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Optimizing the Use of Soil-Applied Neonicotinoids for Control of Diaphorina Citri Kuwayama (Hemiptera

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Title:
Optimizing the Use of Soil-Applied Neonicotinoids for Control of Diaphorina Citri Kuwayama (Hemiptera Liviidae) in Young Citrus Trees
Creator:
Langdon, Kevin W
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (132 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
ROGERS,MICHAEL E
Committee Co-Chair:
NOLING,JOSEPH W
Committee Members:
STELINSKI,LUKASZ LECH
SCHUMANN,ARNOLD WALTER
LOVELADY,CLARK

Subjects

Subjects / Keywords:
citrus -- insecticide -- neonicotinoid -- psyllid -- resistance
Entomology and Nematology -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Entomology and Nematology thesis, Ph.D.

Notes

Abstract:
Diaphorina citri Kuwayama is the insect vector of Candidatus Liberibacter asiaticus (CLas), the presumed cause of huanglongbing (HLB) in citrus (Rutaceae). Following the 2005 discovery of HLB in Florida, management strategies were developed using soil-applied neonicotinoids to protect young trees from the deadly disease. In such programs, neonicotinoids are applied to the soil allowing uptake through xylem channels from the roots into the foliage. Diaphorina citri are exposed to the insecticide through feeding. Despite implementation of neonicotinoid-intensive management programs, infection continues to spread among even the most intensively managed groves. A series of studies conducted in the laboratory, greenhouse, and field attempted to: 1) Quantify the spatial and temporal distribution of neonicotinoids in citrus foliage following soil-application; 2) Determine how tree size and application rate affect expression and D. citri incidence and abundance; 3) Determine the concentration needed to kill D. citri by ingestion and compare with residues required to kill by contact; 4) Use electropenetrography to investigate how dosage administered in diet affects feeding behavior. Higher concentrations occurred in the bottom 10% of the canopy compared to other regions, yet no difference occurred between the bottom and center, and levels peaked up to five weeks after application. Tree size and application rate affected titer in leaf tissue. Approximately 64.63 ppm thiamethoxam was required to reach a one percent probability of encountering a flush shoot with at least one adult D. citri. The ingestion LC50 of the laboratory strain was approximately 10-fold greater than the contact LC50. Neonicotinoid resistance was found in various field populations. No effect on feeding behavior was observed after 0.55 ppm imidacloprid was administered, however 5.5 and 55 ppm imidacloprid reduced various probing, pathway, and salivation/ingestion behaviors. Overall, neonicotinoid titers observed in the field failed to reach lethal levels quantified in the lab. Exposure to sublethal dosages is of concern for HLB management, as well as development of neonicotinoid resistance. Based on comprehensive results, the use of neonicotinoids may be better applied by foliar rather than soil application to maintain the utility of this chemical class in future insecticide management programs in Florida citrus. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2017.
Local:
Adviser: ROGERS,MICHAEL E.
Local:
Co-adviser: NOLING,JOSEPH W.
Statement of Responsibility:
by Kevin W Langdon.

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Rights Management:
Applicable rights reserved.
Classification:
LD1780 2017 ( lcc )

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1 OPTIMIZING THE USE OF SOIL APPLIED NEONICOTINOIDS FOR CONTROL OF DIAPHORINA CITRI KUWAYAMA (HEMIPTERA: LIVIIDAE) IN YOUNG CITRUS TREES By KEVIN WILLIAM LANGDON 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 2017

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2 2017 Kevin William Langdon All Rights Reserved

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3 To my mom and dad

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4 ACKNOWLEDGMENTS I wish to express sincere gratitude to my colleagues and associates with whom I worked through the course of my degree at the University of Florida. First, I wish to thank my major advisor, Dr. Michael E. Rogers, for the opportunity to work under his direc tion. His guidance allowed me to advance as a professional scientist and excel in academic rigor. I would also like to thank my manager within Syngenta, Dr. Clark Lovelady, for it is solely he who encouraged me to return to the university to pursue this de gree. His perpetual support as a manager and mentor was the key to completion of the degree while under his employ. I would also like to thank the balance of my academic advisory committee, Dr. Lukasz Stelinski, Dr. Arnold Schumann, and Dr. Joe Noling, for their infinite support and continuous guidance through the extent of my academic tenure. Perhaps the most critical component of my research program was Mrs. Rhonda Schumann. I offer my sincerest of gratefulness to her for the many hours logged running le af tissue samples, the invaluable office conversations, and for becoming a dear friend through the course of my research. I also wish to thank the members of the Rogers lab, Mr. Harry Anderson, Dr. Timothy Ebert, Ms. Gouping Liu, and Ms. Percivia Mariner f or research support in crushing leaf samples, collecting insects, and EPG expertise. I would like to thank my colleagues, Dr. Gurpreet Brar, Mr. Nick Ryan, and Ms. Ada Snyder for continuous support in the lab and field. I would also like to thank Mr. John Taylor for being a mentor and endless supply of citrus industry knowledge that provided conceptual support and industry guidance to keep my research grounded to the needs of Florida citrus growers. I must also thank Mr. James Colee for statistical consulti ng during my time at the University of Florida. In addition, I would like to thank the citrus growers that allowed me to conduct my research in their groves.

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5 I wish to thank my parents for the constant words of encouragement and high moral upbringing, for those are what instilled in me, a mindset of drive and determination. I wish to also me at the University of Florida. Go Gators! And in saving the best for last, I must thank my wife, Mrs. Barbara Lee Langdon for her unwavering support, unconditional love, and the many sacrifices made over the course of recent years, particularly in having a husband whose time was consumed with work ing toward a higher degree while being employed full time. Her continuous words of encouragement, alone, pushed me to this great achievement.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ....................... 10 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................................ ... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 14 Diaphorina citri Life History ................................ ................................ .......................... 14 Florida Citrus and Management of Diaphorina citri ................................ ...................... 15 2 SPATIAL AND TEMPORAL DISTRIBUTION OF SOIL APPLIED NEONICOTINOIDS IN CITRUS ................................ ................................ .......................... 24 Justification ................................ ................................ ................................ ............................. 24 Materials and Methods ................................ ................................ ................................ ........... 29 Spatial and Temporal Neonicotinoid Distribution ................................ .......................... 29 Effect of Leaf Maturity on Neonicotinoid Expression ................................ .................... 31 Extraction and Leaf Tissue Analysis ................................ ................................ ............... 32 Statistical Analyses ................................ ................................ ................................ .......... 33 Results ................................ ................................ ................................ ................................ ..... 34 In Leaf Distribution of Neonicotinoids ................................ ................................ ........... 34 Temporal Expression of Neonicotinoids ................................ ................................ ......... 36 Spatial Distribution of Admire Pro Analytes throughout the Tree Canopy .................... 38 Effect of Leaf Maturity on Neonicotinoid Expression. ................................ ................... 39 Discussion ................................ ................................ ................................ ............................... 40 3 INFLUENCE OF TREE SIZE AND APPLICATION RATE ON EXPRESSION OF THIAMETHOXAM IN CITRUS AND ITS EFFICACY AGAINST DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) ................................ ................................ ........................ 58 Justification ................................ ................................ ................................ ............................. 58 Materials and Methods ................................ ................................ ................................ ........... 62 Insect icide Application and Citrus Leaf Sampling ................................ .......................... 62 Extraction and Leaf Tissue Analysis ................................ ................................ ............... 64 Insect Biological Assays ................................ ................................ ................................ .. 65 Statistical Analyses ................................ ................................ ................................ .......... 67 Results ................................ ................................ ................................ ................................ ..... 68

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7 Chemical Titer in Leaf Tissue ................................ ................................ ......................... 68 Baseline Susceptibility of Field D. citri Population ................................ ........................ 69 Relationship Between D. citri Incidence and Chemical Titer ................................ ......... 70 Discussion ................................ ................................ ................................ ............................... 71 4 NEONICOTINOID INDUCED MORTALITY OF DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) IS AFFECTED BY ROUTE OF EXPOSURE ......................... 88 Justification ................................ ................................ ................................ ............................. 89 Materials and Methods ................................ ................................ ................................ ........... 93 Lab Cu lture ................................ ................................ ................................ ...................... 93 Field Collection ................................ ................................ ................................ ............... 93 Adult Ingestion Assay ................................ ................................ ................................ ..... 94 Adult Contact Assay ................................ ................................ ................................ ........ 95 Statistical Analyses ................................ ................................ ................................ .......... 96 Results ................................ ................................ ................................ ................................ ..... 96 Discussion ................................ ................................ ................................ ............................... 97 5 EVALUATING THE EFFECT OF IMIDACLOPRID ADMINISTERED IN ARTIFICIAL DIET ON FEEDING BEHAVIOR OF DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) USING ELECTROPENETROGRAPHY .............................. 105 Justification ................................ ................................ ................................ ........................... 106 Materials and Methods ................................ ................................ ................................ ......... 111 Electropenetrography Assays ................................ ................................ ........................ 111 Insect Culture ................................ ................................ ................................ ................. 113 Statistical Analysis ................................ ................................ ................................ ........ 113 Results and Discussion ................................ ................................ ................................ ......... 113 6 CONCLUSIONS ................................ ................................ ................................ .................. 121 LIST OF REFERENCES ................................ ................................ ................................ ............. 124 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 131

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8 LIST OF TABLES Table page 2 1 Neonicotinoid product description and use rates for greenhouse and field studies ........... 48 2 2 Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application of Admire Pro (1.48 mL per tree) to the soil in the green house and in the field ................................ ................................ ................................ ................................ .... 49 2 3 Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application o f Platinum 75SG (0.37g per tree) to the soil in the greenhouse and in the field ................................ ................................ ................................ ................................ .... 50 2 4 Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application of Belay 2.13SC (1.27 mL per tree) to the soil in the greenhouse and in the field ................................ ................................ ................................ .............................. 51 2 5 Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Admire Pro (1.48 mL per tree) to the soil in the greenhouse ................................ ............ 52 2 6 Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Admire Pro (1.48 mL per tree) to the soil in the field at two commercial Florida citrus groves ................................ ................................ ................................ ....................... 53 2 7 Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Platinum 75SG (0.37g per tree) to the soil in the greenhouse ................................ ........... 54 2 8 Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Belay 2.13SC (1.27 mL per tree) to the soil in the greenhouse ................................ ......... 55 3 1 Mean parts per mi llion (ppm) of thiamethoxam (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments ................................ ................................ ........... 80 3 2 Mean parts per million (ppm) of clothianidin (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments ................................ ................................ ........... 81 3 3 Mean parts per million (ppm) of TZMU (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments ................................ ................................ ....................... 82 3 5 Respons e of laboratory susceptible and field collected Diaphorina citri to thiamethoxam (ppm) administered by ingestion and contact ................................ ............ 84 3 6 Nonparametric Spearman correlation between mean number of adult or nymph Diaphorina citri per terminal and chemical titer (ppm) during 2016 and 2017 field seasons ................................ ................................ ................................ ............................... 85

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9 3 7 Probability of encountering a Diaphorina citri adult on young citrus trees based on thiamethoxam titer (ppm) in leaf tissue ................................ ................................ ............. 86 3 8 Probability of encountering a Diaphorina citri nymph on young citrus trees based on thiamethoxam titer (ppm) in leaf tissue ................................ ................................ ............. 87 4 1 Response of laboratory susceptible Diaphorina citri strain to three neonicotinoid insecticides by ingestion and contact routes of exposur e. ................................ ............... 103 4 2 Response of laboratory and field collected Diaphorina citri to imidacloprid by ingestion and contact routes of exposure in 2016 ................................ ............................ 104 5 1 Description of adult Diaphorina citri feeding behavior by EPG model abbreviation. .... 118 5 2 LSMeans SEM for each behavioral parameter following exposure of adult Diaph orina citri to artificial diet with and without 0.55 ppm imidacloprid .................... 119 5 3 LSMeans SEM for each behavioral parameter following exposure of adult Diaphorina citri to artificial diet with 0, 5.5, or 55 ppm imidacloprid ............................ 120

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10 LIST OF FIGURES Figure page 2 1 Comparison of chemical titer between seven tree regions during 2015 and 2016 field seasons ................................ ................................ ................................ ............................... 56 2 2 Comparison of 5 OH titer between seven tree regions at two locations during 2015 and 2016 field seasons ................................ ................................ ................................ ..... 57 3 1 Predicted probability for incidence of insects based on thiamethoxam titer (ppm) in citrus leaf tissue ................................ ................................ ................................ ................. 77 3 2 Comparison of thiamethoxam titer (ppm) and percentage insect control in trees of the large size during 2016 and 2017 field seasons. ................................ ................................ .. 78 3 3 Comparison of thiamethoxam titer (ppm) and percentage insect control in trees of the large size during 2016 and 2017 field seasons. ................................ ................................ .. 79

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11 LIST OF ABBREVIATIONS 5 OH 2 Chloro 5[(4,5 dihydro 2 nitroamino) 1 H imidazole 1 yl] methyl 3 pyridinol ACP Asian citrus psyllid C Las Candidatus Liberibacter asiaticus FL Florida HLB Huanglongbing LC ## Lethal concentration at the ## percent mortality level L:D Light:Dark LOD Limit of detection LOQ Limit of quantification LS Laboratory susceptible Olefin 1 [(6 Chloro 3 pyidinyl )methyl] 1,3 dihydro N nitro 2H imidazol 2 imine QuEChERS Quick easy cheap effective rugged safe RH Percent relative humidity PCR polymerase chain reaction TZMU N (2 chloroth iazol 5 ylmethyl) N methylurea TZNG N (2 chlorothiazol 5 ylmethyl) N nitroguanidine UHPLC MS Ultra high performance liquid chromatography mass spectrometry

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12 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 OPTIMIZING THE USE OF SOIL APPLIED NEONICOTINOIDS FOR CONTROL OF DIAPHORINA CITRI KUWAYAMA (HEMIPTERA: LIVIIDAE) IN YOUNG CITRUS TREES By Kevin William Langdon December 2017 Chair: Michael Rogers Major: Entomology and Nematology Diaphorina citri Kuwayama is the insect vector of Candidatus Liberibacter asiaticus ( C Las), the presumed cause of huanglongbing (HLB) in citrus (Rutaceae) Following the 2005 discovery of HLB in Florida, management strategies were developed using soil applied neonicotinoids to protect young trees from the deadly disease. In such programs, n eonicotinoid s are applied to the soil allowing uptake through xylem channels from the roots into the foliage. Diaphorina citri are exposed to the insectic ide through feeding. Despite implementation of neonicotinoid intensive management programs, infection continues to spread among even the most intensively managed groves. A series of studies conducted in the laboratory, greenhouse and field attempted to: 1 ) Quantify the spatial and temporal distribution of neonicotinoids in citrus foliag e following soil application; 2) Determine how tree size and application rate affect expression and D. citri incidence and abundance ; 3) Determine the concentration needed t o kill D. citri by ingestion and compare with residues required to kill by contact ; 4) Use electropenetrography to investigate how dosage administered in diet a ffects feeding behavior. Higher concentrations occurred in the bottom 10% of the canopy compared to other regions, yet

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13 no difference occurred between the bottom and center and levels peaked up to five weeks after application. Tree size and application rat e affected titer in leaf tissue. Approximately 64.63 ppm thiamethoxam was required to reach a one percent probability of encountering a flush shoot with at least one adult D. citri The ingestion LC 50 of the laboratory strain was approximately 10 fold grea ter than the contact LC 50 Neonicotinoid resistance was found in various field populations. No effect on feeding behavior was observed after 0.55 ppm imidacloprid was administered, however 5.5 and 55 ppm imidacloprid reduced various probing, pathway, and s alivation/ingestion behaviors. Overall, neonicotinoid titers observed in the field failed to reach lethal levels quantified in the lab. Exposure to sublethal dosages is of concern for HLB management, as well as development of neonicotinoid resistance. Base d on comprehensive results, the use of neonicotinoids may be better applied by foliar rather than soil application to maintain the utility of this chemical class in future insecticide management programs in Florida citrus.

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14 CHAPTER 1 INTRODUCTION The Asian citrus psyllid, Diaphorina citri Kuwayama (Hempitera: Liviidae) is the insect vector of Candidatus Liberibacter asiaticus ( C Las), the presumed cause of huanglongbing (HLB) or citrus greening disease T hroughout the world HLB reduces health and p roductivity of citrus trees (Rutaceae) (Halbert and Manjunath 2004, Bov 2006, Gottwald 2007, Ichinose et al. 2010a, 2010b; Grafton Cardwell et al. 2013). Candidatus Liberibacter asiaticus is a phloem limited bacteria that negatively impacts r oot density and fuction, leading to a decline in the tree canopy, including twig dieback, mottled leaves, misshapen fruit, decreased fruit quality, increased fruit drop, and subse quent death of infected trees (Halbert and Manjunath 2004, Bov 2006, Grafton Cardwell et al. 2013). Diaphorina citri was first discovered in Florida in 1998 (Halbert and Manjunath 2004), followed by HLB in 2005 (Halbert 2005). Since, HLB has spread throug h the southern United States and was recently discovered in California ( Kumagai et al. 2013). The Florida citrus industry was valued at nearly 9.9 billion dollars during 2014 and 2015 (Hodges and Spreen 2015) and is greatly threatened by the spread of HLB Since HLB was discovered in Florida in 2005, the use of insecticides, particularly neonicotinoids, has increased substantially and play s a vital role in the management of the insect vector, and thus HLB (Rogers 2008). Diaphorina citri Life History Diapho rina citri reproduce and develop rapidly, l aying up to 800 eggs per female primarily on young, unexpanded flush. The eggs hatch after two to four days, and the insect completes 5 nymphal stages within 11 to 15 days, continuously feeding on the phloem sap of the young, growing flush shoots. B etween 15 to 47 days are required for completion of one life cycle (Liu and Tsai 2000, Grafton Cardwell et al. 2013). Optimum temperature conditions for

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15 development are 25 28C, temperatures that occur often during the months of May, June, and September in Florida, which also coincide with peak populations of D. citri and tree flush availability (Liu and Tsai 2000, Tsai et al. 2002, Hall and Albrigo 2007, Hall et al. 2008). Both adult and nymphal stages actively feed on phloem sap (Boina et al. 2009). Diaphorina citri prefer to feed on young leaf tissue, however, they will feed on mature leaves in the absence of young flush (Tsai et al. 2002, Serikawa et al. 2012). When eggs are deposited on the flush of C Las infected tre es, the developing nymphs ingest the bacteria upon feeding on bacteria containing phloem sap (Pelz Stelinski et al. 2010). Pelz Stelinski et al (2010) found that C Las acquisition was greater for D. citri nymphs that fed on C Las infected tissue than for D. citri adults feeding on infected tissue When a newly infected nymph completes development, the adult disperses and inoculates uninfected trees Bonani et al (2010) determined that successful C Las acquisition by adults could occur after 1h of phloem inge stion (EPG waveform E2). Flori da Citrus and Management of Diaphorina citri In Florida, mature trees typically follow a synchronized annual flush sequence characterized with a major flush in early spring, another major flush in summer, and minor flushes during late summer and fall. In contrast, young trees tend to flush asynchrono usly and more often throughout the year (Hall and Albrigo 2007). Young flush shoots emit volatiles attractive to D. citri adults (Patt and Setamou 2010). Serikawa et al (2012) found that D. citri conducted more feeding probes and probed for a longer durat ion per event on young citrus leaves compared to mature leaves. This result indicates that D. citri adults prefer young leaf tissue to mature leaf tissue. Because young trees flush more frequently, they are presumably at greater risk of contracting C Las (S tansly and Rogers 2006). Although D. citri prefer young tissue, adults can survive on mature leaves (Tsai et al. 2002). Following the discovery of C Las in Florida, researchers attempted to develop more efficient sampling methods using flush ing events to

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16 pr edict D. citri population densities (Hall et al. 2008). As a result, the use of a year round scouting program was recommended to justify control and align foliar insecticide sprays with D. citri egg hatch and nymph development. Stansly and Rogers (2006) su ggested the use of clean nursery stock, a program of soil applied systemic insecticides for protection of growing nursery stock, and the rapid removal of any trees that displayed evidence of HLB infection. They also suggested using minimal foliar sprays in the field to reduce negative impacts on Tamarixia radiata Waterston (Hymenoptera: Eulophidae), a key parasitic wasp of D. citri Despite early control measures, the incidence and severity of HLB in Florida increased rapidly and quickly warranted the evolu tion of new management recommendations. In addition to vector management, increased attention was given to the overall health of mature trees; the reduction of stressors associated with HLB was studied as a means of maintaining existing mature citrus grove s (Inserra et al. 2003, Obreza and Morgan 2008, Graham et al. 2013, 2014; Gottwald et al. 2012). For example, Phytophthora spp. targeted fungicide applications and the use of resistant rootstocks were found essential to promoting fibrous root health (Graha m et al. 2013, 2014). Also, the selection of nematode resistant rootstocks became more important in the management of nematode infestations (Inserra et al. 2003). Moreover the use of macro nutrient fertilizers were found to increase yields of previously s tressed trees (Obreza and Morgan 2008). Each of these management strategies can play an important role in overall grove health as a component of circumvent ing the impact of HLB. However, some expressed concerns of increased disease spread following impleme ntation of nutrient management strategies due to an increase in flushing frequency and abundance (Gottwald et al. 2012). Nonetheless, insecticidal control remains arguably the most important component of D. citri management, and thus, the management of HLB (Stansly and Rogers 2006, Rogers 2008, Boina et al. 2009, Qureshi and

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17 Stansly 2009, Qureshi et al. 2011, Ichinose et al. 2010a, 2010b; Rogers 2012, Stansly et al. 2012, Hall et al. 2013a, 2013b; Qureshi et al. 2014). As mature groves accumulated HLB infe ction over time, it was believed that t o maintain grower production sustainability, unproductive, HLB infected citrus trees would need to be replaced with new trees (Rogers 2012). As a result, an emphasis was placed on young tree care to maintain the indus try. Because young trees flush often, and because flush is preferred by D. citri young trees were believed to require more protection than mature trees (Hall and Albrigo 2007, Serikawa et al. 2012, Rogers et al. 201 5 Qureshi et al. 2014). Rogers (2012) noted that the goal of D. citri management in young trees is to prevent HLB until after the trees reach fruit bearing age which generally occurs between two and five years after planting. If HLB is prevalent around a new planting or if D. citri control is lacking in surrounding groves, it can be difficult to protect young trees from the disease (Hall et al. 2013b). A n area wide approach to vector control was implemented as the citrus health management area (CHMA) prog ram in 2010 (Rogers et al. 2012). The impact of the CHMA program from a pest management point of view is two fold: 1) T o reduce the overall psyllid population in Florida citru s (Rogers et al. 2012), and; 2) T o manage potential resistance problems from the repeated use of the same ins ecticide (Tiwari et al. 2011a). The area wide sprays are conducted between applications of neonicotinoid insecticides to the soil. Soil applied neonicotinoids have been a key management tool in the attempt to control D. citri a nd thus help to mitigate the risk of HLB infection in young citrus (Rogers 2012, Rogers et al. 201 5 ). The translocation of insecticides within plant tissue was first noted in 1947 (Jeppson et al. 1952). In 1950, researchers found that when the organophosph ate insecticides, paraoxon and octamethyl pyrophosphoramide, were applied to the soil, each moved into the foliage and killed feeding

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18 mites on citrus (Metcalf and Carlson 1950a, 1950b; Jeppson et al. 1952). A relatively new class of chemistry that is also highly systemic and mobile within plant tissue are neonicotinoids. The Insecticide Resistance Action Committee (IRAC) classifies neonicotinoids within the chemical sub group 4A, which act on the Nicotinic acetylcholine receptor (nAChR) (IRAC 2009). Neonico tinoid s mimic acetylcholine and bind to the nAChR, causing the nerve to continuously fire. Acetylcholinesterase cannot hydrolyze the acetylcholine mimic, resulting in over exhaustion of the insect nervous system paralysis, and eventual death. Neonicotinoi d insecticides can be applied to plant foliage and translocate throughout the plant, or applied to the soil, and transported through the xylem channels from the roots into the plant foliage (Elbert et al. 2008). While systemic insecticides are effective at controlling targeted pests, when applied to the soil, they help to minimize direct contact with pollinators and other beneficial insects (Stansly and Qureshi 2008). Currently, three neonicotinoid insecticides are labeled for use in Florida citrus: thiamet hoxam (Platinum 75 SG) (Syngenta Crop Protection, Inc., Greensboro, NC), imidacloprid (Admire Pro 4.6F) (Bayer CropScience, Research Triangle Park, NC), and clothianidin (Belay 2.13 SC) (Valent USA Corporation, Walnut Creek, CA) (Rogers et al. 201 5 ). A num ber of studies have addressed the use of neonicotinoids as a means of protecting young citrus trees from feeding with residual control effects reported between six and eleven weeks after application (Qureshi and Stansly 2007, Qureshi and Stansly 2009, Ichi nose et al. 2010a, Setamou et al. 2010, Byrne et al. 2012, Rogers 2012). The use of soil applied neonicotinoids on newly planted seedlings at a two month interval was found to reduce psyllid infestation and disease (Ichnose et al. 2010a). Moreover thiamet hoxam and imidacloprid each provided better D. citri control than other systemic insecticides; clothianidin was not tested as it was not registered in citrus at the time (Rogers and Shaw e r 2007). Transport of chemistry through the plant xylem is critical f or

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19 the effectiveness of neonicotinoids when applied to the soil. Water solubility of each chemistry can influence rate of ground leaching and successful uptake (Rogers 2012). Thiamethoxam is the most water soluble, imidacloprid is less water soluble, and c lothianidin is the least water soluble, comparatively. Castle et al (2005) found that the concentration of thiamethoxam in citrus tissue increased more rapidly than imidacloprid, and Qureshi and Stansly (2009) found that thiamethoxam provided rapid D. cit ri control compared to imidacloprid, which suggests that rapid uptake is directly related to water solubility. Rogers (2012) proposed the use of the most water soluble compound (thiamethoxam) during the dry winter months, and the least water soluble compou nd (clothianidin) during the rainy summer months to maximize uptake of each insecticide in Florida. Because the three effective soil applied insecticides comprise one mode of action, their use must be accomplished with great care, such to pre vent resistanc e (Rogers 2012). One effective resistance management strategy is to rotate soil applied neonicotinoids with foliar sprays of a different mode of action (Rogers 2008) as done with the CHMA program Tiwari et al (2011 a ) reported neonicotinoid resistance in field collected D. citri populations in Florida in 2009, but subsequent studies conducted in 2013, after the implementation of the CHMA program did not detect resistance, indicating that reversion had occurred (Coy et al. 2016) Even in the most intensively managed citrus groves, trees continued to accumulate HLB infection over time at an estimated rate of one to three percent each year (Rogers 2013). A more thorough understanding of the use of soil applied neonicotinoids is critical to furth er reduce the spread of HLB. Limited knowledge regarding the movement and distribution of soil applied neonicotinoids through citrus tissue subsists to date. Boina et al (2009) proposed that uneven temporal and spatial distribution in citrus tissue may ca use exposure of D. citri to sublethal doses of insecticide. Uneven uptake of systemic insecticides by the root system could make it possible

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20 for D. citri to develop (Rogers 2012). Previous studies that attempted to quantify neonicotinoid concentration in c itrus sampled either xylem fluid or entire leaves and quantified concentrations using enzyme linked immunosorbent assay ( ELISA) (Castle et al 2005, Garlapati 2009, Setamou et al. 2010). Castle et al (2005) stated that the potential for cross reactivity w ith plant metabolites of imidacloprid was assumed as positive detection of the parent compound, imidacloprid. The use of Ultra High Performance Liquid Chromatography Mass S pectrometry ( UHP LC MS) is a more reliable method for quantifying the concentration of neonicotinoids in leaf tissue, as the method can distinguish metabolites from parent compounds. Neonicotinoid insecticides metabolize into various analytes over time though the effect of any one resulting metabolite on D. citri mortality is unknown (B yrne et al. 2017). For example, thiamethoxam metabolizes into clothianidin (Nauen et al. 2003) and clothianidin further metabolizes into TZNG ( N (2 chlorothiazol 5 ylmethyl) N nitroguanidine ) and TZMU ( N (2 chlorothiazol 5 ylmethyl) N methylurea) ( Kim et al. 2012). Likewise, imidacloprid metabolizes into 5 OH ( 2 Chloro 5[(4,5 dihydro 2 nitroamino) 1 H imidazole 1 yl] methyl 3 pyridinol) and then 5 OH further metabolizes into olefin ( 1 [(6 Chloro 3 pyidinyl )methyl] 1,3 dihydro N nitro 2H imidazol 2 imine) (Sur and Stork, 2003) Nevertheless, Castle et al (2005) found no difference in the spatial distribution of the parent compounds of imidacloprid or thiamethoxam within a citrus tree as it related to the glassy winged sharpshooter, Homalodisca coagu lata (Say). The authors sampled xylem fluid from branch shoots, which is not consistent with the phloem feeding patterns of D. citri In addition, the authors applied insecticides through a micro irrigation system, which evenly distributes material around the tree trunk In contrast, Florida growers typically use a drench application device mounted to a four wheel utility vehicle to apply a solution of approximately 2 37 m L (water + insecticide) to the soil below only one side of

PAGE 21

21 each tree This application method may result in an uneven distribution of insecticide within a tree canopy Moreover, differences within a single leaf may occur, causing uneven distribution within a leaf. Whole leaf samples, as used in Garlapati (2009) and Setamou et al (2010), qua ntified a mean titer within a leaf, which did not account for potential differences within a leaf If the lowest concentration within a leaf coincide with where D. citri feeding occurs and that concentration is sub lethal, successful C Las acquisition and/or inoculation may result. In addition to potential differences in spatial distribution, t emporal expression over time as it relates to residual control may result in sublethal dosages. Temporal expression of soil applied neonicotinoids may be influenced by factors such as soil type, leaf maturity, application volume, irrigation, tree age and size, addition of adjuvants, and environmental conditions. While six to eleven weeks of control have been reported following application of neonico tinoids to the soil (Qureshi and Stansly 2007, Qureshi and Stansly 2009, Ichinose et al. 2010a, Setamou et al. 2010, B yrne et al. 2012, Rogers 2012), studying factors that influence uptake and expression over time would allow great improvement to suggested management plans. Concentration of neonicotinoid insecticide required to kill by ingestion is an additional major unknown. Serikawa et al (2012) determined that D. citri can undergo phloem ingestion (EPG waveform E2) for at least 1h with imidacloprid in the plant tissue; one hour is enough time for successful C Las acquisition. Although eventual death did occur in D. citri individuals that ingested imidacloprid, it is possible for those individuals to move to an uninfected tree and salivate within the phl oem before death, thus inoculating the new tree. In Florida, an estimated 80 100% of D. citri are C Las positive ( Coy and Stelinski 2015) and therefore, a single successful feeding event on an uninfected tree cannot be tolerated. Setamou et al (2010) ident ified the lethal concentration of imidacloprid for D. citri as between 200 and 250 parts per billion (ppb).

