMENTS I thank Dr. Tim Ebert for advice on the statistical analysis used in this dissertation. I also thank Guoping Liu for technical assistance with EPG, Percivia Mariner for managing the psyllid colony used in this work, Rhonda Schumann for greenhouse assistan ce, and Shelley Jones, Foad El Ramawy , and Faraj Hijaz for technical work on metabolite analysis .

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 14 CHAP T E R 1 INTRODUCTION TO THE PROBING BEHAVIOR OF DIAPHORINA CITRI .......... 16 Electropenetrography as a Tool to Study Hemipteran Probing Behavior ................ 16 History of Electropenetrography Technology ................................ .......................... 17 Comparison of AC and DC EPG System Signal Processing ................................ .. 17 EPG Setting Choices in This Dissertation ................................ ............................... 19 Overview of Hemipteran Probing Behavior ................................ ............................. 20 Psylloidea Probing Behavior ................................ ................................ ................... 23 Taxonomic Classification of Psyllids ................................ ................................ . 23 Histological Evidence of Psyllid Probing Behavior ................................ ............ 23 Diaphorina citri the Asian Citrus Psyllid ................................ ................... 26 Bactericera cockerelli the Potato Psyllid ................................ .................. 26 Cacopsylla pyri the European Pear Psylla ................................ ............... 27 Pd Waveform ................................ ................................ ............................. 27 Psyllid Mouthpart Morphology ................................ ................................ .......... 29 Candidatus Liberibacter Diseases of Agricultural Crops ................................ .. 29 Diaphorina citri as a Putative Vector of Candidatus Liberibacter asiaticus ................................ ................................ ................................ .. 29 Other Psyllid Vectors of Liberibacter Plant Pathogenic Bacteria ................ 30 Huanglongbing Disease of Citrus Caused by the Bacterial Pathogen, Candidatus Liberibacter asiaticus ................................ ................................ . 31 Research Questions and Objectives in This Dissertation ................................ ....... 33 2 PSYLLIDS ARE NOT APHIDS A STUDY OF COMBINED INTERCELLULAR AND INTRACELLULAR PROBING TACTICS OF THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI (HEMIPTRA: LIVIIDAE), AND SOME HISTOLOGICAL CORRELATIONS OF STYLET TIP LOCATION DURING THE PERF ORMANCE OF SELECTED WAVEFORMS ................................ .................. 37 Introduction ................................ ................................ ................................ ............. 37 Histological Evidence of Psyllid Probing Behavior ................................ ............ 38 Diaphorina citri Waveform Correlations ................................ ............................ 39

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6 M aterials and Methods ................................ ................................ ............................ 40 Plants ................................ ................................ ................................ ............... 40 Insects ................................ ................................ ................................ .............. 41 Electropenetrography Equipment and Settings ................................ ................ 41 Histological Processing ................................ ................................ .................... 42 R esults ................................ ................................ ................................ .................... 44 Waveform C Correlation ................................ ................................ ................... 44 Waveform D Correlation ................................ ................................ ................... 45 Early interrupte d waveform D ................................ ................................ ..... 45 Late interrupted waveform D ................................ ................................ ...... 45 Waveforms E1 and E2 Correlation ................................ ................................ ... 46 Waveform G Correlation ................................ ................................ ................... 46 Combined Intercellular and Intracellular Probing Tactic ................................ ... 47 D iscussion ................................ ................................ ................................ .............. 48 Wavform C Correlation ................................ ................................ ..................... 48 Waveform D Correlation ................................ ................................ ................... 48 Stylet Tips are Inside Phloem Tissues During Waveforms D, E1, and E2 ........ 49 Waveform G is Correlated with Xylem Ingestion ................................ .............. 49 Aphids Make a pd Waveform ................................ ................................ ........... 49 Psyllids Do not Make a pd Waveform ................................ ............................... 50 Combined Intercellular and Intracellular Probing Tactic ................................ ... 51 3 COMPARISON OF THE PROBING BEHAVIOR OF THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI , ON SWEET ORANGE, CITRUS SINENSIS VAR. VALENCIA, AND SOUR ORANGE, CITRUS AURANTIUM , IN RELATION TO HOST PLANT PHLOEM AND XYLEM METABOLIC PROFILES ..................... 63 Introduction ................................ ................................ ................................ ............. 63 Materials and Methods ................................ ................................ ............................ 65 Plants and Insects ................................ ................................ ............................ 65 Electropenetrography Equipment and Settings ................................ ................ 66 Statistical Analysis Programs and Variables ................................ .................... 68 Chemical Analysis of Phloem and Xylem ................................ ......................... 69 Extraction of phloem and xylem ................................ ................................ . 69 Deriviti zation ................................ ................................ ................................ ..... 70 Gas chromatography mass spectrometry ................................ ................. 70 Peak analysis ................................ ................................ ............................. 71 Results ................................ ................................ ................................ .................... 71 Sequential Variables ................................ ................................ ........................ 71 Non sequential Var iables ................................ ................................ ................. 72 Cohort level variables ................................ ................................ ................ 73 Insect level variables ................................ ................................ .................. 73 Waveform event level variables ................................ ................................ . 74 Waveform event transitions ................................ ................................ ........ 75 Gas Chromatography Mass Spectrometry Analysis ................................ ........ 76 Phloem Metabolite Concentration ................................ ................................ .... 77 Xylem Metabolite Concentration ................................ ................................ ....... 78

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7 Discussion ................................ ................................ ................................ .............. 80 4 PROBING BEHAVIOR OF DIA PHORINA CITRI (KUWAYAMA) (HEMIPTERA: LIVIIDAE) ON FOUR CLEOPATRA MANDARIN, CITRUS RETICULATA , HYBRID SELECTIONS AND PUMMELO, CITRUS MAXIMA , IN RELATION TO WHOLE LEAF METABOLITES ................................ ................................ ............... 97 Introduction ................................ ................................ ................................ ............. 97 Materials and Methods ................................ ................................ .......................... 100 Plants and Insec ts ................................ ................................ .......................... 100 Electropenetrography Equipment and Settings ................................ .............. 101 EPG Data Analysis Programs and Variables ................................ .................. 102 Whole Leaf Metabolites ................................ ................................ .................. 103 Metabolite Statistical Analysis ................................ ................................ ........ 104 Di aphorina citri Oviposition and Survivorship Comparison ............................. 104 Results ................................ ................................ ................................ .................. 105 Sequential Variables ................................ ................................ ...................... 105 Ranking Mandarin and Pummelo Using Sequential Variables ....................... 106 Non sequential Variables ................................ ................................ ............... 108 Cohort level variables ................................ ................................ .............. 1 08 Insect level variables ................................ ................................ ................ 108 Waveform event level variables ................................ ............................... 109 Metabo lic Profiles ................................ ................................ ........................... 110 Diaphorina citri Oviposition and Survivorship Comparison ............................. 111 Discussion ................................ ................................ ................................ ............ 112 5 CONCLUSIONS ................................ ................................ ................................ .... 133 REFERENCES ................................ ................................ ................................ ............ 137 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 151

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8 LIST OF TABLES Table page 3 1 Differences in timing and duration of Diaphorina citri on Sour Orange and Valencia. ................................ ................................ ................................ ............. 85 3 2 Diaphorina citri probing behaviors on Sour Orange and Valencia. ..................... 86 3 3 Phloem percent metabolite concentration in Valencia and Sour Orange. ........... 87 3 4 Xylem percent metabolite concentration in Valencia and Sour Orange. ............. 88 3 5 Diaphorina citri waveform event transitions. ................................ ....................... 89 4 1 Probing behavior of Diaphorina citri on Cleopatra Mandarin hybrid selections and Pummelo summary of non phloem sequential variables. .......................... 117 4 2 Probing behavior of Diaphorina citri on Cleopatra Mandarin hybrid selections and Pummelo summary of phloem associated sequential variables. .............. 118 4 3 Using sequential variables to rank Mandarin selections and Pummelo for resistance. ................................ ................................ ................................ ........ 119 4 4 Summary of resistance ranking for Mandarin selections and Pummelo. .......... 119 4 5 Probing behavior of Diaphorina citri on Cleopatra Mandarin hybrid selections and Pummelo. ................................ ................................ ................................ .. 120 4 6 Metabolites in 0.1g leaf tissue in Cleopatra Mandarin hybrid selections an d Pummelo organic acids, fatty acids, and amino acids. ................................ .... 121 4 7 Metabolites in 0.1g leaf tissue in Cleopatra Mandarin hybrid selections and Pummelo sugars. ................................ ................................ ............................ 122 4 8 Metabolites in 0.1g leaf tissue in Cleopatra Mandarin hybrid selections and Pummelo sugar alcohols and su gar acids. ................................ ...................... 123 4 9 Diaphorina citri oviposition and survivorship to adult on Cleopatra Mandarin hybrid selections, Citrus reticulata , and Pummelo, Citrus grandis . ................... 124

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9 LIST OF FIGURES Figure page 1 1 Images of Diaphorina ci tri in various life stages ................................ .................. 36 2 1 Feeding site histology and waveform traces corresponding to medium to long probes terminated during waveform C. ................................ ............................... 53 2 2 Feeding site histology and waveform traces corresponding to short probes terminated during waveform C ................................ ................................ ............ 54 2 3 Waveform D correlation with stylets in phloem and phloem salivation ............... 55 2 4 Waveform D correlation with stylets in phloem and phloem cell filled with saliva ................................ ................................ ................................ .................. 56 2 5 Waveform D correlation with stylets in phloem ................................ ................... 57 2 6 Waveform G is correlated with xylem ingestion ................................ .................. 58 2 7 Waveform E1 corresponds with stylets in phloem ................................ .............. 59 2 8 Waveform E2 corresponds with stylets in phloem ................................ .............. 60 2 9 Scanning electron micrographs of Diaphorina citri intercellular and intracellular probing ................................ ................................ ............................ 61 2 10 Scanning electron microscopy preparation of slides ................................ ........... 62 3 1 Waveform duration per insect, in hours, and total number of waveform events produced by Diaphorina citri probing Sour Orange and Valencia. ...................... 90 3 2 Percent concentration of phloem metabolites metabolite groups and organic acids. ................................ ................................ ................................ .................. 91 3 3 Percent concen tration of phloem metabolites sugars, sugar alcohols, and sugar acids. ................................ ................................ ................................ ........ 92 3 4 Percent concentration of phloem metabolites fatty acids and amino acids. ...... 93 3 5 Percent concentration of xylem metabolites metabolite groups and organic acids. ................................ ................................ ................................ .................. 94 3 6 Percent concentration of xylem metabolites sugars, sugar alcohols, and sugar acids. ................................ ................................ ................................ ........ 95 3 7 Percent concentration of xylem metabolites fatty acids and amino acids. ......... 96

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10 4 1 Diaphorina citri probing durations on Mandarin hybrid selections and Pummelo waveform duration per insect (WDI) in hours. ................................ . 125 4 2 Percentage composition of metabolic groups found in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ ................ 126 4 3 Percentage composition of organic acids in Mandarin selections, Citrus reticulata, and Pummelo, Citrus grandis. ................................ .......................... 127 4 4 Percentage composition of fatty acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ .......................... 128 4 5 Percentage composition of amino acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ .......................... 129 4 6 Percentage composition of sugars in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ ................................ .......... 130 4 7 Percentage composition of sugar alcohols in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ .......................... 131 4 8 Percentage composition of sugar acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . ................................ .......................... 132

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11 LIST OF ABBREVIATION S Abb Abbreviation ANOVA Ca Analysis of Variance statistical test Candidatus CtoFrstG Time from beginning of waveform C to first waveform G event Deriv Derivative DF Degrees of freedom, sample size minus 1 Dur Duration DurFirstE Duration of first E waveform event (mean) DurG Mean duration of G waveform DurNnprbBfrFrstD Duration of non probing before first waveform D event DurNnprbBfrFrstE1 Duration of non probing before first E1 wf event (mean) DurNnprbBfrFrstG Duration of n on probing before first wf G event (mean) G Gram GC MS Gas Chromatography Mass Spectrometry HLB Huanglongbing disease of Citrus Hr/h Hour LS Least squares maxD Longest waveform D event maxE2 Longest waveform E2 event meanD Average duration of waveform D events Min Minute(s) MnDurC Shortest duration of waveform C (mean)

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12 MnDurE1 Minimum duration of E1 waveform events MnDurE2 Minimum duration of E2 waveform events NumLngD Number of long waveform D events (mean) NumLngE2 Number of long E2 wf events (mean), greater than 10 min NumLngG Number of long G wf events (mean), greater than 1 hour NWEI Number of waveform events per insect (mean) PDI Probing duration per insect, mean duration per treatment PrcntPrbC Mean percent probing spent as waveform C PrcntPrbD Mean percent of probing spent as waveform D PrcntPrbE2 Mean percent of probing spent performing waveform E2 PrcentPrbG Mean percent of probing spent as waveform G PrcntE2SusE2 Mean percent of probing spent as sustained waveform E2 Sec Second(s) sdPrbs Standard deviation of mean probe duration ShrtCbfrE1 Duration of the shortest C waveform before E1 SE Standard error TmBegPrbFrstD Time from beginn ing of probe to first D wf event (mean) TmBegPrbFrstE Time from beginn ing of probe to first E wf event (mean) TmFrmFrstPrbFrst D Time from first probe to first D waveform event (mean) TmFrmFrstPrbFrstE Time from first probe to first E waveform event (mean) TmFrstE2FrmFrstPrb Time from first E2 eve nt from time of start of first probe TmFrstE2FrmPrbStrt T ime from first E2 event from start of probe with E2 event TmFrstE2StrtEPG Time to first E2 waveform event from start of recording (mean)

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13 TmFrstPrbFrmStrt Time to first probe from start of recording (mean) TmFrstSusDFrstPrb Time to first sustained D from first probe (mean) TmFrstSusE2 Time to first sustained waveform E2 event (mean) TmFrstSusE2FrstPrb Time from f irst sustained E2 wf from time of start of probe TmFrstSusE2StrtPrb Time from first sustained E2 event from the start of probe TmFrstSusGFrstPrb Time to first sustained wf G from start of first probe (mean) TmLstE2EndRcrd Time to start of last waveform E2 event (mean) TNP Total number of probes per cohort TPD Total probing duration f or cohort, all insects in a treatment TtlDurC Total duration of C waveform events TtlDurE Total duration spent performing both E1 and E2 waveforms TtlDurE1FllwdE2PlsE2 Total duration of waveform E1 by waveform E2event TtlDurE2 Total duration of E2 waveform events TtlDurNnPhlmPhs Total duration of non phloem phase, sum of non phloem events TtlDurNP Total duration of time spent not probing (mean) TtlPrbTm Total probing time, mean duration spent probing Unk Unk n own WDI Waveform duration per insect, mean duration per treatment Wf Waveform

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy P ROBING BEHAVIOR AND HOST PREFERENCE IN THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) By Holly Shugart May 2019 Chair: Michael Rogers Major: Entomology and Nematology The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus worldwide. D. citri is a vector of the plant pathogenic bacterium Candidatus Liberibacter asiaticus, causal agent of Huanglongbing disease affecting citrus growing regions worldwide. Current management practices in citrus groves involve copious use of pesticides to eliminate psyllids with the hope of preventing transmission of C a . L. asiaticus. In an effort to minimize pesticide use, researchers are working to develop citrus varieties, selections, or rootstocks which are either resistant to psyllid feeding and transmission or which are resistant to the development of Huanglongbin g disease . In order to develop citrus varieties resistant to psyllid feeding, we must know the details of psyllid feeding behavior and transmission of Ca . L. asiaticus. The work outlined here aims to address the gaps in knowledge of psyllid feeding behavio r in Citrus with an aim of identifying potential characteristics of resistant Citrus species, varieties, or selections which may be used to develop additional resistant varieties or selections. This work employs techniques such as electropenetrography, pla nt histology and microscopy, phloem and xylem chemistry, and whole leaf composition on a variety of susceptible and resistant

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15 Citrus species, selections, and varieties with aim of identifying which attributes contribute to psyllid host preference.

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16 CHAPTER 1 INTRODUCTION TO THE PROBING BEHAVIOR OF DIAPHORINA CITRI Electropenetrography as a T ool to S tudy H emipteran P robing B ehavior This dissertation investigates various aspects of the probing behavior and host preferen ces of the Asian citrus psyllid, Diaphorina citri (Kuwayama), (Hemiptera: Liviidae). Psyllids and other hemipterans have piercing, sucking mouthparts called stylets which cannot be observed during host plant feeding. As such, hemipteran feeding cannot simply be observed by watching or video recording what test insects do while on host pla nts. Rather, researchers must employ a variety of complex research tools in order to answer the question of what insects are doing when their stylets are embedded in plant tissue. Electropenetrography (EPG) allows researchers to visualize hemipteran prob ing behavior in real time by connecting the insect as part of an electrical circuit and measuring the voltage changes generated when the insect inserts the stylets and performs a wide range of behavi ors within host plant tissues. Since the waveforms genera ted as part of EPG research are only generated with electrical contact of the stylets with host plant tissues, EPG is a measure of probing behavior and not feeding behavior. While it is true that a small amount of electrical contact is made when an insect is placed on an electrified host plant, EPG is really only a measure of probing versus non probing behavior. In the absence of additional types of monitoring of the insect behavior prior to stylet insertion, for example, video recordings, EPG is blind to the majority of the activities carried out by the insect prior to the insertion of the stylets. This dissertation uses EPG to address many aspects of psyllid probing behavior in relation to host plant resistance to psyllid probing in a variety of citrus sp ecies, var ieties, and hybrid selections. The waveforms generated by psyllid probing can represent a

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17 variety of probing behaviors, including salivation, stylet movements , puncturing of host plant cell walls and plasma membranes , ingestion, and behaviors rel evant to the transmission of plant pathogens, such as acquisition and inoculation of plant pathogens (Walker, 2000) . History of E lectropenetrography T echnology The original electropenetrograph (EPG) monitor was developed by McLean and Kinsey (1964) and used alternating current (AC) to record the probing behavior of aphids. The AC monitor design was later expanded upon by Backus and Bennett (1992) . Prior to 2009, AC EPG systems all operated using an input impedance ranging between 10 4 to 10 6 Ohms (Backus, 1994) . Schaefers (1966) developed an EPG system using direct cu rrent (DC) which was expanded upon by W. F. Tjallingii (1978) . DC EPG systems operate using input impedance settings ranging from 10 8 to 10 11 Ohms (Backus, 1994) . Recent improvements by Backus and collaborators have expanded the potential application for recording many different types of insects and arthropods by adding a switchable input impedance function and flexible AC or DC recording options . The most current terminology for this technology is as follows. The instrument is termed an electropenetrograph, th e waveform output is an electropenetrogram, while electropenetrography refers to the overall technology used for the study of insect probing behaviors () . Previous naming conventions have included electrical penetration graph or electrical penetra tion graph monitor (Backus, 2016) , and electronic monitoring system (Walker, 2000) . Comparison of AC and DC EPG S ystem S ignal P rocessing Over the course of the last fifty five years since the invention of the EPG monitor technology (Backus, 1994) , two primary circuit pro cessing technologies have been

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18 utilized in EPG monitors either AC or DC. The original signal processing used by McLean and Kinsey (1964) was an AC signal processing design and since that time the AC EPG system has unde rgone many changes, with as many as seven different system designs reported in the literature (McLean & Kinsey, 1964; Backus et al. , 2000) . Early designs of the AC EP G monitors were best suited to working with larger Hemiptera, including leafhoppers (Backus et al. , 2005; Backus & Bennett, 2009) , planthoppers (Wayadande & Nault, 1993) , and heteropterans (Velusamy & Heinrichs, 1986; Bonjour et al. , 1991) . AC EPG systems and the associated input imped ance range of between 10 4 to 10 6 Ohms were better suited to recording larger auchenorrhynchan and heteropteran insects due to the way in which the applied voltage combined with the generated electromotive forces (emf) pass through the food canal and other portions of the insect foregut. The theoretical construct of how the electrical current passes through recorded insects and the impact on waveform appearance have been illustrated using R/emf responsiveness curves, first posited by Backus et al. (2007) and further explained as to how these electrical components relate generally to insect size in W. F. Tjallingii (1985a) . DC EPG systems have been primarily used to record the probing behavior of sternorrhynchans, primarily aphids (W. F. Tjallingii, 1985b; W. F. Tjallingii & Hogen Esch, 1993; W. Fred Tjallingii et al. , 2010; Backus et al. , 2019 ) , but also a few whiteflies et al. , 2012) , and mealybugs (Walker & Perring, 1994) . There is growing evidence that some hemipterans are sensitive to DC signals, especially large auchenorrhynchans, including sharpshooter leafhoppers (Cicadellidae: Cicadellinae) (Calatayud et al. , 1994) , but also heteropterans, such as Lygus spp. (Cervantes &

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19 Backus, 2018) . Although some hemipterans have a demonstrated sensitivity to DC applied during EPG recording, not all tested hemipterans appear to be sensitive to this type of signal. Cervantes et al. (2016) e xposed D. citri to a wide range of DC EPG voltages from 20 to 600 mV an d found no effect on probing behavior below 600 mV. EPG S etting C hoices in T his D issertation The EPG recordings made as a part of this dissertation used DC EPG applied votage of 150 mV, following protocols of previous research done on D. citri in the sam e lab space (Serikawa, 2011; Serikawa et al. , 2012; Ebert & Rogers, 2016; Ebert et al. , 2018) , using the same EPG monitors (Backus & Bennett, 2009; Serikawa et al. , 2013) . Additionally, most EPG recordings on psyllids have been done using DC EPG, including those on D. citri (Backus & Bennett, 2008; Bonani et al. , 2010; Cen et al. , 2012; Serikawa et al. , 2012, 2013; Luo et al. , 2015; Ebert & Rogers, 2016; Ebert et al. , 2018) , Bactericera cockerelli (Butle r et al. , 2012; Sandanayaka et al. , 2014; Miranda et al. , 2016) , and Cacopsylla pyri (Civolani et al. , 2013; Pearson et al. , 2014) . Another important setting for the EPG recordings made as a part of this body of work is the input impedance setting. The AC DC monitors used in this body of work have flexible, switchable input impedance settings (10 6 , 10 7 , 10 8 , 10 9 , 10 10 , and 10 13 ), allowing users to choose this important setting based on the recorded insect species and the goal of the EPG research (Backus & Bennett, 2009; Civolani et al. , 2011) . In this dissertation, EPG recordings made to correlate EPG waveform meaning were done at an input impedance of 10 8 Ohms, with an aim to highlight the R component of waveforms, making waveform type determination easier during real time EPG recording so as to increase the accuracy of waveform determination during the interruption of probing (methods described in Chapter 2). Also in Chapter 2, EPG recording s were

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20 made at various input impedances (10 7 , 10 8 , 10 10 , and 10 13 ) in an effort to determine if a pd waveform could be found in recordings made at input impedances other than those published in the literature to date (10 9 Ohms). EPG recordings made as a part of Chapters 3 and 4, used DC applied voltage of 150 mV and 10 9 input impedance to be more in line with what previous studies have used and with the expectation that these settings did not have an unnecessary negative i mpact on D. citri probing behavior (Backus & Bennett, 2008) . Overview of H emipteran Probing B ehavior Hem ipterans perform complex, and often invisible, behaviors deep within plant tissues. The unique terminology associated with these complex behaviors is complicated and often terms are used synonymously within the literature when they each have distinct meani ngs (Ebert & Rogers, 2016) . In reference to hemipteran insects, the terms feeding, probing and ingestion are not synonymous and should be defined before exploring the topic further. Feeding behavior represents the broadest category of these terms. Feedi ng behavior is defined here as all of the activities performed with the express goal of acquiring and ingesting food (Backus, 2000) . Feeding behaviors may include the interpretation of visual or olfactory cues by the insect that might cause them to orient towards o r land on a particular host plant or olfactory or visual cues encountered on the surface of the plant, prior to the insect making a decision to insert their mouthparts into the plant tissues. The interpretation of, and response to, sensory stimuli, both fr om the air prior to landing on a plant and those perceived while on the plant surface, combined with stimuli encountered by sensory organs on the mouthparts, termed stylets, or within the foregut, all encompass behaviors that fall within the broad category of feeding behavior (Backus, 2000;

