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Morphology, Biology and Distribution of Corn-Infesting Ulidiidae

Permanent Link: http://ufdc.ufl.edu/UFE0042445/00001

Material Information

Title: Morphology, Biology and Distribution of Corn-Infesting Ulidiidae
Physical Description: 1 online resource (219 p.)
Language: english
Creator: Goyal, Gaurav
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alternate, annonae, chaetopsis, corn, development, distribution, eluta, euxesta, fecundity, florida, hosts, identification, immature, maize, massyla, oviposition, period, pest, reproductive, spatial, stigmatias, sweet
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Corn silk fly, Euxesta stigmatias Loew is serious pest of sweet corn in Florida. . Experiments were conducted to determine the pest status of three most common ulidiids, Chaetopsis massyla (Walker), Euxesta eluta Loew, and E. stigmatias. All three species were reared from both uninfested and infested ears, indicating their primary as well as secondary mode of attack. More flies of all three species emerged from corn ears infested with S. frugiperda than uninfested corn ears, indicating that ulidiid adults are attracted to damaged ears by the presence of olfactory cues in ears damaged by Lepidoptera larvae, or that previously damaged corn ears are more suitable for larval survival. Fewer adults of E. stigmatias and C. massyla emerged from ears infested with E. eluta in no-choice tests, suggesting the presence of visual or olfactory cues that negatively affect the oviposition. Surveys were conducted throughout Florida to evaluate species richness and distribution of corn-infesting Ulidiidae. Adults were sampled using sweep nets and reared from fly larvae-infested corn ears collected from representative corn fields in 16 and 27 counties in 2007 and 2008, respectively. Euxesta eluta and C. massyla were found infesting field and sweet corn throughout Florida. Euxesta stigmatias was only found in Martin, Miami-Dade, Okeechobee, Palm Beach and St. Lucie Counties on field and sweet corn. Euxesta annonae (F.) was found in sweet corn in Miami-Dade, Okeechobee, and Palm Beach Counties, but field corn was not sampled in these counties. Euxesta eluta, E. stigmatias and C. massyla were collected from corn throughout the corn reproductive stage. Raising adults from fly larvae-infested ears provided the best method for assessing rates of ear infestation and species richness. Spatial and temporal distributions of flies in corn fields were determined by sampling flies caught on sticky traps arranged in a certain pattern in small and large-scale corn fields from corn at first silk appearance to corn harvesting. Four corn ears were sampled close to each trap location for presence of immatures and held for immature development to adults under laboratory conditions to determine the number and species of adults obtained. Morisita?s index showed the distribution of the flies to be aggregated in both small and large-scale fields at most of the sampling dates. More flies were found on the sides of the field than in the center in most of the large scale fields. More flies were found in field sides bordered by sugarcane, fallow or residences than sides bordered by corn. The proportion of infested corn ears to total number of sampled corn ears was found to be strongly correlated with the flies caught on sticky traps. Field surveys and laboratory evaluations were conducted to evaluate crop and non-crop plants for their potential to act as developmental hosts and determine developmental and survivorship rates for the immatures of these flies. Several commodities and weeds (bell pepper, spiny amaranth, cattail, sugarcane, and johnsongrass) were found in field surveys to provide satisfactory food sources for successful development from eggs to adults. In laboratory evaluations, all three species were able to complete development on alternative commercial crops and weedy species (bell pepper, cabbage, radish, papaya, hass avocado, sugarcane, little hogweed, haban tildeero pepper, tomato, southern cattail, spiny amaranth, johnsongrass). Morphology of the immature stages was examined for three species of Ulidiidae using light and scanning electron microscopy. Many egg, larval, and pupal characters were measured and described, but only a few traits in each stage could be used to separate the species due to considerable range overlap. Pores were restricted to the posterior end of C. massyla eggs, but distributed over the entire surface of E. eluta and E. stigmatias eggs. A distinct tooth on the ventral surface of the mouth hooks separates C. massyla larvae from the other species. Euxesta eluta and E. stigmatias had more oral ridges than C. massyla. The two Euxesta species could be separated based on the length of the 2nd and 6th creeping welts. The first row of spinules was discontinuous on the 4th - 8th creeping welts in larval C. massyla while it was continuous in Euxesta species. The larval posterior spiracles were black on E. eluta, dark brown to black on E. stigmatias and brown on C. massyla. Chaetopsis massyla pupae were reddish brown while E. eluta and E. stigmatias pupae were light brown to black. The posterior spiracular plates in C. massyla pupae were trapezoidal in shape while those in E. eluta and E. stigmatias were ovoid. Studies were conducted to determine and compare biological parameters (i.e., adult longevity, developmental periods, and survival of different immature stages) of the three species of these flies on artificial diet and corn ears. Euxesta eluta lived longer, deposited more eggs and had shorter pre-oviposition period compared to other species. Flies of all three species developed faster in corn ears than in artificial diet. The development periods in corn ears, in general, were shortest in May, followed by in March, followed by December. Survival of immature stages was not significantly different among three species. The percentage pupal survival was greatest, followed by egg survival, and then larval survival. Field studies were conducted to determine the flies? period of oviposition by examining sweet corn ears for eggs naturally deposited by Ulidiidae every 2-h periods of the day. Chaetopsis massyla deposited more eggs in young ears whereas E. stigmatias deposited more eggs in ears during and after peak silk extension. Chaetopsis massyla deposited eggs throughout the day whereas E. eluta deposited more eggs and in more corn ears from 1100-1700 h than during other times of the day. Euxesta stigmatias deposited more eggs and in more ears from 1100-1500 h than during other times of the day. In studies to understand the intra ear resource competition among these flies, it was found that more larvae were in ear tips than middle and bottom segments. This indicates that flies may compete for their resources, forcing the larvae to go deeper in the corn ears. Moreover, differences in the frequency of ears with individual versus multiple species indicated the presence of behavioral interactions among these species in oviposition preference, and development within the corn ears.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gaurav Goyal.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Nuessly, Gregg S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042445:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042445/00001

