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Ecology and Management of the American Serpentine Leafminer, Liriomyza Trifolii (Bugress) (Insecta

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Title:
Ecology and Management of the American Serpentine Leafminer, Liriomyza Trifolii (Bugress) (Insecta Diptera: Agromyzidae) on Five Major Vegetable Crops
Creator:
Devkota, Shashan
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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Language:
english
Physical Description:
1 online resource (118 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
SEAL,DAKSHINA R
Committee Co-Chair:
LIBURD,OSCAR EMANUEL
Committee Members:
FERGUSON,SCOTT
WADDILL,CHRISTINE TAYLOR
Graduation Date:
8/8/2015

Subjects

Subjects / Keywords:
Adult insects ( jstor )
Beans ( jstor )
Insecticides ( jstor )
Larvae ( jstor )
Leafminers ( jstor )
Leaves ( jstor )
Parasitoids ( jstor )
Planting ( jstor )
Pupae ( jstor )
Squashes ( jstor )
Entomology and Nematology -- Dissertations, Academic -- UF
abundance -- botanical -- distribution -- microbial -- polyphagous
Miami metropolitan area ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Entomology and Nematology thesis, M.S.

Notes

Abstract:
American serpentine leafminer, Liriomyza trifolii, a polyphagous fly, is an important insect pest of vegetable crops. High L. trifolii infestations can considerably reduce the rates of photosynthesis by reducing leaf mesophyll and stomatal conductance thus seriously diminishing plant growth, yield, and causing premature plant death. Major objectives of this study included studying the abundance of leafminers and their parasitoids on five vegetable crops including snap bean, squash, cucumber, tomato, and cabbage, their distribution on bean and squash, and the management of leafminers on snap beans. Among five vegetable crops, snap beans had the highest number of leafminers and their parasitoids and cabbage had the lowest. Leafminer activity was highest at 2 weeks after planting on bean and at 3 weeks after planting on squash in 3 plantings. The abundance of leafminer parasitoids was dependent on the numbers of leafminers. Leafminers showed seasonal preference on beans and squash. Leafminer abundance was higher during the plantings in November, May, and September, when average daily temperatures were 21 to 26 degree Centigrade. However, fewer leafminers seemed to occur in the December planting when the average daily temperature was below 18 degree Centigrade. In addition, leafminers had mostly aggregated distributions on most sample dates of the four plantings on bean and squash. Parasitoids distributions were similar to those of their host leafminers. Plants treated with abamectin and spinosad had significantly fewer leafminer mines, larvae, and pupae than non treated control plants on all sample dates. However, cyromazine treated plants had more mines compared to abamectin and spinosad, but the mines were small and often aborted. Hence, no pupae were recovered from plants treated with cyromazine on most sample dates. Similarly, plants treated with azadirachtin had significantly fewer mines, larvae, and pupae compared with non treated control plants on most sample dates. However, plants treated with and Isaria fumosorosea did not have significant differences in numbers of mines, larvae, or pupae compared with non treated control plants on most sample dates. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2015.
Local:
Adviser: SEAL,DAKSHINA R.
Local:
Co-adviser: LIBURD,OSCAR EMANUEL.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-02-29
Statement of Responsibility:
by Shashan Devkota.

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UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
2/29/2016
Classification:
LD1780 2015 ( lcc )

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1 ECOLOGY AND MANAGEMENT OF THE AMERICAN SERPENTINE LEAFMINER, LIRIOMYZA TRIFOLII ( BURGESS ) (INSECTA: DIPTERA: AGROMYZIDAE ) ON FIVE MAJOR VEGETABLE CROPS By SHASHAN DEVKOTA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2015

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2 © 2015 Shashan Devkota

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3 To Mandu didi, my parents and my w ife

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4 ACKNOWLEDGMENTS I express sincere gratitude to my advisor Dr. Dakshina R. Seal for his invaluable support and encouragement during my graduate study and research. I extend my thanks to my committee members , Dr. Oscar E. Liburd , Dr. Scott Ferguson and Dr. Christine Waddill, for their generous advice, assistance and encouragement during my research and my thesis. I thank C. Sabines, J. Teyes, C. Carter, and B. Panthi of Vegetable IPM Laboratory for their help in planting and main taining crops and collecting data. I would like to thank Dr. Cliff Martin for his help during my thesis preparation. I would also like to thank my aunt (Mandavi), my parents and my brother, sister, brother in law and my niece for their love, support and encouragement. Above all, I sincerely thank my wife, Radhika, for helping me collecting and processing data and for he r love, support and motivation at all times. I would also like to thank all my friends from Nepal and USA for their help and support. Finally, I would like to thank Bill Bussey and Syngenta Seeds Inc. for providing the seeds for my research.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST O F TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRA CT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Nomencla ture ................................ ................................ ................................ ......... 15 Host Range ................................ ................................ ................................ ............. 15 Geographic Distribution ................................ ................................ .......................... 15 Morphology ................................ ................................ ................................ ............. 16 Biology and Development ................................ ................................ ....................... 17 Host Selection by L. trifolii ................................ ................................ ....................... 18 Ecology ................................ ................................ ................................ ................... 19 Management Practices ................................ ................................ ........................... 20 Chemical Control ................................ ................................ .............................. 20 Biological Control ................................ ................................ ............................. 21 Research Objectives ................................ ................................ ............................... 23 3 ABUNDANCE OF AMERICAN SERPENTINE LEAFMINER, LIRIOMYZA TRIFOLII AND ITS PARASITOIDS ON FIVE VEGETABLE CROPS IN SOUTH FLORIDA. ................................ ................................ ................................ ............... 28 Materials and Methods ................................ ................................ ............................ 29 Field Preparation, Planting, and Crop Management ................................ ......... 29 Sampling ................................ ................................ ................................ .......... 30 Abundance of Mines, Larvae, and Pupae ................................ ........................ 31 Statistical Analyses ................................ ................................ .......................... 31 Results ................................ ................................ ................................ .................... 31 Abundance of Mines on Vegetable Crops ................................ ........................ 31 Abundance of Larvae on Vegetable Crops ................................ ....................... 33 Abundance of Pupae on Vegetable Crops ................................ ....................... 34 Abundance of Adult Leafminer on Vegetable Crops ................................ ......... 34 Parasitoid Abundance ................................ ................................ ...................... 35 Discussion ................................ ................................ ................................ .............. 35

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6 4 SEASONAL ABUNDAN CE AND SPATIAL PATTERN OF DISTRIBUTION OF LIRIOMYZA TRIFOLII (BURGESS) (DIPTERA: AGROMYZIDAE) AND ITS PARASITOID ON BEAN AND SQUASH IN SOUTH FLORIDA .............................. 49 Materials and Methods ................................ ................................ ............................ 51 Field Preparation, Planting, and Crop Management ................................ ......... 51 Seasonal Abundance of Leafminer Mines, Larvae, Adults, and Parasitoids .... 52 Planting 1 (October November 2013) ................................ ...................... 52 Planting 2 (May June 2014) ................................ ................................ ....... 53 Planting 3 (September October 2014) ................................ ....................... 53 Planting 4 ( November 2104 January 2015) ................................ ............... 53 Statistical a nalyses ................................ ................................ .................... 54 Spatial Distribution ................................ ................................ ........................... 54 Statistical a nalysis ................................ ................................ ...................... 54 Results ................................ ................................ ................................ .................... 55 Seasonal Abundance of Leafminer Mines, Larvae, Adults, and Parasitoids .... 55 Planting 1 (October November 2013) ................................ ...................... 55 Planting 2 (May June 2014) ................................ ................................ ....... 57 Planting 3 (September October 2014) ................................ ....................... 58 Planting 4 ( November 2014 January 2015) ................................ ............... 59 Spatial Distribution ................................ ................................ ........................... 60 Planting 1 (October November 2013) ................................ ...................... 60 Planting 2 (May June 2014) ................................ ................................ ....... 62 Planting 3 (September October 2014) ................................ ....................... 63 Planting 4 ( November 2104 January 2015) ................................ ............... 65 Discussion ................................ ................................ ................................ .............. 66 5 RESPONSE OF AMER ICAN SERPENTINE LEAFMINER, LIRIOMYZA TRIFOLII , TO CHEMICAL, BOTANICAL AND MICROBIAL INSECTICIDE. ........... 89 Materials and Methods ................................ ................................ ............................ 91 Result ................................ ................................ ................................ ...................... 93 Number of Mines ................................ ................................ .............................. 93 Number of La rvae ................................ ................................ ............................. 95 Number of Pupae ................................ ................................ ............................. 96 Discussion ................................ ................................ ................................ .............. 97 6 CONCLUSIONS ................................ ................................ ................................ ... 103 LIST OF REFERENCES ................................ ................................ ............................. 106 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 118

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7 LIST OF TABLES Table page 2 1 Global distribution of Liriomyza trifolii Burgess adopted from (CABI, 2014) ....... 27 4 1 distribution of L. trifolii mines on beans and squash sampled in Planting 1 (Oct Nov 2013) ................................ ................................ ................................ 81 4 2 distribution of parasitoids of L. trifolii on bean and squash sampled in Planting 1 (Oct Nov 2013) ................................ ................................ ............... 82 4 3 distribution of L. trifolii mines on beans and squash sampled in Planting 2 (May June 2014) ................................ ................................ .............................. 83 4 4 distribution of parasitoids of L. trifolii on bean and squash samp led in Planting 2 (May June 2014) ................................ ................................ ............. 84 4 5 distribution of L. trifolii mines on beans and squash sampled in Planting 3 (Sep Oct 2014) ................................ ................................ ................................ 85 4 6 distribution of parasitoids of L. trifolii on bea n and squash sampled in Planting 3 (Sep Oct 2014) ................................ ................................ ............... 86 4 7 dis tribution of L. trifolii mines on beans and squash sampled in Planting 4 ( Nov 2014 Jan 2015) ................................ ................................ ....................... 87 4 8 distribution of parasitoids of L. trifolii on bean and squash sampled in Planting 4 ( No v 2014 Jan 2015) ................................ ................................ ...... 88 5 1 Efficacy of different chemical and biological insecticides vs non treated (Control) on number of mines/5 leaves made by L. trifolii in bean leaves ......... 100 5 2 Efficacy of different chemical and biological insecticides vs non treated (Control) on number of L. trifolii larvae/5 leaves in bean leaves ....................... 101 5 3 Efficacy of different chemical and biological insecticides vs non treated (Control) on number of pupae of L. trifolii / 5 leaves collected from bean plants ................................ ................................ ................................ ................ 102

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8 LIST OF FIGURES Figure page 2 1 Map showing distribution of Liriomyza trifolii in Europe (CABI 2014). ................. 25 2 2 Map showing distribution of Liriomyza trifolii in North America. (CABI 2014) ..... 26 3 1 Mean (± SE) number of L. trifolii mines per five leaves on five vegetable crops during season 1 (May June 2014).. ................................ ........................ 39 3 2 Mean (± SE) number of L. trifolii mines per five leaves on five vegetable crops during season 2 (September October 2014).. ................................ ......... 40 3 3 Mean (± SE) number of L. trifolii larvae per five leaves on five vegetable crops during season 1 (May June 2014).. ................................ ........................ 41 3 4 Mean (± SE) number of L. trifolii larvae per five leaves on five vegetable crops during season 2 (September October 2014).. ................................ ......... 42 3 5 Mean (± SE) number of L. trifolii pupae per five leaves on five vegetable crops during season 1 (May June 2014).. ................................ ........................ 43 3 6 Mean (± SE) number of L. trifolii pupae per five leaves on five vegetable crops during season 2 (September October 2014).. ................................ ......... 44 3 7 Mean (± SE) number of L. trifolii adults per five leaves on five vegetable crops during season 1 (May June 2014).. ................................ ........................ 45 3 8 Mean (± SE) number of L. trifolii adults per five leaves on five vegetable crops during season 2 (September October 2014).. ................................ ......... 46 3 9 Mean (± SE) number of L. trifolii parasitoids per five leaves on five vegetable crops during season 1 (May June 2014).. ................................ ........................ 47 3 10 Mean (± SE) number of L. trifolii parasitoids per five leaves on five vegetable crops during season 2 (September October 2014).. ................................ ......... 48 4 1 Planting 1 (26 Oct to 30 Nov 2013), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves).. ................................ .............................. 70 4 2 Planting 1 (26 Oct to 30 Nov 2013), Site 2 (squash) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves). ................................ ........................ 71 4 3 Planting 2 (10 May to 14 June 2014), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves). ................................ ........................ 72 4 4 Planting 2 (10 May to 14 June 2014), Site 2 (squash) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves). ................................ ........................ 73

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9 4 5 Planting 3 (6 Sep through 11 Oct 2014), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves). ................................ ........................ 74 4 6 Planting 3 (6 Sep through 11 Oct 2014), Site 2 (squash) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves ). ................................ ............... 75 4 7 Planting 4 (28 Nov 2014 through 2 Jan 2015), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 l eaves). ................................ ............... 76 4 8 Planting 4 (28 Nov 2014 through 2 Jan 2015), Site 2 (squash) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves). ................................ ........... 77 4 9 Seasonal abundance (mean ± SE / 5 leaves) of L. trifolii mines, larvae, pupae, adults and its parasitoids on bean during 4 plantings. ............................ 78 4 10 Seasonal abundance (mean ± SE / 5 leaves) of L. trifolii mines, l arvae, pupae, adults and its parasitoids on squash during 4 plantings. ........................ 79 4 11 Comparison of average daily temperature (°C) and abundance of L. trifolii mines, larvae, and parasitoids on bean during the four plantings (26 Oct 2013 2 January, 2015). ................................ ................................ .................... 80

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10 LIST OF ABBREVIATIONS a.i. Active Ingredient ANOVA Analysis of variance cm Centimeters df Degree of freedom gm Grams gm /l gram per liter Km Kilometers kpa kilopascals m Meters P Statistical probability value RH Relative humidity SAS Statistical Analysis Software SE Standard error

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11 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 ECO LOGY AND MANAGEMENT OF THE AMERICAN SERPENTINE LEAFMINER, LIRIOMYZA TRIFOLII (BURGESS) ( INSECTA: DIPTERA: AGROMYZIDAE) ON FIVE MAJOR VEGETABLE CROPS By Shashan Devkota August 2015 Chair: Dakshina R. Seal Major: Entomology and Nematology American serpentine leafminer, Liriomyza trifolii (Burgess) , a polyphagous fly, is an important insect pest of vegetable crops. High L. trifolii infestations can considerably reduce the rates of photosynthesis by reducing leaf mesophyll and stomata l conductance th us seriously diminish ing plant growth, yield, and causing premature plant death . Major objectives of this study included studying the abundance of leafminers and their parasitoids on five vegetable crops including snap bean, squash, cucumber, tomato, and cabbage; their distribution on bean and squash; and the management of leafminers on snap beans. Among five vegetable crops, snap beans had the highest number of leafminers and their parasitoids and cabbage had the lowest. There was no difference among the parasitoids complex found in all five crops. Among the parasitoids recorded, Opius dissitus was the most abundant. Leafminer activity was highest at 2 weeks after planting on bean and at 3 weeks after planting on squash i n 3 plantings. The abundance of leafminer parasitoids was dependent on the number of leafminers. Leafminers showed seasonal preference on beans and squash. Leafminer abundance was higher during the plantings in November,

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12 May, and September, when average da ily temperatures were 21 26 o C. However, fewer leafminers seemed to occur in the December planting when the average daily temperature was below 18 o C. In addition, leafminers had mostly aggregated distributions on most sample dates of the four plantings on bean and squash. Parasitoids distributions were similar to those of their host leafminers. Plants treated with abamectin and spinosad had significantly fewer leafminer mines, larvae, and pupae than non treated control plants on all sample dates. However , cyromazine treated plants had more mines compared to abamectin and spinosad, but the mines were small and often aborted. Hence, no pupae were recovered from plants treated with cyromazine on most sample dates. Similarly, plants treated with azadirachtin had significantly fewer mines, larvae, and pupae compared with non treated control plants on most sample dates. However, plants treated with and Isaria fumosorosea did not have significant differences in numbers of mines, larvae, or pupae compared with non treated control plants on most sample dates.

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13 CHAPTER 1 INTRODUCTION Florida has one of the largest acreages of vegetable production in the United States, where it ranks first in snap bean production with 44% of the total national production. Florida also produces nearly all the winter fresh market snap beans with Miami Dade ranking # 1 among the 67 cou nties in the state (Elwakil and Mossler, 2012). Snap bean , Phaseolus vulgaris L. production in Florida faces challenges from various pests that cause considerable economic losses. T he American serpentine leafminer, Liriomyza trifolii ( Bu rg ess ) (Diptera: Ag romyzidae), is considered one of the most serious pest of snap bean (Spencer 1965 , Stegmaier 1966 , Seal et al. 2002) . I t damages the host plant s by making feeding punctures and mining the leaves. C hemical insecticides belonging to various modes of action are used to control leafminers. In 1999 2000, Florida vegetable growers spent an average of $131.32 per acre on insecticides ( Elwakil and Mossler 2012 ). However, heavy insecticide use may kill natural enemies resulting in increased leafminer populations . B esides vegetable crops, leafminers also cause economic damage to ornamentals and fruit crops. Exact economic losses caused by L. trifolii is difficult to quantify especially in vegetable crops , h owever, the losses that occur in ornamental crops are more ae sthetic rendering them easier to quantify. From 1981 to 1985, leafminers cause d losses of $95 million in the chrysanthemum industry (Parrella 198 7, Heinz et al. 1993). High infestations of L. trifolii often considerably reduce rates of photosynthesis and mesophyll and stomatal conductance, hence, seriously reducing plant growth (Parrella et al. 1985) and yield eventually resulting in plant death (Trumble et al. 1985). Severe mining also causes destruction of seedlings and premature leaf drop, which may res ult in sunburn on

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14 tomato fruits (Musgrave et al. 1975 , Minkenberg and van Lenteren 1986). In ornamental crops, leafminers reduce plant aesthetic values resulting in economic losses through lack of sales (Parrella et al. 1985). In addition, punctures caused by stippling and mining may serve as entry points for bacterial and fungal pathogens (Zitter and Tsai 1977 , Minkenberg and Van Lenteren 1986 , Broadbent and Matteoni 1990 , Hernandez et al. 2011). The ratio of cost to benefit for control measures and determining economic returns ha ve continued to be frequent subject s of discussion for many vegetable growers (C h andler and Gilstrap 1987 ) . The research goal was to study the abundance of L. trifolii and its parasitoid on five vegetable crops including beans ( Phaseolus vulgaris L. ), cabbage ( Brassica oleracea L . var capitata L . ), tomato ( Solanum lycopersicum L .), squash ( Cucurbita pepo L.) and cucumber ( Cucumis sativus L.), commonly grown in South Florida. In addition, the study was focus ed on testing the efficacy of selected insecticides against L. trifolii .