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21 Wenninger et al. , 2009; Youn et al. , 2011) . Due to the piercing sucking mouthpart anatomy of hemipterans, the term probing is used to discern activities occurring while the mouthparts are embedded in host tissues. However, not all probing activities are specifically involved in d irectly taking up plant cell contents as food, or ingestion. Much of the time spent probing is expended salivating, tasting, and making decisions about stylet location within host plant tissues. Ingestion represents the narrowest category of feeding behavi or and specifically refers to behaviors performed while plant cell contents are being taken up into the foregut and swallowed into the esophagus (Arras et al. , 2012) . Hemipteran probing on plants occurs in two broad categories, termed feeding strategies (Garzo et al. , 2012) . Hemipteran feeding strategies are classified as either salivary sheath fe eding or cell rupture feeding. The salivary sheath feeding strategy may occur as intercellular, intracellular or as som e combination of both tactics. Intercellular feeding describes a stylet path that passes in between the middle lamellae of adjacent cells such that the stylets do not break plasma m embranes or interact with cell contents for many of the cells as they pass en route to the preferred feeding tissues (Backus et al. , 2005) . During intercellular probing, th e middle lamella is enzymatically dissolved so the stylet s may pass through this space. The middle lamella is made up of pectin and thus pectinases must be a large portion of the salivary product of insects that employ the intercellular probing tactic (McAllen & Adams, 1961; W. F. Tjallingii & Hogen Esch, 1993) . During intercellular probing, the four stylets remain together as they pass thr ough host plant tissues until they enter a phloem sieve element, when only the maxillary stylets enter the cell during phloem salivation (E1) and phloem ingestion (E2) (Dreyer &

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22 Campbell, 1984) . One benefit to the insect of the intercellular probing tactic is that the stylets are kept separate from cell contents and any potential toxins or deterrents that may exist to protect the cells aga inst herbivores (W. F. Tjallingii & Hogen Esch, 1993) . As insects probe intercellularly, they only interact with cell contents when the in sect chooses to taste selected cells along this pathway in an effort to determine stylet location, cell type or to make decisions about how to proceed within the plant or whether to ingest cell contents. Most Sternorrhyncha have been documented as using t he intercellular probing tactic (see below for m ore on this topic) (Goggin, 2007) . In contrast, most Auchenorrhyncha and Heteroptera who employ the sheath probing strategy have been shown to use the int racellular tactic (Backus et al. , 2005) . Auchenorrhynchans probe directly through the middle of cells as they pass through plant tissues and thus encounter a mix of their own saliva and plant cell contents at the tip of their stylets. One important advantage to the intracellular probing tactic is that the insect passes through tissues very quickly and is able to begin ingestion from preferred tissues often within a matter of a few minutes (Backus et al. , 2005) rather than the hours long time frame before the intercellular prober begins sustained ingestion from preferred tissues (Backus et al. , 2009) . The cell rupture feeding strat egy may occur as one of many tactics. Cell rupture feeding tactics include lacerate and sip, lacerate and flush, or lance and ingest (Ebert et al. , 2018) . Each of these tactics involves intracellular stylet penetration, variable and mostly unconnected salivary deposits, a nd cell emptying of parenchyma tissues in addition to some disruption of the phloem tissue. The Coreidae (Heteroptera) use an osmotic pump feeding strategy which does not fit well into either the salivary sheath or

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23 cell rupture strategies. This strategy involves making a shallow salivary sheath which does not reach the ingestion tissues, injecting enzymatic saliva into the intracellular fluid thus changing the osmotic concentration of the fluid, which allows the insects to imbibe fluid from the vascular t issues without the stylets actually reaching the vascular tissues (Mitchell, 2004; Backus et al. , 2005) . Psylloidea Prob ing B ehavior Taxonomic Classification of Psyllids Psyllids are classified within the order Hemiptera (Miles & Taylor, 1994) and into the suborder Sternorrhyncha. Other insects classified as Sternorrhyncha include, aphids (Aphoidea), mealybugs (Pseudococcidae), whiteflies (Aleyrodoidea), scale insects (Coccidea), phylloxera and woolly con ifer aphids (Phylloxeroidea). All other Sternorrhyncha examined are reported in the literature to probe using the intercellular salivary sheath tactic, including: aphids (Brennan et al. , 2001; Forero, 2008) , mealybugs (Hewer et al. , 2011) , whiteflies (Calatayud et al. , 1994) , scale insects (Walker & Perring, 1994) . Psyllids are classified into a superfamily named Psylloidea (Brennan et al. , 2001) . Burckhardt and Ouv rard (2012) classify psyllids into eight families within the superfamily Psylloidea. This new classification involved a family shift for the psyllid studied in this dissertation. Formerly, D iaphorina citri was classified within the family Psyllidae, and is now classified within the family Liviidae. Histological E vidence of Psyllid Probing Behavior Most Sternorrhyncha investigated thus far have been reported to feed using the intercellular feeding tactic (Burckhardt & Ouvrard, 2012) . However, aphids represent the bulk of research on sternorrhynchan feeding behavior. Aphids have been shown to enzymatically dissolve the middle lamellae between host plant parenchyma cells en

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24 route to vascula r tissues (W. F. Tjallingii & Hogen Esch, 1993) . A careful analysis of the literature demonstrates that psyllids employ the salivary sheath feeding strategy but are highly variable in the performance of feeding tactics. Some psyllids appear to utilize the intercellular feeding tactic while others utilize the intracellular tactic and still others seem to use both tactics even wi thin the same salivary sheath (discussed in the next para graph). The literature does not discuss this variability in psyllid feeding strategies and usually psyllids are reported to feed intercellularly in the same manner as aphids . Diaphorina citri does not appear to feed in the same manner as aphids. The app earance of salivary sheaths of D. citri is mostly intercell ular. However, D. citri salivary sheaths also sometimes pass through cells clearly away from the cell walls in a manner consistent with intracellular penetration, (W. F. Tjallingii & Hogen Esch, 1993; Ammar & Hall, 2012) images 3H and 3I show saliv ary sheath with mixed inter and intracellular penetration. For the Asian citrus psyllid, D. citri , and the potato psyllid, Bactericera cockerelli , only light or fluorescent micrographs of relatively thick sections of salivary sheaths have been published : D. citri , 12 µm (Ammar & Hall, 2012) , D. citri , hand sections 50 70 µm, (Bonani et al. , 2010) , and B. cockerelli , 10 µm , (Ammar & Hall, 2012) . Electron microscopy or high resolution confocal microscopy is needed to be sure of the position of salivary sheaths in relation to the cell walls and to be certain whether plasma membranes have been br oken . Pearson et al. (2014) published images of the salivary sheaths of four psyllid pests of eucalyptus. Psyllids studied included, Cardiaspina albitextura , Creiis costatus , Lasiopsylla rotundipennis , and Glycaspis spp . These four psyllids are all classified within th e family Psyllidae. The light micrographs taken using a compound light

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25 microscope appear to depict intracellular feeding. One unique aspect of this study compared to other histological studies of psyllid salivary sheaths is that the authors sectio n ed the salivary sheaths of nymphs only and no adult psyllids were included as a part of this study. Woodburn and Lewis (1973); Brennan and Weinbaum (2001) studied the saliv ary sheaths of three psyllid species ( Ctenarytaina spactulata, C. eucalypti, and Glycaspis brimblecombei ) all of which are classified within the family Psyllidae and all pests of eucalyptus. Again , the light micrographs taken using a compound light microsc ope appear to depict intracellular feeding. The highest magnification images of psyllid feeding were published by Brennan and Weinbaum (2001) on the European pear psylla, Cacopsylla pyri . The images ar e primarily transmission electron micrographs (including some with cut stylets) and include a single light micrograph showing the overview of a salivary sheath. The overview of the path of the salivary sheath in relation to the parenchyma published in this paper show s that C. pyri employs the intrac ellular feeding tactic ( Figure 7a ) , (Civolani et al. , 2011) . (Civolani et al. , 2011) Additional evidence of intracellular penetration in C. pyri includes parenchyma cells filled with salivary sheath material along t he stylet pathwa y ( Figure 7b ) (Civolani et al. , 2011) . The only way saliva can fill a cell in this m anner is if the insect punctures the plasma membrane so that the stylets and/or saliva ente r the living portion of the cell, the symplast (Civolani et al. , 2011) . Known EPG Waveforms and Waveform Correlations of Psyllid Probing Behavior

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26 Diaphorina citri the Asian C itrus P syllid Diaphorina citri waveforms have been partially correlated histologically with salivary sheath termini (Hewer et al. , 2011) . Bonani et al. (2010) interrupted feeding D. citri as particular waveforms were produced and collected the feeding site of the citrus leaf for histological processing and microscopic imaging of t he correlated salivar y sheath. In D. citri , the C waveform has been correlated with salivary sheath termini in parenchyma, D waveform with sheath termini at the edge of the phloem, E1 in phloem, and E2 i n phloem (Bonani et al. , 2010) . The G waveform was observed but not histologically correlated. The G waveform is extrapolated by the authors to represent xylem ingestion, based on its likeness with the G waveform appearance for aphids (Spiller et al. , 1990; Bonani et al. , 2010) . As part of this dissertation work, the G w aveform has been correlated with salivary sheath termini in xylem (Chapter 2). Bactericera cockerelli the P otato P syllid Powell and Hardie (2002) used both histological observ ations of salivary sheath termini and the electrical origins of the EPG signal to correlate the probing behaviors of the potato psyllid, B. cockerelli (Triozida e). Pearson et al. (2014) named B. cockerelli waveforms following the model of waveforms names used for the European pear psylla, Cacopsylla pyri , by Pearson et al. (2014) . Civolani et al. (2011) histologically correlated B. cockerelli waveform A with sheath termini terminating on the plant surface, waveforms B, C1, and C2 in t he parenchyma, waveforms E1 and E2 in the phloem, and waveform G in the xylem vessel elements. Pearson et al. (2014) did not histologically correlate wavefor m D for B. cockerelli .

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27 Cacopsylla pyri the European P ear P sylla Pe arson et al. (2014) histologically correlated the EPG waveforms produced by Cacopsylla pyri on pear. Wavefo rms A and B were correlated on the plant surface. Waveform C1 was correlated with parenchyma and C2 with vascular parenchyma. Waveform D, E1, and E2 were all correlated with stylet tips in the phloem sieve elements. Waveform G was correlated with xylem vessels and with pericyclic fibers. P d W aveform The pd (potential drop) waveform of aphids is well correlated with the act of puncturing a plasma membrane , salivation and tasting behaviors within the intracellular space of this cell, followed by stylet withdrawal back into the extra cellular space (Civolani et al. , 2011) . The pd waveform can represent the tasting and rejection of any cell adjac ent to the salivary sheath or of an individual phloem sieve element by an aphid, or, if followed by E1 and E2 waveforms, the pd waveform represents the tasting and acceptanc e of the phloem sieve element. The pd waveform should be found in EPG studies of he mipterans which utilize intercellular feeding in the manner described for aphids (W. F. Tjallingii, 1985b) . One important difference between the probing behavior of psylli ds and aphids is a lack of pd waveforms observed in the best representative psyllid EPG studies (W. F. Tjallingii, 1985b; Bonani et al. , 2010; Civolani et al. , 2011) . As a part this dissertation, D. citri has been recorded at a variety of input impedances in order to visualize multiple possible waveform outcomes. Recordings at lower input impedances (10 7 and 10 8 ) emphasize behaviors representing salivation, and large scale stylet movements. These waveforms are generated as a result of the conductivity of the environ ment at the tip of the stylets. Salivation, for example, generates a rapid rise in conductiv ity at the tip of the stylets. Recordings at higher input

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28 impedances (10 9 and 10 10 and 10 13 ) emphasize behaviors repre senting tasting and ingestion. These waveforms are generated as a result of tiny muscular movements within the head of the insect and from streaming potentials (Pearson et al. , 2014) . No waveform is consistently produced by D. citri which could correspond to a pd waveform in psyllids. However, the D waveform of D. citri has been shown to represent the tasting and subsequent acceptance or rejection of a phloem sieve element ( (W. F. Tjallingii, 1985b) and Chapter 2). While aphids perform pd waveforms at many points along thei r path to the phloem, tasting several parenchyma cells in the performance of each probe (Bonani et al. , 2010) , psyllids do not perform pd waveforms in parenchyma cells. Ra ther, psyllids only perform the pd like D waveform in the phloem. In order for a waveform like the pd or D waveform to be generated, there must be separation of the apoplast (outside of the cell) and symplast (inside of the cell). This separation can only be maintained during intercellular probing. When aphids probe parenchyma cells, they pass through the apoplast in between adjacent cells. During tasting behaviors, aphids puncture a cell wall and plasma membrane, such that they only insert the tip of the ir maxillary stylets into the living portion of the cell (W. F. Tjallingii, 1985b; W. F. Tjallingii & Hogen Esch, 1993) . If the cell is rejected, the aphid rem oves its stylets, seals the hole they made, and repositions their stylets to continue along the original path. An aphid, probing in this manner, does not mix the contents of parenchyma cells with their own saliva as they pass through the apoplast en route to the phloem. So, it makes sense that D. citri and other psyllids do not make pd waveforms in parenchyma, because they do not maintain a separation of the apoplast and symplast due to their

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29 combined inter and intracellular probing. It appears, even thou gh they sometimes perform intercellular probing, that psyllids encounter a mix of saliva and leaked plant cell cytoplasm at the tip of the stylets, creating a combined source of conductivity to generate the C waveform. Psyllids certainly taste parenchyma c ells en route to the phloem so they can make decisions about their location within the plant. However, their mixed inter and intracellular probing tactic makes for a complicated electrical origin of the C waveform. Likely, the delicate tasting behaviors p erformed within parenchyma cells are lost within the voltages generated by the saliva and leaked plant cell cytoplasm that also surrounds their stylet tips. Psyllid M outhpart M orphology Diaphorina citri have sufficiently long stylets to wind their style ts along an indirect path through the apoplast rather than via a direct route (intracellularly through the symplast) in order to reach the vascular tissues. This is accomplished, in part, by the psyllids ability to retract the stylets into a chamber, term ed a crumena, located within the head, near the base of the labium (W. F. Tjallingii & Hogen Esch, 1993) . It is unclear whether psyll ids, like aphids, have an acrostyle at the tip of the stylets (Uzest et al. , 2010; Garzo et al. , 2012) . The ac rostyle may play an important role in the binding of viruses transmitted by aphids in a non persistent manner within the common canal, where the food and salivary canals join at the distal tip of the mandibular stylets (Garzo et al. , 2012) . Candidatus Liberibacter D iseases of A gricultural C rops Diaphorina citri as a P utative V ector of Candidatus Liberibacter asiaticus Diaphorina citri is an invasive insect to North America and transmits the phloem infecting fastidious bacterium Candidatus Liberibacter asiaticus. In the United States,

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30 Candidatus L. asiaticus is transmitt ed in a persistent manner by D. citri as well as through grafting of citrus trees (Blanc et al. , 2011) . D iaphorina citri can acquire Ca. L. asiaticus within a 15 minute acquisition access period (Batool et al. , 2007) . Diaphorina citri reared from eggs on infected citrus plants acquired Ca. L. asiaticus at a rate of 60% as newly emerged adults after an acquisition access period of 35 days (Pelz Stelinski et al. , 2010) . Under lab conditions a single infected adult D. citri averages a 5% inoculation rate with an infection that can be PCR verified (Pelz Stelinski et al. , 2010) . In an attempt to estimate transmission rates of D. citri under representative field conditions, Pelz Stelinski et al. (2010) cag ed 200 psyllids reared from eggs on infested citrus onto uninfected citrus and found a 73% transmission rate of Ca. L. asiaticus. Additionally, D. citri can sexually transmit Ca. L. asiaticus between sexual partners and transovar ially from mother to offspr ing. Diaphorina citri mothers can transovarially transmit Ca. L. asiaticus to their offspring at a low rate of 3.6% (Pelz Stelinski et al. , 2010) and Ca. L. asiaticus can be transmitted sexually during copulation at a low level of 4% from males to fema les (Pelz Stelinski et al. , 2010) . Other P syllid V ectors of Liberibacter P lant P athogenic B acteria Diaphorina citri is not the only psyllid known to transmit Liberibacter bacteria to their host plants. A variety of other psyllids are known to transmit Liberibacter plant pathogenic bacteria including the potato/tomato psyllid, B . cockerelli , the African citrus psyllid, Trioza erytreae , and the carrot psyllid, Trioza apicalis . B actericera cockerelli can transmit Ca . L. solanacearum to crops in the plant family Solanaceae, including potato and tomato (Mann et al. , 2011) . Buchman et al. (2011) showed that 20 infected adult psyllids could produce symptoms of zebra chip disease in a potato plant with an

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31 inoculation access period of only one hour. Additionally, a single infected adult B. cockerelli could inoculate Ca . L. solanacearum to potato within an inoculati on access period of six hours. Bactericera cockerelli is thought to be a result of a salivary toxin injected during feeding (Buchman et al. , 2011) . Buchman et al. (2011) corr elated B. cockerelli probing into phloem and acquisition of Ca . L. solanacearum from infected tomato with waveforms D, E1, and E2, all behaviors known to occur in phloem tissues. Transmission of Ca . L. solanacearum by B. cockerelli was correlated with all probing waveforms, though inoculation efficiency increased as the insect spent more time probing plant tissues, specifically phloem tissues (Sandanayaka et al. , 2014) . Trioza erytreae , transmits Ca . L. africanus, putative causal agent of citrus greening disease in Africa (Aubert, 1987; Sandanayaka et al. , 2014) . Additionally, T. apicalis transmits Ca . L. solana cearum, the putative causa l agent of carrot proliferation disease in Europe (Garnier et al. , 2000; Munyaneza et al. , 2010) . Hua nglongbing D isease of Citrus C aused by the B acterial P athogen, Candidatus Liberibacter asiaticus Candidatus Liberibacter bacteria are gram negative alpha proteobacteria within the family Rhizobiacea (Aubert, 1987) . Huanglongbing (HLB) of citrus is associated with three species of Candidatus Liberibacter. These include, Ca . L. asisaticus, Ca . L. africanus, and Ca . L. americanus (da Graca et al. , 2016) . Ca . Liberibacter bacteria can be transmitted by two citrus psylids, D. citri and T. erytreae (Bove, 2006) . Diaphorina citri and Ca. L. asiaticus can now be found in all citrus producing states within the continental United States, including Florida (da Graca et al. , 2016) , Texas (Halbert & Manjunath, 2004) , California (Kunta et al. , 2012) , South Carolina, Georgia, and Louisiana (Kumagai et al. , 2013) .

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32 HLB or citrus greening disease has quickly become the most ec onomically important disease of production citrus in North America since the accidental introduction of the psyllid to Florida in 1998 and the subsequent arrival of the disease in 2005 (Halbert & Manjunath, 2004; Halbert et al. , 2010) . The disease can also be found in many important citrus produ cing regions around the world. These regi ons include, Brazil since 2004 (Manjunath et al. , 2008) , Argentina and Paraguay (Teixeira et al. , 2005) , Cuba (Lopez et al. , 2013) , Jamaica (Luis et al. , 2009) , Belize (Oberheim et al. , 2011) , and Mexico (Manjunath et al. , 2010) Whole plant and organ symptoms of HLB include, yellow shoot, leaf mottling, as ymmetrical fruit, unripe fruit at harve st, fruit drop, and mature fruit that is bitter tasting (Trujillo Arriga et al. , 2010) . Internal tissue and cellular level symptoms of HLB include, the deposition of callose around infected tissues to prevent further spr ead of the bacteria (da Graca et al. , 2016) , thi cker phloem cell walls, and thicker and more disordered cambial tissues (Jones & Dan gl, 2006; Kim et al. , 2009; Folimonova & Achor, 2010) , and an accumulation of starch in phloem and necrotic phloem tiss ues (Aritua et al. , 2013) . There is currently no cure for HLB and management practices includes removal of infected limbs and trees, over watering and f ertilizing trees to compensate for dehydration and malnutrition resulting from the disease (Folimonova & Achor, 2010) . These tactics are used in addition to alternating application of insecticides with differing modes of action since D. citri has been shown to develop resistance to insecticides (Grafton Cardwell et al. , 2013) .

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33 Currently, researchers are unable to grow Liberibacter in pu re culture, impeding progress in the study of t he microbe plant interactions that lead to a lethal HLB infection. Specifically, the inability to culture Ca . Liberibacter pathogenic bacteria makes it impossible for researchers to genetically modify the pathogen. In order to better understand the microbe plant interaction as a result of an active HLB infection, several researchers have employed multiple molecular techniques on both uninfected and Libe ribacter infected citrus plants. These techniques include, comparisons of transcription profiles, protein expression profiles (Tiwari et al. , 2011) , and RNA expression profiles in order to better understand metabolic changes that occur in host plants as a result of HLB infection (Killiny & Nehela, 2017; Kruse et al. , 2017) . There is also some evidence of native immunity within rutac eous host plants to certain Ca. Liberibacter species, which might eventually lead to identifying host plant species that migh t be used for cross protection. A lthough there is some native immunity, there have yet to be any citrus seedlings or scion rootstoc k combination showing complete resistance to Ca . Liberibacter. There is some recent evidence that Murraya koenigii (Rutaceae) is immune to Ca . L. asiaticus infection (da Graca et al. , 2016) . Currently, we have no definitive defense against Ca . L. asiaticus with which to protect the citrus industry. Research Q uestions and O bjectives in T his Dissertation Our ability to maximize the data acquired though the recording of psyllid probing behavior utilizing the EPG technique is limited by our understanding of what probing behaviors the resulting waveforms actually represent. This lack of a comprehensive unders tanding of where stylets are located, including cell type and position of the stylets outside or within the living portion of the cells, or what the stylets are doing during the

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34 performance of each waveform, impedes the quality of the analyses resulting fr om EPG recordings of probing behavior. The electrical environment at the tip of the stylet bundle informs the electrical origin and the voltage of recorded waveforms and determines important portions of the waveform shape. If researchers are to translate w aveform shapes into meaningful behaviors, we benefit greatly from the quality and quantity of thorough waveform correlations. Chapter 2 aimed to fill in several gaps in our knowledge of how D. citri stylets move through Citrus tissues and correlate previ ously uncorrelated waveforms with stylet locations in specific tissue types. Of specific interest was whether D. citri stylets pass inter or intracellularly through tissues en route to phloem sieve elements, the primary target cells for ingestion behavior s. The research outlined in Chapter 2 also aims to improve the tissue correlations for important psyllid waveforms, including waveform G (putative xylem ingestion previously uncorrelated), and waveform D (phloem contact with a single, low image quality correlation). This research also contributes an increase in image quality by using scanning electron micrographs in combination with compound light micrographs to elucidate D. citri probing behaviors. The experiments in Chapters 3 and 4 both explored the levels of resistance or susceptibility in a range of Citrus species to the probing of D. citri using EPG with an effort to relate this resistance or tolerance to D. citri probing with host plant metabolite profiles. Chapter 3 investigates D. citri probing behavior on Sweet Orange, Citrus sinensis , var. Valencia, compared to Sour Orange, Citrus aurantium . The metabolic profiles of extracted phloem and xylem from each host species were determined using gas chromatography mass spectrometry. This dissertation seeks to link important

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35 metabolic aspect of host plants to the probing behavior outcomes of D. citri on each species. Do the metabolic profiles in the phloem and xylem of different host species impact psyllid probing behavior, and contribute to host plant resistance or tolerance to the psyllid? Chapter 4 investigated D. citri probing behavior on, four Cleopatra Mandarin hybrid selections, Citrus reticulata x Citrus ichangensis and Pummelo, Citrus grandis . The whole leaf metabolic profiles from each host s pecies were determined using gas chromatography mass spectrometry. Citrus reticulata and C. ichangensis have both demonstrated resistance to psyllids in previous studies, but have never been fully investigated using a combined EPG and GC MS study. Does the whole leaf metabolite profile of each species or selection contribute or determine the resistance of that species or selection to D. citri probing?