PAGE 22

22 This lethal threshold was developed by correlating percent age control of D. citri and leaf tissue residue analysis using enzyme linked immunosorbent assay (ELISA). When evaluating insecticides under field conditions percent age control, or efficacy, is most often defined by the absence of a particular insect pest as compared to some untreated control. In the case of systemic insecticides, efficacy cou ld be a result of mortality, repellency, feeding deterrence, or a combination thereof. In this case, repellenc e can be defined as olfactory avoidance behavior of aversive volatiles, associated with feeding sites and deterrence can be defined as gustatory avoidance of less or non suitable f eeding sources Dosages of imidacloprid between 200 to 250 ppb associated with imidacloprid efficacy observed by Setamou et al. (2010) may have result ed from a combination of mortality, repellency, and/or feeding deterren ce caused by imidacloprid rather than mortality only Because mortality was not quantified in the aforementioned study, the concentration of imidacloprid required to kill D. citri through feeding remains unknown. Quantifying uptake and distribution patter ns of neonicotinoids in citrus leaf tissues when applied to the soil is paramount to understanding how to control D. citri with soil applied neonicotinoids and to further reduce or stop the spread of HLB in Florida citrus. Moreover, it is important to unde rstand how tree size and application rate a ffects expression in citrus leaf tissues. By quantifying expression of neonicotinoids in citrus leaf tissues, we can map the pattern of sublethal neonicotinoid titers within a tree or leaf and develop strategies t o minimize exposure to those dosages, or improve uptake efficiency to maximize expression when applied to the soil. Quantification of the lethal concentration of neonicotinoids by ingestion coupled with studying feeding behavior under exposure to neonicoti noids using electropenetrography will allow us to understand how dosages observed in field and greenhouse studies impact D. citri behavior and mortality and will allow us to better understand the potential for C Las transmission under D.

PAGE 23

23 citri managed syst ems. Moreover, understanding the relationship between neonicotinoid dosage and mortality levels will help identify the potential for development of resistance and help develop sound resistance management strategies. The overarching goal of this research is to allow us to refine current vector management programs which will help e ither maximize the reduction or perhaps optimistically, prevent the spread of C Las in Florida citrus.

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24 CHAPTER 2 SPATIAL AND TEMPORAL DISTRIBUTION OF SOIL APPLIED NEONICOTINO IDS IN CITRUS Diaphorina citri Kuwayama is the insect vector of Candidatus Liberibacter asiaticus ( C Las), the presumed cause of huanglongbing (HLB) in citrus (Rutaceae) Soil applied neonicotinoids are used to manage vector populations and thus reduce the spread of HLB in Florida citrus. A series of studies conducted in the greenhouse and field attempted to quantify the spatial distribution within a single leaf and throughout the tree canopy, as well as tem poral expression following soil application of three neonicotinoid insecticides. No difference in parent material titer was observed between leaf middle and leaf margin following application of Platinum 75SG or Belay 2.13SC, however, imidacloprid titer was higher in the leaf margin follo wing application of Admire Pro in the field. TZMU and TZNG accumulated in leaf margins after application of Platinum and Belay. The bottom tree region contained higher levels of imidacloprid compared with other regions, but was not different from the spher ical center. In the greenhouse, imidacloprid and clothianidin titers peaked at five weeks following application of Admire and Belay, respectively, and thiamethoxam titer peaked at three weeks after application of Platinum. We were unable to quantify tempor al expression in the field due to existing imidacloprid titers at the time of application. Overall, titers observed in the field failed to reach lethal levels quantified in previous studies. Exposure to sublethal dosages is of concern for HLB management, as well as development of neonicotinoid resistance. Based on our results, subsequent work should realign focus on foliar use patterns to maintain the utility of neonicotinoids in citrus. Justification The Florida citrus (Rutaceae) industry has come under severe decline over the last decade, due to the combined introductions of the Asian citrus psyllid, Diaphorina citri

PAGE 25

25 Kuwayama (Hemiptera: Liviidae) and the presumed causal agent of citrus greening disease, Candidatus Liberibacter asiaticus ( C Las) (Halber t and Manjunath 2004, Bov 2006). Citrus greening disease, or Huanglongbing (HLB) was first detected in the state in 2005, seven years after the discovery of the insect vector, D. citri (Halbert and Manjunath 2004). The citrus industry in Florida was value d at 9.9 billion dollars during 2014 and 2015 and is the single largest agricultural commodity in the state (Hodges and Spreen 2015). When trees succumb to HLB through feeding by C Las positive D. citri the bacteria moves through the phloem from the infect ion site to the roots, which negatively impacts the root system. In turn, the tree canopy is starved for nutrients, causing leaf and fruit drop, thereby reducing yield in the near term, and eventually resulting in tree death (Halbert and Manjunath 2004, Bo v 2006, Grafton Cardwell et al. 2013). Various methods of HLB management have been investigated, including routine releases of the biological control agent, Tamarixia radiata Water ston (Hymenoptera: Eulophidae), nursery sanitation, and rogueing of infecte d trees in the field, among other strategies (Stansly and Rogers 2006 Hall and Albrigo 2007, Hall et al. 2008). Given the potential impact of the disease and an estimated 80 100 percent of D. citri in the state that are infected with C Las (Coy and Stelins ki 2015), insecticides have become the primary method for slowing the spread of HLB throughout Florida grove space, particularly through use of soil applied neonicotinoids in young tree plantings (Rogers 2008, 2013). Young trees do not bear fruit and are typically categorized as those less than eight feet tall (Hall and Albrigo 2007, Rogers 2012). Unlike mature citrus trees, non bearing trees flush often throughout the year, which places them at great risk of HLB infection (Stansly and Rogers 2006). D iapho rina citri adults are attracted to volatiles emitted by actively growing flush shoots, which are preferred for oviposition (Patt and Setamou 2010). Newly hatched nymphs feed on

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26 phloem sap of the developing flush shoots where nymphs acquire the bacterium if on infected plant material (Pelz Stelinski et al. 2010). As newly infected nymphs reach adulthood, they disperse and inoculate uninfected citrus tissue. In attempt to intercept this cycle, Rogers (2008) developed a program approach of rotating between neo nicotinoids applied to the soil and foliar sprays of alternate modes of action. Neonicotinoids are highly systemic, xylem mobile insectic ides within the Insecticide Resistance Action Committee (IRAC) sub group 4A and are often applied to the soil for transport to the plant foliage (Elbert et al. 2008). Three neonicotinoid insecticides are labeled for use in non bearing citrus in Florida: thiamethoxam (Platinum 75 SG Syngenta Crop Protection, Inc., Greensboro, NC ), imidacloprid (Admire Pro 4.6F Bayer CropScience, Research Triangle Park, NC), and clothianidin (Belay 2.13 SC Valent USA Corporation, Walnut Creek, CA). Previous investigations documented residual D. citri adult and / or nymph control of six to elev en weeks after neonicotinoids were applied to the soil (Qureshi and Stansly 2007 2009; Ichinose et al. 2010 ; Setamou et al. 2010 ; B yrne et al. 2012; Rogers 2012). However, even in the most intensively managed citrus groves, trees continued to accumulate H LB infection over time at an estimated rate of one to three percent each year (Rogers 2013). Little is known regarding the movement and distribution of soil applied neonicotinoids through citrus tissue s Boina et al (2009) proposed that uneven temporal a nd spatial distribution in citrus tissue may cause exposure of D. citri to sublethal doses of insecticide. Furthermore, uneven uptake of systemic insecticides by the root system make it possible for D. citri to develop (Rogers 2012). Previous studies that quantified neonicotinoid concentration in citrus sampled either xylem fluid or entire leaves and quantified parent material concentrations using enzyme linked immunosorbent assay ( ELISA) (Castle et al 2005, Garlapati 2009, Setamou et al. 2010).

PAGE 27

27 When quant ifying chemical constituents using ELISA, one cannot differentiate between parent material and resulting metabolites. Nevertheless, Castle et al (2005) found no difference in the spatial distribution of imidacloprid or thiamethoxam throughout citrus tree canopy xylem fluid for control of the glassy winged sharpshooter, Homalodisca coagulata (Say) They sampled x ylem fluid from branch shoots, which is not consistent with the phloem feeding patterns of D. citri In addition, the authors applied insecticides through a micro irrigation system, which evenly distributes insecticide around the tree trunk at the time of application. In contrast, Florida growers typically use a drench application device mounted to a four wheel utility vehicle to apply a solution of approximately 2 37 m L (water + insecticide) to the soil below only one side of each tree This application method may result in an uneven distribution of insecticide within a tree canopy In the case of Florida citrus, quantifying the spatial distribution o f insecticide within a tree as well as within a single leaf, is essential to understanding the potential of D. citri exposure to sublethal dosages. Setamou et al. (2010) correlated percentage control with imidacloprid concentration in citrus leaf tissues. They determined that between 200 and 250 parts per billion (ppb) was required to provide D. citri control in citrus. More recent studies found that 62.19 parts per million (ppm) imidacloprid was required to kill 90 percent of a laboratory D. citri population when administered by ingestion (Langdon and Rogers 2017). They suggested that control observed by Setamou et al. (2010) may have been a result of feeding deterrence at sublethal dosages. In addition, a series of more recent studies determined that 64.63 ppm thiamethoxam was required to achieve a one percent probability of encountering a flush shoot with at least one adult D. citri in the field, as determined by liquid c hromatography mass spectrometry (LC MS) (Langdon 2017). The highest mean thiamethoxam titer observed in their field study was 33.39

PAGE 28

28 ppm, more than 30 ppm below the threshold for a tree predicted to be free of D. citri adults. The magn itude of difference between dosages required to achieve high mortality levels or perceived control, and observed thiamethoxam titer may explain why HLB infection incidence continues to rise despite deliberate soil applied neonicotinoid use in the field. Wh ile spread of HLB is of great concern, sublethal dosages resulting from u neven spatial and temporal distribution is also likely to increase the potential for development of resistance to neonicotinoids. Tiwari et al. (2011a) documented resistance to neonic otinoids in the field in 2009, but no resistance was detected in subsequent studies conducted in 2014 (Coy et al. 2016). This shift was thought to be due to the implementation of area wide spray programs that used non neonicotinoid insecticides applied ove r broad landscapes (Rogers et al. 2012). However, resistance was again detected in 2016 to imidacloprid in various field populations throughout the state, albeit resistance appeared to be isolated to specific areas at the time; tests evaluating resistance to thiamethoxam or clothianidin were not conducted (Langdon and Rogers 2017). Quantifying uptake and distribution patterns of neonicotinoids in citrus leaf tissues when applied to the soil is paramount to understanding how to control D. citri and to furth er reduce or stop the spread of HLB in Florida citrus. In addition, we must quantify exposure of insects to sublethal insecticide dosages to minimize the potential for resistance development to key insecticide classes, such as neonicotinoids. The purpose o f this study was to quantify the spatial distribution and temporal expression of all analytes in citrus leaf tissues resulting from the soil application of three neonicotinoid insecticides using an application method commonly implemented by Florida citrus growers. By quantifying expression of neonicotinoids in citrus leaf tissues, we can map the pattern of sublethal neonicotinoid titers within a tree or leaf and

PAGE 29

29 develop strategies to minimize exposure to those dosages, or improve uptake efficiency for maxim um expression when applied to the soil. Materials and Methods Spatial and Temporal Neonicotinoid Distribution Greenhouse study. A greenhouse study was conducted to evaluate the uptake of three neonicotinoid insecticides following application to the soil T he distribution of insecticide residue within citrus leaves was evaluated Small citrus trees (ca. 0.08m 3 canopy volume) were planted to 11.4L pots containing a blend of 50% sand and 50% potting media. Plots consisting of four trees were arranged in a ra ndomized complete block design (RCBD) with 4 treatments and 4 replicates. Treatments consisted of an untreated control, Platinum 75SG, Admire Pro 4.6F, and Belay 2.13SC applied at the recommended rate for non bearing citrus trees based on 346 trees / hecta re (140 trees / acre) ( Table 2 1 ). A single insecticide application was made by applying 237 m L of insecticide solution (deionized water + insecticide) into each pot. Leaf tissue samples were collected prior to the application of insecticides and then weekly for 1 3 weeks following the application. At each sample date, four leaves across each of the four trees within a plot were harvested Each leaf was excised into two sections: 1. Middle (area inclusive of 0.5cm on either side of the mid vein extending from leaf petiole to 0.5cm from leaf tip), and 2. Margin within a plot was wrapped separately in labeled heavy duty aluminum foil and collectively stored by treatment in a plastic re sealable bag at 2 0C until residue analyses were conducted. Two season by two location field study. A field study was conducted at two commercial grove locations across two seasons to evaluate the spatial and temporal distribution of three neonicotinoid insecticides in citrus trees following application to the soil Non bearing (v. Hamlin / r.s. Swingle) trees of similar size and age (ca. 1.3m 3 canopy volume and field planted

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30 approximately 18 months prior to the first application ) were identified in two commercial groves, each of which represent a major citrus production area of Florida ( p ine latwoods and entral r idge ). The low lying flatwoods location was a continuous solid set planting of trees of t he same age. The trees were planted to sandy soils comprised of 96.4% sand, 2% clay, and 1.6% silt with 0.68% organic matter and cation exchange capacity (CEC) of 14.2 meq/100g The central ridge location contained a mature grove with random spans of betwe en two and fifteen young trees planted among the mature trees. This grove was comprised of sandy soils with 98.4% sand, 1.6% clay, and 0% silt with 0.59% organic matter and CEC of 4.1 meq/100g The central ridge site was centrally located on the North Sout h running ridge through central Florida spanning from near Orlando to south of Lake Placid. At each site, plots were arranged in a randomized complete block design with 4 treatments and 4 replicates. Treatments consisted of an untreated control, Platinum 7 5SG, Admire Pro 4.6F, and Belay 2.13SC applied at the recommended rate for non bearing citrus trees based on 346 trees / hectare (140 trees / acre) ( Table 2 1 ). At each location, t he first season insecticide application was made on 19 VIII 2015 and the second season application was made on 13 I 2016. At the time of application, 237 m L of insecticide solution (deionized water + insecticide) was applied to the soil at the base of each tree trunk. At the flatwoods location, tree rows were oriented north south, and the application was made on the west side of the tree trunk. At the central ridge location, tree rows were oriented east west, and the application was made on the south side of the tree t runk. Leaf tissue samples were collected prior to the application of insecticides and then weekly for 1 2 weeks following the application. Trees were divided into 7 tree regions: bottom (lower 10% of canopy), spherical center, top (upper 10% of canopy), and 4 cardinal sides (east, west, south, north). At each sample date, four leaves from each of the seven tree regions across each of the four trees within a plot were

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3 1 harvested Each leaf was excised into two sections: 1. Middle (area inclusive of 0.5cm on ei ther side of the mid vein extending from leaf petiole to 0.5cm from leaf tip), and 2. Margin section and each tree region within a plot were wrapped individuall y in labeled heavy duty aluminum foil and stored collectively in a re sealable plastic bag at 20C until residue analyses were conducted. To evaluate distribution of analytes within a leaf, only leaf tissues from the within leaf residue distribution observed in the greenhouse. To evaluate temporal expression differences and to determine the distribution of Effect of Leaf M aturity on Neonicotinoid Expression Two season field study. A field study was conducted across two seasons to determine the effect of leaf maturity on expression of each of three neonicotinoids following application to the soil. Untreated non bearing citr us trees (v. Hamlin / r.s. Swingle) (ca. 1. 5 m 3 canopy volume) were used in the study T rees were field planted approximately 22 months prior to the first insecticide application to sandy soil comprised of 96.8% sand, 1.6% silt, and 2% clay, with 1.04% orga nic matter and CEC of 6.7 meq/100g. Trees were planted using a 2.4m in row spacing and 2.4m between row spacing, which provided sufficient separation to eliminate uptake of insecticides applied to an adjacent tree, confirmed by analysis of trees in the unt reated control The study was arranged in a randomized complete block design with 8 treatments and 4 replicates. Treatments consisted of an untreated control, Platinum 75SG, Admire Pro 4.6F, and Belay 2.13SC applied at the recommended rate for non bearing citrus trees based on 346 trees / hectare (140 trees / acre) ( Table 2 1 ). Approximately 14 d p rior to each insecticide application, a gas powered hedge trimmer was used to trim the tree canopy to a mean canopy volume (MCV) of appr oximately 1.3 m 3 to promote flushing. The first season insecticide application was made

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32 on 5 V 2017 and the second season application was made on 21 VI 2017, each when flush shoots were approximately 2.5cm long. At the time of application, 237 mL of insecticide solution (deionized water + insecticide) was applied to the soil at the base of each tree trunk. Leaf tissue samples were collected prior to the application of insecticides and then weekly for 4 weeks following the application. At each samp le date, four mature leaves and four flush shoots were harvested across each of the four trees within a plot. Mature leaves and flush shoots were placed into separate labeled paper bag s and collectively stored by treatment in a plastic re sealable bag at 20C until residue analyses were conducted. The same cohort of flush shoots were sampled each week to control for potential differences in expression values in flush that had not yet formed at the time of application in later sampling dates. Extraction and Leaf Tissue Analysis Leaf material from each plot was ground to a fine powder using liquid nitrogen and morter and pestal. A ca. five gram subsample of leaf powder was weighed and transferred to a 20 m L glass vial with a Teflon lined cap and stored at 20C until extraction; the exact weight of each sample was recorded for conversion of analyte concentration to the fresh leaf weight basis. Extraction was conducted using QuEChERS in 15 m L acetonitrile using pre weighed reagent sachets (United Chemical Technologies, #ECQUEU7 MP). A cleanup step was then conducted in which chlorophyll was removed from the acetonitrile extract using ChloroFiltr polymeric based sorbent tubes (United Chemical Technologies, # ECMPSGG15CT). The supernatant from cleanup was t hen filtered through a 20 m Teflon filter into an auto sampler vial. Separation and quantification of analytes was accomplished using Ultra High Performance Liquid Chromatography with a C The aqueous mobile phase was 0.1% formic acid in water and the polar modifying phase was 0.1% formic acid in acetonitrile. Samples were run against standards to construct a five point

PAGE 33

33 linear curve in a concentration range of 0.5 50 ppm and then against a five point standard curve in the range of 5 300 ppb. The concentration represented by the curve (in extract solution) was then converted back to g/g leaf tissue using the exact sample weight. Statistical Analyses In leaf distribution of neonicotinoids. Chemic al titer data for greenhouse leaf section means were averaged over replicate and subjected to a general linear mixed model to account for the experimental design using SASv9 .4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for sample date by leaf section inte ractions. For leaf section field data, only chemical titer leaf section were used and averaged over replicate, then subjected to a general linear mixed model to account for experimental design using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for sample date by leaf section interactions ; location was treated as a random effect and the model was adjusted for cumulative rainfall M ean separations indicate differences between leaf sections Temporal expres sion of neonicotinoids Chemical titer data for greenhouse means were averaged over replicate and subjected to a general linear mixed model to account for experimental design using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for sample date by leaf section interactions. For field data, c hemical titer section were averaged over replicate and subjected to a general linear mixed model to account for experimental design using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for sample date by location and sample date by tree region interactions ; the model was adjusted for cumulative rainfall M eans were square root transformed prior to analysis to achieve homogeneity of variance and meet the assumptions of the model M ean separations indicate differences between sample date

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34 Spatial distribution of Admire Pro analytes throughout the tree canopy Chemical titer data were averaged over replicate and subjected to a general linear mixed model to account for ex perimental design using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for location by tree region and sample date by tree region interactions ; the model was adjusted for cumulative rainfall M eans were square root transformed prior to analysis to ach ieve homogeneity of variance meet ing the assumptions of the model. M ean separations indicate differences between tree region Effect of Leaf Maturity on Neonicotinoid Expression. Chemical titer data were averaged over replicate and subjected t o a general linear mixed model to account for experimental design using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for sample date by leaf maturity interactions ; season was treated as a random effect and the model was adjusted for cumulative rainfall M eans were square root transformed prior to analysis to achieve homogeneity of variance meet ing the assumptions of the model. Results In Leaf Distribution of Neonicotinoids Following the application of Admire Pro in the greenhouse, no sample da te by leaf section interaction was observed for imidacloprid (F 11, 1 = 2.14; p = 0.4914), 5 OH (F 12, 4.107 = 1.2; p = 0.4682), or olefin (F 12, 18.4 = 2.42; p = 0.0532). Furthermore, no significant difference in titer was observed between leaf margin and le af center for imidacloprid (F 1, 8.654 = 1.97; p = 0.1950; Table 2 2 ), 5 OH (F 1, 7.393 = 1.82; p = 0.2175; Table 2 2 ), or olefin (F 1, 12.57 = 1.37; p = 0.2643; Table 2 2 ). When Admire Pro was applied to the soil in the field, no sample date by leaf section interaction was observed for imidacloprid (F 9, 276 = 0.19; p = 0.9948), 5 OH (F 9, 275.9 = 0.27; p = 0.9832), or olefin (F 9, 275.9 = 0.64; p = 0.7631). A significant difference in titer between leaf sections was observed for imidacloprid (F 1, 276 = 4.19; p = 0.0415; Table 2 2 ) and 5 OH (F 1,

PAGE 35

35 275.9 = 12.27; p = 0.0005; Table 2 2 ) where the leaf margin contained a higher concentrations than the leaf center. No difference in titer was observed between leaf sections for olefin (F 1, 275.9 = 1.22; p = 0.2699; Table 2 2 ). Following the application of Platinum 75SG in the greenhouse, no sample date by leaf section interaction was observed for thiamethoxam (F 12, 1 = 1.38; p = 0.5894), clothianidin (F 12, 1 = 6.04; p = 0.3088), or TZMU (F 12, 27.2 = 0.94; p = 0.5267). No significant difference was observed in titer between leaf margin and leaf center for t hiamethoxam (F 1, 23.67 = 1.05; p = 0.3158; Table 2 3 ) or for clothianidin (F 1, 2.981 = 4.11; p = 0.1363; Table 2 3 ), however, the leaf margin contained more TZMU than the leaf center (F 1, 17.12 = 12.44; p = 0.0026; Table 2 3 ). A sample date by leaf interaction was observed for TZNG (F 12, 14.4 = 4.52; p = 0.0042), but the order between leaf sections remained constant over time with the exception of 11 weeks following application Nevertheless, no significant difference was observed in titer between leaf margin and leaf center for TZNG (F 1, 12.83 = 4.41; p = 0.0561). Following application of Platinum 75SG to the soil in the field, no sample date by leaf section interaction was obs erved for thiamethoxam (F 7, 191.1 = 0.08; p = 0.9992) or for clothianidin (F 7, 189.9 = 0.02; p = 1.000). Furthermore, no significant difference in titer was observed between leaf margin and leaf center for thiamethoxam (F 1, 191.1 = 0.16; p = 0.6938; Table 2 3 ) or for clothianidin (F 1, 189.9 = 0.33; p = 0.5668; Table 2 3 ). In contrast to observations from the greenhouse, TZMU and TZNG were not detected in the field. Following the application of Belay 2.1 3SC in the greenhouse, no sample date by leaf section interaction was observed for clothianidin (F 9, 1 = 3.49; p = 0.3944) and no significant difference in leaf section was observed for clothianidin (F 1, 6.418 = 2.28; p = 0.1785; Table 2 4 ). A sample date by leaf section interaction was observed for TZMU (F 11, 12.9 = 3.01; p = 0.0315), yet