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15 C HAPTER 2 LITERATURE REVIEW Nomenclature Different scientific names have been suggested at different tim es for American serpentine leafminer. They include Liriomyza trifolii (Burgess 1980), Oscinis trifolli ( Burgess 1980 ) , Liriomyza alliovora ( Frick 1955 ) , and Agromyza phaseolunulata ( Frost 1943 ) . Common names used in various geographical locations are American serpentine leafminer, serpentine leafminer, broad bean leafminer, California leafminer, celery leafminer, and chrysanthemum leafminer. Host Range Like most vegetable pests, Liriomyza leafminer is polyphagous and feeds on different vegetable, ornamental, weedy, and native plants including more than 400 plant species in 25 families ( Stegmaier 1966 , Parrella 1987) . Feeding on weeds allow the pest to survive in the absence of cultivated c rops (Stegmaier 1966 , Minkenberg and van Lenteren 1986 , Schuster et al. 1991 ). However, the most important crops attacked by Liriomyza leafminers include bean, celery, chrysanthemum, cotton, lettuce, pepper, potato, tomato, onion, and cotton (Leibee 1984 , Parrella et al. 19 83, Seal et al. 2002). Geographic Distribution The place of origin of L. trifolii is believed to be Florida, where it can be found all year round (Spencer 1965 , Stegmaier 1966). Until the 1970s, L. trifolii was found only in the Caribbe an islands and in the southern and eastern USA, but its range has subsequently expanded to other parts of the world by floral export (Minkenberg and van Lenteren 1986 , Parrella 1987 , Minkenberg 1988 a ). American serpentine leafminer is now most abundant in many tropical and subtropical regions throughout the world

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16 (Figure 2 1, 2 2) , but can survive in areas with freezing temperatures in protected areas such as greenhouses (Capinera 2001 , Table 2 1 ). Morphology The grayish black mesonotum and eyes with yello w hind margins are distinguishing characteristics of L. trifolii adults (Capinera 2001). L . trifolii adults have a yellow head, specifically with a yellow frons, orbit, and third antennal segment, and red eyes. Adults of L. trifolii are usually < 2 mm long and have transparent wings 1.2 1.9 mm long. They have a gray and black thorax and abdomen, while the legs and ventral surfaces are yellow. The yellow femur is interrupted with brown striations. The aedeagus is distiphallus with one distal bulb bearing a marked constriction between the upper and lower halves (Capinera 2001). Further information on the description of L. trifolii can be found in Spencer (1965, 1973), Dempewolf (2004), Malipatil et al. (2004), and Shiao (2004 ). Eggs of L. trifolii are whi te and small ( 1.0 mm long and 0.2 mm wide) . The eggs become larger with increased oviposition period, which is affected by imbibition of fluids and temperature (Leibee 1984; Parrella 1987). The larval shape is cylindrical and color is yellow. The four larv al instars are distinguishable by the size of mouthparts and body. Mouthparts for first instars are 0.10 mm (0.08 0.11 mm), 0.17 mm (0.15 0.18 mm) for second instars, and 0.25 mm (0.22 0.31 mm) for third instars ( Capinera 2001). The corresponding bod y lengths are 0.39 mm (0.33 0.53 mm) for first instars, 1.00 mm (0.55 1.21 mm) for second instars, and 1.99 mm (1.26 2.62 mm) for third instars (Capinera 2001). The fourth instar does not feed and occurs between formation of the puparium and pupa. Th e pupa is initially brown but darkens with time.

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17 Biology and Development Liriomyza trifolii females begin oviposition within 24 48 hours of emergence from pupae. The female oviposits into one of every four punctures it makes while feeding (Parrella et al. 1983). A female can lay 35 39 eggs per day for a total of 180 400 eggs per lifetime depending on temperature (Leibee 1984). Liriomyza trifolii completes development in 21 28 days but this duration is dependent on temperature (Leibee 1984 , Parrella 1984 , Minkenberg 1988b ). When caged at temperatures of 25 30 °C, adult longevity is 15 20 days for females and 10 15 days for males. Because of the short development time, these leafminers c an produce several generations per year. The most suitable temperatures for L. trifolii growth and development are between 20 30 °C, and temperatures above 35 °C are lethal (Leibee 1984 , Lanzoni et al. 2002). As the name suggests, leafminer larvae create serpentine shaped mines within the leaves of host crops (Parrella 1987). Injury is done by adult females and larvae feeding on the host plant leaf tissue. Adult females initially puncture the leaf using its ovipositor and then feed on the plant sap ( Bethk e and Parrella 1985, Minkenberg and van Lenteren 1986 , Parrella 1987). The size of a feeding puncture depends on the size of the female. Females can make about 100 feeding punctures per day and prefer palisade mesophyll tissue (Parrella et al. 1983). Adult males cannot make punctures and tend to feed on punctures made by females (Parrella 1987). Larvae of L. trifolii are within leaves and cannot choose preferred leaves for feeding. Hence, they, depend on the initial oviposition choice by the adult female for suitable food. Before laying eggs, females check the suitability of leaves for offsprin g development (Minkenberg and van Lenteren 1986 , Parrella 1987). Immediately after hatching , the larva begins mining the leaf mesophyll layer until it completes feed ing and

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18 emerges from the leaf (Fagoonee and Toory 1984). Larval development time is inversely related to temperature (Leibee 1984 , Parrella 1987 , Lanzoni et al., 2002). At 30 °C, larval development time is 4 6 days depending on host plant (Leibee 1984 , Lan zoni et al. 2002). In addition to temperature, larval development depends on the age and nutritional content of the host leaf (Parrella 1987). The size of the mine and rate of mining increase with larval development stage. Third instar larvae consume 50 ti mes as much leaf material as first instars (Fagoonee and Toory 1984 , Parrella 1987). Once the mature larva completes feeding, it emerges from the leaf in the morning and searches for a dark place to pupate. Although prolonged if exposed to light, the prepupal stage is short lived and lasts for up to 5 hours (Leibee 198 6 ). Larval transformation to pupa usually occurs in the mid afternoon (Leibee 1984). The pupa generally requires 7 11 days to complete development. The pupa can be kept at 4 °C for severa l days without impairing its viability (Miller and Isger 1985). Host Selection by L. trifolii After selecting the host leaf, the female generally oviposits after a series of host inspections and involving a sequence of visual, chemical, and tactile stimul i (Parrella, 1987). Acceptance may result from certain host plant characteristics, while rejection may be caused by others (Singer 1986 , Martin et al. 2005). Distribution and density of leaf trichomes plays a vital role in host selection by the adult , with high trichome density acting as a physical deterrent ( Parrella 1987 ). Plant nutrients, chemical attractants, and the age and life history of a plant may all affect host selection. After the host is chosen, a female positions its ovipositor and makes a dee p puncture followed by egg laying (B ethke and Parrella 1985 ) .

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19 Ecology To develop an effective integrated pest management ( IPM ) program, it is important to know the appropriate method of management and time of its application. Therefore, information on th e seasonal population dynamics and field distribution of an insect pest gives an idea of pest populations at different times of year . Abundance and distribution of an insect may differ with host plant species, developmental stage, and the presence of other species of flora and fauna (Seal et al. 1992, Seal 2001, Kakkar et al. 2012) . Physical environment including temperature, humidity, and rainfall affect leafminer development as do changes in biotic and abiotic factors over time (Leibee 1985). In Lebanon for example, leafminer populations were reported to be reduced because of high mean temperatures in September and October (Hammad and Nemer 2000). Li et al. (2011) also recorded increased leafminer populations in December and January, when mean temperatures were relatively low ( 21 23 o C ) . Shepard et al. (1998 ) found that leafminer populations on potato were relatively low during the dry season. Liriomyza trifolii differs in infestation levels depending on location of leaves on a host plant. On beans and potatoes, the infestation begins on lower leaves and proceeds to the middle and upper leaves as the host plant develops. Leafminers on older leaves of potato showed higher survival rates than on younger leaves. Leaves that are older and thicker have increased mesophyll, hence can supply more food and space (Facknath, 2005) .

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20 Management Practices Chemical Control Insecticide application is the most commonly used control metho d for leafminers because of their potential to cause economic losses to many crops even in low densities (Ferguson 2004 , Cox et al . 1995). Using insecticides for leafminer management in Florida has a long history. Insecticide applications began as early as 1946, and Wolfenbarger (1958) reported that between 1946 and 1957, many kinds of insecticides were used in potato and tomato to control leafminers. The insecticides included Carbamates, D ichlorodiphenyltrichloroethane , Benzene hexachloride , Chloradane, Li ndane, Parathion, T oxaphene, N icotine sulphate, M rthoxychlor, Dieldrin, Aldrin, Metacide, D emeton, M alathion, M ethyl demeton, S chradan, H eptachlor, Isodrin, D ilan, Perthane, S trobane, D iazinon, T hiodan, C hlorthion, etc (Wolfenbarger 1958). Most of the insecticides were ineffective after a few seasons. Similarly, oxamyl was recommended for use on celery in 1975, but after two years of use, it failed to achieve control rendering leafminers almost impossible to control in Florida (Poe and Strandberg 1979 , Leibee and Capinera 1995). Later, growers used mathamidophos and permethrin . M athamidophos was considered effective for leafminer control in 1977, but leafminer soon developed resistance against to this insecticide (Leibee and Capinera 1995). The development of resistance in L. trifolii populations to different groups of insecticides in different crops resulted from extensive insecticide use (Leibee 1981 , Mason et al . 1987 , Keil and Parrella 1990 , Ferguson 2004). In 1982, c yromazine provided a welcome relief to growers by controlling L. trifolii populations on celery in Florida b ut by 1989, resistance to cyromazine was reported from the Everglades area . L. trifolii larval mortality w as very hard to achieve even at the highe st dose (Leibee and Capinera

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21 1995). However , in 1982 abamectin was also shown to control L. trifolii effectively (Trumble 198 5, Leibee 1988 , Parrella et al . 1988 , Cox et al . 1995). Hence, the use of abamectin to control leafminers in celery was permitted b ut only for two consecutive applications during a growing period, which prompted the suggestion of a rotation program using abamectin and cyromazine (Leibee and Capinera 1995). Seal et al . (2002) later found that abamectin and spinosad provided better cont rol of L. trifolii than on non treated control plants . In an experiment, SpinTor ® (spinosad) and Proclaim ® (emamectin benzoate) were reported to be effective in controlling L. trifolii and were relatively benign to natural enemies (Webb 2002). Bensultap ( Bancol ® ) was effective in controlling high larval densities of L. trifolii in Turkey (Civelek and Weintraub 2003). Ferguson (2004) reported that abamectin, cyromazine, and spinosad were effective against L. trifolii and suggested that cyromazine resistance on celery was limited to only one place in Florida. Resistant strains of L. trifolii to spinosad, cyromazine, and abamectin became susceptible in the absence of pesticide selection pressure which was not the same for permethrin and chlorpyrifos (Parrella and Trumble 1989 , Ferguson 2004). Biological C ontrol Biological control theory states that if natural enemy species are sufficiently abundant within terrestrial communities, they can limit herbivore populations, which can allow plant communities to grow u ntil they are limited by competition (Rosenheim et al. 1993 , Sher et al. 2000 , Colfer and Rosenheim 2001 ). Traditionally, L. trifolii has been considered a secondary pest i n vegetable and ornamental production systems . It gained the status of primary pest due to the absence of natural enemies that usually regulate L. trifolii populations . Biological control often plays a pivotal role in managing L. trifolii

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22 populations , which have hosted more than 40 parasitoid species (Waterhouse and Norris 1987 , Patel et al . 2003). In Florida, 14 species of L. trifolii parasitoids have been recorded and the most common belong to the families Braconidae, Eulophidae, and Pteromalidae (Capinera 2001). From snap beans growing in south Florida, Li (2011 ) recorded 13 parasitoid species from 3 families of Hymenoptera (Braconidae, Eulophidae, and Pteromalidae). He reported that Opius dissitus (Muesebeck ) ( Hymenoptera: Braconidae ) , a solitary, larval pupal endoparasitoid, was very abundant in bean crops causing 63% parasitism to L. trifolii . Diglyphus intermedius (Girault) ( Hymenoptera: Eulophidae ) , an ectoparasitoid, prefers to attack third instar larvae and is one of the most common L. trifolii parasitoids in tomato (Schuster and Wharton 1993 , Patel et al . 2003). Similarly, D. begini (Ashmead), may also help suppress L. trifolii populations (Heinz et al . 19 93 ); it can be mass reared in a controlled environment and then released to manage L. trifolii populations, such as on marigolds (Parrella et al. 1989). Diglyphus begini paral yzes L. trifolii larvae by injecting venom, then oviposits on or near the larva. Parasitoid larvae typically consume leafminer larvae and emerge from the mine as adults (Sher et al. 2000 ). Microbial agents have also shown great potential for controlling L . trifolii . The entomopathogenic nematodes Steinernema carpocapsae and Heterohabditis bacteriophora have shown promising results when used against Liriomyza leafminers in the greenhouse (Olthof and Broadnet 1990 , Hara et al. 1993, Lebeck et al. 1993). Ste inernema carpocapsae was as effective as abamectin in controlling leafminers on greenhouse chrysanthemums (Lebeck et al . 1993). Sher et al . ( 2000 ) evaluated parasitoid wasp , Diglyphus begini (Ashmead), and a nematode , Steinernema

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23 carpocapsae (Weiser), for control of leafminer and concluded that control by using both the agents ( parasitoid wasps and nematodes ) together was better than when either agent was used individually. With increased concern s about environmental and human health hazards from pesticides in recent years, considerable research has focused on using biorational pesticides as an alternative to synthetic pesticides for control of insect pests. For instance, n eem based biopesticides h ave been investigated for the control of Liriomyza leafminers (Webb et al . 198 3, Larew et al. 1985, Schmutterer 1990 , Banchio et al . 2003). Neem based pesticides degrade quickly in the environment and are low risk to the environment, humans, and natural en emies; and are relatively low cost yet good control for key pests. However in south Florida, relatively few studies have tested the effectiveness of neem based pesticides as an alternative to synthetic pesticides. Research Objective s Population estimation of leafminer is important for implementing management practices. Estimation of live mine s present in the leaves provide information about present damage and future infestations of L. trifolii in the field. P opulation estimate s of adults also provide knowle dge on the present status of the pest . Alternatively, the information s on population abundance help in making decision on when to apply control measures rather than applying control on calendar schedule (Jones and Parrella 1986). This study was conducted to understand the abundance of L. trifolii and its parasitoid on five vegetable crops, beans ( Phaseolus vulgaris L. ), cabbage ( Brassica oleracea L . var capitata L . ), tomato ( Solanum lycopersicum L .), squash ( Cucurbita pepo L.) and cucumber ( Cucumis sativ us L.), commonly grown in South Florida. In addition, the study

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24 was focus ed on testing the efficacy of selected insecticides against L. trifolii and their compatibility with parasitoids . The specific objectives of my research were: 1. To s tudy the abundance of leafminer and its parasitoid on 5 vegetable crops (bean, cabbage, squash, cucumber, and tomato) in South Florida. 2. To determine the seasonal abundance and spatial pattern of distribution of L. trifolii and its parasitoids on squash and snap bean. 3. To e val uat e the effect of chemical and bio rational insecticides on leafminer and

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25 Figure 2 1. Map showing distribution of Liriomyza trifolii in Europe (CABI 2014).