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36 Figure 1 1. Images of Diaphorina citri in various life stages. A)

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37 CHAPTER 2 PSYLLID S ARE NOT APHIDS A STUDY OF COMBINED INTERCELLULAR AND INTRACELLULAR PROBIN G TACTICS OF THE ASI AN CITRUS PSYLLID, DIAPHORINA CITRI (HEMIPTRA: LIVIIDAE) , AND SOME HISTOLOGI CAL CORRELATIONS OF STYL ET TIP LOCATION DURI NG THE PERFORMANCE O F SELECTED WAVEFORMS Introduction Hemipteran probing on plants occurs in two broad categories, termed feeding strategies (Beloti et al. , 2018) . Hemipteran feeding strategies are classified as either salivary sheath fe eding or cell rupture feeding. The salivary sheath feeding strategy may occur as intercellular, intracellular or as som e combination of both tactics. Intercellular feeding describes a stylet path that p asses in between the middle lamellae of adjacent cells such that the stylets do not break plasma membranes or interact with cell contents for many of the cells as they pass en route to the preferred feeding tissues (Backus et al. , 2005) . During intercellular feeding the middle lamella is enzymatically dissolved so the stylet s may pass through this space. Intracellular feeding describes a tactic where the stylets pass directl y through the living portion of cells, breaking cell walls and Psyllids are classified within the order Hemiptera (W. F. Tjallingii & Hogen Esch, 1993) and into the suborder Sternorrhyncha. Forero (2008) classify psyllids into eight families within the superfamily Psylloidea. This new classification involved a family shift for the psyllid s tudied in this dissertation. Formerly, the Asian citrus psyllid, Diaphorina citri (Kawayama), was classified within the family Psyllidae, and is now classified within the family Liviidae.

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38 Histological E vidence of Psyllid Probing Behavior Most Sternorrhy ncha investigated thus far have been reported to feed using the intercellular fee ding tactic (Burckhardt & Ouvrard, 2012) . However, aphids represent the bulk of research on sternorrhycnhan feeding behavior. Aphids have been shown to enzymatically dissolve the middle lamellae between host plant parenchyma cells en route to vascular tissues (W. F. Tjallingii & Hogen Esch, 1993) . A careful analysis of the literature demonstrates that psyllids employ the salivary sheath feeding strategy but are highly variable in the p erformance of feeding tactics. Some psyllids appear to utilize the intercellular feeding tactic while others utilize the intracellular tactic and still others seem to use both tactics even within the same salivary sheath (W . F. Tjallingii & Hogen Esch, 1993; Brennan & Weinbaum, 2001; Brennan et al. , 2001; Civolani et al. , 2011; Pearson et al. , 2014) . The literature does not discuss this variability in psyllid feeding strategies and usually psyllids are reported to feed intercellularly. Diaphorina citri does not appear to feed in the same manner as aphids. The appearance of salivary sheaths of D. citri is mostly intercellul ar. However, D. citri salivary sheaths also sometimes pass through cells clearly away from the cell walls in a manner consistent with intracellular penetration (personal o bservation, and Bonani et al. (2010) , ( i mages 3H and 3I are mixed inter and intracellular). For D. citri , and the potato psyllid, Bactericera cockerelli , only light or fluorescent micrographs of relatively thick sections of salivary sheaths have been published (Ammar & Hall, 2012) , D. citri , 12 µm, (Bonani et al. , 2010) , D. citri , hand sections 50 70 µm, and (Ammar & Hall, 2012) , B. cockere lli , 10 µm). Electron microscopy or high resolution confocal microscopy is needed to be sure of the position of salivary sheaths in relation to the cell walls.

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39 The highest magnification images of psyllid feeding were published by Pearson et al. (2014) on Cacopsylla pyri . The images are primarily transmission electron micrographs (including some with cut stylets) and include a single light micrograph showing the overview of a sali vary sh eath. The overview published in this paper show that C. pyri at times employs the intracellular feeding tactic. However, Civolani et al. (2011) did not discuss the use of this feeding tactic by C. pyri . Civolani et al. (2011) acknowledge the intracellular appearance of the single light micrograph overview of a salivary sheath (Fi gure 7a), but consistently refer the C waveform as being produced in the extracellular s pac e (i.e., the middle lamellae). Additional evidence of intracellular penetration in C. pyri includes parenchyma cells filled with salivary sheath material along t he stylet pathway (Figure 7b). The only way saliva could fill a cell in this manner is if th e insect punctures the cell membrane so that the stylets and/or saliva enter the living portion of the cell. Diaphorina citri W aveform C orrelations Diaphorina citri waveforms have been partially correlated histologically with salivary sheath termini (Civolani et al. , 2011) . Bonani et al. (2010) interrupted feeding D. citri as particular waveforms were produced and collected the fed upon portion of the citrus leaf for histological processing and microscopic imaging of t he correlated salivary sheath. In D. citri , the C waveform has been correlated with salivary sheath termini in parenchyma, D waveform with sheath termini at the edge of the phloem, E1 in phloem, and E2 in phloem (Bonani et al. , 2010) . The G wavefo rm is observed but not histologically correlated. The G waveform is extrapolated by the authors to represent xylem ingestion, based on its likeness with the G waveform appearance for aphids (Bonani et al. , 2010) .

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40 The purpose of this C hapter is to summarize what is known about psyllid probing behavior and to fill in several gaps in the scientific literatu re in respect to what is known about D. citri probing on Citrus . The known biological meaning of D. citri EPG waveforms were expanded upon and some uncorrelated waveforms were correlated. Diaphorina citri feeding sites histologically processed and visualiz ed using both compound light microscopy and scanning electron microscopy, expanding on the level of detail with which D . citri salivary sheaths have been shown in the literature. Additionally, the absence of a pd waveform in D. citri has been confirmed in this work. Importantly, this work demonstrates that D. citri employs a unique probing tactic, using both inter and intra cellular probing through host plant tissues. M aterials and Methods Plants Plants were 1 2 years old during the course of this experiment and were maintained in a single greenhouse environment. Citrus sinensis var. Midsweet scions grafted to Kuharsky rootstock were used for all EPG recordings and histological processing of probed tissues. Plant s were maintained at near ambient temperature with fans that turn on at 30 °C and a natural day light cycle with no supplemental lighting. 3 Lakela nd, FL, USA) or Mira cle Gro all purpose plant food (Scotts Miracle Gro, Marysville, OH, USA). Plants were pruned regularly to maintain leaf flush. Test plants were chosen for use in the experime nt from a larger pool of plants (approximately 100) based on having available leaf flush on which to test psyllids.

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41 Insects The psyllids used for this experiment were taken from a laboratory colony began in 2006. Insects were originally collected from Polk County, Florida, USA, and maintained consistently in a growth room at the Cit rus Research and Education Center in Lake Alfred, FL, USA. Colony psyllids are CLas free and have been maintained on Curry Leaf, Murraya bergera, formerly Murraya koenigii , a s Curry Leaf has shown strong resistance to CLas infections (Spiller et al. , 1990) . Prior to recording on Midsweet, psyllids were maintained in an acclimation colony on Midsweet plants for several months. Electropenetrography E quipment and S ettings g old wire (Sigmund Cohn Corp., Mount Vernon, NY, USA) to the pronotum of the insect using water based s ilver glue. Psyllids were allowed to d angle f rom their wires for 30 minutes to 1 hour before they were set up on a test plant at the beginning of the recording period. Psyllids were given access to the abaxial leaf surface during the recording and were initially positioned near the midvein, as this is where they prefer to probe. Psyllid probing progress was watched carefully, and psyllids were quickly removed during the perform ance of waveforms of interest. The EPG recording was marked as to when the psyllid was removed so the waveform could be correlated with the histological sample generated by that recording. Recording s were made using a four channel analog AC DC EPG monitors built by EPG Technologies, Inc. Gainesville, FL, USA per the design outlined in (Backus & Bennett, 2009; Beloti et al. , 2018) . Recordings made for the purp ose of waveform correlations were recorded at an input impedance of 10 8 Ohms with an applied DC

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42 voltage of 150 mV. Recordings made to investigate the presence of a pd waveform were made at various input impedances, including: 10 7 , 10 8 , 10 10 , and 10 13 Ohms. Signal gain was adjusted on the analog control box as needed. The gain is a post acquisition change to the signal such tha t it does not affect the insect but can modify the waveform appearance to account for minor variation in conductivity due to wiring and other variables. Signals were digitized using a DI 710 converter (Dataq Instruments, Akron, OH, USA) and waveforms were recorded and measured using Windaq software (Dataq Instruments, Windaq Lite for acquisition and Windaq Waveform Browser for post ac quisition visualization and measurement). The lighting environment of the room included overhead fluorescent fixtures kept on during recording (24:0 light:dark ratio) with no outside light input because the windows are completely covered. The temperature w as maintained between 25 28°C. Histological P rocessing The probing sites were excised (midvein, 3 4 mm) and processed for histological and microscopic examination. Initially, samples were processed for examination using brightfield microscopy and placed in 6% paraformaldehyde in HEPES buffer for fixation. Samp les were then processed through a standard tert butanal ethanol dehydration series, infiltrated with and embedded in paraffin wax. Wax embedded blocks were sectioned to a thickness of 8 µm using a rotary microtome (Microm HM355, Waldorf, Germany). Sections were arranged serially on slides and allowed to dry at 42°C. Sections were de waxed using 100% xylene, stained using 0.5% aqueous safranin, and counter stained using 0.01% ethanolic fast green, then coverslipped using Permount mounting medium (Thermo Fish er, Waltham, MA, USA). Slides were visualized using brightfield settings on an Olympus BX61 compound microscope. Digital images were

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43 captured using a 14 megapixel OMAX model A35140U camera (Irvine, CA, USA), and ToupViewX software (Hangzh ou ToupTek Photoni cs Co., Ltd; Zhejiang, P.R.China). Following acquisition of brightfield images, samples were further processed for examination of the same probing sites for scanning electron microscopy (SEM). Rather than using photons (light) to illuminate the specimen, the SEM uses electrons to illuminate the specimen and much greater detail in tissue and salivary sheath structure can be achieved. In order to transition these specimens to visualization on the SEM, the coverslip had to be removed. To accomplish this, t he slides were soaked for 24 hours in 100% xylene until the coverslips could then be loosened with forceps and removed. The slides were then allowed to soak a little longer, 1 2 hours, until the remaining mounting medium was dissolved from the sections. Secti ons of interest, with salivary deposits, were identified and the slides were then broken, preserving the sections of interest, and then placed on a luminum SEM stubs (Figure 2 10) . To break the slides, they were scored using a diamond tip pen, and using a f lat edge screw driver, and a small hammer to apply pressure at the score mark. The now smaller portions of slides were affixed to the SEM stub using a c arbon sticker, coated with a g old p alladium mix using a 30300 Model Ladd sputter coater (Williston, VT, USA) . The slide pieces could now fit into the SEM vacuum chamber and be imaged at a higher magnification than can be achieve d using brightfield microscopy. All scanning electron micrographs in this dissertation were taken using a Hitachi S 4000 microscope (Hitachi High Technologies America, Schaumburg, IL , USA).

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44 R esults Waveform C Correlation There is much diversity in the appearance of waveform C for D. citri . Waveform C was correlated by Backus and Bennett (2009) with salivary sheath termini ending in parenchyma cells. Bonani et al. (2010) provided no data on wa veform appearance or the duration of waveform C prior to each interruption and histological correlation. Waveform appearance and probing durations are important details missing from previous studies and this dissertation aims to provide some of these data in an effort to make more complete correlations. In the current study, twelve waveform C correlations were made. These correlations were taken during the performance of a wide variety of waveform C appearances, during very early performance of waveform C, and after 30 minutes or longer performance of waveform C with the expectation that many differences would be observed in the outcome of stylet location and activities. While the overall appearance of salivary sheaths was variable, with some portions passin g intercellularly and others intracellularly, the histological correlations were very similar. Every interruption of the C waveform occurred in parenchyma with very little diversity of salivary sheath structure at the end (the point of interruption), no ma tter what the waveform appearance during the moment of interruption. Generally, shorter probing durations during the performance of waveform C were correlated with shorter salivary sheaths penetrating fewer parenchyma cells (Figure 2 2) while longer probin g durations of waveform C or those following the performance of other probing waveforms prior to returning to waveform C were associated with longer salivary sheaths penetrating more parenchyma cells (Figure 2 1). While this was an unexpected that salivary sheath structure would explain little of the diversity of the waveform C appearances, it explains

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45 much about the electrical origin of this waveform. Waveform C must be generated by activities that are largely invisible to microscopic analysis of plant fee ding sites, for example, stylet movements (Bonani et al. , 2010) . Waveform D C orrelation The correlation of waveform D is very important for interpretation of EPG recording generated by D. citri probing. Previo usly, Walker (2000) correlated D. citri salivary sheath at the very edge of the phloem. No data was provided on how many seconds, or which waveform sub types within waveform D had been allowed occur, prior to the interruption and correlation of waveform D. The current study aimed to improve on the correlation of waveform D by Bonani et al. (2010) . Early interrupted waveform D This work determined D. citri salivary sheaths, even when interrup ted very early during the performance of waveform D (6 seconds), terminate in phloem tissues. The plasma membrane has been punctured at the beginning of waveform D, allowing sheath saliva to partially fill the initial phloem cell contacted. Only one correl ation was made at the very beginning of waveform D ( Figure 2 5). Late interrupted waveform D It was much easier to correlate the later occurring portions of waveform D. Like with the early interrupted waveform D, the later interrupted waveform D correl ations show the salivary sheath terminating in phloem, with the terminal cell partially filled with sheath saliva. Eight waveform D correlations were made in total. Figure 2 3 summarizes a correlation with the later occurring portions of waveform D. The wa veform was interrupted after 31.55 seconds. Figure 2 4 summarizes a correlation with the later occurring portions of waveform D and was interrupted after 24.70 seconds of the

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46 waveform had occurred. The waveforms summarized in Figures 2 3 and 2 4 show all o f the same components occurring. These components were simply carried out faster by the psyllid correlated with Figure 2 4 compared to the psyllid correlated with Figure 2 3. In my study, the D. citri waveform D was correlated with stylets in phloem a tota l of eight times. One correlation was made at the very beginning of waveform D (Figure 2 5) and seven others were correlated near the end of the waveform. Waveforms E1 and E2 C orrelation Previously Bonani et al. (2010) correlated D. citri waveforms E1 and E2 with stylets located in phloem. However, no data was provided on how far into the waveform event the correlation was made or how many correlation s were made for each waveform type. My study correlated the D. citri waveform E1 10 times and waveform E2 6 times. Figure 2 7 summarizes two waveform E1 correlations. Figure 2 7 panels A, C, and E correspond to a correlation interrupted after 48.10 seconds of E1 had passed. Figure 2 7 panels B, D, and F correspond to a correlation interrupted after only 16.55 seconds of E1 occurred. Both E1 correlations in Figure 2 7 show salivary sheaths terminating in the phloem. Figure 2 8 summarizes a correlation of D. citri waveform E2. The E2 correlation was made after 8,842 seconds of the E2 waveform occurred. During the performance of the E2 waveform, the stylets were located within phloem cells. W aveform G C orrelation When D. citri probes a preferred host plant a nd is given access to preferred probing tissues, they spend relatively little time ingesting from xylem. For this reason, Bonani et al. (2010) was unable to correlate waveform G when conducting other D. citri waveform correlations. Bonani et al. (2010) provided test psyllids with young leaves of a preferred host plant, Citrus sinensis var. Pera, which to probe. In the current study, D.

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47 citri was given access to both young and mature C. sinensis var. Midsweet leaves on which to probe. This difference of providing access to mature leaves as a comp arison to young leaves made it possible to achieve many waveform G correlations. Nine waveform G correlations were made on mature leaves and one correlation was made on a young leaf. Figure 2 6 summarizes the waveform G correlation captured while a psyllid was probing a young C. sin ensis leaf. Each waveform G correlation clearly shows that D. citri stylets have penetrated a xylem cell and have fully or partially filled that xylem cell with sheath saliva. The probe that was interrupted to provide the correla tion summarized in Figure 2 6 only contained 70.40 seconds of waveform G, and yet the small xylem cell penetrated is filled with sheath saliva. Combined Intercellular and Intracellular Probing T actic Diaphorina citri does not probe in the same manner as aphids. Figure 2 9 summarizes scanning electron micrographs showing how D. citri variably uses the intercellular and intracellular probing tactics. In this F igure, red arrows are used to illustrate all of the salivary deposits representing intracellular pr obing, where saliva has partially filled cells adjacent to the main path of the salivary sheath or where the entire salivary sheath passes directly through the center of the cell. Saliva can only fill adjacent cells when cell walls and plasma membranes hav e been broken. Cell walls and plasma membranes are only broken during the performance of a probing tactic that more closely resembles intracellular probing and stylets are passing into the living portions of cells en route to phloem and xylem tissues.

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48 D is cussion Wavform C Correlation The results from this study indicate that the appearance of w aveform C is not determined by the cell type found at the stylet tip location. Rather, the electrical origin of the diversity of waveform C appearance is likely du e to stylet movements which would not leave visible traces within host plant tissues to be found using microscopic analysis of the probing site. During the performance of waveform C, the stylets are moving forward, propelled by muscular contractions that are generated by neural impulses (Dugravot et al. , 2008; Bonani et al. , 2010) . The psyllid also saws through portions of hardened saliva and t hrough cell walls as it passes through parenchyma cells (Walker, 2000) . In order the visualize stylet movements and sawing behaviors durin g probing, a diet correlation study should be carried out using EPG and simultaneous video recording of stylet movements (Joost et al. , 2006) . Waveform D Correlation A clear and detailed correlation of every aspect of Waveform D is key to understanding the probing behavior of D. citri , specifically the act of accessing the phloem. Prior to this work, wavef orm D had not been well correlated. Joost et al. (2006) correlated waveform D with phloem tissue using brightfield light microscopy. It is very diffic ult to determine phloem cell type using transverse sections at the level of detail available using light microscopy. Bonani et al. (2010) were able to ass ociate stylet tips at the edge of the phloem but could not determine if phloem cells were punctured. Additionally, Bonani et al. (2010) did not provide da ta on when during the performance of waveform D that probing was interrupted. There is still much to learn about the tasting, salivating, and stylet movements of D. citri that contribute to the electrical origin

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49 of waveform D. In my study, D waveforms have been interrupted at the very beginning, middle and near the end, and have all shown that stylet tips were in phloem cells, with cells filled with saliva, indicating that the plasma membrane was punctured and that the stylets had entered the symplast of th e cell. Stylet Tips are Inside Phloem Tissues During Waveforms D, E1, and E2 Correlations of waveforms E1 and E2 found phloem cells partially or fully filled with sheath saliva even very early during the performance of the waveform. Enzymatic saliva cou ld also be present but would not be visible without more detailed microscopic work, namely the immunolocalization of enzymatic proteins (Bonani et al. , 2010) . Waveform G is Correlated with Xylem Ingestion Previous work extrapolated waveform G as xylem ingestion simply based on the notion that psyllids probably probe plants much like aphids (Bonani et al. , 2010; Backus et al. , 2012) . The current study proved this to be true; the psyllid wavefor m G does occur with stylets in xylem just like the aphid waveform G. Salivary sheath termini from 10 correlated samples were found to end in xylem elements. Aphids Make a pd W aveform The pd waveform of aphids is well correlated with the act of puncturing a cell membrane, salivation and tasting behaviors within the intracellular space of this cell, which may be followed by stylet withdrawal back into the extracellular space or the acceptance and ingestion from that cell (Spiller et al. , 1990) . Short duration pd waveforms can be made into a variety of cell types encountered en route to the phloem or within the phloem. These waveforms are generally less than five seconds long and always end with stylet withdrawal back into the apoplast (W. F. Tjallingii, 1 985b) . Some pd waveforms are followed by a bout of the E1 waveform, for a duration of five seconds

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50 to 30 minutes. When the pd waveform is followed by the E1 waveform, stylets remain in the symplast of the cell and the aphid salivates into the symplast of the cell for a period of time (W. F. Tjallingii, 1985b) . At times, this behavior terminates with the performance of E1 and the cell is rejected by the aphid. Other tim es, the bout of E1 is followed by the E2 waveform. During the performance of the E2 waveform, the aphid ingests from the symplast of phloem sieve elements for a period of time (W. F. Tjallingii, 1985b) . The pd waveform can represent the tasting and rejection of an individual cell by an aphid, or, if followed by E1 and E2 waveforms, the pd waveform represents the tasting and acceptanc e of the phloem sieve element. The pd wa veform should be found in EPG studies of hemipterans utilizing intercellular feeding in the manner descr ibed for aphids (W. F. Tjallingii, 1985b) . Psyllids D o n ot M ake a pd W aveform One of the most significant difference s between the probing behavior of psyllids and aphids is a lack of pd waveforms observed in the best representative psyllid EPG studies (W. F. Tjallingii, 1985b; Bonani et al. , 2010; Civolani et al. , 2011) . The lack of a pd waveform during psyllid probing implies a major difference in how psyllids probe compared to aphids, the most thoroughly s tudied sternorrhynchan. As a part of this research, D. citri was recorded at a variety of input impedances in order to visualize multip le possible waveform outcomes in order to be sure pd waveforms do not occur at any input impedance. All previous report s of psyllids not reporting a pd waveform were recorded at 10 9 input impedance, the same setting commonly used to record aphids (Kimmins & Tjallingii, 1985; Pearson et al. , 2014) . Recordings at lower input impedances (10 7 and 10 8 ) emphasize behaviors representing salivation, and large scale stylet movements (Prado & Tjallingii, 1994) . These

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51 waveforms are generated as a result of the conductivity of the environm ent at the tip of the stylets. Salivation, for example, generates a rapid rise in conductiv ity at the tip of the stylets. Recordings at higher input impedances (10 9 , 10 10 , and 10 13 ) emphasize behaviors repre senting tasting and ingestion. These waveforms are generated as a result of tiny muscular movements within the head of the insect. No wavefo rm which could correspond to the pd waveform in aphids was performed by D. citri at any observed input impedance level . Combined I ntercellular and I ntracellular P robing T actic Diaphorina citri and other psyllid vectors of Candidatus Liberibacter bacteri al pathogens have only recently become important subjects of research surrounding their probing behavior (Bonani et al. , 2010; Pearson et al. , 2014; Sandanayaka et al. , 2014; Backus et al. , 2018) . Early in this body of research, assumptions and extrapolations were made to fill in the gaps in knowledge surrounding how psyllids probed plant tissues using what was known for other closely related insects, especially aphids. However, researchers need to work to fill in these gaps in knowledge with data based on psyllid probing rather than extrapolations from the much larger body of work that has been carried out using aphid subjects over the last nearly 60 years since the invention of EPG recording of hemipteran probing (Ebert et al. , 2018) . The current work seeks to address misconceptions about the similarities of psyllid and aphid probing behaviors. The most basic assumption, that psyllids employ the inte rcellular probing tactic like aphids and other studied Sternorrhyncha, has been shown to be false. Psyllids do, at times, probe intercellularly. However, they are flexible in how they execute their probing behaviors, using intercellular and intracellular p robing tactics variably and interchangeably. More research is required to fully understand the nuances of psyllid

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52 probing behavior. However, the first step to accomplish this task is to acknowledge that psyllids do not probe exactly like aphids.

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53 Figure 2 1. Feeding site histology and waveform traces corresponding to medium to long probes terminated during waveform C. A) Interrupted during C following E2 waveform, phloem ingestion. B) Waveform trace corresponding to panel A. Probe length 29,591.32 seconds. C) Interrupted after performing only C waveform, probe length 5,018 sec. D) Waveform trace corresponding to panel C. E) Waveform trace preceding panel D, showing the waveform shape preceding interruption. F) Salivary sheath in parenchyma. G) Waveform interrupted during waveform C, corresponding to panel F, probe length 2,528 seconds. Panels A., C., and F are compound light micrographs at 200x magnification. 0.4 sec/div 0.2 sec/div 0.2 sec/div 0.2 sec/div

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54 Figure 2 2. Feeding site histology and waveform traces corresponding to short probes terminated during waveform C. A) Salivary sheath resulting from probing interrupted during wavefor m C after 783.90 seconds. B) Waveform trace corresponding to panel A, showing drop in voltage when psyllid was removed. C) Salivary sheath resulting from probing interrupted during waveform C after 376. 85 sec. D) Waveform trace corresponding to panel C. E ) Salivary sheath resulting from probe interrupted during waveform C, after 171.35 sec. F) Waveform trace corresponding to panel E. G) C waveform trace preceding interruption of panel F, showing shape of waveform. Panels A., C., and E are compound light mi crographs at 200x magnification.

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55 Figure 2 3. Waveform D correlation with stylets in phloem and phloem salivation. A) Overview of waveform D showing point of interruption. B) Expanded views of early waveform D subcomponents. C) Expanded views of middle waveform D subcomponents. D) Expanded views of late waveform D subcomponents. E) Brightfield micrograph showing salivary sheath terminus corresponding to above waveform, 200x. The terminal phloem cell is filled with sheath saliva. F) View of salivar y sheath passing through parenchyma tissues. Salivary deposits fill the center of cells, showing stylets passed through the symplast en route to the phloem.