PAGE 36

36 the order between leaf sections remained constant over time with the exception of five weeks following application. However, a significant diff erence in TZMU titer was observed between leaf margin and leaf center (F 1, 1.29 = 81.54; p = 0.0402; Table 2 4 ) where the leaf margin had higher TZMU concentrations than the leaf center. Likewise, a sample date by leaf section in teraction was observed for TZNG (F 12, 3.657 = 8.12; p = 0.0356), but the order between leaf section concentration remained constant across all sample dates. Furthermore, a significant difference in TZNG titer was observed (F 1, 5.897 = 102.05; p < 0.0001; Table 2 4 ) where the leaf margin had a higher TZNG titer than the leaf center. When Belay 2.13SC was applied to the soil in the field, no sample date by leaf section interaction was observed for clothianidin (F 7, 146 = 0.64; p = 0.7256) or for TZNG (F 7, 145.9 = 1.21; p = 0.3019). No difference was detected between leaf margin and leaf center for clothianidin (F 1, 146 = 3.37; p = 0.0685; Table 2 4 ), yet higher levels of TZNG occurred in the l eaf margin than the leaf center (F 1, 145.9 = 10.05; p = 0.0019; Table 2 4 ). Dissimilar to observations in the greenhouse, TZMU was not detected in the field. Temporal Expression of Neonicotinoids When Admire Pro was applied to the soil in the greenhouse, a significant effect of sample date was observed for imidacloprid (F 12, 78 = 7.4; p < 0.0001; Table 2 5 ), 5 OH (F 12, 17.25 = 10.71; p < 0.0001; Table 2 5 ), and olefin (F 12, 1 8.4 = 11.6; p < 0.0001; Table 2 5 ). The titer of each analyte peaked at five weeks following application and persisted through 13 weeks following application. The highest mean imidacloprid titer observed was 192.060 ppm, while th e highest mean titer for 5 OH and olefin w as 33.673 ppm and 8.134 ppm, respectively. Following the application of Admire Pro in the field, a location by sample date interaction was observed for imidacloprid (F 9, 935 = 18.54; p < 0.0001), and imidacloprid t iter was affected by sample date (F 9, 935 = 48.84; p < 0.0001; Table 2 6 ). The highest mean imidacloprid titer was observed one week following application at the flatwoods location (1.052 ppm) and just before application at the

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37 c entral ridge location (1.246 ppm) ( Table 2 6 ). Low levels (< 0.090 ppm) of imidacloprid were detected up to eight weeks following application at the flatwoods location and 10 weeks following application at the central ridge locat ion. A location by sample date interaction was also observed for 5 OH following the application of Admire Pro to the soil in the field (F 9, 935 = 15.26; p < 0.0001), and a significant effect of 5 OH titer was observed by sample date (F 9, 935 = 45.85; p < 0 .0001; Table 2 6 ) where at the flatwoods location, 5 OH titer remained relatively constant to two weeks following application before decreasing, while the 5 OH titer at the central ridge location was highest prior to application and continuously decreased over time. Furthermore, a location by sample date interaction was observed for olefin (F 9, 935 = 2.52; p = 0.0076) following the application of Admire Pro, and olefin titer was affected by sample date (F 9, 935 = 23.66; p < 0.0001 ; Table 2 6 ). At each location, olefin persisted for up to six weeks following application of Admire Pro. When Platinum 75SG was applied to the soil in the greenhouse, sample date had a significant effect on thiamethoxam titer (F 12, 5.146 = 7.94; p = 0.015; Table 2 7 ), where thiamethoxam expression was highest at three weeks (271.140 ppm) following application. Similarly, sample date had a significant effect on clothianidin titer (F 12, 5.068 = 6.16; p = 0.0274; Table 2 7 ) and TZMU titer (F 12, 34.62 = 5.92; p < 0.0001; Table 2 7 ) following the soil application of Platinum 75SG, which also peaked at 3 weeks (99.379 ppm and 3.019 ppm, respectively) foll owing application. While sample date significantly affected TZNG expression (F 12, 12.65 = 134.66; p < 0.0001; Table 2 7 ) following the soil application of Platinum 75SG, no clear TZNG peak was observed at a single time point; TZN G titer fluctuated over the weeks following application. In contrast to expression levels observed in the greenhouse, when Platinum 75SG (0.37g / tree) was applied to the soil in the field, limited quantifiable thiamethoxam titers, or

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38 resulting metabolite titers were detected in citrus leaf tissues, thus residue analyses for these citrus leaf tissues were ceased. Following the soil application of Belay 2.13SC in the greenhouse, a significant effect was observed by sample date for clothianidin (F 12, 12.61 = 14.25; p < 0.0001; Table 2 8 ), TZMU (F 11, 14.9 = 3.86; p = 0.0087; Table 2 8 ), and TZNG (F 12, 15.67 = 25; p < 0.0001; Table 2 8 ). The maximum mean clothianidin titer (62.226 ppm) was observed at five weeks following application. The mean TZMU titer exhibited two distinct peaks, the first (0.543 ppm) at four weeks following application, and the second (0.833 ppm) at 11 weeks following application. A continual increase was obser ved for TZNG through eight weeks (peak mean 11.430 ppm) after application. Like following the application of Platinum 75SG to the soil in the field, limited quantifiable analytes were observed after application of Belay 2.13SC to the soil (1.27 m L / tree) in the field, therefore residue analyses for these citrus leaf tissues were discontinued. Spatial Distribution of Admire Pro Analytes throughout the Tree Canopy When Admire Pro was applied to the soil in the field, we observed no sample date by tree regi on interaction (F 54, 935 = 0.61; p = 0.9877) and no location by tree region interaction for imidacloprid (F 6, 84 = 2.16; p = 0.0555). Tree region had a significant effect on imidacloprid titer (F 6, 84 = 8.86; p < 0.0001; Fig. 2 1A ), in which the bottom tree region contained a significantly higher mean imidacloprid titer than the top or four cardinal side regions; no difference was observed between the spherical center region and the bottom region. Likewise, the spherical center r egion contained a higher mean imidacloprid titer than the top, north, or east tree regions, but was not different from the west or south tree regions. Furthermore, no difference was observed between the top tree region and the four cardinal side regions. N o sample date by tree region interaction (F 54, 935 = 1.04; p = 0.3982) or location by tree region interaction (F 6, 84 = 0.32; p = 0.9249) was observed for olefin following application of Admire Pro to the soil in the field.

PAGE 39

39 Furthermore, a significant eff ect of tree region was observed for olefin (F 6, 84 = 7.41; p < 0.0001; Fig. 2 1B ) in which the bottom tree region contained a higher mean olefin titer than the top tree region or the four cardinal side regions; no difference was observed between the bottom tree region and the spherical center region. No difference was observed in mean olefin titer between the top tree region and the four cardinal side regions, and no difference was observed between the spherical center region and the west and top tree region. In contrast, for the analyte 5 OH, no sample date by tree region interaction (F 54, 935 = 1.3; p = 0.0777) was observed, yet a location by tree region interaction was observed (F 6, 84 = 3.94; p = 0.0016). Tree region had a sig nificant effect on mean 5 OH titer (F 6, 84 = 16.65; p < 0.0001; Fig. 2 2 ) at the flatwoods location, where the bottom tree region contained higher 5 OH levels than all other tree regions. No difference in 5 OH titer was observed between the spherical center, west, and south tree regions, and no difference was observed between the top and four cardinal side regions. At the central ridge location, no difference was observed between the bottom, spherical center, west, north or east tree regions, and no difference was observed between the spherical center, top, and four cardinal side tree regions. Effect of L eaf M aturity on N eonicotinoid E xpression After application of Admire Pro to the soil, there was no sample date by leaf maturi ty interaction for expression of imidacloprid (F 4, 55 = 0.84; p = 0.5053), olefin (F 4, 55 = 0.77; p = 0.5500), or 5 OH (F 4, 55 = 1.00; p = 0.4178). Moreover, no difference in titer was observed between flush shoots and mature leaves for imidacloprid (F 1, 7 = 0.74; p = 0.4191), olefin (F 1, 7 = 1.95; p = 0.2057), or 5 OH (F 1, 7 = 2.55; p = 0.1543). Following application of Platinum 75SG to the soil, no sample date by leaf maturity interaction was observed in expression of thiamethoxam (F 4, 55 = 2.14; p = 0.0879), clothianidin (F 4, 55 = 1.07; p = 0.3823), or TZNG (F 4, 55 = 0.21; p = 0.9318). Furthermore, no difference was observed between flush shoots and

PAGE 40

40 mature leaves in expression of thiamethoxam (F 1, 7 = 2.08; p = 0.1929), clothianidin (F 1, 7 = 0 .01; p = 0.9419), or TZNG (F 1, 7 = 0.04; p = 0.8531). No TZMU was detected following the application of Platinum 75SG to the soil in this study. In contrast, a sample date by leaf maturity interaction was observed in clothianidin titer following applicati on of Belay 2.13SC to the soil (F 4, 55 = 3.36; p = 0.0156). Although an interaction did occur, no difference was observed in clothianidin titer between flush shoots and mature leaves during each sample date or when sample date data were pooled (F 1, 7 = 5 .26; p = 0.0554). No sample date by leaf maturity interaction was observed in TZNG titer following application of Belay 2.13SC to the soil (F 4, 55 = 1.28; p = 0.2908) and no difference was observed between flush shoots and mature leaves (F 1, 7 = 3.16; p = 0.1189). No TZMU was detected following the application of Belay 2.13SC to the soil in this study. Discussion The goal of this study was to quantify the spatial distribution and temporal expression of three currently labeled neonicotinoid insecticides in the citrus tree canopy to elucidate why trees continue to succumb to HLB infection despite intensive management efforts by growers. Previous studies have discussed the possibility of uneven expression of neonicotinoids in citrus resulting in potential exp osure of D. citri to sublethal dosages (Boina et al. 2009, Rogers 2012). This study was the first to use UHPLC MS to quantify the temporal expression and spatial distribution of neonicotinoids and resulting metabolites in citrus following application to t he soil. High parent material titers were observed following applications of Admire Pro (imidacloprid), Platinum 75SG (thiamethoxam), and Belay 2.13SC (clothianidin) in the greenhouse (max. mean 192 ppm imidacloprid; max. mean 240 ppm thiamethoxam; max. me an 62 ppm clothianidin). In contrast, low parent material titers (max. mean 1.246 ppm) of imidacloprid and very low titers (thiamethoxam max. mean 0.008; clothianidin max. mean 0.159) of thiamethoxam and

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41 clothianidin were detected after application in the field. As a result, only the Admire Pro treatment in the field was used to evaluate expression of neonicotinoids and resulting metabolites leaf distribution for all three insecticides in th e field, despite very low detection levels of thiamethoxam and clothianidin. While we did not find a difference in imidacloprid concentration between leaf sections in the greenhouse, we did discover that the leaf margin contained elevated levels of imidac loprid following application of Admire Pro in the field. This difference was inconsistent with our findings for thiamethoxam and clothianidin following the application of Platinum 75SG and Belay 2.13SC, respectively; no difference in active ingredient expr ession was observed between leaf sections for either insecticide. Mendel et al. (2000) found low levels of 14 C labeled imidacloprid around leaf vascular bundles when compared with the leaf margins. While we found one event that exhibited a difference betwe en leaf sections, it is possible that our excision method failed to fully account for intricate vascular bundle related expression patterns within a citrus leaf, resulting in a failure to detect differences. A number of the metabolites were detected at a h igher concentration in the leaf margin compared with the leaf middle, however, because the concentration of each metabolite is directly dependent on the concentration of the associated parent material, we cannot d etermine how any one metabolite may affect D. citri mortality in this study. Understanding the role of each metabolite is beyond the scope of this investigation, though it is possible that a combination of multiple metabolites has an additive effect on D. citri mortality. Furthermore, because radiolabeled parent material cannot be differentiated from metabolites through radiographic imaging, it is possible that patterns related to the vascular bundles observed by Mendel et al. (2000) were actually accumulations of metaboli tes (metabolized imidacloprid constituents) carrying the 14 C marker instead of accumulations of the

PAGE 42

42 parent material, imidacloprid. Nevertheless, inconsistent expression within a leaf remains of concern as it relates to potential expression of sublethal neo nicotinoid dosages. Understanding how neonicotinoids are expressed over time is important for a number of reasons: 1) determining when a subsequent non neonicotinoid foliar spray must be applied, 2) determining the time it takes to reach peak expression l evels as related to application timing, and 3) understanding the persistence of neonicotinoids in leaf tissues at sub lethal levels. While lethal levels of each compound as determined by Langdon and Rogers (2017) were observed in the present greenhouse s tudy, sublethal levels of all analytes were detected in citrus leaf tissues in the field following insecticide application to the soil. Between six and eleven weeks of D. citri control have been reported following soil application of neonicotinoids to youn g trees in the field ( Qureshi and Stansly 2007 2009 ; Ichinose et al. 2010 ; Setamou et al. 2010 ; B yrne et al. 2012; Rogers 2012). Percentage control is often quantified by comparing a mean number of insects across replicates in a given treatment with a mea n number of insects across replicates within an untreated control. In this case, for percentage control to be reduced, insects must re infest the treated area and develop to stages that make detection possible. Given the life cycle of D. citri there is a presumed delay between when the expression level drops below the oviposition deterrence threshold (currently unknown) and the time that nymphs are detected in insect counts. The result is that while some number of weeks control may have been observed in th e field, titers likely fell below lethal or deterrent levels before drops in percentage control were detected. Nevertheless, at the flatwoods location, we found a peak mean titer of 1.098 ppm imidacloprid at one week following application of Admire Pro, an d at the central ridge location, a peak mean titer of 1.246 ppm imidacloprid at the time of application of Admire Pro. Because trees at each location contained imidacloprid at the time of application, we cannot draw

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43 definitive conclusions based on temporal expression observed in the current study. We allowed seven weeks from the last known soil application of Admire Pro and the start of our field studies each season. Given the current state of HLB infection in commercial groves, we opted to not allow more t ime due to the risk of developing HLB infection in cooperator groves. Interestingly, the levels of imidacloprid and metabolites observed in the pre application samples were not statistically different from the highest mean titer observed following the appl ication for our study. Nevertheless, while the temporal expression objective was compromised, the objective outcomes addressing within leaf concentration gradient and spatial canopy distribution remain valid. Mapping the distribution of neonicotinoids thr oughout the tree canopy following application to the soil is critical to identifying likely inoculation sites and for determining where to sample on the tree in future research studying systemic neonicotinoids in citrus. We found higher imidacloprid concen trations in the bottom 10% of the tree canopy compared to other tree canopy regions, although no difference was observed between the lower canopy and the spherical center. Neonicotinoids are highly systemic and move through the plant xylem (Elbert et al. 1 991, Maienfisch et al. 2001). A common characteristic of xylem mobile herbicides applied to the soil is injury accumulation in the oldest leaves. This is in contrast to phloem mobile herbicides, which cause injury near the growing point of the plant, or in the newest leaf tissue. Triazine herbicides, which like neonicotinoid insecticides are xylem mobile, result in h igher concentrations within older leaves compared to new leaves when applied to the soil (Stoller 1970). The lower tree canopy contains the old est set of leaves and our findings are consistent with movement patterns and accumulation of known xylem mobile herbicides. Castle et al. (2005) sampled xylem fluid to study spatial distribution of thiamethoxam and imidacloprid in

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44 citrus tree canopies, but found no spatial difference in expression. They divided the tree into upper and lower halves and divided each half into quadrants. They sampled xylem fluid from two branches within each quadrant. The sampling technique used may have contributed to the dif ferential outcome of their study compared to the present study. In addition, we wanted to determine if our one sided application technique influenced distribution within the tree canopy. Insecticide solution was applied to the west side of the tree trunk a t the flatwoods location, and the south side of the tree trunk at the central ridge location. We did not observe a location by tree region interaction for imidacloprid expression, which indicates that the pattern of spatial distribution was not different b etween locations. This suggests that the drench application site did not affect the distribution of imidacloprid within the tree canopy. Because we found no difference between the four tree canopy sides and the tree top, subsequent studies evaluating citru s leaf tissues should sample from this area to achieve a consistent sampling pattern. Young, non bearing trees flush more often than mature trees throughout the year and gravid adults are attracted to the volatiles emitted by flush shoots (Stansly and Rog ers 2006, Patt and Setamou 2010). Nymphs are also more likely to acquire C Las as they develop on flush shoots (Pelz Stelinski et al. 2010). Although D. citri are more attracted to flush shoots, much of the leaf tissue subjected to analytical evaluation of chemical titers to date have utilized only mature leaves, largely due to the constant availability of leaves within the same cohort over a long period of time following a single application (Langdon 2017). It is important to determine whether insecticide e xpression levels differ between mature leaves and flush shoots to allow one to predict whether better or worse control would be expected in flush shoots based on known titers in mature leaves. We found no difference in titer between flush shoots and mature leaves for any chemical evaluated following application of each of the three neonicotinoids included in

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45 our study. At the time insecticide applications were made, flush shoots had already emerged and were actively growing, and the same cohort was sampled across the four post ap plication sample events S ubsequent studies should be conducted to evaluate the relation between bud break timing and ti ming of application to the soil, such that r esultant titers are maximized in flush shoots. P erhaps if one were to apply neonicotinoids to the soil two weeks prior to bud break, expression levels in flush shoots would have been less than in mature leaves due to limited availability of i nsecticide by the time flush shoots emerg ed. Nevertheless, in the present study, neonicotinoid expression in mature leaves did not differ from expression in the more attractive flush shoots when the application was made after bud break. The overarching goal of this research was to identify the temporal expression and spatial distribut ion of neonicotinoids in citrus leaf tissues following application to the soil. While we successfully mapped the distribution of three neonicotinoids and resulting metabolites within individual leaves and one neonicotinoid and resulting metabolites throug hout the tree canopy, we unexpectedly observed low levels of expression of all compounds tested, much lower than what was identified as lethal through ingestion by Langdon and Rogers (2017). Contributing factors to low level expression observed in our stud y may have been that application rates were not high enough for the size of tree tested or while unlikely, it is possible that HLB infection negatively impacted uptake of our treatment applications as trees were not tested for C Las infection. Tree size and application rate have been shown to directly impact uptake and expression of thiamethoxam following application to the soil in citrus (Langdon 2017). Furthermore, Langdon (2017) found that 0.55 ppm imidacloprid did not reduce in any feeding behavior inclu ding probing, pathway, or salivation / ingestion activities when monitoring using electropenetrography. However, they did find that 5.5 ppm imidacloprid caused significant

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46 reductions in pathway and salivation / ingestion behaviors. Nevertheless, it is like ly that the highest mean titer observed for any neonicotinoid in the present study offered no reduction in feeding activity, and thus no interception of C Las inoculation. Because non neonicotinoid foliar sprays were routinely applied to the study area, no attempt was made to correlate insect incidence or abundance with neonicotinoid titer levels. In addition to potential risk of the spread of HLB, neonicotinoid resistance following exposure to sublethal dosages is of significant concern. Diaphorina citri r esistance to neonicotinoids was recently detected in Florida (Langdon and Rogers 2017), which may have been exacerbated by sublethal neonicotinoid expression like that observed in the present study. Tiwari et al. (2011a) originally discovered D. citri resi stance to neonicotinoids in Florida in 2009, but likely due to intensive area wide spray programs implemented in 2010, reversion was believed to have occurred by 2013 (Coy et al. 2016). Tiwari et al. (2011a) found that imidacloprid resistant D. citri popul ations expressed higher levels of detoxifying enzymes, including general esterase, gluta t hione S transferase, and cytochrome P 450 monooxygenases. Subsequent research identified five family 4 cytochrome P 450 genes that were induced by exposure to imidaclopr id (Tiwari et al. 2011b). Although elevated levels of detoxifying enzymes were found in insecticide resistant populations, Tiwari et al. (2011a) suggested that reduced penetration, target site insensitivity, and mutations in detoxifying enzymes may also im pact development of neonicotinoid resistance. Nevertheless, development of resistance to neonicotinoids by D. citri has occurred in the field, and therefore, applications of neonicotinoids must be carefully administered such that D. citri exposure to suble thal dosages is minimized. The present study quantifies the concentration of imidacloprid, thiamethoxam, and clothianidin in citrus leaf material in space and over time following application to the soil. While

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47 imidacloprid accumulated at higher levels in the lower portion of the tree canopy, we did not find titers near the lethal range quantified by Langdon and Rogers (2017), therefore even the bottom tree region which exhibited the highest imidacloprid levels would expose D. citri to sublethal dosages. Langdon and Rogers (2017) found that lethal activity from contact exposure to neonicotinoid insecticides occurs at very low concentrations compared with ingestion. To potentially maximize the activity of neonicotinoids and permit the longevity of their use subsequent work should investigate neonicotinoid residues over time following foliar application. Presumably, foliar application would result in much higher acute residues following application, with a more rapid residue degradation, which is more suitab le within the scope of insecticide resistance management. Given the recent findings of neonicotinoid resistance in various field populations of D. citri future implementation of neonicotinoid insecticides in the field should focus on reducing the likeliho od of increasing the incidence and severity of resistance.

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48 Table 2 1 Neonicotinoid product description and use rates for greenhouse and field studies. Product Rate per hectare applied (rate per acre) Rate per tree (based on 346 trees per hectare or 140 trees per acre) Grams active ingredient per tree Resulting analytes Admire Pro 4.6F 511.09 mL /ha (7 fl oz/ac) 1.48 m L /tree 0.814 g/tree imidacloprid* 5 OH olefin Platinum 75SG 128.10 g/ha (1.83 oz wt/ac) 0.37 g/tree 0.324 g/tree thiamethoxam* clothianidin TZMU TZNG Belay 2.13SC 438.07 mL /ha (6 fl oz/ac) 1.27 m L /tree 0.278 g/tree clothianidin* TZMU TZNG Active ingredient of listed formulated product.

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49 Table 2 2. Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application of Admire Pro (1.48 m L per tree) to the soil in the greenhouse and in the field. Study Leaf section imidacloprid 5 OH Olefin Mean 95% CI Mean 95% CI Mean 95% CI greenhouse center 109.930a (44.819 175.041) 15.399a (8.777 22.021) 3.571a (1.738 5.404) margin 129.310a (64.199 194.421) 21.217a (14.595 27.839) 4.959a (3.126 6.791) p value = 0.1950 p value = 0.2175 p value = 0.2643 field center 0.412b (0.295 0.528) 0.078b (0.062 0.095) 0.015a (0.009 0.022) margin 0.528a (0.406 0.650) 0.110a (0.092 0.127) 0.019a (0.012 0.026) p value = 0.0415 p value = 0.0005 p value = 0.2699

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50 Table 2 3 Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application of Platinum 75SG (0.37g per tree) to the soil in the greenhouse and in the field. Study Leaf Section thiamethoxam clothianidin TZMU TZNG mean 95% CI mean 95% CI mean 95% CI mean 95% CI Green house center 94.162a (54.080 134.244) 45.201a (29.866 60.536) 0.796a (0.081 1.512) 3.637a (2.875 4.399) margin 104.660a (64.578 144.742) 54.230a (38.895 69.565) 1.052b (0.340 1.765) 4.776a (4.014 5.538) p value = 0.3158 p value = 0.1363 p value = 0.0026 p value = 0.0561 field center 0.006a (0 .000 0.012) 0.002a (0 .000 0.004) 0 0 margin 0.008a (0.002 0.014) 0.003a (0 .000 0.005) 0 0 p value = 0.6938 p value = 0.5668

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51 Table 2 4. Chemical titer (ppm) in citrus leaf tissue across two leaf sections following application of Belay 2.13SC (1.27 mL per tree) to the soil in the greenhouse and in the field. Study Leaf section clothianidin TZMU TZNG mean 95% CI mean 95% CI mean 95% CI greenhouse center 38.425a (27.890 48.960) 0.340a (0.191 0.488) 6.595a (5.536 7.653) margin 48.306a (37.771 58.841) 0.522b (0.374 0.669) 9.649b (8.592 10.707) p value = 0.1785 p value = 0.0402 p value < 0.0001 field center 0.138a (0.115 0.160) 0 0.033a (0.020 0.046) margin 0.159a (0.137 0.182) 0 0.055b (0.042 0.068) p value = 0.0685 p value = 0.0019

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52 Table 2 5. Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Admire Pro (1.48 m L per tree) to the soil in the greenhouse. Weeks following application imidacloprid 5 OH Olefin mean 95% CI mean 95% CI mean 95% CI 0 0 0.000 d 0cd 0bc 1 60.148cd ( 0 0 .000 128.178) 3.548c (0 0 .147 6.948) 0.269b (0.137 0.402) 2 153.770ab (85.279 222.261) 14.474abc (10.940 18.007) 1.796b (1.402 2.191) 3 167.060ab (87.238 246.882) 25.178a (20.784 29.571) 4.375ab (3.523 5.227) 4 171.690a (102.554 240.826) 27.001a (21.031 32.972) 5.191ab (4.033 6.349) 5 192.060a (122.523 261.597) 33.673a (25.500 41.845) 8.134a (5.917 10.350) 6 164.640ab (91.245 238.035) 26.616a (19.821 33.411) 5.091ab (3.286 6.897) 7 137.020abc (61.172 212.868) 22.544ab (14.249 30.838) 5.339ab (2.891 7.786) 8 101.190abcd (31.965 170.415) 16.796abc (10.968 22.625) 4.896ab (2.853 6.940) 9 102.810abcd (33.698 171.922) 18.309ab (11.883 24.734) 5.920ab (2.776 9.064) 10 64.766bcd (0 0.000 134.007) 9.376bc ( 0 4.023 14.729) 2.390b (1.642 3.138) 11 105.670abcd ( 0 7.877 203.463) 14.260abc ( 0 3.686 24.834) 1.956b (0.952 2.961) 12 93.438abcd (10.572 176.303) 18.015abc ( 0 5.753 30.277) 6.250ab (0.600 11.900) 13 40.810d ( 0 0 .000 108.649) 8.219bc ( 0 3.094 13.343) 3.834ab (1.197 6.470) p value < 0.0001 p value < 0.0001 p value < 0.0001

