Integrated Management Strategies for Spotted Wing Drosophila, Drosophila suzukii, in Southern Highbush Blueberries

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Integrated Management Strategies for Spotted Wing Drosophila, Drosophila suzukii, in Southern Highbush Blueberries
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english
Creator:
Iglesias, Lindsy Erin
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University of Florida
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Interdisciplinary Ecology
Committee Chair:
Liburd, Oscar Emanuel
Committee Members:
Stelinski, Lukasz Lech
Mcsorley, Robert T

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Subjects / Keywords:
blueberry -- drosophila -- florida -- monitoring -- oviposition -- pesticide -- survey -- suzukii
Interdisciplinary Ecology -- Dissertations, Academic -- UF
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Interdisciplinary Ecology thesis, M.S.
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Abstract:
Spottedwing drosophila (SWD), Drosophila suzukii(Matsumura), an invasive pest threatening Florida’s blueberry industry,causes direct injury to healthy fruit by ovipositing eggs under the fruit skinwhere larvae develop.  A survey wasconducted in 9 and 8 blueberry growing counties in 2012 and 2013, respectively,using a clear cup trap baited with apple cider vinegar (ACV).  Spotted wing drosophila was found in allcounties in both years except for the southernmost DeSoto County.  Oviposition preferences were investigatedusing the two Florida-grown blueberry species, southern highbush and rabbiteye,and on blueberry ripening stages. Spotted wing drosophila appears to prefer southern highbush overrabbiteye, and blue fruit over other stages of fruit development.  However, all stages of fruit development weresusceptible to SWD infestation.  In orderto develop effective monitoring techniques for SWD, different traps with andwithout a yellow visual stimulus, baited with ACV were evaluated inblueberries.  Results indicated thatadding a yellow band (visual stimulus), odorless dish detergent, and/or ayellow sticky card inside the trap, did not increase captures.  A clear cup trap baited withyeast-sugar-water was more attractive to SWD than ACV traps.  In a subsequent study, four bait treatmentswere evaluated to investigate the attraction of SWD in blueberries.  Treatments included 1) ACV, 2)yeast-sugar-water, 3) yeast-sugar-water with whole wheat flour, dish detergent,and ACV, and 4) rice vinegar and red grape wine with dish detergent.  The two yeast baits captured significantlymore SWD than the vinegar baits. Finally, a field-based laboratory bioassay was used to identify chemicaltools for managing SWD in blueberries. Seven treatments including 1) Belay® (high and low rate), 3) Danitol®(high and low rate), 5) Mustang Max®, 6) Delegate®, and 7) a water-treated controlwere evaluated against SWD.  Belay® (bothrates) was ineffective at reducing SWD adult activity throughout the 14 day experiment.  Danitol® (both rates), Mustang Max®, andDelegate® were equally effective at reducing adult activity up to sevendays.  Danitol® (both rates) also reducedlarval emergence.  Successful tacticswill be integrated into an IPM program for management of SWD in southernblueberries.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Lindsy Erin Iglesias.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Liburd, Oscar Emanuel.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 INTEGRATED MANAGEMENT STRATEGIES FOR SPOTTED WING DROSOPHILA, DROSOPHILA SUZUKII IN SOUTHERN HIGHBUSH BLUEBERRIES By LINDSY E. IGLESIAS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Lindsy E. Iglesias

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3 To my mom who gave me strength To my dad for continuous encouragement Y para mi amor, q ue me hizo sonrer en cada paso de este viaje

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4 ACKNOWLEDGMENTS I thank my major advisor Oscar E. Liburd for his support, encouragement, and guidance throughout my endeavor. I also thank my committee members Robert McSorley and Lukasz Stelinski for their f eedback and patience during the writing process. I appreciate the assistance of all participating growers, extension agents, and consultants. I am indebted to Janine Razze for the sacrifice of her valuable time in conducting the survey studies and to Elk e Weibelzahl and the members of the Small Fruit and Vegetable IPM Laboratory for their help during my field season. I also would like to thank my family for their loving support and my boyfriend Sean for his constant reassurance and laughter throughout th is journey

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5 TABLE OF CONTENTS page ACKNOWLEDGMEN TS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 The Blueberry Industry in the United States and Florida ................................ ........ 12 Blueberry Production in Florida ................................ ................................ ............... 12 Insect Pests of Blueberry in Florida ................................ ................................ ........ 13 Objectives ................................ ................................ ................................ ............... 15 Justification ................................ ................................ ................................ ............. 16 2 LITERATURE REVIEW ................................ ................................ .......................... 19 Spotted Wing Drosophila ................................ ................................ ........................ 19 Identification ................................ ................................ ................................ ..... 19 Biology ................................ ................................ ................................ .............. 20 Spotted Wing Drosophila as a Pest of Blueberries ................................ ................. 22 Management Practices of Spotted Wing Drosophila ................................ ............... 23 Monitoring ................................ ................................ ................................ ......... 23 Control ................................ ................................ ................................ .............. 25 3 SURVEY FOR SPOTTED WING DROSOPHILA IN FLORIDA BLUEBERRIES .... 32 Materials and Methods ................................ ................................ ............................ 34 Survey Sites ................................ ................................ ................................ ..... 34 Site Characteristics ................................ ................................ .......................... 34 Monitoring Traps ................................ ................................ .............................. 35 S ample Collection ................................ ................................ ............................ 36 Data Analysis ................................ ................................ ................................ ... 37 Results ................................ ................................ ................................ .................... 37 Discussion ................................ ................................ ................................ .............. 38 4 OVIPOSITION PREFERENCE OF SPOTTED WING DROSOPHILA IN SOUTHERN HIGHBUSH AND RABBITEYE BLUEBERRIES ................................ 54 Materials and Methods ................................ ................................ ............................ 55 Source of Flies ................................ ................................ ................................ 55

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6 Blueberry Bushes ................................ ................................ ............................. 56 Suitability of Rabbiteye and Souther n Highbush Blueberry Species for SWD Larval Development ................................ ................................ ...................... 57 Berry Maturity Stage ................................ ................................ ......................... 58 Results ................................ ................................ ................................ .................... 59 Suitability of Rabbiteye and Southern Highbush Blueberry Species for SWD Larval Development ................................ ................................ ...................... 59 SWD Oviposition Events and Preferences for Different Ripening Stages of Southern Highbush Blueberries ................................ ................................ .... 60 Discussion ................................ ................................ ................................ .............. 60 5 EVALUATION OF TRAPS AND BAITS FOR MONITORING OF SPOTTED WING DROSOPHILA IN BLUEB ERRIES ................................ ............................... 70 Materials and Methods ................................ ................................ ............................ 73 Experimental Design ................................ ................................ ........................ 73 Trap Comp arison Study ................................ ................................ .................... 73 Bait Study ................................ ................................ ................................ ......... 75 Sample Collection ................................ ................................ ............................ 75 Data Analysis ................................ ................................ ................................ ... 76 Results ................................ ................................ ................................ .................... 76 Trap Comparison Study ................................ ................................ .................... 76 Bait Study ................................ ................................ ................................ ......... 77 Discussion ................................ ................................ ................................ .............. 78 6 EVALUATING ALTERNATIVE CHEMICAL TOOLS FOR CONTROL OF SPOTTED WING DROSOPHILA ................................ ................................ ............ 93 Materials and Methods ................................ ................................ ............................ 94 Flies ................................ ................................ ................................ .................. 94 Field Setup ................................ ................................ ................................ ....... 95 Labor atory Setup ................................ ................................ .............................. 96 Data Collection and Analysis ................................ ................................ ............ 97 Adult activity ................................ ................................ ............................... 97 La rval survival (emergence) ................................ ................................ ....... 98 Results ................................ ................................ ................................ .................... 98 Adult Activity ................................ ................................ ................................ ..... 98 Larval Su rvival ................................ ................................ ................................ .. 99 Discussion ................................ ................................ ................................ .............. 99 7 CONCLUSION ................................ ................................ ................................ ...... 108 LIST OF REFERENCES ................................ ................................ ............................. 111 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 119

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7 LIST OF TABLES Table page 3 1 Counties and sites included in 2012 and 2013 survey for SWD, including number of traps deployed at each site. ................................ ............................... 43 3 2 Total number of SWD captured in 2012 and 2013 survey seasons. ................... 43 3 3 Mean male, female, and SWD capture per sample for year 2012 and 2013 of SWD survey. ................................ ................................ ................................ ....... 44 3 3 Mean number of SWD captured in each county in the 2012 and 2013 survey study. ................................ ................................ ................................ .................. 45 4 1 Mean oviposition events per observation on different berry maturity stages in choice study. ................................ ................................ ................................ ....... 63 5 2 Bait study treatments and m ixtures. ................................ ................................ ... 81 6 1 Insecticide treatments for efficacy study 2012. ................................ ................. 102 6 2 Mean activity level per fly between treatments within each day after application. ................................ ................................ ................................ ....... 102 6 3 Mean activity level per fly for female SWD between treatments within each day after application. ................................ ................................ ........................ 103 6 4 Mean activity level per fly for male SWD between treatments within each day post treatment. ................................ ................................ ................................ 103 6 5 Mean emergence per berry. ................................ ................................ ............. 103

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8 LIST OF FIGURES Figur e page 2 1 Male spotted wing drosophila ................................ ................................ ............ 27 2 2 Female spotted wing drosophila. ................................ ................................ ........ 28 2 3 Serrated ovipositor of the f emale spotted wing drosophila. ................................ 29 2 4 SWD larval instars. Clockwise from right, first, second, and third. ...................... 30 2 5 Three pupal instars. From left, first, second, and third instars. ........................... 31 3 1 Survey sites and counties fo r 2012 2013 blueberry seasons. ............................ 46 3 2 Plastic cup trap with yellow stimulus used during the 2012 survey year. .......... 47 3 3 Plastic cup trap without yellow stimulus used for 2013 survey .......................... 48 3 4 Mean SWD captured by county for 2012 and 2013 survey years.. ..................... 49 3 5 Mean female and male SWD captured in each county for 2012. ........................ 50 3 6 Mean female and male SWD captured in each county for 2013. ........................ 51 3 7 Mean temperatures in survey areas for 2012 and 2013 ................................ .... 52 3 8 Mean precipitation in survey areas in 2012 and 2013. ................................ ....... 53 4 1 Bioassay chamber for no choice experiment. ................................ ..................... 64 4 2 Bioassay chamber for choice experiment. ................................ .......................... 65 4 3 Mean SWD emerged from southern highbush and rabbiteye species in no choice bioassay. ................................ ................................ ................................ 66 4 4 Mean SWD emergence from different berry maturity stages in no choice tests. ................................ ................................ ................................ ................... 67 4 5 Mean oviposition events per observation on differ ent maturity stages in choice study. ................................ ................................ ................................ ....... 68 4 6 Percent oviposition on different stages of blueberry maturity stages in choice bioassays. ................................ ................................ ................................ ........... 69 5 1 Mean spotted wing drosophila (SWD) captured per trap in 2012 trapping study, experiment 1. ................................ ................................ ........................... 82

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9 5 2 Mean spotted wing drosophila (SWD) per trap in 2012 trapping study, experiment 2. ................................ ................................ ................................ ...... 83 5 3 Mean male and female spotted wing drosophila (SWD) captured in 2012 trapping study, experiment 1. ................................ ................................ ............. 84 5 4 Mean male and female spotted wing drosophila (SWD) captured in 2012 trapping study, experiment 2. ................................ ................................ ............. 85 5 5 Mean spotted wing drosophila (SWD) captured per trap in 2 013 trapping study, experiment 3 ................................ ................................ ........................... 86 5 6 Mean spotted wing drosophila (SWD) captured per trap in 2 013 trapping study, experiment 4 ................................ ................................ ........................... 87 5 7 Mean male and female spotted wing drosophi la (SWD) captured per trap in the 2 013 trapping study, experiment 3 ................................ .............................. 88 5 8 Mean male and female spotted wing drosophila (SWD) captured per trap in the 2 013 trapping study, experiment 4 ................................ .............................. 89 5 9 Mean spotted wing drosophila (SWD) captured during 2013 bait study. All treatments used the basic cup trap design. ................................ ........................ 90 5 10 Mean female and male spotted wing drosophila (SWD) captured during 2013 bait study ................................ ................................ ................................ ............ 91 5 11 Mean Drosophila spp. and Zaprionus spp. captured per trap in the 2013 bait study. ................................ ................................ ................................ .................. 92 6 1 Field setup at UF IFAS PSREU in Citra, Florida ................................ ............... 104 6 2 Bioassay chamber for efficacy study. ................................ ............................... 105 6 3 Daily mean relative humidity (%) and temperature (C) at the UF IFAS PSREU in Citra, Florida for during of pesticide efficacy study 2012. ................ 106 6 4 Total daily rainfall (cm) at the UF IFAS PSREU in Citra, Florida for duration of pesticide efficacy study 2012. ................................ ................................ ....... 106 6 5 Average Female and Male Activity per Day by Treatment. ............................... 107

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INTEGRATED MANAGEMENT STRATEGIES FOR SPOTTED WING DROSOPHILA, DROSOPHILA SUZUKII IN SOUTH ERN HIGHBUSH BLUEBERRIES By Lindsy E. Iglesias August 2013 Chair: Oscar E. Liburd Major: Interdisciplinary Ecology Spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) an invasive thy fruit by ovipositing eggs under the fruit skin where larvae develop A survey was conducted in 9 and 8 blueberry growing counties in 2012 and 2013, respectively, using a clear cup trap baited with apple cider vinegar (ACV). Spotted wing drosophila wa s found in all counties in both years except for the southernmost DeSoto County. Oviposition preferences were investigated using the two Florida grown blueberry species, southern highbush and rabbiteye, and on blueberry ripening stages. Spotted wing dros ophila appears to prefer southern highbush over rabbiteye, and blue fruit over other stages of fruit development. However, all stages of fruit development were susceptible to SWD infestation. In order to develop effective monitoring techniques for SWD, d ifferent traps with and without a yellow visual stimulus, baited with ACV were evaluated in blueberries. Results indicated that adding a yellow band (visual stimulus), odorless dish detergent, and/or a yellow sticky card inside the trap, did not increase captures. A clear cup trap baited with yeast sugar water was more attractive to SWD than ACV traps. In a subsequent study, four bait treatments were evaluated to investigate the attraction of

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11 SWD in blueberries. Treatments included 1) ACV, 2) yeast suga r water, 3) yeast sugar water with whole wheat flour, dish detergen t, and ACV, and 4) rice vinegar and red grape wine with dish detergent. The two yeast baits captured significantly more SWD than the vinegar baits. Finally, a field based laboratory bioas say was used to identify chemical tools for managing SWD in blueberries. Seven treatments including 1) Belay (high and low rate), 3) Danitol (high and low rate), 5) Mustang Max, 6) Delegate, and 7) a water treated control were evaluated against SWD. Belay (both rates) was ineffective at reducing SWD adult activity through out the 14 day experiment Danitol (both rates), Mustang Max, and Delegate were equally effective at reducing adult activity up to seven days. Danitol (both rates) also reduced larval emergence. Successful tactics will be integrated into an IPM program for management of SWD in southern blueberries.