Material Information

Title: Morphology, Biology and Distribution of Corn-Infesting Ulidiidae
Physical Description: 1 online resource (219 p.)
Language: english
Creator: Goyal, Gaurav
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alternate, annonae, chaetopsis, corn, development, distribution, eluta, euxesta, fecundity, florida, hosts, identification, immature, maize, massyla, oviposition, period, pest, reproductive, spatial, stigmatias, sweet
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Corn silk fly, Euxesta stigmatias Loew is serious pest of sweet corn in Florida. . Experiments were conducted to determine the pest status of three most common ulidiids, Chaetopsis massyla (Walker), Euxesta eluta Loew, and E. stigmatias. All three species were reared from both uninfested and infested ears, indicating their primary as well as secondary mode of attack. More flies of all three species emerged from corn ears infested with S. frugiperda than uninfested corn ears, indicating that ulidiid adults are attracted to damaged ears by the presence of olfactory cues in ears damaged by Lepidoptera larvae, or that previously damaged corn ears are more suitable for larval survival. Fewer adults of E. stigmatias and C. massyla emerged from ears infested with E. eluta in no-choice tests, suggesting the presence of visual or olfactory cues that negatively affect the oviposition. Surveys were conducted throughout Florida to evaluate species richness and distribution of corn-infesting Ulidiidae. Adults were sampled using sweep nets and reared from fly larvae-infested corn ears collected from representative corn fields in 16 and 27 counties in 2007 and 2008, respectively. Euxesta eluta and C. massyla were found infesting field and sweet corn throughout Florida. Euxesta stigmatias was only found in Martin, Miami-Dade, Okeechobee, Palm Beach and St. Lucie Counties on field and sweet corn. Euxesta annonae (F.) was found in sweet corn in Miami-Dade, Okeechobee, and Palm Beach Counties, but field corn was not sampled in these counties. Euxesta eluta, E. stigmatias and C. massyla were collected from corn throughout the corn reproductive stage. Raising adults from fly larvae-infested ears provided the best method for assessing rates of ear infestation and species richness. Spatial and temporal distributions of flies in corn fields were determined by sampling flies caught on sticky traps arranged in a certain pattern in small and large-scale corn fields from corn at first silk appearance to corn harvesting. Four corn ears were sampled close to each trap location for presence of immatures and held for immature development to adults under laboratory conditions to determine the number and species of adults obtained. Morisita?s index showed the distribution of the flies to be aggregated in both small and large-scale fields at most of the sampling dates. More flies were found on the sides of the field than in the center in most of the large scale fields. More flies were found in field sides bordered by sugarcane, fallow or residences than sides bordered by corn. The proportion of infested corn ears to total number of sampled corn ears was found to be strongly correlated with the flies caught on sticky traps. Field surveys and laboratory evaluations were conducted to evaluate crop and non-crop plants for their potential to act as developmental hosts and determine developmental and survivorship rates for the immatures of these flies. Several commodities and weeds (bell pepper, spiny amaranth, cattail, sugarcane, and johnsongrass) were found in field surveys to provide satisfactory food sources for successful development from eggs to adults. In laboratory evaluations, all three species were able to complete development on alternative commercial crops and weedy