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53 Table 2 6. Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Admire Pro (1.48 m L per tree) to the soil in the field at two commercial Florida citrus groves. Location Weeks following application Imidacloprid 5 OH Olefin Mean 95% CI Mean 95% CI Mean 95% CI Flatwoods 0 0.926bc (0.777 1.075) 0.440bcd (0.375 0.505) 0.142abc (0.091 0.193) 1 1.098ab (0.989 1.207) 0.436bcd (0.388 0.483) 0.096bcd (0.059 0.133) 2 1.052ab (0.958 1.147) 0.447bc (0.405 0.488) 0.143ab (0.111 0.175) 3 0.902bc (0.812 0.992) 0.390cd (0.351 0.429) 0.055cd (0.024 0.085) 4 0.539e (0.449 0.630) 0.270ef (0.231 0.310) 0.106abc (0.076 0.137) 5 0.491ef (0.400 0.583) 0.223efg (0.183 0.263) 0.103bcd (0.072 0.134) 6 0.301fg (0.208 0.394) 0.174fgh (0.133 0.215) 0.048cd (0.016 0.079) 8 0.090gh (0 .000 0.185) 0.045ij (0.004 0.087) 0 .000 efg 10 0 .000 h 0 .000 ij 0 .000 efg 12 0 .000 h 0 .000 j 0 .000 fg Central Ridge 0 1.246a (1.097 1.395) 0.606a (0.541 0.672) 0.210a (0.160 0.261) 1 1.117ab (0.988 1.246) 0.539ab (0.482 0.595) 0.126abc (0.082 0.169) 2 0.885bc (0.788 0.982) 0.434bcd (0.391 0.476) 0.141abc (0.109 0.174) 3 0.784cd (0.694 0.873) 0.393cd (0.354 0.432) 0.014def (0 .000 0.045) 4 0.561de (0.470 0.653) 0.296de (0.256 0.335) 0.096abc (0.065 0.127) 5 0.484ef (0.390 0.577) 0.255efg (0.214 0.296) 0.066bcd (0.035 0.098) 6 0.398ef (0.302 0.494) 0.289de (0.246 0.331) 0.038cde (0.006 0.071) 8 0.149gh (0.036 0.262) 0.157gh (0.108 0.207) 0 .000 fg 10 0.006h (0 .000 0.125) 0.069hi (0.018 0.121) 0 .000 g 12 0 .000 h 0 .000 ij 0 .000 g p value < 0.0001 p value < 0.0001 p value < 0.0001

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54 Table 2 7. Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Platinum 75SG (0.37g per tree) to the soil in the greenhouse. Weeks following application thiamethoxam clothianidin TZMU TZNG mean 95% CI mean 95% CI mean 95% CI mean 95% CI 0 0 0.000 cd 0 0 .000 cd 0 .000 b 0 .000 bc 1 69.801abc (26.922 112.679) 12.293c ( 0 0 .000 27.877) 0 .000 b 0.943b (0.762 1.123) 2 240.070ab (152.601 27.539) 66.470abc (36.160 96.780) 1.039b (0.149 1.928) 4.028ab (1.873 6.182) 3 271.140a (180.863 361.417) 99.379a (74.223 124.534) 3.019a (2.129 3.908) 5.358a (5.121 5.594) 4 92.293abc (51.673 132.912) 40.939abc (24.869 57.008) 0.701b (0 .000 1.591) 2.503b (2.249 2.756) 5 118.970abc (68.429 169.511) 58.515abc (37.710 79.320) 0.855b ( 0 .000 1.744) 3.830ab (2.864 4.796) 6 86.693abc (47.322 126.063) 51.415abc (35.267 67.563) 0.478b (0 .000 1.367) 2.795ab (2.233 3.357) 7 114.820abc (61.219 168.421) 68.703ab (46.374 91.031) 0.692b (0 .000 1.581) 5.228ab (4.014 6.441) 8 58.246abc (17.836 98.656) 61.495abc (31.895 91.095) 1.025b (0.136 1.914) 5.313ab (3.636 6.989) 9 62.636abc (23.276 101.997) 43.769abc (25.739 61.799) 0.886b (0 .000 1.775) 2.690ab (1.268 4.112) 10 57.736abc (18.481 96.992) 39.940abc (24.144 55.736) 0.969b (0.079 1.858) 2.728ab (1.664 3.791) 11 44.153bc (0 0 .517 87.788) 27.114bc (11.430 42.797) 1.176b (0.287 2.066) 3.528ab (2.760 4.295) 12 49.399abc ( 0 7.219 91.579) 43.935abc (19.780 68.090) 1.011b (0.121 1.900) 4.940ab (1.777 8.103) 13 26.421c (26.421 65.464) 32.336bc (16.303 48.370) 0.453b (0 .000 1.342) 3.400ab (2.344 4.456) p value = 0.0150 p value = 0.0274 p value < 0.0001 p value < 0.0001

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55 Table 2 8. Chemical titer (ppm) in citrus leaf tissue during the weeks following application of Belay 2.13SC (1.27 m L per tree) to the soil in the greenhouse. Weeks following application clothianidin TZMU TZNG mean 95%CI mean 95%CI mean 95%CI 0 0 0.000 cd 0 .000 bc 0 .000 de 1 12.714c (8.009 17.418) 0 .000 bc 1.019d (0 .000 2.465) 2 39.171ab (33.239 45.103) 0.084b (0 .000 0.332) 2.813cd (1.313 4.312) 3 49.430ab (42.742 56.118) 0.419ab (0.171 0.667) 6.349bc (4.894 7.803) 4 55.218a (47.285 63.150) 0.543ab (0.295 0.790) 5.903bc (4.445 7.360) 5 62.225a (46.816 77.634) 0.460ab (0.212 0.708) 9.045ab (7.551 10.539) 6 51.076ab (42.718 59.434) 0.232b (0 .000 0.480) 9.605ab (8.135 11.075) 7 54.576a (43.322 65.831) 0.129b (0 .000 0.377) 10.933a (9.361 12.504) 8 48.741ab (39.853 57.629) 0.451ab (0.203 0.699) 11.430a (9.880 12.980) 9 42.701ab (35.428 49.975) 0.564ab (0.316 0.812) 9.848ab (8.329 11.366) 10 36.338abc (21.613 51.062) 0.613ab (0.365 0.860) 7.733abc (5.042 10.423) 11 45.959ab (35.325 56.593) 0.833a (0.585 1.080) 10.649a (9.141 12.157) 12 36.048abc (23.594 48.501) 0.545ab (0.266 0.824) 10.267ab (8.044 12.489) 13 29.558bc (17.487 41.628) 0.295ab (0.034 0.556) 9.994ab (6.920 13.067) p value < 0.0001 p value = 0.0087 p value < 0.0001

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56 Figure 2 1. Comparison of chemical titer between seven tree reg ions during 2015 and 2016 field seasons. A. Imidacloprid titer in citrus leaf tissues resulting from soil application of Admire Pro in the field. B. Olefin titer in citrus leaf tissues resulting from soil application of Admire Pro in t he field. Bars sharin g the same letter do not differ c bc ab ab a a a 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 bottom center west south top north east Imidacloprid titer (ppm) ( 95% CI) following application of Admire Pro to the soil in the field tree region F 6, 84 = 8.86; p < 0.0001 A c bc ab a ab a a 0 0.02 0.04 0.06 0.08 0.1 0.12 bottom center west south top north east Olefin titer (ppm) ( 95% CI) following application of Admire Pro to the soil in the field Tree Region F 6, 84 = 7.41; p < 0.0001 B

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57 Figure 2 2. Comparison of 5 OH titer between seven tree regions at two locations during 2015 and 2016 field seasons. A. Tree region comparison at the flatwoods location following soil application of Admire Pro in the field. B. Tree region comparison at the central ridge location following soil application of Admire Pro in the field. Bars sharing the same letter do not differ a b bc bc c c c a ab ab b b ab ab 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 bottom (a) center (b) west (drench) south (g) top (c) north (e) east (f) bottom (a) center (b) west (e) south (drench) top (c) north (f) east (g) A. Flatwoods B. Central Ridge 5OH titer (ppm) ( 95% CI) following application of Admire Pro to the soil in the field Tree region based on cardinal direction (region corresponding to drench) F 6, 84 = 3.94; p = 0.0016

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58 CHAPTER 3 INFLUENCE OF TREE SIZE AND APPLICATION RATE ON EXPRESSION OF THIAMETHOXAM IN CITRUS AND ITS EFFICACY AGAINST DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) Neonicotinoids are a key group of insecticides used to manage Diaphorina citri Kuwayama in Florida citrus. Diaphorina citri is the vector of Candidatus Liberibacter asiaticus ( C Las), the presumed causal agent of huanglongbing (HLB), a worldwide disease of citrus. A two season field study was conducted to evaluate the effect of tree size and application rate on expression of thiamethoxam in young citrus following application to the soil. Diaphorina citri adult and nymph abundance was also correlated with thiamethoxam titer in leaves. Tree size and application rate each significantly affected thiamethoxam titer i n leaf tissue. The highest mean thiamethoxam titer observed (33.39 ppm) in small trees (mean canopy volume = 0.08m 3 ) occurred after application of the high rate (0.74 g Platinum 75SG per tree) tested. There was a negative correlation between both nymph and adult abundance with increasing thiamethoxam titer in leaves. A concentration of 64.63 ppm thiamethoxam was required to reach a one percent probability of encountering a flush shoot with at least one adult D. citri while 19.05 ppm was required for the sa me probability of encountering nymphs. The LC 99 for the field population was 147.91 ppm by ingestion and 0.33 ppm by contact. Because thiamethoxam titer failed to reach a lethal level (>147.91 ppm), D. citri were presumably exposed to sublethal thiamethoxa m doses, likely exacerbating resistance potential. Based on our results, we suggest the use of neonicotinoids by foliar rather than soil application to maintain the utility of this chemical class in future insecticide management programs in Florida citrus. Justification Citrus (Rutaceae) is the largest agricultural commodity in Florida ; approximately one quarter million hectares valued at nearly 9.9 billion dollars were cultivated in 2 015 ( Hodges and

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59 Spreen 2015 ) This crop has come under severe decline in recent years due to the spread of the devastating citrus disease, huanglongbing (HLB). Huanglongbing is presumably the result of infection by the phloem limited bacterium, Candidatus Liberibacter asiaticus ( C Las), transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) (Halbert and Manjunath 2004, Bov 2006, Grafton Cardwell et al. 2013). Following inoculation into a tree by a feeding C Las infected psyllid bacteria move from the infection site, through the vascular phloem to compromise the root system, which in turn deprives the tree canopy and reduces fruit yield (Halbert and Manjunath 2004, Bov 2006, Grafton Cardwell et al. 2013). H ua nglongbing was first detected in Florida in 2005 (Halbert 2005), just seven years after the discovery of D. citri (Halbert and Manjunath 2004). Vector management using insecticides quickly became the key strategy to reduce D. citri populations and consequent spread of HLB (Rogers 2008). Diaphorina citri develop and reproduce rapidly, requir ing as little as 1 5 days to complete the egg to adult life cycle under optimal environmental conditions (25 28C) (Liu and Tsai 2000, Grafton Cardwell et al. 2013). Adults typically seek volatile emitting flush shoots as sites for oviposition (Patt and Set amou 2010) Eggs hatch in two to four days, where newly emerged nymphs feed on phloem sap, thus acquirin g C Las from the infected tree (Pelz Stelinski et al. 2010). Because the probability of successful C Las acquisition by D. citri is higher for nymphs than for adults, the greatest risk of spread is from insects that acquire the bacteria during the nymph stage (Pelz Stelinski et al. 2010) As nymphs become adults, they disperse and spread the bacteria to uninfected trees. An estimated 80 100 percent of D. ci tri found in Florida are C Las positive (Coy and Stelinski 2015); therefore, both nymphs developing on infected citrus tissue, as well as, infected adults feeding on new, uninfected plant material must be targets for successful vector suppression.

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60 Young t rees are defined as less than eight feet in height that flush asynchronously and more often than mature trees (Hall and Albrigo 2007 Rogers 2012 ) Because young trees produce attractive flush shoots often throughout the year, they are at high risk of becoming infected with C Las throughout the year (Stansly and Rogers 2006). Insecticides have been important in mitigating C Las infection of young trees, especially before trees reach fruit bearing age (Rogers 2012). Extension recommendations by the Univer sity of Florida suggest a rotation between soil applied neonicotinoids and non neonicotinoid foliar sprays to reduce D. citri populations in young tree groves (Rogers 2012, Rogers et al. 201 5 ). Neonicotinoids are within the Insecticide Resistance Action Co mmittee (IRAC) sub group 4A, and act on the insect nicotinic acetylcholine receptor (nAChR) (IRAC 2017) Neonicotinoids are highly systemic and when applied to the soil, are taken up by the root system and transported to the foliage via xylem channels (El bert et al. 2008). Three neonicotinoid insecticides are currently labeled for use in non bearing citrus in Florida : thiamethoxam (Platinum 75 SG Syngenta Crop Protection, Inc., Greensboro, NC), imidacloprid (Admire Pro 4.6F Bayer CropScience, Research Triangle Park, NC), and clothianidin (Belay 2.13 SC Valent USA Corporation, Walnut Creek, CA). B etween six and eleven weeks of D. citri control have been documented following the application of neonicotinoids to the soil (Qureshi and Stansly 2007, Qures hi and Stansly 2009, Ichinose et al. 2010, Setamou et al. 2010, Rogers 2012). Residual activity of insecticides applied to the soil are likely influenced by factors such as soil type, application volume, irrigation/rainfall, tree age and size, and environm ental conditions. Moreover, neonicotinoid insecticides metabolize into various analytes, though the effect of any one resulting metabolite on D. citri mortality is unknown (Byrne et al. 2017). For example, thiamethoxam metabolizes into clothianidin (Nauen et al.

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61 2003) and clothianidin further metabolizes into TZNG and TZMU (Kim et al. 2012). Nevertheless, t o date, there has been scant information on movement, distribution, and persi stence of soil applied neonicotinoids or metabolites in citrus tissue in the field Previous i nsecticide trials evaluating soil applied neonicotinoids used percent control relative to the untreated check or mean number of D. citri per sample size to assess efficacy (Qureshi and Stansly 2007, Qureshi and Stansly 2009, Ichinose et al. 2010, Setamou et al. 2010, Byrne et al. 2012, Rogers 2012). Additional studies specifically quantified the concentration of neonicotinoids in leaf tissue following soil application using enzyme linked immunosorbent assay ( ELISA ) (Castle et al 2005, Garlapati 2009, Setamou et al. 2010 Byrne et al. 2017 ) H owever, only one attempted to compare percent control with chemical titer within citrus leaf tissues The lethal concentration of imid acloprid for D. citri was estimated between 200 and 250 parts per billion (ppb) by correlating percentage control of D. citri with imidacloprid titer (Setamou et al. 2010). Because virtually all D. citri in Florida are assumed to be C Las positive, growers cannot tolerate any feeding on uninfected tree s. In 2013, an estimated one to three percent of trees succumb to HLB infection a nnually in Florida groves, despite deliberate use of soil applied neonicotinoids (Rogers 2013). Langdon and Rogers (2017) found t hat 62.19 ppm of imidacloprid was required to kill 90 percent of D. citri from a laboratory susceptible population through ingestion and hypothesized that the 200 to 250 ppb efficacy threshold set by Setamou et al. ( 2010 ) may have been the result of sublet hal feeding deterrence as opposed to lethal activity. Nevertheless, u neven uptake or distribution of neonicotinoids in citrus tissue may also result in exposure of D. citri to sublethal doses (Boina et al. 2009, Rogers 2012), which may aid in development o f resistance to this particular chemistry The problem of uneven distribution is compounded as trees grow, requiring increasing application rate. As rates are increased to match

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62 tree age and size annual use limits become increasingly restrict ing for the development of effective year long management strategies. Resistance to neonicotinoids was discovered in the field in 2009 (Tiwari et al. 2011 a ) ; however reversion to susceptibility was reported by 2014 (Coy et al. 2016) This was attributed to increa sed rotations with foliar applied insecticides through area wide spray programs. A more thorough understanding of the use of soil applied neonicotinoids and factors that may stimulate the development of resistance is critical to maintaining effective use o f this mode of action for manag ement of HLB in Florida citrus. The purpose of this study was to quantify thiamethoxam expression over time within citrus tissue following soil application, to evaluate efficacy of Platinum 75SG applied at two rates to young citrus trees of two sizes against D. citri and to quantify susceptibility of the D. citri field population to thiamethoxam by exposure through ingestion and contact. By quantifying the temporal distribution of thiamethoxam in citrus leaf tissue with dist inct tree sizes and application rates, we can more effectively refine management recommendations for the use of neonicotinoids in young citrus within the context of resistance management. Materials and Methods Insecticide Application and Citrus Leaf Sampli ng A two season field study was conducted during 2016 and 2017 to determine the concentration of thiamethoxam and resulting metabolites in citrus leaves following application of Platinum 75SG to the soil over time, based on application rate and tree size. We also determined the influence of thiamethoxam concentration on incidence and abundance of D. citri on treated trees over time. Untreated citrus (v. Hamlin / r.s. Swingle) trees of two non bearing size classes were used in the study and defined as : larg e (mean canopy volume (MCV) approx. 1.34m 3 ) and small (MCV approx. 0.08m 3 ) in size. Large trees used in the study were field planted approximately 18 months prior to the first insecticide application and small trees were field

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63 planted approximately 1 month prior to the first insecticide application. The same cohort of trees used during 2016 were used during 2017. Trees were planted to sandy soil comprised of 96.8% sand, 1.6% silt, and 2% clay, with 1.04% organic matter and cation exchange capacity (CEC) of 6.7 meq/100g. Although rate calculations were based on the most common plant density of 140 trees per acre, due to space constraints, t rees for this study were planted using a 2.4m in row spacing and 2.4m between row spacing, which provided sufficient separation to eliminate uptake of insecticides applied to an adjacent tree, confirmed by analysis of trees in the untreated control The study was arranged in a randomized complete block design with six treatments and four replicates and each plot consisted of four trees Prior to each insecticide application, tree canopy volume was measured, but no attempt was made to account for differences in individual tree size when identifying treatment plots. The first season insecticide application was made on 8 IX 2016 and the second season application was made on 20 I 2017. At the time of application, 237 m L of insecticide solution (deionized water + insecticide) was ap plied to the soil at the base of each tree trunk which is the common application volume in the commercial setting The high application rate was 0.74 g Platinum 75SG per tree (equiv. 3.67 oz wt product/ac on 140 trees/ac) and the low application rate was 0.37 g Platinum 75SG per tree (equiv. 1.83 oz wt product/ac on 140 trees/ac). Leaf tissue samples were collected prior to the application of insecticides and then weekly for 12 weeks following the application. At each sample date, four mature, fully expand ed leaves (ca. 5 15 grams) were randomly harvested from the outer canopy across each of the four trees within a plot Leaves were placed into labeled paper bag s and collectively stored by treatment in a plastic re sealable bag at 20C until residue anal yses were conducted. Additionally, trees were evaluated weekly for the incidence and abundance of D. citri nymphs and adults. At each sample date, the number of D. citri nymphs and adults across ten

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64 flush terminals per plot and the number of nymph and adu lt infested terminals within each plot were counted and reported separately. Extraction and Leaf Tissue Analysi s Leaf material from each plot was ground to fine powder using liquid nitrogen with morter and pestl e A ca. five gram subsample of leaf powder was weighed and transferred to a 20 m L glass vial with a Teflon lined cap and stored at 20C until extraction; the exact weight of each sample was recorded for conversion of analyte concentration to fresh leaf weight. Extraction was conducted with QuEChE RS (Anastassiades 2003) in 15 m L acetonitrile using pre weighed reagent sachets (United Chemical Technologies, #ECQUEU7 MP). A cleanup step was then conducted in which chlorophyll was removed from the acetonitrile extract using ChloroFiltr polymeric based sorbent tubes (United Chemical Technologies, Horsham, PA, # ECMPSGG15CT). The supernatant from cleanup was filtered through a 20m T eflon filter into an auto sampler vial. Separation and quantification of analytes was accomplished using Ultra High Perform ance Liquid Chromatography with a C 18 column coupled to a Thermo TSQ Quantum mass spectrometer (UHPLC MS) The LOQ was 5 ng/g for imidacloprid, 10 ng/g for clothianidin, thiamethoxam, and 5 OH, and 25 ng/g for olefin, TZMU, and TZNG. The LOD was 1.5 ng/g for imidacloprid, 3.2 ng/g for clothianidin and thiamethoxam, 3.0 ng/g for 5 OH, 8.0 ng/g for olefin, and 8.3 ng/g for TZMU and TZNG. The aqueous mobile phase was 0.1% formic acid in water and the polar modifying phase was 0.1% formic acid in acetonitrile. Samples were run against standards to construct a five point linear curve in a concentration range of 0.5 50 ppm, and then against a five point standard curve in the range of 5 300 ppb. The concentration represented by the curve (in extract solution) was then converted back to g/g leaf tissue using the exact sample weight. The standards were matrix matched to compensate for signal suppression effects of the matrix. Plant tissue free of all four analytes was extracted using

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65 QuEChERS as outlined above in or der to obtain a blank matrix for mixing working standards. Primary standards were made using technical grade material (97.6 99.9%) in acetonitrile; technical grade material was obtained from either Syngenta Crop Protection, Inc., Greensboro, NC or Valent USA Corporation, Walnut Creek, CA. A set of working standards encompassing the linear range of concentrations was prepared from the primary standards, again in acetonitrile. To prepare a range of working standards in blank plant matrix, a 1000 L aliquot o f blank plant matrix was dry evaporated under nitrogen. The residue was then reconstituted with a 1000 L aliquot of standard acetonitrile. The solution was sonicated to ensure a homogeneous product and passed through a 0.2 m PTFE filt e r prior to injection, as were unknowns. Insect Biological Assays Lab Culture The laboratory susceptible (LS) strain was reared in continuous culture at the University of Florida Citrus Research and Education Center in Lake Alfred on Murraya koenigii mai ntained at 27C with RH 65% with a photoperiod of 14:10 L:D. The colony did not receive any exposure to insecticides following establishment in 2005 and routine quantitative real time (qPCR) testing as described in Pelz Stelinski et al. (2010) was used to confirm the colony was C Las free Adult D. citri were aspirated from host rearing plants and used in laboratory bioassays during the same day to reduce unintended mortality. Field Collection of D. citri Diaphorina citri adults were collected prior to the first field application to establish baseline susceptibility of the field population to thiamethoxam in the lab. Adults were aspirated from citrus foliage in the field and transported to the lab within labeled plastic aspirator vials. Laboratory assays we re conducted during the same day that D citri were collected from the field to reduce unintended mortality. Ingestion and Contact Bioassays. The ingestion and contact assays have been comprehensively described in Langdon and Rogers (2017). Briefly, for t he ingestion assay, a

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66 30% sucrose solution was used as the base artificial diet. Serial dilution was conducted to form eight doses of spiked diet using formulated Platinum 75SG (750 g thiamethoxam kg, Syngenta Crop Protection, Greensboro, NC). The caps of 5 m L snap cap centrifuge tubes (Eppendorf Tubes, Hamburg, Germany, Cat. No.: 0030119401) w ere filled with 0.7 m L of each prepared dose Parafilm M (Bemis, Neenah, WI, Cat. No.: PM 992) was stretched over each diet filled cap. Four to six adult D. citri were loaded into each centrifuge tube and the diet filled cap was reinstalled Tubes were held upright in a tube tray at 27C, 70% relative humidity, with a 14:10 L:D photoperiod for 72h. One replicate was defined as a single tube and 10 replicates were used for each dose. Between 40 and 60 adults were tested for each dose. After 72h, i nsects were scored as alive (full function), moribund (insects lacking coordinated movement), or dead (no movement upon disturbing). Moribund in sects were classified as dead for data analysis. A serial dilution using a nalytical grade thiamethoxam (> 99.5% purity) (Chem Service, Inc, West Chester, PA) and acetone (Fisher Scientific, Fair Lawn, NJ, Cat. No.: A929 4) was used to prepare eight doses for the contact assay. A 1.5 m L aliquot of each dose, including the acetone control, was pipetted into individually labeled 16 m L g lass vials (Wheaton, Millville, NJ, Cat. No.: 224746) and vials were rolled on an electric hot dog roller until all acetone e vaporated (approx. 1 2hr) Treated vials were stored at room temperature conditions in a closed cardboard container overnight Eight to twelve adult D. citri were aspirated into each vial and a cap was installed. Tubes were held horizontally at 27C, 70% r elative humidity, with a photoperiod 14:10 L:D for 24h. Each replicate consisted of one vial and five replicates were used for each dose Between 40 and 60 adults were tested for each dose After 24h, insects were scored as alive (full function), moribund (insects lacking coordinated movement), or dead (no movement upon disturbing). Moribund insects were classified as dead for data analysis.

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67 Statistical Analyses Chemical titer data were averaged over replicate and subjected to a general linear mixed model using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for year by treatment interactions. Means were square root transformed prior to analysis to achieve homogeneity of variance meet ing the assumptions of the model, as checked by visual examination of the residuals to ensure constant variance and normality Additionally, chemical titer data were averaged over replicate and year and subjected to a general linear mixed model using SASv9.4 (Proc GLIMMIX SAS Institute, 20 13 ) to test for tree size by appli cation rate interactions. Means were square root transformed prior to analysis to achieve homogeneity of variance and meet the assumptions of the model For tests of differences between treatments, data were subjected to a non parametric multiple compariso ns test where mean separations indicate differences between treatments within the same sample week Insect count data were in monotonic distribution ; therefore data were subjected to Spearmans rank order correlation using JMP (JMP Version 13, SAS Institute, 2007 ) to determine if concentration of thiamethoxam, or the metabolites clothianidin, TZNG ( N (2 chlorothiazol 5 ylmethyl) N nitroguanidine ) or TZMU ( N (2 chlorothiazol 5 ylmethyl) N methylurea) influen ced D. citri incidence on lea ves C orrelations were estimated using the Restricted Maximum Likelihood (REML) method. Additionally, nymph and adult incidence data were subjected to Probit analysis using SAS v9.4 (Proc Probit, SAS Institute, 2013) to determine the probability of encountering a flush terminal containing at least one nymph or one adult based on thiamethoxam titer.