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26 Figure 2 2. Map showing distribution of Liriomyza trifolii in North America. (CABI 2014)

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27 Table 2 1. Global distribution of Liriomyza trifolii Burgess adopted from ( CABI, 2014) Region Countries North America Canada, Mexico, USA Caribbean Bahamas, Barbados, British Virgin, U.S. Virgin Islands, Trinidad and Tobago Central America Argentina, Bahamas, Barbados, Bermuda, Brazil, Chile, Columbia, Costa Rica, Cuba, Dominican Republic, Ecuador, French Guiana, Guadeloupe, Guatemala, Guyana, Martinique, Netherlands Antilles, Peru, Puerto Rico, South America Brazil, Colombia, French Guiana, Guyana, Peru, Venezuela Asia China, Cyprus, India, Iran, Israel, Japan, Jordan, Korea Republic, Lebanon, Oman, Philippines, Saudi Arabia, Taiwan, , Vietnam, Yemen Europe Austria, Belgium, Bulgaria, Central Russia, Croati a, Cyprus, , France, Greece, Ireland, Israel, Italy, Lebanon, Malta, Netherlands, Norway, Poland, Portugal, Portugal, Romania, Slovakia, Slovenia, Spain, Switzerland, Turkey, Yugoslavia Africa r, Mauritius, Mayotte, Morocco, Nigeria, Réunion, Senegal, South Africa, Sudan, Tanzania, Tunisia, Zambia, Zimbabwe

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28 CHAPTER 3 ABUNDANCE OF AMERICAN SERPENTINE LEAFMINER, LIRIOMYZA TRIFOLII AND ITS PARASITOIDS ON FIVE VEGETABLE CROPS IN SOUTH FLORIDA. American serpentine leafminer, Liriomyza trifolii (B urgess ) (D iptera : A gromyzidae ) , is a polyphagous fly and is an important insect pest of vegetable crops (Seal et al. 2002, Minkenberg 1988a). The larva of a leafminer causes major damage as it tunnels int o and feeds on the leaf mesophyll layer (Parrella 1987). High L. trifolii infestations can cause serious economic losses due to reduce d rates of photosynthesis (Parrella et al. 1985, Trumble et al. 1985). L . trifolii feeds on more than 400 cultivated and w ild host plant species in more than 25 plant families and is found in almost all geographical regions (Spencer 1973, Parrella 1987, Wei et al. 200 6 ). Due to its polyphagy feeding habits and the ability to develop resistance to chemical insecticides, L. tri folii poses a considerable threat to vegetable growers. Host plant selection in L. trifolii is performed by the adult female as the larvae are limited to host plant leaves. Hence, the performance of the larva is solely dependent on host plant selection by females. The female L. trifolii generally oviposit after a series of inspections of the host in a sequence of visual, chemical, and tactile stimuli. Acceptance of a host plant by an ovipositing female may result from a different set of characteristics than those causing rejection (Singer 1986, Martin et al. 2005). The choice of a host by L. trifolii is dependent on many factors including host plant allelochemicals, nutritional chemistry, plant morphology, and the activities of natural enemies (Thompson and Pellmyr 1990). When given a choice, L. trifolii feeds and oviposits more eggs on a plant that is its preferred host, similarly , the leafminer parasitoids also have differential preference s on leafminer s on different host plants (Johnson and Hara 1987). Inf ormation on

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29 infestation levels of L. trifolii and activities of its parasitoids on different host crops provides input for developing effective management plans. The present study was conducted to determine infestation levels of L. trifolii and parasitism on five vegetable crop species grown in south Florida. Materials and Methods Field P reparation, P lanting, and C rop M anagement All studies were conducted at the Tropical Research and Education Center, Homestead, FL. The soil type was Krome gravelly loam (33 % soil and 67% limestone pebbles). The study utilized five crops: snap bean ( Phaseolus vulgaris P , squash ( Curcurbita pepo E , cucumber ( Cucumis sativus D , cabbage ( Brassica oleracea E , and tomato ( Solanum lycopersicon R . This study was conducted at two sites (Sites 1 & 2). At site 1, planting was done in May 2014 with samples collected until July, 2014. At site 2, planting was done in September, 2014 and samples were collected until October 2014. Bean, squas h and cucumber were directly seeded on beds raised 15 cm that were 91 cm wide, and covered with polythene mulch (1.5 ml thick) . Seeds were sown within holes cut in the plastic mulch dug into the soil . Depending on the vegetable 3 5 seeds were planted to ensure germination. Plants were 25 cm apart within the row with 91 cm between rows. After germination, seedlings were thinned so that one healthy plant was left in each hole. Four week old seedlings of cabbage and to mato transplants were planted on the raised beds. All crops were planted in the field on the same date (May 1 2014 in season 1 and September 12 2014 in season 2) to maintain consistency of growth and development of experimental plants.

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30 All crops were grow n in Randomized Complete Block designs each with four replications. Each plot was 11.5 m long with 12.5 cm of non planted space between plots. Twenty one days before planting, the pre emergent herbicide Halosulfuron methyl (Sandea®, Gowan Company LLC., Yu ma, AZ) was applied at 51.9 g / ha, to control weeds. Granular fertilizer 6:12:12 (N: P: K ) was applied at 1345 kg / ha in a 10 cm wide band on both sides of the center of each raised bed and was mixed with the soil before covering with plastic mulch. Liqu id fertilizer 4: 0: 8 (N: P: K ) was applied at 0.56 kg of N / ha / day through the drip system at 3, 4, and 5 weeks after planting. Plants were irrigated using drip tubes (T systems, DripWorks, Inc., Willits, California) with two parallel lines spaced 30 c m apart within and parallel to each bed and having 2 mm wide openings each 13 cm. The fungicides chlorothalonil (Bravo ® , Syngenta Crop Protection, Inc., Greensboro, NC) at 1.75 liters / ha and copper hydroxide (Kocide ® 3000, BASF Ag Products, Research Tria ngle Park, NC) at 0.8 liters / ha, were sprayed every two weeks. To control melonworms, pickleworms and diamondback moths, the Bacillus thuringiensis insecticides, Dipel DF ® ( B. thuringiensis var. kurstaki) at 1.1 kg / ha and Xentari DF ® ( B. thuringiensis var. kurstaki) at 1.2 liters / ha (Valent Biosciences Co., Libertyville, IL), were sprayed once every 2 w ee k s beginning 21 days after planting. Sampling Sampling began 15 days after planting , when all plants had two primary (non cotyledonus) leaves fully unfolded . Five plants from each plot were selected randomly for sampling. From each plant, one leaf was randomly collected and placed in a plastic pot (15 cm diameter x 20 cm deep).

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31 Abundance of M ines, L arvae, and P upae Numbers of mines and larvae per plot were recorded. Sample l eaves were then maintained at room temperature (28 ± 1.5 o C, 75 ± 5 % RH, and 14:10 ( L: D ) h photoperiod) until the larvae pupated. The resulting number of pupae per sample was recorded. The pupae were maintained in a Petri dish (5 mm x 60 mm) marked with plot number and observed for further L. trifolii development. Numbers of leafminer adults were counted every day along with its parasitoids. All parasitoids were stored for further identification. Statistical A nalyses Abundance data were analyzed by one way analyses of variance (ANOVAs) (PROC MIXED, SAS Institute 2013). To normalize the error variances, all data were square root transformed. Repeated measures in P ROC MIXED w as used for ANOVAs because the same multiple treatments were surveyed on different dates and this method compensates for possible errors resulting from picking the same plant on more than one sampling date. For abundance data, crop, date, and th eir interaction were modeled. Comparisons between the mean number of mines, larvae, pupae, adults, and parasitoids were performed following the ANOVAs using the Tukey Kramer procedure ( P < 0.05) (SAS Institute 2013). Results Abundance of M ines on V egetabl e C rops The mining activity of L. trifolii , in season 1 was significantly affected by the crop ( F 4, 59 = 96.27, P < 0.0001), date ( F 3, 59 = 61.40, P < 0.0001), and the interaction between crop and date was also significant ( F 12, 59 = 14.9, P < 0.0001).

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32 In season 1 (May June 2014), mean numbers of mines at 2 and 3 w eeks after planting were significantly higher in snap beans than in squash, cucumber , tomato or cabbage (Figure 3 1). However, there w ere no significant difference between mean numbers of mine s among squash, cucumber, tomato, and cabbage at 2 w eeks after planting. However, at 3 weeks after planting, mean numbers of mines on squash and cucumber were not significantly different , but were significantly higher than mean number of mines on tomato an d cabbage. The mean number of mines on beans decreased steeply at 4 weeks after planting and was not significantly different than squash and cucumber ( Figure 3 1). The number of mines on bean at that time was significantly higher than tomato and cabbage , h owever, the mean number of mines on squash, cucumber and tomato, were not significantly different. At 5 weeks after planting, cabbage had no mines and was significantly lower than on all other crops (bean, squash, cucumber and tomato). Overall in season 1 , bean had significantly more mines than squash, cucumber, tomato and cabbage. The mean number of mines on squash and cucumber was not significantly different but was higher than tomato and cabbage. Cabbage had significantly lower mean number of mines than all other crops ( Figure 3 1). Similar results were obtained in season 2 (September October, 2014) as well. The mining activity of L. trifolii , in season 2, was also significantly affected by crop ( F 4, 59 = 338.03, P < 0.0001) and date ( F 3, 59 = 65.50, P < 0.0001) , and the interaction between crop and date was significant ( F 12, 59 = 28.90, P < 0.0001). For the seasonal average, bean had significantly more mines than squash, cucumber, tomato, or cabbage ( Figure 3 2). The mean number of mines on squash and cu cumber was not significantly

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33 different but was higher than tomato and cabbage. Cabbage had significantly lower mean number of mines (less than one mine per leaf) than all other crops ( Figure 3 2). Abundance of L arvae on V egetable C rops The mean number of larvae among crops showed similar results to mean numbers of mines. In season 1, larval populations of L. trifolii were also significantly affected by crop ( F 4, 59 = 111.56, P < 0.0001), date ( F 3, 59 = 60.77, P < 0.0001), and the interac tion of crop and date was also significant ( F 12, 59 = 15.64, P < 0.0001). The mean number of larvae was significantly higher on bean than on cabbage during the entire sampl e period ( Figure 3 3). Similarly, the mean number of larvae was significantly higher on squash and cucumber than on cabbage for 50% of the sampling period. 2 and 5 weeks after planting the number of L. trifolii larvae on squash, cucumber and tomato were not significantly different to cabbage . At 2 and 3 weeks after planting, bean had sign ificantly higher mean numbers of L. trifolii larvae than squash, cucumber , tomato or cabbage . The seasonal average for mean numbers of larvae were significantly higher in bean than in squash or cucumber, which were significantly higher than tomato or cabba ge. Mean numbers of larvae on squash and cucumber did not differ significantly within any sample period or for the seasonal average ( Figure 3 3). Similar results were observed in season 2 (September October 2014, Figure 3 4), when the larval density of L. trifolii was significantly affected by crop ( F 4, 59 = 303.00, P < 0.0001), date ( F 3, 59 = 71.03, P < 0.0001), and the interaction of crop and date ( F 12, 59 = 29.94, P < 0.0001). The mean number s of larvae w ere higher on bean than squash, cucumber, tomato o r cabbage at 2 and 3 weeks after planting and for the seasonal average ( Figure 3 4). For the seasonal average, mean numbers of larvae were not significantly different between squash and cucumber, which were significantly higher

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34 than on tomato or cabbage. Cabbage had significantly lower mean number of mines (less than one mine per leaf) than all the other crops in s eason 2 ( Figure 3 4). Abundance of P upae on V egetable C rops Mean numbers of pupae from leaf samples of different vegetable crops were generally similar to previous results for mines and larvae. In s easons 1 and 2, mean numbers of pupae were highly influenced by crop ( F 4, 59 = 103.96, P < 0.0001; F 4, 59 = 252.76, P < 0.0001), date ( F 3, 59 = 54.33, P < 0.0001; F 4, 59 = 68.95, P < 0.0001); and the interaction of crop and date ( F 12, 59 = 14.57, P < 0.0001; F 4, 59 = 30.71, P < 0.0001). Numbers of pupae from leaf samples in season 1 were significantly higher on bean than squash, cucumber, tomato, and cabbage at 2, 3, and 4 weeks after planting. Cabbage had lowest mean number of pupae at 3 and 4 weeks after planting ( Figure 3 5). Similar results were found in s eason 2 except for cabbage. Cabbage had lowest mean number of pupae at 3, 4, and 5 weeks after planting ( Figure 3 6). Abundance of A dult L eafminer on V egetable C rops Leafminer adult numbers were similar to previous results. In s easons 1 and 2, mean numbers of leafminer adults emerging from collected pupae were highly influenced by crop ( F 4, 59 = 88.56, P < 0.0001; F 4, 59 = 210.98, P < 0.0001), dat e ( F 3, 59 = 47.99, P < 0.0001; F 4, 59 = 51.25, P < 0.0001) and the interaction of crop and date ( F 12, 59 = 11.72, P < 0.0001; F 4, 59 = 22.18, P < 0.0001). Numbers of leafminer adults emerging from pupae collected in Season 1 were significantly higher on bean at 2 and 3 weeks after planting than on squash , cucumber, tomato, and cabbage . Cabbage had the lowest L. trifolii adults at 3 weeks after planting ( Figu re 3 7). In season 2 , bean had highest leafminer adults a t 2 and 3 weeks after planting and cabbage had the lowest leafminer adults a t 3 weeks after planting ( Figure 3 8).

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35 Parasitoid A bundance Counts of relative numbers of parasitoid s emerging from samples were not different from the previous results. In s easons 1 and 2, mean numbers of emerged parasitoids were highly influenced by crop ( F 4, 59 = 32.74, P < 0.0001; F 4, 59 = 32.07, P < 0.0001), date ( F 3, 59 = 47.38, P < 0.0001; F 4, 59 = 19.25, P < 0.0001), and the interaction of crop and date ( F 12, 59 = 11.72, P < 0.0001; F 4, 59 = 22.18, P < 0.0001). Mean numbers of parasitoids from samples collected in s eason 1 were significantly higher on bean at 2 and 3 weeks after planting than on squash , cucumber, tomat o and cabbage . Cabbage had the lowest number of parasitoids at 3 weeks after planting ( Figure 3 9). However, except for week 3 mean numbers of parasitoids from tomato were not significantly different from squash or cucumber. I n season 2 , bean had highest m ean number of parasitoids for seasonal average ( Figure 3 10). There were no differences between the parasitoids complex found between different crops. Opius dissitus was the most abundant parasitoid in all the crops and comprised of more than 60% of the to tal population recorded. There were other parasitoids but were few in numbers. The other parasitoids found were Diglyphus sp. , and Diaulinopsis callichroma . Discussion In each season, mean numbers of mines, larvae, and pupae were higher on snap bean followed by cucumber, squash, tomato, and were lowest on cabbage ( Figure 3 ) . It can be conclude d that bean was the most preferred and cabbage the least preferred host in this test. Thompson and Pellmyr (1991) stated that a polyphagous insect in a choice test will deposit most eggs on the host plant most preferred and fewer eggs on the other plant spe cies. An adult female will select a particular host plant species if it is best for the performance of its offspring (Scheirs and De Bruyn, 2002, Valladares and

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36 Lawton 1991, Bethke and Parrella 1985) or for the ovipositing female adult (Jaenike 1986, Schi ers et al. 2000, Scheirs and De Bruyn 2002). Because leafminer larvae cannot move from leaf to leaf, larval performance is highly influenced by the initial host plant selection by the adult female. Several factors affect host selection by adult females: visual, chemical, acoustic, and touch (Bernays and Chapman 1994, Kang et al . 2009). Insects respond to a specific ratio and blends of plant volatiles produced from their host plants (Bruce et al . 2005). One factor affecting leafminer preference for bean ma y be bean plant aromas. Zhao and Kang (2002) reported that leafminers show different electroantennogram (EAG) responses to different host plants, hence, they were more attracted to odors of tomato than of cotton plants. Zhao and Kang (2003) found that leaf miner EAG responses were higher on bean and tomato than on squash and cucumber plants with the lowest responses on Chinese rose, tobacco, and morning glory. Similarly, bean and tomato were the most preferred host by Liriomyza leafminers in Kenya (Foba et a l . 2015). In the present study, however, lower numbers of mines, larvae, and pupae on tomato leaves than on bean, cucumber, or squash may have resulted from lower leaf area in tomato samples compared with the other crops used in this study. Similarly, Pang et al . (2006) reported that the preference of leafminer adult flies was highest on plants in the Fabaceae followed by Asteraceae, Cucurbitaceae, Solanaceae, Apiaceae with the lowest on Brassicaceae. Furthermore, Pang et al . (2006) reported that the accept ance and rejection of host resulted from levels of tannin and flavone in the leaf in addition to the densities of leaf trichomes. Activity of l eafminer was negatively correlated with the levels of tannin and flavone in the leaf. Because

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37 cabbage has the hi gher flavone content, it had the fewest leafmines. Similar results were observed in our study. In both seasons, cabbage had the fewest leaf mines, larvae, and pupae on each sample date. Another reason for this low infestation on cabbage may be the presenc e of leaf waxes. When leaves have high amounts of e picuticular wax, i nsect herbivores often have greater difficulty adhering to leaf surfaces for feeding or oviposition compared with plants with less leaf wax (Eigenbrode 200 4 ). The lower leafminer activity on cucumber and squash than bean may be because of the presence of cucurbitacin, a substance present in cucurbit plants that acts as feeding deterrent in many insect species ( Tallamy et al . 1997). L . trifolii adults avoided ovi position on plants in the Cucurbitaceae with high contents of cucurbitane glucosides and triterpenoids (Mekuria et al . 2006). Differences in oviposition and feeding preferences in the present study may have also been affected by differences in leaf nutriti ve value. Oviposition, feeding, and fecundity of L. trifolii females is positively correlated with the leaf nitrogen content (Minkenberg and Fredrix 198 9, Minkenberg and Ottenheim 1990 ). Therefore, L. trifolii often prefers plants with high nitrogen conten t over those with lower nitrogen (Minkenberg and Fredrix 198 9 , Minkenberg and Ottenheim 1990 ). Snap bean, cucumber, and squash are major vegetable crops grown in Miami Dade County, where they have been grown for a very long time (DRS, personal communication). These three crops may have high er leafminer infestations because th is sp e cies have been feeding continuously on these crops for many generations ; furthermore , adults of many pests tend to prefer the crop where they began feeding when they were larvae (Zhao and Kang 2002 a ).

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38 There is a tritrophic interaction between host plants, herbivorous insects and their natural enemies. Volatile compounds produced by plants can help influence the success of natural enemies in finding their host insects (Vet and Dicke 1992, Wei et al . 200 7 ). Plants from the families Fabaceae, Solanaceae (eg. Capsicum annuum ), Cucurbitaceae, Apiaceae, Rosaceae, and Vitaceae have been found to release fewer volatiles in lower concentrations whe n they are healthy (Wei et al . 2007). However, when plants such as bean become s injured, they produce volatiles, which may attract parasitoids (Wei et al . 200 6 ). Opius dissitus (Hymenoptera: Braconid), a leafminer parasitoid, which was more attracted to od ors of leafminer infested lima bean plants than to odors of leafminer infested eggplant or cotton plant ( Petitt et al . 1992) , which supports our results from finding significantly more parasitoids in bean leaves infested with leafminer than squash, cucu mbe r, tomato or cabbage. Similar ly, Zhao and Kang ( 2002 b ) reported that Diglyphus isaea (Hymenoptera: Eulophidae ) we re attracted more to plants infested with leafminers . The results show a preference of L. trifolii towards bean . F urther s tudies should be conducted to analyze the chemicals found in the five vegetable crops tested in the present study. Because cabbage plant had the fewest mines, chemical analysis of cabbage leaves may be an interesting way to find if the chemicals present may act as oviposi tion or feeding deterrents of L. trifolii .