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12 CHAPTER 1 INTRODUCTION T he Blueberry Industry in the United States and Florida The United States is the largest producer of blue berries in the world, producing over 196 ,000 kg in 2011 (FAO STAT 2013). T he U.S. blueberry market includ es both cultivated and wild species and is currently valued at approximately 880 mil USD annually, an increase of 37 percent from 2010 (644 mil USD) a nd 371 percent since 2001 (187 mil USD) (NASS USDA 2013). U nited S tates fresh blueberries first reach the global market as early as late March to early April and continue through October. Florida is a major producer of early season blueberries from March to May producing approximately 8 0 percent of the national total. Although the acreage is considered small at about 2,300 ha in 2012 the industry is greatly valued due to high market prices when berries begin ripening as early as March. The average pric e for fresh blueberries in Florida at 7.18 USD kg 1 is well abo ve the national average of just 4.87 USD kg 1 and has remained steady since 2008 (ERS USDA 2012b). Revenues for bluebe rries in Florida increased from 47 mil USD in 2010 to approximately 70 mi l USD in 2011 (ERS USDA 2012a). Florida also has a growing organic blueberry industry with over 43,545 kg of certified organic blueberries produced in 2008 (NASS USDA 2012). Blueberry Production in Florida Blueberry ( Vaccinium spp.) is one of the only c ultivated crops native to North America and is grown as a deciduous crop. The two most commonly grown species of blueberry in Florida are the s outhern h ighbush (SHB) and the r abbiteye (RE) Southern highbush, Vaccinium corymbosum L. x V. darrowi Camp, is a cross be tween highbush

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13 and a lowbush evergreen species native to Florida (Lyrene and Ballington 2006). The SHB differs from the northern highbush species primarily due to the lower chill requirements which allow it to thrive in warm south eastern win ters (Darnell 2006). Rabbiteye, Vaccinium virgatum Aiton, is a native species to the southeastern U.S. making it hardier than SHB and more tolerant to drought and soils with low organic matter. Rabbiteye also flowers in late spring so is le ss susceptible to freezes Alt hough RE is more adapted to climate in Florida, it has a longer fruit development time of 60 to 135 days (Birkhold et al. 1992) whereas SHB is much shorter ranging from 55 to 60 day s (Maust et al. 1999). As a result, SHB from Florida are the first domestic berries on the U.S. market ripening in early March and continuing through May. Rabbiteye however, do not begin producing ripe fruit until May when market prices are much reduced The reduced fruit development time for SHB is part of the reason why it accounts for more than 75 percent of the blueberries grown in Florida. Blueberries are grown in either single or double, raised rows. Beds are raised t o provide well drained medium for the short root system of the bluebe rry bushes in many of the low l ying locations in Florida. A layer of pine bark is either incorporated into the bed soil or added as a top layer to increase moisture retention, increase soil organic matter, and maintain optimum pH of 4.2 to 5.2 (Williamson et al. 2006). Southern highbush growers generally space their bushes 0.6 to 1.2 m apart with 2.7 to 3.5 m between rows whereas the vigor of RE requires a spacing of 1.2 to 1.8 m between bushes and 3.0 to 3.7 m between rows (Williamson et al 2006). Insect Pests of Blueberry in Florida Since blueberries are a deciduous crop, pest issues can occur throughout the year before, during and after the season Principal arthropod pests of blueberries in

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14 Florida include blueberry gall midge, Dasineura oxycoccana (Johnson), Florida flower thrips, Frankliniella bispinosa (Morgan), blueberry leaf beetle, Colaspis pseudofavosa Riley and the new invasive spotted wing drosophila, Drosophila s uzukii (Matsumura). The blueberry maggot, Rhagoletis mendax Curran is problematic only for growers who live in the counties north of Live Oak, Florida. Blueberry gall midge is an early season pest of the buds of blueberries, Vaccinium spp. It was previously referred to as cranberry tipworm outside of the southern U.S. but recent evidence suggests that these are cryptic species determined by reproduction isolation and differences in mitochondrial genes (Cook et al. 2011, Mathur et al. 2012). Female s oviposit within the vegetative and floral buds where the larvae hatch and feed on the bud tissues (Sarzynski and Liburd 2003, Dernisky et al. 2005). Lyrene and Payne (1992) found that feeding can cause yield loss of up to 80 percent in fields of severe infestation in southeastern blueberries. Until recently, the Florida flower thrips, Frankliniella bispinosa (Morgan ) was the key arthropod pest in SHB blueberry production (Liburd and Arvalo 2006 Arvalo and Liburd 2007 ). Thrips feeding and ovipositio n result s in scarring of the fruit, rendering it unmarketable (Arvalo 2006). Monitoring for thrips consists of hanging white sticky traps within the canopy and collecting flower buds in alcohol (Arvalo and Liburd 2007 Liburd et al. 2009 ). The blueb erry leaf beetle, Colaspis pseudofavosa Riley is the primary post harvest pest in SHB (Nyoike and Liburd 2009). Adults prefer to feed on summer (new) Leaf beetles are known to be attracted to the black weed fabric tha t is commonly in southern blueberry production (Krewer et al. 2009). A

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15 high population of leaf beetles can interfere with plant (bush) vigor the following year and severely affect marketable yield. The blueberry maggot, Rhagoletis mendax Curran, is fou nd in the eastern U.S. from Nov a Scotia, Canada south to northern Florida in highbush and rabbiteye blueberries (Payne and Berlocher 1995). In Florida and Georgia adults emerge in late May and continue through July. When they reach sexual maturity, adult female flies oviposit into ripening berries where larvae hatch and feed, rapidly degrading the fruit and rendering it unmarketable (Liburd et al. 1998). The spotted wing drosophila (SWD) Drosophila suzukii (Matsumura), is a new invasive pest threatening the blueberry industry. Spotted wing drosophila females oviposit into the ripening fruit leaving a blemish on the skin of the berry (Mitsui et al. 2006) Eggs hatch inside the berry where the larvae develops, causing the berry to become soft and degrade rapidly (Walsh et al. 2011). Consequently the berries become unmarketable. Additionally, berries found containing larvae are immediately rejected upon inspection. Estimated crop loss due to SWD injury in Florida blueberries in 2012 wa s 10 to 15 per cent, a value of 7.8 mil to 11.7 mil USD (eFly 2012). Objectives Since SWD is quickly becoming an important pest in Florida, more information is needed on the extent of its infestation, fruit susceptibility to oviposition, monitoring, and control tools. The first objective of this study was to determine the magnitude and level of SWD infestation in Florida blueberries by conducting a statewide survey in the major blueberry growing regions of Florida The second objective was to de velop and refine monitoring programs for SWD including trap design and bait attraction Third, we wanted to determ ine if oviposition host selection by SWD varied by blueberry species or

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16 fruit maturity stage. Finally, we wanted to identify tools that gro wers can use for managing SWD in Florida blueberries. Justification round growing season, SWD has the potential to spread rapidly throughout the state (Lee et al. 2011, Walsh et a l. 2011). A survey was conducted in the major blueberry growing regions in Florida to help determine the extent to which SWD is present in blueberries. This information will help growers understand the potential threat SWD may pose to their crops. Grow ers are recommended to monitor for SWD prior to implementing control tactics as part of an integrated pest management (IPM) program. However, since SWD is a new pest, monitoring techniques are still being developed. Commonly recommended techniques includ e a plastic cup trap made from a deli container with a lid and multiple entry holes along the upper rim (Wu et al. 2007, Beers et al. 2011, Birmingham et al. 2011, Lee et al. 2012 ,Walsh et al. 2011, Cini et al. 2012). Some modifications of this trap inclu de features to prevent SWD from escap ing such as a yellow sticky card hanging inside of the trap. Since SWD is in the family Drosophilidae also known as the vinegar or fruit flies, many of the recommended baits are vinegars, alcohols, fruits, yeast mixtures and combinations of the like (Kanzawa 1939, Steck et al. 2009, Walsh et al. 2011, Landolt et al. 2012a, 2012b). One of the goals of our research was to evaluate the effectiveness of new and commonly recommended trap designs and baits for ca pturing SWD in blueberry fields. Effective monitoring programs that detect SWD early in the season will help to determine when to implement control actions and evaluate the effectiveness of control techniques. In addition to

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17 effectiveness (trap captures) the ease of implementing the trap and lure system will be discussed. An effective monitoring program is dependent upon knowledge of the pest biology, ecology, and behavior. Some host plant s of SWD are more preferred for oviposition than others. Burra ck et al. (2013) found that SWD preferred to lay eggs on different fruits in the following order from most preferred to least: blackberries, raspberries, strawberries, blueberries, and grapes. Factors that play a role in oviposition site selection have ye t to be determined but larval presence, texture, firmness, brix levels, and pH have been suggested (Chess and Ringo 1985, Lee et al. 2011 Burrack et al. 2013). Major injury to blueberries occurs after the eggs hatch and the larvae begin to develop in t he berry. Therefore control techniques must be implemented prior to oviposition In order to investigate SWD oviposition behavior, we examined SHB and RE species to determine the most preferred host. The first objective of our oviposition study was to determine whether the two most commonly grown species of blueberries in Florida ( SHB and RE ) were suitable oviposition hosts that supported larval development. Second ly we aimed to evaluate SWD preference for different blueberry maturity (ripening) stages for ovi position. Results from these studies will help to determine where and when to establish traps for an effective SWD monitoring program. I t is general practice for growers to use insecticides for SWD control. Insecticides target the adult stage s ince the immature stages occur within the berry. Common effective conventional chemicals are in the classes of organophosphates and pyrethroids, reduced risk chemicals in the spinosyn (spinetoram) class, and organic

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18 chemicals in the pyrethrin and spinosyn (spinosad) classes (Liburd and Iglesias 2013). The se chemicals have shown effectiveness in the lab and the field (Bruck et al. 2011). However, the limited number of control tools available results in increasingly more applications of the same chemicals. Given the rapid development time of SWD it is a highly potential candidate for pesticide resistance. Therefore, it is important for growers to have multiple effective tools available for SWD control. One of the objectives of this study was to compare the efficacy of reduc ed risk tools to commonly used tools for SWD control. New tools provide growers with alternatives options for rotational control programs, aid in resistance management, and reduce risk to worker and environmental health.

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19 CHAPTER 2 LITERATURE REVIEW Spo tted Wing Drosophila The spotted wing drosophila (SWD) D rosophila suzukii (Diptera: Drosophilidae) is an invasive pest species native to parts of East Asia (Kanzawa 1939, Markow and n in 1916 and it was described by Matsumura in 1931 (Kanzawa 1939). Spotted wing drosophila was first recorded in the western hemisphere in Hawaii in 1980 (Kaneshiro 1983) and detected in Calif ornia in 2008 (Bolda et al. 2010 ). Subsequent captures occurred in Washington, Oregon, and Florida in 2009, and Wisconsin, Michigan, Utah, Kentucky, Louisiana and the Carolinas in 2010 (Steck et al. 2009, Walsh et al. 2011). By the end of 2012 SWD had spread to 37 states in the U.S. Spotted wing dros ophila has also been found in parts of Canada and Europe (BCMA 2012, Calabria et al. 2012). Since its first record in Hillsborough County, Florida in 2009 SWD has quickly spread throughout Florida becoming a concern for all berry growers in the state (Steck et al. 2009). Identification The family Drosophilidae is also known as the vinegar, fruit, or pomace flies. Spotted wing drosophila has several similar morphological characteristics to the common vinegar fly, Drosophila melanogaster Meigen th at frequents over ripe and damaged fruits. The abdomen of SWD is round, light brown to pale yellow and has unbroken, ho rizontal bands on the dorsal sid e (Figures 2 1, 2 2). They have large, red compound eyes and a posteromedial ocellar triangle ( Tripleho rn and Johnson 2005,

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20 In addition, SWD has sponging mouthparts with which they suck up their food. The male flies can be easily distinguished from most other vinegar flies by the single dark spot at the distal end of the R 2+3 vein on the wing s (Figure 2 2 ). The wing spots can be seen with the naked eye or a hand lens for easy identification in the field. The males of the sister species, D rosophila biarmipes and close relative D. subpulchrella also have the wingspots which can m ake distin guishing between them difficult (Takamori et al. 2006, Ometto et al. 2013). D rosophila biarmipes however, does which only allows her to oviposit in overrip e or damaged fruit. D rosophila suzukii can be distinguished from D. biarmipes by the two rows of sex combs on the forelegs, one on the first tarsal segment and one on the second, whereas D. biarmipes has both rows of sex combs on the first tarsal segment (Hauser 2011). The set of two sex combs appear as two black horizontal stripes on the forelegs when using a hand lens for identification. Female SWD are slightly larger than the males and do not posses s the dark wingspots (Figure 2 2 ). Females look ver y similar to other drosophilids except for the large, dark, and heavily serrated ovipositor that is use to pierce the skin of u nripe and ripe host fruits (Figure 2 3 ). When ovipositors of other drosophilids are compared to that of SWD, they appear rounded faintly colored, and blunt with small, ligh t teeth, limiting oviposition to soft, overripe, or damaged host fruits only. Biology The lifecycle of SWD includes an egg, larva, pupa, and adult. The eggs are laid under the skins of thin skinned fruits tha t will hatch in 1 to 3 d inside the berry (Kanzawa 1939). Eggs a re mil ky white, oblong and approximately 0.5 mm by 0.2 mm (Kanzawa

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21 1939). The eggs have two long respiratory spiracles that protrude from the skin of the blueberry. Larvae of S WD are thin, white, and soft bodied with pointed anterior and posteri or ends (Fig. 2 4 ). The mouthparts appear b lack on the anterior end. The three larval instars generally develop in 4 to 5 d inside of the berry (Kanzawa 1939). Spotted wing drosophila p upae are oblong and range from light brown to dark brown as they deve lop through three stages (Fig. 2 5 ). Spiked spiracles are located at the anterior end whereas pointed caudal spiracles at the posterior end. The red eyes and wing pads of the adult fly can be seen through the pupal case in the final stage. Pupation can occur in the soil, inside or outside the fruit and generally occurs within 5 to 7 d from egg hatch (Kanzawa 1939, Walsh et al. 2011). Kanzawa (1939) observed that SWD lifecycle can be co mpleted in 10 to 24 d whereas a recent study showed th at the lifecycle can be complete d in 12 to 15 d at 18.3C (Walsh et al. 2011). Spotted wing drosophila has been shown to have as many as 15 generations per year when observed in captivity (Kanza wa 1939) It has been predicted to complete 3 to 9 generations in the western U.S. and Canada based on degree day models (Walsh et al. 2011). The spotted wing drosophila prefer s temperature between 20 and 25C (Kanzawa 1939). It is tolerant to temperat ures as low as 1.6C for females and 0.1C for males and as high as 32.6C for females and 32.2C for males (Kimura 2004). Adult male SWD can survive for 88 d at a constant 10C E ven when exposed to a 7 d freeze ( 0.2C) adults were predicted to survive up to 103 d at 10C (Kimura 2004). In addition, SWD overwinters or enters into a state of reduced activity during periods of severe cold temperatures and short daylength, allowing it to continue its lifecycle the

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22 following season (Kimura 2004, Da lton et al. 2011). Males show signs of sterility above 30C (Kanzawa 1939). Whereas the lower s terility threshold for SWD male s has not been studied, D. melanogaster shows signs of sterility at 12 and 30C (Petavy et al. 2001, Chakir et al. 2 tolerance in SWD is one reason why SWD has become such a damaging pest throughout North America from C anada south to Florida. Spotted Wing Drosophila as a Pest of Blueberries Spotted wing drosophila has become a major threat to the blueberry industry in Florida and the world (Walsh et al. 2011) Injury from SWD results in depressed scars on the fruit left upon insertion of the ovipositor. The more economically important injury occurs as a result of larval development inside the berries, causing rapid deterioration and soft ening that render the fruit unmarketable. In addition, the fruit becomes more susceptible to subsequent infection by f ungal or bacterial pathogens (Walsh et al. 2011) F inally, there is a zero tolerance from consumers for larvae in fes ted fruit in the fresh blueberry market. Economic data analyses in California showed yield losses from SWD in strawberries and raspberries accounted for 20 and 50 percent of crop value, resp ectively (Goodhue et al. 2011) and 40 percent for blueberries (Bolda et al. 2010). Furthermore, a recent SWD working group in the eastern U.S. evaluated potential and current loss estimates in the east for the first time. Potential loss in blueberries fo r 2012 from reporting e astern states was estimated at 1 38.7 mil USD (eFly 2012). Estimated crop loss in Florida blueberries in 2012 was 10 to 1 5 percent, a value of 7.8 to 11 7 mil USD (eFly 2012). S potted wing drosophila certainly requires attention for the protection of the blueberry industry.

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23 Unlike most vinegar flies that typically prefer damaged or decaying fruit, female SWD lays eggs in healthy, ripening fruit. Female SWD lays 1 to 3 eggs per oviposition site, with an average of 380 eggs througho ut her lifetime (Kanzawa 1939, Mitsui et al. 2006). Multiple female SWD can lay eggs on a single fruit resulting in several larvae injuring one berry. Recent research investigating oviposition as a factor of fruit maturity stage has shown that females pr efer to lay eggs in ripe blueberries over unripe, green berries and that a greater number of SWD developed in the ripe berries over the unripe stages in both blueberries and blackberries (Lee et al. 2011 ). However, in the same study, no significant differ ences were found in eggs laid or larvae developed among the remaining stages. Higher brix levels seemed to have a positive correlation with oviposition and development whereas firmness had a negative correlation. Penetration force may also be a possible contributing factor to host selection When greater force was needed to penetrate the host, fewer eggs were laid or fewer larvae develop ed on multiple different berry crops (Burrack et al. 2013). Management Practices of Spotted Wing Drosophila Most Dros ophila spp. are not agricultural pests but rather nuisance pests in human habitats. As a result, few management techniques for control of SWD were available for the U.S. before 2008. As SWD has become an increasingly damaging pest to the U.S. berry indus try research on management tools and techniques is developing rapidly. Monitoring Monitoring for SWD is a crucial integrated pest management step to determine where and when populations entered a field and when to begin control. Trap designs used for capt uring SWD are based o n other commercial fruit fly traps The standard

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24 SWD trap that is used in blueberries consist s of a plastic deli container or jar with a lid and entry holes along the sides (Wu et al. 2007, Beers et al. 2011, Birmingham et al. 2011, L ee et al. 2012 ,Walsh et al. 2011, Cini et al. 2012). Commercial traps are also being used including a dome trap (Trappitt trap, Agrisense Ltd., Pontypridd, United Kingdom), cylindrical gate trap (Fruit Fly Trap, Contech Enterprises Inc., Victoria, Canada ) traps have shown slightly better results compared to a cup trap with 4 holes along the rim h owever, the large unmeshed entry hole in the bottom of the dome traps allow s a greater n umber of larger non target captures that must be sifted through for SWD identification (Landolt et al. 2012). A recent study showed that the related D rosophila melanogaster is more attracted to d arker colors such as black and red especially in high light co nditions (Kagawa et al. 2012). Similarly, Edwards et al. (2012) showed that black and red were more attractive to SWD when used in the field. Other studies in the western U.S. showed that the color of the trap had less of an impact but rather the greater the total entry size the greater the number of SWD captured in the field (Lee et al. 2012 Basoalto et al. 2013). One disadvantage is that larger entry size increases the potential for water to enter the trap via rain or irrigation. Traps with shades or roofs have been slightly more successful at capturing SWD but ineffective in windy conditions as the shades were blown over and bait spilled (Lee et al. 2012 ). Yellow sticky traps hung inside the trap are thought to increase capture s by helping to prevent SWD escape and are commonly use d in the field. Traps are generally baited with a liquid that acts as a drowning solution. Drosophila are also called vinegar flies because they are attracted to overripe, decaying