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56 Figure 2 4. Waveform D correlation with stylets in phloem and phloem cell filled with saliva. A) Overview of waveform D showing point of interruption. B) Expanded views of early waveform D subcomponents. C) Expanded views of middle waveform D subcomponents. D) Expanded views of late waveform D subcomponents. E) Brightfield micrograph showing salivary sheath terminus corresponding to above waveform, 200x. F) Scanning electron micrograph showing salivary sheath terminus in greater detail, 1300x. The terminal phloem cell is filled with sheath saliva.

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57 Figure 2 5. Waveform D c orrelation with stylets in phloem. A) Waveform C preceding waveform D that is interrupted. Red line indicates typical waveform C, performed as stylets pass through parenchyma cells. Blue line indicates C waveform directly preceding D waveform. B) Expanded view of beginning of waveform D that is interrupted. C) Scanning electron micrograph showing stylet path through the parenchyma, 700x . D) Brightfield micrograph showing stylet path through parenchyma . E) Brightfield micrograph showing salivary sheath term inus in phloem (red arrow), 200x . F) Brightfield micrograph showing salivary sheath terminus in phloem (red arrow), 400x . G) Scanning electron micrograph showing salivary sheath terminus in phloem (red arrow), 700x

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58 Figure 2 6. Waveform G is correlated with xylem ingestion. A) Waveform G prior to interruption of probing. Psyllid was only allowed 70.40 seconds of xylem ingestion before it was interrupted. B) Waveform G interrupted. C) Brightfield micrograph showing salivary sheath very near xylem, 200x . D) Brightfield micrograph showing salivary sheath has penetrated xylem and saliva partially fills small xylem cell, 200x . E) Scanning electron micrograph showing salivary sheath passing through the middle of parenchyma cells, 2000x . F) Scanning electron mi crograph showing salivary sheath penetrating xylem and saliva filling xylem cell, 1500x

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59 Figure 2 7. Waveform E1 corresponds with stylets in phloem. A) Waveform D preceding interrupted waveform E1 . B) Waveform D preceding interrupted waveform E1 . C) Probing interrupted after 48.10 seconds of waveform E1 . D) Probing interrupted after 16.55 seconds of waveform E1 . E) Brightfield micrograph showing salivary sheath terminating in phloem, 400x. Sheath corresponds with waveforms from panels A and C. F) Brig htfield micrograph showing salivary sheath terminating in phloem, 400x. Sheath corresponds with waveforms from panels B and D.

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60 Figure 2 8. Waveform E2 corresponds with stylets in phloem. A) Waveform D preceding interrupted waveform E2. B) Interrupted waveform E2, after 8,842 seconds. C) Brightfield micrograph showing stylets were in phloem during the performance of waveform E2, 400x.

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61 Figure 2 9. Scanning electron micrographs of Diaphorina citri intercellular and intracellular probing. A) Salivary sheath passing through parenchyma cells. Sheath path appears intercellular. 2,500x. B) Higher magnification view of a neighboring section showing the same area as panel A. Sheath path is not entirely intercellular as evidenced by saliva partia lly filling parenchyma cells, indicated by red arrows. 3,500x. C) Intracellular probing, with sheath passing through middle of parenchyma cell, at arrow.1,500x. D) Higher magnification view of panel C, area of red box. Intracellular probing with saliva par tially filling parenchyma cells at red arrows. Saliva partially filling parenchyma cells at red arrow. 3,000x. E) Salivary sheath passing through parenchyma cells. 2,000x. F) Higher magnification view of panel E, area of red box. Saliva partially filling p arenchyma cells at red arrows. 6,000x

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62 Figure 2 10. Scanning electron microscopy preparation of slides . A) Slides are broken so only sections of interest can be fit onto aluminum stubs for scanning electron microscopy (SEM) imaging. B) Broken slides pie ces of interest. Note marked areas indicating sections of interest. C) Broken slide pieces mounted onto aluminum stubs and coated with a gold palladium mix as preparation for SEM imaging.

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63 CHAPTER 3 COMPARISON OF THE PR OBING BEHAVIOR OF THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI, ON SWEET ORANGE, CITRUS SINENSIS VAR. VALENC IA, AND SOUR ORANGE, CIT RUS AURANTIUM, IN RE LATION TO HOST PLANT PHLOEM AND XYLEM MET ABOLIC PROFILES Introduction The Asian citrus psyllid, Diaphorina citri (K uway ama) (Hemiptera: Liviidae) is an invasive insect in North America and primary vector of the phloem infecting Gram proteo bacterium , Candidatus Liberibacter asiaticus (McLean & Kinsey, 1964) . Diaphorina citri feeds on and can transmit Ca . L. asiaticus to many Citrus species and varieties as well as to many other non agricultural or ornamental plants within the plant family Rutaceae (da Graca et al. , 2016) . Diaphorina citri and Ca . L. asiaticus can now be found in all citrus producing states within the continental United States, including Florida, Texas, Arizona, and Cali for nia (Sétamou et al. , 2016) as well as most other citrus producing countries worldwide (Halbert & Manjunath, 2004) . Hua nglongbing (HLB), caused by Ca . L. asiaticus (CLas), is a devastating and incurable disease of citrus. HLB has quickly become the most economically important disease of production cit rus in North America since the initial introduction of the psyllid to Florida in 1998 and the subsequent arrival of the pathogen in 2005 (da Graca et al. , 2016) . Symptoms of HLB include yellow discolorations in asymmetric islands on leaves, immature fruit that remains green rather than developing a yellow or orange color, asymmetric fruit, aborted seeds, e ventual branch die back, dropped fruit, and fruit that remains on the tree is bitter and never develops the expected level of sweetness (Manjunath et al. , 2008) . These symptoms eventually kill the tree and during the slow decline, the tree produces few, low quality fruit (da Graca et al. , 2016) . Growers cu rrently attempt to reduce disease incidence through spraying insecticides aimed to

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64 reduce psyllid numbers, removing infected trees, and replanting them with disease free seedlings (Alvarez et al. , 2016) . Overall, the acreage of citrus groves has decreased by 40% while the production of citrus has decli ned by 49% in Florida over the last 20 years (Alvar ez et al. , 2016) . Alvarez et al. (2016) estimated a loss of $1.7 billion in citrus production in Florida for the 5 year range of 2006 2011. Thes e losses are largely due to HLB disease in citrus. Many Citrus species and varieties are susceptible to D. citri probing and to HLB disease. There is, however, a marked difference in response to D. citri probing and HLB infection among Citrus species and other host species within the Rutaceae (Hodges & Spreen, 2012) . Several studies have investigated olfactory or visual cues associated with D. citri host preference (Wenninger et al. , 2009; Moghbeli Gharaei et al. , 2014; Sétamou et al. , 2016) or host suitability for survival or reproduction (Stockton et al. , 2016) . However, few studies have focused on the specifics of D. citri probing behavior on a range of preferred and non preferred citrus species and varieties. The most rigorous method for quantifying probing behavior in order to determine host preference of D. citri is through the use of the method of electropenetrography (EPG). It is impossible to quantify probing behavior of D. citri through simple observatio ns of psyllids on host plants. Diaphorina citri , as with other insects within the order Hemiptera, has piercing sucking mouthparts, and the activities of their mouthparts, termed stylets, are invisible when they probe plant tissues. EPG allows researcher s to observe these otherwise invisible behaviors as a waveform when the psyllid inserts their stylets into the host plant tissues. During EPG recording, a small electrical signal is applied to the plant and the waveform is conveyed via an attached

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65 conducti (Hall & Hentz, 2016) . Even fewer studies have employed EPG methodology to determine the susceptibility of Citrus species (Walker, 2000; Ebert et al. , 2018; George & Lapointe, 2018) and other rutaceous hosts to psyllid probing (George et al. , 2017) . This study investigated D. citri probing behavior on two closely related and generally preferred host plants ( Citrus sinensis var. Valencia; and Citrus aurantium , Sour Orange) and compared the resulting behavior to the chemical composition of the phloem and xylem of both host plant species in an effort to better understand the exact mechanisms of host preference. Herein we relat e important differences in metabolite profiles in phloem and xylem and how these differences may impact psyllid host preference in Valencia and Sour Orange. Materials and Methods Plants and Insects All plants used for this experiment were purchased from Southern Citrus Nurseries (Dundee, FL, USA) and certified CLas free. Plants were 3 5 years old during the course of this experiment and were maintained in a single greenhouse environment. Sweet orange , Citrus sinensis , . Sour orange, Citrus aurantium , were not grafted to any rootstock. Plants were maintained at near ambient temperature with fans that turn on at 30 °C and a natur al day light cycle with no supplemental lighting. Plants were fertilized variably 3 Gro A ll P urpose plant food (Scotts Miracle Gro, Marysville, OH, USA). Plants were pr uned regularly to maintain leaf flush. Test plants were chosen for use in the

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66 experiment from a larger pool of plants (50 Sour Orange and 150 Valencia) based on having available leaf flush on which to test psyllids. The psyllids used for this experiment were taken from a laboratory colony began in 2006. Insects were originally col lected from Polk County, FL , USA, and maintained consistently in a growth room at the Citrus Research and Education Center in Lake Alfred, FL, USA. Colony psyllids are CLas free and have been maintained on Curry Leaf, Murraya bergera, (formerly Murraya koenigii ) as Curry Leaf has shown strong resistance to CLas infections (Cen et al. , 2012) . Test psyllids w ere removed from the Curry Leaf based colony and set up on the host on which they would be tested on for an acclimation period of at least one week. Sour Orange and Valencia psyllid colonies w ere maintained under natural lighting conditions within an outdoor screen cage just a few feet from the greenhouse in which the test plants were maintained. Colony plants and insects were exposed to natural rainwater and given supplemental water as needed. The experiment began one week after the Sour Orange and Valencia colonies were established and continued for two months. Psyllids can be found in seve ral color morphs across Florida (Beloti et al. , 2018) . The brown gray color morph was not used in this expe riment as they are small and sens itive to handling during the wiring process of EPG. Electropenetrography Equipment and Settings g old wire (Sigmund Cohn Corp., Mount Vernon, NY, USA) to the pronotum of the insect using water b ased s ilver glue . The silver glue is made in the lab as needed using roughly 1v:1v:1w water, water based glue, and silver flakes (cat # 327077 Sigma Aldrich, St. Louis, MO, USA) . Psyllids were allowed to dangle from their wires for 30 min 1 h before the y were set up

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67 on a test plant at the beginning of the recording period. Psyllids were given access to the abaxial leaf surface during the recording and were initially positioned near the midvein, as this is where they prefer to probe (Wenninger & Hall, 2008) . Psyllids wer e recorded for 24 hours each. Recordings were not used if the insect jumped off of the leaf part way through the recording period or if there was some other issue that complicated interpretabili ty of the resulting waveforms. The final sample size for each treatment was 23 psyllids recorded on Sour Orange and 22 psyllids recorded on Valencia. Recording were made using two four channel analog AC DC EPG monitors built by EPG Technologies, Inc. ( Gainesville, FL, USA ) per the design outlined in Arredondo de Ibarra (2009) . Recordings were made using an input impedance of 10 9 Ohms a nd an applied DC voltage of 150 mV. Signal gain was adjusted on the analog control box as needed. The gain is a post acquisition change to the signal such tha t it does not affect the insect but can mo dify the waveform appearance to account for minor varia tion in conductivity due to wiring and other variables. Signals were digitized using a DI710 converter (Dataq Instruments, Akron, OH, USA) and waveforms were recorded and measured using Windaq software (Dataq Instruments, Windaq Lite for acquisition and Wi ndaq Waveform Browser for post acquisition visualization and measurement). The lighting environment of the room included overhead fluorescent fixtures kept on during recording (24:0 light:dark ratio) with no outside light input because the windows are comp letely covered. The temperature was maintained between 25 28°C. Waveforms were measured according to the Backus and Bennett (2009) naming conventions. Waveforms measured inclu ded C, D, E1, E2, G, and non probing (NP).

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68 Waveforms C, D, E1, E2, and G are all probing waveforms performed when the psyllid inserts its stylets into host tissue. Waveform C occurs in epidermis, parenchyma, and cambial tissues and represents biological a ctivities such as salivating, tasting of cell contents, and stylet movements including sawing and puncturing through cell walls. Waveform D occurs at the edge of the phloem and represents all of the behaviors involved in tasting and either accepting or rej ecting a phloem sieve element. Waveform E1 occurs with stylets located in a phloem sieve element and includes tasting and salivation into a phloem sieve element. Waveform E2 occurs with stylets located in a phloem sieve element and represents ingestion fro m a phloem sieve element. Waveform G occurs with stylets in a xylem element and represents ingestion from a xylem element. Waveform NP included as movements and behaviors performed by the psyllid on the surface of the plant with stylets remaining outside o f host plant tissues. Waveform NP can represent walking (Bonani et al. , 2010) or other movements that generate an electrical connection with the plant during EPG recording. Statistical Analysis Programs and Variables Waveform data was analyzed usi ng two different analysis programs in order to provide the mo st detailed analysis possible. Waveform data was first analyzed using the Ebert 2.0 SAS program (Youn et al. , 2011) . The Ebe rt program is freely available on the web at ( https://crec.ifas.ufl.edu/extension/epg/ ). SAS version 9.4TS1M was used within the SAS enterprise guide version 7.15HF3 for this analysis . The Ebert p rogram calculates 89 variables [ a complete list can be found in the supplemental information of Ebert et al. (2015) ] using a proc GLIMMIX model ANOVA. Means were separate d using an LSD test, with the P value set to 0.05. All means reported herein result from u ntransformed data. Waveform data were also analyzed using the variables outlined in

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69 Ebert et al. (2015) . The Backus 2.0 SAS program is also freely available to download from the web ( https://crec.ifas.ufl.edu /extension/epg/ ) . SAS version 9.4TS1M3 run in SAS Enterprise Guide version 7.15HF3 was used for this analysis and also ran a proc GLIMMIX with P set at 0.05. All w aveform data using the Backus variables are presented as untransformed means. Transitional pr obabilities (Table 3 5) were calculated by the Backus 2.0 SAS program. Chemical Analysis of Phloem and Xylem Extraction of phloem and xylem Phloem and xylem were extracted from five Sour Orange and five Valencia seedlings using the centrifugation method outlined in Backus et al. (2007) . Data from the five sampled plants were pooled for each treatment. For the chemical analysis, treatments included: Valencia phloem, Va lencia xylem, Sour Orange phloem, and Sour Orange xylem. Phloem and xylem collection occurred from the same plants used within the time that EPG portion of the experiment was ongoing so the resulting chemistry would be representative for the plants used fo r EPG recordings. Lengths of stem (10 20 cm) were excised from Sour Orange and Valencia p lants. The phloem and xylem were then separated from the lengths of stem using a sharp, sterile, single edge blade (# 71980, Electron Microscopy Sciences, Hatfield, PA , USA). Once separated, the outer portion of th e stem was comprised of the epidermis, parenchyma, cambium, and phloem, while the inner portion was co mprised of the xylem and pith. The pieces of stem were rinsed with deionized water and blotted dry using a Kimwipe. The phloem and xylem portions were cut to fit into 0.5 ml Eppendorf tubes which had been cut using a single edge blade so there was a small hole at the base of the tube. The smaller 0.5 ml tubes were then placed within intact 1.5 ml tubes so the l arger tubes could collect the

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70 extracted fluids. The tubes were then placed within a centrifuge and spun at 12,000 rpm for 1 5 minutes at room temperature. The fluid pooled at the bottom of the larger tubes during centrifugation. The anatomical structure of the phloem and xylem (narrow tubes stacked vertically without complete separation of cell walls) allowed for the phloem and xylem to be pulled from the stem pieces while the cellular contents of the other plant cells (irregularly arranged with each cell c ompletely separated by a cell wall) remained in place within the tissues remaining within the smaller tube. The extracted fluids were stored at 80°C until chemical analysis was performed. Derivitization Phloem and xylem fluids were processed via trime thylsilylation (TMS) derivatization using the protocol outlined in Hijaz and Killiny (2014b) and modified Roessner et al. (2000) and Hijaz and Killiny (2014a) . Phloem and xylem fluids were processed overnight using 30 µL methoxamine hydrochloride in pyridine (MOX) at 25 °C and then processed for two hours at using 80 µL N methyl N trimethysilyl trifluoroacetamide (MSTFA ) at 25 °C (Thermo Scientific, Waltham, MA, USA). Azelaic acid was used as an internal standard (Sigma Aldrich, St. Louis, MO, USA). Gas chromatography mass spectrometry Phloem and xylem fluids were injected into a Phenomenex ZB 5 mass spectrometry col umn, 30 mm x 0.25 mm, ID x 0.25 µm (Torrence, CA, USA). Phloem and xylem fluids were analyzed using a PerkinElmer Autosystem XL gas chromatography mass spectrometer (Shelton, CT, USA). Settings follow the protocol outlined in Killiny (2018).

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71 Peak analysi s Ion chromatograms were analyzed with Turbomass software. Peaks were idientified by their relative retention time using an ion spectrum comparison and external standards (Wiley 9 th ed, NIST 2011 mass spectral libraries ) . Compounds were converted from pe ak area to millimolar concentrations. Millimolar concentrations reported herein are the mean of five individual technical replicates from pooled extracts of 5 plants of both Sour Orange and Valencia. Results Sequential Variables Fifteen sequential varia bles were significantly different between Sour Orange and Valencia at P < 0.05 (T able 3 1 ) (Killiny, Valim , et al. , 2018) . These variables largely focus on the sequence an d timing of when psyllids reach the phloem and xylem and how long they spend performing phloem and xylem behaviors. I n general, D. citri make many test probes prior to accessing or ingesting from the phloem. A probe can be defined as an uninterrupted perio d of time within host plant tissues thus making strong electrical contact, and therefore raisin g the waveform voltage levels. During a probe the psyllids may be navigating toward the vascular tissues by alternately s awing through plant cell walls and salivating to create a salivary sheath or they may be salivating into or ingestin g from either phloem or xylem. Psyllids generally take at least a few hours before they access the phloem for the firs t time and perform w aveform D. Psyllids reached the phloem sooner, and ingested from the phloem for longer durations when probing Valencia. The mean time to the first probe containing a D waveform event was shorter on Valencia (4.675 h ) compar ed to on Sour Orange (9.23 h ) (Tm FrmFrstPrbFrstD, P= 0.015). The time from the first probe

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72 within a recording to the first E2 waveform was shorter on Valencia (7.55 hours) compared to on Sour Orange (11.63 h ) (TmFrstE2FrstPrb, P= 0.04074). The mean duration of waveform E2 per formed per p syllid was (4.94 h) for Valencia and (1.98 h ) for Sour Orange (MnDurE2, P= 0.0345). The total duration each insect spent not performing phloem associated waveforms wa s lower in Valencia (12.66 h ) comp ared to Sour Orange (16.46 h ) (TotDurNnPhlmPhs, P= 0.034 8) (Table 3 1.) . While psyllids consistently accessed phloem more quickly and spent more time ingesting from phloem on Valencia, psyllids probing Sour Orange spent longer durations and a larger percentage of the recording period ingesting from xylem compa red to psyllids probing Valencia. The total duration of time spent ingesting from xylem (waveform G) wa s lower for Valencia (1.60 h ) com pared to Sour Orange (5.17 h ) (DurG, P= 0.0282). The percent of the recording period spent ingesting from xylem was lowe r for Valencia (5%) compared to Sour Orange (17%) (PrcentPrbG , P= 0.0306 ) (Table 3 1.) . Non s equential Variables Non sequential variables offer information complimentary to the sequential variables. The Ebert SAS program variables are more sequential in nature, which fits with the sequential natur e of psyllid probing behavior. However, the hierarchical and non sequential variables provided by the Backus analysis program offer a distinct and useful overview of the waveform data (Ebert et al. , 2015) . The Backus analysis program calculates a series of variables at the cohort (or treatm ent) level, the insect level (both as means and for each individual insect), the probe level (not discussed herein), and the waveform event level (see Fig 1 in Backus et al ., 2007 ) for hierarchical arrangement of variables).

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73 Cohort l evel v ariables Coho rt level variables include the total number of probes (TNP) and the total number of waveform events (TNWE). These variables represent all of the probes (TNP) or a count of the total waveform events (TNWE) made by all of the psyllids within each treatment. The total number of probes (TNP) for psyllids probing Sour Orange was 178 compared to 801 for psyllids probing Valencia . This value can also be calculated per insect for each treatment by dividing by the number of psyllids recorded for each treatment . T he mean number of probes per psyllid probing Sour Orange is 7.74 and the number of probes performed by psyllids probing Valencia is 36.41. T he total number of waveform events per waveform type (TNWE) is presented graphically in Figure 3 1 (as pie charts) with each waveform type representing a portion of the total of all waveform events and total access time of each recording. Insect l evel v ariables Insect level variables include the number of waveform events per insect (NWEI) for each wav eform category and the waveform duration per insect (WDI) for each waveform category. The duration data are summarized in Table 3 2 as seconds and in Figure 3 1 as a pie chart presented in hours. The original measurement of the data was completed using the unit of seconds. However, the nature of the very long recording duration (24 hours) and often the long duration waveforms such as phloem ingestion (E2) or xylem ingestion (G) are often better represented as minutes or hours, while other variables are expr essed simply as counts, or the number of times that event type occurred, (NWEI). The number of waveform events per insect (NWEI) yielded some significant differences between the treatments, including the occurrence of C and NP. The number of times C was pe rformed on Sour Orange was 18.20 and 32.43 for

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74 Valencia (P= 0.0062), while the occurrence of NP (non probing) was 12.20 for Sour Orange and 26.97 for Valencia (P= 0.0014). The waveform duration per insect (WDI) for each waveform category also yielded impor tant significant differences between the treatments. The durations in seconds of C (Sour Orange 21,043.00, Valencia 32,065.00, P= 0.0001), D (Sour Orange 722.72, Valencia 277.37, P= 0.0487), E2 (Sour Orange 24,527.00, Valencia 29,352.00, P= 0.0001), and G (Sour Orange 27,942.00, Valencia 5,412.87, P= 0.0001) were all significantly different between psyllids probing Sour Orange and Valencia. Waveform e vent l evel v ariables The waveform event level variables expressed as durations (WDE) herein are expresse d as seconds in Table 3 2 as seconds and in Figure 3 1 in hours. The waveform duration per event (WDE) for phloem associated waveforms, xylems ingestions, and non probing yielded important significant differences between treatments. Waveform E1 was perform ed for 50% longer by psyllids probing Sour Orange compared to those probing Valencia. Waveform E2 occurred for 66% longer durations for psyllids probing Valencia compared to psyllids probing Sour Orange. Psyllids probing Sour Orange spent five times longer ingesting from xylem (waveform G) compared to psyllids probing Valencia. Psyllids probing Sour Orange spent more than twice as long not probing compared to psyllids probing Valencia. The waveform durations per event per insect (WDEI) in seconds were si gnificantly different between treatments for the waveform categories of E2, G, and NP. Psyllids ingested from phloem more than twice as long on Valencia compared to those probing Sour Orange (Sour Orange 7,033.79, Valencia 15,155.00, P= 0.0001). Psyllids probing Sour Orange spent four times more time ingesting from xylem compared to

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75 psyllids probing Valencia (Sour Orange 10,262.00, Valencia 2,470.03, P= 0.0023). Psyllids spent nearly three times more time not probing on Sour Orange compared to psyllids tes ted on Valencia (Sour Orange 2,334.33, Valencia 804.42, P= 0.0421). Waveform e vent t ransitions The probability that each waveform event will transition to another specific waveform event is represented in Table 3 5. In this T able, the first two column s of data (frequency and percent) represent the number of time or the percent of occurrence of transitions from one waveform to another, for example C to D, out of all of the transitions to occur in this dataset, including all represented waveform types. Whereas, the last two columns (cumulative frequency and percent) represent the number of times or the percent of occurrence of transitions from one waveform to another, for example C to D, out of the possible transitions for a particular waveform, for exam ple C. The cumulative frequency and percent transition data also take into account other possible transitions form waveform C, which also includes waveforms G and NP. These data represent counts of occurrences and are not appropriate for t tests or other statistical analyses comparing treatments (Backus et al. , 2007) . However, several interesting trends are evident and consistent with other EPG waveform data comparing D. citri probing on Valencia to probing on Sour Orange. Among these are several compariso ns highlighting the trend that overall many waveform events occurred in the Valencia recordings (total number of all waveform events 2,213) compared to the Sour Orange recordings (total number of all waveform events 698). While probing on Valencia, psyll ids transitioned from waveform C to waveform D 112 times compared to 78 times when probing Sour Orange. Psyllids transitioned from waveform E1 to waveform E2 (phloem salivation to phloem ingestion) on Valencia 51 times compared to 72 times on