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68 Results Chemical Titer in Leaf Tissue No year by treatment interaction was observed for thiamethoxam (F 1, 5 = 0.9982; p value = 0.4328), clothianidin (F 1, 5 = 1.4132; p value = 0.2429), or TZMU (F 1, 5 = 2.1454; p value = 0.0822) ; however a year by treatment interaction was observed for TZNG (F 1, 5 = 3.5969; p value = 0.0097). For TZNG, a larger magnitude of difference was observed in 2016 compared with 2017 ; however, the order of the treatment effects was the same across years. T herefore data w ere combined across years for each of the four chemicals to evaluate the effect of treatment on chemical titer. A tree size by application rate interaction was observ ed for thiamethoxam (F 1, 27 = 15.11; p value = 0.0006), clothianidin (F 1, 27 = 11.66; p value = 0.0020), and TZMU (F 1, 27 = 43.60; p value < 0.0001) ; however, no tree size by application rate interaction was observed for TZNG (F 1, 27 = 0.8936; p value = 0. 3529). Main effects of tree size and application rate were therefore analyzed separately. Tree size influenced thiamethoxam titer (F 1, 27 = 180.5; p value < 0.0001), where higher thiamethoxam titers were expressed in small trees compared to large trees. Tr ee size also influenced titer of clothianidin (F 1, 27 = 187.2; p value < 0.0001), TZMU (F 1, 27 = 233.6; p value < 0.0001), and TZNG (F 1, 27 = 263.3; p value < 0.0001). Additionally, rate of application affected thiamethoxam titer (F 1, 27 = 44.24; p value < 0.0001), where application of the high rate resulted in significantly more thiamethoxam in leaf tissue than at the low rate. Application rate also affected clothianidin (F 1, 27 = 39.35; p value < 0.0001), TZMU (F 1, 27 = 58.02; p value < 0.0001), and TZNG (F 1, 27 = 27.03; p value < 0.0001) by increasing measured titer with increased application rate The high rate (0.74g Platinum 75SG per tree) applied to the small tree size resulted in the highest thiamethoxam titer observed during each week following app lication; the peak mean

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69 concentration occurred two and three weeks following application at 33.39 and 33.29 ppm thiamethoxam, respectively ( Table 3 1 ). M ean thiamethoxam titer peaked at 12.53 ppm by three weeks after application following the low rate application (0.37g ) of Platinum 75SG per small tree; however no significant difference in titer was observed between rates in small tree s three weeks following application (X 2 = 36.53, p < 0.0001 Table 3 1 ). Peak thiamethoxam titer was observed at two (2.86 ppm) and four (2.47 ppm) weeks after the high rate (0.74g Platinum 75SG per tree) application to large tree s and at two weeks (0.69 ppm) following the low rate (0.37g Platinum 75SG per tree) applicatio n to large tree s At two and four weeks after application, significantly more thiamethoxam was found after the high rate rather than the low rate application to large tree s (X 2 = 40.27, p < 0.0001 and X 2 = 43.91, p < 0.0001, respectively Table 3 1 ). The thiamethoxam metabolite, clothianidin, peaked at five weeks post application of both the high rate (15.44 ppm) and the low rate (6.29 ppm) of Platinum 75SG in trees of the small size ( Tab le 3 2 ). Following application to the large size trees, clothianidin titer peaked at four weeks with the high rate (1.69 ppm) and the low rate (0.48 ppm). Very low levels (<2 ppm) of the clothianidin metabolites, TZMU ( Table 3 3 ) and TZNG ( Table 3 4 ) were detected in leaf tissues from trees following the application of Platinum 75SG to the soil. Baseline Susceptibility of Field D. citri P opulation The lethal concentration required to kill half of the f ield collected (Vero Beach) population (LC 50 ) by ingestion was 0.20 ppm of thiamethoxam, while the LC 50 was 0.11 ppm for the lab susceptible population ( Table 3 5 ). A comparison of these results indicates that the field populatio n investigated during this experiment was fully susceptible to thiamethoxam, exhibiting a resistance ratio (RR 50 ) of 1.82. Furthermore, the lethal concentration required to kill 99 percent of the field collected population by ingestion (LC 99 ) was 147.91 pp m thiamethoxam. In contrast,

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70 the LC 50 of the field population when exposed to thiamethoxam through contact was determined at 0.01 ppm, which was identical to that of the laboratory susceptible population. Moreover, the LC 99 by contact was 0.33 ppm, approximately 450 fold less than by ingestion. Relationship B etween D. citri I ncidence and C hemical T iter A dult incidence was negatively correlated with increasing thiamethoxam (Spearmans = 0.6440; T able 3 6 ), clothianidin (Spearmans = 0.6320; Table 3 6 ), TZMU (Spearmans = 0.5429; Table 3 6 ), and TZNG (Spearmans = 0.6117; Table 3 6 ) titer Likewise, n ymph inciden ce was also negatively correlated with increasing thiamethoxam (Spearmans = 0.7010; Table 3 6 ), clothianidin (Spearmans = 0.6913; Table 3 6 ), TZMU (Spearmans = 0.6051; Table 3 6 ), and TZNG (Spearmans = 0.6655; Table 3 6 ) titer. An estimated 64.63 ppm (95% CL: 34.40 147.16) of thiamethoxam was required to achieve a one percent probability of encountering a flush ter minal with one D. citri adult ( Fig. 3 1A ). Additionally, a thiamethoxam titer of 0.329 ppm (329 ppb) yielded a 50 percent probability of encountering a flush terminal with one D. citri adult ( Table 3 7 ). In contrast, an estimated 19.05 ppm of thiamethoxam was required to achieve a one percent probability of encountering a flush terminal with one D. citri nymph ( Fig. 3 1B ); 0.715 ppm of thiamethoxam (715 ppb) yielded a 50 perc ent probability of encountering a flush terminal with one D. citri nymph ( Table 3 8 ). A pproximately five weeks of nymph and adult control was observed in 2016 and 2017, respectively ( Figs. 3 2A and 3 2B respectively) following application of the low rate of Platinum 75SG to the soil beneath small trees i s defined 100 percent compared to the untreated check. In contrast, ten and n ine weeks of nymph and eight and seven weeks of adult control was observed in 2016 ( Fig. 3 2C ) and 2017 ( Fig. 3 2D ), respectively, following the high rate application to trees of the small size The low rate applied to the large tree size did not offer complete control of nymphs or adults at any time during 2016 or 2017 ( Figs. 3 3A and 3

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71 3B respectively). During 2016, complete adult control was observed only during wee ks one through three following the high rate application to trees of the large size ( Fig. 3 3C ), but the same level of adult control was not observed during 2017 ( Fig. 3 3D ); n ymph control reached 100 percent only at two weeks after application in 2016 ( Fig. 3 3C ) Discussion The goals of this study were to quantify uptake and expression of thiamethoxam in young citrus trees of two sizes following the application of Platin um 75SG to the soil at two rates, as well as, to evaluate residual efficacy against D. citri nymphs and adults, and quantify the inherent susceptibility of the field population of D. citri to thiamethoxam to compare lethal concentration values with perceiv ed control. This was the first formal investigation to use UHPLC MS for quantification of the four analytes of Platinum 75SG (thiamethoxam, clothianidin, TZNG, and TZMU) in citrus leaf tissues following soil application in the field. Thiamethoxam is a neon icotinoid precursor to clothianidin (Nauen et al. 2003) and clothianidin is known to metabolize into TZNG and TZMU (Kim et al. 2012). While c lothianidin is effective against D. citri little is known about the influence of specific dosage on D. citri feeding behavior or mortality (Byrne et al. 2017). Langdon and Rogers (2017) found that the LC 50 of thiamethoxam and clothianidin by ingestion was 0.11 and 0.09 ppm, respectively, while the LC 90 was 4.94 and 9.35 ppm, respectively. Because a higher dose o f clothianidin was required to achieve the same level of mortality at the 90 percent lethal concentration, it is unlikely that mortality observed in the current study is the result of exposure to the metabolite, clothianidin alone. Furthermore, t he concent ration of each of the three metabolites is directly dependent upon the concentration of thiamethoxam. Consequently, we cannot determine how any one metabolite, including clothianidin a ffects D. citri mortality in this study While understanding the role o f each metabolite is beyond the scope of this investigation, it is possible that the combination of

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72 multiple metabolites has an additive effect on D. citri mortality as suggested by Byrne et al. (2017). Byrne et al. (2017) demonstrated a strong correlation between thiamethoxam and the metabolite, clothianidin, but were unable to determine whether mortality was the result of either thiamethoxam or clothianidin, or a combination of the two. Nevertheless, earlier studies used ELISA to quantify expression of ne onicotinoids, but did not quantify metabolites of thiamethoxam nor manipulate tree size or application rate to study effects on thiamethoxam titer ( Castle et al 2005, Garlapati 2009, Setamou et al. 2010 ). The target concentration threshold of imidaclopri d following application to the soil was 200 to 250 ppb based on the report by Setamou et al. (2010). Since a correlation between D. citri abundance and clothianidin or thiamethoxam titer did not exist, 200 to 250 ppb became the assumed efficacy threshold c oncentration for all neonicotinoids (K. W. Langdon, personal observation). In the current investigation, peak levels of nearly 3 ppm (3000 ppb) and 0.7 ppm (700 ppb) of thiamethoxam were measured when the respective high and low rates tested were applied t o trees of the large size. When the high and low rates were applied to trees of the small size, peak concentrations were nearly 18 and 11 fold, respectively, higher than when applied to the large tree size. Each titer was well above the 250 ppb upper thres hold set for imidacloprid, suggesting that efficacy should be expected in both tree sizes at the rates tested. However, despite the high titers observed in our study, we failed to reach a mean dose high enough to provide a 95 percent confidence (34.40 ppm) of only a one percent probability of encountering a flush shoot with at least one adult D. citri We did, however, observe a mean concentration high enough in only the small tree size at both rates, to achieve a 95 percent confidence (12.17 ppm) of only a one percent probability of encountering a flush shoot with at least one nymph, albeit

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73 that titer was reached only during the third week of the investigation in the low rate treatment; the high rate treatment exceeded 12.17 ppm between weeks one and six. The LC 99 for the field population was 147.91 ppm. This was 4.5 fold higher than the highest mean dose observed when the high rate was applied to trees of the small size. Given that nearly 100 percent control of adults was observed for up to eight weeks fol lowing application and that the highest observed concentration was 4.5 times lower than the lowest lethal dose (LC 99 ), a dose of less than 34.40 ppm remains likely to deter D. citri from feeding and therefore, may offer non lethal control of D. citri in yo ung citrus trees. Langdon and Rogers (2017) defined insecticide mediated feeding deterrence of D. citri exposed to citrus tissue containing less than 34.40 ppm of thiamethoxam adults may move to find a leaf surface containing a lower concentration within the same plant or move to a new host plant that contains a concentration sufficiently low to be suitable for feeding. This movement away from the treated foliage or tree may result in a perception of control upon visual assessment, but should not be confused with mortality. While control may be perceived at titers less than 34.40 ppm, any c oncentration below 147 .91 ppm should be assumed as sublethal and therefore exposure to a dose below 147.91 ppm is likely to result in exposed survivors, which may promote the development of resistance in populations of D. citri to thiamethoxam and likely o ther chemistries within the same mode of action. Thiamethoxam is known to metabolize into clothianidin, which acts on the same receptor site as imidacloprid (Nauen et al. 2003), supporting a high likelihood of cross resistance. Im idacloprid resistant D. ci tri express higher levels of detoxifying enzymes, including general esterase, glutathione S transferase, and cytochrome P 450 monooxygenases (Tiwari et al. 2011a) R educed penetration, target site insensitivity, and mutations in detoxifying

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74 enzymes may also play a role in resistance (Tiwari et al. 2011a) Tiwari et al. (2011b) found five family 4 cytochrome P 450 genes induced by imidacloprid exposure. Imidacloprid and thiamethoxam are within the same chemical sub group (4A); therefore cross resistance betwe en the two chemistries is of concern. Additionally, thiamethoxam persisted at very low levels (ca. 0.05 0.80 ppm) during the final evaluation of this investigation (12 weeks following application). The duration at which sublethal doses in this range will persist is unknown; if doses in this range do not inhibit feeding activity it may further increase the likelihood and rate of resistance development. Diaphorina citri resistance to neonicotinoids has been recently documented in Florida ( Tiwari et al. 201 1 a, Kanga et al. 2016); therefore, a deeper understanding of soil applied neonicotinoids wa s warranted for develop ment of future resistance management strategies. We observed a number of effects that are of significant concern regarding use of neonicotinoi ds by soil application in Florida citrus: 1) failure to achieve lethal concentrations (those that exceed LC 99 ) in leaf tissue following application to the soil; 2) persistence of thiamethoxam concentrations at low levels (less than 1 ppm) through 12 weeks following application; 3) failure of the highest allowable annual rate to achieve acceptable D. citri control following application to trees 18 months of age (MCV = 1.34m 3 ); 4) expression level relative to dose applied (e.g. high rate of 0.74g Platinum 75S G in 237 m L water per tree is equivalent to 2370 ppm thiamethoxam applied to the soil, and low rate of 0.37g Platinum 75SG in 237 m L water per tree is equivalent to 1185 ppm thiamethoxam applied to the soil); and 5) higher sensitivity of D. citri to thiamethoxam through contact exposure compared to ingestion The foremost strategy for stewardship and future implementation of neonicotinoids in citrus must be resistance management. Therefore, the current results suggest that foliar use of

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75 neonicoti noids may be more appropriate than their applications to the soil, particularly in trees with a canopy larger than 0.08m 3 to mitigate resistance development and preserve efficacy of this mode of action Because contact sensitivity in our field population w as approximately 450 fold greater than by ingestion at the LC 99 level, labeled rates of thiamethoxam applied to the foliage would be sufficient (foliar applied dose of 68.65 ppm at 1400 L/ha; Contact LC 99 = 0.33 ppm; Table 3 5 ) t o effectively kill D. citri Subsequent investigations should evaluate factors including coverage uniformity, peak residue levels, persistence / degredation, and resulting efficacy following foliar applications of neonicotinoid insecticides. The significan t reduction in titer between small trees and large trees after soil application may simply be a result of dilution of chemical due to an increase in canopy size, application method in relation to root distribution under the canopy, or perhaps due to compro mise of the root system caused by C Las infection resulting in reduced uptake of available compound. If the latter is true, soil applied neonicotinoids may work better when applied to trees not compromised by HLB. Because the trees used in this study were i nsecticide free prior to each application, it is likely that all trees, particularly of the large size, had some level of C Las infection, which may have negatively influenced uptake efficiency. Follow up comparative investigations quantifying the concent ration of thiamethoxam imidacloprid, and clothianidin required to interrupt and manipulate feeding behavior of D. citri that utilize electropenetrography are warranted to further improve use of these management tools for D. citri and HLB in citrus. Moreover, alternative soil application methods should be investigated that attempt to increase uptake efficiency, particularly in trees 18 months and older. Nonetheless, future work on foliar application of neonicotinoids should investi gate temporal residue and breakdown/ metabolism as related to the probability of sublethal exposure of D. citri

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76 Given the dynamic nature of susceptibility of D. citri to insecticides, we must remain diligent in research efforts with a keen focus on resista nce management and be willing to adjust insecticide use patterns to ensure the longevity of each available chemical class.

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77 Figure 3 1. Predicted probability for incidence of insects based on thiamethoxam titer (ppm) in citrus leaf tissue A. Incidence of D iaphorina citri adults B. Incidence of D iaphorina citri nymphs Predicted probability for incidence of D. citri adults A Predicted probability for incidence of D. citri nymphs B Thiamethoxam expression (ppm) in leaf tissue (ppm)

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78 Figure 3 2. Comparison of thiamethoxam titer (ppm) and percentage insect control in trees of the large size during 2016 and 2017 field seasons. A. Low rate applied to small (0.08m 3 ) trees in 2016. B. Low rate applied to small (0.08m 3 ) trees in 2017. C. High rate applied to small (0.08m 3 ) trees in 2016. D. High rate applied to small (0.08m 3 ) trees in 2017 0 20 40 60 80 100 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 10 11 12 Thiamethoxam titer (ppm) in citrus leaf tissue Small tree 0.37g Platinum 75SG per tree 2016 0 20 40 60 80 100 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 10 11 12 Percentage control of adults or nymphs compared to untreated check Small tree 0.74g Platinum 75SG per tree 2016 0 20 40 60 80 100 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 10 11 12 Thiamethoxam titer (ppm) in citrus leaf tissue Weeks following application Small tree 0.37g Platinum 75SG per tree 2017 0 20 40 60 80 100 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 10 11 12 Percentage control of adults or nymphs compared to untreated check Weeks following application Small tree 0.74g Platinum 75SG per tree 2017 nymphs adults A B C D

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79 Figure 3 3. Comparison of thiamethoxam titer (ppm) and percentage insect control in trees of the large size during 2016 and 2017 field seasons. A. Low rate applied to large (1.34m 3 ) trees in 2016. B. Low rate applied to large (1.34m 3 ) trees in 2017 C. High rate applied to larg e (1.34m 3 ) trees in 2016. D. High rate applied to large (1.34m 3 ) trees in 2017 0 20 40 60 80 100 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 11 12 Thiamethoxam titer (ppm) in citrus leaf tissue Large tree 0.37g Platinum 75SG per tree 2016 0 20 40 60 80 100 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 11 12 Percentage control of adults or nymphs compared to untreated check Large tree 0.74g Platinum 75SG per tree 2016 0 20 40 60 80 100 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 11 12 Thiamethoxam titer (ppm) in citrus leaf tissue Large tree 0.37g Platinum 75SG per tree 2017 0 20 40 60 80 100 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 11 12 Percentage control of adults or nymphs compared to untreated check Weeks following application Large tree 0.74g Platinum 75SG per tree 2017 nymphs adults A B C D

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80 Table 3 1. Mean parts per million (ppm) of thiamethoxam (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments. Tree Size Application rate per tree Weeks after application 0 1 2 3 4 5 6 7 8 9 10 11 12 Small 0.08m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 7.53bc (3.46 11.61) 6.92b (3.20 10.63) 12.53b (8.27 16.79) 7.51c (3.56 11.45) 7.03b (4.29 9.77) 3.27b (2.14 4.39) 2.39c (1.03 3.76) 1.42b (0.95 1.88) 1.14b (0.72 1.56) 0.56c (0.30 0.82) 0.37b (0.24 0.49) 0.30bc (0.13 0.46) 0.74g Platinum 75SG 0 13.58c (6.07 21.09) 33.39c (16.54 50.23) 33.29b (17.20 49.39) 26.90d (15.37 38.44) 28.10c (17.01 39.19) 16.29c (9.08 23.51) 11.03d (6.18 15.87) 6.09c (2.96 9.23) 3.96c (2.09 5.83) 2.90d (1.48 4.33) 1.52c (0.48 2.57) 0.80c (0.43 1.15) Large 1.34m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0.61ab (0.00 3.14) 0.69a (0.12 1.27) 0.53a (0.00 2.03) 0.39a (0.18 0.60) 0.17a (0.00 2.03) 0.17a (0.00 0.89) 0.16ab (0.04 0.27) 0.11a (0.00 0.58) 0.09a (0.00 0.31) 0.03ab (0.00 0.06) 0.03a (0.00 0.11) 0.05ab (0.00 0.10) 0.74g Platinum 75SG 0 1.59abc (0.00 4.58) 2.86b (1.00 4.72) 1.90a (0.63 3.17) 2.47bc (0.00 5.70) 0.67a (0.00 2.44) 0.70a (0.01 1.39) 0.39b (0.20 0.59) 0.30a (0.00 0.80) 0.28ab (0.03 0.53) 0.19bc (0.08 0.30) 0.14ab (0.04 0.25) 0.08ab (0.03 0.14) p value < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 X 2 28.95 40.27 36.53 43.91 35.18 35.65 40.89 35.23 34.89 41.27 35.08 32.74 a. Mean volume of citrus tree canopy. Mean separations within columns indicate differences between treatments within weekly samples.

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81 Table 3 2. Mean parts per million (ppm) of clothianidin (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments. Tree Size Application rate per tree Weeks after application 0 1 2 3 4 5 6 7 8 9 10 11 12 Small 0.08m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 1.99b (0.77 3.21) 2.45cd (0.81 4.09) 5.82c (4.07 7.56) 4.50cd (2.85 6.15) 6.29b (5.07 7.52) 4.33b (2.45 6.21) 2.65b (1.57 3.73) 1.84b (1.39 2.29) 1.46b (0.94 1.98) 0.85b (0.52 1.17) 0.55b (0.43 0.66) 0.40b (0.22 0.58) 0.74g Platinum 75SG 0 3.27b (1.46 5.08) 8.62d (4.27 12.96) 12.12c (6.77 17.47) 13.09d (8.34 17.85) 15.44b (7.32 23.57) 14.02c (7.95 20.09) 11.53c (7.67 15.39) 7.25c (3.63 10.88) 5.01c (3.00 7.02) 5.02c (3.04 6.99) 2.21c (1.05 3.37) 1.45c (0.84 2.05) Large 1.34m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0.26ab (0.00 0.97) 0.39b (0.00 0.82) 0.38b (0.17 0.58) 0.48b (0.10 0.87) 0.21a (0.00 0.78) 0.19a (0.00 0.81) 0.18a (0.00 0.66) 0.14a (0.00 0.81) 0.09a (0.00 0.56) 0.04a (0.00 0.38) 0.03a (0.00 0.22) 0.05a (0.00 0.14) 0.74g Platinum 75SG 0 0.68ab (0.00 1.42) 1.15bc (0.45 1.85) 1.06b (0.38 1.73) 1.69bc (0.00 3.75) 0.86a (0.37 1.36) 0.74a (0.26 1.23) 0.40a (0.00 0.90) 0.33a (0.00 0.98) 0.30ab (0.00 0.77) 0.29ab (0.00 0.60) 0.17a (0.00 0.36) 0.09a (0.00 0.18) p value < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 X 2 29.17 40.86 42.45 43.16 32.66 36.60 35.43 35.16 32.25 32.20 34.93 35.47 a. Mean volume of citrus tree canopy. Mean separations within columns indicate differences between treatments within weekly samples.

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82 Table 3 3. Mean parts per million (ppm) of TZMU (95% CI) found in citrus leaf tissue during 2016 and 20 17 field experiments. Tree Size Application rate per tree Weeks after application 0 1 2 3 4 5 6 7 8 9 10 11 12 Small 0.08m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0.04a (0.02 0.06) 0.05a (0.01 0.08) 0.11b (0.04 0.17) 0.09b (0.05 0.13) 0.05a (0.00 0.09) 0.07ab (0.02 0.12) 0.01a (0.00 0.03) 0.01a (0.00 0.03) 0.01ab (0.00 0.02) 0a 0a 0a 0.74g Platinum 75SG 0 0.08a (0.02 0.15) 0.44b (0.22 0.65) 0.38c (0.20 0.57) 0.52c (0.32 0.72) 0.40b (0.25 0.54) 0.38b (0.19 0.57) 0.23b (0.10 0.36) 0.15b (0.09 0.22) 0.10b (0.05 0.15) 0.06a (0.00 0.12) 0.02a (0.00 0.04) 0.01a (0.00 0.02) Large 1.34m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0.74g Platinum 75SG 0 0a 0a 0a 0.01ab (0.00 0.04) 0.01a (0.00 0.01) 0a 0a 0a 0a 0a 0a 0a p value 0.0008 < 0.0001 < 0.0001 < 0.0001 0.0005 < 0.0001 0.0014 < 0.0001 0.0030 0.0911 0.0174 0.6178 X 2 21.13 27.40 33.82 39.89 22.11 26.59 19.80 25.37 17.93 9.49 13.73 3.54 a. Mean volume of citrus tree canopy. Mean separations within columns indicate differences between treatments within weekly samples.

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83 Table 3 4. Mean parts per million (ppm) of TZNG (95% CI) found in citrus leaf tissue during 2016 and 2017 field experiments. Tree Size Application rate per tree Weeks after application 0 1 2 3 4 5 6 7 8 9 10 11 12 Small 0.08m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0.17ab (0.07 0.27) 0.25bc (0.13 0.37) 0.68c (0.50 0.87) 0.66c (0.51 0.81) 0.65b (0.46 0.83) 0.87c (0.53 1.21) 0.69c (0.57 0.80) 0.59b (0.45 0.74) 0.57b (0.42 0.72) 0.39b (0.32 0.47) 0.33b (0.24 0.41) 0.22b (0.15 0.29) 0.74g Platinum 75SG 0 0.24b (0.12 0.36) 0.41c (0.28 0.55) 0.77c (0.60 0.95) 0.70c (0.51 0.89) 1.39c (0.92 1.86) 1.36c (0.87 1.86) 1.27c (0.78 1.75) 1.19b (0.66 1.73) 0.81b (0.40 1.23) 0.89c (0.68 1.10) 0.66b (0.41 0.90) 0.52c (0.42 0.63) Large 1.34m 3 MCV a Untreated 0 0 0 0 0 0 0 0 0 0 0 0 0 0.37g Platinum 75SG 0 0.02ab (0.00 0.08) 0.08ab (0.00 0.18) 0.12b (0.02 0.23) 0.15ab (0.00 0.30) 0.04a (0.00 0.13) 0.09b (0.05 0.13) 0.06ab (0.01 0.11) 0.04a (0.00 0.12) 0.02a (0.00 0.10) 0.01a (0.00 0.05) 0.01a (0.00 0.04) 0.01a (0.00 0.03) 0.74g Platinum 75SG 0 0.06ab (0.00 0.13) 0.12ab (0.01 0.22) 0.20b (0.06 0.35) 0.33bc (0.10 0.56) 0.16a (0.05 0.28) 0.19b (0.08 0.30) 0.13b (0.07 0.19) 0.12a (0.04 0.20) 0.11a (0.03 0.19) 0.09a (0.04 0.14) 0.04a (0.00 0.10) 0.03a (0.00 0.06) p value 0.0004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 X 2 22.64 30.73 41.08 37.89 38.01 40.59 40.03 34.70 35.01 37.49 34.14 34.95 a. Mean volume of citrus tree canopy. Mean separations within columns indicate differences between treatments within weekly samples.