species (bell pepper, cabbage, radish, papaya, hass avocado, sugarcane, little hogweed, haban tildeero pepper, tomato, southern cattail, spiny amaranth, johnsongrass). Morphology of the immature stages was examined for three species of Ulidiidae using light and scanning electron microscopy. Many egg, larval, and pupal characters were measured and described, but only a few traits in each stage could be used to separate the species due to considerable range overlap. Pores were restricted to the posterior end of C. massyla eggs, but distributed over the entire surface of E. eluta and E. stigmatias eggs. A distinct tooth on the ventral surface of the mouth hooks separates C. massyla larvae from the other species. Euxesta eluta and E. stigmatias had more oral ridges than C. massyla. The two Euxesta species could be separated based on the length of the 2nd and 6th creeping welts. The first row of spinules was discontinuous on the 4th - 8th creeping welts in larval C. massyla while it was continuous in Euxesta species. The larval posterior spiracles were black on E. eluta, dark brown to black on E. stigmatias and brown on C. massyla. Chaetopsis massyla pupae were reddish brown while E. eluta and E. stigmatias pupae were light brown to black. The posterior spiracular plates in C. massyla pupae were trapezoidal in shape while those in E. eluta and E. stigmatias were ovoid. Studies were conducted to determine and compare biological parameters (i.e., adult longevity, developmental periods, and survival of different immature stages) of the three species of these flies on artificial diet and corn ears. Euxesta eluta lived longer, deposited more eggs and had shorter pre-oviposition period compared to other species. Flies of all three species developed faster in corn ears than in artificial diet. The development periods in corn ears, in general, were shortest in May, followed by in March, followed by December. Survival of immature stages was not significantly different among three species. The percentage pupal survival was greatest, followed by egg survival, and then larval survival. Field studies were conducted to determine the flies? period of oviposition by examining sweet corn ears for eggs naturally deposited by Ulidiidae every 2-h periods of the day. Chaetopsis massyla deposited more eggs in young ears whereas E. stigmatias deposited more eggs in ears during and after peak silk extension. Chaetopsis massyla deposited eggs throughout the day whereas E. eluta deposited more eggs and in more corn ears from 1100-1700 h than during other times of the day. Euxesta stigmatias deposited more eggs and in more ears from 1100-1500 h than during other times of the day. In studies to understand the intra ear resource competition among these flies, it was found that more larvae were in ear tips than middle and bottom segments. This indicates that flies may compete for their resources, forcing the larvae to go deeper in the corn ears. Moreover, differences in the frequency of ears with individual versus multiple species indicated the presence of behavioral interactions among these species in oviposition preference, and development within the corn ears.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gaurav Goyal.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Nuessly, Gregg S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042445:00001


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1 MORPHOLOGY, BIOLOGY AND DISTRIBUTION OF CORN INFESTING ULIDIIDAE By GAURAV GOYAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF D OCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Gaurav Goyal

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3 To my loving and wonderful wife, Dr. Harsimran Gill for her unconditional support and everlasting love