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25 fruit, yeasts and other volatile s of fermentation ( Hunter et al. 1937, B echer et al. 2010). Kanzawa (1939) tested many different vinegars and wines including molasses, rice wine, grape wine, fruit juices, and in numerous combinations. In the U.S. apple cider vinegar ( ACV ) was the first successful bait recommended for use in SWD traps (Steck et al. 2009, Walsh et al. 2011). Apple cider vinegar is clear and allows for easy identification has longevity in the field and is inexpensive when compared to other bait s such as wine (personal experience). Additional baits used inclu de yeast sugar water mixture, wines, other vinegars, fruit purees, and volatile components of the above compounds such as acetic acid and ethanol (Walsh et al. 2011). Adding dish soap to any of the above baits is thought to decrease surface tension and in crease fly capture (Walsh et al. 2011). In addition, combinations of various vinegars with wines have shown synergistic effects on SWD attraction (Landolt et al. 2012a, 2012b). Control Cultural control techniques include sanitation to reduce areas for SWD repr oduction and short harvest intervals to prevent larval infestation in marketable fruit. Netting with mesh 0.98 mm or smaller, will prevent D. suzukii from reaching the berries entirely (Kawase and Uchino 2005). However, installation of nettin g may not be an economically feasible control technique for growers with large operations. The most effective control techniques are insecticides that target the adult stage. There are no insecticides that target the larval stage inside of the berries. The most common chemicals used by conventional blueberry growers for SWD control are Delegate 25 WG (spinetoram, Dow AgroSciences LLC, Indianapolis, IN), Mustang Max (zeta cypermethrin, FMC Corp, Philadelphia, PA), and Malathion 8 EC (malathion, Arysta LifeScience North America, LLC, Cary, NC). A direct spray of all

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26 these chemicals resulted in 100 percent mortality of adult SWD (Bruck et al. 2011). However, not all SWD will be killed on contact in a blueberry field Mustang Max provided 100 percent adult mortalit y after 10 d of residual activity, Malation provided signific ant adult mortality up to 7 d whereas Delegat e provided control up to 3 d (Bruck et al. 2011). Organic growers have a limited number of chemicals available to them including Entrust (spinosad, Dow AgroSciences LLC, Indianapolis, IN) and Pyganic (pyrethrin, McLaughlin Gormley King Co., Minneapolis, MN). Very few tools are available for chemical control resulting in multiple applications of the same chemicals as SWD populations reach damaging levels. Furthermore, SWD is at a high risk of pesticide resistance due to its rapid development time.

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27 Figure 2 1 Male spotted wing drosophila. Photo courtesy of L. E. Iglesias.

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28 Figure 2 2 Female spotted wing drosophila. Photo courtesy of L. E. Iglesias.

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29 Figure 2 3 Serrated ovipositor of the female spotted wing drosophila. Photo courtesy of L. E. Iglesias.

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30 Figure 2 4 SWD larval instars. Clockwise from right, first, second, and third. Photo courtesy of L. E. Iglesias.

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31 Figure 2 5 Three pupal instars. From left, first, second, and third instars. Photo courtesy of L. E. Igles ias.

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32 CHAPTER 3 SURVEY FOR SPOTTED WING DROSOPHILA IN FLORIDA BLUEBERRIES The spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) is an invasive fruit fly pest from East and Southeast Asia (Kanzawa 1935, Markow and first described in Japan by Dr. Shounen Matsumura in 1931 as Leucophenga suzukii then amended by Kanzawa to Drosophila suzukii cherry fruit fly (Kanzawa 1935). Since then, SWD has been recorded in Korea, Canada, Thailand, Russia, China, India, Spain, and Italy (Haus er e t al. 2009, Calabria et al. 2012 Walsh et al. 2011). In the U.S. SWD has been established in Hawaii since at least 1980 and its first continental appearance was in California in 2008 (Kaneshiro 1983, Steck et al. 2009, Walsh et al. 2011). It has since reach ed 37 U.S. states. The ecology of SWD and the climate of Florida may contribute to the potential threat of SWD to the blueberry industry The SWD has a wide host range that includes all thin skinned fruits and some stone fruits (Walsh et al. 2011) Given humid climate and occasional hard freezes in the north and north central regions, commercial SWD hosts grow throughout most of the year. Strawberry ( Fragaria ananassa Duchesne) harvest extends from December through March, blueberry season is late March through July, peaches and nectarines ( Prunus spp.) from April until late May or early June, blackberries ( Rubus spp.) are May through July, and grapes [ Vitis spp. (bunch and muscadine) ] occur June through September. Additionally, potential alternative non crop hosts may serve as hosts between cropping seasons including wild blackberry, nightshade ( Solanaceae spp.) and wild holly ( llex spp.) Weather conditions during highbush blueberry season are ideal for SWD reproduction. In mid April as the southern highbush blueberry harvest season begins to

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33 peak in north Florida, average temperatures were 20 and 23 C in Alachua County in 2012 and 2013 respectively (FAWN 2013). An earlier report indicated that SWD was found to be most active at 20C (Ka nzaw a 1935 ). The SWD also has a rapid development time and can comple te its lifecycle in 12 to 15 d at 18.3C Development time can be even shorter averagi ng 9 d 15 h at 25C ( Kanzawa 1939 Walsh et al. 2011 ). Early studies reported u p to 13 generations per year for SWD when observed in captivity (Kanzawa 1939) The SWD is predicted to complete 3 to 9 generations in the western U.S. and Canada based on degree day models (Walsh et al. 2011). Given potentially experience up to 10 or more generations as predicted in California (Walsh et al. 2011). There have been multiple confirmed and unconfirmed reports of SWD throughout Florida in blueberries and other fruits since its first detection in Florida in 2009 (Steck et al. 2009) In a recent moni toring study, 409 SWD adults were captured in six Florida counties in blueberry plantings during the 2011 blueberry growing season (Liburd 2011 unpublished). A n extensive survey in the major blueberry growing regions will better help understand the po tential risk posed by SWD to the Florida blueberry industry. Furthermore, effective monitoring for SWD will guide control actions and protect s the crop during the production season The ultimate goal of this study was to determine the extent to which SWD populations were present in commercial Florida blueberry plantings. The specific objective was to survey for SWD in commercial and non commercial blueberry plantings using the current ly recommended trap and bait system.

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34 Materials and Methods Survey Sites The survey w as conducted over two consecutive blueberry growing seasons, 2012 and 2013. In 2012 we s urveyed nine Florida counties from north to south including, Suwannee Alachua Putnam Marion, Citrus, Lake Orange Polk and DeSoto (Figure 3 1). During 2013 we su rveyed eight counties. The counties included all of those surveyed for 2012 except DeSoto County. This county is the southernmost county surveyed and it was excluded from the 2013 survey because no SWD was found in 2012. The counties selected represented g reater than 70 percent of harvested blueberry acreage in Florida in 2007 according to the most recent survey ( ERS USDA 2012). A total of 28 survey sites were selected in the various counties during 2012 and 2013 (14 in each year). Among the 28 sites ther e were 24 conventionally managed sites, 2 organically managed, and 2 with separate plantings dedicated to both conventional and organic management onsite (Table 3 1) Sites in the southern most counties were chosen where farm managers or extension agents offered assistance in sample collection to help reduce travel required for the weekly sample collections. Training on proper sample collection techniques was provided to all farm managers and extension agents to eliminate variance due to procedural differ ences and to minimize errors in the data. Site Characteristics Blueberry bushes at survey sites were between 4 and 8 y old and approximately 1.5 m high. In the northern region of the state (Suwannee, Alachua, Putnam, and

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35 Marion counties) sites were surrounded mostly by rural residential or small farm lands. All location s in Putnam and Alachua counties were on low lying lands prone to flooding as a result of heavy rains and were equipped with freeze protection. Suwannee and Marion county sites were at research centers and were bordered by forest or agricultural plots. In the central region (Citrus, Lake and Orange counties) surrounding areas were small berry or vegetable farms or natural landscapes consisting of oak hammocks. Many of the sites we re within 1 km of a lake or low lying wetlands. Sites in the southern region of the survey (Polk and DeSoto counties) in general were surrounded by rural, non agricultural residenti al areas with mostly landscap e vegetation. Monitoring Traps Since SWD causes injury to berries as a result of larval development and upon insertion of the ovipositor into the skin of the berry, traps were set in each field during the full green fruit stage just as they were changing to pink. Lee at al. (2011) showed that SW D had a higher percentage of larval development in the fully ripe blueberries than in the earlier stages. Initial trap set date for each site for both seasons varied based on the ripening stages of the blueberry fruit. During 2012 t he first traps were s et on 7 February and trap setup con tinued until all traps were set by 11 April 2012. In 2013 the first traps were set on 6 February and con tinued until all traps were set at the field sites by 17 April 2013. Traps remained in the field through the peak harvest period and were removed by 31 May in 2012 and 29 May in 2013. Two to six traps were establis hed at each site with at least one trap set in the center of the planting and the remaining along the perimeter. The number of traps assigned to the perimet er or the center w as dependent on the size of the planting Sites that were gre ater than 20.2 ha had at least two traps placed within the center of the

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36 planting. Perimeter traps were hung within 3.1 m of the edge of the field. Traps were hung in the shade in the center of the blueberry bush using a twist tie since Walsh et al. (2011) showed traps in shady areas perform better than traps placed in the direct rays of the sun. Traps were made with 0.95 L clear plastic cups with lids (Solo, Urbana, IL) wit h eight t o ten 6.35 mm holes along the upper rim of the cup (Liburd and Iglesias 2013) The trap used in 2012 included a yellow band ( visual stimulus ) inserted inside the trap whereas the trap for the 2013 survey did not (Figures 3 2, 3 3). The yellow band was tho ught to increase visual attraction to the trap. However o ur trapping study in year one ( C hapter 5 ) showed that the yellow band did not increase captures of SWD. Each trap contained 150 mL of apple cider vinegar (ACV) [ 5% acetic acid ( Winn Dixie, Jacksonville, FL) ]. The apple cider vinegar was used as bait since previous research had shown it to be attractive to SWD (Steck et al. 2009, Walsh et al. 2011) O dorless, colorless dish soap (Palmolive Pure and Clear, Colgate Palmolive Com pany, New York, NY) was added to the bait to act as a surfactant to help reduce the surface tension of the ACV and prevent fly escape. Sample Collection Samples were collected weekly and transported back to the University of Florida Small Fruit and Vegetable IPM (SFVIPM) l ab oratory in Gainesville, Florida for processing. Sample collection consisted of pouring all liquid bait into a collection container in the field and refilling the trap with fresh bait to the 150 mL level pre marked on the trap. Male and female SWD were counted a nd identified using keys from and Vlach (2010)

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37 Data Analysis Data were square root transformed before analysis to stabilize the variances A one way analysis of variance ( ANOVA ) was used to dete rmine whether there were differences among means of SWD captured for each county per year. Mean were considered significant when P test was used to compare mean differences for female, male, and total SWD captured between 2012 and 2013 (JMP, SAS Institute 2013) test was used because variances were considered unequal by for equal variances a mong female, male, and total SWD captured for each year of the survey (JMP, SAS Institute 2013) Results T he mean SWD captured in 2012 was significantly higher than in 2013 ( t = 3.95; df = 100 0.9 ; P < 0.0001, Table 3 3 ). There were a total of 844 and 49 8 SWD captured in 2012 a nd 2013, respectively (Table 3 2 ). There were significant differences in SWD captures among counties in 2012 ( F = 22.02; df = 8, 584; P < 0.0001) and 2013 ( F = 2.52; df = 7, 645; P < 0.0001, Table 3 4 ). Citrus County had the high est mean captures in 2012 ( 4.81 0 .31). Marion County ( 1.44 0.37) and Alachua County ( 1.34 0.20) were significantly higher than Orange (0.53 0.17) and Suwannee County (0.05 0.49) in 2013. DeSoto County did not have any captures during the 2012 s eason and therefore was not included in the 2013 survey. Sl ight differences were found when evaluating differences between years for each county. Citrus County had significantly higher SWD captured in 2012 ( t = 7.46 ; df = 144.3 ; P < 0.0001) compared wit h 2013. Alternatively, in Marion County there were significantly more SWD captured in 2013 compared with 2012 ( t = 2.81 ; df = 44.3 ; P =

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38 0.0074 ) (Figure 3 4) All other counties had similar captures between the two years in the study. Twice as many fema les as males were captured throughout the study. There were a total of 911 females and 431 males capture d throughout the 2 y study (Table 3 2 ). The mean number of females captured in 2012 was significantly greater than the number captured in 2013 ( t = 3.85 ; df = 100 2.8 ; P = 0.0001, Table 3 3 ). Likewise, the mean number of males captured in 2012 were also higher than in 2013 ( t = 3.01; df = 991.4 ; P < 0.0027 ). The overall ratio of female to male SWD was 2.06 (568:276) in 2012 and 2.21 (343:155) in 201 3. In 2012, there were significantly more females captured than males in Alachua ( t = 3.83; df = 260 ; P = 0.0002 ), Putnam ( t = 3.72 ; df = 86.8 ; P = 0.001 6 ), and Citrus counties ( t = 3.34 ; df = 196 ; P = 0.0010 ) (Figure 3 5) A similar trend was seen in t he 2013 survey year (Figure 3 6). Significantly more females were captured in Alachua ( t = 4.34 ; df = 177 ; P 0.0001 ) Putnam ( t = 3.47 ; df = 58.9 ; P = 0.0010 ) Citrus ( t = 2.41; df = 284 ; P = 0.0167 ) and Polk counties ( t = 1.99; df = 91.6 ; P = 0.0493 ). Discussion Our result s showed that SWD is present in the major blueberry producing regions of Florida. The differences between SWD population in 2012 and 2013 may be the result of a number of factors including temperature differences between the years and Weekly mean temperatures averaged below 20C until the first week in May 2013 ; conversely during the early part of 2012 temperatures averaged between 20 and 23C. The SWD has shown decreased activity at temperatures below 20C and above 25C (Kanzawa 1935, Kimura 2004).

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39 W hen the survey began in 2012 many growers were hearing about SWD for the first time or were unaware of its presence in damaging numbers in Florida. As a result growers were not im plementing management programs for this pest. As we obtained weekly capture data the growers were notified and management actions were taken based on our recommendations As the 2013 season began, blueberry growers implemented control plans for SWD earl y in the season especially if SWD was recorded at the site during the previous year. This shift in awareness and proactive management approach of growers may have contributed to the decrease in the numbers of SWD during 2013. Further statewide surveys wi ll help to answer whether the current management programs are effective at reducing SWD populations to the next season Spotted wing drosophila captures varied by county throughout the state. The highest mean captures were in the central (Citrus Co.) and north central (Alachua Co.) parts of the state. Citrus County is a unique location with many factors that can contribute to higher numbers of SWD. Citrus County has a large number of organically managed blueberries. The tools available for control of SW D for organic blueberry growers are extremely limited. Chemical control options are only limited to Entrust ( S pinosad, Dow AgroSciences, Indianapolis, IN) and Pyganic (pyrethrin, MGK, Minneapolis, MN). Entrust has been shown to be an effective tool aga inst SWD adults in lab and field trials (Bruck et al. 2011). Alternatively, lower activity on SWD has been recorded for the insecticide Pyganic In fact only direct spray s against SWD adults result ed in reduced activity without any form of residual effe ctiveness in field trials (Bruck et al. 2011). Many of these organic growers resorted to cultural techniques

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40 including sanitation methods such as removing culls and stripping the bushes entirely of ripe fruit, with only partially successful results. Secon dly, there are several growers in Citrus County who grow strawberries adjacent to blueberries. Strawberries are a known host of SWD and the crop seasons overlap to a limited extent in the field. Blueberries begin harvesting in March and the strawberry season cl imax es in March/ April depending on market price, temperature and pest pressure (Nyoike and Liburd 2013 ). Spotted wing drosophila is an opportunistic species and moves from one fruit crop to another as the season progresses allowing population numbers to increase rapidly. Alachua County had the second highest mean SWD capture s in both years of our study and has the largest number of blueberry growing hectares in the state. The greater the number of hecta res the greater risk Alachua is for SWD losses. In addition, Alachua County has a large number of blueberries being grown under high tunnels. High tunnels cover the blueberries during the winter for protection from freezes and cold temperatures. The tunnels act as greenhouses, creating artificial warm environments that promote SWD development Blueberries grown in high tunnels ripen much sooner than field berries and as a result are the first U.S. berries on the market each season. Early ripening varieties of host fruits have been shown to be at a g reater risk to in jury by SWD (Burrack et al. 2013 ) ; however, it is unclear whether this is the case for Florida Spotted wing drosophila populations are high er in the tunnels than in the field early in the season potentially increasing the risk for injur y. If these populations are not managed they may pose a greater risk to field berries if and when they migrate toward new resources (Coyne et al. 1982).