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76 Sour Orange. These transition comparisons are interesting in the context of the total number of waveform events for each treatment. Even though psyllids performed more than three times as many overall waveform events on Valencia, they were more successful at accessing the phloem (waveform D) and ingesting from the phloem (waveform E2). Psyllids probing Sour Orange were less successful at accessing and ingesting from the phloem and therefore and spent longer durations ingesting from xylem. The transitions from waveform C to waveform G are similar for both treatments (Valencia 74, Sour Orange 77) even though many more waveform events occurred while probing on Valencia and more time was spent probing xylem (waveform G) on Sour Orange, 17% of total access time compared to 5% of tot al access time on Valencia ( Table 3 1). Another important aspect of waveform transition data is that is can be used as a check for the accuracy of the dataset. Each waveform type has a range of possible transitions based on what we know about t he biological activities associated with each waveform. To use waveform C as an example again, waveform C can transition to waveform D (stylets moving from parenchyma/cambial tissues into phloem), to waveform G (stylets moving from parenchyma/cambial tiss ue to xylem), or to NP, where the stylets begin in parenchyma/epidermal tissues and are withdrawn from the plant. Waveform C cannot transition to E1 or E2 (phloem waveforms) without first transitioning to waveform D. The specific behaviors of waveform D ar e required before the psyllid can salivate in phloem (E1) or ingest from phloem (E2). Gas Chromatography Mass Spectrometry Analysis Thirty three metabolites were found in both Valencia and Sour Orange phloem and xylem, including metabolites from the following broad categories: organic acids,

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77 fatty acids, simple sugars, sugar alcohols, sugar acids, and amino acids. Phloem metabolites a re summarized in T able 3 3 and F igures 3 2 , 3 3, and 3 4. Xylem metabolites are summarized in Table 3 4 and F igures 3 5, 3 6, and 3 7. Phloem Metabolite Concentration Organic acids made up 22% of the metabolites found in Valencia phloem compared to 27% in Sour Orange phloem. Fatty acids (steric, palmitric, and oleic acids) made up 33% of the total phloem metabolites in Valencia, and only 15% of the phloem metabolites in Sour Orange. Sugars constituted 16% of the phloem metabolites in Valencia and 23% of the phloem metabolites in Sour Orange. Sugar alcohols made up 20% of phloem metabolites in Valencia and 25% of phloem metabolites in Sour Orange. Sugar acids made up 3% o f phloem metabolites in Valencia and 4% of phloem metabolites in Sour Orange. Amino acids made up 5% of phloem metabolites in Valencia and 4% of phloem metabolites in Sour Orange (Table 3 3 and Figure 3 2) Eight metabolites were found in significantly di fferent (P= 0.05) quantities in Valencia and Sour Orange phloem (Table 3 3). Among these were organic acids, fatty acids, simple sugars, sugar alcohols, and sugar acids. The organic acids, malic acid and quinic acid, were found in higher concentrations in Sour Orange compared to Valencia. Malic acid was found in 22% the concentration in Valencia phloem compared to Sour Orange phloem (Sour Orange 1.86 mM, Valencia 0.42 mM, P= 0.017). Quinic acid was found at four times higher concentrations in Sour Orange ph loem compared to in Valencia phloem (Sour Orange 8.34 mM, Valencia 2.28 mM, P= 0.00001). Palmitic acid, a fatty acid, was found in 66% the concentration in Sour Orange phloem compared to in Valencia phloem (Sour Orange 1.90 mM, Valencia 3.08 mM, P= 0.03 6).

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78 The sugars glucose and sucrose were found in higher concentrations in Sour Orange. Glucose was found in three times higher concentrations in Sour Orange phloem compared to Valencia phloem (Sour Orange 1.01 mM, Valencia 0.33 mM, P= 0.024). Sucrose w as found in 28% the concentration in Valencia phloem that it was found in Sour Orange phloem (Valencia 2.20 mM, Sour Orange 7.84 mM, P= 0.00037). Sugar alcohols were also generally found in higher concentrations in Sour Orange phloem compared to Valenci a phloem. Chiro Inositol and Scyllo Inositol were both found in roughly three times the concentration in Sour Orange phloem that it was found in Valencia phloem ( Chiro Inositol: Sour Orange 5.88 mM, Valencia 2.22 mM, P= 0.00079; Scyllo Inositol: Sour Ora nge 3.10 mM, Valencia 1.03 mM, P= 0.0509). The sugar acid, threonic acid, was found in a 15 fold higher concentration in Sour Orange phloem (1.53 mM) compared to Valencia phloem (0.10 mM) (P= 0.0045). Xylem Metabolite Concentration Organic acids made u p 14% of the metabolites found in Valencia xylem compared to 22% in Sour Orange xylem. Fatty acids (steric, palmitric, and oleic acids) made up 33% of the total xylem metabolites in Valencia, and only 16% of the xylem metabolites in Sour Orange. Sugars con stituted 26% of the xylem metabolites in Valencia and 32% of the xylem metabolites in Sour Orange. Sugar alcohols made up 19% of xylem metabolites in Valencia and 21% of xylem metabolites in Sour Orange. Sugar acids made up 2% of xylem metabolites in Valen cia and 4% of xylem metabolites in Sour Orange. Amino acids made up 5% of xylem metabolites in Valencia and 5% of xylem metabolites in Sour Orange (Table 3 4 and Figure 3 5).

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79 Twelve metabolites were found in significantly different (P= 0.05) quantities i n Valencia and Sour Orange xylem (Table 3 4). Three organic acids, phosphoric, malic, and quinic acids) were found in significantly higher concentrations in Sour Orange compared to Valencia. Phosphoric acid was found in a seven fold higher concentration i n Sour Orange xylem compared to Valencia xylem (Sour Orange 0.44 mM, Valencia 0.06 mM, P= 0.0335). Malic acid was found in a 5 fold higher concentration in Sour Orange xylem compared to in Valencia xylem (Sour Orange 1.87 mM, Valencia 0.37 mM, P= 0.003 3). Quinic acid was found at 38% the concentration in Valencia xylem compared to in Sour Orange xylem (Valencia 1.74 mM, Sour Orange 4.52 mM, P= 0.00006). Simple sugars were found in significantly higher concentrations in Sour Orange xylem compared to V alencia xylem. Fructose and Glucose were both found at 70% the concentration in Valencia xylem that they were found in Sour Orange xylem. Sucrose was found at only 43% the concentration in Valencia xylem that it could be found in Sour Orange xylem. Sucrose was found at 1.13mM in Sour Orange and 0.49 mM in Valencia (P= 0.0488). Sugar alcohols were found in significantly higher concentrations in Sour Orange compared to Valencia. The sugar alcohols, Chiro inositol and Scyllo inositol, were both found in roughl y twice the concentration in Sour Orange xylem compared to Valencia xylem ( Chiro inositol: Sour Orange 4.53 mM, Valencia 2.45 mM, P= 0.000464; Scyllo inositol: Sour Orange 2.16 mM, Valencia 1.11 mM, P= 0.00006882). Sugar acids, threonic acid and sacc haric acid, were found in significantly higher concentrations in Sour Orange compared to Valencia. Threonic acid was found in an 8 -

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80 fold higher concentration in Sour Orange xylem compared to Valencia xylem (Sour Orange 0.42 mM, Valencia 0.05 mM, P= 0.0247 ). Saccharic acid was found in 17 fold higher concentrations in Sour Orange xylem compared to Valencia xylem (Sour Orange 0.88 mM, Valencia aminobutyric acid, was found in twice the concentration in Sour Orange xyl em compared to Valencia xylem (Sour Orange 0.41 mM, Valencia 0.21 mM, P= 0.0508). Discussion Diaphorina citri prefer to ingest from phloem and receive most of their required nut rition from phloem (Dytham, 2003) . However, D. citri also regularly ingest from xylem (A. Sharma & Raman, 2017) . While xylem is less nutritionally important to D. citri , citr us x ylem is not devoid of nutrients. Many of the same nutrients found in citrus phloem can also be found in xylem in lower concentrations (Hijaz & Killiny, 2014b; Ebert et al. , 2018) . Ingestion from xylem may also play an important role in water bala nce for phloem ingesting hemipteran insects (Killiny, 2017) . The EPG results herein demonstrate a strong preference for Valencia over Sour Orange by D. citri , with shorter times to access the phloem and longer overall ingestion of phloem on Valencia while more time is required to reach phloem a nd more time spent ingesting from xylem on Sour Orange. These outcomes are consistent with previous studies in other hemipteran insects. Many Hemiptera who engage in phloem ingestion will spend more time ingesting from xylem when tested on a non preferred host or feeding site compared to when they are tested on a preferred host or feeding site. On several resistant selections of soybean, Glycine max , Pompon et al. (2011) found that the aphid, Aphis glycines , spent more time ingesting from xylem compared to a tested susceptible variety. On a non preferred feeding site, mature leaves, on another C.

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81 sinensis variety (var. Midsweet), D. citri spent significantly more time ingesting from xylem compared to when they were tested on a preferred feeding site, immature leaves, of the same host (Diaz Montano et al. , 2007) . Diaphorina citri preference for immature leaves may be li nked to an avoidance of plant defensive compounds as has been shown in other species. The aphid, Myzus persicae , also prefers immature leaves on two Brassica sps. ( Brassica oleracea and Brassica rapa ) and Ebert et al. (2018) link this preference to an avoidance of higher glucosinolates levels in mature leaves, despite mature leaves also having a higher concentration of nutritious metabolites including sugars and amino acids. In the current study, Sour Orange was found to contain higher concentrations of important metabolic nutrients in both phloem and xylem compared to the levels found in Valencia phloem and xylem, including: simple sugars, sugar alcohols, sugar acids, and fat ty acids compared to Valencia. Sour Orange was also found to contain higher concentrations of organic acid metabolites several of which can be classified as plant defensive compounds or which are precursor molecules to plant defensive compounds, such as al kaloids or benzenoids (Cao et al. , 2018) . Interestingly, Sour Orange was found to contain higher concentrations both of nutritious metabolites (sugars and sugar alcohols) an d of plant defensive compounds (mostly organic acids), making Sour Orange both more nutritious to psyllids and better defended against them compared to Valencia. While many of the metabolites found in higher concentrations in Sour Orange phloem and xylem can be associated with a role in plant defense, this role cannot be assigned to the higher concentrations of sugars, especially sucrose, which are known to play a role in hemipteran nutrition (Friend, 1958; Ashford et al. , 2000; Guo et al. , 2014) .

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82 Sucrose was found in a four fold higher concentration in Sour Orange phloem compared to Valencia phloem and was found in a seven fold higher concentration in the phloem of Sour Orange compared to the concentration within the xylem. One possible explanation for the observed reduction of psyllid probing durations in phloem on Sour Orange could be simply that the four times higher concentration of sucrose, and resulting higher nutritional quality of Sour Orange phloem allowed the psyllids to spend less time in this tissue. Plants produce a number of secondary metab olites which are not required for growth or reproduction and which may act as defensive compounds against herbivores (A. Sharma & Raman, 2017) . A variety of organic acids, including: malic acid, quinic acid, and phosphoric acid, were found in concentrations ranging from 3 5 times higher in Sour Orange compared to Valencia. Organic acids have been assoc iated with host plant resistance in a variety of plants. Malic acid has associated with an upregulation in the production of reactive Oxygen species and increased lignin production in response to host wounding in Tobacco (Agrios, 2005) . Quinic acid can be converted to shikimic acid, which is an important precursor molecule to several plant defensive compounds, including alkaloids and benzenoids (Schaaf et al. , 1995) . (Guo et al. , 2014) . Maeda and Dudareva (2012) found quinic acid as an important molecule for the upregulati on of lignin production. Lignin can be upregulated as a response in plants to herbivore attack or to pathogen infection (Sabella et al. , 2018) . Karban and Baldwin (1997) found quinic acid in higher concentrations in varieties of chrysanthemum, Dendranthema grandiflora , resistant to feeding by Western flower thrips, Franklinella occidentalis .

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83 aminobutyric acid (GABA), was found in the xylem of both Sour Orange and Valencia. GABA was found in higher concen trations in Sour Orange phloem and xylem, at twice the concentrations as in Valencia phloem and xylem. The presence of GABA has been identified as a plant metabolite associated as a stress response to a variety of abiotic factors and pathogen infection (Bown et al. , 2006; Leiss et al. , 2009; Killiny, 2017) . Scholz et al. (2015) identified GABA as a powerful inhibitory neurotransmitter in herbivore pests and that herbivory induces GABA accumulation in soybean, Glycine max , and tobacco , Nicotiana tobacum . Few studies have linked the specific probing behaviors of a pest insect with the metabolite profile of the host plant. Shelp et al. (2003) performed an EPG study of the aphid, Brevicoryne brassicae , on mustard, Sina pis alba , plant feeding sites with differing levels of glucosinolates and found that the glucosinolate concentration affected aphid probing behavior. Grabrys et al. (1997) , through a combined EPG and metabolomics study, found concentrations of the secondary metabolite tricin in resistant and sus ceptible rice, Oryza sativa , to be a key factor in inhibition of phloem ingestion by the rice brown planthopper, Nilaparvata lugens . This study combined two powerful techniques, electropenetrography (EPG) and gas chromatography mass spectrometry (GC MS ), to elucidate and correlate the probing behavior of Diaphorina citri with the metabolic profile of phloem and xylem of Citrus s i nensis (var. Valencia) and C. aurantium (Sour Orange) in an effort to determine specific probing differences linked to the met abolic profiles of each host. Diaphorina citri strongly prefers Valencia to Sour Orange. In fact, in every quantifiable EPG parameter assessed in this study, D. citri demonstrated preference for Valencia; reaching the

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84 phloem sooner, spending more overall time in phloem, and more quickly and more frequently performing long bouts of phloem ingestion. Diaphorina citri readily probes Sour Orange and eventually accesses the phloem. However, when D. citri probes Sour Orange, it takes longer to access the phloem , phloem ingestion durations are reduced, and more time is spent ingesting from xylem compared to when D. citri probes Valencia. The correlations between D. citri probing behavior and phloem and xylem metabolic profiles herein can be applied to other less preferred and likely more resistant Citrus and Rutaceous hosts of D. citri in an effort to elucidate specific host preference and chemical resistance factors which can be used to screen for species and varieties potentially resistant to D. citri probing.

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85 Table 3 1. Difference s in timing and duration of Diaphorina citri on Sour Orange and Valencia. Waveform Variable Sour Orange LS Mean + SE Valencia LS Mean + SE P Value DF Probe Level C D E E2 G TotDurNnPhlPhs SdPrbs ShrtCBfrE1 TmFrmFrstPrbFstD TmBegPrbFrstD TmFrstSusDFrstPrb TmFrmFrstPrbFrstE TmBegPrbFrstE TmFrstSusE2FrstPrb TmFrstSusE2FrmStrtPrb TmFrstE2FrmFrstPrb TmFrstE2FrmPrbStrt MnDurE2 DurG PrcntPrbG 16.46 + 3.76 2.99 + 2.99 0.73 + 1 .11 9.23 + 5.92 2.07 + 3.61 21.74 + 5.32 11.44 + 7.73 1.50 + 2.18 11.76 + 7.98 1.37 + 2.11 11.63 + 7.81 1.37 + 2.08 1.98 + 1.07 5.17 + 6.07 0.17 (17%) + 1.74 12.66 + 6.09 1.57 + 1.48 0.28 + 0.33 6.68 + 3.85 0.42 + 0.58 14.31 + 7.70 7.60 + 7.43 0.37 + 0.34 7.67 + 7.42 0.36 + 0.29 7.55 + 7.20 0.36 + 0.29 4.94 + 5.36 1.60 + 1.53 0.05 (5%) + 0.93 0.0348 0.0228 0.0079 0.015 0.0018 0.0457 0.043 0.002 0.0421 0.0035 0.0407 0.0032 0.0345 0.0282 0.0306 36 41 36 37 37 12 43 36 43 34 43 34 34 37 37 Variables generated and named using Ebert et al. 2015 . Means and standard errors are not transformed. Durations are reported in hours.

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86 Table 3 2 . Di a phorina citri probing behaviors on Sour Orange and Valencia . Waveform d uration per e vent per i nsect (WDEI) , means (seconds) Waveform Sour Orange Valencia DF P value C 1,294 ± 343 1,286 ± 242 43 0.9845 D 214 ± 81 77 ± 61 37 0.1889 E1 260 ± 62 194 ± 45 36 0.4014 E2 7,033 ± 2,404 15,155 ± 2,404 34 0.0001* G 10,262 ± 1,977 2,470 ± 1,318 37 0.0023* NP 2,334 ± 596 804 ± 421 43 0.0421* Wav eform d uration per i nsect (WDI), means (seconds) Waveform Sour Orange Valencia DF P value C 21,043 ± 2,471 32 ,065 ± 2,471 43 0.0001* D 722.72 ± 174.93 277 ± 130 37 0.0487* E1 1,094 ± 195 734 ± 140 36 0.1437 E2 24,527 ± 2,779 29,352 ± 2,779 34 0.0001* G 27,942 ± 2,204 5,412 ± 2,204 37 0.0001* Number of w aveform e vents per i nsect (NWEI) , means Waveform Sour Orange Valencia DF P value C 18.2 ± 4.03 32.43 ± 2.85 43 0.0062* D 5.71 ± 1.14 4.4 ± 0.85 37 0.3626 E1 5.46 ± 1.33 5.52 ± 0.96 36 0.9718 E2 3.75 ± 0.64 3.25 ± 0.45 34 0.5249 G 3.833 ± 0.92 3.89 ± 0.61 37 0.9601 NP 12.2 ± 3.52 26.97 ± 2.49 43 0.0014* Waveform d uration per e vent (WDE) , means Waveform Sour Orange Valencia DF P value C 1,156 ± 106.67 988 ± 56 1244 0.1654 D 126 ± 31 63 ± 26 188 0.1231 E1 200 ± 20 133 ± 14 207 0.0089* E2 6,540 ± 958 9,031 ± 958 121 0.0001* G 7,289 ± 937 1,391 ± 620 149 0.0001* NP 1,443 ± 181 663 ± 86 990 0.0001* Mean values are untransformed ± standard error . * Indicates P value < 0.05 Waveform s analyzed according to Backus et al., (2007)

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87 Table 3 3 . Phloem p ercent m etabolite c oncentration in Valencia and S our O range . Metabolite Valencia Phloem (mM) Sour Orange Phloem (mM) Benzoic acid 0.09±0.03 0.11±0.01 Phosphoric acid 0.06±0.02 0.09±0.03 L proline 0.32±0.09 0.63±0.30 Glycine 0.16±0.00 0.16±0.00 Succinic acid 0.08±0.03 0.06±0.02 Fumaric acid 0.09±0.06 0.09±0.03 Malic acid 0.42±0.03 1.86±1.11 Aspartic acid 0.28±0.10 0.26±0.05 -Aminobutyric acid 0.24±0.04 0.35±0.09 Threonic acid 0.10±0.03 1.53±0.64 Erythrose 0.20±0.01 0.19±0.00 L phenylalanine 0.16±0.02 0.18±0.02 Shikimic acid 0.05±0.03 0.09±0.05 Citric acid 0.11±0.09 0.11±0.07 Quinic acid 2.28±0.16 8.34±1.31 Fructose 1 0.18±0.01 0.18±0.03 Fructose 2 0.12±0.01 0.15±0.07 Glucose 1 0.33±0.16 1.01±0.14 Glucose 2 0.20±0.16 0.23±0.05 chiro Inositol 2.22±0.06 5.88±2.39 Galactonic acid 0.08±0.03 0.15±0.04 Saccharic acid 0.13±0.08 0.52±0.14 scyllo Inositol 1.03±0.13 3.10±0.78 Galactaric acid 0.44±0.26 0.31±0.15 Palmitic acid 3.08±0.69 1.90±0.41 myo Inositol 1.18±0.08 1.71±0.39 Unknown disaccharide 0.12±0.01 0.13±0.01 Oleic acid 0.76±0.02 0.76±0.01 Stearic acid 3.52±1.46 3.61±2.11 Adipic acid 1.65±1.35 0.96±0.09 Unknown 1 0.16±0.02 0.17±0.03 Sucrose 2.20±1.20 7.84±3.39 Turanose 0.11±0.01 0.19±0.05 Note: identification and quantification of the detected metabolites w ere performed using external standards. Samples were pooled from 5 plants (n=5). Data presented as means ± standard error.

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88 Table 3 4 . Xylem p ercent m etabolite c oncentration in Valencia and S our O range . Metabolite Valencia Xylem (mM) Sour Orange Xylem (mM) Benzoic acid 0.07±0.02 0.07±0.01 Phosphoric acid 0.06±0.02 0.44±0.09 L proline 0.22±0.04 0.72±0.58 Glycine 0.16±0.00 0.17±0.01 Succinic acid 0.06±0.03 0.08±0.03 Fumaric acid 0.06±0.01 0.18±0.03 Malic acid 0.37±0.16 1.87±0.50 Aspartic acid 0.24±0.07 0.25±0.03 -Aminobutyric acid 0.21±0.00 0.41±0.04 Threonic acid 0.05±0.01 0.42±0.08 Erythrose 0.19±0.00 0.19±0.00 L phenylalanine 0.16±0.01 0.17±0.01 Shikimic acid 0.03±0.03 0.04±0.00 Citric acid 0.02±0.01 0.11±0.05 Quinic acid 1.74±0.24 4.52±0.43 Fructose 1 0.80±0.09 2.51±0.24 Fructose 2 0.53±0.09 1.69±0.25 Glucose 1 3.59±0.82 5.28±0.47 Glucose 2 0.52±0.25 0.84±0.07 chiro Inositol 2.45±0.36 4.53±0.31 Galactonic acid 0.11±0.08 0.10±0.01 Saccharic acid 0.05±0.01 0.88±0.11 scyllo Inositol 1.11±0.18 2.16±0.12 Galactaric acid 0.21±0.16 0.19±0.05 Palmitic acid 3.64±1.51 2.62±0.83 myo Inositol 1.15±0.10 1.31±0.04 Unknown disaccharide 0.11±0.01 0.12±0.00 Oleic acid 0.77±0.02 0.76±0.01 Stearic acid 3.68±1.60 2.80±0.65 Adipic acid 0.99±0.15 1.08±0.18 Unknown 1 0.18±0.02 0.17±0.01 Sucrose 0.49±0.15 1.13±0.23 Turanose 0.14±0.02 0.20±0.03 Note: identification and quantification of the detected metabolites were performed using external standards. Samples were pooled from 5 plants (n=5). Data presented as means ± standard error.

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89 Table 3 5. Diaphorina citri w aveform e vent t ransitions . Sour Orange Wf Transition Frequency Percent Cumulative Cumulative f requency p ercent C to D 112 8.02 112 8.02 C to G 77 5.51 189 13.53 C to NP 400 28.63 589 42.16 D to C 16 1.15 605 43.31 D to E1 96 6.87 701 50.18 E1 to C 35 2.51 736 52.68 E1 to E2 72 5.15 808 57.84 E1 to NP 1 0.07 809 57.91 E2 to C 48 3.44 857 61.35 E2 to E1 18 1.29 875 62.63 E2 to NP 5 0.36 880 62.99 G to C 56 4.01 936 67 G to NP 20 1.43 956 68.43 NP to C 441 31.57 1,397 100 Valencia Wf Transition Frequency Percent Cumulative Cumulative f requency p ercent C to D 78 5.31 78 5.31 C to G 74 5.04 152 10.35 C to NP 488 33.22 640 43.57 D to C 7 0.48 647 44.04 D to E1 71 4.83 718 48.88 E1 to C 36 2.45 754 51.33 E1 to E2 51 3.47 805 54.8 E1 to NP 3 0.2 808 55 E2 to C 15 1.02 823 56.02 E2 to E1 24 1.63 847 57.66 E2 to NP 11 0.75 858 58.41 G to C 54 3.68 912 62.08 G to NP 19 1.29 931 63.38 NP to C 538 36.62 1,469 100

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90 Figure 3 1. Waveform duration per i nsect, i n hours, and total number of waveform events produced by Diaphorina citri probing Sour Orange and Valencia . C 12.15 D 0.09 E1 0.23 E2 8.89 G 1.85 NP 6.77 Waveform Duration per Insect (WDI) in hours C 3.81 D 0.12 E1 0.17 E2 3.55 G 4.05 NP 3.19 Waveform Duration per Insect (WDI) in hours C 273 D 80 E1 71 E2 45 G 46 NP 183 Total Number of Waveform Events (TNWE) C 973 D 110 E1 138 E2 78 G 105 NP 809 Total Number of Waveform Events (TNWE)

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91 Figure 3 2. Percent concentration of phloem metabolites metabolite groups and organic acids.