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39 Figure 3 1. Mean (± SE) number of L. trifolii mines per five leaves on five vegetable crops during season 1 (May June 2014). Means with same letters within a sampl e date do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b ab a b b b ab a b b c b a c b c c b d 0 10 20 30 40 50 60 70 80 90 100 110 120 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of mines / 5 laeves Bean Squash Cucumber Tomato Cabbage

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40 Figure 3 2. Mean (± SE) number of L. trifolii mines per five leaves on five vegetable crops during season 2 (September October 2014). Means with same letters within a sample date do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b ab ab b b b ab ab b b b b b c b c c c d 0 10 20 30 40 50 60 70 80 90 100 110 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of mines / 5 laeves Bean Squash Cucumber Tomato Cabbage

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41 Figure 3 3. Mean (± SE) number of L. trifolii larvae per five leaves on five vegeta ble crops during season 1 (May June 2014). Means with same letters within a sample date do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b a ab b b b a ab b b c a ab c b c b b d 0 10 20 30 40 50 60 70 80 90 100 110 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of larvae / 5 laeves Bean Squash Cucumber Tomato Cabbage

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42 Figure 3 4. Mean (± SE) number of L. tr ifolii larvae per five leaves on five vegetable crops during season 2 (September October 2014). Means with same letters within a sample date do not significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal averag e. a a a a a b b ab ab b b b b ab b b b b b c b c c c d 0 10 20 30 40 50 60 70 80 90 100 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of larvae / 5 laeves Bean Squash Cucumber Tomato Cabbage

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43 Figure 3 5. Mean (± SE) number of L. trifolii pupae per five leaves on five vegetable crops during season 1 (May June 2014). Means with same letters within a sample dates do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b b a b b b b a b b b b a c b c c a d 0 10 20 30 40 50 60 70 80 90 100 110 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of pupae / 5 laeves Bean Squash Cucumber Tomato Cabbage

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44 Figure 3 6. Mean (± SE) number of L. trifolii pupae per five leaves on five vegetable crops during season 2 (September October 2014). Means with same letters within a sample dates do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b b a b b b b a b b b b ab c b c c b d 0 10 20 30 40 50 60 70 80 90 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of pupae / 5 laeves Bean Squash Cucumber Tomato Cabbage

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45 Figure 3 7 . Mean (± SE) number of L. trifolii adults per five leaves on five vegetable crops during season 1 (May June 2014). Mea ns with same letters within a sample date do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b ab a b b b ab a b b bc ab a b b c b a c 0 10 20 30 40 50 60 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of adults / 5 laeves Bean Squash Cucumber Tomato Cabbage

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46 Figure 3 8. Mean (± SE) number of L. trifolii adults per five leaves on five vegetable crops during season 2 (September October 2014). Means with same letters within a sample date do not differ significantly based on T ukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b b ab a b b b ab a b b b bc ab c b c c b d 0 10 20 30 40 50 60 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of adults / 5 laeves Bean Squash Cucumber Tomato Cabbage

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47 Figure 3 9. Mean (± SE) number of L. trifolii parasitoids per five leaves on five vegetable crops during season 1 (May June 2014). Means with same letters within a sample date do not differ significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasona l average. a a a a a b b ab a b b b abc a b b c abc a bc b c c a c 0 5 10 15 20 25 30 35 40 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of parasitoids/ 5 laeves Bean Squash Cucumber Tomato Cabbage

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48 Figure 3 10. Mean (± SE) number of L. trifolii parasitoids per five leaves on five vegetable crops during season 2 (September October 2014). Means with same letters within a sample date do not significantly based on Tukey Kramer test ( P > 0.05). WAP = weeks after planting; Season = seasonal average. a a a a a b ab ab a b b ab abc a b b bc abc a bc b c c a c 0 5 10 15 20 25 2 WAP 3 WAP 4 WAP 5 WAP Season Mean ( ± SE) number of parasitoids/ 5 laeves Bean Squash Cucumber Tomato Cabbage

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49 CHAPTER 4 SEASONAL ABUNDANCE AND SPATIAL PATTERN OF DISTRIBUTION OF LIRIOMYZA TRIFOLII (BURGESS) (DIPTERA: AGROMYZIDAE) AND ITS PARASITOID ON BEAN AND SQUASH IN SOUTH FLORIDA American serpentine leafminer, Liriomyza trifolii , is a polyphagous insect pest that feeds on a wide range of vegetable and ornamental plants around the world (Seal et al. 2006, Parrella 1987, Spencer, 1973 ). Damage is caused by adult female s and larvae . Adult female s make several punctures in the leaf, using their ovipositor. The punctures are made for feeding and egg laying. Major damage to the plant is caused by larval feeding. Leafminer larvae feeds on the mesophyll layer of leaves, which reduces the photosynthetic area. L . trifolii complete s development from egg to adult in 19 days at 25 o C (Leibee 1984). Because of a short development time, leafminer can produce multiple generations per season. Biological control often plays a major role in managing L . trifolii (Burgess) , which hosts more than 40 parasitoid species (Waterhouse and Norris 1987 , Patel et al. 2003). In vegetable and ornamental production systems, L. trifolii is often considered a secondary pest, but its status has been raised to primary pest because excessive use of pesticides have reduced the natural enemies that usually regulate its population. I f natural enemy species are sufficiently abundant, they can limit herbivore populations, which can al low plant communities to grow until they are limited by competition (Rosenheim et al. 1993 , Colfer and Rosenheim 1995 , Sher et al. 2000). Hence, information on parasitoid density and composition throughout the year, and their effects on leafminer density c an help in developing an IPM program. Changes in environmental factor s , both biotic and abiotic, over time strongly affect leafminer development (Leibee 1985). For example, a female leafminer fly may

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50 lay up to 300 eggs per lifetime, which is over a span of 17 days at 25 °C (Charlton & Allen 1981). This rapid egg production may facilitate population increase (Jong and Rademaker 1991). Weather conditions including temperature, humidity, precipitation, and wind have are some of the most important causes of dra matic changes in pest abundance in an ecosystem (Risch 1987, Nestel et al. 1994). Changes in weather parameters may directly influence the physiology and behavior (locomotion and dispersal) of an insect and indirectly affect the insect population because o f changes in the host plants and the behavior of its natural enemies (Martinat 1987, Nestel et al. 1994). In Lebanon, leafminer populations were reported to be reduced because of high mean temperatures in September and October (Hammad and Nemer 2000). Alternatively , Li et al. (2011) recorded increased leafminer populations in December and January, when mean temperatures were relatively low (21 23 o C). Rainfall and humidity may also affect leafminer population. For instance, Shepard et al. (1998) found th at leafminer populations on potato were relatively low during the dry season. Cultivated crops are principal reproductive and feeding hosts of leafminer. However, when cultivated hosts are absent, leafminer tend to invade alternate weed hosts typically f ound nearby in the fields and returns to the main crops after they are re planted. Knowledge of crop biology and ecology is also important in developing an IPM program for managing leafminer population . Therefore, to develop an effective IPM program, infor mation on the seasonal field distribution and population dynamics of leafminer and its parasitoids is very important . The objectives of this study were to observe the seasonal abundance, and the distribution of L. trifolii and its parasitoids. Specifically , I studied:

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51 Seasonal abundance of leafminer mines and other development stages on bean and squash planted on four dates (Oct Nov 2013, May June 2014, Sep Oct 2014, Nov 2014 Jan 2015 ). Abundance of leafminer parasitoids on above four plantings on b ean and squash. Within field distribution of leafminer and its parasitoids in four plantings. Materials and Methods Field P reparation , P lanting , and C rop M anagement The study was conducted at two sites separated by 1 Km within the Tropical Research and Education Center, Homestead, FL. Snap bean ( Phaseolus vulgaris L. Cucurbita pepo at site 2. Each crop was planted on four dates at the respective sites: Oct 26 ( Oct Nov 2013 ) , May 10 ( May June 2014 ) , Sep 6 ( Sep Oct 2014 ) , and Nov 28 (Nov 2014 Jan 2015). Snap bean and squash seeds (Syngenta Seeds Inc., Othello, WA) were directly seeded on raised beds ( 1 m wide, 0.15 m high) covered with 1.5 ml thick black and white polyethylene mulch . 3 5 seeds were sown in a hole, 1.5 cm deep. Planting holes were spaced 25 cm within the row and 1 m between adjacent rows. A pre plant herbicide, Halosulfuron methyl (Sandea®, Gowan Company LLC., Yuma, Arizona) was applied at 51.9 g / ha 21 days before planting to control weed emergence. Crops were fertilized applying granular fertilizer 6:12:12 (N: P: K ) at 1345 kg/ha in a 10 cm wide band on both sides of the raised bed center and was incorporated before placement o f plastic mulch. Additionally, liquid fertilizer 4: 0: 8 (N: P: K ) was also applied at 0.56 kg N / ha / day through a drip system at 3, 4, and 5 weeks after planting. Plants were irrigated every day for one hour to deliver water ( 1.25 cm ) using two paralle l lines of drip tube (T systems, DripWorks, Inc., Willits, California),

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52 spaced 30 cm apart and parallel to the bed center, having an opening at every 13 cm. The fungicides, Chlorothalonil (Bravo ® , Syngenta Crop Protection, Inc., Greensboro, NC) at 1.75 lit er/ha and Copper hydroxide (Kocide ® 3000, BASF Ag Products, Research Triangle Park, NC) at 0.8 l/ha were sprayed every two weeks , using 655 l/ha at 207 kpa, to prevent fungal diseases. To control melonworms and pickleworms in squash, Bacillus thuringiensis based insecticides, Dipel DF ® (var. kurstaki) at 1.1 Kg/ha and Xentari DF ® ( B. thuringiensis var. kurstaki) at 1.2 liters/ha (Valent Biosciences Corporation, Libertyville, IL), were used in weekly rotation. Seasonal A bundance of L ea fminer M ines , L arvae , A dults , and P arasitoids Seasonal abundance of leafminers w as studied using snap beans and squash in four plantings. Field preparation, planting, and crop management were performed following the methods as previously described as abov e . Planting 1 (October November 2013) Snap bean seeds were planted at Site 1 and squash seeds were planted at Site 2. Each crop site was 552 m 2 which was divided into six beds, each 92 m long and 1 m wide. Both crops were planted on October 26, 2013. Ea ch bed was divided into 8 equal plots, each 11.5 m 2 . Thus, the field was equally divided into 48 plots. Sampling began 15 days after planting , when bean plants had two primary leaves fully unfolded. Five plants from each plot were randomly selected and one full grown leaf from the bottom stratum of each plant was sampled . Thus, five leaves were collected from each plot. All leaves from a plot were placed in a plastic pot (10 cm diameter and 15 cm depth) which was marked with date and plot number. The sample s were then transported to the IPM laboratory and checked under a binocular microscope at 10X to record numbers of mines and larvae per leaf. Leaves were returned to the same pot and placed at room

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53 environment 25 ± 5 o C, 75 ±5 % RH, and 14 h : 10 h ( L: D ) for further studies. All samples were checked at 24 h intervals for larvae and pupae and continued until the emergence of the last pupae. Pupae from each samples were placed separately in a Petri dish (10 cm diameter) marked with date and plot number and l ined at the bottom with a moisten filter paper to prevent desiccation. Petri dishes with pupae were observed daily for adult and parasitoid emergence. Numbers of adult leafminer flies and parasitoids were recorded by date and plot number. Planting 2 (May J une 2014) Snap bean seeds were planted at Site 1 and squash seeds were planted at Site 2. Plot sizes, planting and sample collection were same as Planting 1. Planting 3 (September October 2014) As in the previous two plantings, snap bean was planted at site 1 and squash at site 2 on September 6, 2014, where each site consisted of 368 m 2 . Each site was divided into four 92 m long and 1 m wide beds each having equally divided eight plots of 11.5 m 2 . Thus, there were 32 plots in Planting 3. Sampling began when bean plants had two primary leaves fully unfolded ( 15 days after planting ) using the same methods described for bean at Site 1, Planting 1. Planting 4 ( November 2104 January 2015) In the fourth planting, each site consisted of 276 m 2 which was divided into three 92 meter long beds. Each bed was then divided into eight equal sections of 11.5 meters. Thus, there were 24 equal sections, each 11.5 m 2 . Snap beans were planted at site 1 and squash at site 2. Both bean and squash seeds were planted on November 28, 2014, and sampling began when bean plants had two primary leaves fully unfolded ( 15 days after planting ) using same methods described in Planting 1.

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54 Statistical a nalys e s Seasonal a bundance data were analyzed independently for each planting by one way analyses of variance (ANOVAs) using PROC MIXED in the SAS System (PROC MIXED, SAS Institute 2013). This system provides a very flexible modelling environment for handling a variety of p roblems involved with using subjects repeatedly. To normalize the error variances, all data were square root transformed ( x+0.25) before the analyses . Repeated measures ANOVAs were used (PROC MIXED) because the same multiple treatments were surveyed on d ifferent dates. ANOVAs c ompari ng mean number s of mines, larvae, pupae, adults, and parasitoids were followed by Tukey Kramer procedure s for mean separation ( P < 0.05) (SAS Institute 2013). Spatial Distribution S patial distribution for L. trifolii and its parasitoid s were studied in the same field where abundance was studied . The data collected for abundance were also used for determining spatial distribution of L . trifolii and its parasitoid. In the present study, I used three different plot sizes to comp are distribution pattern. The plot sizes were: 1) 23 m 2 plots which were the combination of two adjacent initial sections; 2) 46 m 2 plots which were the combination of four adjacent initial sections; and 3) 92 m 2 plots which were the combinations of eight adjacent initial sections. Accordingly, data were pooled from 2, 4 and 8 initial sections, respectively. Statistical a nalysis Spatial distribution was determin Equation 3 1 ( Taylor 1961) an 2 ( Iwao 1968). log s 2 = b a (3 1)

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55 + (3 2) In Equation 3 1, S lope ( b ) is the index of aggregation, s 2 is the variance, is the mean number of leafminer s, and a is the factor related to variability of sample size. In Equation 3 2 , m is mean crowding index given by Lloyd (1967) which is ratio of sample variance (s 2 , which is similar to b value i power law, is the density of the contagiousness coefficient. (intercept) is an i ndex of basic contagion or t endency of insects towards crowding . Both b and , respectively, are ind ices of aggregation. Aggregate distributions resulted w hen b or were significantly greater than 1, random w hen b and were not significantly different from 1 , and uniform (regular) w hen b and values were significantly less than 1. The significance of slo pe b and was determined by using s tudent t test s . Estimation of regression pattern s were done by PROC GLM (SAS Institute Inc. 201 3 ). Evaluation of the goodness of fit of the data for each linear model was done by an r 2 value. Results Seasonal A bundance of L eafminer M ines , L arva e , A dults , and Parasitoids Planting 1 (October November 2013) Site 1 (bean) : In Site 1 of Planting 1, t he mining activity of L. trifolii on bean was significantly affected by sampling date ( F 3, 141 = 187.53 , P < 0.0001) ( Figure 4 1). The mean number s of mines (53.33 ± 2.32 mines / 5 leaves) at 2 weeks after planting (Nov 9) were significantly higher than at 3 weeks (Nov 16), 4 weeks (Nov 23), or 5 weeks (Nov 30) after planting. The mean numbers of mines were the lowest (8.58 ± 0. 46 mines / 5 leaves) when plan t s were 5 week old.

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56 Similarly, the mean number s of larvae, pupae, emerged adults, and parasitoids were significantly affected by sampling dates ( Larvae: F 3, 141 = 288.63 , P < 0.0001 ; Pupa: F 3, 141 = 280.83 , P < 0.0001 ; Adults: F 3, 141 = 151.59 , P < 0.0001 ; and p arasitoids: F 3, 141 = 191.99 , P < 0.0001 , respectively ) ( Figure 4 1). The m ean number s of larvae, pupae , emerged adults , and parasitoids were highest at 2 weeks (43.37 ± 2.08, 37.37 ± 1.83, 22.89 ± 1.42 , and 8.8 5 ± 0.59 / 5 leaves, respectively ) and lowest at 5 weeks after planting ( 2.31 ± 0.26 , 1.91 ± 0.22 , 1.43 ± 0.18, and 0.20 ± 0.24 ) . The parasitoids recorded from leaf samples at site 1 were Opius dissitus , Diglyphus sp. , Euopius sp. and Diaulinopsis callichroma . O. dissitus was the most abundant parasitoid and was about 70% of total population. Site 2 (squash) : In Site 2 of Planting 1, the mean number s of mines on squash w ere significantly affected by sampling date s ( F 3, 141 = 81.13, P < 0.0001) ( Figure 4 2). The mean number s of mines on squash (8.22 ± 0.692 mines / 5 leaves) w ere significantly lowe r at 2 weeks after planting (Nov 9). The mean number s of mines (34.18 ± 1.69 mines / 5 leaves) increased significantly and reached the peak at 3 weeks a fter planting (Nov 16). Relative to the third week, numbers of mines then dropped significantly by 4 weeks after planting (23.79 ± 1.357 mines / 5 leaves, Nov 23). Again, at 5 weeks relative to the fourth week, the numbers of mines (11.29 ± 1.035) decreas ed significantly ( Figure 4 2). Similarly, sample dates significantly affected mean numbers of larvae, pupae, emerged adults, and parasitoids ( Larvae: F 3, 141 = 85.74, P < 0.0001; Pupae: F 3, 141 = 87.35, P < 0.0001; Adults: F 3, 141 = 79.57, P < 0.0001; and Parasitoids: F 3, 141 = 59.79, P < 0.0001) ( Figure 4 2). Mean numbers of larvae, pupae, and parasitoids were highest at