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84 Table 3 5. Response of laboratory susceptible and field collected D iaphorina citri to thiamethoxam (ppm) administered by ingestion and contact. Strain Assay Method N a Slope + SE X 2 LC 50 b 95% CL LC 90 b 95% CL LC 99 b 95% CL RR 50 c Vero Beach Ingestion 356 0.35 + 0.04 84.53 0.20 (0.10 0.34) 7.62 (4.10 17.73) 147.91 (52.34 693.09) 1.82 Contact 351 0.69 + 0.11 41.78 0.01 (0.01 0.02) 0.07 (0.05 0.14) 0.33 (0.16 1.13) 1.00 Lab Susceptible Ingestion 404 0.34 + 0.04 73.58 0.11 (0.05 0.21) 4.94 (2.63 11.75) 106.45 (36.17 555.23) Contact 405 0.75 + 0.12 38.69 0.01 (0.01 0.02) 0.05 (0.04 0.11) 0.23 (0.12 0.78) a. Number of adult D iaphorina citri tested. b. Parts per million (ppm) of active ingredient. c. Ratio of Vero Beach LC 50 divided by Lab Susceptible LC 50

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85 Table 3 6. Nonparametric Spearman correlation between mean number of adult or nymph D iaphorina citri per terminal an d chemical titer (ppm) during 2016 and 2017 field seasons. Variable by Analyte Spearman p value Mean number of D. citri adults per terminal (n = 10 terminals) thiamethoxam 0.6440 <0.0001 clothianidin 0.6320 <0.0001 TZMU 0.5429 <0.0001 TZNG 0.6117 <0.0001 Mean number of D. citri nymphs per terminal (n = 10 terminals) thiamethoxam 0.7010 <0.0001 clothianidin 0.6913 <0.0001 TZMU 0.6051 <0.0001 TZNG 0.6655 <0.0001

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86 Table 3 7 Probability of encountering a Diaphorina citri adult on young citrus trees based on thiamethoxam titer (ppm) in leaf tissue. Probability Concentration (ppm) 95% Fiducial Limits Probability Concentration (ppm) 95% Fiducial Limits 0.01 64.62813 34.40416 147.16423 0.55 0.24763 0.18915 0.31486 0.02 34.81426 19.88874 72.07353 0.60 0.18534 0.13803 0.23892 0.03 23.51246 14.03654 45.85806 0.65 0.13737 0.09920 0.18047 0.04 17.50123 10.79389 32.65360 0.70 0.10019 0.06976 0.13481 0.05 13.76460 8.71368 24.78236 0.75 0.07127 0.04754 0.09874 0.06 11.21976 7.25962 19.60355 0.80 0.04877 0.03093 0.07002 0.07 9.37867 6.18388 15.96622 0.85 0.03135 0.01868 0.04705 0.08 7.98808 5.35517 13.28972 0.90 0.01797 0.00988 0.02861 0.09 6.90326 4.69711 11.25013 0.91 0.01571 0.00846 0.02538 0.10 6.03539 4.16204 9.65293 0.92 0.01358 0.00716 0.02229 0.15 3.46039 2.51446 5.13735 0.93 0.01157 0.00595 0.01932 0.20 2.22395 1.67583 3.12827 0.94 0.00967 0.00484 0.01648 0.25 1.52197 1.17661 2.05542 0.95 0.00788 0.00382 0.01374 0.30 1.08264 0.85119 1.41831 0.96 0.00620 0.00290 0.01111 0.35 0.78960 0.62631 1.01252 0.97 0.00461 0.00206 0.00855 0.40 0.58525 0.46475 0.74073 0.98 0.00312 0.00131 0.00604 0.45 0.43802 0.34567 0.55145 0.99 0.00168 0.0006408 0.00350 0.50 0.32934 0.25652 0.41537

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87 Table 3 8. Probability of encountering a Diaphorina citri nymph on young citrus trees based on thiamethoxam titer (ppm) in leaf tissue. Probability Concentration (ppm) 95% Fiducial Limits Probability Concentration (ppm) 95% Fiducial Limits 0.01 19.05254 12.16790 34.07621 0.55 0.59899 0.49336 0.72459 0.02 12.96913 8.64334 21.89294 0.60 0.50024 0.40916 0.60505 0.03 10.16081 6.95350 16.54363 0.65 0.41525 0.33608 0.50386 0.04 8.45671 5.90171 13.40459 0.70 0.34126 0.27223 0.41687 0.05 7.28366 5.16330 11.29901 0.75 0.27613 0.21615 0.34088 0.06 6.41433 4.60705 9.77162 0.80 0.21812 0.16664 0.27335 0.07 5.73789 4.16809 8.60486 0.85 0.16570 0.12263 0.21204 0.08 5.19299 3.81004 7.68013 0.90 0.11725 0.08306 0.15463 0.09 4.74248 3.51067 6.92674 0.91 0.10786 0.07556 0.14335 0.10 4.36242 3.25549 6.29960 0.92 0.09850 0.06817 0.13204 0.15 3.08692 2.37788 4.25992 0.93 0.08914 0.06086 0.12066 0.20 2.34503 1.84788 3.12933 0.94 0.07974 0.05361 0.10913 0.25 1.85240 1.48480 2.40760 0.95 0.07023 0.04638 0.09735 0.30 1.49887 1.21695 1.90723 0.96 0.06048 0.03911 0.08514 0.35 1.23180 1.00945 1.54090 0.97 0.05034 0.03170 0.07224 0.40 1.02251 0.84303 1.26208 0.98 0.03944 0.02396 0.05809 0.45 0.85394 0.70607 1.04352 0.99 0.02685 0.01540 0.04125 0.50 0.71519 0.59118 0.86814

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88 CHAPTER 4 NEONICOTINOID INDUCED MORTALITY OF DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) IS AFFECTED BY ROUTE OF EXPOSURE The use of neonicotinoids in citrus has increased substantially to help manage the Asian citrus psyllid, Diaphorina citri Kuwayama, a vector of the devastating citrus disease, huanglongbing (HLB). In citrus pest management programs, n eonicotinoid s are most often applied to the soil as a drench and move through xylem channels from the roots into the foliage. We developed a novel assay to quantify the d ose required to kill D. citri following ingestion and compare it with the dose required to kill by contact. The LC 50 of the laboratory strain for ingestion of imidacloprid, thiamethoxam, and clothianidin were each approximately 10 fold greater than the res pective LC 50 by contact exposure. Four field populations were tested to validate comparative exposure of the laboratory strain to imidacloprid and determine the relative susceptibility of field populations to imidacloprid by exposure through ingestion and contact. The contact assay exhibited low (<10) RR 50 values for the Vero Beach and Labelle populations when compared to the ingestion assay method. High (>10) RR 50 values were observed for the Lake Placid and Lake Alfred populations using the contact and th e ingestion method. This research demonstrates that the ingestion assay method described herein is more sensitive in detection of low level resistance and should be the standard meth odology used in monitoring for lower than expected susceptibility to neoni cotinoids in the field, which warrants the implementation of resistance management practices to preserve the utility of soil applied neonicotinoids in citrus. Used with permission from: Langdon, K. W., M. E. Rogers. 2017. Neonicotinoid induced mortality of Diaphorina citri (Hemiptera: Liviidae) is affected by route of exposure. J. Econ. Entomol 110: 2229 2234.

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89 Justification The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae ), is a major pest of citrus (Rutaceae) throughout the world negatively impacting productivity and yield (Halbert and Manjunath 2004, Bov 2006, Gottwald 2007, Ichinose et al. 2010a, 2010b; Grafton Cardwell et al. 2013). Diaphorina citri serves as the vector of the bacterium, Candidatus Liberibacter asiaticus ( C Las ), the presumed causal agent of huanglongbing (HLB), or citrus greening disease. Candidatus Liberibacter asiaticus is a phloem limited bacteri um that negatively impacts the root system leading to a decline in the tree canopy, including twig dieback, mot tled leaves, misshapen fruit, decreased fruit quality, increased fruit drop, and subsequent death of infected trees (Halbert and Manjunath 2004, Bov 2006, Grafton Cardwell et al. 2013). Diaphorina citri was first discovered in Florida in 1998 (Halbert and Manjunath 2004), followed by HLB in 2005 (Halbert 2005). HLB was recently discovered in California ( Kumagai et al. 2013). The Florida citrus industry was valued at nearly 9.9 billion dollars during 2014 and 2015 (Hodges and Spreen 2015) and is greatly thr eatened by the spread of HLB Since HLB was discovered in Florida in 2005, the use of insecticides, particularly neonicotinoids, has increased substantially and play s a vital role in the management of the insect vector, and thus HLB (Rogers 2008). Followi ng the discovery of C Las in Florida, investigations of a wide array of management strategies to reduce the spread of HLB in Florida citrus was initiated The use of biological control agents such as Tamarixia radiata Water ston (Hymenoptera: Eulophidae), nu rsery sanitation, rogueing of infected trees in the field, and scouting based sprays were each suggested as methods for management of HLB (Stansly and Rogers 2006 Hall and Albrigo 2007, Hall et al. 2008). Given the severity and potential impact of the dis ease, vector control

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90 through use of insecticides remained the fundamental tool for slowing the spread of HLB in Florida citrus (Rogers 2008, Boina et al. 2009, Qureshi and Stansly 2009). Largely due to the increased frequency of insecticide applications i n citrus following the onset of HLB it was recognized that growers could not rely solely on foliar applied insecticides to protect young trees (Rogers 2012). As growers removed infected trees for replanting, protection of young trees from HLB for the firs t three to five years of growth to bearing age became highly important (Rogers 2012). As a result, soil applied neonicotinoids were identified as a very effective tool for reducing D. citri populations; they remain a key component of management programs t hat allow growers to mitigate the risk of HLB infection in young citrus typically defined as trees less than eight feet in height ( Rogers and Shawer 2007, Rogers 2012, Rogers et al. 201 5 ). University of Florida recommendations suggested an intensive progr am in which neonicotinoids are applied to the soil at six week intervals, with supplemental non neonicotinoid foliar applications made between soil application events (Rogers 2012). Neonicotinoids are characterized as highly systemic and mobile within plan t tissue. The Insecticide Resistance Action Committee (IRAC) classifies neonicotinoids within the chemical sub group 4A, which act on the n icotinic acetylcholine receptor (nAChR). N eonicotinoid insecticides often are applied to the soil where they are abso rbed through the roots and transported to the foliage through xylem channels (Elbert et al. 2008). Systemic insecticides applied to the soil effective ly target insect pests, while minimizing direct contact with pollinators and other beneficial insects (Sta nsly and Qureshi 2008). Currently, three neonicotinoid insecticides are labeled for use in Florida citrus: thiamethoxam (Platinum 75 SG Syngenta Crop Protection, Inc., Greensboro, NC), imidacloprid (Admire Pro 4.6F Bayer CropScience,

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91 Research Triangle Park, NC), and clothianidin (Belay 2.13 SC Valent USA Corporation, Walnut Creek, CA) (Rogers et al. 201 5 ). A number of studies have addressed the use of neonicotinoids as a means of protecting young citrus trees from feeding with residual control effect s reported between 6 and 11 weeks after application (Qureshi and Stansly 2007, Qureshi and Stansly 2009, Ichinose et al. 2010a, Setamou et al. 2010, Byrne et al. 2012, Rogers 2012). Serikawa et al (2012) used electropenetrography to demonstrate that adult D. citri exhibited a reduced number and duration of phloem related feeding behaviors on citrus plants receiving soil applications of imidacloprid compared to untreated plants. Despite the use of soil applied neonicotinoids, 2013 reports estimated one to t hree percent of trees becoming infected annually in intensively managed groves in Florida (Rogers 2013). Boina et al (2009) proposed that uneven temporal and spatial distribution of imidacloprid in citrus tissue following a soil application may permit exp osure of D. citri to sublethal doses of imidacloprid. Uneven uptake of systemic insecticides by the root system make it possible for D. citri to develop on treated trees (Rogers 2012). If D. citri feed on C Las infected citrus tissue with sublethal imidaclo prid concentrations which do not inhibit feeding, acquisition and /or inoculation of C Las is possible. In Florida, roughly 80 100% of all D. citri individuals are C Las positive ( Coy and Stelinski 2015) and therefore, a single successful feeding event on an uninfected tree cannot be tolerated. Setamou et al (2010) identified the lethal concentration of imidacloprid for D. citri as between 200 and 250 parts per billion (ppb). This lethal threshold was developed by correlating percent age control of D. citri an d leaf tissue residue analysis using enzyme linked immunosorbent assay (ELISA). When evaluating insecticides under field conditions percent age control, or efficacy, is most often defined by the absence of a particular insect pest as compared to some untreated control. In the case of systemic

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92 insecticides, efficacy could be a result of mortality, repellency, feeding deterrence, or a combination t hereof. In this case, repellenc e can be defined as olfactory avoidance behavior of aversive volatiles, associated with feeding sites and deterrence can be defined as gustatory avoidance of less or non suitable f eeding sources Dosages of imidacloprid betwe en 200 to 250 ppb associated with imidacloprid efficacy observed by Setamou et al. (2010) may have result ed from a combination of mortality, repellency, and/or feeding deterrence caused by imidacloprid rather than mortality only Because mortality was not quantified in the aforementioned study, the concentration of imidacloprid required to kill D. citri through feeding remains unknown. To date, resistance monitoring efforts in citrus utilize only contact style assay methods for comparing susceptibility lev els of field collected populations to that of laboratory susceptible culture s (Tiwari et al. 2011 a 2013, IRAC 2009, 2011, 2014, Kanga et al. 201 6, Coy et al. 2016). Three distinct methodologies are among the contact style assay methods cited: 1) topical; 2) vial; and 3) leaf dip. Topical assays are used to evaluate only contact exposure by administration of a small volume of insecticide directly to the insect thorax (Coy et al. 2016, IRAC 2011, Tiwari 2011a, 2013). Vial assays are also used to evaluate onl y contact exposure by coating the inside walls of a glass vial with insecticide, aspirating insects into the treated vial, and allowing them to traverse the treated glass surface (Kanga et al. 2016). Unlike the topical and vial assays, leaf dip assays enco mpass both contact and ingestion routes of exposure, where insects are permitted to walk on and feed upon insecticide covered leaf material (IRAC 2009, 2014, Tiwari et al. 2011a). While contact assays are effective for determining shifts in susceptibility over time and if resistance exists in some field population, contact values are not equivalent to ingestion concentration s required to kill D. citri In the case of systemic insecticides, such as neonicotinoids applied to soil, ingestion is the primary ro ute of insecticide exposure, and thus the

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93 concentration of insecticide required to cause mortality exclusively through ingestion should be quantified. The purpose of this study was to determine the concentration of systemic insecticide within citrus tissu e required to kill D. citri through ingestion and to validate the lethal concentration using various field populations within citrus production areas of Florida. By determining the lethal concentration of systemic insecticide by ingestion, we will advance our understanding of the interaction between D. citri as a vector of C Las and citrus treated with soil applied systemic neonicotinoid insecticides. Materials and Methods Lab Culture The laboratory susceptible (LS) strain was reared in continuous culture at the University of Florida Citrus Research and Education Center in Lake Alfred on Murraya koenigii maintained at 27 C with RH 65 % with a photoperiod of 14:10 L:D. The LS strain was m aintained C Las free, confirmed by routine quantitative real time (qPCR) testing as described in Pelz Stelinski et al. (2010) and did not receive any exposure to insecticides following establishment of the colony in 2005. Adult D. citri were collected dire ctly from plants through oral aspiration. Adult D citri were collected and used during the same day to minimize negative effects from storage and to reduce unintended mortality. Field Collection Four citrus groves were sampled for D. citri each represen ting a major citrus production area in the state: 1) Vero Beach, east coast flatwoods, collected 24 VIII 2016 ; 2) Lake Placid, southern central ridge, collected 6 IX 2016 ; 3) Lake Alfred, northern central ridge, collected 19 IX 2016 ; and 4) Labelle, southe rn pine flatwoods, collected 21 IX 2016 Adult D. citri were collected by two methods: 1) aspirati on directly from citrus foliage or 2) by swee p net and

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94 aspirat ion of trapped adults. Diaphorina citri adults were transported from the field within labeled p lastic aspirator vials placed in to a small cooler containing one cold pack wrapped in paper towel s D iaphorina citri collected from the field were assayed during the same day to minimize negative effects of storage and to reduce unintended mortality. In the case of the Labelle, FL population, a limited number of adult D. citri were available in the grove at the time of collection. Instead of collecting adults during the grove visit, f lush infested with fourth and fifth instar D. citri nymphs were collecte d into small paper bags and transported to the lab. Flush stems were inserted into floral foam placed in a plastic tray and wetted with d eionized water. Each plastic tray containing foam and flush was held in a small mesh insect cage with two Mur raya koe nigii plants. The cage was stored in a greenhouse cubicle set to 27C under ambient lighting and humidity conditions. After nine days, adult D. citri were abundant and thus collected for assay as done with the direct field collected population s Adult Ing estion Assay The ingestion assay method used was a modification to that described in Huseth et al. (2016 ). A 30% sucrose solution similar to that described in Hall et al. (2010) was prepared to achieve a final volume of 600 m L in the following order of mix ture steps : 300 m L deionized water, 180 g sucrose (30% w/v; Sigma Life Science, St. Louis, MO, Cat. No.: S0389 5KG), 0.6 m L green food dye (0.1% v/v; McCormick & Co., Inc. Hunt Valley, MD), and 2.4 m L yellow food dye (0.4% v/v; McCormick & Co., Inc. Hunt Valley, MD). This mixture was lightly heated to dissolv e sucrose. Once the sucrose was in solution, deionized water was added to reach a final volume of 600 m L Aliquots of th e stock sucrose solution were then used to perform a serial dilution of one of th ree formulated neonicotinoid insecticides of seven to eight doses : Admire Pro 4.6F ( 550 g imidacloprid L 1 Bayer CropScience, Research Triangle Park, NC), Platinum 75SG ( 750 g thiamethoxam kg, Syngenta Crop Protection Greensboro, NC), or Belay

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95 2.13 SC ( 255 g clothianidin L 1 Valent USA Corporation, Walnut Creek, CA) The cap was removed from 5 m L snap cap centrifuge tubes (Eppendorf Tubes, Hamburg, Germany, Cat. No.: 0030119401) and appropriately labeled by treatment. Each centrifuge tube cap was fille d with 0.7 m L sucrose solution with or without insecticide. A two cm 2 piece of Parafilm M (Bemis, Neenah, WI, Cat. No.: PM 992) was stretched and placed over the diet filled cap and excess was wrapped around the cap. Depending on availability of insects, four to six adult D. citri were aspirated into individual centrifuge tubes and a diet filled cap was reinstalled for feeding through the thin Parafilm M membrane. Tubes were placed upright in a tube tray and held at 27C, 70% relative humidity, with a p hotoperiod 14:10 L:D for 72h. One replicate consisted of one tube and 10 replicates were used for each of seven to eight doses in each ingestion assay. A total of 40 to 60 adults were tested for each dose. Insects were assessed at 72 hours for mortality. I nsects were scored as alive (full function), moribund (insects lacking coordinated movement), or dead (no movement upon disturbing). Moribund insects were classified as dead for data analysis. The lab susceptible culture was tested against each of the thre e insecticides and each field population was tested against only imidacloprid due to the lack of availability of field collected insects. Adult Contact Assay To test contact activity, the vial roll method similar to that described in Kanga et al. (2016) was used due to similar insecticide exposure properties to that of a foliar spray, while excluding the possibility of ingestion activity. Analytical grade insecticides (> 99.5% purity) of each imidacloprid, thiamethoxam, and clothianidin were obtained from Chem Service (Chem Service, Inc, West Chester, PA). An initial stock insecticide solution was prepared using acetone (Fisher Scientific, Fair Lawn, NJ, Cat. No.: A929 4). A serial dilution was utilized to achieve seven to eight doses for each assay. Indiv idual pre labeled 16 mL glass vial s (Wheaton,

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96 Millville, NJ, Cat. No.: 224746) were each treated with 1.5 mL insecticide solution and placed onto an electric hot dog roller within a fume hood. Vials were rolled for 1 2 hours or until all acetone evaporated from within the glass vial. Control vials were treated with acetone only and subjected to the same rolling process. Treated vials were stored in a dark cardboard container at room temperature conditions for no more than 24h until use in an assay. Dependin g on availability of insects, eight to twelve adult D. citri were aspirated into individual vials using a small medical vacuum (Invacare, Elyria, OH, Model: IRC1135) and a cap was installed. Tubes were placed horizontally onto a cafeteria tray and held at 27C, 70% relative humidity, with a photoperiod 14:10 L:D for 24h. One replicate consisted of one vial and five replicates were used for each of seven to eight doses in each contact assay. A total of 40 to 60 adults for each dose were tested. Insects were assessed at 24 hours for mortality. Insects were scored as alive (full function), moribund (insects lacking coordinated movement), or dead (no movement upon disturbing). Moribund insects were classified as dead for data analysis. The lab susceptible cultu re was tested against each of the three insecticides and each field population was tested against only imidacloprid due to the lack of availability of field collected insects. Statistical Analyses Concentration mortality data were subjected to Probit analy sis using SAS v9.4 (Proc Probit, SAS Institute, 2013). Mean separations between D. citri populations within each exposure route were based on mortality at the mean dose level using Tukey Kramer Least Squares Means where means differed significantly at 0.05. Results A fully susceptible laboratory D. citri strain (LS) was tested to determine baseline susceptibilities to imidacloprid, thiamethoxam, and clothianidin when exposed to each insecticide by ingestion and contact ( Table 4 1 ). The LC 50 for ingestion was 0.39, 0.11, and 0.09

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97 parts per million (ppm) for imidacloprid, thiamethoxam, and clothianidin, respectively. In contrast, the LC 50 for contact exposure was 0.04, 0.01, and 0.01 ppm for imidacloprid, thiamethoxam, and cloth ianidin, respectively. The relative difference in LC 50 values were compared using a ratio of LC 50 via ingestion divided by the LC 50 via contact for each insecticide and is described as IC 50 in Table 4 1 The IC 50 for imidacloprid indicates that the LC 50 by ingestion was 9.75 fold greater than by contact; the IC 50 for thiamethoxam 11 fold greater and the IC 50 for clothianidin 9 fold greater. Four field populations of D. citri were tested to validate comparative ex posure observations of the laboratory D. citri strain to imidacloprid exposure and to determine the relative susceptibility of field populations to imidacloprid by exposure through ingestion and contact ( Table 4 2 ). LC 50 values w ere greater by ingestion than by contact in each field population investigated. Resistance ratios were also generated to compare susceptibility levels of field populations to the LS strain within each exposure route. Resistance ratios at the 50 percent mor tality level (RR 50 ) were calculated by dividing the LC 50 of the field population by the LC 50 of the LS strain. All field populations tested expressed some level of resistance as compared to the LS strain. The contact assay exhibited low level RR 50 values f or the Vero Beach and Labelle populations (3.06 and 5.77, respectively) when compared with RR 50 values generated using the ingestion assay method (10.57 and 26.36, respectively). High RR 50 values were observed for the Lake Placid and Lake Alfred population s using the contact method (18.75 and 42.21, respectively), and the ingestion method (20.39 and 33.43, respectively). Discussion This study is the first to quantify the lethal concentration of neonicotinoid insecticides required to effectively kill D. citr i when ingested in the absence of contact exposure All lethal concen trations developed to date utili zed only an assay method that permit s physical contact

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98 between the insect and insecticide where insects cannot escape exposure (Tiwari et al. 2011a, 2013, IRAC 2009, 2011, 2014, Kanga et al. 2016, Coy et al. 2016). Neonicotinoid insecticides are most often applied to young citr us trees as a soil drench, absorbed by the roots and expressed in leaf tissue. Because D. citri are only exposed to these insecticide s by ingesting insecticide inclusive plant sap, there was a need to determine insecticide concentrations required to kill D. citri upon ingestion. This research is also the first to document the magnitude of difference in mortality between ingestion and co ntact exposure. A concentration of 9 to 11 fold higher, depending on active ingredient, was required to kill 50 percent of the LS strain through ingestion when compared to contact for imidacloprid, thiamethoxam, and clothianidin. Similarly, the lowest imid acloprid concentration difference between ingestion and contact for the field populations tested was 8.51 fold higher. These results document that a higher neonicotinoid concentration is required to kill the same number of D. citri individuals through ingestion than by contact. The observed difference between mortality by ingestion and by contact may be explained by the following factors: 1) Volume of diet consumed determines the amount of insecticide exposure; 2) a portion of inges ted insecticide is evacuated through the digestive tract and rendered unavailable to the insect before absorption into the body occurs; and 3) higher metabolic activity in the gut may impact insecticide toxicity compared with absorption through the cuticle via contact. High mortality observed in the negative control (no available diet) suggests that observed survivors within the ingestion assay did successfully feed, therefore complete avoidance of the insecticide diet was unlikely. The ingestion assay also likely better approximates field exposure of adult D. citri to systemically occurring imidacloprid, since these hemipterans must alight on plant material and initiate feeding prior to exposure. Upon insertion of stylets into the plant material, D. citri c an choose whether or not to feed. Individuals that do

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99 not feed in the field can move to new host plants in search of more acceptable food sources. Presumably, if feeding deterrence occurred in the ingestion assay, those individuals would have died prior to evaluation, further reducing the LC 50 values for ingestion. This would reduce the magnitude of difference between insecticidal activity with the ingestion and contact assays. In previously published studies, b etween 200 and 250 parts per billion (ppb) (0. 2 0.25 ppm) of imidacloprid was determined as the (presumed) lethal concentration needed to kill D. citri by correlating insecticide efficacy with imidacloprid titer (Setamou et al. 2010). In the present study, a concentration of 0.39 ppm (390 ppb) imida cloprid was required to kill half (LC 50 ) of the LS strain by ingestion and 62.19 ppm (62190 ppb) imidacloprid was required to kill 90% (LC 90 ) of the LS strain by ingestion. The higher than expected values observed indicates that the imidacloprid concentra tion threshold required to kill D. citri in the field is likely much higher than previously assumed Because Setamou et al. ( 2010 ) found that 200 250 ppb of imidacloprid provide strong efficacy against D. citri field populations and the current study fou nd 62.19 ppm to kill just 90% of the laboratory susceptible population, it is likely that 200 250 ppb corresponds to a sublethal dose as a result of feeding deterrence rather than mortality. In the case of systemic insecticides where feeding is required fo r insecticide exposure, insect mortality is likely not required to achieve perceived high levels of control. Additional work is warranted to investigate the feeding behavioral response of D. citri when exposed to various neonicotinoid concentrations. Whil e the foremost goal of this study was to compare the difference between ingestion vs contact mortality our results indicate a second event of reduced susceptibility to neonicotinoid s in field populations of D. citri at our selected study sites not unlike that documented for populations in similar regions of Florida in 2010 (Tiwari et al. 2011a) Resistance ratios

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100 generated using the contact assay suggest that low levels of resistance exist in the Vero Beach and Labelle populations. Interestingl y, resistance ratios calculated using the ingestion assay method for the same populations are higher, demonstrating that the ingestion assay method is more sensitive in detection of low level resistance development Populations from Lake Alfred and Lake Pl acid exhibited high resistance ratios by both the contact assay method and the ingestion assay method. Perceived product failures have been observed at or near the Lake Alfred and Lake Placid collection sites in previous years (M. E. Rogers, personal obser vation). Results from this study illustrate the importance of matching each specific insecticide with the route of insecticide exposure in the field when undertaking resistance monitoring efforts. This match of exposure is especially important in the detec tion of low level resistance in the field before product failures occur. Tiwari et al. (2011a) found that imidacloprid resistant field populations of D. citri expressed higher levels of detoxifying enzymes, including general esterase, glutahione S transfer ase, and cytochrome P 450 monooxygenases. Later work discovered five family 4 cytochrome P 450 genes that were induced by imidacloprid exposure (Tiwari et al. 2011b). Tiwari et al. (2011a) advised that despite elevated levels of detoxifying enzymes in insect icide resistant populations, other mechanisms of resistance may play a role in the development of resistance in D. citri populations. Suggested mechanisms were reduced penetration, target site insensitivity, and mutations in detoxifying enzymes. Nonetheles s, because D. citri are most often exposed to neonicotinoids in citrus through ingestion and that D. citri likely encounter sub lethal concentrations of this insecticide more frequently than lethal ones (Boina et al. 2009) it is possible that behavioral r esistance as a single mechanism has thus far been incorrectly ignored as possibly a primary concern given the need for ingesting neonicotinoids by D. citri following soil applied treatments The most recent resistance

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101 monitoring work to occur in Florida re ported a reversion of insecticide resistance to imidacloprid and thiamethoxam in 2013 and 2014 D. citri populations (Coy et al. 2016). This work was completed using a topical contact assay and reemphasizes the dynamic susceptibility shifts described by Tiw ari et al. (2013). Nevertheless resistance monitoring efforts that utilize contact assay methods may underestimate neonicotinoid resistance or fail to detect mechanisms specific to neonicotinoid resistance that are related to ingestion exposure pathways The present study quantifies the concentration of imidacloprid, thiamethoxam, and clothianidin in citrus leaf material required to effectively kill D. citri and identifies the utility of an ingestion assay in monitoring for neonicotinoid resistance in fi eld populations of D. citri Although we determined the lethal dose required to kill D. citri upon feeding, this study d id not determine the insecticide concentration threshold at which feeding is deterred relative to pathogen transmission disruption Seri kawa et al. (2012) demonstrated that a small portion of D. citri tested were able to undergo phloem ingestion (E2) for more than one hour on citrus tissue assumed to contain lethal levels of imidacloprid. While one hour of ingestion (E2) is sufficient for C Las acquisition to occur (Bonani et al. 2010), Serikawa et al. (2012) explained that subsequent inoculation of nearby uninfected citrus plants following C Las acquisition was not likely due to lethal effects of imidacloprid. While lethal levels of imidaclo prid may prevent successful C Las transmission, sublethal levels that do not deter feeding may allow successful C Las acquisition from infected tissue and subsequent inoculation into new, uninfected trees The dose required to deter feeding, as it relates to pathogen transmission, remains unknown. Future work should utilize tools such as electropenetrography to determine the dose at which feeding activity is interrupted to determine the minimum neonicotinoid dose required to significantly reduce pathogen tran smission Since 2009, insecticide resistance to neonicotinoids has been a

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102 reoccurring phenomenon in D. citri ( Tiwari et al. 2011a, 2013 ; Kanga et al. 2016; Coy et al. 2016). Because of these acute shifts in susceptibility to neonicotinoids, growers must re main cognizant of the potential for resistance. Furthermore, our finding of potentially neonicotinoid resistant D. citri populations in the field in 2016 warrants the development and implementation of resistance management practices directly aimed to prese rve the utility of soil applied neonicotinoids in citrus.