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92 Figure 3 3. Percent concentration of phloem metabolites sugars, sugar alcohols, and sugar acids.

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93 Figure 3 4. Percent concentration of phloem metabolites fatty acids and amino acids.

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94 Figure 3 5. Percent concentration of xylem metabolites metabolite groups and organic acids.

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95 Figure 3 6. Percent concentration of xylem metabolites sugars, sugar alcohols, and sugar acids .

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96 Figure 3 7. Percent concentration of xylem metabolites fatty acids and amino acids.

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97 CHAPTER 4 PROBING BEHAVIOR OF DIAPHORINA CITRI (KU WAYAMA) (HEMIPTERA: LIVIIDAE) ON FOUR CL EOPATRA MANDARIN, CI TRUS RETICULATA, HYB RID SELECTIONS AND PUMME LO, CITRUS MAXIMA, I N RELATION TO WHOLE LEAF METABOLITES Introduction The Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Liviidae) is an invasive insect in North America and primary vector of the phloem infecting gram proteo bacterium , Candidatus Liberibacter asiaticus (Zhang et al. , 2015) . Diaphorina citri feeds on and can transmit Ca . L. asiaticus to many Citrus species and var ieties grown in Florida as well as many other non agricultural or ornamental plants within the plant family Rutaceae (da Graca et al. , 2016) . Hua nglongbing (HLB), caused by Ca . L. asiaticus (CLas), is a devastating and incurable disease of citrus. HLB has quickly become the most economically important disease of production cit rus in North America since the initial introduction of the psyllid to Florida in 1998 and the subsequent arrival of t he pathogen in 2005 (Sétamou et al. , 2016) . Many Citrus species an d varieties are susceptible to D. citri probing and to HLB disease. There is, however, a marked difference in response to D. citri probing and C Las infection among Citrus species and other host species within the Rutaceae (Manjunath et al. , 2008; Sétamou et al. , 2016) . One of the most important technologies available to citrus growers to combat disease in groves is the use of rootstock resistant to the disease or pest of concern. The rootstock type has been shown to impact the scion in a number of chemic al and morphological aspects. Rootstock can impact the nutritional composition, defensive metabolite concentrations, and plant size (Cano & Bermejo, 2011; Killiny, 2017) . Changes in chemical and morphological parameters of the scion via the rootstock may impact the feeding behavior of insect pests. Soares e t

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98 al. (2015) addressed this question by assessing reproduction and survival of D. citri on several four Citrus scion and three rootstock combinations. This study found that the rootstock impacted reproduction and survival outcomes of the grafted scions (Alves et al. , 2017) . The probing behavior of hemipteran pests cannot be easily observed and quantified since the insects insert their piercing sucking mouthparts into opaque plant tissues such that the subsequent probing activities are invisible to observers. The most rigorous method of quantifying the probing behavior of hemipteran pests is electropenetrography (EPG). EPG requires the attachment of a conductive gold wire to the p ronotum of the insect and the application of a small electrical signal to the substrate to complete an electrical circuit which is interpreted as a waveform by an EPG monitor (Alves et al. , 2017) . Probing waveforms are produced any time the insect inserts the stylets into plant tissues and waveforms are consistent and reproducible such that salivation, stylet movements, and ingestion behaviors can all be observed and quantified (Backus & Bennett, 2009) . As the impact of D. citri and CLas on the citrus industry became more apparent, more in depth research has been carried out on D. citri probing behavior, including a few EPG studies on a variety of citrus species. Most EPG studies have been carried out on the most susceptible species, Citrus sinensis (sweet orange) (Walker, 2000; George et al. , 2017; Ebert et al. , 2018; George & Lapointe, 2018) . Bonani et al. (2010) performed an EPG study on healthy and C andidatus Liberibacter asiatius infected Mandarin. Cen et al. (2012) found that psyllids ingested more from xylem as the severity

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99 of the Liberibacter infection increased and that psyllids preferred to ingest more from phloem on healthy Mandarin. Host plant met abolites have been shown to impact pest insect behavior and host preference in hemipteran pests (Cen et al. , 2012) . Plant metabolites can be constitutive or induced (Hopkins et al. , 2017) and can have diverse functions, including: defense against herbivores or pathogens (Wittstock & Gershenzon, 2002) , as a response to mechanical damage (Albrecht et al. , 2016; Asai et al. , 2017) , and as a response to environmental stresses (Chen et al. , 2018) . The Cleopatra Mandarin hybrid selections used in this study have variable Cleopa tra Mandarin, Citrus reticulata , mothers while pollen from a single Citrus ichangensis plant was used to fertilize and create hybrid selections. Citrus ichangensis was first brought to the attention of Western growers and scientists in 1913 by Swingle (Obata & Fernie, 2012) . It can be found in high elevations and is very hardy to cold and dry environmental condit ions. The potential of this species as a hardy, resistant candidate for use as a scion or rootstock was recognized, but the species has not been widely studied. Swingle (1913) carried out a molecular phylogeny on C. ichangensis providing a better understanding of the relatedness of more widely used Citrus scions and rootstocks. Some metabolic data is known for C. ichangensis , including citric acid, ascorbic acid, and phenolics (Yang et al. , 2017) . The rootstock used for the Mandarin hybrid selection in the current study, Carrizo citrange , has been more widely used and studied. Carrizo citrange is known to be resistant to HLB disease (Ruiz et al. , 2018; R. Sharma et al. , 2018) and salinity tolerant (Killiny, Jones , et al. , 2018) .

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100 O nly a handful of studies have combined probing behavior of the pest insect with the metabolic profile of the host plant (Garcia Sanches & Syvertsen, 2006) . The current study aims to quantify D. citri probing behavior on four Cleopatra Mandarin hybrid selections which might be candidates as rootstock resistan t to D. citri probing. In addition, whole leaf metabolite concentrations were investigated in an effort to explain how variation in leaf metabolites might contribute to differences in D. citri probing behavior on these Mandarin hybrid selections. Pummelo was investigated as a less resistant out group as it is a parent plant to the Mandarin maternal plants used to generate the Cleopatra hybrids in this study (Hu et al. , 2018) . Materials and Methods Plants and Insects Plants were 5 6 years old during the course of this experiment and were maintained in a single greenhouse environment , under the same conditions as described in Chapter 3 of this dissertation . The Cleopatra Mandarin hybrid selections used for this experiment were developed and grafted onto Carrizo citrange rootstock by the Gmitter lab at the Citrus Research and Education Center in Lake Alfred, Florida, USA. The Citrus ichangensis pollen donor to the hybrid selections was located at the Florida Citrus Arboretum in Winter Haven, FL, USA. Pummelo plants were not grafted to a rootstock. Test plants were chosen for use in the experiment from a larger pool of plants ( 10 12 plants of each Mandarin selec tion, and 30 Pummelo plants ) based on having available leaf flush on which to test psyllids. Test psyllids w ere removed from the Curry Leaf based colony (the same colony and same rearing conditions as described in Chapter 3) and set up on the host they would be tested on for an acclimation period of at least one week. Mandarin and

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101 Pummelo psyllid colonies were maintained under natural lighting conditions within an outdoor screen cage just a few feet from the greenhouse in which the test plants were maint ained. Colony plants and insects were exposed to natural rainwater and given supplemental water as needed. The experiment began one week after the Mandarin and Pummelo colonies were e stablished and continued for three months. Several of the Mandarin coloni es had to be re stocked with Murraya colony psyllids because psyllids could not successfully reproduce on the plants. So, when all of the adult psyllids were used or had died, new psyllids were added and the experiment was halted to allow for a one week a cclimation period. Psyllids were only recorded after they had been acclimated to the test plant for at least one week. Psyllids can be found in seve ral color morphs in Florida (Wu et al. , 2018) . The brown gray color morph was excluded from use in this expe riment as they are small and sensitive to handling during the wiring process of EPG. Electropenetrography Equipment and Settings g old wire (Sigmund Cohn Corp., Mount Vernon, NY, USA) to the pronotum of the insect using water based s ilver glue. Psyllids were allowed to dangle from their wires for 30 minutes 1 hour before they were set up on a test plant at the beginning of the recording period. Psyllids were recorded for 24 hours each. The final sample size for each treatment was as follows: Pummelo 21 psyllids, Mandarin 2 22 psyllids, Mandarin 19 23 psyllids, Mandarin 26 20 psyllids, and M31 21 psyllids. Some recordings for each treatment were not used if the insect jumped off of the leaf part way through the recording period o r if there was some other issue that complicated interpretabili ty of the resulting waveforms. Psyllids were given access to the abaxial leaf surface during the recording

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102 and were initially positioned near the midvein, as this is where they prefer to probe (Wenninger & Hall, 2008) . Recording were made using two four channel analog AC DC EPG monitors built by EPG Technologies, Inc. Gainesville, FL, USA per the design outlined in (Arredondo de Ibarra, 2009) . Eight recordings were completed each day of the experiment. Care was taken that at least one replication of each of the five treatments was recorded each day, with some treatments being replicated twice daily. R ecordings were made using an input impedance of 10 9 Ohms with an applied DC voltage of 150 mV. Signal gain was adjusted on the analog control box as needed. The gain is a post acquisition change to the signal such that it does not affect the insect, but can mo dify the waveform appearance to account for minor variation in conductivity due to wiring and other variables. Signals were digitized using a DI710 converter (Dataq Instruments, Akron, OH, USA) and waveforms were recorded and measured using Windaq software (Dataq Instruments, Windaq Lite for acquisition and Windaq Waveform Browser for post acquisition visualization and measurement). Waveforms were measured according to the Backus and Bennett (2009) naming conventions. Waveforms measured included C, D, E1 , E2, G, and non probing (NP). The lighting environment of the room included overhead fluorescent fixtures kept on dur ing recording (24:0 light:dark ratio) with no outside light input because the windows are completely covered. The temperature was maintained between 25 28°C. EPG Data Analysis Programs and Variables Waveform data was analyzed using two different analys is programs in order to provide the most detailed analysis possible. Waveform data was first analyzed using the Ebert 2.0 SAS program (Bonani et al. , 2010) . The Ebert program is free ly available

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103 on the web at ( https://crec.ifas.ufl.edu/extension/epg/ ). SAS version 9.4TS1M was used within the SAS enterprise guide version 7.15HF3 for this analysis . The Ebert program calculates 89 variables (a complete list can be found in the supplemental information of Ebert et al. (2015) ) using a proc GLIMMIX model ANOVA. Means were separated using an LSD test, with the P value set to 0.05. U ntransformed means are reported herein. Waveform data were also analyzed using the Backus variables outlined in Ebert et al. (2015) . The Backus 2.0 SAS program is also freely available to download from the web ( https://crec.ifas.ufl.edu/extension/epg/ ) . SAS version 9.4TS1M3 run in SAS Enterprise Guide version 7.15HF3 was used for this analysis and also ran a pr oc GLIMMIX with P set at 0.05. Waveform data analyzed using the Backus variables are presented as untransformed values . Whole Leaf Metabolites Th ree leaves (one young, one medium, one old) were excised from 5 plants of each of the Ma ndarin selections and Pummelo. Leaves were sampled three days after the completion of the EPG portion of the experiment with the aim of acquiring a representative snaps hot of what the metabolites were during the course of the experiment, but not link the specific metabolite profiles of individual plants with the probing behavior s of specific tested psyllids. Sampled leaves were place immediately in a cooler with dry ice and then walked from the greenhouse and direc tly placed in a 80°C freezer. M etabolites were analyzed from 0.1 g (fresh leaf weight), from three pooled leaf samples from five biological replicates of each treatment for metabolite analysis (n= 5). Leaf tiss ue was homogenized in liquid Nitrogen, then extracted with 1 ml extraction solvent (8:1:1 MeOH, Chloroform, water) and derivatized prior to GC MS. Injection volume was 0.5 µL. All peak areas were normalized to the mean area of the

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104 internal standard and con verted to µg/g using calibration curves of authentic standards derivatized and injected into the GC MS in the same way as experimental samples. Compounds not detected were noted as ND and zeros (0.0) were removed from the means calculations. Data were gene rated from 5 biological samples from each treatment without duplicate injections. Metabolite Statistical Analysis Plant metabolite concentrations were determined for sixty six compounds. Mean concentrations were analyzed with SAS analysis software, us ing ANOVA and means were separated using and LSD test set to P= 0.05. Missing values, because a few of the compounds were missing from one treatment, were treated as zeros. All data are presented as untransformed. The degrees of freedom for all whole lea f metabolite data is 4, because 5 plants (biological replicates) were sampled to generate these data, with 3 leaves pooled from each plant. Diaphorina citri Oviposition and Survivorship Comparison In an effort to determine if oviposition and survivorship mirrored probing ability on each hybrid selection or species, a no choice oviposition study was performed and the survivorship of eggs to adulthood was performed on the four Mandarin hybrid selection and Pummeo. The ps yllids used for this experiment are CLas free and have been maintained on Curry Leaf, Murraya berger a in a laboratory colony started in 2006 . Plants were maintained in a greenhouse environment at near ambient temperature with fans that turn on at 30 °C and a natural day light cycle with no su pplemental lighting. Plants were chosen for use in this experiment based on the availability of young leaf flush, as this is where D. citri prefers to oviposit (Backus et al. , 2007) . Three plants were selected with a range of sizes of young flush, placed in a Bugdorm cage (Bioquip

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105 Products, Rancho Dominguez, CA, USA) with 25 females and 25 males, allowing psyllids to move freely and choose the size of flush preferred for oviposition. Tests were performed in a growth room with artificial lighting (14:10 light:dark ratio), with temper ature maintained between 25 30°C, and humidity kept between 60 80%. Psyllids were allowed to mate and oviposit freely for 10 days, at which point the adult psyllids were removed. This ensured that the eggs and developing nymphs were from the same cohort and could be followed through their development. During development, young psyllid nymphs (first through third instars) are not very mobile, allowing for quantification of nymphs from each flush as nymphs were not moving from one flush to another. During the fourth and fifth instars, D. citri can be mobile. However, they were not observed moving between different sets of leaf flush. The leaf flush was separated by large, woody stem in most cases. The immobility of the nymphs in the current study allowed f or the quantification of the development of nymphs on individual sets of leaf flush. As adults emerged, they were removed daily to prevent mating and further oviposition. Results Sequential Variables Thirty seven of the Ebert program sequential variables were signi ficantly different between the Mandarin hybrid selections and Pummelo at P = 0.05 (Tables 4 1 and 4 2). These variables largely focus on the sequence and timing of when psyllids reach the phloem and xylem and how long they spend performing phloe m and xylem behaviors. Psyllids prefer to ingest from phloem and receive most of their neces sary nutrition from the phloem (Cifuentes Arenas et al. , 2018) . However, ps yllids also ingest from xylem, though it is less preferred compared to phloem. Psyllid waveform names were

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106 established and partially correlated by A. Sharma and Raman (2017) and the analysis herein follows the naming convention, assigned behaviors, and stylet tip locations establish ed in Bonani et al. (2010) and further investigated in this dissertation (Chapter 2) . Diaphorina citri make many test probes prior to accessing or ingesting from the phloem. A probe is an mbedded within host plant tissues thus making strong electrical contact, and therefore raisin g the waveform voltage levels. Psyllids also generally take at least a few hours before they access the phloem for the first time and perform waveform D. Psyllids reached the phloem sooner, and ingested from the phloem for longer durations when probing Mandarin 19 and Pummelo compared to the other treatments (Tables 4 1, 4 2, and 4 3). Psyllids began sustained phloem ingestion (variable TmFrstSusE2) more quickly on Pummelo (fastest at 27,100 seconds) compared to Mandarin 2 (slowest at 54,180 seconds Table 3 1) (P= 0.0001). Psyllids spe nt more time ingesting from phloem (variable TltDurE2) on Mandarin 19 (longest at 37,489 seconds) compared to Mandarin 31 (shortest at 16,829 seconds Table 3 1) (P= 0.0001). When psyllids are less successful at accessing the phloem, they will often spen d more of their access time ingesting from xylem (Bonani et al. , 2010; A. Sharma & Raman, 2017) . Psyllids spent an average duration ingesting from xylem (variable DurG) of 10,914 seconds on Mandarin 2 (longest duration) compared to 5, 101.99 seconds on Mandarin 31 (shortest duration) (P= 0.0159). Ranking Mandarin and Pummelo Using Sequential Variables In an effort to make sense of these data and assign a relative resistance value to each selection or species, a subset of twenty three variables from the Ebert program

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107 that were significantly different (at P= 0.05) between the treatments were used to assign resistance rankings. These variables were selected because they could be associated with resistance or susceptibility in some way. For example, any variable associated with accessing the phloem was ranked susceptible for short durations and resistant for long durations. Variables associated with spending more time ingesting from phloem were ranked as susceptible for higher durations and resistant for lower durations. More time spent ingesting from xylem (rather than phloem) was ranked as resistant for high durations and susceptible for low durations. The treatments with the highest and lowest values were ranked as either resistant or susceptible. The full list and associated ranks is summarized in Table 4 3 and 4 4. Each treatment was then given a resistance ranking in accordance with how many times it was the highest or lowest value and whether each of those values was assigned to a category as either resistant or susceptible. Of the twenty three variables used for resistance ranking, Mandarin 31 was scored as most resistant eleven times and most susceptible two times. Mandarin 2 was scored as most resistant nine times and most sus ceptible three times. Mandarin 26 was scored as most resistant two times and most susceptible two times. Mandarin 18 was never scored as most resistant and was scored as most susceptible six times. Pummelo was never scored as most resistant and was scor ed as most susceptible eleven times. These resistance and susceptibility scores were used to rank the treatments from most resistant (1) to least resistant (5). The resistance rankings are as follows: Mandarin 31 1, Mandarin 2 2, Mandarin 26 3, Mandarin 19 4, and the least resistant is Pummelo, ranked as 5.

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108 Non s equential Variables The Backus SAS program and the associated range of non sequential variables offer complimentary information co mpared to the Ebert program mostly sequential variables. The Eb ert program variables are more sequential in nature, which fits with the sequential natur e of psyllid probing behavior. The Backus analysis program calculates a series of variables at the cohort (or treatment) level, the insect level ( means only are discus sed herein ), and the waveform event level . Cohort l evel v ariables Cohort level variables include the total number of probes (TNP) and the total probing duration (TPD). These variables represent all of the probes (TNP) and the sum of the probing duration (TPD) completed by all of the p syllids within each treatment. The TNP for each treatment is as follows: Mandarin 2 465, Mandarin 19 546, Mandarin 26 488, Mandarin 31 682, and Pummelo 690. The total probing duration in hours for each treatment is as follows: Mandarin 2 391.38, Mandarin 19 404.83, Mandarin 26 324.74, Mandarin 31 311.37, and Pummelo 347.59. Insect l evel v ariables Insect level variables include the waveform duration per insect (WDI) for each waveform category, and the probing duration per insect (PDI). The waveform duration per insect (WDI) values were significantly different among treatments for waveforms C, E2, G, and NP (non probing). For waveform E2, representing phloem ingestion, shorter durations can be associated with host plant resistance to probing while longer durations can be associated with host susceptibility to D. citri probing. Waveform durations per i nsect in seconds for waveform E2 are as follows: Mandarin 2 21,485.05 (5.97 hours), Mandarin 19 37,497.60 (10.42 hours), Mandarin 26 17, 325.16 (4.81 hours), Mandarin

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109 31 16,828.70 (4.67 hours), and Pummelo 24,575.49 (6.83 hours) (P= 0.0001). The wavef orm duration per insect (WDI) data are summarized in Table 3 4 as seconds and in Figure 3 1 as a pie chart presented in hours. The original measurement of the data was completed using the unit of seconds. However, the nature of the very long recording dura tion (24 hours) and often the long duration waveforms such as phloem ingestion (E2) or xylem ingestion (G) are often better represented as hours (Figure 4 1). The probing duration per insect (PDI) variable represents the total probing duration per insect p er treatment. Overall, longer probing durations are associated with probing on a susceptible host plant, while shorter probing durations are associated with probing a resistant host plant. Probing durations per insect (PDI) per treatment in hours are as follows: Mandarin 2 17.79, Mandarin 19 17.60, Mandarin 26 16.24, Mandarin 31 14.83, and Pummelo 16.55 (P= 0.0001). Waveform e vent l evel v ariables The waveform event level variables include waveform duration per event (WDE) and waveform duration per event per insect (WDEI). WDE and WDEI values are expressed seconds in Table 4 5. The waveform duration per event (WDE) variable was significantly different among treatments for the waveforms C, D, E1, and E2. For waveform E2, representing phloem ingesti on, shorter durations can be associated with host plant resistance to probing while longer durations can be associated with host susceptibility to D. citri probing. Waveform durations per insect in seconds for waveform E2 are as follows: Mandarin 2 5,476. 58, Mandarin 19 7,119.80, Mandarin 26 4,950.05, Mandarin 31 3,109.65, and Pummelo 5, 020.80 (P= 0.0004) (Table 4 5). Waveform duration per event per insect (WDEI) values were significantly different among treatments for waveforms C, D, and E1. For wa veform D, representing

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110 initial access of phloem, longer durations can be associated with host plant resistance to probing while shorter durations can be associated with host susceptibility to D. citri probing. Waveform durations per insect per event in sec onds for waveform D are as follows: Mandarin 2 65.80, Mandarin 19 74.29, Mandarin 26 63.16, Mandarin 31 45.58, and Pummelo 45.90 (P= 0.0019) (Table 4 5). Metabolic Profiles A total of sixty six metabolites were found in tested Cleopatra Mandarin hyb rid selections and Pummelo. A total of eight metabolites were found in statistically different concentrations among treatments ( Table s 4 6, 4 7, and 4 8) . The amino acid, serine, was found in the highest concentration in Pummelo, 187.76 mM, and in the lowe st concentration in Mandarin 19, 18.02 mM (P= 0.0173). Two organic acids, succinic acid and quinic acid, were found in statistically different concentrations. Succinic acid was found in the highest concentration in Pummelo, 12.99 mM, and in the lowest conc entration in Mandarin 19, 1.18 mM (P= 0.0297). Quinic acid was found in the highest concentration in Mandarin 2, 9.75 mM, and in the lowest concentration in Mandarin 26, 4.17 mM (P= 0.0203). The fatty acid, oleic acid, was found in the highest concentratio n in Mandarin 19, 2.44 mM, and in the lowest concentration in Mandarin 31, galactose, were found in significantly different concentrations. Xylose 1 was found in the highest concentration in Pummelo, 16.78 mM, galactose was found in the highest concentration in Mandarin 26, 10.13 mM, and in the lowest concentration in Pummelo, 1.71 mM (P= 0.039). Two sugar acids, glycerol and another identif ied as 204/333, were found in significantly different concentrations. Glycerol was found in the highest concentration in Mandarin 26, 5.06 mM, and in the

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111 lowest concentration in Mandarin 2, 1.97 mM (P= 0.0267). The sugar acid 204/333 was found in the high est concentration in Mandarin 26, 73.84 mM, and in the lowest concentration in Mandarin 31, 9.22 mM (P= 0.0247). Diaphorina citri Oviposition and Survivorship Comparison Diaphorina citri was variably successful in both the number of eggs laid and the sur vivorship of each egg to adulthood (Table 4 9). Psyllids were provided with sufficient flush for each Mandarin selection and Pummelo so they could choose the development level of the leaf flush for probing and oviposition. Each cohort of psyllid was provid ed with plants bearing at least ten appropriately sized leaf flushes (Mandarin 2 n= 16, Mandarin 19 n= 10, Mandarin 26 n= 10, Mandarin 31 n= 12, and Pummelo n= 16. This is an important distinction because a lack of available flush does not explain th e variability of observed oviposition differences. Mandarin 2 and Pummelo both had high oviposition levels with variable survivorship to adult. Mandarin 2 survivorship was generally low at 40.83% with 236 of the total 578 eggs laid surviving to adulthood . Psyllids survived to the adult stage at much higher rates on Pummelo, with 81.18% (371) of the 457 eggs laid surviving to the adult stage. Oviposition rates and survivorship to adult were both low on Mandarin 19 and Mandarin 26. Psyllids laid eggs in mod erate levels on both selections with 87 eggs found on Mandarin 19 and and 76 eggs found on Mandarin 26. However, in both of these cases most of the nymphs did not survive past the second instar, with only four psyllids surviving to the adult stage (4.6%) o n Mandarin 19 and only three surviving to the adult stage (3.95%) on Mandarin 26. Interestingly, the highest survival rate was found on Mandarin 31 along with the lowest oviposition rate. Psyllids only laid 29 eggs on Mandarin 31, but 27 (93%) of these su rvived to the adult stage.