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41 Marion County had the highest mean SWD captures in 2013 but was among the lowest counties in 2012 (T able 3 4). The field site located in Marion County was at the University of Florida Plant Science Research and Education Unit in Citra, Florida. This location is not a commercially managed planting and is used for research purposes only. During 2012, th ese plantings had severe problems with cedar wax wing ( Bombycilla cedrorum Vieillot ) pests. As a result many of the berries were lost to feeding injury or as fallen culls. In 2013, however, the cedar wax wings were not a concern and there was a la rge amount of fruit on the bushes throughout the season. Therefore, there was a much greater amount of food and reproductive resources available to SWD in 2013 than in 2012. This may explain the significantly higher number of SWD in Marion County in 2013 than in 2012. DeSoto and Suwannee Counties had the lowest SWD captured during the survey, which may have been influence d by cl imate. DeSoto was the southern most county in the study and as such experien ced the warmest temperatures earlier in the year c ompared with other sites High temperatures in DeSoto typically reach 30 C in early to mid April (FAWN 2013). Conversely, t he survey sites located in Suwannee County grew mostly rabbiteye blueberries. Rabbiteye blueberries ripen dur in g the summer month s when temperatures are much warmer. The spotted wing drosophila is most active at 20 C ( Kanzawa 1935). In addition, SWD males are believed to become sterile in temperatures above 30 C ( Kanzawa 1935); however, m ore research is needed to determine wheth er this is valid in Florida conditions More females than males were captured during 2012 and 2013. One possible explanation for this trend is that the apple cider vinegar bait used in the traps for the

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42 survey is more attractive to females than males. B echer et al. (2012) found that female D. melanogaster were more attractive to food related odors, ethyl acetate and balsamic vinegar, than were their male counterparts. Furthermore, f emale flies require more food than males due to egg production and are constantly sea rching for oviposition sites from which the food related odors originate ( Becher et al. 2012). It is possible that using food related odors as baits for SWD such as apple cider vinegar may result in a greater number of female flies captured. The re sults of this study indicate that SWD is present in 8 or the 9 major blueberry growing counties in Florida. round growing season, SWD is likely to become a concern for blueberry growers in the seasons to co me. Growers are encouraged to continue to monitor for this invasive pest to remain current on the possi ble threat to their blueberries and to be proactive in their management programs for this pest.

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43 Table 3 1 : Counties and sites included in 2012 and 2013 survey for SWD, including number of traps deployed at each site. 2012 County Number of Sites Surveyed Total Traps Deployed Suwannee 1 7 Alachua 3 16 Putnam 1 6 Marion 1 5 Citrus 2 13 Lake 2 8 Orange 1 5 Polk 2 12 DeSoto 1 3 2013 Suwannee 1 3 Alachua 3 17 Putnam 1 6 Marion 1 3 Citrus 2 12 Lake 2 6 Orange 2 7 Polk 2 12 DeSoto 0 0 Table 3 2 Total number of SWD captured in 2012 and 2013 survey seasons. Year Samples Female Male F:M Ratio Total SWD 2012 593 568 276 2.06 844 2013 653 343 155 2.21 498 Total 1246 911 431 2.11 1342

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44 Table 3 3 Mean male, female, and SWD capture per sample for year 2012 and 2013 of SWD survey. Year 2012 2013 Female Mean SEM 0.96 0.10 0.53 0.06 t value 3.85 df 10 02.8 P value 0.0001* Male Mean SEM 0.47 0.06 0.24 0.04 t value 3.01 df 991.4 P value 0.0027 SWD Total Mean SEM 1.42 0.01 0.76 0.09 t value 3.95 df 1000.9 P value <0.0001*

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45 Table 3 3 Mean number of SWD captured in each county in the 2012 and 2013 survey study. Mean SEM* County 2012 2013 Suwannee 0.29 0.47c 0.05 0.49b Alachua 1.65 0.25b 1.34 0.20a Putnam 0.47 0.41bc 0.41 0.33ab Marion 0.21 0.44cA 1.44 0.37aB Citrus 4.81 0.31aA 0.90 0.19abB Lake 0.20 0.46c 0.41 0.27ab Orange 1.23 0.48bcA 0.53 0.17bB Polk 0.16 0.31cA 0.33 0.31abB DeSoto 0 0.68c --*SEM = standard error of the mean. No survey sites in DeSoto County in the 2013 survey season. Means with the same letter are not signifi cantly different ( P 0.05). Lowercase letters represent differences within columns. Capital letters represent differences across rows.

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46 Figure 3 1 Survey sites and counties for 2012 2013 blueberry seasons. Yellow place marks denote survey sites for 2012 and 2013. Yel that was only surveyed in 2012, not 2013. Color overlays are surveyed Florida counties.

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47 Figure 3 2 Plastic cup trap with yellow stimulus used during the 2012 survey year Photo courtesy of L. E. Iglesias.

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48 Figure 3 3 Plastic cup trap without yellow stimulus used for 2013 survey Photo courtesy of L. E. Iglesias.

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49 Figure 3 4 Mean SWD captured by county for 2012 and 2013 survey years. Asterisk (*) indicates significant differences at P 0.05 among survey years. 0 1 2 3 4 5 6 Moean SWD per Sample CountY (North to South) 2012 2013

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50 Figure 3 5 Mean female and male SWD captured in each county for 2012. Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean SWD County (North to South) Female Male

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51 Figure 3 6 Mean female and male SWD captured in eac h county for 2013 Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean SWD County (North to South) Female Male *

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52 Figure 3 7 Mean temperatures in survey areas for 2012 and 2013. Week number begins February 1, 2012 to June 1, 2012. 0.00 5.00 10.00 15.00 20.00 25.00 30.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean Temperature ( C) Week 2013 2012

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53 Figure 3 8 M ean precipitation in survey areas in 2012 and 2013. Week number begins February 1, 2013 to June 1, 2013. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean Precipitation (cm) Week 2013 2012

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54 CHAPTER 4 OVIPOSITION PREFERENCE OF SPOTTED WING DROSOPHILA IN SOUTHERN HIGHBUSH AND RABBITEYE BLUEBERRIES The ability to appropriately time insectic ide applications is critical for the effective management of a key pest. Therefore, determining the stage when the s potted wing drosophila (SWD) Drosophila suzukii (Matsumura) will oviposit into blueberries will help to improve the timing of pesticide a pplications against adults. The SWD is known to oviposit in ripening fruits (Kanzawa 1935, Lee et al. 2011, Walsh et al. 2011, Burrack et al. 2013, Liburd and Iglesias 2013); however, information on the stage of ripening in blueberries when SWD will oviposit is still unclear. The SWD has a wide host range that includes thin skinned fruits such as blueberries ( Vaccinium spp.) strawberries ( Fragaria ananassa Duchesne ), caneberries ( Rubus spp.) grapes ( Vitis spp.), and cherries ( Prunus spp.) as well as some known stone fruits like peaches ( Prunus persica L.) nectarines [ P. persica (L.) Batsch ] and plums ( Prunus salicina Lindl.) (Steck et al. 2009, Walsh et al 2011, Burrack et al. 2013). It is a frugivore pest that utilizes host f ruit s for feeding and reproduction. Frugivores generally lay t he i r eggs in locations that provide the best opportunity for larval survival. Lee et al. (2011) found that SWD preferred to oviposit in blackberries, blueberries, cherries, raspberries, and strawb erries over grapes in laboratory choice tests. Fu r ther investigations show that host preference for SWD increased as brix level rises (Lee et al. 2011) In a recent study with no choice tests, Burrack et al. (2013) found no differences in eggs laid in va rious blackberry maturity stages. We hypothesized that SWD will show host preference to fully ripe blue berries over green and pink fruit.

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55 We chose to conduct assays mainly on southern highbush (SHB) blueberry because it has a higher risk than rabbiteye (RE) species of loss due to injury by SWD in Florida. Rabbiteye has characteristics dissimilar to SHB varieties, such as a grittier texture, larger seeds, and tougher skins (Lyrene and Moore 2006). Some potential factors influencing host preference are brix levels, pH, skin thickness and firmness (Lee et al. 2011 Burrack et al. 2013). Given the characteristics of RE we predict it will be less susceptible to SWD than SHB In addition, RE ripens later in the summer months i n Florida than SHB and may be less at risk for infestation by SWD. B erry stage preference helps determine when pest management tactics need to be implemented including monitoring for SWD and application of pesticides The objectives of the study wer e to : 1) evaluate the suitability of RE and SHB blueberry species (at different berry maturity stages ) for SWD larval development and 2) to evaluate SWD oviposition preference s for different ripening stages of SHB blueber ries. To add r ess these objectives we conducted no choice and choice laboratory bioassay experiments. Materials and Methods Source of Flies Spotted wing drosophila flies were obtained from a laboratory colony reared in an environmental chamber (Model I36VL Percival Scientific, Inc., Perry, IA) at the University of Florida, Small Fruit and Vegetable IPM ( SFVIPM) l aboratory in Gainesville, Florida. Chamber conditions were maintained at 23C with 16:8 h light:dark cycle and RH ~65%. Flies were reared on Formula 4 24 (Caroli na Biological Supply, Burlington, NC) in 0.25 L polypropylene bottles (Applied Scientific, Kalamazoo, MI) with foam stoppers (Jaece, North Tonawanda, NY). Flies used in both experiments were sexually

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56 mature (4 to 7 d old ) They were removed from polyp ropylene bottles using an air pu mp and placed into 50 mL plastic vials with screw caps The plastic vials were placed in the freezer for 90 s to immobilize the flies before they were transferred to bioassay chambers. Blueberry Bushes The blueberries use d in both studies were selected from a b lueberry planting located at the University of Florida, Plant Scien ce Research and Education Unit ( PSREU ) in Citra, Florida Blueberry branches were transported back to the SFVIPM laboratory in Gainesville, Florida There were four block s of SHB blueberry bushes and four block s of RE blueberry bushes (approximately 4 to 6 years old and 1 m tall) plan ted 1 m apart Blocks were blocked by surrounding vegetation. Each block contained 8 rows eac h with 25 blueberry bushes. Each row had 5 different varieties each with 5 bushes Pine bark was used as mulch for blueberry rows. Blueberries were watered daily with drip irrigation, and no other chemicals had been used in the plantings for pest manage ment. Prior to the start of laboratory bioassays, larval tests were performed in salt solution to ensure that berries were maggot free ( Hueppelsheuser 2010) Larval salt tests were completed by randomly collect ing at least 30 ripe berries from each block and placing th em in a resealable plastic bag. Berries were l ightly crush ed in the bag and covered with a salt solution ( 68.25 g salt to 0.95 L water ) The fruit were allowed to sink to the bottom of the bag for approximately 10 to 15 min and larvae, if present, to flo at to the top of the bag. None of the berries tested were infested with larvae.

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57 Suitabi lity of Rabbiteye and Southern Highbush Blueberry Species for SWD Larval D evelopment A no choice laboratory bioassay was used to determine the suitability of RE and SHB blueberry species f or SWD larval development The experiment was a two way factorial design with 4 replicates. B lueberries species and berry maturity stage were the effects The two blueberries species tested were southern highb ush ( Vaccinium corymbosum L. x V. darrowi Camp) and rabbiteye ( Vaccinium virgatum Aiton ). The 4 berry maturity stages evaluated were : 1) green, 2) green pink, 3) pink, and 4) blue. Thirty two be rries were randomly selected from each of RE and SHB s pecies from a planting at the PSREU in Citra, Florida. Two berries were selected from each of the 4 maturity stages. Maturity stages and species were separated into individual bioassay containers (32 containers with 2 berries each) Ten mated females were introduced into the chambers using the freezing method described above The bioassay chamber consisted of a 0.5 L clear plastic deli container (Solo, Urbana, IL) with a mesh lid (Figure 4 1). A 30 mL container with a cotton wick was filled with 15 mL of 1 M of sugar water to feed the flies and placed in the treatment container. Bioassay chambers were placed on the laboratory bench under grow lights on a 16:8 h light:dark cycl e at a mean temperature of 22.8 C. Female flies remained in the bioassay chamber for 96 h before they were removed. Berries were kept for 14 d after flies were removed to check for emergence Suitability of host was measured by the number of adult SWD that emerge from the berries. The mean number of eme rged SWD per berry was calculated. A two way analysis of variance ( ANOVA ) was used to determine effects of blueberry species,

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58 maturity stage and their interaction on SWD emergence if any. Significant means were (JMP, SAS Insti tute 2013) Berry Maturity Stage A choice laboratory bioassay was used to evaluate SWD oviposition preference s for different ripening stages of SHB blueberries The experimental was completely randomized design with 5 treatments and 24 rep licates Treatments represented berry maturity stages and include d : 1) blue, 2) pink blue, 3) pink, 4) green pink, and 5) green Forty eight branches were randomly selected from two SHB blueberry varieties Emerald and Jewel Branches were selected that had stems at least 7.6 cm long and at least one berry for each treatment stage. Branches were placed in re sealable plastic bags in an ice cooler, and transported back to the S FVIPM labora tory in Gainesville Florida Two branches, one branch from each variety w ere placed into a bioassay chamber. Two varieties were used in this study to provide SWD with an alternative in case females had a preference for one variety over another and to ensure enough berries were available for the study. Each b ioassay chamber consisted of a 1 L clear plasti c deli container and a mesh lid (Figure 4 2 ). The chambers had a 35 mL plastic vial filled with tap water in which a foam stopper and two branches were placed. T he vial was secured in a 30 mL container to prevent movement within the chambe r and risk killing flies. A 30 mL container with a cotton wick was filled with 15 mL of 1 M sugar water to feed the flies and placed in the treatment container. Bioassay chambe rs were placed on the laboratory bench under grow lights on a 16:8 h light:dark cycle at a mean temperature of 22.8 C.

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59 Once the branches were secured in the bioassay chamber 10 mated females were introduced following freezing procedures detailed above. Females rema ined in the chambers for 72 h. Oviposition events were measured by observing and counting the number of females ovipositing on berries. Females were observed at least two times daily for 5 min One oviposition event was counted when the abdomen of the f emale was curled and her ovipositor was extended into the skin of the blueberry. A female was recorded as having oviposited multiple times during one observation only if she moved to a different berry before ovipositing again. The mean number of oviposi tion events per treatment was calculated per observation. A one way ANOVA was used to evaluate mean differences among berry (JMP, SAS Institute 2013) Results Suitability of R abbiteye and Southern H ighbush Blueberry Species for SWD Larval D evelopment The mean number of SWD that emerged from the RE blueberry species was 0.63 0.18 per berry compared with 1.06 0.18 from SHB blueberries (Figure 4 3) Although SHB blueberry appeared almost two times more suitable than RE the data were not significantly different at P ( F = 3.02; df = 1, 24; P = 0.10). T he effect of berry maturity (ripening) stage was highly significant ( F = 9.25; df = 3, 24; P = 0.00 03) (Figure 4 4) The mean emergence from blue berries was significantly higher t han all other stages No differences were found among the other stages. Additionally, color* blueberry species interaction effects were not significant ( F = 0.36; df = 3 24 ; P = 0.79)

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60 SWD O viposition E vents and P references for Different Ripening Stages of Southern Highbush B lueberries Overall, t he mean number of oviposition events per observation for all treatments was low (Table 4 1) but there were significant differen ces among treatments ( F = 3.38; df = 4, 325; P = 0.0098). T here were a significantly higher number of oviposition events on blue berries than there were on p ink blue berries (Figure 4 5). More than half of the events ( 50 %) occurred on blueberries, 38 % on green pink berries, 6 % on both pink and green berries, and zero events took place on pink blue berries (Figure 4 6 ). Discussion T here were no significant differences between number of emerged SWD from the RE and SHB blueberry species at P SWD from SHB was two times greater than that of RE Considering the h igh degree of variability among biological organisms the difference between species at P = 0.10 could be considered biologically significant These results suggest that the characteristics of RE blueberry that make it different from SHB (grittier texture, firmer skin, and larger seeds) may play a role in host suitability. Similarly results from Burrack et al. (2013) found in choice and no choice assays with agar medium, SWD would not oviposit at the highest tested firmness (in their study penetration force) of 52.00 cN. These results may suggest that SWD will reject a host if the firmness is too high. N either firmness nor other possible characteristics were quantified in this study and therefore cannot necessarily be assumed to be different between species. The firmness and brix content of these two species will need further research before fir m conclusions can be drawn. Subsequent experiments should include quantifying possible related factors.

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61 T he number of female flies to berries in the bioassay chambers does not mimic the conditions in a blueberry field, where the number of female SWD is likely to be much lower than the number of blueberries present. Intraspecific competition plays a key role in oviposition site selection in some Drosophila species (Chess and Ringo 1985) and may have played a similar role in this study In a study by Che ss and Ringo ( 1985 ) Drosophila melanogaster and Drosophila simulans laid s ignificantly more eggs on media that had not been pre conditioned with Drosophila larvae suggesting that these species prefer oviposition sites that have not been oviposited on p rev iously In addition, the same study showed that larval survival w as significantly lower in media with larvae present. Due to the high female to berry ratio in my study it is possible that more than one larva could have been developing inside the berries As discussed by Chess and Ringo (1985) it is possible that the survival of larvae may have been reduced due to intraspecific larval competition for resources. In the choice tests, female flies preferred to oviposit on fully ripe blue berries over pink b lue berries however, no clear pattern was present among ripening stages Conversely, i n no choice tests significantly more flies emerged from blue blueberries than all other stages. The s e finding s suggest that though oviposition preference may not differ drastically among stages, suitability of each stage for larval development might In a study by Lee et al. ( 2011 ) the mean number of oviposition events was only slightly lower for green pink blue berries than it was for blue berries. Furthermore, the same s tudy found no significant differences between eggs laid on blue berries and green pink berries. Since larval development may vary depending on medium and competition, larval survivorship should be i ncluded in subsequent studies.