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4 ACKNOWLEDGMENTS I express my sincere appreciation for my mentor and major advisor, Dr. Gregg S. Nuessly, for his encouragement, guidance, constructive criticism, intellectual stimulation, and encouragement throughout my tenure at the University of Florida. His expertise and advice in this endeavor have been indispensable to my success. I would also like to thank the other members of my committee, Dr. John L. Capinera, Dr. Gary J. Steck, Dr. Dakshina Seal, and Dr. Kenneth J. Boote, for their guidance and valuable suggestions for the improvement of dissertation project. I would like to thank Dr. Donald Hall, Dr. John Capinera, Debbie Hall, and Dr. Heather J. McAuslane for all of their administrative help and Dolly Hand group and Miami Dade Agri council scholarships for the financial support for my studies I would like to thank the staff and workers at the Everglades Research and Education Center, University of Florida for their constant support and timely help in growing and maintaining plants for my research. I would like to acknowledge the help from Nick, Bijeyta, Kimbe rly, Hardy, Faira, Minerva, and Israel for assisting me with laboratory and field experiments. I would also thank Lyle Buss for assisting me with photographing insect specimens and Paul Skelley and Jim Wiley at Division of Plant industry (DPI) for assisti ng me with scanning electron microscopy and critical point drying I want to acknowledge the help of Dr. McSorley with data analysis, and providing laboratory space. Words fail to convey the depth of my feelings and gratitude to my friends Gurpreet Hayer, Jaskirat, Jaya, Thais, Jango, and Olawale for their encouragement, generosity and memorable association. I would also like to thank my friends Deepak, Megan,

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5 Chandra, Bijey i ta, Sunil, Gungeet, Navneet, Pedro, Gurpreet, Amit, a nd Teresia for supporting me at every moment and for everlasting memories. I am e ternally indebted to my parents, Mr. Ashok Kumar and Mrs. Lalita Goyal, who have been a constant source of encouragement and support throughout this work. I seize the opportunity to express my moral obl igations to my brother Saurav Goyal and sister in law Neha Goyal for their encouragement and moral support. I would also like to thank my inlaws family for their support and encouragement. It is truly hard to acknowledge the love, sacrifice, encouragemen t, and everything one could provide extended by my loving wife Harsimran Gill. It would have been impossible for me to complete this strenuous task without her support. Above all, I thank my God for his guidance, grace and unending blessings.

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6 TABLE OF C ONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 12 ABSTRACT ................................................................................................................... 13 CHAPTER 1 REVIEW OF LITERATURE .................................................................................... 17 Introduction ............................................................................................................. 17 History ..................................................................................................................... 17 Damage .................................................................................................................. 17 Life Cycle ................................................................................................................ 18 Chemical Control .................................................................................................... 18 Plant Resistance ..................................................................................................... 19 Biological Control .................................................................................................... 20 Additional Ulidiidae Species That Damage Corn in America South of the United States ...................................................................................................................... 21 Problems with Correct Species Identification .......................................................... 21 Identification of Immature Stages ........................................................................... 22 Biology and Ecology ............................................................................................... 23 Research Objectives ............................................................................................... 27 2 PEST STATUS OF PICTURE WINGED FLIES ...................................................... 28 Materials and Methods ............................................................................................ 29 Colony Maintenance ......................................................................................... 29 No choice Tests ............................................................................................... 30 Choice Tests .................................................................................................... 33 Statistical Analysis ............................................................................................ 34 Results and Discussion ........................................................................................... 35 No choice Tests ............................................................................................... 35 Choice Tests .................................................................................................... 36 3 DISTRIBUTION OF CORN INFESTING PICTURE WINGED FLIES IN FLORIDA ................................................................................................................ 42 Materials and Methods ............................................................................................ 43 Statistical Analysis ............................................................................................ 46 Results and Discussion ........................................................................................... 47 2007 Field Survey ............................................................................................ 48