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112 Discussion The EPG results herein outline a range of behavioral responses in D. citri probing among the Cleopatra Mandarin hybrid selections and Pummelo tested in this study. While no one selection was consistently the most resistant or the most susceptible to D. citri probing, there was a clear trend. Mandarin 31 was the selection most r esistant to psyllid probing tested herein. Psyllids performed poorly when probing Mandarin 31, making longer D waveforms, spending more time navigating through parenchyma tissues while performing waveform C, spending longer durations not probing before rea ching phloem, ingesting from phloem for the shortest total duration of tested selections, and making only short bouts of ingestion from phloem rather than engaging in long durations of sustained phloem ingestion (Table 4 2). Mandarin 2 scored as only slig htly less resistant compared to Mandarin 31. Psyllids probing Mandarin 2 spent the most overall time navigating the parenchyma tissues and spent the most time performing the C waveform, spent the most time ingesting from xylem rather than phloem, and spen t the longest duration before performing phloem salivation and phloem ingestion (Table 4 3 and 4 4). Mandarin 26 was intermediate, with psyllids exhibiting some resistant behaviors and some susceptible behaviors while probing this selection. Among the b ehaviors exhibited associated with resistance, psyllids took longer to reach the phloem from the time they first probed compared to all other selections, and spent the most time overall not performing phloem associated behaviors, waveforms D, E1, and E2. Among the behaviors associated with Mandarin 26 susceptibility was the lowest total duration of waveform D, associated with initial entry into phloem.

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113 The most susceptible host plants tested, Mandarin 19 and Pummelo (most susceptible), did not rank highe st in any EPG variable category which could be associated with resistance to D. citri probing. Psyllids probing Mandarin 19 exhibited the shortest duration of non probing before performing waveform E1, the highest total duration of phloem ingestion, the lo ngest single bout of phloem ingestion, the highest frequency of E1 (phloem salivation) followed by E2 (phloem ingestion), and the shortest total duration not spent with their stylets in phloem (Table 4 2). Pummelo was ranked as the most susceptible host pl ant tested. Psyllids probing Pummelo exhibited many behaviors linked with EPG variables (11 in total) which could be interpreted as Pummelo being highly susceptible to D. citri probing. Behaviors associated with susceptibility to D. citri include, the fas test initial probe, the lowest total duration of C (meaning the psyllid gets to the phloem much faster), the shortest duration of nonprobing before performing the first D waveform, the highest number of long bouts of phloem ingestion, the shorted duration before the first bout of phloem ingestion, and the shortest duration before starting a long sustained bout of phloem ingestion (Table 4 3). As with the EPG results of this study, the metabolite concentrations do not highlight one Mandarin hybrid selectio n as being consistently the most resistant or the most susceptible to D. citri probing. Rather, a range of resistance can be observed among the metabolite profiles associated with the Mandarin hybrid selections and Pummelo tested herein. Interestingly, the organic acids, succinic and quinic acids, were not necessarily found in the highest levels in selections most resistant to D. citri probing (Mandarin 31 and Mandarin 2). Succunic acid was found in the highest concentration in Pummelo, the species most sus ceptible to D. citri probing. The other concentrations of

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114 succinic acid do not line up neatly with the resistance spectrum defined by EPG variables and D. citri probing behavior. As an organic acid, succinic acid was expected to be found in the highest con centrations in the most resistant Mandarin, selections 31 and 2. Mandarin 2 was found to contain the highest concentration of quinic acid (next to most resistant, 9.75 mM), with the next highest in Mandarin 19 (generally susceptible, 9.09 mM) and the next highest concentration found in Mandarin 31 (most resistant, 9.08 mM). Pummelo exhibited a far higher concentration of the amino acid serine (187.76 mM) compared to other selections, with the next highest concentration found in Mandarin 26 at 76.78 mM and all other selections showing concentration under 26.26 mM. Pummelo also exhibited a far higher concentration of the sugar xylose ( 16.78 mM) compared to other selections, with the next highest concentration found in Mandarin 26 at 6.30 mM. Pummelo was al so generally higher than average in other sugars and sugar acids compared to the Mandarin hybrid selections, indicating that Pummelo is more nutritious to psyllids compared to the tested Mandarin hybrid selections. Paradoxically, Pummelo is relatively def ended against psyllid and other pests, having generally high levels of succinic acid and quinic acids found in leaf tissues. The Cleopatra Mandarin hybrid selections and Pummelo metabolites outlined herein do not explain all of the probing behavior of D . citri observed in this study. This is due, in part, to the incredibly complex nature of how and when plants manage their arsenal of defensive compounds. We do not know which types of metabolites are constitutive and which are induced by D. citri probing in each of the plants tested.

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115 Another possible angle might be that Pummelo, the most susceptible plant tested, may not have a complex arsenal of constitutive defensive metabolites, but rather upregulated the production of succinic acid and quinic acid in response to D. citri probing, while the other more resistant Mandarin hybrid selections, 31 and 2, had generally high constitutive levels of these compounds. Defensive plant metabolites can be induced in response to pest attack (Ebert et al. , 2018) , pathogen attack (Hijaz et al. , 2013) , and as a wound response (Killiny, 2017) . Another interpretation of the metabolite concentrations found in Mandarin hybrid selections and Pummelo and the ir impact on D. citri probing behavior might focus on psyllid nutrition rather than defensive metabolites. Portillo Estrada and Niinemets (2018) found sugar concentrations to be a limiting factor of HLB disease development in Cleopatra Mandarin and found defensive compounds were not tightly associated with resistance to HLB. Lower concentratio ns of sugars and sugar acids in Mandarin 31 and Mandarin 2 might be more important variables linking D. citri probing behavior and resistance rather than high levels of defensive metabolites. The small oviposition study tracking nymphal survival to adult w as performed to assess the quality of the Mandarin hybrid selections and Pummelo as hosts for D. citri in conjunction with probing data and host plant metabolic concentrations. We found that psyllids readily oviposited on and survived well on Pummelo (81% survival to adult), while psyllids struggled to oviposit on some Mandarin hybrid selections (19, 26, and 31), and oviposited readily but struggled to survive to the adults stage (Mandarin 2) with only 41% of eggs surviving to adulthood. We do not yet hav e a complete understanding of the complex interactions between plant metabolites, psyllid probing behavior and development on Citrus host.

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116 Th e current study aims to provide data on psyllid probing behaviors and plant metabolic contents and attempts to elu cidate some of the contributing factors to psyllid host preference. The detailed quantification of D. citri probing behavior, oviposition, survivorship, and identification of Cleopatra Mandarin hybrids more resistant to psyllid probing combined with the de tection of many important metabolic compounds summarized in this study provides new insights which can be used for the selection of new rootstock candidates.

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117 Table 4 1. Probing behavior of Diaphorina citri on Cleopatra Mandarin h ybrid selections and Pummelo s ummary of non phloem sequential variables. Data are means and standard errors generated by ANOVA using a SAS program from Ebert et al ., 2015. Means with different letter designations are statistically different from one another. P= 0.05. Variable Mandarin 2 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P value DurFrstPrb 3,028.69 ± 1,060.94 b 7,004 ± 1,060.94 b 3,777.27 ± 1,060.94 b 652.35 ± 1,060.94 a 3,960.04 ± 1,060.94 a 0.0001 DurScndPrb 6,131.48 ± 1,038.06 b 5,677.06 ± 1,038.06 a 2,978.65 ± 1,038.06 b 415.72 ± 1,038.06 c 4,667.99 ± 1,038.06 b 0.0001 TtlPrbTm 64,043 ± 1,461.24 a 63,364 ± 1,461.24 ab 58,454 ± 1,461.24 b 53,379 ± 1,461.24 c 59,587 ± 1,461.24 b 0.0001 TtlDurNP 17,086 ± 1,477.84 b 18,294 ± 1,477.84 a 25,139 ± 1,477.84 b 28,616 ± 1,477.84 c 23,141 ± 1,477.84 a 0.0001 TmFrstPrb FrmStrt 894.38 ± 365.78 bc 1,394.91 ± 357.74 b 994.22 ± 383.63 b 2,142.6 ± 374.39 a 636.31 ± 374.39 c 0.0493 DurNnprb BfrFrstE1 10,595 ± 1,402.11 b 7,714.37 ± 1,402.11 c 13,003 ± 1,402.11 a 13,576 ± 1,402.11 a 9,498.31 ± 1,402.11 b 0.0001 TtlDurC 39,726 ± 1,468.56 a 31,001 ± 1,468.56 bc 36,203 ± 1,468.56 ab 33,904 ± 1,468.56 b 28,496 ± 1,468.56 c 0.0001 MnDurC 1,549.03 ± 208.16 a 1,486.29 ± 203.58 a 1,307.32 ± 218.32 a 1,041.82 ± 213.05 ab 740.48 ± 213.05 b 0.045 PrcntPrbC 62.01% ± 0.21 b 50.75% ± 0.2 c 61.77% ± 0.22 b 66.15% ± 0.21 a 48% ± 0.21 c 0.0502 DurG 10,914 ± 1,174.94 a 7,236.19 ± 1,174.94 b 7,784.82 ± 1,232.29 b 5,010.99 ± 1,232.29 c 8,746.03 ± 1,232.29 b 0.0159 CtoFrstG 2.55 ± 0.97 c 3.18 ± 0.97 c 4.8 ± 1.02 b 6.75 ± 1.02 a 7.5 ± 1.02 a 0.0016 DurNnprb BfrFrstG 2,690.05 ± 911.11 c 2,925.42 ± 911.11 c 4,228.46 ± 955.58 b 7,799.86 ± 955.58 a 3,481.51 ± 955.58 b 0.0013 NumLngG 4.64 ± 0.38 a 3.32 ± 0.38 bc 2.95 ± 0.4 c 3.4 ± 0.4 b 3.7 ± 0.4 b 0.0362 TmFrstSus GFrstPrb 5,290.69 ± 1,176.53 c 9,859 ± 1,176.53 b 10,260 ± 1,176.53 ab 12,593 ± 1,176.53 a 9,158.57 ± 1,176.53 b 0.0001

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118 Table 4 2. Probing behavior of Diaphorina citri on Cleopatra Mandarin h ybrid selections and Pummelo s ummary of p hloem a ssociated s equential v ariables. Variable Mandarin 2 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P value meanD 65.8 ± 9.01 b 74.29 ± 7.62 b 63.16 ± 8.01 b 89.86 ± 8.01 a 43.28 ± 7.8 c 0.0019 DurNnprbBfrFrstD 10,414 ± 1,137.58 a 7,100.46 ± 1,137.58 b 10,375 ± 1,137.58 a 9,858.38 ± 1,137.58 a 6,525.98 ± 1,137.58 b 0.0001 TmFrmFrstPrbFrstD 25,391 ± 2,533.24 a 22,434 ± 2,533.24 b 28,086 ± 2,533.24 a 21,247 ± 2,533.24 b 23,052 ± 2,533.24 b 0.0001 NumLngD 1.07 ± 0.75 b 2.14 ± 0.64 a 0.32 ± 0.67 c 2.42 ± 0.67 a 0.15 ± 0.65 c 0.0497 TmFrstSusDFrstPrb 50,847 ± 3,956.87 b 55,335 ± 3,956.87 a 24,223 ± 3,956.87 c 49,283 ± 3,956.87 b 55,939 ± 3,956.87 a 0.0001 maxD 97.55 ± 22.99 b 129.85 ± 19.43 a 91.53 ± 20.43 b 141.95 ± 20.43 a 61.04 ± 19.91 c 0.0426 PrcntPrbD 6.18% ± 0.2 b 8.48% ± 0.19 a 6.35% ± 0.21 b 10.29% ± 0.2 a 8.10% ± 0.2 c 0.05032 MnDurE1 76.39 ± 9.68 a 50.17 ± 8.19 b 68.92 ± 8.61 a 45.58 ± 8.61 b 45.9 ± 8.39 b 0.0463 TmFrmFrstPrbFrstE 44,248 ± 2,791.3 a 27,104 ± 2,791.3 bc 30,938 ± 2,791.3 b 27,109 ± 2,791.3 bc 25,995 ± 2,791.3 c 0.0001 DurFirstE 4,0 49.22 ± 1,614.45 a 6,805.83 ± 1,364.46 c 24,30.27 ± 1,434.48 b 25,33.66 ± 1,434.48 b 6,919.58 ± 1,398.16 c 0.05011 NumLngE2 1.27 ± 0.46 a 2.7 ± 0.45 b 2.2 ± 0.49 b 2.38 ± 0.47 b 3.57 ± 0.47 a 0.0175 TtlDurE2 21,485 ± 1,410.43 a 37,498 ± 1,410.43 a 17,325 ± 1,410.43 c 16,829 ± 1,410.43 c 24,575 ± 1,410.43 b 0.0001 TmFrstSusE2FrstPrb 53,957 ± 2,879.65 a 31,589 ± 2,879.65 c 40,353 ± 2,879.65 b 40,329 ± 2,879.65 b 26,581 ± 2,879.65 c 0.0001 TmFrstE2StrtEPG 49,854 ± 2,878.41 a 32,586 ± 2,878.41 bc 39,348 ± 2,878.41 b 38,777 ± 2,878.41 b 26,581 ± 2,878.41 c 0.0001 TmFrstE2FrmFrstPrb 48,960 ± 2,855.3 a 31,191 ± 2,855.3 bc 38,354 ± 2,855.3 b 36,634 ± 2,855.3 b 25,945 ± 2,855.3 c 0.0001 TmLstE2EndRcrd 20,057 ± 3,474.06 b 9,265.65 ± 3,474.06 c 21,138 ± 3,474.06 b 29,78.84 ± 3,474.06 a 6,688.05 ± 3,474.06 c 0.0001 maxE2 12,665 ± 1,968.22 b 17,409 ± 1,832.31 a 9,979.22 ± 1,774.13 c 8,391.74 ± 1,721.16 c 10,803 ± 1,628.05 c 0.0081 PrcntPrbE2 29.23% ± 0.41 c 56.71% ± 0.38 a 19.47% ± 0.37 c 22.44% ± 0.36 c 37.05% ± 0.34 b 0.0149 PrcntE2SusE2 43.78% ± 0.35 c 69.21% ± 0.29 a 72.31% ± 0.33 a 50.25% ± 0.27 b 72.11% ± 0.27 a 0.0184 TtlDurE 19,240 ± 1,587.1 b 27,282 ± 1,587.1 a 14,962 ± 1,587.1 c 15,619 ± 1,587.1 c 23,729 ± 1,587.1 a 0.0001 TtlDurE1Fllwd E2PlsE2 21,784 ± 1,420.42 b 37,813 ± 1,420.42 a 17,584 ± 1,420.42 c 17,095 ± 1,420.42 c 24,801 ± 1,420.42 b 0.0001 TotDurNnPhlPhs 62,111 ± 1,554.42 ab 55,186 ± 1,554.42 c 68,613 ± 1,554.42 a 66,372 ± 1,554.42 a 58,941 ± 1,554.42 b 0.0001 TmFrstSusE2 54,180 ± 2,843.84 a 32,642 ± 2,843.84 c 41,154 ± 2,843.84 b 41,630 ± 2,843.84 b 27,100 ± 2,843.84 c 0.0001

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119 Table 4 3. Using s equential v ariables to r ank Mandarin s elections and Pummelo for r esistance . Variable Abb. Variable Rank Highest Value Lowest Value TtlPrbTm High= Susceptible/ Low= Resistant Mandarin 2 Mandarin 31 TtlDurNP High= Resistant/ Low= Susceptible Mandarin 31 Mandarin 2 TmFrstPrbFrmStrt High= Resistant/ Low= Susceptible Mandarin 31 Pummelo DurNnprbBfrFrstE1 High= Resistant/ Low= Susceptible Mandarin 31 Mandarin 19 TtlDurC High= Resistant/ Low= Susceptible Mandarin 2 Pummelo PrcntPrbC High= Resistant/ Low= Susceptible Mandarin 31 Pummelo DurG High= Resistant/ Low= Susceptible Mandarin 2 Mandarin 31 meanD High= Resistant/ Low= Susceptible Mandarin 31 Mandarin 26 DurNnprbBfrFrstD High= Resistant/ Low= Susceptible Mandarin 2 Pummelo TmFrmFrstPrbFrstD High= Resistant/ Low= Susceptible Mandarin 26 Mandarin 31 PrcntPrbD High= Resistant/ Low= Susceptible Mandarin 31 Mandarin 2 TmFrmFrstPrbFrstE High= Resistant/ Low= Susceptible Mandarin 2 Pummelo NumLngE2 High= Susceptible/ Low= Resistant Pummelo Mandarin 2 TtlDurE2 High= Susceptible/ Low= Resistant Mandarin 19 Mandarin 31 TmFrstSusE2FrstPrb High= Resistant/ Low= Susceptible Mandarin 2 Pummelo TmFrstE2StrtEPG High= Resistant/ Low= Susceptible Mandarin 2 Pummelo TmFrstE2FrmFrstPrb High= Resistant/ Low= Susceptible Mandarin 2 Pummelo TmLstE2EndRcrd High= Resistant/ Low= Susceptible Mandarin 31 Pummelo maxE2 High= Susceptible/ Low= Resistant Mandarin 19 Mandarin 31 TtlDurE High= Susceptible/ Low= Resistant Mandarin 19 Mandarin 26 TtlDurE1FllwdE2PlsE2 High= Susceptible/ Low= Resistant Mandarin 19 Mandarin 31 TotDurNnPhlPhs High= Resistant/ Low= Susceptible Mandarin 26 Mandarin 19 TmFrstSusE2 High= Resistant/ Low= Suscept ible Mandarin 2 Pummelo * Italics denotes most resistant response for that particular variable Table 4 4. Summary of r esistance r anking for Mandarin s elections and Pummelo . Host Plants Resistance Score Susceptibility Score Overall Score 1= Most Resistant Mandarin 31 11 2 1 Mandarin 2 9 3 2 Mandarin 26 2 2 3 Mandarin 19 0 6 4 Pummelo 0 11 5

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120 Table 4 5. Probing behavior of Diaphorina citri on Cleopatra Mandarin hybrid selections and Pummelo. Data are means and standard errors generated by ANOVA using a SAS program from Backus et al ., 2007. Means with different letter designations are statistically different from one another. P= 0.05. Waveform d uration per e vent (WDE) in seconds Wf Mandarin 2 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value C 1, 279.62 ± 79.04 a 933.27 ± 62.11 b 1,120.82 ± 75.17 a 800.89 ± 49.19 bc 684.70 ± 43.61 c 0.0001* D 72.09 ± 4.03 b 90.71 ± 7.10 a 63.87 ± 5.62 b 88.78 ± 4.28 a 40.99 ± 2.19 c 0.0001* E1 68.32 ± 5.23 a 53.66 ± 4.26 b 55.76 ± 6.32 ab 49.38 ± 3.32 b 45.00 ± 4.34 b 0.0092* E2 5,476.58 ± 912.22 ab 7,119.80 ± 830.69 a 4,950.05 ± 760.92 bc 3,109.65 ± 429.29 c 5,020.80 ± 533.54 b 0.0004* G 1,752.61 ± 178.05 1,693.58 ± 202.92 1,831.72 ± 282.86 1,252.75 ± 93.78 1,604.78 ± 264.75 0.4403 NP 791.37 ± 111.34 759.50 ± 78.92 1,009.60 ± 184.82 872.17 ± 83.04 697.22 ± 101.78 0.3507 Waveform d per e vent per i nsect (WDEI) in seconds Wf Mandarin 2 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value C 1,549.03 ± 203.59 a 1,486.29 ± 320.55 a 1,307.32 ± 188.53 ab 1,041.82 ± 164.62 ab 740.48 ± 53.46 b 0.045* D 65.80 ± 5.52 bc 74.29 ± 10.15 ab 63.16 ± 9.05 ac 89.86 ± 8.53 a 43.28 ± 3.92 c 0.0019* E1 76.39 ± 10.15 a 50.16 ± 5.32 b 68.92 ± 14.12 ab 45.58 ± 5.42 b 45.90 ± 5.95 b 0.0463* E2 7,050.43 ± 1,484.56 8,826.80 ± 1,642.97 5,423.26 ± 850.94 4,263.67 ± 898.17 6,394.92 ± 1,245.20 0.1189 G 1,999.73 ± 206.73 1,738.44 ± 278.56 2,002.33 ± 470.14 1,331.00 ± 104.59 2,014.61 ± 536.17 0.6 NP 1,374.57 ± 486.89 1,496.96 ± 520.94 2,032.19 ± 666.50 1,347.90 ± 363.23 1,737.58 ± 890.02 0.9288 Waveform d uration per i nsect (WDI) in seconds Wf Mandarin 2 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value C 39,726.34 ± 3,415.73 a 31,000.63 ± 2,854.98 b 36,202.63 ± 3,897.54 c 33,904.28 ± 3,316.37 d 28,496.46 ± 2,940.51 e 0.0001* D 418.13 ± 155.91 583.11 ± 218.12 265.55 ± 50.73 630.81 ± 145.03 170.13 ± 24.36 0.0917 E1 619.45 ±132.33 498.26 ± 122.84 372.74 ± 75.03 561.38 ± 124.80 382.48 ± 68.98 0.427 E2 21,485.05 ± 3,223.37 a 37,497.60 ± 3,149.74 b 17,325.16 ± 2,818.96 c 16,828.70 ± 3,121.45 d 24,575.49 ± 3,277.74 e 0.0001* G 10,913.95 ± 1,139.76 a 7,236.19 ± 1,198.01 bc 7,784.82 ± 1,359.79 abc 5,010.99 ± 599.97 c 8,746.03 ± 1,542.43 ab 0.0159* NP 17,086.40 ± 2,812.32 a 18,294.12 ± 2,389.30 b 25,139.14 ± 4,475.46 c 28,615.53 ± 3,119.83 d 23,141.09 ± 3,649.61 e 0.0001*

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121 Table 4 6. Metabolites in 0.1g l eaf tissue in Cleopatra Mandarin hybrid selections and Pummelo o rganic a cids, f atty a cids, and a mino a c ids. Metabolite Mandarin 02 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value Ferulic Acid 338 7.06 ± 6.81 26.87 ± 32.38 45.78 ± 40.40 33.77 ± 37.14 7.76 ± 8.95 0.096 Gluconic Acid 11.26 ± 14.76 7.33 ± 7.70 31.29 ± 30.03 4.06 ± 3.21 3.90 ± 6.62 0.7348 Citric Acid 3,989.68 ± 5,276.66 2,316.82 ± 4,308.36 5,901.28 ± 4,018.27 3,287.54 ± 6,973.44 8,546.84 ± 11,588.80 0.23 Quinic Acid 9.75 ± 0.84 a 9.09 ± 0.84 a 4.17 ± 0.84 b 9.08 ± 0.84 a 8.48 ± 0.84 a 0.0203 * Malic Acid 729.46 ± 1,459.26 119.33 ± 165.17 275.41 ± 280.76 1,465.81 ± 869.77 422.74 ± 249.38 0.0864 Synephrine 228.52 ± 365.65 274.02 ± 357.05 246.44 ± 337.61 141.95 ± 218.21 0 0.3256 Succinic Acid 4.24 ± 2.46 ab 1.18 ± 2.46 b 4.49 ± 2.46 ab 3.43 ± 2.46 ab 12.99 ± 2.46 a 0.0297 * Fumaric Acid 0.84 ± 1.10 2.41 ± 4.02 2.98 ± 3.39 1.22 ± 0.94 1.22 ± 1.24 0.834 Phosphoric Acid 13.76 ± 19.06 0 0 0 66.54 ± 17.25 0.2055 Maleic Acid 21.86 ± 22.15 20.79 ± 24.03 110.88 ± 144.76 76.40 ± 59.14 129.57 ± 149.87 0.3013 Lactic Acid 4.00 ± 7.89 8.90 ± 13.51 26.46 ± 53.07 10.24 ± 15.38 5.85 ± 5.52 0.7579 Oxalic Acid 4.14 ± 2.66 2.46 ± 3.04 3.92 ± 3.3 52.28 ± 88.07 27.03 ± 50.68 0.3798 Palmitic Acid 171.63 ± 146.79 62.77 ± 131.28 204.87 ± 250.98 172.55 ± 209.70 45.40 ± 33.12 0.4819 Oleic Acid 1.11 ± 0.72 bc 2.44 ± 0.72 a 1.85 ± 0.72 ab 0.4 ± 0.72 abc 1.47 ± 0.8 c 0.0072 * Stearic Acid 4.27 ± 2.47 2.82 ± 4.78 18.59 ± 21.38 120.75 ± 256.19 19.72 ± 26.09 0.327 L Alanine 24.96 ± 25.13 22.81 ± 20.35 2.84 ± 2.01 22.08 ± 36.15 43.70 ± 77.58 0.3133 Glutamic Acid 8.13 ± 11.47 0.97 ± 1.11 23.93 ± 41.10 22.07 ± 36.68 77.57 ± 95.77 0.6388 L Aspartic Acid 18.05 ± 31.34 5.03 ± 7.42 19.66 ± 17.27 8.77 ± 9.81 20.48 ± 22.38 0.8808 Aminobutyric Acid 360.04 ± 664.31 590.50 ± 715.28 725.34 ± 717.59 974.47 ± 529.83 79.91 ± 93.21 0.6335 L Threonine 12.69 ± 9.53 10.16 ± 13.50 41.98 ± 57.13 18.63 ± 21.13 35.38 ± 35.41 0.6296 Serine 24.21 ± 36.44 b 18.02 ± 36.44 b 76.78 ± 36.44 ab 26.26 ± 36.44 b 187.76 ± 36.44 a 0.0173 * L Isoleucine 5.28 ± 3.35 2.45 ± 3.85 2.15 ± 4.21 1.29 ± 0.54 6.75 ± 6.69 0.3506 L Proline 155.95 ± 111.91 13.57 ± 9.52 40.55 ± 26.16 10.80 ± 8.45 128.26 ± 206.64 0.0669 Glycine 0 1.93 ± 2.38 20.81 ± 17.85 25.00 ± 35.82 18.09 ± 17.96 0.2206 L Valine 2.62 ± 2.28 2.82 ± 4.78 18.59 ± 21.38 4.02 ± 7.02 1.10 ± 1.06 0.324 2 Aminopropanol 37.54 ± 38.67 52.81 ± 36.33 51.34 ± 41.49 15.73 ± 4.03 11.93 ± 11.12 0.297 Data are means and standard errors generated by ANOVA in SAS. Means with different letter designations are statistically diff erent from one another. P= 0.05.