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62 The re sults of our study s uggest that both species of blueberries that are grown in the southeastern U.S. are susceptible to larval infestation by SWD Southern highbush blueberries appear 50% more suitable than RE blueberries ( P = 0.10) Additionally, SWD will oviposit on earl ie r ripening stages including mature green berries. Blue berries appear to be the most susceptible stage but growers should begin their management programs (monitoring and spraying) early in the season when berries are full green since flies are capa ble of ovipositing in these berries. It will be important for growers to manage both RE and SHB in a similar fashion until more information becomes available.

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63 Table 4 1. Mean oviposition events per observation on different berry ma turity stages in choice study. Stage Mean SEM* Green 0.02 0.03ab Green Pink 0.10 0.03ab Pink 0.02 0.03ab Pink Blue 0 0.03b Blue 0.12 0.03a Mean s followed by the same letters are not significantly different at P

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64 Figure 4 1 : Bioassay chamber for no choice experiment Photo courtesy of L. E. Iglesias.

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65 Figure 4 2. Bioassay chamber for choice experiment. Photo courtesy of L. E. Iglesias.

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66 Figure 4 3: Mean SWD emerged from s outhern highbush and rabbit eye species in no choice bioassay. Bars with the same letters are not significantly different at P 0.05. 0 2 4 6 8 10 12 14 Mean SWD Emerged per Berry Blueberry Species Southern Highbush Rabbiteye

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67 Figure 4 4 Mean SWD emergence from different berry maturity stages in no choice tests. Bars with the same letters are not significantly different at P 0.05. b b b a 0 1 2 3 4 5 6 7 8 9 Green Green-Pink Pink Blue Mean SWD Emerged per Berry Maturity Stage Rabbiteye Southern Highbush

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68 Figure 4 5 Mean oviposition events per observation on different maturity stag es in choice study. Bars with the same letters are not significantly different at P 0.05. 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Green Green-Pink Pink Pink-Blue Blue Mean Oviposition Event per Observation Maturity Stage a b ab ab ab

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69 Figure 4 6 Percent oviposition on different stages of blueberry maturity stages in choice bioassays Blue 50% Pink Blue 0% Pink 6% Green Pink 38% Green 6%

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70 CHAPTER 5 EVALUATION OF TRAPS AND BAITS FOR MONITORING OF SPOTTED WING DROSOPHILA IN BLUEBERRIES Effective monitoring is the cornerstone for a successful i ntegrated pest management (IPM) program for a key invasive pest. Monitoring involves collecting biological information on pests and beneficials, as well as da ta on climate and crop phenology (Flint and Gouveia 2001). Traps are important tools that are frequently used in pest management program s to guide management actions The effectiveness of a trap is dependent on several factors including attractant, siz e, color, and ease of handling. The most effective traps detect pest populations before the action threshold is reached so that growers can implement control measures before economic damage occurs The spotted wing drosophila (SWD), Drosophila suzukii (Mat sumura) (Diptera: Drosophilidae), is an invasive pest, causing devastating economic loss es in blueberries up to 40 percent in California in 2008 (Bolda et al. 2010) and 10 to 15 percent in Florida in 2012 (eFly 2012). Due to its rapid development time it s populations can increase quickly (Walsh et al. 2011) Therefore effective m anagement programs must be developed to combat this invasive pest. Current monitoring methods for SWD employ the use of a trap and lure system. Trap designs have been based on traps for other Drosophila spp. and generally use a container with a lid, entry holes, and a liquid bait that also acts as a drowning solution. In the past, several types of traps have been tested for SWD including handmade plastic cup s with 2 to 10 entr y holes, traps with mesh entries, and plastic cups with tents to provide shade and prevent water from entering (Kanzawa 1939, Wu et al. 2007, Lee

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71 (Basoalto et al. 2013) wit h varying degrees of success Insects are attracted to colors that have the same or similar spectral reflectance patterns as their host (Prokopy and Owens 1983). Differences in the attractiveness of colors for some insects are related to their reflectan ce values and the ability of the insect to perceive reflected light at certain wavelengths (Roessingh and Stadler 1990). Color is an easily manipulated feature of a trap that could potentially affect trap attractiveness. Recent studies investigating diff erences in color preference of SWD showed a higher affinity toward darker colors such as red and black (Lee et al. 2012, Basoalto et al. 2013). However, these studies also indicated that clear was just as attractive as colored traps Therefore we chose to employ clear traps for our trap comparison studies. Yellow has been shown to be attractive for many insects including plant feeding dipterans because it depicts a foliage type band (Prokopy and Owens 1983). M any traps used for monitoring SWD population s employ a yellow sticky card inside of the trap to act as a visual stimulus and prevent flies from escap ing (Beers et al. 2011, Walsh et al. 2011, Klick et al. 2012, Bellamy et al. 2013). Preliminary trapping studies by Landolt et al. (2012a) indicated t hat a dome type trap with a yellow bottom captured more SWD than a clear trap We wanted to determine whether the color yellow would act as a visual stimulus and improve trap captures for SWD. Drosophilids are able to walk on the surface of a liquid due to special structures on the distal tarsal segments (Walker 1992, Triplehorn and Johnson 2005). Odorless dish detergent is added to liquid baits to break the surface tension, causing the flies to

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72 drown. Adding dish detergent could be an inexpensive and e asy method for increasing captures ( if effective ) In our treatment design, w e included a trap baited with apple cider vinegar with and without a drop of odorless dish detergent to test the effect of the dish detergent on SWD captures Baits developed for SWD have been based on previous studies on the attractiveness of fermentation products such as ethanol, acetic acid, and methanol to other Drosophila spp. (Reed, 1938, Becher et al. 2010, Lebreton et al. 2012). The first recommended traps used for SWD mo nitoring in the U.S. were vinegars and wines. However, there is variability between different kinds of vinegars and wines (Kanzawa 1935 ; Landolt et al. 2012a, 2012b). Kanzawa (1935 ) found that molasses mixed with a home brewed red wine was more attractiv e than t he two components on their own as well as cherry juice, rice wine, sugar water, and a variety of botanical oils. In a recent study, Landolt et al. (2012a ) found that apple cider vinegar was more attractive than red grape wine (Merlot). C ombinatio ns of w ines and vinegars show ed promise when used as bait for SWD. For instance, r ice vinegar and red wine vinegar when added to a red grape wine (Merlot) were significantly more attractive than the red grape wine alone ( Landolt et al. 2012b). We chose t o include the most attractive combination of rice Drosophila spp. has been associated with multiple yeast species found during gut dissection. Drosophila feed on the yeasts of fermenting food sour ces as well as the juices from the source itself. Hamby et al. (2012) found that Drosophila suzukii also shows this association with yeasts from fermenting berries. In addition, results from Becher et al. (2012) suggest that SWD is attracted to the yeast s associated with

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73 fermentation, not the fermentation volatiles themselves. Recent baits for SWD have been a yeast sugar water mixture and one with the addition of whole wheat flour and this bait study. A pple cider vinegar remains a common bait because it is clear, has longevity in the field, preserves specimens, and is inexpensive. The objective of this study was to determine the relative attractiveness of various types of trap designs and commonly recom mended baits in blueberry fields. Additionally, the ease SWD identification and servicing associated with each bait is discussed. Materials and Methods Experimental D esign The experimental sites were located within the center of commercial blueberry plant ings. Experimental designs were completely randomized with 4 replicates (rows) Each replicate consisted of one double planted row approximately 1 m wide and at least 60 m long. A buffer zone of a t least 15 m separated each replicate Each row was plante d with multiple varieties of southern highbush (SHB) blueberries. Pine bark was used as mulch for blueberry rows. Blueberries were watered daily with drip or overhead irrigation. Bushes were planted approximately 1 m apart and the height ranged from 1 to 1.5 m. Trap Comparison Study Two experiments were conducted during the 2012 trapping study. Experiment 1 was conducted in high tunnels at a conventional blueberry operation located in Alachua County. The experiment ran from 25 January until 10 April 20 12. Experiment 2 was conducted from 19 April to 10 May 2012 in the fields of an organically managed plan ting located in Citrus County.

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74 Two experiments were conducted for the 2013 trapping study (experiments 3 and 4) Experiment 3 was conducted in a field planting at a conventional blueberry operation located in Alachua County. The experiment ran from 18 April to 23 May 2013. Exper iment 4 was conducted at the same organically managed planting in Ci trus County as the 2012 study and took place from 3 May t o 22 May 2013. All locations in 2012 and 2013 were included in our survey study (C hapter 3) and were being monitored closely for SWD presence. The trapping studies began when SWD populations were high and the berries were fully ripe and therefore startin g dates differed at each location. In 2012 five trap designs were evaluated all baited with apple cider vinegar (ACV). Four o f the traps were made from 0.95 L clear plastic deli containers with lids (Solo, Urbana, IL) and ten 0.64 cm holes along the upper rim. Two of the cup treatments had yellow visual stimuli (30 cm wide foamboard) placed around the rim of the cup, one of which had odorless, colorless dish soap (Palmolive Pure and Clear, Colgate Palmolive Company, New York, NY) added to the bait to help reduce the surface tension of the ACV and prevent fly escape. The last two cup treatments were one with a small yellow sticky card (7.6 x 7.6 cm) hanging inside and a standard transparent cup (without the yellow sticky card) The final trap was a standard yellow sticky card (15.2 x 20.3 cm) used for monitoring flying insects (Great Lakes IPM, Vestaburg, MI), acting as the control. Plastic c up traps were baited with 150 mL of ACV that also acted as a drowning soluti on. The yellow sticky card had a 7.4 mm vial containing ACV attached as a lure. Based on results from the 2012 trap ping study, 4 trap designs were evaluated in 2013. Three of the traps used in 2012 were re evaluated in 2013: basic cup, cup with

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75 yellow band around the rim and soap, and cup with yellow sticky card inside. The cup trap with the yellow band around the rim (without soap) and the yellow sticky card were excluded from the treatments (based on 2012 results). A fourth trap similar to the one u sed in 2012 with the yellow sticky card inside was adopted, however this trap was baited with a 150 mL yeast sugar water mixture. T he yeast bait was made with 14.8 g 39.4 g of white sugar, and 710 mL of tap water (Table 5 1). With the excep tion of the fourth trap all other traps were baited with 150 mL ACV. Bait Study The bait study was conducted at an organically managed blueberry planting located in Citrus County, FL. The study ran from May 8 to May 27, 2013. There were 4 bait treatmen ts evaluated using the standard plastic cup trap ( 0.95 L clear plastic deli containers with lids and ten 0.64 cm holes along the upper rim) Treatments evaluated were: 1) ACV plus dish detergent 2) yeast sugar water mixture and 4) Landolt 1). Sample Collection Samples were collected weekly and transported back to the University of Florida Small Fruit and Vegetable IPM (SFVIPM) l ab oratory in Gainesville, Florida for processing. Sample collection consisted of pouring all l iquid bait into a collection container in the field and refilling the trap with fresh bait to the 150 mL level pre marked on the trap. Yellow sticky cards in the trapping studies were replaced weekly with fresh cards. Traps were rotated each week to prev ent positional bias. Male and female SWD were identified and counted for each sample collected (Triplehorn and Johnson 2005,

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76 SWD, other Drosophila spp. were collected in the traps. These specimens were also recorded. Data Analysis In the trapping studies, a one way analysis of variance ( ANOVA ) was used to determine whether there were differences among means of the SWD captured for each treatment for each experiment. Mean com (JMP, SAS Institute 2013). Data were square root transformed to standardize the variances. Means were considered significant ly different when P Results from each experiment are presented separately for the 2012 and 2013 trapping studies. The data from the bait study were square root transformed to standardize the variances. A one pare the means of SWD captured (JMP, SAS Institute 2013) test was used to compare mean differences among female and male SWD captured in all experiments (JMP, SAS Institute 2013) test was used s test for equal variances (JMP, SAS Institute 2013) Means were considered significant ly different when P Results Trap Comparison Study In 2012, e xperiments 1 and 2 showed similar trends for mean SWD captured among treatments (Figures 5 1, 5 2 ). The yellow sticky card (control) did not capture any SWD through out the duration of the experiments. The basic cup, cup with the yellow band and cup with the yellow band and the soap captured significantly more SWD than the yellow sticky card in experiment 1 ( F = 6.28 ; df = 4 95 ; P = 0.000 2) T he cup with the yellow sticky card was not significantly differ ent than the other cup traps or

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77 the sticky card In experiment 2, all of the cup treatments captured significantly more SWD than the yellow sticky card ( F = 6.28 ; df = 4 95 ; P = 0.000 2) There were no significant differences among the four cup treatment s in either experiment 1 or 2. The number of females was significantly greater than the number of males in the basic cup ( t = 1.9, df = 37.1, P = 0.0617 ) cup with the yellow band ( t = 2.7, df = 42.1, P = 0.0097 ) and cup with the yellow band and the d ish soap ( t = 2.2, df = 47.8, P = 0.0321 ) in experiment 1 (Figure 5 3 ). In experiment 2, no significant differences were found among number of female and male SWD captured for each treatment (Figure 5 4). In 2013, t he results from both experi ments were si milar (Figures 5 5, 5 6 ). The commonly recommended trap design with the yellow sticky card and the yeast mixture captured significantly greater number of SWD in both experiments ( F = 10.12; df = 3, 108 ; P < 0.0001 ) The yeast trap captured a mean of 1.44 and 10.83 in experiments 1 and 2, respectively. There were no differences among any of the cup traps baited with ACV in both experiments. The trap with the yellow band did not cap ture any SWD during experiment 3 No significant differe nces ( P 0.05 ) in number of females and males captured were observed in any of the traps in 2013 (Figures 5 7, 5 8). Bait Study Results of the bait study showed significant differences among t he bait treatments ( F = 11.7; df = 3, 43, P < 0.0001 ) (Figure 5 9 ). Both mean = 12.91) and the yeast sugar water bait (mean = 6.36) had significantly greater mean captures than the ACV bait ( mean mean = 0.67). None of the treatments captured different number s of females and males (Figure 5 1 0 ).

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78 The bait treatments also attracted other dipteran species, specifically other Drosophila spp. and the recent invasive fig fruit fly, Zaprionus indianus Gupta (Figure 5 11 ). The number of Drosophila spp. captured were significantly different ( F = 10. 3; df = 3, 43, P < 0.0001 ) number of Drosophila spp. ( mean = 59.17) Significant differences were found in Z. indianus ( F = 7.32; df = 3, 43, P = 0.0005) capturing significantly greater numbers of Z. indianus ( mean = 21.33) Discussion The results of our 2012 trapping study support previous findings that the color yellow does not increase trap captures of SWD (Lee et al. 2011 and Basoalto et al. 2013). The ACV traps with the yellow sticky card inside were not significantly different from other ACV traps (without the yellow sticky card) T he trap with the yellow sticky card and the yeast captured significantly more SWD than the same trap with ACV This may suggest that the bait is a more important factor in attracting SWD flies than the visual stimulus of the yellow sticky card. The yellow sticky card may catch a few SWD; however, this tends to complicate matters since identification o f SWD can be difficult when flies land on the sticky card We noticed that specimens tend to desiccate rapidly, requiring immediate identification. sugar water mixes) were s ignificantly more attractive than the vinegar baits (ACV and associated yeasts and bacteria as well as the fruit material from damaged or fallen 2008, Hamby et al. 2012). This may explain the

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79 attractiveness of the yeast baits. In addition, odors of fermenting fruit baits such as wine or vinegar may be masked by the odors from the surrounding fruit. All baits come with their advantages and disadv antages. The vinegar baits are generally clear for easy identification in the field or lab, have longevity in the field, and act as decent preservatives for collected specimens. The wine baits may be clear if using white wine but red wines will quickly s tain the flies. The yeast sugar water mixtures are cloudy and have sediments that make identification difficult and timely. and timely because it was the same color as t he flies and the grains were too large to be sifted out. In addition, none of the baits tested in the study are specific to SWD but to many other species of insects attracted to sweet or fermenting odors (Landolt 1995). Other drosophilids are especially a ttracted to the odors of fermenting fruit and yeasts (Reed et al. 2010, Becher et al. 2012, Landolt et al. 2012a, Landolt et al. 2012b, Lebreton et al. 2012, Steck et al. 201 Drosophila spp. with a mean of 59.17 per trap. Traps with high numbers of non target species make identifying SWD difficult and timely. The size of the entry holes can be designed small enough to prevent entry of larger insects such as hemipterans and lepidopterans but unless a specific bait for SWD is developed, other vinegar flies will likely be found in SWD traps. The effectiveness of the trap and bait system for capturing SWD is more dependent upon the attr activeness of the bait than the cup trap modifications evaluated