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57 3 weeks after planting. Opius dissitus , Diglyphus sp. , Euopius sp. and Diaulinopsis callichroma were the parasitoids recorded from site 2. O. dissitus was the most abundant parasitoid and was about 60% of total population. Planting 2 (May June 2014) Site 1 (bean): Activity of L. trifolii in Planting 2 was similar to Planting 1. The mean number s of mines, larvae, pupae, emerged adults and parasitoids were significantly affected by sample dates ( Mines : F 3, 141 = 396.23 , P < 0.0001 ; Larvae: F 3, 141 = 516.41 , P < 0.0001 ; Pupae: F 3, 141 = 517.30 , P < 0.0001 ; Adults: F 3, 141 = 392.71 , P < 0.0001 ; Para sitoids: F 3, 141 = 258.48 , P < 0.0001) ( Figure 4 3). The mean number s of mines , larvae, pupae , emerged adults, and parasitoids (80.20 ± 3.63 , 71.77 ± 3.41 , 66.58 ± 3.24, 45.60 ± 2.68, and 16.10 ± 1.08 / 5 leaves; respectively ) were highest at 2 weeks after planting (May 24) ( Figure 4 3). These numbers were the highest among all sample dates across all seasons. There was a steep drop in mean number s of mines, larvae, pupae , emerged adults, and parasitoids (27.89 ± 1.42 , 20.52 ± 1.06, 16.77 ± 0.82, 11.31 ± 0. 67, and 3.16 ± 0. 26 / 5 leaves ; respectively) at 3 weeks after planting (May 31). The mean number s of mines, larvae, pupae, adults and parasitoids gradually decreased and were the lowest ( 7.33 ± 0.63; 2.43 ± 0.28; 2.04 ± 0.22; 1.47 ± 0.20; and 0.27 ± 0. 07 / 5 leaves ; respectively ) at 5 weeks after planting (June 14) ( Figure 4 3). Opius dissitus , Diglyphus sp. , and Diaulinopsis callichroma were the parasitoids recorded at site 1 in planting 2. O. dissitus was the most abundant among all the parasitoids recorded. Site 2 (squash): Similar to Planting 1, the mean number s of mines on squash, in Planting 2, w ere significantly affected by sample date s ( F 3, 141 = 52.76, P < 0.0001) ( Figure 4 4). At 3 weeks after planting (May 31), the mean number s of mines (25.41 ±

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58 1.83 mines / 5 leaves) w ere significantly higher than at other sample dates ( Figure 4 4). At 4 weeks after planting (June 7), the mean number s of mines had dropped significantly (14.77 ± 1.04 mines / 5 leaves) compared to the third week, and at 5 weeks after planting (June 14), number of mines again dropped significantly (6.64 ± 0.84 mines / 5 leaves) compared to week 4 reaching lowest value. ( Figure 4 4). Similarly, sample date significantly affected mean number s of larvae, pupae, emerged adults, and parasitoids ( Larvae: F 3, 141 = 65.04, P < 0.0001; Pupae: F 3, 141 = 68.98, P < 0.0001; Adults: F 3, 141 = 63.17, P < 0.0001; and Parasitoids: F 3, 141 = 52.41, P < 0.0001) ( Figure 4 4). The mean number s of larvae, pupae, adults, and parasitoids were each the highest at 3 weeks and the lowest at 5 weeks after planting. The parasitoids found at site 2 were similar to that found at site 1 and O. dissitus was the most abundant among all parasitoids. Plantin g 3 (September October 2014) Site 1 (bean) : In Planting 3, the mean numbers of min es , larvae, pupae , and adults of L. trifolii and the mean number s of its parasitoids on bean were significantly affected by sample dates ( F 3, 93 = 127.58, P < 0.0001 ; Larvae: F 3, 93 = 128.04, P < 0.0001 ; Pupae: F 3, 93 = 122.40, P < 0.0001 ; Adults: F 3, 93 = 102.89, P < 0.0001 ; and Parasitoids: F 3, 93 = 91.09, P < 0.0001) ( Figure 4 5). The mean numbers of mines , larvae, pupae , emerged adults , and parasitoids (56.03 ± 4.1 2 , 50.28 ± 4.07 , 45.40 ± 3.56 , 29.93 ± 2.93 , and 9.71 ± 0.89 / 5 leaves ; respectively ) were all highest at 2 weeks after planting (Sep 20) ( Figure 4 5). Mean numbers of min es , larvae, pupae , and adults of L. trifolii and mean number s of its parasitoids decreased significantly at 3 , 4 , and 5 weeks after planting with lowest values at 5 weeks after planting (Oct 11) (4.78 ± 0.69 ; 3.37 ± 0.54 ; 2.59 ± 0.39 ; 1.84 ± 0.30 ; and 0.50 ± 0.12 / 5 leaves ; respectively ) ( Figure 4 -

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59 5). The parasitoi ds recorded at site 1 were Opius dissitus , Diglyphus sp. , Euopius sp. and Diaulinopsis callichroma . O. dissitus was the most abundant parasitoid and was about 75% of total population. Site 2 (squash): Consistent with the two previous plantings, the mean number s of L. trifolii mines, larvae, pupae, adults and parasitoids on squash in Planting 3 w ere significantly affected by sample date s ( Mines: F 3, 93 = 29.59, P < 0.0001; Larvae: F 3, 93 = 26.49, P < 0.0001; Pupae: F 3, 93 = 23.79, P < 0.0001; Adults: F 3, 93 = 16.7, P < 0.0001; and Parasitoids: F 3, 93 = 37.27, P < 0.0001) ( Figure 4 6). The mean number s of mines, larvae, pupae, and adults on squash were significantly higher at 3 and 4 weeks after planting than 2 and 5 weeks after planting ( Figure 4 6). Simi larly, mean numbers of parasitoids were significantly higher at 3 weeks after planting (5.00 ± 0.49 parasitoids / 5 leaves) than on other sample dates and were lower (0.93 ± 0.18 parasitoids / 5 leaves) at 5 weeks after planting. Opius dissitus , Diglyphus sp. , Euopius sp. and Diaulinopsis callichroma were the parasitoids recorded at site 2. O. dissitus was the most abundant parasitoid and was about 70% of total population. Planting 4 ( November 2014 January 2015) Site 1 (bean): The m ean number s of L. trifolii m in es , larvae, pupae , adults and parasitoids on bean w ere significantly affected by sample date s ( Mines: F 3, 93 = 12.04, P < 0.0001; Larvae: F 3, 93 = 10.49, P < 0.0001; Pupae: F 3, 93 = 6.73, P = 0.0005; Adults: F 3, 93 = 3.66, P = 0.0165; and Paras itoids: F 3, 93 = 7.71, P = 0.0002) ( Figure 4 7). However, the pattern of population density was not consistent and was different from the previous 3 plantings. The mean number s of mines w ere significantly higher at 4 weeks after planting (11.37 ± 0.986 mines / 5 leaves, Dec 26) compared with 2 w eeks ( 4.75 ± 0.90 mines / 5 leaves ) and 3 weeks after planting (6.79 ± 1. 0 3 mines / 5 leaves ) ( Figure 4 7).

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60 The m ean number s of larvae, pupae, adults and parasitoids also wer e significantly higher at 4 weeks compared with 2 weeks and 3 weeks after planting ( Figure 4 7). Opius dissitus , Diglyphus sp. , Euopius sp. and Diaulinopsis callichroma . O. dissitus was the most abundant parasitoid and was about 50% of total population. Site 2 (squash): Unlike the other 3 plantings, Planting 4 had different population density trends (Figure 4 8). The mean number s of mines, larvae, pupae, adults, and parasitoids on squash were significantly affected by sample date s ( Mines: F 3, 141 = 69.66, P < 0.0001; Larvae: F 3, 141 = 68.76, P < 0.0001; Pupae: F 3, 141 = 57.84, P < 0.0001; Adults: F 3, 141 = 48.80, P < 0.0001; and p arasitoids: F 3, 141 = 54.22, P < 0.0001) ( Figure 4 8). At 2 weeks after planting the squash (Dec 12), the mean number s of mines (2.70 ± 0.44 mines / 5 leaves) w ere significantly lower than the three other sample dates and was the lowest of the planting. However, the mean number s of mines at 4 weeks after planting w ere significantly higher than on the other dates (27.54 ± 2.39 mines / 5 leaves) ( Figure 4 8).. Similar results were observed for mean numbers of larvae, pupae, adults, and parasitoids with minima and maxima at 2 week s and 4 we ek s after planting, respectively ( Figure 4 8). O. dissitus was the most abundant parasitoid and was about 50% of total population of parasitoids. Spatial Distribution Planting 1 (October November 2013) Site 1 (bean) : Based on the number s of mines, leafminer distributions on bean were uniform in all plot sizes ( 23, 4 6 , and 92 m 2 ) at 2 weeks after planting. S lope s b and from s patchiness regression models were significantly < 1 (Table 4 1, P < 0.05) . However, the distribution changed to aggregated for all plot sizes at 3 and 4 weeks a fter planting. S lope s b and were significantly > 1 (Table 4 1 , P

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61 < 0.05) . But at 5 weeks after planting, the distribution pattern of mines in the smallest plots (23 m 2 consistent with s patchiness s provided better fit of data because of the higher r 2 . Hence, the overall mine distribution pattern for 23 m 2 plots was apparently aggregated. The distribution of parasitoids, however, did not show consistent resu lts. At 2 weeks after planting (Nov 9), both the s patchiness regression models contradicted with each other in 23 and 46 m 2 patchiness regression model had a higher r 2 value and provided a better fit to th e data and exhibited random and aggregated distribution, respectively. But for the largest plots, both the linear regression models yielded aggregated distributions (Table 4 2). At 3 weeks after planting (Nov 16), both linear regression models showed aggre gated distributions for 23 and 46 m 2 plots, but were uniform for large (92 m 2 ) plots. However at 4 weeks after planting (Nov 23), the parasitoid distributions in all plot sizes were uniform based on both models (Table 4 2). At 5 weeks after planting, howe ver (Nov 30), both higher r 2 values for each plot size, it provided better fits to the data, and the overall parasitoid distributions were aggregated, uniform , and random for plot sizes 23, 46, and 92 m 2 , respectively (Table 4 2). Site 2 (squash) : s patchiness regression models agreed at 2 weeks after planting and exhibited uniform distributions for leafminers based on numbers of mines in all plot sizes (23, 46 and 92 m 2 ) ( Table 4 1). At 3 weeks after planting, the distributions remained uniform in 23 and 46 m 2 plot, but

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62 the distribution for largest plots (92 m 2 ) was aggregated. The slope s b and were significantly > 1 (Table 4 1 , P < 0.05) . Distributions remained aggregated in all plot sizes at 4 weeks after planting, but changed to uniform at 5 weeks ( Table 4 1). Although both the regression models applied to parasitoid distribution were generally in agreement, there were some inconsistenc ies (Table 4 2). The distribution was mostly uniform for 2, 3, and 4 weeks after planting, but was aggregated at 5 weeks. Planting 2 (May June 2014) Site 1 (bean) : Based on the mean number s of mines, leafminer distribution s on bean were dissimilar among r egression model s at 2 weeks after planting (May 24) (Table 4 higher r 2 value s and better fit s to the data , which therefore exhibited uniform, random, and aggregated distributions for 23, 46, and 92 m 2 plots, respectively. Similarly, at 3 weeks after planting (May 31), values of the indices contradicted each other for distributions in 46 and 92 m 2 plots. higher r 2 value s and provided better fit s to the data , wh ich exhibited random distributions. However for the smallest plots (23 m 2 ), both the indices showed aggregated distributions (Table 4 3). At 4 weeks after planting (June 7), based on random, an d aggregated for plot sizes of 23, 46 and 92 m 2 , respectively. At 5 weeks after planting (June 14), both modes yielded aggregated distributions for all plot sizes. For distribution of parasitoids, both indices agreed for all times and plot sizes (Table 4 4). At 2 weeks after planting, parasitoids showed aggregated, uniform, and uniform distribution for 23, 46 , and 92 m 2 plots, respectively. However, at 3 and at 4 weeks after planting, parasitoids yielded uniform, uniform, and aggregated distributions

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63 in 23 , 46, and 92 m 2 plots, respectively. At five weeks after planting, distributions were aggregated for all plot sizes (Table 4 4). Site 2 (squash) : Leafminer distributions on squash were not consistent, and the regression models often disagreed with each oth er (Table 4 patchiness regression model yielded higher r 2 value s and provided a better fit s to the data , I chose its resulting distribution patterns. Distributions of mines at 2 weeks after planting in 23, 46 and 92 m 2 plots were uniform, aggregated, and aggregated. But at 3 weeks after planting, the distributions changed to aggregated, aggregated, and random in 23, 46, and 92 m 2 plots, respectively. However, at 4 weeks after planting, distribution of mines were aggreg ated, uniform, and uniform in 23, 46, and 92 m 2 plots, respectively. At 5 weeks after planting, however, distributions of mines were aggregated for all plot sizes. Parasitoids distributions were mostly aggregated for all plot sizes and sample dates (Table 4 4). However, at 5 weeks after planting, parasitoid distributions were aggregated in 23 m 2 plot sizes and uniform in 46 and 92 m 2 plot sizes. Planting 3 (September October 2014) Site 1 (bean): Distributions of leafminer mines were not consistent through out Planting 3 and were contradicting among the regression models at 2 weeks after planting (Sep 20) (Table 4 5). The plot sizes 23, 46, and 92 m 2 exhibited uniform, regression models yielded higher r 2 value s, thus provided better fit s to the se data . However, at 3 weeks after planting (Sep 27), the models found aggregated distributions for all plot sizes (Table 4 5). Similarly, at 4 weeks after planting (Oct 4), both indices sug gested the distributions were uniform, uniform, and aggregated for 23, 46, and 92

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64 m 2 plots (Table 4 5). At 5 weeks after planting (Oct 11), leafminer distributions for 23, 46and 92 m 2 plots were aggregated, random, and uniform, respectively, based on Iwao F or parasitoid distributions, both indices were in agreement 2 weeks after planting with aggregated distributions for all plot sizes (Table 4 6 ). A t 3 weeks after planting, however, parasitoid distributions disagreed among models. P arasitoids had aggregated, aggregated, and uniform distribution s in 23, 46 , and 92 m 2 plots , respectively based on which yielded higher r 2 value s, thus provided better fit s to the se data . Similarly, at 4 weeks af ter planting, based uniform distribution s in 23, 46 , and 92 m 2 plots , respectively However , at 5 weeks after planting, distribution s were uniform for all plot sizes (Table 4 6 ). Site 2 (squash). Both regression models agreed and yielded uniform distributions of leafminer mines in squash at 2 weeks after planting for all plot sizes (Table 4 5 ) . But 3 weeks after planting, distributions changed to aggregated, aggregated, and random in 23, 46, and 92 m 2 plots, respectively. Similarly, distributions of mines were aggregated, aggregated, and uniform in 23, 46, and 92 m 2 plots, respectively at 4 we eks after planting. Distributions changed to uniform, uniform, and aggregated in 23, 46, and 92 m 2 plot sizes at 5 weeks after planting (Table 4 5 ) . Leafminer parasitoid distributions on squash were uniform at 2 weeks after planting for all plot sizes (Ta ble 4 6). At 3 weeks, the distributions changed to aggregated, uniform, and aggregated for 23, 46 , and 92 m 2 plots . Based on both regression models, mines exhibited aggregated, uniform, and uniform for 23, 46 , and

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65 92plots , respectively at 4 weeks after pl anting. But, at 5 weeks, distributions were aggregated, aggregated, and uniform for 23, 46 , and 92 m 2 plots , respectively (Table 4 6). Planting 4 ( November 2104 January 2015) Site 1 (bean): s models for distribution of L. trifolii mines were in agreement for all plot sizes and sample dates except for the smallest plots ( 23 m 2 ) at 4 week after planting (Table 4 s . Distributions in larger plots ( 46and 92 m 2 ) a t 4 weeks were aggregated. At 2 weeks after planting (Dec 12), distributions were uniform for the 23 m 2 plots based on both models. Distributions on larger plots of 46and 92 m 2 were aggregated. At 3 weeks after planting, distributions aggregated, uniform , and uniform for 23, 46 , and 92 m 2 plots , respectively. At 5 weeks after planting, distributions in 23 m 2 plots were random , but were uniform in 46and 92 m 2 plots (Table 4 7). s patchiness regression models were in agreement for distributions of parasitoids for all plot sizes and sample dates except for the smallest plots ( 23 m 2 ) at 2 week after planting (Table 4 8). Here, the s . Distributions i n larger plots ( 46and 92 m 2 ) at 2 weeks were aggregated. At 3 weeks after planting, the parasitoids exhibited aggregated, uniform, and uniform distributions for 23, 46 , and 92 m 2 plots , respectively. However, the distributions were uniform for all plot siz es at 4 weeks after planting which changed to aggregated at 5 weeks after planting. Site 2 (squash) : Distributions of L. trifolii based on numbers of mines according to both the regression models were consistent throughout all sample dates for all plot sizes, except for the plots of 23 and 46 m 2 at 5 weeks after planting (Table 4 7). Since,

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66 higher r 2 value s, thus provided better fit s to the se data and exhi bited uniform and random, respectively. Distribution for 92 m 2 plots were aggregated. The distribution of parasitoids was mostly uniform except at 2 and 5 weeks after planting (Table 4 8). At 2 weeks after planting, the distribution was aggregated, aggrega ted, and uniform for 23, 46 , and 92 m 2 plots , respectively . At 3 weeks, distributions were uniform. At 4 weeks, distributions were uniform except for 46 m 2 plots , which were s , which had the higher r 2 suggesting it was the most valid result for 46 m 2 plots . At 5 weeks, results were uniform for 23 m 2 plots and aggregate for 92 m 2 plots . Plots of 46 m 2 were random d s , which had a higher r 2 suggesting it was the mos t valid result for 46 m 2 plots (Table 4 8). Discussion The mean number s of mines, larvae, pupae, and adult L. trifolii on bean were observed to be highest at 2 weeks after planting for all trials except for Planting 4 ( Nov 2014 Jan 2015) (Figures 4 1, 4 3, 4 5, and 4 7). Similarly, on squash, mean numbers of mines, larvae, pupae, and adults were highest at 3 weeks after planting for all trials, except for Planting 4 ( Nov 2014 Jan 2015) (Figures 4 2, 4 4, 4 6, and 4 8). With beans, leafminer activity p eaked few days after the plants had two primary leaves fully unfolded, and there was significant decrease in leaf miner activity thereafter. In contrast, leafminer activity on squash gradually increased, tending to peak 3 weeks after planting, then it grad ually decreased 4 and 5 weeks after planting. These leafminer activity patterns may have resulted from factors such as leaf nutrients, defensive compounds, trichome presence, and cuticle thickness. Many reports have demonstrated the

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67 differential leaf utili zation by leafminer based on these leaf characteristics (Stiling et al. 1982, Fagoonee and Toory 1983, Nuessly and Nagata 1994, Li et al. 199 7 , Scheirs et al. 2001, Facknath, 2005, Digweed 2006, Ayabe and Shibata 2008). Results from our study suggest that leafminers on bean prefer the first pair of leaves, which are cotyledonus. Similarly, Chandler and Gilstrap (1987) working with L. trifolii on peppers observed an initial period of increased damage during the cotyledon growth phase. Many other studies con firm the exclusive utilization of new (young) leaves of host plants by different leafminer species (Auerbach & Simberloff 1984, Hespenheide 1991, Ayabe and Shibata 2008 ). The a verage across all plantings for leafminer mines, larvae, pupae, and adults on b eans were inconsistent (Figure 4 9) . Planting 1 (Oct Nov 2013) and Planting 3 (Sep Oct 2014) had similar seasonal averages for numbers of mines, larvae, pupae, and adults. However, Planting 2 (May June 2014) had apparently higher seasonal averages fo r mean number s of mines, larvae, pupae, and adults than Plantings 1 or 3, whereas Planting 4 ( Nov 2014 Jan 2015) had the lowest. However on squash, seasonal average s for mines, larvae, pupae, and adults were similar (comparable) in Plantings 2, 3 and 4 , and slightly higher in Planting 1 than the other plantings (Figure 4 10 ) . Because there was a large temperature decrease in December 2014 during the 4 th planting, this low temperature may have helped to minimize the leafminer infestations (<1 mines per le af). The temperature at 2 weeks after the fourth planting dropped to 17 o C, which was relatively low compared to other sample dates in the planting, when temperatures were generally between 20 26 o C ( FAWN 2015) (Figure 4 11) .