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122 Table 4 7. Metabolites in 0.1g l eaf tissue in Cleopatra Mandarin hybrid selections and Pummelo s ugars . Metabolite Mandarin 02 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value Xylose 1 3.37 ± 2.35 b 4.15 ± 2.35 b 6.30 ± 2.35 b 3.08 ± 2.35 b 16.78 ± 2.35 a 0.0473 * Xylose 2 48.96 ± 58.79 13.06 ± 17.08 32.76 ± 58.84 36.09 ± 48.58 45.15 ± 43.79 0.8393 Arabinose 1.50 ± 1.04 7.56 ± 5.72 4.46 ± 3.34 5.25 ± 6.92 3.77 ± 2.01 0.5822 Erythrose 156.54 ± 78.21 239.49 ± 64.14 254.54 ± 100.14 260.12 ± 119.96 181.15 ± 5.04 0.2415 Fructose 414.94 ± 471.91 648.13 ± 864.54 2,827.34 ± 4,297.50 1,183.59 ± 1,404.23 1,162.75 ± 1,412.64 0.3747 Mannose 104.01 ± 177.97 128.47 ± 149.63 140.10 ± 125.65 192.22 ± 103.78 59.57 ± 71.99 0.433 Glucose 7001.43 ± 12,025.90 1,569.44 ± 2,011.27 5,770.29 ± 12,120.85 1,037.07 ± 1,240.04 2,398.38 ± 4,143.17 0.9915 Glucopyranose 11.19 ± 16.33 32.25 ± 35.77 19.94 ± 21.38 18.18 ± 15.79 3.29 ± 3.95 0.3198 Threose 8.13 ± 11.20 12.64 ± 15.14 17.22 ± 10.86 19.24 ± 13.40 8.61 ± 10.88 0.6122 Pyranoside 204/338 1.18 ± 0.44 5.16 ± 6.12 1.15 ± 1.39 0.16 ± 0.06 0.24 ± 0.45 0.8232 galactoside 5.06 ± 1.93 ab 2.95 ± 1.93 ab 10.13 ± 1.93 a 2.50 ± 1.93 ab 1.71 ± 1.93 b 0.039 * Deoxy galactoside 393.40 ± 869.20 74.87 ± 102.35 59.01 ± 43.88 19.72 ± 25.23 8.58 ± 15.46 0.2983 Glucoheptose 932.98 ± 1,318.23 1,350.10 ± 1,316.29 1,300.50 ± 2,053.14 1,404.91 ± 2,826.17 356.07 ± 566.44 0.2401 Sucrose 24,586.78 ± 20,814.29 10,144.57 ± 9,511.408 26,319.23 ± 56,240.63 11,322.82 ± 15,132.80 10,305.66 ± 10,812.49 0.1514 Maltose 66.46 ± 50.97 332.82 ± 376.90 31.92 ± 26.38 69.59 ± 54.02 229.71 ± 157.41 0.7644 Unk Disaccharide 361 218.88 ± 222.90 94.45 ± 152.11 188.48 ± 340.13 283.68 ± 261.95 67.99 ± 111.31 0.1241 Unk Disaccharide 87.59 ± 69.97 11.82 ± 12.26 19.89 ± 20.037 53.94 ± 50.23 69.23 ± 86.47 0.5592 Data are means and standard errors generated by ANOVA in SAS. Means with different letter designations are statistically diff erent from one another. P= 0.05.

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123 Table 4 8. Metabolites in 0.1g l eaf tissue in Cleopatra Mandarin hybrid selections and Pummelo s ugar a lcohols and s ugar a cids. Metabolite Mandarin 02 Mandarin 19 Mandarin 26 Mandarin 31 Pummelo P Value Inositol 2 Phosphate 15.89 ± 21.34 10.65 ± 7.55 18.13 ± 23.89 11.55 ± 14.80 1.79 ± 1.86 0.946 Scyllo Inositol 218.96 ± 167.70 193.23 ± 156.54 326.86 ± 411.38 281.57 ± 189.51 197.42 ± 256.13 0.7775 Myo Inositol 351.59 ± 546.51 2.46 ± 3.04 3.92 ± 3.30 93.55 ± 184.35 205.76 ± 227.12 0.1965 Phytol 143 13.94 ± 18.45 0 45.89 ± 56.93 9.72 ± 3.68 0.02 ± 0.04 0.3732 Glycerol 1.97 ± 0.67 b 4.9 ± 0.67 a 5.06 ± 0.67 a 4.32 ± 0.67 ab 4.46 ± 0.67 ab 0.0267 * Xylitol 87.91 ± 181.95 252.24 ± 251.94 382.21 ± 346.67 110.18 ± 137.42 117.65 ± 150.62 0.318 Glucitol 11.87 ± 16.82 5.53 ± 6.40 7.27 ± 10.61 3.35 ± 3.11 0.96 ± 1.57 0.6759 Mannitol 34.73 ± 38.52 37.67 ± 55.35 48.29 ± 39.73 17.44 ± 21.41 97.69 ± 108.58 0.3695 Chiro Inositol 730.71 ± 818.83 1,337.19 ± 1,033.19 1,334.80 ± 2,109.64 803.14 ± 720.79 0 0.4593 Sugar Alcohol 217/319 6.17 ±4.01 5.78 ± 6.52 11.96 ± 10.08 11.03 ± 8.31 3.31 ± 5.84 0.4336 Ribonic Acid 93.48 ± 112.08 34.67 ± 43.88 64.91 ± 78.58 140.54 ± 121.14 71.06 ± 78.88 0.2806 Saccharic Acid 14.12 ± 24.50 5.88 ± 4.38 20.83 ± 23.03 12.21 ± 21.97 8.61 ± 5.06 0.6895 Sugar Acid 204/333 17.10 ± 14.16 b 19.12 ± 14.16 b 73.84 ± 14.16 a 9.22 ± 14.16 b 14.37 ± 14.16 b 0.0247 * Glucuronic Acid 3.37± 3.71 2.59 ± 2.88 7.06 ± 11.11 6.43 ± 5.96 1.19 ± 2.67 0.5053 Threonic Acid Deriv. 66.70 ± 123.42 15.65 ± 15.43 33.12 ± 22.20 67.12 ± 81.69 31.79 ± 37.23 0.1031 Threonic Acid 48.34 ± 34.19 193.47 ± 193.11 95.82 ± 65.08 100.25 ± 114.15 974.05 ± 1,182.63 0.8799 2 Ketoglutaric Acid 2.04 ± 2.02 2.24 ± 0.75 2.08 ± 2.61 6.50 ± 7.55 5.70 ± 5.44 0.5845 Arabino Hexaric Acid 5.59 ± 5.12 7.28 ± 10.57 5.44 ± 2.67 3.30 ± 3.88 4.52 ± 3.49 0.5592 Unk Sugar Acid 333 75.24 ± 92.05 26.66 ± 38.38 132.95 ± 202.05 165.58 ± 185.82 34.11 ± 42.22 0.2685 Glycerol Glycoside 170.89 ± 251.21 256.32 ± 375.03 180.57 ± 177.95 33.23 ± 55.60 34.83 ± 27.99 0.3235 Data are means and standard errors generated by ANOVA in SAS. Means with different letter designations are statistically diff erent from one another. P= 0.05.

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124 Table 4 9. Diaphorina citri oviposition and survivorship to ad ult on Cleopatra Mandarin hybrid selections, Citrus reticulata , and Pummelo , Citrus grandis . Selection/ Species Eggs l aid Adults c ounted Total a dults e merged % Adults e merged from e gg Day 10* Day 24 Day 27 Day 29 Mandarin 2 578 12 139 66 19 236 40.83% Mandarin 19 87 0 3 1 0 4 4.60% Mandarin 26 76 0 0 3 0 3 3.95% Mandarin 31 29 2 3 6 6 27 93.10% Pummelo 457 42 241 78 11 371 81.18% * Adults were removed at day 10 and as they emerged to avoid additional oviposition

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125 Figure 4 1. Diaphorina citri probing durations on Mandarin hybrid selections and Pummelo waveform duration per insect (WDI) in hours. C 10.06 D 0.07 E1 0.10 E2 4.81 G 2.16 NP 6.98 Mandarin 26 C 11.04 D 0.12 E1 0.17 E2 5.97 G 3.03 NP 4.75 Mandarin 02 C 8.61 D 0.16 E1 0.14 E2 10.42 G 2.01 NP 5.08 Mandarin 19 C 9.42 D 0.18 E1 0.16 E2 4.67 G 1.39 NP 7.95 Mandarin 31 C 7.92 D 0.05 E1 0.11 E2 6.83 G 2.43 NP 6.43 Pummelo

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126 Figure 4 2. Percentage composition of metabolic groups found in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . Organic Acids 14% Fatty Acids 0% Amino Acids 3% Sugars 71% Sugar Alcohols 9% Sugar Acids 3% Mandarin 19 Organic Acids 12% Fatty Acids 0% Amino Acids 2% Sugars 81% Sugar Alcohols 4% Sugar Acids 1% Mandarin 2 Organic Acids 21% Fatty Acids 1% Amino Acids 5% Sugars 65% Sugar Alcohols 6% Sugar Acids 2% Mandarin 31 Organic Acids 14% Fatty Acids 0% Amino Acids 2% Sugars 78% Sugar Alcohols 5% Sugar Acids 1% Mandarin 26 Organic Acids 35% Fatty Acids 0% Amino Acids 2% Sugars 56% Sugar Alcohols 2% Sugar Acids 5% Pummelo

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127 Figure 4 3. Percentage composition of organic acids in Mandarin selections, Citrus reticulata, and Pummelo, Citrus grandis. Ferulic Acid 338 1% Citric Acid 65% Malic Acid 29% Synephrin e 3% Maleic Acid 1% Oxalic Acid 1% Mandarin 31 Ferulic Acid 338 1% Citric Acid 83% Quinic Acid 1% Malic Acid 4% Synephrin e 10% Maleic Acid 1% Mandarin 19 Citric Acid 79% Malic Acid 15% Synephrine 5% Maleic Acid 1% Mandarin 2 Ferulic Acid 338 1% Citric Acid 89% Malic Acid 4% Synephrine 4% Maleic Acid 2% Mandarin 26 Citric Acid 93% Malic Acid 5% Phosphori c Acid 1% Maleic Acid 1% Pummelo

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128 Figure 4 4. Percentage composition of fatty acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . Palmitic Acid 92% Oleic Acid 4% Stearic Acid 4% Mandarin 19 Palmitic Acid 97% Oleic Acid 1% Stearic Acid 2% Mandarin 2 Palmitic Acid 91% Oleic Acid 1% Stearic Acid 8% Mandarin 26 Palmitic Acid 68% Oleic Acid 2% Stearic Acid 30% Pummelo Palmitic Acid 59% Oleic Acid 0% Stearic Acid 41% Mandarin 31

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129 Figure 4 5. Percentage composition of amino acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . L Alanine 3% L Aspartic Acid 1% Aminobutyr ic Acid 82% L Threonine 2% Serine 3% L Proline 2% 2 Aminoprop anol 7% Mandarin 19 L Alanine 2% Glutamic Acid 2% L Aspartic Acid 1% Aminobutyric Acid 86% L Threonine 2% Serine 2% L Proline 1% Glycine 2% 2 Aminopropano l 2% Mandarin 31 L Alanine 4% Glutamic Acid 1% L Aspart ic Acid 3% Aminobuty ric Acid 55% L Threonine 2% Serine 4% L Isoleucine 1% L Proline 24% 2 Aminoprop anol 6% Mandarin 2 Glutamic Acid 2% L Aspartic Acid 2% Aminobuty ric Acid 71% L Threonine 4% Serine 8% L Proline 4% Glycine 2% L Valine 2% 2 Aminoprop anol 5% Mandarin 26 L Alanine 7% Glutamic Acid 13% L Aspartic Acid 3% Aminobutyr ic Acid 13% L Threonine 6% Serine 31% L Isoleucine 1% L Proline 21% Glycine 3% 2 Aminopropa nol 2% Pummelo

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130 Figure 4 6. Percentage composition of sugars in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . Erythrose 2% Fructose 4% Mannose 1% Glucose 11% Deoxy galactosid e 1% Glucohepto se 9% Sucrose 69% Maltose 2% Unk Disaccharide 361 1% Mandarin 19 Erythrose 2% Fructose 7% Mannose 1% Glucose 7% Glucohep tose 9% Sucrose 71% Unk Disaccharid e 361 2% Mandarin 31 Fructose 1% Glucose 21% Deoxy galactosid e 1% Glucohept ose 3% Sucrose 72% Unk Disacchari de 361 1% Mandarin 2 Erythrose 1% Fructose 8% Glucose 16% Glucohept ose 4% Sucrose 71% Unk Disacchari de 361 1% Mandarin 26 Erythrose 1% Fructose 8% Glucose 16% Glucohepto se 2% Sucrose 69% Maltose 2% Pummelo

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131 Figure 4 7. Percentage composition of sugar alcohols in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . Inositol 2 Phosphat e 1% Scyllo Inositol 11% Xylitol 14% Mannitol 2% Chiro Inositol 72% Mandarin 19 Inositol 2 Phosphate 1% Scyllo Inositol 21% Myo Inositol 7% Phytol 143 1% Xylitol 8% Mannitol 1% Chiro Inositol 60% Sugar Alcohol 217/319 1% Mandarin 31 Inositol 2 Phosphate 1% Scyllo Inositol 15% Phytol 143 2% Xylitol 18% Mannitol 2% Chiro Inositol 61% Sugar Alcohol 217/319 1% Mandarin 26 Scyllo Inositol 31% Myo Inositol 33% Glycerol 1% Xylitol 19% Mannitol 16% Pummelo Inositol 2 Phospha te 1% Scyllo Inositol 15% Myo Inositol 24% Phytol 143 1% Xylitol 6% Glucitol 1% Mannitol 2% Chiro Inositol 50% Mandarin 2

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132 Figure 4 8. Percentage composition of sugar acids in Mandarin selections, Citrus reticulata , and Pummelo, Citrus grandis . Ribonic Acid 6% Saccharic Acid 1% Sugar Acid 204/333 3% Glucuronic Acid 1% Threonic Acid Deriv. 3% Threonic Acid 34% Arabino Hexaric Acid 1% Unk Sugar Acid 333 5% Glycerol Glycoside 46% Mandarin 19 Ribonic Acid 26% Saccharic Acid 2% Sugar Acid 204/333 2% Glucuronic Acid 1% Threonic Acid Deriv. 12% Threonic Acid 18% 2 Ketoglutaric Acid 1% Arabino Hexaric Acid 1% Sugar Acid 333 31% Glycerol Glycoside 6% Mandarin 31 Ribonic Acid 19% Saccharic Acid 3% Sugar Acid 204/333 4% Glucuronic Acid 1% Threonic Acid Deriv. 13% Threonic Acid 10% Unk Sugar Acid 333 15% Glycerol Glycoside 34% Mandarin 2 Ribonic Acid 11% Saccharic Acid 3% Sugar Acid 204/333 12% Glucuronic Acid 1% Threonic Acid Deriv. 5% Threonic Acid 16% Arabino Hexaric Acid 1% Unk Sugar Acid 333 22% Glycerol Glycoside 29% Mandarin 26 Ribonic Acid 6% Saccharic Acid 1% Sugar Acid 204/333 1% Threonic Acid Deriv. 3% Threonic Acid 83% Unk Sugar Acid 333 3% Glycerol Glycoside 3% Pummelo

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133 CHAPTER 5 CONCLUSIONS ON THE P ROBING BEHAVIOR OF DIAPHHORINA CITRI IN RELATION TO HOST PLA NT RESISTANCE, HOST PLANT METABOLITES, A ND PROBING TACTIC This body of work demonstrates that Citrus host plant resistance indices to Diaphorina citri probing can be rigorously established using electropenetrograph y (EPG) to record probing behavior, complex statistical analysis, and ranking of statisti cal variables. When EPG data are combined with phloem and xylem chemistry and whole leaf metabolite profiles of host plant tissues, much can be learned about the inte rsection of insect plant interactions and insect probing behaviors associated with insect host preference and host plant resistance . Chapter 2 expands our knowledge of the histological correlations of D. citri probing behavior on Sweet Orange, C. sinensis , and makes a case that D. citri and other psyllids have their own distinctive probing tactic that is unique and separate from what we know of aphids. All of the waveform types performed by D. citri were correlated with salivary sheath termini, and plant cell type at the terminus. Waveform G, with salivary sheaths ending in xylem, was correlated for the first time in D. citri . Previous studies on psyllid probing behavior extrapolated the meaning of waveform G from aphid studies with no data confirmi ng this tissue location of stylets during the performance of waveform G by psyllids. Additionally, early and late interrupted D waveforms were correlated with stylets located in phloem tissues. All observed variations of waveform C were correlated with sal ivary sheath production in the parenchyma. This research built on the work of Bonani et al ., (2010), expanding the views of salivary sheath termini by imaging tissues using scanning electron microscopy (SEM) combined with brightfield imaging on a compound microscope. Previously, no scanning electron micrographs

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134 existed for D. citri salivary sheaths. Additionally, t he microscopy work completed as a part of this dissertation shows that D. citri employs a unique combination of probing tactics, using both intercellular and intracellular probing within a single probe . The inter or intracellular probing tactic has important implications for waveform appearance and type. Specifically, the result tha t D. citri does not explicitly utilize intercellular probing, as aphids do, easily explains the lack of a pd waveform in psyllids. The pd waveform is an important behavioral marker of the tasting of cell contents by aphids. Previous studies on psyllids ack nowledge the absence of a pd waveform, but could not explain why it was missing. We now know the answer to this question as a result of this dissertation. Psyllids do not make pd waveforms because they do not isolate their stylets tips within the apoplast between the living portions of cells prior to puncturing cells. Rather, psyllids readily move both between cells and within the living portion of cells as they penetrate tissues, making for a complex, and somewhat messy electrical origin of the waveform vo ltage. The voltage generated by plasma membrane punctures by psyllids is simply lost in the complex electrical environment surrounding the tips of the stylets during the performance of these behaviors. This behavio ral plasticity allows D. citri to optimize its probing and overcome anatomical host plant defenses and host plant defensive chemicals as needed to access their preferred ingestion tissue, the phloem . Chapter 3 explores the probing behavior of D. citri on sweet orange, Citrus sinensis , (var. Val encia) and Sour Orange, C . aurantium , in relation to the phloem and xylem metabolic profiles of each host plant in an effort to determine specific probing differences linked to the metabolic profiles of each host. In order to accomplish this, two powerful techniques, electropenetrography (EPG) and gas chromatography mass spectrometry (GC MS), were used to elucidate and correlate the probing behavior of D. citri with the metabolic

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135 profile of the phloem and xylem of both Citrus species. Diaphorina citri stron gly prefers Valencia to Sour Orange. In fact, in every quantifiable parameter assessed in this study, D. citri demonstrated preference for Valencia; reaching the phloem sooner, spending more overall time in phloem, and performing long bouts of phloem inges tion. Diaphorina citri readily probes Sour Orange and eventually accesses the phloem. However, when D. citri probes Sour Orange, it takes longer to access the phloem, phloem ingestion durations are reduced, and more time is spent ingesting from xylem compa red to when D. citri probes Valencia. The correlations between D. citri probing behavior and phloem and xylem metabolic profiles herein can be applied to other less preferred and likely more resistant Citrus and rutaceous hosts of D. citri in an effort to elucidate specific host preference and chemical resistance factors which can be used to screen for species and varieties potentially resistant to D. citri probing. Chapter 4 investigates the probing behavior of D. citri on four Cleopatra Mandarin hybrids and Pummelo in relation to the whole metabolite profiles of each host plant. The methodology of electropenetrography (EPG) and gas chromatography mass spectrometry (GC MS) were used to generate these data. The EPG results herein define a range of resistanc e to D. citri probing among the Cleopatra Mandarin and Pummelo tested in this study. While no one selection was consistently the most resistant or the most susceptible to D. citri probing, there was a clear trend. Mandarin 31 was most resistant to D. citri probing, with Mandarin 2, 19, and 26 representing the next highest resistance, consecutively. The most susceptible host plant tested herein was Pummelo, Citrus grandis , exhibiting no true resistance in probing variables compared to the Mandarin hybrids. In all host plants tested, D. citri was able to access and ingest from the phloem, the preferred ingestion tissue. In the most resistant hosts, D. citri was less successful in ingesting phloem, with it taking longer to reach the phloem and with shorter ove rall durations of time spent ingesting phloem. As with

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136 the EPG results of this study, the metabolic compound profiles do not highlight one host plant as being consistently the most resistant or the most susceptible to D. citri probing. Rather, a range of r esistance can be observed among the metabolite compound profiles associated with the Mandarin selections and Pummelo tested herein. The research completed as a part of this dissertation sought to test host plant resistance through the correlation of D. ci tri probing behaviors with host plant metabolite profiles on several Citrus species and selections. This research also expanded our understanding of D. citri probing through visualization of salivary deposits correlated with specific, interrupted probing b ehaviors. Additionally, this research identified a new, and wholly unique probing tactic in D. citri , a combined intercellular and intracellular tactic, giving D. citri the flexibility to navigate through host plant tissues in as efficient a manner as poss ible.

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151 BIOGRAPHICAL SKETCH Holly Shugart completed her Bachelor of Arts degree with a double major in b iology and the c lassics at Westminster College, Fulton, Miss ouri, USA. Her Master of Arts degree was completed with a major in b iology at the University of Missouri, Columbia, Missouri, USA.