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80 in SHB blueberry fields. Therefore the basic cup trap would be recommended as it requires less time to construct and is less ex pensive than its modified counterparts. An effec tive bait is not only attractive to the target pest but also promotes quick and easy construction, handling, and pest identification. Benefits also include longevity of the bait in the field and preservation of specimens. Due to the highly destructive po tential of SWD, intense monitoring programs in blueberries and other thin skinned fruits are recommended. Growers must consider whether they value a highly attractive bait such as a yeast sugar g and

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81 Table 5 2 : Bait study treatments and mixtures. Bait Ingredient Manufacturer Amount Trap amount ACV ACV 5% acetic acid, Winn Dixie, Jacksonville, FL 150 mL 150 mL (ACV) Dish detergent Palmolive Pure and Clear, Colgate Palmolive Company, New York, NY drop Yeast Sugar Water Yeast Cordova, TN 14.8 g 150 mL (YSW) Sugar white granulated, Publix, Lakeland, FL 39.4 g Water tap 710 mL Rich's Mix (Rich) Yeast Cordova, TN 29.6 g 150 mL Sugar white granulated, Publix, Lakeland, FL 118.3 g Wheat flour King Arthur Flour Co ., Inc., Norwich, VT 59.1 g ACV 5% acetic acid, Winn Dixie, Jacksonville, FL 29.6 mL dish detergent Palmolive Pure and Clear, Colgate Palmolive Company, New York, NY few drops water tap 710 mL Landolt's Mix Rice Vinegar 25% acetic acid, Korea 280 mL 150 mL (Land) Red Grape Wine Merlot 12% ethanol, Carlo Rossi, Modesto, CA 420 mL Dish detergent Palmolive Pure and Clear, Colgate Palmolive Company, New York, NY few drops

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82 Figure 5 1 : Mean spotted wing drosophila (SWD) captured per t rap in 2012 trapping study, experiment 1. Treatments included the basic cup trap (cup), cup trap with a yellow band (cup+yell), cup trap with yellow band and odorless dish soap (cup+yell+soap), cup trap with a yellow sticky card (cup+YSC), and a yellow st icky card (YSC). Bars with the same letters are not significantly different at P 0.05. a a a ab b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean SWD per Trap Treatment

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83 Figure 5 2 : Mean spotted wing drosophila ( SWD ) per trap in 2012 trapping study, experiment 2. Treatments included the basic cup trap (cup), cup trap with a yello w band (cup+yell), cup trap with yellow band and odorless dish soap (cup+yell+soap), cup trap with a yellow sticky card (cup+YSC), and a yellow sticky card (YSC). Bars with the same letters are not significantly different at P 0.05. a a a a b 0 0.5 1 1.5 2 2.5 3 3.5 Mean SWD per Trap Treatment

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84 Figure 5 3 : Mea n male and female spotted wing drosophila ( SWD ) captured in 2012 trapping study, experiment 1. Treatments included the basic cup trap (cup), cup trap with a yellow band (cup+yell), cup trap with yellow band and odorless dish soap (cup+yell+soap), cup trap with a yellow sticky card (cup+YSC), and a yellow sticky card (YSC). Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean SWD per Trap Treatment Female Male

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85 Figure 5 4 : Mean male and female spotted wing drosophila ( SWD ) captured in 2012 trapping study, experiment 2. Treatments included the basic cup trap (cup), cup trap with a yellow band (cup+yell), cup trap wi th yellow band and odorless dish soap (cup+yell+soap), cup trap with a yellow sticky card (cup+YSC), and a yellow sticky card (YSC). Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured. 0 0.5 1 1.5 2 2.5 Mean SWD per Trap Treatment Female Male

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86 Figure 5 5 : Mean spotte d wing drosophila ( SWD ) captured per trap in 2 013 trapping study, experiment 3 Treatments included the basic cup trap with a yellow sticky card baited with a yeast sugar water mixture (yeast+YSC), cup trap with a yellow sticky card with apple cider vineg ar bait (ACV+YSC), cup trap with a yellow band with ACV and odorless dish soap (ACV+yell), and a basic cup trap with ACV and odorless dish soap (ACV). Bars with the same letters are not significantly different at P 0.05. a b b b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Yeast+YSC ACV+YSC ACV+Yell ACV Mean SWD per Trap Treatment

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87 Figure 5 6 : Mean spotted wing drosophila ( SWD ) captured per trap in 2 013 trapping study, experiment 4 Treatments included the basic cup trap with a yellow sticky card baited with a yeast sugar water mixture (yeast+YSC), cup trap with a yellow sticky card with apple cider vinegar bait (ACV+YSC), cup trap with a yellow band with ACV and odorless dish soap (ACV+yell), and a basic cup trap with ACV and odorless dish soap (ACV). Bars with the same letters are not significantly different at P 0.05 a b b b 0 2 4 6 8 10 12 14 Yeast+YSC ACV+YSC ACV+Yell ACV Mean SWD per Trap Treatment

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88 Figure 5 7 : Mean male and female spotted wing drosophila ( SWD ) captured per trap in the 2 013 trapping study, experiment 3 Treatments included the basic cup trap with a yellow sticky card baited with a yeast sugar water mixture (yeast+YSC), cup trap with a yellow sticky card with apple cider vinegar bait (ACV+YSC), cup trap with a yellow band with ACV and odorless dish soap (ACV+yell), and a basic cup trap with ACV and odorless dish soap (ACV). Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Yeast+YSC ACV+YSC ACV+Yell ACV Mean SWD per Trap Treatment Female Male

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89 Figure 5 8 : Mean male and female spotted wing drosophila ( SWD ) captured per trap in the 2 013 trapping study, experiment 4 Treatments included the basic cup trap with a yellow sticky card baited with a yeast s ugar water mixture (yeast+YSC), cup trap with a yellow sticky card with apple cider vinegar bait (ACV+YSC), cup trap with a yellow band with ACV and odorless dish soap (ACV+yell), and a basic cup trap with ACV and odorless dish soap (ACV). Asterisk (*) in dicates significant differences at P 0.05 among female and male SWD captured. 0 1 2 3 4 5 6 7 8 Yeast+YSC ACV+YSC ACV+Yell ACV Mean SWD per Trap Treatment Female Male

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90 Figure 5 9 : Mean spotted wing drosophila ( SWD ) captured during 2013 bait study. All treatments used the basic cup trap design. Treatments included apple cider vinegar (ACV), yeast sugar water mixture (YSW), Ri Bars with the same letters are not significantly different at P 0.05. b a a b 0 2 4 6 8 10 12 14 16 18 ACV YSW Rich Land Mean SWD Captured Treatment

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91 Figure 5 10 : Mean female and male spotted wing drosophila ( SWD ) captured during 2013 bait study. Treatments included apple cider vinegar (ACV), yeast sugar Asterisk (*) indicates significant differences at P 0.05 among female and male SWD captured. 0 1 2 3 4 5 6 7 8 9 10 ACV YSW Rich Land Mean SWD Captured Treatment Female Male

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92 Figure 5 11 : Mean Drosophila spp. and Zaprionus spp. captured per tra p in the 2013 bait study. Treatments included apple cider vinegar (ACV), yeast sugar water Bars with the same letters are not significantly different among treatments at P 0.05. b b a b b ab a b 0 10 20 30 40 50 60 70 80 ACV YSW Rich Land Mean Flies per Trap Treatment Drosophila spp. Zaprionus spp.

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93 CHAPTER 6 EVAL UATING ALTERNATIVE CHEMICAL TOOLS FOR CONTROL OF SPOTTED WING DROSOPHILA The spotted wing drosophila (SWD), Drosophila suzukii (Matsumura), causes direct injury to blueberry plants by scarring the berry upon insertion of the ovipositor and by the rapid det erioration of berries as a result of larval development. Y ield losses in Florida blueberries in 2012 due to SWD injury have been estimated at 7.8 to 11.7 mil USD (eFly 2012). Therefore, developing an effective management program is essential for growers to protect their crops and their livelihoods. An integrated pest management (IPM) program is comprised of a number of strategies and techniques based on knowledge of the pest, specific site characteristics, and market values. Currently Florida blueberry growers have several cultural control techniques available for SWD management. Sanitation by removing fallen, damaged or overripe berries from the fields and destroying them reduces areas for SWD reproduction. Short harvest intervals help to keep ripe fr uit off of the bushes during the time of the season when SWD populations are likely to increase. In addition, exclusion netting with mesh size less than 0.98 mm can prevent SWD entry by 100 percent (Kawase and Uchino 2005). However, cultural controls hav e their limitations as they can be costly and timely for large blueberry operations and are most effective when coupled with chemical control strategies. Chemical controls that target the adult stage of SWD are currently the most effective technique for controllin g SWD populations. Conventional growers most commonly use Delegate 25 WG (spinetoram, Dow AgroSciences LLC, Indianapolis, IN), Mustang Max (zeta cypermethrin, FMC Corp, Philadelphia, PA), and Malathion 8 EC (malathion, Arysta LifeScience North America, LLC, Cary, NC) (Liburd and

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94 Iglesias 2013). A direct spray of all these chemicals in laboratory tests resulted in 100 percent mortality of adult SWD (Bruck et al. 2011). However, some flies will not be in contact with a direct spray in the field. Tests on residual activity showed that Mustang Max provided 100 perce nt adult mortality after 10 d Malation provided signific ant adult mortality up to 7 d and Delegat e provided control up to 3 d (Bruck et al. 2011). A chemical toolbox full of ava ilable alternatives will give a grower the flexibility to choose the best option for their operation by taking into consideration chemical cost, the chemical class to prevent resistance by rotating chemicals, reentry and pre harvest intervals and worker, c onsumer, and environmental safety. The purpose of this study was to provide growers with additional tools to use for SWD control. To do this we conducted a field based laboratory bioassay to evaluate the residual effects of various chemicals on 1) adult SWD mortality as a result of contact on blueberry branches and 2) larval survival to the next generation in blueberries. Materials and Methods A field based laboratory bioassay was conducted during the spring 2012. Various insecticides from different clas ses were applied recommended rate to blueberries in the field. Blueberry branches with fruit were transported bac k to the laboratory. Spotted wing drosophila adults were exposed to treated branches in bioassays chambers and adult mortal ity and larval emergence were recorded. Source of Flies Drosophila suzukii flies were obtained from a laboratory colony reared in an environmental chamber (Model I36VL, Percival Scientific, Inc., Perry, IA) at the University of Florida, Small Fruit and V egetable IPM (SFVIPM) l aboratory in Gainesville,

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95 Florida. Environmental conditions were maintained at 23C with 16:8 h light:dark cycle and RH ~65%. Flies were reared on Formula 4 24 instant Drosophila medium (Carolina Biological Supply, Burlington, NC ) in 0.25 L polypropylene bottles (Applied Scientific, Kalamazoo, MI) with foam stoppers (Jaece, North Tonawanda, NY). Spotted wing drosophila used in both experiments were 4 to 7 d old. Flies were transferred from culture bottles using an air pump in to plastic vials. Flies were immobilized by placing the plastic vials in the freezer for 90 s and then introduced into the bioassay chambers. Field Setup The experimental field plot was located at t he University of Florida Plant Science Research and Ed ucation Unit ( PSREU ) in Citra, Florida. The experimenta l area was 62.2 by 59.4 m with four blocks blocked by surrounding vegetation Each block consisted of 8 rows 27.4 m long and 1 m wide. A 1.9 m wide grass buffer zone was established between each row, a 7.32 m buffer between the north and south blocks, and 13.72 m buffer between the east and west blocks. Each row had 25 bushes plan ted 1 m apart Each row was made up of 5 different varieties of southern highbush (SHB) blueberries each with 5 bushes Bushes were approximately 4 to 6 years old and 1 m tall. Pine bark was used as mulch for blueberry rows. Blueberries were watered daily with drip irrigation, and no other chemicals had been used in the plantings for pest management. The experiment was conduct ed in two phases between 10 April and 23 May 2012. The experimental field design was a randomized complete block with four replicates and seven treatments. Treatments were formulated insecticide products

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96 registered for use on small fruits Florida (Table 6 1). Treatments were appli ed using a back pack sprayer. A ll treatments were randomly assigned to a row in each block. T here was an additional buffer row with no treatment between the control and the other treatments. The additional buffer row was included ad jacent to the control to prevent any risk of spray drift between treatment rows and the control row. Prior to the start of laboratory bioassays, larval tests were performed to ensure that berries were free from infestation ( Hueppelsheuser 2010). First, larval salt tests were completed by randomly collecting at least 30 ripe berries from each block and placing them in a resealable plastic bag. The berries were lightly cr ushed in the bags and some salt solution ( 59 mL salt to 0.95 L water ) was added. The fruit were allowed to sink to the bottom of the bag for approximately 10 to 15 min. No larvae floated to the top of the solution and were therefore considered uninfested by the salt test. Laboratory Setup One d ay after insecticide application, two branches were selected from blueberry varieties (Emerald and Jewel) in each treatment row, placed in resealable plastic bags in an ice cooler, and transported back to the SFVIPM laboratory in Gainesville Florida Branches were selected that had stems of at least 7.6 cm l ong and at least five berries at different ripening stages. One branch from each variety was placed into a bioassay chamber for males and the other two branches into a chamber for females. This procedure was repeated for all treatments 3, 7, and 14 d aft er insecticide application. Bioassay chamber consisted of 1 L transparent plastic deli container with a mesh lid (Figure 6 1). Ea ch treatment container had a 35 mL plastic vial filled with tap water in which a foam stopper and two branches were placed. The vial was secured in a 30

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97 mL container to prevent movement within the chambe r and risk killing fli es. A 30 mL container with a cot ton wick was filled with 1 M of sugar water placed in the treatme nt container to feed the flies Bioassay chambers w ere placed on the laboratory bench in a completely randomized design under grow lights with a 16:8 h light:dark cycle at a mean temperature of 22.8 C. Once the branches were secured in the bioassay chamber 10 mated females and 10 males were introduced into separate containers following freezing procedures detailed above. Flies remained in the chambers for 72 h. Data Collection and Analysis Mean daily temperature ( C), relative humidity (%), and total rainfall (cm) were collected using FAWN (Florida Autom ated Weather Network, Gainesville, FL) for the duration of the study (Figures 6 2, 6 3) Adult activity Adult activity measurements were recorded 72 h after the flies were introduced into the bioassay containers, just prior to removal. Data were taken by picking up the container and gently tapping the sides to elicit an activity response from the flies. Fly activity was measured on a scale of 0 to 3, using methods described in Liburd et al. (2003). A score of 3 indicated unaltered fly activity (fly in i ts natural state). A score of 2 indicated decreased responsiveness to tapping. A score of 1 indicated no responsiveness to tapping and a general inverted, twitching appearance. Fly death was designated a score of 0. Flies that died unrelated to the ins ecticides such as drowning in sugar solution, vial movement, or berry drop, were omitted from analysis. Contai ners were o bserved for 5 min

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98 The total number of flies in each activity category (0 through 3) was weighted based on their categorical numbe r. The weighted total was then averaged by the number of responding flies in each container (total flies minus omitted flies). Data were square root transformed to standardize the variances. Means were separated using analysis of variance ( ANOVA ) and Du Differences were considered significant when P Larval survival (emergence) Larval survival (emergence) was measured by counting the number of flies that emerged from the berries in each chamber containing f emale flies. Emergence was recorded 48 h after the first fly emerged in each container. Therefore, the start of the 48 h emergence period differed slightly for each container. The total number of emerged flies was averaged by the number of berries in ea ch container. Data were square root transformed to account for unequal variances. Analysis of variance ( ANOVA ) was used (JMP, SAS Institute 2013). Differences were considere d significant when P Results Adult Activity There was a significant treatment effect on SWD activity on 1 d ( F = 8.14, df = 6, 47; P <0.0001), 3 d ( F = 2.21; df = 6, 47; P = 0.05), and 7 d post treatment ( F = 2.49; df = 6, 47; P = 0.04). On 1 d Delegate Mustang Max Danitol high, and Danitol low significantly reduced SWD activity level below the control whereas no significant differences were found between Belay high, Belay low and the control. On 3 d post treatment Danitol high reduced acti vity more than Belay low and the control. Additionally, Danitol high, Danitol low, and Mustang Max significantly reduced

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99 SWD activity below the control. Treatment effects were significant whereas gender effects were not ( F = 1.61; df = 1, 47; P = 0.2 1) on 7 d post treatment Both Danitol treatments reduced activity significantly more than Delegate Belay low and the control. On 14 d post treatment neither gender ( F = 1.28; df = 1, 47; P = 0.26) nor treatment effects ( F = 0.89; df = 6, 47; P = 0.51) were significant. Wh en evaluating means among days within treatments neither Belay treatments showed any significant differences. Both Danitol trea tments reduced activity on 1 d and 3 d more than 14 d post treatment whereas 1 d was lower than 3 d post treatment (Figure 6 4). Mustang Max and Delegate reduced activity on 1 d post treatment more than all other days. Only Danitol low had significant treatment differences between gender ( F = 12.86; df = 1, 24; P = 0.0015); activity was reduced more for males than it was for females. On 1 d post treatment Dan itol low, Dan itol high, and Mustang Max reduced male activity greater than female activity. On 3 d post treatment no significant differences were found. Danitol low reduced male activity significantly greater than female activity on 7 d post treatment Belay hig h showed a significant reduc tion in male activity on 14 d post treatment Larval Survival On 1 d post treatment all treatments had lower number of emerged flies per berry than did the control ( F = 3.47; df = 6, 20; P = 0.02). There were no significant differences between trea tments and the control on 3 d ( F = 0.57; df = 6, 21; P = 0.75), 7 d ( F = 1.38; df = 6, 21; P = 0.27), or 14 d post treatment ( F = 1.29; df = 6, 21; P = 0.31). Discussion Our study identified effective insecticides for SWD management. Re sults indicate that Mustang Max Delegate and Danitol high and low rates were effective