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103 Table 4 1. Response of laboratory susceptible D iaphorina citri strain to three neonicotinoid insecticides by ingestion and contact routes of exposure Insecticide Assay Method Strain N a Slope + SE LC 50 b 95% CL LC 90 95% CL X 2 IC 50 c NC d Imidacloprid Ingestion LS 546 0.25 + 0.03 0.39 (0.19 0.72) 62.19 (30.36 164.74) 96.21 9.75 100 Contact LS 320 1.03 + 0.10 0.04 (0.03 0.04) 0.13 (0.10 0.18) 100.80 Thiamethoxam Ingestion LS 404 0.34 + 0.04 0.11 (0.05 0.21) 4.94 (2.63 11.75) 73.58 11.00 100 Contact LS 405 0.75 + 0.12 0.01 (0.01 0.02) 0.05 (0.04 0.11) 38.69 Clothianidin Ingestion LS 402 0.28 + 0.03 0.09 (0.03 0.19) 9.35 (4.55 25.15) 69.74 9.00 100 Contact LS 393 0.51 + 0.07 0.01 (0.01 0.02) 0.16 (0.10 0.34) 46.59 a. Number of adult D. citri tested. b. Parts per million (ppm) active ingredient. c. Ratio of ingestion LC 50 divided by contact LC 50 d. Percent mortality in negative control containing no diet at 72h.

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104 Table 4 2. Response of laboratory and field collected D iaphorina citri to imidacloprid by ingestion and contact routes of exposure in 2016. Method Population N a Slope + SE LC 50 bc 95% CL LC 90 c 95% CL X 2 RR 50 Lab Susc RR 50 Field Susc IC 50 d NC e Ingestion LS 546 0.25 + 0.03 0.39a (0.18 0.71) 62.19 (30.36 164.74) 96.21 0.09 9.75 100 Vero Beach 284 0.39 + 0.04 4.13b (2.43 6.77) 109.19 (55.27 284.22) 83.96 10.57 37.55 97.5 Lake Placid 282 0.31 + 0.04 7.97bc (4.35 14.42) 522.58 (204.31 2150) 69.85 20.39 1.93 11.72 95 Lake Alfred 440 0.29 + 0.03 13.10c (8.04 21.61) 1077 (455.13 3622) 104.01 33.54 3.17 8.51 98.3 Labelle 359 0.34 + 0.03 10.28bc (6.36 16.60) 425.46 (201.83 1206) 99.76 26.36 2.49 48.95 98.0 Contact LS 320 1.03 + 0.10 0.04 a (0.03 0.04) 0.13 (0.10 0.18) 100.80 0.36 Vero Beach 418 0.34 + 0.03 0.11 b (0.06 0.18) 4.87 (2.59 11.11) 116.08 3.06 Lake Placid 320 0.33 + 0.03 0.68 c (0.40 1.17) 31.81 (14.65 92.08) 102.44 18.75 6.18 Lake Alfred 496 0.19 + 0.02 1.54 c (0.80 3.07) 1232 (313.60 9480) 83.67 42.21 14.00 Labelle 408 0.30 + 0.03 0.21 b (0.12 0.35) 14.61 (6.96 39.69) 111.49 5.77 1.91 a. Number of adult D. citri tested. b. estion: 97.7 ppm). c. Parts per million (ppm) active ingredient. d. Ratio of ingestion LC 50 divided by contact LC 50 by location. e. Percent mortality in negative control containing no diet at 72h.

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105 CHAPTER 5 EVALUATING THE EFFECT OF IMIDACLOPRID ADMINISTERED IN ARTIFICIAL DIET ON FEEDING BEHAVIOR OF DIAPHORINA CITRI (HEMIPTERA : LIVIIDAE) USING ELECTROPENETROGRAPHY The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Huanglongbing (HLB) a global disease of citrus. Following the discovery of HLB in Florida citrus, management strategies were developed using soil applied neonicotinoids to protect young trees. Despite the implementation of intense management programs, infection continued to spread among even the most intensively managed groves. In the present study, we used electopenetrography ( EPG) to test three sublethal imidacloprid doses administered in artificial diet to approximate the dosage required to reduce feeding activity and prevent salivation / ingestion activity altogether. We failed to detect a significant effect of 0.55 ppm imida cloprid on probing behavior, pathway (C), or salivation/ingestion (E1E2) activity when compared to the untreated control. Conversely, we observed a significant reduction in the number of probes and the number of C with both 5.5 and 55 ppm imidacloprid. Fur thermore, we detected a significant reduction in the number of E1E2 events at both 5.5 ppm and 55 ppm imidacloprid (57 and 54 percent, respectively) compared to the untreated control, and a reduction in number of sustained (>600 sec) E1E2 (NumLngE1E2) at 5 5 ppm. Reductions in feeding activity were apparent at dosages of at least 5.5 ppm, which likely helps reduce HLB spread. However, we were unable to prevent E1E2 with dosages of up to 55 ppm, indicating that titers of 55 ppm imidacloprid following applicat ion to the soil may not be completely effective in preventing C Las inoculaton, and thus spread of HLB in Florida citrus.

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106 Justification The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) was first detected in Florida in 1998 (Halb ert and Manjunath 2004) and is known to transmit the phloem limited proteobacterium, Candidatus Liberibacter asiaticus ( C Las), the presumed cause of citrus greening disease, or Huanglongbing (HLB) (Halbert and Manjunath 2004, Bov 2006, Grafton Cardwell et al. 2013). Huanglongbing was discovered in Florida in 2005 (Halbert 2005) and has citrus production (Hodges and Spreen 2015). Upon inoculation of C Las into plant phloem, the bacteria moves downward into th e roots where the root system is sev erely compromised In turn, the canopy is starved of vital nutrients resulting in dead limbs and leaf drop, followed by a reduction in fruit quality and yield, with eventual tree death (Halbert and Manjunath 2004, Bov 2 006, Grafton Cardwell et al. 2013) Following the discovery of HLB in Florida citrus, management strategies were quickly developed and focused on tree health and vector management to aid in reducing the spread of the serious disease (Rogers 2008). Despite the implementation of intense management programs, virtually all D. citri are currently infected with C Las, and tree infection continues to spread among even the most intensively managed groves (Rogers 2013, Coy and Stelinski 2015). As a result, we must ev aluate current vector management practices to elucidate why spread of the pathogen continues in order to develop and deliver improved management tactics to growers. Diaphorina citri are characterized as insects with high fecundity and rapid development, undergoing completion of the egg to adult life cycle in as little as 15 days during periods of optimal environmental conditions (Liu and Tsai 2000, Grafton Cardwell et al. 2013). Adult D. citri are attracted to volatiles emitted by newly formed flush shoo ts where they lay up to 800 eggs per female (Patt and Setamou 2010). If egg lay occurs on HLB infected host tissue, newly hatched nymphs feed on phloem sap and acquire C Las (Pelz Stelinski et al. 2010). Acquisition

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107 efficiency is increased for nymphs develo ping on infected host tissue compared with D. citri acquiring the pathogen in the adult stage (Pelz Stelinski et al. 2010). Nonetheless, dispersal of infected adults from a point source of inoculum results in the spread of the pathogen within and among groves. Much of HLB vector management has maintained focus on young tree programs (Rogers 2008, Rogers 2013). The key objectiv e of the young tree management program is to maintain HLB free trees until trees reach fruit bearing age. Young trees flush asynchronously and frequently relative to mature trees in Florida (Hall and Albrigo 2007, Rogers 2012). Because adult D. citri seek young flush for e gg lay or feeding, young trees are presumably at great est risk of acquiring C Las (Stansly and Rogers 2006). V ector management programs in young trees advise an approximate three week alternation between soil applied neonicotinoids and non neonicotinoid foliar sprays aimed to maintain D. citri populations at low levels in young tree groves (Rogers 2012, Rogers et al. 201 5 ). Neonicotinoids are a unique group of systemic insecticides that when applied to the soil, are absorbed by the roots, an d transported through xylem vascular bundles to the foliage (Elbert et al. 2008). According to the Insecticide Resistance Action Committee (IRAC) neonicotinoids are within the insecticide sub group 4A, and bind to the insect nicotinic acetylcholine recepto r (nAChR) resulting in hyper excitation, paralysis, and eventual death (IRAC 2017) Three neonicotinoid insecticides are currently labeled for use in non bearing citrus in Florida: thiamethoxam (Platinum 75 SG Syngenta Crop Protection, Inc., Greensboro, NC), imidacloprid (Admire Pro 4.6F Bayer CropScience, Research Triangle Park, NC), and clothianidin (Belay 2.13 SC Valent USA Corporation, Walnut Creek, CA). A number of studies have investigated the residual activity of neonicotinoids applied to the s oil and reported between six and eleven weeks control (Qureshi

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108 and Stansly 2007, Qureshi and Stansly 2009, Ichinose et al. 2010, Setamou et al. 2010, Byrne et al. 2012, Rogers 2012). While influential factors that affect neonicotinoid expression levels in leaf tissue are likely related to the environment (e.g soil type, application volume, irrigation / rainfall, tree age and size, and climatic and / or weather conditions), uneven insecticide distribution within a plant and / or over time is likely to resul t in areas of sublethal concentrations within leaf tissue at any time following application to the soil (Boina et al. 2009, Rogers 2012). Electropenetrography (EPG) is a highly effective method used to study and quantify specific feeding behaviors of pier cing sucking hemipterans ( Prado and Tjallingii 1994, Joost et al. 2006, Bonani et al. 2010, Butler et al. 2012, Jacobson and Kennedy 2014) and rasping sucking Thysanoptera (Harrewijn et al. 1996, Groves et al. 2001 Kindt et al. 2003 Joost and Riley 2005 ). Each feeding behavior is identified through a behavior specific waveform captured by an EPG monitor. Bonani et al. (2010) correlated repetitive waveforms for D. citri with six specific feeding behaviors including non probing (NP), pathway (C), xylem ing estion (G), phloem contact (D), phloem salivation (E1), and phloem ingestion (E2). Occurrence, frequency, and duration of specific waveforms can be used to study insect feeding behavior in response to various stimuli. For example, D iaphorina citri phloem f eeding activities E1 and E2 have been significantly reduced through the use of soil applied imidacloprid in citrus, however neither salivaton nor ingestion has been prevented to date (Serikawa et al. 2012, Miranda et al. 2016). Understanding the response of particular feeding behaviors, such as salivation or ingestion, can have major implications in pathogen transmission. Coy and Stelinski (2015) speculated that between 80 and 100 percent of D. citri in Florida are infected with C Las. Because not all grove s are adequately managed for the vector, particularly mature groves and abandoned groves

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109 preventing the inoculation component of the transmission cycle is key to averting the spread of the deadly disease. Pathogen transmission is largely a two component phenomenon, 1. Acquisition, and 2. Inoculation. A number of EPG studies reported a focus of feeding behaviors associated with phloem ingestion (E2) activity as related to C Las acquisition (Bonani et al. 2010, Miranda et al. 2016, Serikawa et al. 2012, Luo et al. 2015). Bonani et al. (2010) determined that D. citri were able to acquire C Las when ingestion behavior (E2) was sustained for one hour, albeit acquisition success was low (ca. 6 percent) In contrast, Luo et al. (2015) demonstrated nearly 96 percent successful C Las acquisition by adult D. citri with a phloem ingestion (E2) period of as little as two minutes. Moreover, Serikawa et al. (2012) found that D. citri were able to undergo phloem ingestion (E2) for more than one hour on citrus tissue containi ng assumed lethal levels of imidacloprid, yet Miranda et al. (2016) determined that both thiamethoxam and imidacloprid disrupted probing behaviors related to phloem ingestion. Each of the aforementioned studies and resultant conclusions maintained focus on the acquisition / ingestion component of the transmission cycle. While a reduction in acquisition (and subsequent inoculation) of C Las is likely to reduce the spread of HLB and could be helpful to the industry, given that citrus is a perennial crop where cumulative effects of disease spread are compounded annually, a simple C Las may no longer be economically acceptable to a grower. Moreover, many groves have become abandoned over recent years throughout Florida, and that space serves as an unmanaged source of inoculum to neighboring groves that are intensively managed and still in production. Consequently, perhaps the neonicotinoid dose required to deter and / or prevent salivation into the phloem as related to inoculation is mo re critical today than the

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110 neonicotinoid dose required to reduce or deter ingestion activity (bacterial acquisition) as studied in the past. T he t wo investigations discussed above used EPG to study feeding behavior in response to imidacloprid exposure (Ser ikawa et al. 2012, Miranda et al. 2016). These studies each have a single key limitation: imidacloprid dosages in which D. citri were exposed are unknown. In both Serikawa et al. (2012) and Miranda et al. (2016), various rates of Admire Pro ranging from 0. 25 0.35 g plant 1 were applied to the soil of varying plant sizes up to 80 cm tall. While the amount of imidacloprid applied to the soil is known, application rate and plant size can each have a significant impact on expression in leaf tissues (Langdon 2 017). Moreover, expression in leaf tissue can only be quantified after the EPG monitoring period using analytical methods such as enzyme linked immunosorbent assay ( ELISA ) (Castle et al 2005, Garlapati 2009, Setamou et al. 2010 ) or liquid c hromatography m ass spectrometry (LC MS) (Langdon 2017). One must chemically analyze the leaf tissue following each EPG monitoring period to develop a mean imidacloprid titer across the test leaf, which likely would not accurately emulate the imidacloprid concentration wi thin the phloem due to potential in leaf concentration gradients as proposed by Boina et al. (2009), as well as potential changes in concentration during the EPG monitoring period. Because phloem feeding activity is of most interest to researchers studying transmission of C Las, knowing the concentration of imidacloprid expressed specifically within the phloem sap is paramount to behavioral studies regarding the C Las D. citri transmission matrix. Despite demonstrations of changes in feeding behavior under t he influence of imidacloprid, the imidacloprid dosage required to elicit a particular behavioral response remains unknown (Serikawa et al. 2012, Miranda et al. 2016). The ability to study feeding behavior during ingestion of a range of known imidacloprid d osages would allow us to develop an

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111 improved understanding of the effects of sublethal imidacloprid exposure to D. citri feeding behavior. Herein, we used electropenetrography to evaluate D. citri feeding behavior during exposure to a sucrose based liquid diet spiked with varying, known concentrations of imidacloprid. The overarching goal of this research was to determine the concentration of imidacloprid in citrus leaf tissue required to reduce feeding activity and the concentration required to prevent sal ivation / ingestion. Langdon and Rogers (2017) defined [feeding] of less or non suitable f imidacloprid concentration required to deter and / or prevent D. citri salivation / ingestio n in phloem will allow us to refine current vector management programs which will help either maximize the reduction or perhaps prevent the spread of C Las in Florida citrus. Materials and Methods Electropenetrography Assays Two electropenetrography exper iments were conducted to determine the imidacloprid dosage required to reduce feeding activity and prevent salivation / ingestion feeding behaviors when exposed via ingestion. Three sublethal imidacloprid dosages were administered across two experiments using a combination of Admi re Pro 4.6F and a 30% sucrose based artificial diet described in detail within Langdon and Rogers (2017) The first experiment tested 0.55 ppm imidacloprid against an untreated control, and the second experiment tested 5.5 ppm and 55 ppm imidacloprid again st an untreated control To monitor insect feeding behavior, the sucrose based diet, with or without insecticide, was used to fill a polystyrene petri dish 3.5 cm in diameter by 1 cm deep (Corning Glass Works, Corning NY 14831, part #25050 35) A 26 AWG c opper wire was inserted into the diet, with the tag end folded over the outer rim of the petri dish. Parafilm M (Pechiney Plastic Packaging, Menasha WI 54952) was then stretched over the diet filled petri dish in a manner tha t prevented

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112 air gaps between t he undersurface of stretched Parafilm M and top concave surface of liquid diet. The equipment and its set up was described in detail elsewhere (Ebert and R ogers 2016). In brief, two 4 channel AC DC monitors (Backus & Bennett 2009) custom built by William H. Bennett (EPG Equipment Co., Otterville, MO) were used in DC mode with 150 mV substrate voltage. Data was acquired through a DI710 AD converter (Akron, OH) using Windaq software at a sampling rate of 100 Hz/channel. Diaphorina citri adults were tethered using a 2 cm long by 25.4 m diameter gold wire (Sigma Cohn Corp., Vernon, NY) attached to the thoracic tergites using silver glue (1:1:1 w : w : w, white glue:water:silver flake [8 10 m, Inframat Advanced Materials, Manchester CT]). The opposite end of the g old wire was connected to the unit head amp set to an impedance of 10 9 he copper wire from the petri dish was connected to the electrode from the monitor. Test insects were subjected to a starvation period of 30 minutes from the time the insects were removed from the colony until they were placed on the plant. All insects were wired during this period without being c hilled or anesthetized with CO 2 Recording began before D. citri were placed on the Parafilm M covered petri dish to e ns ure that all recordings started in the NP behavior and recordings were made over a 23 h period. The insects, diet, and head amp were contained in a Faraday cage to minimize electronic noise. Light was provided by overhead fluorescent lights (24:0 L:D) and r oom tem perature was maintained at 26.6 o C. When on a plant, D. citri are known to exhibit at least six waveforms: non probing (NP), pathway (C), phloem contact (D), phloem salivation (E1), phloem ingestion (E2), and xylem ingestion (G) (Bonani et al. 2010) When exposed to artificial diet in this study, three waveforms were identified : non probing (NP), pathway (C), and salivation / ingestion ( E1 E2). In the first experiment, 28 adult D. citri were monitored in the control treatment and 27 adult D. citri wer e monitored in the 0.55

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113 ppm imidacloprid treatment. In the second experiment, 26 adult D. citri were monitored in the control treatment, 27 in the 5.5 ppm imidacloprid treatment, and 31 in the 5 5 ppm imidacloprid treatment. For each run of the experiment, all treatments were run at least once. Multiple replicates of the same treatment within a single run were evenly split between two monitors. The position in the room for any one treatment was rotated between runs to e nsure that any potential room effects w ere evenly distributed between all treatments. Insect Culture A continuous culture of laboratory susceptible (LS) D. citri was reared at the University of Florida Citrus Research and Education Center in Lake Alfred on Murraya koenigii maintained at 27C with RH 65% with a photoperiod of 14:10 L:D. Following establishment in 2005, the LS strain did not receive any exposure to insecticides and routine quantitative real time (qPCR) testing as described in Pelz Stelinski et al. (2010) was used to confirm the colony was C Las free. Statistical Analysis Data analysis used an adaptation of Ebert 2.01 (Ebert and Rogers 2016) that was simplified to deal with a psyllid exhibiting only three waveforms (non probing (NP), pathway (C), and salivatio n / ingestion (E2)). There was no clear separation between salivation (E1) and ingestion (E2), therefore all salivation and ingestion behaviors were pooled into one unit: salivation / ingestion (E1E2). Count data were square root transformed, duration data were log e transformed, and percentage data were logit transformed prior to analysis. Analyses were performed using Proc Glimmix in SAS 9.4M4 running under SAS Enterprise Guide 7.13. A detailed description of each measured parameter can be found in Table 5 1 Results and Discussion In the present study, we tested a range of three sublethal imidacloprid doses across two experiments to approximate the dosage required to : 1) Reduce feedi ng activity, and 2) Prevent

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114 salivation / inge stion activity. Electropenetrography has been used to study feeding behavior for a number of insect species on artificial media (Joost et al. 2006, Jin et al. 2012, Trebicki et al. 2012). This was the first formal study to use EPG to monitor the feeding be havior of D. citri against an insecticide spiked liquid diet. During the first experiment, we failed to detect a significant effect of 0.55 ppm (550 ppb) imidacloprid on D. citri probing behavior, pathway, or salivation / ingestion activity when compared t o the untreated control ( Table 5 2 ). These results indicate that a concentration of 0.55 ppm may not deter D. citri feeding activity or prevent E1E2, resulting in a failure to intercept bacterial transmission. Conversely, imidacl oprid doses of 5.5 (5500 ppb) and 55 ppm (55,000 ppb) generally influenced a majority of probing and pathway parameters ( Table 5 3 ). A significant reduction in the number of probes (NumPrbs) and the number of pathway events (Nmbr C) was observed with both 5.5 and 55 ppm of imidacloprid compared to the untreated control. Similarly, Miranda et al. (2016) found that significantly fewer probing and pathway events occurred on plants treated with imidacloprid compared to untreated plants at 35 days following insecticide application to the soil, although the precise dosage received by the insect was unknown. Furthermore, we failed to detect a reduction in the duration of the first (DurFrstPrb) probe event, a reduction in the percentage of probe events that resulted in pathway (PrcntPrbC), nor a reduction in the percentage of probe events that resulted in E1E2 (PrcntPrbE1E2), which may indicate that D. citri adults were unable to detect imidacloprid at concentrations up to 55 ppm. Miranda et al. (2016) determined that D. citri were able to detect imidacloprid treated plants only following a short period of ingestion (E2), and went on to conclude that imidaclo prid likely acts as a feeding deterrent when applied to the soil. In addition, the total duration of non probing (TtlDurNP) and mean duration of non probing (MnDurNP) was significantly longer at 55 ppm imidacloprid compared to the untreated control, and th e total

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115 duration of pathway (TtlDurC) was significantly reduced at 5.5 ppm, and further reduced at 55 ppm. Similarly, Butler et al. (2012) reported longer periods of non probing activity for the potato psyllid, B actericera cockerelli ( S ulc) on potato plant s treated with imidacloprid We detected an effect of imidacl oprid on two E1E2 parameters: 1) the number of E1E2 events (NumE1E2), and 2 ) the number of sustained (>600 sec) E1E2 events (NumLngE1E2) ( Table 5 3 ). A significant redu ction in the number of E1E2 events was observed at both 5.5 ppm and 55 ppm of imidacloprid (57 and 54 percent, respectively) compared to the untreated control. In addition, the number of sustained (>600 sec) E1E2 events was significantly reduced (ca. 61 pe rcent) at only 55 ppm of imidacloprid relative to the untreated control. However, we failed to detect a difference between treatments in the total (TtlDurE1E2) or mean (MnDurE1E2) duration of E1E2. These results clearly demonstrate a reduction in feeding a ctivity (ie. salivation / ingestion), which presumably would equate to a reduction in bacterial acquisition from C Las infected leaf material in the field, yet a number of D. citri were able to successfully salivate in / ingest imidacloprid spiked diet at o ur highest dose of 55 ppm for a period that exceeded 10 minutes. An inoculation access period (IAP) of as little as 15 minutes is known to result in inoculation of C Las into uninfected citrus tissue (Capoor et al. 1974, Grafton Cardwell et al. 2013), there fore, it remains possible that sustained salivation / ingestion activity exhibited in our study may result in inoculation of C Las into uninfected tissue. We failed to detect a significant difference between 0, 5.5, and 55 ppm imidacloprid in the percent of E1E2 events that resulted in sustained (>600 sec) E1E2, time to first E1E2 from start of probe ( TmFrstE1E2FrmPrbStrt), nor time to first sustained E1E2 from start of probe (TmFrstSusE1E2StrtPrb), indicating that D. citri adults that did undergo salivation / ingestion, did not stop feeding due to imidacloprid detection. Nevertheless, in two separate whole plant studies where small potted citrus plants were drenched

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116 with some rate of imidacloprid, a reduction in the number of E1 events was observed (Serikawa et al. 2012, Miranda et al. 2016), yet neither investigation indicated that E1 occurrence was prevented. While insecticide did influence feeding behavior in the present study, our highest imidacloprid dose of 55 ppm did not completely prevent E1E2, there fore inoculation of C Las into uninfected leaf material remains possible at 55 ppm imidacloprid. Despite intensive D. citri management programs implemented by growers that utilize frequent soil applications of neonicotinoid insecticides, groves continue to succumb to C Las infection. We revealed a reduction in a number of probing activities, an increase in non probing behaviors (NP), a reduction in pathway behaviors (C), and a reduction in salivation / ingestion behaviors (E1E2) under oral exposure of at least 5.5 ppm imidacloprid spiked artificial diet using EPG. Reductions in feeding activity observed in the present study conf irm findings of previous studies (Serikawa et al. 2012, Miranda et al. 2016), and are likely to elucidate a reduction in the spread of HLB within and among commercial citrus groves, demonstrating some level of value in the use of neonicotinoids applied to the soil. Langdon and Rogers (2017) found that the LC 90 of imidacloprid following ingestion ranged from 62.19 ppm in the lab population to as much as 522.58 ppm in a potentially resistant field collected population, indicating that 55 ppm is a sublethal im idacloprid dose when administered through ingestion. In addition, they found increased activity when imidacloprid was administered through contact (laboratory susceptible population LC 90 = 0.13 ppm imidacloprid) than by ingestion (laboratory susceptible po pulation LC 90 = 62.19 ppm imidacloprid) Nevertheless, while reductions in feeding activity are apparent following ingestion of imidacloprid, because we were unable to prevent salivation / ingestion feeding behavior by oral administration of imidacloprid d oses of up to 55 ppm, and because imidacloprid titer following the soil application of Admire Pro in commercial groves is not

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117 known to exceed 55 ppm (Langdon, 2017), we are certain that soil applied imidacloprid is not capable of completely preventing C Las inoculaton, and thus preventing the spread of HLB in Florida citrus. Future work should investigate imidacloprid residues following foliar application and resulting D. citri feeding behaviors at those concentrations in the attempt to find an effective use for imidacloprid that is likely to prevent inoculation of C Las into uninfected citrus.