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68 Similar trends of temperature dependent fluctuation in population density have been shown in other studies (Johnson et al. 1980, Nestel et al. 1994, Palumbo et al. 1994, Hammad and Nemer 2000, Park et al. 2001, Weintraub 2001, Tran et al. 2005, Arida et al . 2013 ) . Nestel et al. (1994) reported that leafminer populations peaked during intermediate temperatures. They suggested that dynamics of tropical insect populations can be changed with slight variations in climatic conditions in tropical regions. Palum bo et al. (1994) reported decreased leafminer populations in December and January (when temperatures were low) and increased populations in September and October, when temperatures were above 23 °C . This pattern of population density was concurrent with our results. Park et al. (2001) reported a similar temperature dependent population trend for L. trifolii in Korea. They observed that populations of leafminer adults increased immediately after transplanting Gerbera jamesonii in April, and the population wa s still higher in mid May, early September, and late October while the population decreased in December . Similarly, Tran et al. (2005) reported that leafminer populations in Vietnam were highest in November with densities as high as 38 larvae/leaf. Simila rly, Hammad and Nemer (2000) reported that leafminer population densities were lower with temperatures above 28 °C, but were relatively high with temperatures of 20 27 °C. In our study, the leafminer populations were highest within a similar temperature r ange (24 26 °C ) . Population densities of L. trifolii parasitoids showed similar trends as leafminers on both bean and squash. Population densities of the parasitoids were highest when leafminer population densities were also high. Occurrence of parasitism and its

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69 magnitude varies with leafminer densities ( Palumbo et al. 1994) . Hence, this parasitism by leafminer parasitoids may be density dependent parasitism , which may warrant further investigation. Based on numbers of larvae sampled, L. trifolii populations on squash and bean did not show a particular d istribution pattern on all the sample dates. The distribution patterns appeared similar on both crops . L . trifolii exhibited mostly aggregated distributions on each crop on most sample dates. Similar results were reported by Beck et al. (1981) Jones & Par rella (1986), and Hammad and Nemer (2000). Distributions of parasitoids and their leafminer hosts were similar on bean and squash. Therefore, these results do not provide enough evidence to conclude that distribution of parasitoids is affected by weather p arameter s instead we can conclude that the distribution is depended on leafminer density. Results of the present study indicated that leafminer preferred certain chronologies of bean and squash plantings over others. Perhaps additional studies should inves tigate differences in the physical and chemical properties of bean and squash leaves at different periods after planting and any differential effects they may have on leafminer parasitism.

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70 Figure 4 1. Planting 1 ( 26 Oct to 30 Nov 2013), Site 1 (bean) abundance of L. trifolii and its parasitoi ds (mean ± SE / 5 leaves). Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b b c a b c d a b c d a b c d a b c d 0 5 10 15 20 25 30 35 40 45 50 55 60 2 3 4 5 Mean ( ± SE) number of insects/ 5 leaves Weeks after Planting mines larvae Pupae parasitoids adults

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71 Figure 4 2. Planting 1 ( 26 Oct to 30 Nov 2013 ), Site 2 ( squash ) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b c a a b c a a b c a a b c d a b c a 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 2 3 4 5 Average daily Temp ( ° C ) Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults Temperature

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72 Figure 4 3 . Planting 2 ( 10 May to 14 June 2014 ), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b c d a b c d a b c d a b c d a b c d 0 10 20 30 40 50 60 70 80 90 2 3 4 5 Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults

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73 Figure 4 4 . Planting 2 ( 10 May to 14 June 2014 ), Site 2 ( squash ) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b c a a b c a a b c d a b a c a b c d 0 5 10 15 20 25 30 0 5 10 15 20 25 30 2 3 4 5 Average daily Temp ( ° C ) Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults Temperature

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74 Figure 4 5 . Planting 3 ( 6 Sep through 11 Oct 2014 ), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b c d a b c d a b c d a b c d a b c d 0 10 20 30 40 50 60 70 2 3 4 5 Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults

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75 Figure 4 6 . Planting 3 ( 6 Sep through 11 Oct 2014 ), Site 2 ( squash ) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b b a a b b a a b b a a b c a a b b a 0 5 10 15 20 25 30 0 5 10 15 20 25 30 2 3 4 5 Average daily Temp ( ° C ) Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults Temparature

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76 Figure 4 7 . Planting 4 ( 28 Nov 2014 through 2 Ja n 2015 ), Site 1 (bean) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a a b b a ab c bc a a b ab a a b a a a b ab 0 2 4 6 8 10 12 14 2 3 4 5 Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults

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77 Figure 4 8 . Planting 4 ( 28 Nov 2014 through 2 Jan 2015 ), Site 2 ( squash ) abundance of L. trifolii and its parasitoids (mean ± SE / 5 leaves) . Mean s with same letters across the sample dates do not differ significantly based on a Tukey Kramer test ( P > 0.05). a b c d a b c d a b c d a a b c a b c d 0 5 10 15 20 25 0 5 10 15 20 25 30 35 2 3 4 5 Average daily Temp ( ° C ) Mean ( ± SE) number of insects/ 5 leaves Weeks after planting mines larvae pupae parasitoids adults Temperature

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78 Figure 4 9 . Seasonal a bundance (mean ± SE / 5 leaves) of L. trifolii mines, larvae, pupae, adults and its parasitoids on bean during 4 plantings . 0 5 10 15 20 25 30 35 40 mines larvae pupae parasitoids adults Mean ( ± SE) number of insects/ 5 leaves Season 1 Season 2 Season 3 Season 4

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79 Figure 4 1 0 . Seasonal a bundance (mean ± SE / 5 leaves) of L. trifolii mines, larvae, pupae, adults and its parasitoids on squas h during 4 plantings . 0 5 10 15 20 25 mines larvae pupae parasitoids adults Mean ( ± SE) number of insects/ 5 leaves Season 1 Season 2 Season 3 Season 4

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80 Figure 4 11 . Comparison of average daily temperature (°C) and abundance of L. trifolii mines, larvae, and parasitoids on bean during the four planting s ( 26 Oct 2013 2 January , 201 5 ). 0 5 10 15 20 25 30 35 0 10 20 30 40 50 60 70 80 90 Average daily ° C (Temp) Mean ( ± SE) number of insects / 5 leaves Sample dates mines parasitoids Temperature

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81 Table 4 L. trifolii mines on beans and squash sampled in Planting 1 (Oct Nov 2013) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Nov 9 23 46 92 0.001 0.005 0.008 0.004 UNI 0.367 UNI 0.007 UNI 0.835 0.934 0.959 0.883 UNI 0.928 UNI 0.927 UNI 0.001 0.211 0.342 0.126 UNI 0.795 UNI 0.729 UNI 0.006 0.005 0.006 0.796 UNI 0.942 UNI 0.361 UNI Nov 16 23 46 92 0.383 0.148 0.446 4.718 AGG 2.301 AGG 2.691 AGG 0.849 0.863 0.917 1.238 AGG 1.141 AGG 1.196 AGG 0.095 0.001 0.382 2.799 UNI 0.187 UNI 1.288 AGG 0.532 0.656 0.964 0.681 UNI 0.902 UNI 1.027 AGG Nov 23 23 46 92 0.152 0.081 0.263 2.215 AGG 1.817 AGG 2.122 AGG 0.708 0.541 0.882 1.227 AGG 1.041 AGG 1.222 AGG 0.007 0.201 1.639 0.525 UNI 2.174 AGG 1.639 AGG 0.772 0.839 0.493 1.062 AGG 1.195 AGG 1.036 AGG Nov 30 23 46 92 0.013 0.104 0.288 0.781 UNI 1.296 AGG 2.266 AGG 0.653 0.862 0.914 1.150 AGG 1.050 AGG 1.108 AGG 0.007 0.009 0.485 0.265 UNI 0.263 UNI 3.306 UNI 0.220 0.438 0.041 0.800 UNI 0.826 UNI 0.318 UNI AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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82 Table 4 2. for distribution of parasitoids of L. trifolii on bean and squash sampled in Planting 1 (Oct Nov 2013) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Nov 9 23 46 92 0.096 0.041 0.408 0.926 UNI 1.034 RAN 2.709AGG 0.878 0.885 0.813 0.972 RAN 1.098 AGG 1.308 AGG 0.032 0.219 0.002 0.203 UNI 0.455 UNI 0.030 UNI 0.429 0.670 0.749 0.643 UNI 0.695 UNI 0.534 UNI Nov 16 23 46 92 0.273 0.377 0.155 1.475 AGG 1.575 AGG 0.551 UNI 0.651 0.803 0.786 1.218 AGG 1.115 AGG 0.849 UNI 0.007 0.110 0.284 0.335 UNI 1.253 AGG 0.999 RAN 0.303 0.808 0.934 0.712 UNI 1.111 AGG 1.023 RAN Nov 23 23 46 92 0.164 0.011 0.014 0.696 UNI 0.316 UNI 0.202 UNI 0.643 0.268 0.577 0.898 UNI 0.626 UNI 0.718 UNI 0.006 0.067 0.323 0.005 UNI 0.854 UNI 2.231 AGG 0.424 0.533 0.563 0.756 UNI 0.863 UNI 1.539 AGG Nov 30 23 46 92 1.000 0.471 0.810 2.001 AGG 0.943 UNI 1.008 RAN 0.152 0.157 0.273 1.001 RAN 1.444 AGG 1.382 AGG 0.554 0.876 0.823 1.521 AGG 1.507 AGG 1.429 AGG 0.652 0.713 0.6299 2.518 AGG 2.297 AGG 1.847 AGG AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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83 Table 4 3. for distribution of L. trifolii mines on beans and squash sampled in Planting 2 (May June 2014) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Nov 9 23 46 92 0.011 0.081 0.250 0.504 UNI 0.704 UNI 1.261 AGG 0.925 0.971 0.973 0.929 UNI 1.001 RAN 1.041 AGG 0.005 0.229 0.489 0.202 UNI 1.295 AGG 1.567 AGG 0.316 0.628 0.644 0.752 UNI 1.296 AGG 1.159 AGG Nov 16 23 46 92 0.270 0.266 0.285 2.225 AGG 1.722 AGG 1.282 AGG 0.943 0.909 0.950 1.056 AGG 1.018 RAN 0.996 RAN 0.371 0.360 0.192 2.621 AGG 2.105 AGG 1.150 AGG 0.663 0.843 0.627 1.032 AGG 1.337 AGG 1.031 RAN Nov 23 23 46 92 0.350 0.412 0.840 1.243 AGG 0.818 UNI 1.558 AGG 0.913 0.960 0.965 1.023 RAN 0.971 RAN 1.146 AGG 0.368 0.005 0.056 2.893 AGG 0.212 UNI 0.566 UNI 0.760 0.685 0.813 1.281 AGG 0.893 UNI 0.923 UNI Nov 30 23 46 92 0.255 0.388 0.442 1.663 AGG 1.324 AGG 2.118 AGG 0.634 0.872 0.468 1.047 AGG 1.122 AGG 1.234 AGG 0.123 0.341 0.859 1.050 AGG 0.933 UNI 1.461 AGG 0.370 0.572 0.943 1.284 AGG 1.112 AGG 1.468 AGG AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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84 Table 4 4. ss regression parameters for distribution of parasitoids of L. trifolii on bean and squash sampled in Planting 2 (May June 2014) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Nov 9 23 46 92 0.338 0.059 0.086 1.923 AGG 0.586 UNI 0.546 UNI 0.797 0.692 0.829 1.148 AGG 0.919 UNI 0.952 UNI 0.167 0.210 0.372 0.814 UNI 1.002 RAN 1.783 AGG 0.378 0.132 0.167 1.481 AGG 1.016 RAN 1.259 AGG Nov 16 23 46 92 0.001 0.096 0.502 0.032 UNI 0.921 UNI 2.012 AGG 0.291 0.495 0.794 0.751 UNI 0.825 UNI 1.139 AGG 0.032 0.436 0.335 0.460 UNI 2.610 AGG 1.364 AGG 0.461 0.696 0.644 0.930 UNI 1.433 AGG 1.275 AGG Nov 23 23 46 92 0.202 0.279 0.61 0.567 UNI 0.577 UNI 1.123 AGG 0.787 0.893 0.877 0.948 UNI 0.866 UNI 1.109 AGG 0.343 0.471 0.493 1.002 RAN 1.269 AGG 1.091 AGG 0.407 0.312 0.462 1.454 AGG 1.384 AGG 1.275 AGG Nov 30 23 46 92 1.000 0.778 0.823 2.001 AGG 1.192 AGG 1.093 AGG 1.000 0.438 0.383 3.000 AGG 1.678 AGG 1.556 AGG 1.000 0.450 0.242 2.000 AGG 0.943 UNI 0.538 UNI 0.571 0.166 0.014 2.478 AGG 0.954 UNI 0.280 UNI AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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85 Table 4 L. trifolii mines on beans and squash sampled in Planting 3 (Sep Oct 2014) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Sep 20 23 46 92 0.078 0.001 0.234 1.424 AGG 0.089 UNI 1.889 AGG 0.867 0.920 0.772 0.970 UNI 0.879 UNI 1.109 AGG 0.075 0.107 0.250 0.684 UNI 1.272 UNI 2.093 UNI 0.272 0.061 0.006 0.874 UNI 0.301 UNI 0.072 UNI Sep 27 23 46 92 0.135 0.226 0.199 2.752 AGG 4.816 AGG 1.264 AGG 0.882 0.850 0.903 1.315 AGG 1.265 AGG 1.052 AGG 0.532 0.371 0.443 2.585 AGG 1.722 AGG 1.027 RAN 0.746 0.889 0.957 1.406 AGG 1.149 AGG 1.003 RAN Oct 4 23 46 92 0.024 0.288 0.699 0.494 UNI 0.893 UNI 1.347 AGG 0.418 0.727 0.928 0.808 UNI 0.942 UNI 1.095 AGG 0.067 0.271 0.284 1.917 AGG 1.929 AGG 7.203 UNI 0.528 0.792 0.049 1.456 AGG 1.243 AGG 0.736 UNI Oct 11 23 46 92 0.430 0.164 0.028 1.910 AGG 1.226 AGG 0.762 UNI 0.438 0.388 0.003 1.232 AGG 0.990 RAN 0.182 UNI 0.056 0.007 0.636 0.947 UNI 0.391 UNI 1.820 AGG 0.286 0.059 0.820 0.958 UNI 0.606 UNI 1.479 AGG AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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86 Table 4 6. chiness regression parameters for distribution of parasitoids of L. trifolii on bean and squash sampled in Planting 3 (Sep Oct 2014) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Sep 20 23 46 92 0.084 0.526 0.950 1.157 AGG 2.251 AGG 1.279 AGG 0.752 0.925 0.995 1.303 AGG 1.208 AGG 1.073 AGG 0.290 0.131 0.535 0.893 UNI 0.522 UNI 0.761 UNI 0.218 0.246 0.596 0.812 UNI 0.723 UNI 0.873 UNI Sep 27 23 46 92 0.134 0.527 0.036 0.864 UNI 6.345 AGG 5.702 AGG 0.796 0.682 0.006 1.132 AGG 3.396 AGG 0.656 UNI 0.052 0.341 0.656 0.677 UNI 0.771 UNI 1.304 AGG 0.593 0.913 0.951 1.093 AGG 0.954 UNI 1.086 AGG Oct 4 23 46 92 0.032 0.284 0.761 0.379 UNI 0.734 UNI 0.815 UNI 0.301 0.284 0.819 1.090 AGG 0.665 UNI 0.852 UNI 0.188 0.121 0.458 1.482 AGG 0.901 UNI 1.640 UNI 0.344 0.343 0.039 1.279 AGG 0.916 UNI 0.167 UNI Oct 11 23 46 92 0.090 0.555 0.101 0.384 UNI 0.799 UNI 0.679 UNI 0.087 0.431 0.014 0.493 UNI 0.754 UNI 0.466 UNI 0.499 0.456 0.022 1.176 AGG 1.134 AGG 0.302 UNI 0.512 0.467 0.053 1.740 AGG 1.624 AGG 0.621 UNI AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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87 Table 4 7. for distribution of L. trifolii mines on beans and squash sampled in Planting 4 ( Nov 2014 Jan 2015) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Dec 12 23 46 92 0.235 0.711 0.743 0.935 UNI 1.663 AGG 1.321 AGG 0.230 0.550 0.757 0.867 UNI 1.435 AGG 1.367 AGG 0.175 0.590 0.848 0.763 UNI 1.454 AGG 1.688 AGG 0.702 0.823 0.900 0.950 UNI 1.312 AGG 1.520 AGG Dec 19 23 46 92 0.296 0.024 0.001 1.838 AGG 0.255 UNI 0.041 UNI 0.350 0.018 0.317 1.156 AGG 0.172 UNI 0.403 UNI 0.031 0.200 0.991 0.486 UNI 1.089 AGG 2.223 AGG 0.343 0.712 0.998 0.925 UNI 1.253 AGG 1.440 AGG Dec 26 23 46 92 0.039 0.356 0.775 1.296 UNI 3.206 AGG 3.360 AGG 0.547 0.874 0.930 1.037 RAN 1.471 AGG 1.428 AGG 0.033 0.418 0.209 0.728 UNI 2.263 AGG 0.014 UNI 0.825 0.831 0.999 0.833 UNI 1.295 AGG 0.793 UNI Jan 2 23 46 92 0.159 0.039 0.129 1.625 AGG 0.286 UNI 0.865 UNI 0.652 0.877 0.740 1.021 RAN 0.861 UNI 0.906 UNI 0.145 0.298 0.641 2.206 AGG 1.185 AGG 1.491 AGG 0.420 0.847 0.947 0.896 UNI 1.002 RAN 1.140 AGG AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P >0.05); UNI , uniform distribution ( b and significantly < 1, P