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100 at reducing SWD activity up to 3 d residual. After 7 d residual, Danitol at the high and low rates was more effective than Mustang Max and Delgate There were slight differences between the effect of the chemicals on female and male activity. On 1 d post treatment, Danitol at both rates, Mustang Max and Delegate reduced male more t han female activity and on 7 d post treatment only Da nitol at the low rate reduced male more than female activity. The efficacy of Danitol in our study supports recent findings (Beers, et al. 2011, Bruck et al. 2011) that Danitol e xhibits residual control up to 7 d In addition, no significant di fferences were found in SWD activity between the high and low rates of Danitol suggesting that the low rate would be an effective alternative tool for controlling SWD in the field. Danitol is a pyrethroid in class 3A like Mustang Max which is one of the most commonly used insecticides against SWD. Growers are recommended to rotate chemical application by chemical class to prevent pest resistance to effective chemicals. Pesticide resistance occurs when resistant individuals survive and produce resist ant offspring. Rotating to a different chemical class will help to ensure no resistant individuals in the population survive to carry on the resistant gene. Although Danitol and Mustang Max are in the same chemical class, they differ in the preferred t iming of application within the season due to their preharvest interval s (PHI). Mustang Max has a PHI of only 1 d making it attractive to growers during peak harv est when harvest intervals are 3 to 4 d In contrast, Danitol has a 3 d PHI and would be most effective during early season before harvest when SWD populations are beginning to increase. Delegate and Belay are labeled as reduced risk chemicals by the EPA because they have lower toxicity to non target organisms, short re sidual ac tivity, and

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1 01 are slower acting Delegate has been shown to be effective at reducing SWD populations up to 3 d on treated cherry leaves (Beers et al. 2011) and up to 7 d on blueberries (Bruck et al. 2011). Delegate also has a 3 d PHI which may limit its use during peak harvest. Belay is a class 4A insecticide, a neonicotinoid. Neonicotinoids are systemic synthetic chemicals based on the chemical nicotine and are absorbed into Neonicotinoid s mimic acetylcholinesterase, the enzyme that breaks down acetylcholine, blocking the binding cites and allowing Though clothianidin [Belay] has not been evaluated on SWD pri or to our study, other neonicotinoids have shown to be less effective than pyrethroids [Danitol and Mustang Max], organophosphates [Malathion], or spinosyns [Delagate] by knockdown (Bruck et al. 2011) and by residual exposure (Beers et al. 2011). Overa ll, Mustang Max and Delegate remain effective tools for control of SWD up to 3 d in the field. Previous studies sug gest their effectiveness up to 7 d (Beers et al. 2011, Bruck et al. 2011). Mustang Max is suggested for use d uring peak ha rvest as it has a 1 d PHI. In addition, Danitol at the low rate of 0.75 L ha 1 is an effective tool for control duri ng the early season due to its 3 d PHI. Belay appears to be an ineffective tool for managing SWD. Future studies may investig ate more effective methods of application such as chemigation (the application of pesticides through overhead irrigation systems), aerial applications or applications baited with a sweet lure such as sugar.

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102 Table 6 1. Insecticide treatments for efficacy s tudy 2012. Trade Name Treatment Code Chemical Manufacturer AI Rate (h 1 ) Belay Insecticide Bel lo clothianidin Valent U.S.A Corporation, Walnut Creek, CA 0.29 L Belay Insecticide Bel hi clothianidin Valent U.S.A Corporation, Walnut Creek, CA 0.44 L Da nitol 2.4 EC Dan lo fenpropathrin Valent U.S.A Corporation, Walnut Creek, CA 0.75 L Danitol 2.4 EC Dan hi fenpropathrin Valent U.S.A Corporation, Walnut Creek, CA 1.17 L Mustang Max Mus zeta cyperpethrin FMC Corp., Philadelphia, PA 0.29 L Delegate 2 5 WG Del spinetoram Dow AgroSciences LLC, Indianapolis, IN 0.42 g Control Cont Table 6 2 Mean activity level per fly between treatments within each day after application. Bel4 Bel6 Dan10.3 Dan16 Mus4 Del6 Cont Day 1 2.5 0.2 2.2 0.2 1.2 0.2* 1.2 0.2* 1.0 0.2* 0.9 0.2* 2.7 0.2 Day 3 2.5 0.2 2.3 0.2 1.9 0.2* 1.8 0.2* 2.1 0.2* 2.1 0.2* 2.6 0.2 Day 7 2.7 0.2 2.6 0.2 2.2 0.2* 2.2 0.2* 2.5 0.2 2.6 0.2 2.8 0.2 Day 14 2.7 0.2 2 .4 0.2* 2.7 0.2 2.6 0. 2 2.3 0.2 2.6 0.2 2.9 0.2 Asterisk (*) indicates significant differences when compared to the control at P < 0.05. Low activity due to replicate losing all male flies to the freezing process.

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103 Table 6 3 Mean activity level per fly for female SW D between treatments within each day after application. Trt Bel4 Bel6 Dan10.3 Dan16 Mus4 Del6 Cont Day 1 2.6 0.1 2.4 0.0 1.8 0.1* 1.7 0.1* 1.7 0.1* 1.0 0.4* 2.7 0.1 Day 3 2.4 0.1 2.5 0.1 2.2 0.1* 2.2 0.1* 2.0 0.2* 2.4 0.1 2.8 0.1 Day 7 2.7 0.0 2.7 0.1 2.5 0.1* 2.3 0.1* 2.6 0.1 2.5 0.0* 2.9 0.0 Day 14 2.9 0.1 3.0 0.1 2.8 0.0 2.9 0.0 2.6 0.1* 2.6 0.0 2.9 0.1 Asterisk (*) indicates significant differences when compared to the control at P < 0.05. Table 6 4 : Mean activity level per fly for male SWD between treatments within each day post treatment. Trt Bel4 Bel6 Dan10.3 Dan16 Mus4 Del6 Cont Day 1 2.6 0.1 2.3 0.1 1.0 0.2* 1.0 0.2* 0.8 0.2* 1.5 0.2* 2.9 0.1 Day 3 2.6 0.1 2.4 0.1 2.3 0.2* 2.0 0.1* 2.5 0.1 2.1 0.2* 2.8 0.1 Day 7 2.7 0.1 2.6 0.1 2.0 0.2* 2.3 0.2 2.4 0.1 2.8 0.1 2.7 0.1 Day 14 2.8 0.1 1.4 0.4* 2.7 0.1 2.6 0.1 2.4 0.2 2.7 0.1 3.0 0.0 Asterisk (*) indicates significant differenc es when compared to the control at P < 0.05. Low activity due to replicate losing all male flies to the freezing process. Table 6 5 : Mean emergence per berry. Trt Bel4 Bel6 Dan10.3 Dan16 Mus4 Del6 Cont Day 1 0.4 0.3b 0.2 0.3b 0.1 0.3b 0.0 0.3 b 0.2 0.3b 0.6 0.3b 1.4 0.3a Day 3 0.5 0.2a 0.4 0.2a 0.3 0.2a 0.2 0.2a 0.2 0.2a 0.5 0.2a 0.3 0.2a Day 7 2.3 0.5a 1.0 0.5ab 0.4 0.5b 0.5 0.5b 0.8 0.5ab 0.9 0.5ab 1.2 0.5ab Day 14 2.9 0.9a 1.1 0.9a 0.4 0.9a 1.2 0. 9a 0.7 0.9a 1.5 0.9a 2.5 0.9a Asterisk (*) indicates significant differences when compared to the control at P < 0.05.

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104 Bel lo Bel hi Dan lo Dan hi Mus Del Cont Buffer Figure 6 1: Field setup at UF IFAS PSREU in Citra, Florida

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105 Figure 6 2 Bioassay chamber for efficacy study Photo courtesy of L. E. Iglesias.

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106 Figure 6 3: Daily mean relative humidity (%) and temperature (C) at the UF IFAS PSREU in Citra, Florida for during of pesticide efficacy study 2012. Figure 6 4: Total daily rainfall (cm) at the UF IFAS PSREU in Citra, Florida for duration of pesticide efficacy study 2012. 0 5 10 15 20 25 30 0 20 40 60 80 100 4/9 4/11 4/13 4/15 4/17 4/19 4/21 4/23 4/25 4/27 4/29 5/1 5/3 5/5 5/7 Temperature ( C) Relative Humidity (%) Date RH avg T avg 0.0 1.0 2.0 3.0 4.0 5.0 6.0 4/9 4/11 4/13 4/15 4/17 4/19 4/21 4/23 4/25 4/27 4/29 5/1 5/3 5/5 5/7 Total Rainfall (cm) Date

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107 Figure 6 5 : Average Female and Male Activity per Day by Treatment. Asterisks (*) indicate significant differences at P < 0.05. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Mean Activity Day 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Day 3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Mean Activity Treatment Day 7 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Treatment Day 14

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108 CHAPT ER 7 CONCLUSION This study confirms the establishment of spotted wing drosophila (SWD) Drosophila suzukii (Matsumura) in Florida blueberries and emphasizes the importance of implementing effective monitoring programs to prevent economic loss. Spotted win g drosophila was found in 8 of 9 major blueberry producing counties in Florida in a 2012 and 2013 survey, from Suwannee County in the north to Polk County in the south. Spotted wing drosophila was not detected in DeSoto County suggesting that high daily t emperatures early in the season many play a role in preventing establishment of populations in more southern areas. Citrus County, which consists of a large number of organic blueberries and farms that produce other multiple SWD host crops, had the highes t SWD captured per trap in 2012 ( 4.81 0.31) Marion County (1.44 0.37) which also grows multiple crops and has organic blueberries, and Alachua County (1.34 0.20) which uses high tunnel production systems, captured significantly higher numbers of SW D than Orange ( 0.53 0.17) and Suwannee c ounties (0.05 0.49) in 2013 These results suggest that locations with limited chemical tools available for SWD control produce multiple host crops in succession, or employ high tunnel systems that support a lo nger growing season may be at a h igher risk of SWD infestation, but additional research is needed to test these conclusions. Th e study confirmed that SWD will oviposit in both species of blueberries grown in Florida, the southern highbush (SHB) and rabbite ye (RE). T he mean number of SWD that emerged from SHB was two times greater than that of RE and could be considered biologically significant. The findings indicate that the characteristics of RE blueberry that make it differen t from SHB (grittier texture, firmer skin, and larger seeds) may play

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109 a role in host suitability. However, more research on oviposition preference s and host is needed before firm conclusions are drawn In addition, SWD prefer to oviposit on ripe blue fruit 50% of the time, 6% on pink, 38% on green pink, and 6% on full green fruit. The se data suggest that monitoring should begin much earlier in the season when berries are in the green pink stage Trapping SWD with clear plastic cup traps bai ted with apple cider vinegar (ACV) performed was better more effective than a yellow sticky card baited with a vial of ACV Modifications of the cup trap baited with ACV included the addition of a yellow band, yellow sticky card hanging inside of the cup, or dish detergent in the bait These trap modifications did not appear to significantly increase trap captures When the modified c u p traps were compared with the standard plastic cup trap with a yellow sticky card inside baited with a yeast sugar w ater mixture, the yeast baited trap captured significantly more SWD than the ACV cup traps. This suggests that the bait contributed more to the capture of SWD than the design of the trap itself. The results of the bait study showed that the yeast baits sugar water mixes) were significantly more attractive to SWD than the vinegar baits attraction may be a result of adults and larvae of SWD and other drosophilids feeding upo n the associated yeasts and bacteria as well as the fruit material from damaged or fallen berries (Markow and In addition, odors of fermenting fruit baits such as wine or vinegar may be masked by the odors emitted from th e surrounding fruit. However, effective baits are also dependent upon ease of handling and SWD identification. Vinegar or wine baits are generally tinted but clear for easy identification

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110 in the field or lab oratory have longevity in the field, and act a s decent preservatives for collected specimens. The yeast sugar water mixtures are cloudy and have sediments that make identification difficult and timely. Additionally, the yeast baits were especially attractive to other Drosophila spp. and with high nu mbers of non target species SWD identification can be difficult and timely. When planning a monitoring program for SWD, growers must consi der whether they value a highly attractive bait such as a yeast sugar l make handling and identification quick and easy such as ACV Mustang Max and Delegate remain effective tools for control of SWD up to 3 d in the field. Previous studies suggest their effectiveness up to 7 d (Beer s et al. 2011, Bruck et al. 2011). Mustang Max is suggested for use during peak harvest due to its 1 d PHI. In addition, Danitol at the low rate of 0.75 L ha 1 is an effective tool for SWD control and is recommended for use during the early season due to its 3 d PHI. Belay appears to be an ineffective tool for managing SWD. Future studies should investigate the mechanisms behind the ineffectiveness of neonicotinoids on SWD as well as more effective methods of application such as chemigation (the a pplication of pesticides through overhead irrigation systems), aerial applications or applications baited with a sweet lure s such as sugar.

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111 LIST OF REFERENCES Arvalo, H. A. 2006. A study of the behavior, ecology, and control of flower thrips in bluebe rries towards the development of an integrated pest management ( IPM ) program in F lorida and southern Georgia Ph.D. dissertation University of Florida Gainesville, Florida. Arvalo, H. A. and O. E. Liburd 2007. Horizontal and vertical distribution of flower thrips in southern highbush and rabbiteye blueberry plantings, with notes on a new sampling method for thrips inside blueberry flowers. J. Econ. Entomol. 100: 1622 1632. Basoalto, E., R. Hilton, and A. Knight. 2013. Factors affecting the efficacy of a vinegar trap for Drosophila suzukii (Diptera; Drosophilidae). J. Appl. Entomol. (in press). (BCMA) British Columbia Ministry of Agriculture. 2012. Spotted wing drosophila (fruit fly) pest alert. British Columbia Ministry of Agriculture. Victoria, B.C., Canada. http://www.agf.gov.bc.ca/cropprot/swd.htm Becher, P. G., M. Bengtsson, B. S. Hansson, and P. Witzgall. 2010. Flying the fly: long range flight behavior of Drosophila melanogaster to attracti ve odors. J Chem. Ecol. 36:599 607. 2012. Yeast, not fruit volatiles mediate Drosophila melanogaster attrac tion, oviposition and development. Funct. Ecol. 26: 822 828. Beers, E. H., R. A. Van Steenwyk, P. W. Shearer, W. W. Coates and J. A. Grante. 2011 Developing Drosophila suzukii management programs for sweet cherry in the western United States. Pest Manag. Sci. 67: 1386 1395. Bellamy, D. E., M. S. Sisterson, and S. S. Walse. 2013. Quantifying host potentials: indexing postharvest fresh fruits for spotted wing drosophila, Drosophila suzukii. PLoS ONE 8: e61227. Birkhold, K. T., K. E. Koch, and R. L. Darnell. 1992 Carbon and nitrogen economy of developing blueberry fruit. J. Amer. Soc. Hort. Sci. 117: 139 145. Birmingham, A. L., E. Kovacs, J. P. LaFontaine, N. Avelino, J. H. Borden, I. S. Andreller, and G. Gries. 2011. A new trap and lure for Drosophila melano gaster (Diptera: Drosophi lidae). J. Econ. Entomol. 104 : 1018 1023. Bolda M., R. E. Goodhue, and R. G. Zalom. 2010. SWD: potential economic impact of a newly established pest. Giannini Found. Agric. Econ. 13: 5 8.

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112 Bruck, D. J., M. Bolda, L. Tanigoshi, J. Kl ick, J. Kleiber, J. DeFrancesco, B. Gerdeman and H. Spitler. 2011. Laboratory and field comparisons of insecticides to reduce infestation of Drosophila suzukii in berry crops. Pest Manag. Sci. 67: 1375 1385. Budick, S. A. and M. H. Dickinson. 2006. Free fl ight responses of Drosophila melanogaster to attractive odors. J. Exp. Biol. 209: 3001 3017. Burrack, H. J., G. E. Fernandez, T. Spivey and D. A. Kraus. 2013. Variation in selection and utilization of host crops in the field and laboratory by Drosophila su zukii Matsumara (Diptera: Drosophilidae), an invasive frugivore. Pest Manag. Sci. (in press). Calabria, G., J. Maca, G. Bchli, L. Serra and M. Pascual. 2012. First records of the potential pest species Drosophila suzukii (Diptera: Drosophilidae) in Europ e. J. Appl. Entol. 136: 139 147. Chakir, M., A. Chafik, B. Moreteau, P. Gibert, and J. R. David. 2002. Male sterility thermal thresholds in Drosophila: D. simulans appears more cold adapted than its sibling D. melanogaster Genetica 114: 195 205. Chess, K. F. and J. M. Ringo. 1985. Oviposition site selection by Drosophila melanogaster and Drosophila simulans Evol. 39: 869 877. Cini, A., C. Ioriatti, and G. Anfora. 2012. A review of the invasion of Drosophila suzukii in Europe and a draft research agenda fo r integrated pest man agement. Bull. Insectology. 65 : 149 160. Cook, M. A., S. N. Ozeroff, S. M. Fitzpatrick, and B. D. Roitberg. 2011 Host associated differentiation in reproductive behaviour of cecidomyiid midges on cranberry and blueberry. Entomol. Exp Appl. 141: 8 14. Coyne, J. A., I. A. Boussy, T. Prout, S. H. Bryant, J. S. Jones, and J. S. Moore. 1982 Long distance migration of Drosophila. Am. Nat. 119: 589 595. Dalton, D. T., V. M. Walton, P. W. Shearer, D. B. Walsh, J. Capriled and R. Isaacs. 201 1 Laboratory survival of Drosophila suzukii under simulated winter conditions of the Pacific Northwest and seasonal field trapping in five primary regions of small and stone fruit production in the United States. Pest. Manag. Sci. 67: 1368 1374. Darnell, R. L. 2006. Blueberry botany/environmental physiology pp. 5 13 In N. F. Childers and P.M. Lyrene (Eds.), Blueberries: f or growers, gardeners, promoters. Dr. Norm F. Childers Horticultural Publications, Gainesville, FL. Dean, D., J. F. Price, G. Steck, an d C. A. Nagle. Development and impact of the spotted wing drosophila, Drosophila suzukii in Florida strawberries. Int. J. Fruit Sci. 13: 67 75.