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118 Table 5 1 Description of adult D iaphorina citri feeding behavior by EPG model abbreviation. Behavior Abbreviation* Behavior Description Probing NumPrbs total number of probing events MnPrbs mean number of probing events DurFrstPrb Duration (sec) of first probe NumNP Total number of non probing events TtlDurNP Sum of duration (sec) of all non probing events MnDurNP Mean duration (sec) of all non probing events DurNpFllwFrstSus E1 E2 duration (sec) of non probing event before first sustained (>600sec) ingestion Pathway NmbrC Number of pathway events TtlDurC Total duration (sec) of pathway events MnDurC Mean duration (sec) of pathway events PrcntPrbC Percent of probe events that result in pathway Salivation / Ingestion** Num E1 E2 Number of salivation / ingestion events NumLng E1 E2 Number of long (>600 sec) salivation / ingestion events TtlDur E1 E2 Total duration (sec) of salivation / ingestion MnDur E1 E2 Mean duration (sec) of salivation / ingestion TmFrstSus E1 E2StrtPrb Time (sec) until first sustained (>600sec) salivation / ingestion from start of probe with the sustained event TmFrst E1 E2FrmPrbStrt duration (sec) of first salivation / ingestion event from start of probe PrcntPrb E1 E2 Percent of probe duration in salivation / ingestion Prcnt E1 E2Sus E1 E2 Percent of salivation / ingestion duration spent in sustained (>600sec) salivation / ingestion TmFrstSus E1 E2 Duration (sec) of first sustained salivation / ingestion event All variables are by insect. Means are counts, durations, or percentages per insect, where durations are expressed in seconds. ** There is no clear separation between E1 and E2 in the artificial diet. The waveforms blend one into the other, and separating them would introduce considerable error into the measurements.

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119 Table 5 2: LSMeans SEM for each behavioral parameter following exposure of adult Diaphorina citri to artificial diet with and without 0.55 ppm imidacloprid. *All variables are by insect. Means are counts, durations, or percentages per insect, where durations are expressed in seconds. **There is no clear separation between E1 and E2 in the artificial diet. The waveforms blend one into the other, and separating them would introduce considerable error into the measurements. Behavior Parameter Control 0.55 ppm p value LSMeans SE LSMeans SE Probing / non probing NumPrbs 7.10 0.48 6.22 0.49 0.2034 MnPrbs 4.70 0.17 5.02 0.17 0.1843 DurFrstPrb 3.94 0.17 3.95 0.18 0.9712 NumNP 7.19 0.47 6.29 0.48 0.1884 TtlDurNP 11.25 0.06 11.15 0.06 0.2503 MnDurNP 7.52 0.17 7.54 0.18 0.9321 DurNpFllwFrstSusE1E2 1008.18 2607.27 6968.35 3057.30 0.1563 Pathway NmbrC 7.22 0.49 6.30 0.50 0.1901 TtlDurC 8.20 0.19 8.13 0.20 0.8112 MnDurC 4.49 0.09 4.52 0.09 0.7777 PrcntPrbC 1.19 2.30 4.84 2.16 0.2567 Salivation / Ingestion* NumE1E2 1.06 0.20 0.86 0.21 0.4883 NumLngE1E2 0.53 0.14 0.48 0.14 0.8102 TtlDurE1E2 7.81 0.49 7.61 0.49 0.7735 MnDurE1E2 6.70 0.49 6.85 0.49 0.8294 TmFrstSusE1E2StrtPrb 5.45 0.32 4.88 0.34 0.2383 TmFrstE1E2FrmPrbStrt 4.77 0.24 4.71 0.24 0.8415 PrcntPrbE1E2 1.19 0.45 0.59 0.45 0.3547 PrcntE2SusE1E2 1.07 0.42 0.23 0.39 0.1780 TmFrstSusE1E2 11.07 0.11 10.93 0.12 0.3993

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120 Table 5 3: LSMeans SEM for each behavioral parameter following exposure of adult D iaphorina citri to artificial diet with 0, 5.5, or 55 ppm imidacloprid. All variables are by insect. Means are counts, durations, or percentages per insect, where durations are in expressed in seconds. **There is no clear separation between E1 and E2 in the artificial diet. The waveforms blend one into the other, and separating them would introduce considerable err or into the measurements. Behavior Parameter Control 5.5 ppm 55 ppm p value LSMeans SE* LSMeans SE* LSMeans SE* Probing / non probing NumPrbs 8.44 0.46 a 6.32 0.45 b 5.31 0.42 b <.0001 MnPrbs 4.93 0.17 a 4.81 0.17 ab 4.28 0.16 b 0.013 DurFrstPrb 3.61 0.16 3.83 0.16 3.49 0.15 0.2901 NumNP 8.49 0.45 a 6.40 0.44 b 5.42 0.42 b <.0001 TtlDurNP 11.17 0.04 b 11.23 0.04 ab 11.33 0.04 a 0.014 MnDurNP 6.97 0.15 b 7.66 0.15 a 8.09 0.14 a <.0001 DurNpFllwFrstSusE1E2 5.59 0.40 b 9.11 0.55 a 10.38 0.45 a <.0001 Pathway NmbrC 8.64 0.46 a 6.37 0.45 b 5.39 0.42 b <.0001 TtlDurC 8.78 0.16 a 7.81 0.16 b 7.23 0.15 c <.0001 MnDurC 4.54 0.09 a 4.26 0.09 ab 4.01 0.09 b 0.0003 PrcntPrbC 1.94 1.78 3.08 2.04 4.44 1.74 0.6061 Salivation / Ingestion** NumE1E2 1.72 0.20 a 0.74 0.19 b 0.79 0.18 b 0.0006 NumLngE1E2 0.75 0.14 a 0.39 0.14 ab 0.29 0.13 b 0.0497 TtlDurE1E2 7.06 0.44 7.02 0.52 6.27 0.45 0.404 MnDurE1E2 5.85 0.38 6.58 0.45 5.94 0.39 0.4243 TmFrstSusE1E2StrtPrb 4.87 0.25 4.95 0.32 4.46 0.31 0.4881 TmFrstE1E2FrmPrbStrt 4.68 0.22 4.76 0.26 4.48 0.22 0.6951 PrcntPrbE1E2 1.94 0.46 0.83 0.54 1.21 0.47 0.2756 PrcntE1E2SusE1E2 1.01 0.37 0.22 1.16 0.80 0.82 0.6098 TmFrstSusE1E2 10.70 0.19 10.87 0.19 10.97 0.18 0.581

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121 CHAPTER 6 CONCLUSIONS The overarching goal of this research was to help refine current vector management programs which will help either maximize the reduction or prevent the spread of C Las in Florida citrus. This research quantifie d the concentration of imidacloprid, thiamethoxam, and clothianidin in citrus leaf material in space and over time following application to the soil. While factors including tree canopy region, leaf section, tr ee size, and application rate each effected expression of neonicotinoids following application to the soil, observed titers were much lower than expected when used in the field The target concentration threshold of imidacloprid following application to th e soil was 200 to 250 ppb based on the report by Setamou et al. (2010). Since a correlation between D. citri abundance and clothianidin or thiamethoxam titer did not exist, 200 to 250 ppb became the assumed efficacy threshold concentration for all neonicot inoids. This research was the first to quantify the lethal concentration of each of the three currently labeled neonicotinoid insecticides required to effectively kill D. citri when ingested in the absence of contact exposure All lethal concen trations dev eloped to date utili zed only an assay method that permit s physical contact between the insect and insecticide where insects cannot escape exposure (Tiwari et al. 2011a, 2013, IRAC 2009, 2011, 2014, Kanga et al. 2016, Coy et al. 2016). Because D. citri are only exposed to these insecticides by ingesting insecticide inclusive plant sap, there was a need to determine insecticide concentrations required to kill D. citri upon ingestion. This research is also the first to document the magnitude of difference in m ortality between ingestion and contact exposure. We found that lethal activity from contact exposure to neonicotinoid insecticides occurs at very low concentrations compared with ingestion. Moreover, we were able to use electropenetrography to study feedin g behaviors under exposure to precise neonicotinoid dosages. We found that 0.55 ppm imidacloprid did not

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122 reduce in any feeding behavior including probing, pathway, or salivation / ingestion activities, yet 5.5 and 55 ppm imidacloprid caused significant red uctions in probing, pathway, and salivation / ingestion behaviors. W hile the highest mean titer observed for any neonicotinoid in the present composition of studies was likely to have reduced the incidence of C Las inoculation in the field, that titer was u nlikely to completely intercept inoculation. We observed a number of effects that are of significant concern regarding use of neonicotinoids by soil application in Florida citrus: 1) failure to achieve lethal concentrations in leaf tissue following applica tion to the soil; 2) persistence of neonicotinoid concentrations less than 1 ppm through 12 weeks following application; 3) failure to achieve acceptable D. citri control following application to trees 18 months of age (MCV = 1.34m 3 ); 4) lack of efficient uptake relative to dose applied (e.g. high rate of 0.74g Platinum 75SG in 237 mL water per tree is equivalent to 2370 ppm thiamethoxam applied to the soil, and low rate of 0.37g Platinum 75SG in 237 m L water per tree is equivalent to 1185 ppm thiamethoxam applied to the soil); and 5) higher sensitivity of D. citri to neonicotinoids through contact exposure compared to ingestion While the potential risk of the spread of HLB is highly important and within the scope of this research, neonicotinoid resistance following exposure to sublethal dosages is of significant concern. O ur results indicate d a second event of neonicotinoid resistance in field populations of D. citri which may have been exace rbated by sublethal neonicotinoid expression following application to the soil. D evelopment of resistance to neonicotinoids by D. citri has occurred in the field, and therefore, applications of neonicotinoids must be carefully administered such that D. cit ri exposure to sublethal dosages is minimized. To potentially maximize the activity of neonicotinoids and permit the longevity of their use, subsequent work should investigate neonicotinoid residues over time following foliar application. Presumably, folia r application

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123 would result in much higher acute residues following application, with a more rapid residue degradation, more suitable within the scope of insecticide resistance management. The foremost strategy for stewardship and future implementation of n eonicotinoids in citrus must be resistance management. Therefore, the current results suggest that foliar use of neonicotinoids may be a superior tactic than their applications to the soil, particularly in trees with canopies larger than 0.08m 3 to mitigate resistance development and thus preserve efficacy of this mode of action Given the dynamic nature of susceptibility of D. citri to insecticides, we must remain diligent in research efforts with a keen focus on resistance management and be willing to adju st insecticide use patterns to ensure the longevity of each available chemical class.

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124 LIST OF REFERENCES Anastassiades, M., S. J. Lehotay, D. Stajnbaher, and F. J. Schenck. 2003. Fast and easy multiresidue method employing 412 31. Boina D. R., E. O. Onagbola, M. Salyani, and L. L. Stelinski. 2009a. Antifeedant and sublethal ef fects of imidacloprid on Asian citrus psyllid, Diaphorina citri. Pest Manag. Sci. 65: 870 877. Bonani, J. P., A. Fereres, E. Garzo, M. P. Miranda, B. Appezzato Da Gloria, and J. R. S. Lopes. 2010. Characterization of electrical penetration graphs of the As ian citrus psyllid, Diaphorina citri, in sweet orange seedlings. Entomologia Experimentalis et Applicata. 134: 35 49. Bov, J. M. 2006. Huanglongbing: a destructive, newly emerging, century old disease of citrus. J. of Plant Pathol. 88. 7 37. Butler C. D. G. P. Walker, J. T. Trumble. 2012. Feeding disruption of potato psyllid, Bactericera cockerelli by imidacloprid as measured by electrical penetration graphs. Entomol. Exp. Applic. 142 : 247 257. Byrne, F. J., A. A. Urena, L. J. Robinson, R. I. Krieger, J. Doccola, and J. G. Morse. 2012. Evaluation of neonicotinoid, organophosphate and avermectin trunk injections for the management of avocado thrips in California avocado groves. Pest Manag. Sci. 68: 811 817. Byrne, F. J., M P. Daugherty, E E. Grafton Ca rdwell, J. A. Bethke, J G. Morse. 2017. Evaluation of systemic neonicotinoid insecticides for the management of the Asian citrus psyllid Diaphorina citri on containerized citrus. Pest Manag. Sci. 73 : 506 514. Capoor, S. P., D. G. Rao, S. M. Visvanth. 197 4. Greening disease of citrus in the Deccan Trap Country and its relationship with the vector, Diaphorina citri Kuwayama." In Proc. 6th Conference of the International Organization of Citrus Virologists. University of California, Richmond 43 49. Castle, S. J., F. J. Byrne, J. L. Bi, and N. C. Toscano. 2005. Spatial and temporal distribution of imidacloprid and thiamethoxam in citrus and impact on Homalodisca coagulata populations. Pest Manag. Sci. 61: 75 84. Coy, M. R. and L. L Stelinski. 2015. Great va Candidatus Liberibacter asiaticus in field populations of Diaphorina citri (Hemiptera: Liviidae) in Florida. Flor. Entomol. 98: 356 357. Coy, M. R., L. Bin, and L. L. Stelinski. 2016. Reversal of insecticide resistance in Florida populations of Diaphorina citri (Hemiptera: Liviidae). Flor. Entomol. 99: 26 32.

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125 Ebert, T. A., M. E. Rogers. 2016. Effect of substrate voltage on EPG recordings of ingestion and probing behavior in Diaphorina citri (Hemiptera: Liviidae). Florid a Entomol. 99: 528 534. Elbert, A., B. Becker, J. Hartwig, and C. Erdelen. 1991. Imidacloprid a new systemic insecticide. Pflanzenschutz Nachrichten Bayer (Germany, FR) Elbert, A., M. Haas, B. Springer, W. Thielert, and R. Nauen. 2008. Applied aspects of neonicotinoid uses in crop protection. Pest Manag. Sci. 64: 1099 1105. Garlapati, S. 2009. Uptake of soil applied neonicotinoids by citrus plants and their impact on selected biological parameters of the asian citrus psyllid, Diaphorina citri Kuwayama. M.S. thesis, Texas A & M University Kingsville, Kingsville. Gottwald, T. R. 2007. Citrus canker and citrus huanglongbing, two exotic bacterial diseases threatening the citrus industries of the Western Hemisphere. Outlooks on Pest Manag. 18: 274. Gottwald, T. R., J. H. Graham, M. S. Irey, T. G. McCollum, and B. W. Wood. 2012. Inconsequential effect of nutritional treatments on huanglongbing control, fruit quality, bacterial titer and disease progress. Crop Prot. 36: 73 82. Grafton Cardwell, E. E., L. L. Ste linski, and P. A. Stansly. 2013. Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Ann. Rev. of Entomol. 58: 413 432. Graham, J. H., L. W. Timmer, and M. M. Dewdney. 2014. Phytophthora Foot Rot and Root Rot. 2014 Fla. Citr. Pest Manag. Guide. PP 156. 1 6. Graham, J. H., E. G. Johnson, T. R. Gottwald, and M. S. Irey. 2013. Presymptomatic fibrous root decline in citrus trees caused by huanglongbing and potential interaction with Phytophthora spp. Plant Dis. 97: 1195 1199. Groves, R. L., C. E. Sorenson, J. F. Walgenbach, G. G. Kennedy. 2001. Effects of imidacloprid on transmission of tomato spotted wilt tospovirus to pepper, tomato and tobacco by Frankliniella fusca Hinds (Thysanoptera: Thripidae). Crop Prot. 20: 439 445. H albert, S. E. 2005. The discovery of huanglongbing in Florida. In Proceedings, 2nd Internat. Cit. Canker and Huanglongbing Res. Workshop. Orlando, FL. Halbert, S. E., and K. L. Manjunath. 2004. Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Flor. Entomol. 87: 330 353. Hall, D. G., and L. G. Albrigo. 2007. Estimating the relative abundance of flush shoots in citrus with implications on monitoring insects associated with flush. Hort. Sci. 42: 364 368.

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127 Jacobson, A. L., G. G. Kennedy. 2014. Electrical penetration graph studies to investigate the effects of cyantraniliprole on feeding behavior of Myzus persicae (Hemiptera: Aphididae) on Capsicum annuum Pest Mana g. Sci. 70: 836 840. Jeppson, L. R., M. J. Jesser, and J. O. Complin. 1952. Tree trunk application as a possible method of using systemic insecticides on citrus. J. of Econ. Entomol. 45: 669 671. Jin, S. Z. M. Chen, E. A. Backus, X. L. Sun, B. Xiao. 2012. Characterization of EPG waveforms for the tea green leafhopper, Empoasca vitis Gthe (Hemiptera: Cicadellidae), on tea plants and their correlation with stylet activities. J. of Insect Physiol. 58: 1235 1244. JMP, Version 13. SAS Institute Inc., Cary, NC, 2007. Joost, P. H., D. G. Riley. 2005. Imidacloprid effects on probing and settling behavior of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) in tomato. J. Econ. Entomol. 98: 1622 1629. Joost, P. H., E. A. Backus, D. Morgan, F. Yan. 2006. Correlation of stylet activities by the glassy winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms. J. of Insect Physiol. 52: 327 337. Kanga, L. H. B., J. Eason, M. Haseeb, J. Qureshi, and P. S tansly. 2016. Monitoring for insecticide resistance in Asian Citrus Psyllid (Hemiptera: Psyllidae) populations in Florida. J. Econ. Entomol. 109: 832 836. Kim, B. M., J. S. Park, J. H. Choi, A. M. Abd El Aty, T. W. Na, J. H. Shim. 2012. Residual determina tion of clothianidin and its metabolites in three minor crops via tandem mass spectrometry. Food Chem. 131: 1546 1551. Kindt, F., N. N. Joosten, D. Peters, W. F. Tjallingii. 2003. Characterisation of the feeding behaviour of western flower thrips in terms of electrical penetration graph (EPG) waveforms. J. of Insect Physiol. 49: 1 83 191. Kumagai LB, LeVasque CS, Bloomquist CL, Madishetty K, Guo YY, Woods PW, Rooney Latham S, Rascoe J, Gallindo T, Schnabel D, Polek M. 2013. First report of Candidatus Liberibacter asiaticus associated with citrus huanglongbing in California. Plant Disease 97: 283 Langdon, K. W. 2017. Optimizing the use of soil applied neonicotinoids for control of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in young citrus trees. P h.D. Dissertation. University of Florida. Langdon, K. W., M. E. Rogers. 2017. Neonicotinoid induced mortality of Diaphorina citri (Hemiptera: Liviidae) is affected by route of exposure. J. Econ. Entomol. 110: 2229 2234.

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128 Liu, Y. H., J. H. Tsai. 2000. Effects of temperature on biology and life table parameters of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Ann. of Appl. Biol. 137: 201 206. Luo, X., A. L. Yen, K. S. Powell, F. Wu, Y. Wang, L. Zeng, Y. Yang, Y Cen. 2015. Feeding behavior of Diaphorina citri Candidatus infected Citrus reticulata leaves of several maturity stages. Florida Entomol. 98: 186 192. Maienfisch, P., M. Angst, F. Brandl, W. Fischer, D. H ofer, H. Kayser, W. Kobel, A. Rindlisbacher, R. Seen, A. Steinemann, H. Widmer, 2001. Chemistry and biology of thiamethoxam: a second generation neonicotinoid. Pest Manag. Sci. 57: 906 913. Mendel, R. M ., U. Reckmann, and F. Fu¨ hr. 2000. Xylem transport of the pesticide imidacloprid in citrus. Acta Hortic. 531: 129 134. Metcalf, R. L., and R. B. Carlson. 1950a. Systemic insecticides: control of plant feeding pests by poisoning plant juices studied. Ca lif. Agric. 3, 10. Metcalf, R. L., and R. B. Carlson. 1950b. Systemic pest control. Citr. Leaves. 30: 12 13. Miranda, M. P., P. T. Yamamoto, R. B. Garcia, J. P. Lopes, J. R. Lopes. 2016. Thiamethoxam and imidacloprid drench applications on sweet orange nu rsery trees disrupt the feeding and settling behaviour of Diaphorina citri (Hemiptera: Liviidae). Pest Manag. Sci. 72: 1785 1793. Nauen, R, U. Ebbinghaus Kintscher, V. L. Salgado, M. Kaussmann. 2003. Thiamethoxam is a neonicotinoid precursor converted to c lothianidin in insects and plants. Pest. Biochem. and Physiol. 76: 55 69. Obreza, T. A., and K. T. Morgan. 2008. Nutrition of Florida citrus trees. Univ. of Fla. Extens. SL. 253. Patt, J. M., and M. Setamou. 2010. Responses of the Asian citrus psyllid to volatiles emitted by the flushing shoots of its rutaceous host plants. Environ. Entomol. 39: 618 624. Pelz Stelinski, K. S., R. H. Brlansky, T. A. Ebert, and M. E. Rogers. 2010. Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J. of Econ. Entomol. 103: 1531 1541. Prado, E., W. F. Tjallingii. 1994. Aphid activities during sieve element punctures. Entomologia experimentalis et applicata 72: 157 165. Qureshi, J. A., and P. A. Stansly. 2007. Integrat ed approaches for managing the Asian citrus psyllid Diaphorina citri (Homoptera: Psyllidae) in Florida. In Proceedings, Fla. State Hort. Soc. 120 : 110 115.

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129 Qureshi, J. A., and P. A. Stansly. 2009. Insecticidal control of Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). In Proceedings, Fla. State Hortic. Soc. 122: 172 175. Qureshi, J. A., B. C. Kostyk, and P. A. Stansly. 2011. Effectiveness of sel ective insecticides to control Asian citrus psyllid and citrus leafminer during leaf flushing. pp. 85 89. In Proceed ings, Fla. State Hort. Soc, 124: 85 89. Qureshi, J. A., B. C. Kostyk, and P. A. Stansly. 2014. Insecticidal suppression of Asian citrus psyl lid Diaphorina citri (Hemiptera: Liviidae) vector of Huanglongbing pathogens. PLoS ONE 9: 1 22. Rogers, M. E. 2008. General pest management considerations. Citr. Indus. 89: 12 17. Rogers, M. E. 2012. Protection of young trees from the Asian citrus psyllid and HLB. Citr. Indus. 93: 10 15. Rogers, M. E. 2013. Asian citrus psyllid management for young trees. Florida Citr. Instit. 2013. Rogers, M. E., and D. B. Shawer. 2007. Effectiveness of several soil applied systemic insecticides for managing the Asian citr us psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae), In Proceedin gs, Fla. State Hortic. Soc, 120: 116 119. Rogers, M. E., G. Carlton, and T. D. Riley. 2012. Results from th Citr. Indus. 93: 12 16. Rogers, M. E., P. A Stansly, and L. L. Stelinski. 201 5 201 5 Florida citrus pest management guide: Asian citrus psyllid and citrus leafminer. UF/IFAS Extens. ENY 734. SAS Institute. 2013. SAS/IML User's Guide, Version 9.4. SAS Institute, Cary, NC. Serikawa, R. H., E. A. Ba ckus, and M. E. Rogers. 2012. Effects of soil applied imidacloprid on Asian citrus psyllid (Hemiptera: Psyllidae) feeding behavior. J. of Econ. Entomol. 105: 1492 1502. Stamou, M., D. Rodriguez, R. Saldana, G. Schwarzlose, D. Palrang, and S. D. Nelson. 20 10. Efficacy and uptake of soil applied imidacloprid in the control of Asian citrus psyllid and a citrus leafminer, two foliar feeding citrus pests. J. of Econ. Entomol. 103: 1711 1719. Stansly, P. A., and M. E. Rogers. 2006. Managing Asian citrus psyllid populations. Citr. Indus. 87: 17 19. Stansly, P., and J. Qureshi. 2008. Controlling Asian citrus psyllids; sparing biological control. Citr. Ind. 89: 18 24. Stansl y, P., J. A. Qureshi, and B. C. Kostyk. 2012. Effectiveness ranking for insecticides against Asian citrus psyllid. Cit. Indus. 93: 6 9.

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130 Stoller, E. W. 1970. Mechanism for the differential translocation of amiben in plants. Plant Physiol. 46 : 732 737. Sur, R., A. Stork. 2003. Uptake, translocation and metabolism of imidacloprid in plants. Bull. Of Insectol. 56: 35 40. Tiwari, S., R. S. Mann, M. E. Rogers, and L. L. Stelinski. 2011 a Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Manag. Sci. 67: 1258 1268. Tiwari, S A. D. Gondhalekar, R S. Mann, M E. Scharf and L L. Stelinski. 2011b. Characterization of five CYP 4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus infected and uninfected psyllids. Insect Mol. Biol. 20: 733 744. Tiwari, S., N. Killiny, and L. L. Stelinski. 2013. Dynamic insecticide susceptibility changes in Florida populations of Diaphorina citri (Hemiptera: Psyllidae). J. Econ. Entomol. 106: 393 399. Trebicki, P., W. F. Tjallingii, R. M. Harding, B. C. Rodoni, K. S. Powell. 2012. EPG monitoring of the probing behaviour of the common brown leafhopper Orosius orientalis on artificial diet and selected host plants. Arthropod Plant Interact. 6: 405 415. Ts ai, J. H., J. Wang, and Y. Liu. 2002. Seasonal abundance of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) in southern Florida. Fla. Entomol. 85: 446 451.

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131 BIOGRAPHICAL SKETCH Kevin William Langdon was born to Mr. and Mrs. Andy William Langdon and was the youngest sibling of three Kevin was raised between his home in rural eastern Wake County and the family swine and horse farm in southwestern Johnston County. He became passionate about the outdoors through his pur suit of fish and game, with a focus on chasing whitetail deer with his bow and bobwhite quail behind his pair of English pointers, Junior and Bailey. Kevin developed a true affinity for plant agriculture by working on a neighboring tobacco farm throughout his boyhood. Kevin was graduated from East Wake High School in 2006, where he was an active member of the FFA under Ms. Janet Harris. He attended North Carolina State University where he earned a Bachelor of Science degree in Agricultural and Environmenta l Technology with a minor in Soil Science in 2010. Kevin worked as a research assistant between 2006 and 2008 in the entomology lab of Dr. George G. Kennedy. In the summer of 2009, Kevin moved to Vero Beach, Florida to fill an internship position with Syng enta Crop Protection working for Dr. Tony Burd in the Insect Control Lab at the Vero Beach Research Center. It was in Vero Beach where Kevin decided to pursue a graduate degree in entomology and where he met his wife, an intern in the Weed Control Lab, Mis s Barbara Lee Adams. After a love filled summer, Kevin returned to Raleigh where he earned a Master of Science degree in entomology at North Carolina State University under the direction of Dr. Mark R. Abney. Upon graduation in August 2012, he took a posi Scientist, where he conducted research in insecticide development and insecticide resistance. Following encouragement from his manag er, Dr. Clark Lovelady, in 2014 Kevin decid ed to return to academia to pursue a Doctor of Philosophy in entomology at the University of Florida under the direction of Dr. Michael E. Rogers, while maintaining full time employment with

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132 Syngenta. The overarching goal of his research was to provide sol utions to citrus growers to help manage the devastating, insect transmitted citrus disease, huanglongbing. Kevin received his Ph.D. from the University of Florida in the fall of 2017 and continued his career with Syngenta while maintaining his passion for the outdoors.