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88 Table 4 8. for distribution of parasitoids of L. trifolii on bean and squash samp led in Planting 4 ( Nov 2014 Jan 2015) Sample date Plot size Bean Squash r 2 b regression r 2 r 2 b regression r 2 Dec 12 23 46 92 0.221 0.654 0.784 0.823 UNI 1.763 AGG 2.241 AGG 0.294 0.690 0.649 1.408 AGG 1.775 AGG 1.842 AGG 1.000 0.728 0.938 2.000 AGG 1.500 AGG 0.721 UNI 1.000 0.544 0.012 3.000 AGG 2.222 AGG 0.091 UNI Dec 19 23 46 92 0.809 0.002 0.012 1.525 AGG 0.087 UNI 0.189 UNI 0.257 0.049 0.074 1.104 AGG 0.593 UNI 0.494 UNI 0.016 0.006 0.006 0.162 UNI 0.093 UNI 0.035 UNI 0.043 0.397 0.679 0.283 UNI 0.494 UNI 0.292 UNI Dec 26 23 46 92 0.118 0.001 0.059 0.714 UNI 0.091 UNI 0.549 UNI 0.433 0.725 0.689 0.729 UNI 0.741 UNI 0.720 UNI 0.186 0.008 0.388 1.870 UNI 0.352 UNI 0.288 UNI 0.491 0.625 0.987 0.737 UNI 0.943 UNI 0.787 UNI Jan 2 23 46 92 0.944 0.784 0.999 1.552 AGG 1.528 AGG 2.474 AGG 0.839 0.698 0.999 1.523 AGG 1.544 AGG 3.118 AGG 0.001 0.111 0.193 0.141 UNI 1.060 AGG 1.547 AGG 0.196 0.642 0.443 0.805 UNI 1.117 AGG 1.116 AGG AGG, aggregated distribution ( b and significantly >1, P , random distribution ( b and not significantly different from 1, P > 0.05); UNI , uniform distribution ( b and significantly < 1, P

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89 CHAPTER 5 RESPONSE OF AMERICAN SERPENTINE LEAFMINER , LIRIOMYZA TRIFOLII , TO CHEMICAL, BOTANICAL AND MICROBIAL INSECTICIDE . The American serpentine leafminer, Liriomyza trifolii (Burgess), is a major pest of several vegetable crops (Parrella et al.1983, Seal et al. 2002) . L. trifolii damage s the plant when adult female s feeds on the leaf by puncturing holes then laying its eggs in th em . Feeding by larva, on the other hand , damages the mesophyll layer of the leaves causing reduction in the photosynthetic area (Parrella 1987, Schuster and Everett 1983). Snap bean is one of the major vegetable crops produced in south Florida a nd growers in south Florida generally use two translaminar insecticides, abamecti n and cyromazine, for the control of leafminer. Typically, insecticide applications begin after mines are observed in the field (Dakshina R. Seal, personal communication). C ontinuous use of these insecticides can lead to the development of insecticide resistance in the leafminer populations . Leibee (1981) reported that many agricultural chemicals became ineffective against leafminer within a few years of their introduction. T his may be due to the ability of leafminer to undergo rapid development of resistance to new compounds ( Kei l and Parrella 1983) . Leibee and Capinera (1995) reported that some strains of L. trifolii were highly resistant to cyromazine. Later, Ferguson (2004 ) reported that some strains of leafminer showed resistance to abamectin, spinosad and cyromazine in some areas of the United States. Although, these insecticides are providing better control, there remains little doubt that there is potential for leafminer to develop resistance to these insecticide s in the future. Thus there is the need for evaluating reduced risk insecticides for potential control of L. trifolii . Furthermore, to

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90 implement sound integrated management (IPM) approach es to delay the on set of resistance and for effective management. Therefore, the present study focused on the management of leafminers using 3 selective translaminar chemical insecticides (abamectin , spinosad and cyromazin e) having different modes of action and 2 biologi cal insecticides ( Neem based insecticide and entomopathogenic fungus ) . Abamectin is a mixture of two avermectins (B1a and B1b) that are product of naturally fermented Streptomyces avermitilis , a soil bacterium. Abamectin acts on nervous system of insect s a nd hinder neural and neuromuscular transmission ( Ananiev et al. 2002) . Similarly, spinosad is another fermented compound that is mixture of spinosyn A and spinosyn D that are fermented from bacteria Saccharopolyspora spinosa . Spinosad also affects the nerv ous system of insects. It acts on nicotinic acetylcholine receptors (nAChRs) and is active by both contact and ingestion (Salgado 1998) . Cyromazine is a triazine insect growth regulator that interferes with molting, however, the exact mode of action is not known ( El Oshar 1985) . Azadirachtin is a biological insecticide derived from neem tree ( Azadirachta indica ) . It is a growth regulator that inhibits insects from molting and hence is ef fective on immature stages of insect (Koul 1999) . Isaria fumosorosea i s a naturally occurring entomopathogenic fungus . Germinating spores of this fungus parasitize its insect host. These spores multiply inside the body of the insect , which ceases feeding on the host insects and causes death of the insect. The fungus is effective against all the life stages of insect ( Zimmermann 2008) .

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91 Materials and Methods The study was conducted at the Tropical Research and Education Center, Homestead, F L . The soil ty pe was Krome gravelly loam (33% soil and 67% limestone pebbles) . Snap bean (Variety: Prevail) was planted o n 15 October 2014 by directly sowing seeds on raised beds (91 cm wide, 15 cm high) covered with 1.5 ml thick black white polyethylene mulch Solution Co., Cookeville, TN) with white side faced upward . Bean seeds were planted in holes, 1.5 cm deep, spacing 25 cm within the row and 91.4 cm apart between rows. Twenty one days before planting, pre plant herbicide, Halosulfuron methyl (51.9 g / ha, Sandea ® , Gowan Company LLC., Yuma, Arizona) was applied to control the emergence of weed s . The g ranular fertilizer 6:12:12 (N: P: K) at rate of 1345 kg/ha was applied in 10 cm band and incorporated on both sides of the raised bed center before placement of plastic mulch. A l iquid fertilizer 4: 0: 8 (N: P: K) was applied at the rate of 0.56 kg N / ha / day through drip system at 3, 4 and 5 weeks after planting. Plants were irrigated using two parallel lines of drip tube (T systems, DripWorks, Inc., Willits, California), each spaced 30 cm from the center , having an opening at every 13 cm interval. Irrigation was done twice everyday delivering 2.54 cm each time . Fungicides, Chlorothalonil (Bravo ® , Syngen ta Crop Protection, Inc., Greensboro, NC) at 1.75 l/ha and Copper hydroxide (Kocide ® 3000, BASF Ag Products, Research Triangle Park, NC) at 0.8 l/ha, were sprayed every two weeks to prevent fungal diseases. Each treatment plot consist ed of three beds of 8 m long and 1m wide. Treatment plots were arranged in a Randomized Complete Block Design (RCBD) with four replications. Replicates were separated by a 1.5 m long non planted buffer area. Application of insecticide was initiated 12 days after seeds were sow n.

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92 Insecticide treatments evaluated were: 1) Azadirachtin ( 20.82 gm a.i/ha , Neemix ® , Certis USA, L.L.C., Columbia, MD) 2) Cyromazine ( 139.73 gm a.i/ha , Trigard ® , Syngenta Crop Protection, LLC, Greensboro, N.C.) 3) Abamectin ( 9.73 gm a.i/ha , Agri Mek ® , Syngenta Crop Protection, LLC, Greensboro, N.C.) 4) Isaria fumosorosea Apopka strain ( 448.34 gm a.i/ha , PFR Columbia, MD) 5) Spinosad ( 175.98 gm a.i/ha , Spin T or ® , Dow AgroSciences LLC, IN.) and 6) a non treated control. All inse cticide s were sprayed weekly for five weeks using a CO2 back pack sprayer with two nozzles delivering 234 liters per hectare at 172 kpa . Evaluation of treatments for controlling L. trifolii and their effect on parasitoid s was conducted by randomly collecti ng 5 leaves, one leaf per plant, from the central 6 m area of the middle row of each treatment plot. A pre spray sample was collected 24 h before starting insecticide application. Samples were collected 1, 2, 5, and 7 days after each application. Leaves w ere placed in a plastic pot (10 cm diameter and 15 cm depth) marked with blocks and treatments. Plastic pots were transported to the laboratory and were placed on a bench at room temperature (28±1.5 o C), 75 ±5 % RH and 14:10 (L: D) h photoperiod. Leaves were checked daily at 10 A.M. to record number of mines and larvae per leaf, which was kept until all larvae emerge d out as pupae. The n umber of pupae emerged were then counted. Pupae were kept in a Petri dish (6 cm diameter) with a moisten ed filter paper on the bottom to avoid desiccation and marked with blocks and treatments. The Petri dishes were observed daily for adult emergence. The number of leafminer adult s w as counted .

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93 Population density of leafminer was analyzed by the sample mean. To normalize the error variance , all of the data were x+0.25 before analysis. Analysis of variance (ANOVA) was performed and means were separated by Waller Duncan K (SAS Institute Inc. 2013) to compare the infestation between treatments. Result Number of M ines Insecticide treatments were applied four times to determine their effectiveness in reducing leafminers. Residual effect of each insecticide after each application was also determined by counting mines on leaf samples collected at 1, 2, 4 and 7 d after eac h application. First application of insecticides : Because the first spray was applied before leafminer infestation was initiated, no mines were observed on treated leaves, regardless of treatments, two days after the first application (Table 5 1). 5 days after the first application, all treated leaves, irrespective of treatments, showed leaf mines. Mean numbers of mines on all treatment leaves, except Trigard ® , were significantly fewer than the non treated control (3.75 ± 0.47 mines / 5 leaves), although the lowest numbers of mines were observed on the Agri Mek ® (0.50 ± 0.28 mines / 5 leaves) and SpinTor ® (0.50 ± 0.50 mines / 5 leaves) treated leaves ( F = 33.50; df = 5 ; P <0.0001 ) . 7 days after the first application, mean numbers of mines / 5 leaves on plots treated with insecticide treatments were significantly fewer than the non treated control (16.50 ± 1.93 mines / 5 leaves) with significantly lower numbers on Agri Mek ® (0.0 mines / 5 leaves), SpinTor ® (0.25 ± 0.25 mines / 5 leaves) and Neemix ® (0.75 ± 0.75 mines / 5 leaves) treated leaves ( F = 41.42; df = 5 ; P < 0.0001 ) . Mean number of mines / 5 leaves

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94 in these three treatments were fewer than all other treatments on the fifth day sa mple. On the other hand, mean numbers of mines in other treatments at 7 days after spray increased than the previous sample date . Second application of insecticides (7 d ays after the first spray): One day after the 2 nd application of insecticides, al l treatments significantly reduced mean numbers of mines / 5 leaves when compared with the non treated control ( F = 5.17; df = 5 ; P = 0.004 ) (Table 5 1). A similar pattern of reduction of leaf mines was observed on the subsequent sampling dates (5 and 7 d after spray) ( F = 8.65; df = 5 ; P = 0.0002 ) , ( F = 15.59; df = 5 ; P = 0.0001 ) except on the second sample date (2 d after the spray) ( F = 7.33; df = 5 ; P = 0.0007 ) . On the second sample date, only Agri Mek ® and SpinTor ® treated plants had significantly fewer leaf mines than the non treated control. Third application of insecticides (7 d ays after the second spray): All insecticide treatments significantly reduced leaf mines compared to non treated control on all sampling dates ( F = 24.27; df = 5 ; P <0.0001 ) , ( F = 30.77; df = 5 ; P <0.0001 ) , ( F = 9.63; df = 5 ; P <0.0001 ) , except the 7 d sample ( F = 11.07; df = 5 ; P = <0.0001 ) , as compared with the non treated control (Table 5 1). On the 7 d sample, Agri Mek ® , SpinTor ® and Neemix ® treated plants had fewer mines than the non treated control. Fourth application of insecticides (7 d after the third spray): Agri Mek ® , SpinTor ® and Neemix ® consistently reduced leaf mines on all sample dates when compared with the non treate d control. Trigard ® did not differ from the control on the first sample date and PFR 97 differed from the control on 7 days after application . Fifth application of insecticides (7 d ays after the fourth spray): The effectiveness of all treatments in reducing leaf mines was similar to the fourth spray. Agri Mek ® ,

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95 SpinTor ® and Trigard ® consistently had fewer leaf mines than the non treated control (Table 5 1). Number of L arvae After first application, effect of various treatments reveal ed a similar pattern of reduction of larvae as in the instance of leafmines (Table 5 2). Overall, all treatments significantly reduced leafminers larvae when compared with the non treated control on 5 d and 7 d sample ( F = 18.13; df = 5; P <0.0001 ) , ( F = 42.46; df = 5 ; P < 0.0001 ) . Mean number of larvae on Agri Mek ® treated plants did not differ from the SpinTor ® treated plants on all sample dates. Effectiveness of Neemix ® in controlling larvae was lower than Agri Mek ® and SpinTor ® ; but was better than T rigard ® and PFR After the second application, Agri Mek ® and SpinTor ® treated plants did not have any larvae / 5 leaves irrespective of the sample dates when non treated plants had 12 25 larvae / 5 leaves. After the second application, Trigard ® , Ne emix ® and PFR (except the second sample date) also significantly reduced larvae on all sample dates when compared with the non treated plants. After the third application of all insecticides, similar pattern of reduction of leafminer larvae was observe d on all sample dates ( F = 16.47; df = 5; P <0.0001 ) , ( F = 71.16; df = 5; P <0.0001 ) , ( F = 13.50; df = 5; P < 0.0001 ) , ( F = 18.31; df = 5; P < 0.0001 ) as in the instance of first and second application. PFR sampling date (5 day s after application) and Trigard ® on the fourth sample date ( 7 day s after application) did not differ from non treated control in the mean number of larvae. Agri Mek ® and SpinTor ® treated plants did not have any larvae after the fourth spray (Table 5 2). Tr igard ® and Neemix® treated plants significantly differed from non treated control plants in the mean number of larvae on all sample dates except on the

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96 fourth sample date (7 days after application). PFR treated control plan ts. Agri Mek ® , SpinTor ® and Trigard ® treated plants did not have any larvae / 5 leaves after the fifth spray (Table 5 2). Neemix® significantly reduced leafminer larvae on all sample dates except on 2 days after application . PFR t differ from the non treated plants irrespective of sample dates. Number of P upa e After the first application, no pupae emerged from plants treated with Agri Mek ® and SpinTor ® (Table 5 3) . As with larvae, neither treated plants nor control plants had an y pupae on one and two days after first application. 5 days after the first insecticide application, pupae were recorded on Trigard ® , Neemix ® and PFR but the numbers were significantly fewer than the non treated control ( F = 26.20; df = 5; P <0.0001 ) . 7 days after the first insecticide application, mean numbers of pupae / 5 leaves increased on the Trigard ® and PFR differences as compared to the non treated control plants ( F = 23.58; df = 5; P < 0.0001 ) . On this date, larvae in Neemix ® treated plants did not develop into pupae (Table 5 3). After the second spray, Agri Mek ® , SpinTor® and Trigard ® treated plants did not have any pupae irrespective of sample dates when non treated control plants had 10 22 pupae / 5 leaves. Neemix ® and PFR larvae than the non treated control except 2 and days after application , respectively. After the third application, Agri Mek ® , SpinTor ® and Trigard ® treated plants sh owed similar results in pupal emergence as in the second spray. Neemix® and PFR treated control plants.