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113 Dernisky, A. K., Evans, R. C., Liburd, O. E., and Mackenzie, K. 2005. Characterization of early floral damage b y cranberry tipworm ( Dasineura oxycoccana Johnson) as a precursor to reduced fruit set in rabbiteye blueberry ( Vaccinium ashei Reade). Intl. J. Pest Manag 51: 143 148. Edwards, D. L., J. Lee, D. Bruck. 2012. Spotted wing drosophila monitoring: building a better fly trap pp. 30 34 In Proc eedings 71st Annu. Pac. Northwest Insect Manag. Conf. 9 10 January, 2012, Portland, OR. Integrated Plant Protection Center Oregon State University Corvalis, OR. eFly SWD Working Group. 2012. Spotted wing drosophila impacts in the eastern United States. http://www.sripmc.org/WorkingGroups/eFly/Impacts%20of%20SWD%20in%20th e%20Eastern%20US%202012.pdf ( ERS USD A ) Economic Research Service U.S. Department of Agriculture 2012a. Average farm gate value of blueberry production, selected s tates, 1980 2011. ERS USDA, Economics, Statistics and Mar ket Information System (ESMIS) Cornell University, Ithaca, NY. ( ERS USDA ) Economic Research Service U.S. Department of Agriculture 2012b. Cultivated blueberries: c ommercial acreage, yield per acre, production, and season average grower price in Florida, 1992 2011. ERS USDA, Economics, Statistics and Mar ket Information S ystem (ESMIS) Cornell University, Ithaca, NY. (FAO STAT ) Food and Agricultural Organization of the United Nations Statistics 2013 World blueberry production (tonnes) in 2011. Food and Agricultural Organization of the United Nations Rome, Italy. http://www.fao.org/statistics/en/ (FAWN) Florida Automated Weather Network. 2013. University of Florida, Gainesville. http://fawn.ifas.ufl.edu Flint, M. L. and P. Gouve ia. 2001 IPM in practice: principles and methods of integrated pest management. Pub. # 3418. University of California, Davis, California. Goodhue, R. E., M. Bolda, D. Farnsworth, J. C. Williams, and F. G. Zalom. 2011. SWD: infestation of California straw berries and raspberries: e conomic analysis of potential revenue losses and control costs. Pest Manag. Sci. 67: 1396 1402. Hamby, K. A., A. Hernndez, K. Boundy Mills, and F. G. Zalom. 2012. Associations of yeasts with spotted wing drosophila ( Drosophila su zukii ; Diptera: Drosophilidae) in cherries and raspberries. Appl. Environ. Microbiol. 78: 4869 4873.

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114 Hauser, M. 2011. A historic account of the invasion of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) in the continental United States, with remar ks on t heir identification. Pest Manag Sci. 67:1352 1357. Hauser, M. S. Gaimari, and M. Damus. 2009 Drosophila suzukii new to North America In S. D. Gaimari, Fly times. Plant Pest Diagnostics Branch California Department of Food and Agriculture 43: 12 15. Hueppelsheuser, T. 2010. Assessment of solutions used for the purpose of determining spotted wing drosophila larval infestation in blueberry fruit. BC Ministry of Agriculture, Victoria, B.C., Canada. http://whatcom.wsu.edu/ipm/swd/documents/LavalExtraction.pdf Hunter, S. H., H. M. Kaplan, and E. V. Enxmann. 1937. Chemicals attracting Drosophila Am. Nat. 71: 575 581. JMP, Version 9. 2013 SAS Institute Inc., Cary, NC. Kagawa Y., S. Kanematsu, and M. Sugiura. 2012. Color preference change of D rosophila melanogaster (Diptera: Drosophilidae) according to the degree of illuminance. Jp n. J. Appl. Entomol. Zool. 56 : 114 117. Kaneshiro K. Y. 1983. Drosophila (Sophophora) suzukii ( Matsumura). Proc. Hawaii. Entomol. Soc. 24: 179. Kanzawa T. 1935 Research into the fruit fly Drosophila suzukii Mats. (preliminary report). Yamanashi Prefect. Agri. Exp. Stn. Hoshino Printing Kofu Shi, Jpn. Kanzawa T. 1939. Studies on Drosophila suzuk ii Mats. Kofu. Rev. App. Entomol. 29: 622. Kawase, S. and K. Uchino. 2005. Effect of mesh size on Drosophila suzukii adults passing through the mesh. Ann Rep. Kanto Tosan Plant Prot. 52: 99 101. Kimura, M. T. 2004. Cold and heat tolerance of drosophilid f lies with reference to their latitudinal distributions. Ecophysiol. 140: 442 449. Klick, J., W. Yang, J. Hagler, and D. Bruck. 2012. Using protein marker technology to determine spotted wing drosophila movement between border and field, pp. 24 27 In Proc e edings 71st Annu. Pac. Northwest Insect Manag. Conf. 9 10 January, 2012, Portland, OR. Integrated Plant Protection Center Oregon State University Corvalis, OR. Krewer, G., M. Tertuliano,P. Andersen, O. E. Liburd, G. Fonsah, H. Serri, and B. Mullinix. 2009. Effect of mulches on the establishment of organically grown blueberrie s in Georgia. Acta Hort. 810: 483 488.

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115 Landolt, P. J. 1995. Attraction of Mocis latipes (Lepidoptera: Noctuidae) to sweet b aits in t raps Fla. Entomol. 78: 523 530. Landolt, P. J. T. Adams, T. S. Davis, and H. Rogg. 2012a. Spotted wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), trapped with combinations of wines a nd vinegars. Fla. Entomol. 95 :326 332. Landolt, P. J., T. Adams, and H. Rogg. 2012b. Trapping spotted win g drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), with combinations of vinegar and wine, and acetic acid and ethanol. J. Appl. Entomol. 136: 148 154. Lebreton, S., P. G. Becher, B. S Hansson, and P. Witzgall. 2012 Attraction of Droso phila melanogaster males to food related and fly odours. J. Insect Physiol. 58: 125 129. Lee, J. C., D. J. Bruck, H. Curry, D. Edwards, D. R. Haviland, R. A. Van Steenwykd and B. M. Yorgey. 2011. The susceptibility of small fruits and cherries to the spott ed wing drosophila, Drosophila suzukii Pest Manag. Sci. 67: 1358 1367. Lee, J. C., H. J. Burrack, L. D. Barrantes, E. H. Beers, A. J. Dreves, K. A. Hamby, D. R. Haviland, R. Isaacs, T. A. Richardson, P. W. Shearer, C. A. Stanley, D. B. Walsh, V. M. Walton F. G. Zalom, and D. J. Bruck. 2012 Evaluation of monitoring traps for Drosophila suzukii ( Diptera: Drosophilidae ) in North America. J. Econ. Entomol. 105 : 1350 1357. Liburd, O. E ., S. R. Alm, and R. A. Casagrande. 1998. Susceptibility of highbush bluebe rry cultivars to larval infestation (Diptera: Tephritidae). J Econ Entomol 27: 817 821. Liburd, O. E., E. M. Finn, K. L. Pettit, and J. C. Wise. 2003. Response of blueberry maggot fly (Diptera: Tephritidae) to imidacloprid treated spheres and selected i nsecticides. Can. Entomol. 135: 427 438. Liburd, O. E. and H. A. Ar valo. 2006. Insects and mites in blueberries pp. 99 110 In N. F. Childers and P.M. Lyrene (Eds.), Blueberries: f or growers, gardeners, promoters. Dr. Norm F. Childers Horticultural Publi cations, Gainesville, FL. Liburd, O. E., E. M. Sarzynski, H. A. Arvalo, and K. MacKenzie. 2009 Monitoring and emergence of flower thrips species in rabbiteye and southern highbush blueberries. Acta Hort. 810: 251 258. Liburd, O. E. and L. E. Iglesias. 2013. Spotted wing drosophila: pest management recommendatio ns for southeastern blueberries, ENY869 University of Florida, Institute for Food and Agricultural Sciences (IFAS) and Florida Cooperative Extension Service. Gainesville, FL.

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116 Lyrene, P. M. and J. R. Ballington. 2006. Varie ties and their characteristics pp. 26 37 In N. F. Childers and P.M. Lyrene (Eds.), Blueberries: f or growers, gardeners, promoters. Dr. Norm F. Childers Horticultural Publicat ions, Gainesville, FL. Lyrene, P M. and J. N. Moore. 2006. Blueberry breeding, pp. 38 48. In N. F. Childers and P.M. Lyrene (Eds.), Blueberries: for growers, gardeners, promoters. Dr. Norm F. Childers Horticultural Publications, Gainesville, FL. Lyrene, P. M. and J. A. Payne. 1992. Blue berry gall midge: a pest on rabbiteye blueberry in Florida. Proc. Fla. State Hort. Soc. 105: 297 300. Drosophila: a guide to species identification and use. Academic Press, London. Markow, T. A. and P. M 008. Reproductive ecology of Drosophila. Funct. Ecol. 22: 747 759. Mathur, S., M. A. Cook, B. J. Sinclair, and S. M. Fitzpatrick. 2012 DNA barcodes suggest cryptic speciation in Dasineura oxycoccana (Diptera: Cecidomyiidae) on cranberry, Vaccinium macroca rpon and blueberry, V. corymbosum Fla. Entomol. 95: 387 394. Maust, B. E., J. G. Williams, and R. L. Darnell. 1999. Flower bud density affects vegetative and fruit development in field grown southern highbush blueberry. HortSci. 34: 607 610. Mitsui, H., K. H. Takahashi, and M. T. Kimura. 2006. Spatial distributions and clutch sizes of Drosophila species ovipositing on cherry fruits of different stages. Popul. Ecol. 48: 233 237. Murray, D. A., R. D. Kriegel, J. W. Johnson, and A. J. Howitt. 1996. Natural e nemies of cranberry fruitworm, Acrobasis vaccinii (Lepidoptera: Pyralidae) in Michigan highbush blueberr ies. Great Lakes Entomol. 29: 81 86. (NASS USDA) National Agricultural Statis tics Service U.S. Department of Agriculture. 2013. Noncitrus fruits and nuts 2012 preliminary summary ISSN: 1948 2698 NASS USDA, Arlington, VA. http://quickstats.nass.usda.gov/ (NASS USDA) National Ag ricultural Statistics Service U.S. Department of Agriculture. 2012. Quick Stats 2.0: Florida organic blueberry production in acres, 2008. NASS USDA, Arlington, VA. http://quickstats.nass.usda.gov/ Nyoike, T. W. and O. E. Liburd. 2009. Leaf beetle abundance and management in H igh bush blueberries in Florida. In 83rd Annu. Meeting of the Southeastern Branch (SEB) Entomol. Soc. Am. (ESA) Conf. 8 11 March 20 09 Montgomery AL. ESA, Annapolis MD.

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117 Nyoike, T. W. and O. E. Liburd. 2013. Effect of twospotted spider mite, Tetranychus urt icae Koch (Acari: Tetranychidae) on marketable yields of field grown strawberries in north central Florida. J. Econ. Entomol. (in press). Ometto, L., A. Cestaro, S. Ramasamy, A. Grassi, S. Revadi, S. Siozios, M. Moretto, P. Fontana, C. Varotto, D. Pisani, T. Dekker, N. Wrobel, R. Viola, I. Pertot, D. Cavalieri, M. Blaxter, G. An fora, and O. Rota Stabelli. 2013 Linking genomics and ecology to investigate the complex evolution of an invasive drosophi la pest. Genome Biol. Evol. 5 : 745 757. Payne, J. A. and S. H. Berlocher. 1995. Distribution and host plants of the blueberry maggot fly, Rhagoletis mendax (Diptera: Tephritidae) in s outheastern North Amer ica. J. Kans. Entomol. Soc. 68 : 133 142. Pe tavy, G., J. R. David, P. Gibert, and B. Moreteau. 2001. Viabilit y and rate of development at different temperatures in Drosophila : a comparison of constant and alternating thermal regimes. J. Therm. Bio. 26: 29 39. Prokopy, R. J. and E. D. Owens. 1983. Visual detection of plants by herbivorous insects. Annu. Rev. Entom ol. 28: 337 364. Reed, M. R. 1938 The olfactory reactions of D rosophila melanogaster Meigen to the products of fermenting banana. Physiol. Zool. 11: 317 325. Roessingh, P. and E. Stadler. 1990. Foliar form, colour and surface characteristics influence ovi position behavior in the cabbage root fly, Delia radicum Entomol. Exp. Appl. 57: 93 100. Sarzynski, E. M. and O. E. Liburd. 2003 Techniques for monitoring cranberry t ipwo rm (Diptera: Cecidomyiidae) in rabbiteye and s outhern highbush blueberries. J. Econ Entomol. 96: 1821 1827. Steck G. J., W. Dixon, and D. Dean. 2009. Spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosop h ilidae), a fruit pest new to North America. FD OACS Division of Plant Industry, Gainesville, FL. http://www.freshfr omflorida.com/pi/enpp/ento/drosophila_suzukii.html (12 June 2013). Steck, G. J., D Veit, R Grandy, S Bermudez i Badia, Z Mathews, P Verschure B S. Hansson and M Knaden 2012. A high throughput behavioral paradigm for Drosophila olfaction t he Fl ywalk Sci. Rep. 2: 1 9. Takamori, H., H. Watabe, Y. Fuyama, Y. Zhang, and T. Aotsuka. 2006. Drosophila subpulchrella a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: Drosophilidae). Entomol. Sci. 9: 121 128. Tripleh orn, C. A. and N. F. Johnson. 2005. study of insects, 7 th ed. Brooks/Cole, Belmont, CA.

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118 Vlach, J. 2010. Identifying Drosophila suzukii. Oregon Department of Agriculture, Salem, OR. http://www.oregon.gov/ODA/PLANT/docs/pdf/ippm_d_suzukii_id_guide10.pdf Walker, G. 1992. Adhesion to smooth surfaces by insects a review. Int. J. Adhesion and Adhesives. 13: 3 7. Walsh, D. B., M. P. Bol da, R. E. Goodhue, A. J. Dreves, J. Lee, D. J. Bruck, V. M. Walton, S. G. Zalom. 2011 Drosophila suzukii (Diptera: Drosophilidae): invasive pest of ripening soft fruit expanding its geographic range and damage potenti al. J. Integr. Pest Manag. 2 : 1 7. Williamson, J., G. Krewer, G. Pavlis, and C. M. Mainland. 2006. Blueberry soil management, nutrition and irrigation pp. 60 75 In N. F. Childers and P.M. Lyrene (Eds.), Blueberries: f or growers, gardeners, promoters. Dr. Norm F. Childers H orticultural Publications, Gainesville, FL. Wu, S., H. Tai, Z. Li, X. Wang, S. Yang, W. Sun, and C. Xiao. 2007. Field evaluation of different trapping methods of cherry fruit fly, Drosophila suzukii. J. Yunnan Ag. Univ. 22 : 776 778. Zhu, J., K C. Park, a nd T. C. Baker. 2003 Identification of odors from overripe mango that attract vinegar flies, Drosophila melanogaster. J. Chem. Ecol. 29: 899 909.

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119 BIOGRAPHICAL SKETCH Lindsy Iglesias nterdisciplinary Ecology p rogram with a concentration in e ntomology at the University of Florida She graduated with her B.S. in Environmental Science with a minor i n Sustainability Studies from the University of Florida in 2010. Her undergraduate career also included 2 years of study in archi tecture at the University of South Florida where she focused on green building design. She spent the summer of 2010 interning at the Bioenergy Research a nd Education unit at the University of Florida conducting research on the bioremediation of human wast e with algae to produce non potable water and fertilizer. She beg with Oscar Liburd in December of 2011 at the Small Fruit and Vegetable IPM Laboratory at the University of Florida Her program began at a critical time when an inva degree she will continue towards her Ph D with Dr. Liburd researching monitoring methods for management of SWD to help protect the blueberry industry and the livelihoods of its growers.