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97 Mean numbers of pupae / 5 leaves in the non treated control plants ranged from 5 to 15 o n various sample dates. After the fourth spray, Agri Mek ® , SpinTor ® and Trigard ® treated plants showed similar results in having no pupal emergence as in the second and third applications. Although pupal emergence was observed in Neemix ® treated plants on all sample dates, the numbers were significantly fewer than the non treated control. Mean number of pupae in PFR treated control plants in all sample dates. Mean numbers of pupae / 5 leaves in the non trea ted plants ranged from 2.75 to 7.50. After the fifth application of treatments, there was no emergence of pupae in Agri Mek ® , SpinTor ® and Trigard ® treated plants on any of the sample dates (Table 5 3). Mean numbers of pupae / 5 leaves in Neemix ® and PFR differ from the non treated control plants 1 , 2 and 5 days after the fifth application. However, mean numbers of pupae / 5 leaves in the Neemix ® and PFR plants were significantly fewer than the non treated plants 7 d ays after the fifth application. Discussion In previous chapters (Chapter 3 and Chapter 4 ), we observed that the leafminer activity starts 12 days after snap bean planting and peak s ~ two weeks to three weeks after planting, therefore, the first spray was initiated 12 days after planting when the plants had two fully unfolded primary leaves. At that time none of the treated plots showed signs of leafminer infestation. At 5 days after t he first spray, all treated plots except Trigard ® had significantly lower numbers of mines as compared with non treated control . Among all the treatments Agri Mek ® and SpinTor ® had very

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98 few mines, larvae and pupae /5 leaves after each spray and on each sam ple date. This result was in agreement with findings from experiments conducted by Hara (1986), Parrella et al. (1988), Cox et al. (1995) and Seal et al. (2002). However, Trigard ® had more mines as compared with Agri Mek ® and SpinTor ® on almost all the s ampling dates. Although plots treated with Trigard ® had more mean number of mines / 5 leaves, mines were small in size and almost all mines were aborted and hence no pupae were recovered from most of the plots treated with Trigard ® at each sampling dates. These results support the findings of Schuster and Everett (1983), Hara (1986) Saito et al. (1992). These results strongly add to the fact s described by Ferguson (2004) that cyromazine and abamectin, with very little documented cases of resistance , have be en most effective insecticides in controlling leafminers in vegetables and ornamentals for a longer time now . Because azadirachtin does not work as ovipositon deterrent (Webb et al. 1983), Neemix ® treated plots had significantly more mines as compared to A gri Mek ® , SpinTor ® and Trigard ® , but the numbers were significantly fewer than non treated control plots. However, because azadirachtin has strong larvicidal property (Webb et al. 1983, Larew et al. 1985 , Hossain and Poehling 2006), the number of pupae col lected from plots treated with Neemix ® were very few and significantly lower than control and most of the time not significantly different to chemical insecticides; Agri Mek ® , SpinTor ® and Trigard ® . Plots treated with PFR did not show promising result s compared with other treatments. Plots treated with PFR had significantly more mines per 5 leaves on each sampl e date than Agri Mek ® , SpinTor ® , or Trigard ® , but had similar numbers of

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99 mines to the control treatment on many sample dates. Consistency wa s not observed compared to other treatments. Similarly, in case of larval and pupal count, plots treated with PFR was not significantly different than the non treated control plots on most of the sampling dates (Table 5 1, 5 2, 5 3). Since most of the st udies on use of I. fumosorosea as a biological insecticides were done in controlled environment s as in greenhouse and laboratory conditions (Vidal et al . 1998, Wraight et al. 2000, Ali et al . 2010, Wekesa et al. 2011), it is possible that the inability of the fungus I. fumosorosea to control leafminers could be related to fluctuating temperature s and humidity under the field condition. This hypothesis is supported by the fact that endurance of quiescent conidia of I. fumosorosea is highly dependent on temperature and humidity conditions (Bouamama et al. 2010). Fluctuation of relative humidity within 24 of application of fungus I. fumosorosea can significantly lower the rate of development of the fungus (L a nda et al. 199 4 ). This study provides inform ation on the performance of insecticides; abamectin (Agri Mek ® ), spinosad (SpinTor ® ) and cyromazine (Trigard ® ) currently being used by growers for leafminer control. Secondly, this study provides information on performance of botanical insecticide, azadir achtin (Neemix ® ) and microbial insecticide I. fumosorosea (PFR Since it is revealed from this and several other studies that azadirachtin is able to control leafminer population s , azadirachtin can be incorporated in the management plan in rotation wi th other currently used insecticides. This will help to reduce the potential of the development of resistance to insecticides by leafminer.

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100 Table 5 1 . Efficacy of different chemical and biological insecticide s vs non treated (Control) on number of mines/5 leaves made by L . trifolii in bean leaves Treatment Days after treatment 1 2 5 7 First Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0.50±0.28a 0.50±0.50a 3.00±0.41cd 1.00±0.40b 2.50±0.28c 3.75±0.47d 0a 0.25±0.25a 10.75±2.46b 0.75±0.75a 10.00±1.58b 16.50±1.93c Second Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 4.00 ±1.35a 10.25 ±7.98a 7.75 ±2.68a 31.25 ±15.81b 0a 0.75±0.47a 0a 0a 1.00±0.70a 0a 6.50±1.19ab 2.75±0.25ab 2.50±1.19a 3.25±1.70ab 7.25±2.32bc 9.00±2.67b 8.00±1.47b 12.75±3.32c 16.75±4.76b 14.50±7.94b 25.75±7.69d 36.25±8.77c Third Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0.75±0.47a 0.75±0.75a 0a 0.5±0.28a 0.50±0.28a 3.5±1.19b 7.50±1.84b 5.75±0.85bc 6.50±2.25b 12.00±2.27cd 10.75±1.43b 8.25±1.37bc 10.25±1.37bc 9.50±1.32c 9.25±1.25b 12.25±1.79c 15.75±5.39c 16.75±3.06d 26.50±6.65c 24.00±3.02d 25.00±3.39d 14.50±3.96cd Fourth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0.75±0.47a 0a 0.25±0.25a 0a 1.00±0.57a 0.50±0.50a 5.00±1.29bc 2.00±1.41ab 5.75±1.49b 2.00±0.91ab 5.00±3.10b 5.50±1.49bc 8.00±2.00bc 4.00±1.15bc 9.25±2.25bc 13.00±2.28cd 13.00±4.14cd 7.25±1.70c 12.50±3.75c 18.75±1.43d 16.75±2.59d 12.00±3.46d Fifth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0.25±0.25a 0a 0a 0.5±0.5a 1.00±0.40a 1.75±0.25ab 1.50±0.50b 1.25±0.47a 4.50±1.19b 5.75±1.49bc 3.75±0.62c 3.75±0.85b 6.25±1.10b 10.25±2.28cd 6.75±1.03cd 7.25±1.70c 3.75±1.25b 7.00±0.91d 8.50±1.32d 12.00±3.46d

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101 Table 5 2 . Efficacy of different chemical and biological insecticide s vs non treated (Control) on number of L . trifolii larvae/5 leaves in bean leaves Treatment Days after treatment 1 2 5 7 First Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 3.00±0.41c 1.00±0.40b 2.00±0.28c 3.75±0.47d 0a 0.25±0.25a 9.25±2.35b 0.50±0.50a 6.50±1.25b 14.00±1.10c Second Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 2.00 ±0.70a 6.75 ±5.76a 3.50 ±1.25a 24.75 ±13.36b 0a 0a 0a 0a 0a 0a 3.25±0.85ab 0a 0a 1.50±0.86ab 2.50±0.78b 1.75±0.75a 4.25±0.75bc 4.50±0.64b 7.50±2.5b 12.50±6.55c 18.00±7.88c 24.75±7.89c Third Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0.75±0.75a 0a 0a 0a 0.50±0.28a 0.25±0.25a 0.75±0.47a 1.00±0.70a 3.25±0.47bc 1.50±0.95ab 3.25±0.75b 0.75±0.47a 2.50±0.86b 3.00±0.40b 13.25±0.63b 13.25±4.76b 13.75±2.78d 11.75±4.53c 17.25±1.49c 17.50±5.10b 5.75±1.49c Fourth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0.75±0.47a 1.75±0.85ab 1.50±0.95a 2.00±0.70b 3.50±1.55b 2.25±0.75bc 5.25±1.37b 9.25±4.26c 6.50±1.70c 5.00±1.77c 6.00±1.29b 12.25±1.31c 6.50±0.64c 4.50±1.84bc Fifth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 1.75±0.25b 3.50±1.19c 2.25±0.63b 1.25±0.94a 2.75±0.47c 7.00±1.29c 4.00±0.81c 5.50±1.55b 2.00±0.70bc 5.00±0.91bc 4.75±1.10c 9.25±1.93c

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102 Table 5 3 . Efficacy of different chemical and biological insecticide s vs non treated (Control) on number of pupae of L . trifolii / 5 leaves collected from bean plants Treatment Days after treatment 1 2 5 7 First Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 2.50±0.64c 0.75±0.25b 2.00c 2.75±0.47d 0a 0a 7.75±2.56c 0a 3.75±1.60b 12.00±1.47d Second Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 4.50 ± 4.50a 1.00 ±0.57a 21.75 ±12.98b 0a 0a 0a 0a 0a 0a 0a 0a 0a 2.00±2.00ab 0a 0a 0.50±0.50a 1.00±0.40a 6.00±1.58b 10.25±5.87b 10.50±4.40b 16.25±8.34b Third Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0 a 0a 0.25±0.25a 0.50±0.50a 5.00 ±1.35b 3.25±1.10b 11.25±4.21b 11.25±2.84c 12.00 ±4.14c 11.75±0.94c 14.75±3.32b 5.50±1.25b Fourth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 1.00±1.00ab 1.25±0.47ab 1.25±0.47a 2.50±1.19b 2.00±0.70bc 4.50±1.19c 7.75±3.66b 4.25±1.31c 1.75±0.85abc 2.75±1.60bc 7.50±1.19b 5.25±0.78c 2.75±0.63c Fifth Spray Agri M ek Spintor Trigard Neemix PFR 97 Control 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0.50±0.50a 2.25±1.03b 1.50±0.50b 0.50±0.28a 1.00±0.70a 3.00±1.08b 2.25±0.85b 3.50±1.32b 0.75±0.47a 1.50±0.64ab 2.75±0.63b 6.75±2.60c

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103 CHAPTER 6 CONCLUSIONS American serpentine leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) , a polyphagous fly, is an important insect pest of vegetable crops (Seal et al. 2002, Minkenberg 1988a). It was first recorded in the USA but has dispersed to other parts of world. The leafminer larvae cause major damage as they tunnel into and feed on the leaf mesophyll tissue s of the leaves (Parrella 1987). High L. trifolii infestations can considerably reduce photosynthesis rates and conductance by leaf mesophyll and stomat a , and seriously affect plant growth, yield, resulting in plant death . Because of polyphagy and the ability to develop resistance to chemical insecticides, L. trifolii poses a considerable threat to vegetable growers. To develop an effective integrated pest management ( IPM ) program , studies were performed to assess the abundance of leafminers and their parasitoids on five vegetable crops (bean, cucumber, squash, tomato , and cabbage) in south Florida. Leafminer infestations were higher on bean in two crop plantings (May June and Sep Oct 2014). Up to 104 leafminer mines per 5 leaves were found on bean. In contrast, cabbage had the lowest leafminer population with no min es on most sample dates. Cucumber and squash had similar infestation levels, which were much lower than on bean. The abundance of parasitoids were similar to those of their hosts, the leafminer. Numbers of parasitoids were highest on bean and lowest on cab bage. However, there was no any difference in parasitoid community found among the five crops. Opius dissitus was the most abundant parasitoid recorded and comprised of more than 60% of total parasitoids population. The other parasitoids recorded were Digl yphus sp. , and

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104 Diaulinopsis callichroma . From the study presented herein, we can conclude that bean is the most preferred host of L. trifolii among the five vegetable crops in south Florida. Another study was conducted on bean and squash to determine the seasonal abundance and spatial distribution of L. trifolii . Bean and squash were planted 4 times from November 2013 to January 2105. Leafminer activity was highest at 2 weeks after planting on bean and at 3 weeks after planting on squash in 3 plantings (N ov Dec 2013, May June 2014, and Sep Oct 2014). Among the four planting, leafminer abundance was highest during the November, May, and September plantings, when the average daily temperatures were 21 26 o C . Leaf miner populations seemed to be lowest in the December planting, when the average daily temperature was below 18 o C . Similar trends were observed for parasitoids population densities among the four plantings, which were highest when their host leafminers were most abundant and lowest when leafminers we models, distribution patterns exhibited by leafminers were inconsistent. The resulting indices of dispersal on most sample dates for all four bean and squash plantings were significant ly > 1 indicating aggregated distributions. The parasitoid distributions tended to parallel the leafminer distributions and were mostly aggregated. Finally, we tested the effectiveness of chemical and biorational insecticides against leafminers. Plants treated with abamectin and spinosad had significantly fewer leafminer mines, larvae, and pupae than non treated control plants on all sample dates. Usually, numbers of these variables were < 1 per 5 leaves. However, plants treated with cyromazine had more mines than plants treated with abamectin and spinosad. However, the mines from c yromazine were small er and were often aborted. Hence, no

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105 pupae were recovered from plants treated with cyromazine on most sample dates of each planting. Similarly, plants treat ed with azadirachtin had significantly higher numbers of mines and larvae compared with abamectin and spinosad treatments, but the numbers were still significantly lower than non treated control plants. However, numbers of pupae were not significantly diff erent among plants treated with azadirachtin, abamectin, spinosad, and cyromazine. These data suggest that azadirachtin has larvicidal properties. However, numbers of mines, larvae, and pupae did not differ significantly between plants treated with Isaria fumosorosea , an entomopathogenic fungus, and non treated control plants on most sample dates. Results of these studies can provide help in developing an effective IPM program for leafminer management in south Florida.

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106 LIST OF REFERENCES Ali, S., Z. Huang, and S. Ren . 2010 . Production of cuticle degrading enzymes by Isaria fumosorosea and their evaluation as a biocontrol agent against diamondback moth. J. Pest Sci. 83: 361 370. Ananiev, D. E., K. Ananieva, G. Abdulova, N. Christova, and E. Videnova. 2002 . Effects of abamectin on protein and RNA synthesis in primary leaves of Cucurbita pepo L. (Zucchini). Bulg. J. Plant Physiol ogy . 28 ( 1 2 ) : 85 91. Arida, G. S., B. S. Punzal, B. M. Shepard, and E. G. Rajotte . 2013 . Sticky board traps for managing leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae), infestation in onion ( Allium cepa Linn.). Philipp. Entomol. 27: 109 119. Auerbach, M., and D. Simberloff . 1984 . Responses of leaf miner s to atypical leaf production patterns. Ecol. Entomol. 9: 361 367. Ayabe, Y., and E. Shibata . 2008 . Spatial distributions of the leafminer Ophiomyia maura (Diptera: Agromyzidae) in host plant Aster ageratoides. Insect Sci. 15: 343 348. Banchio, E., G. Vall adares, M. Defagó, S. Palacios, and C. Carpinella . 2003 . Effects of Melia azedarach , (Meliaceae) fruit extracts on the leafminer Liriomyza huidobrensis , (Diptera, Agromyzidae): Assessment in laboratory and field experiments. Ann. Appl. Biol. 143: 187 193. Beck, H. W., C. A. Musgrave, J. O. Stra ndberg and W. G. Genung. 1981 . Spatial dispersion patterns of Liriomyza sp on celery. In Proceedings , 2nd IFAS Industry Conference Biology and Control of Liriomyza leafminers. Institute Food Agric. Sci. Univ. Florida, Gainesville . 129 140 pp . Bernays, E. A., and R. F. Chapman . 1994 . Host Plant Selection by Phytophagous Insects. Springer Science & Business Media. Bethke, J. A., and M. P. Parrella . 1985 . Leaf puncturing, feeding and oviposition behavior of Liriomyza trifolii . Entomol. Exp. Appl. 39: 149 154. Bouamama, N., C. Vidal, and J. Fargues . 2010 . Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea. J. Invertebr. Pathol. 105: 139 144. Broadbent, A . B., and J. A. Matteoni . 1990 . Acquisition and transmission of Pseudomonas chichorii by Liriomyza trifolii (Diptera: Agromyzidae). Proc. Entomol. Soc. Ont. 121: 79 84. Bruce, T. J. A., L. J. Wadhams, and C. M. , Woodcock . 2005 . Insect host location: a vola tile situation. Trends Plant Sci. 10: 269 274.

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1 11 Li, Z., W. Jianing, and K. Rongping . 1997 . Biological characteristics and behavior of adult serpentine leafminer Liriomyza huidobrensis (Blanchard). Zool. Res. Dong Wu Xue Yan Jiu Bian Ji Wei Yuan Hu i Bian Ji. 19: 384 388. Lloyd, M. 1967 . Mean crowding'. The Journal of Animal Ecology , 36: 1 30. Malipatil, M. B., P. M. Ridland, A. Rauf, J. Watung, and D. Kandowangko. 2004 . New records of Liriomyza Mik (Agromyzidae: Diptera) leafminers from Indonesia. Formosan Entomol , 24 , 287 292. Martin, A. D., D. Stanley Horn, and R. H. Hallett . 2005 . Adult host Preference and larval performance of Liriomyza huidobrensis (Diptera: Agromyzidae) on selected hosts. Environ. Entomol. 34: 1170 1177. Martinat, P. J. 1987 . The role of climatic variation and weather in forest insect outbreaks. In: Barbosa P, Schultz JC (eds) Insect outbreaks. Academic Press, New York , 241 268. Mason, G. A., M. W. Johnson, and B. E. Tabashnik . 1987 . Susceptibility of Liriomyza sativae and L. t rifolii (Diptera: Agromyzidae) to Permethrin and Fenvalerate. J. Econ. Entomol. 80: 1262 1266. Mekuria, D. B., T. Kashiwagi, S. Tebayashi, and C. S. Kim . 2005 . Cucurbitane triterpenoid oviposition deterrent from Momordica charantia to the leafminer, Liriom yza trifolii . Biosci. Biotechnol. Biochem. 69: 1706 1710. Mekuria, D. B., T. Kashiwagi, S. Tebayashi, an d C. S. Kim . 2006 . Cucurbitane glucosides from Momordica charantia leaves as oviposition deterrents to the leafminer, Liriomyza trifolii . Z. Für Naturfo rschung C. 61: 81 86. Miller, G. W., and M. B. Isger . 1985 . Effects of temperature on the development of Liriomyza trifolii (Burgess) (Diptera: Agromyzidae). Bull. Entomol. Res. 75: 321 328. Minkenberg, O. P. J. M. 1988a . Dispersal of Liriomyza trifolii . E PPO Bull. 18: 173 182. Minkenberg, O. P. J. M. 1988b . Life history of the agromyzid fly Liriomyza trifolii on tomato at different temperatures. Entomol. Exp. Appl. 48: 73 84. Minkenberg, O. P. J. M., and J. C. van Lenteren . 1986 . The leafminers Liriomyza b ryoniae and L. trifolii (Diptera: Agromyzidae), their parasites and host plants: a review. 86 (2) : 1 50. Minkenberg, O. P. J. M., and J. J. G. W. Ottenheim . 1990 . Effect of leaf nitrogen content of tomato plants on preference and performance of a leafmining fly. Oecologia. 83: 291 298.

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118 B IOGRAPHICAL SKETCH Shashan Devkota was born in Rajbiraj, Saptari, Nepal in 1982. He received Bachelor of Science in Agriculture from Institute of Agriculture and Animal Sciences, Tribhuvan University, Nepal in 2009. he work ed as freelancer for different non government organizations in the field of agriculture and social welfare. He started MS degree in the Entomology and Nematology Department, University of Florida in May 2013 under the supervision of Dr. Dakshina R. Seal. He studied biology and management of American serpentine leafminer, Liriomyza trifolii , one of the most serious pest of vegetable crops in south Florida. H e is interested in integrated pest management of vegetables and ornamentals.