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7 2008 Field Survey ............................................................................................ 49 4 SPATIAL AND TEMPORAL DISTRIBUTION OF CORN INFESTING PICTURE WINGED FLIES IN CORN FIELDS ......................................................................... 65 Materials and Methods ............................................................................................ 66 Statistical Analysis ............................................................................................ 68 Results and Discussion ........................................................................................... 69 SmallScale Fields ............................................................................................ 70 LargeScale Fields ........................................................................................... 74 Corn Ear Infestation ......................................................................................... 79 5 ALTERNATE HOSTS OF CORNINFESTING PICTURE WINGED FLIES ............ 93 Materials and Methods ............................................................................................ 94 Laboratory Evaluation ...................................................................................... 94 Field Studies ..................................................................................................... 96 Statistical Analysis ............................................................................................ 97 Results and Discussio n ........................................................................................... 98 Laboratory Evaluation ...................................................................................... 98 Field Evaluation .............................................................................................. 101 6 COMPARATIVE MORPHOLOGY OF THE IMMATURE STAGES OF THREE CORNINFESTING ULIDIIDAE ............................................................................ 114 Materials and Methods .......................................................................................... 115 Egg ................................................................................................................. 115 Larva .............................................................................................................. 116 Pupa ............................................................................................................... 119 Statistical Analysis .......................................................................................... 120 Descriptions .......................................................................................................... 120 Chaetopsis massyla ....................................................................................... 120 Euxesta eluta .................................................................................................. 125 Euxesta stigmatias ......................................................................................... 128 Diagnoses ............................................................................................................. 130 Egg ................................................................................................................. 130 Larva .............................................................................................................. 130 Pupa ............................................................................................................... 133 Discussion ............................................................................................................ 134 7 DEVELOPMENTAL AND LIFETABLE STUDIES OF CORNINFESTING PICTURE WINGED FLIES ................................................................................... 148 Materials and Methods .......................................................................................... 148 Developmental Times ..................................................................................... 149 Adult Longevity ............................................................................................... 152 Survival ........................................................................................................... 153 Reproductive Periods ..................................................................................... 154

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8 Life Table Parameters .................................................................................... 155 Statistical Analysis .......................................................................................... 156 Results and Discussion ......................................................................................... 156 Developmental Times ..................................................................................... 156 Adult Longevity ............................................................................................... 161 Survival ........................................................................................................... 163 Reproductive Periods ..................................................................................... 164 Life Table Parameters .................................................................................... 165 8 ECOLOGICAL STUDIES OF CORNINFESTING P ICTURE WINGED FLIES ..... 182 Materials and Methods .......................................................................................... 183 Daily Pattern of Oviposition ............................................................................ 183 Intra Ear Distribution ....................................................................................... 185 Statistical Analysis .......................................................................................... 186 Results and Discussion ......................................................................................... 187 Daily Pattern of Oviposition ............................................................................ 187 Intra Ear Distribution ....................................................................................... 193 9 SUMMARY ........................................................................................................... 202 LIST OF REFERENCES ............................................................................................. 210 BIOGRAPHICAL SKETCH .......................................................................................... 218

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9 LIST OF TABLES Table page 2 1 ANOVA table of nochoice test for adults obtained per corn ear. ....................... 40 2 2 Mean SEM number of adults that emerged per corn ear in no choice tests, 2007200 8 (n = 17 per fly species per main effects treatment). .......................... 40 2 3 ANOVA table for number of adults that emerged per ear in choice tests. .......... 40 2 4 Mean SEM (range) percent adults that from corn ears previously infested with E. eluta or E. stigmatias or uninfested (n = 24 ears per treatment). ............ 41 3 1 Analysis of variance for sweep net caught flies and corn reared flies on species, corn type, age of corn and year. ........................................................... 58 3 2 Ulidiidae species collected in fields or reared from infested ears in Florida, 2007. .................................................................................................................. 59 3 3 Ulidiidae species collected in fields or reared from infested ears in Florida, 2008. .................................................................................................................. 60 3 4 Percentage of fields wi th Ulidiidae species sweep netted or reared from infested ears by ear age. .................................................................................... 62 4 1 Small and largescale sweet corn fields sampled for determining the spatial and temporal distribution of corn infesting ulidiids, 20082010 ........................... 86 4 2 Borders of small and largescale sweet corn fields, 20082010 .......................... 87 4 3 Morisitas ind ex ( I) for ulidiids in smallscale corn fields, 2008 .......................... 88 4 4 Morisitas index ( I) for ulidiids in large scale corn fields, 20082010 ................. 90 4 5 Mean SEM (range) flies per sticky trap for three Ulidiidae species in small scale and largescale fields, 20082010 ............................................................. 91 5 1 Various commodities/weeds evaluated under laboratory conditions or surveyed in fields in Belle Glade and Homestead, FL. ..................................... 107 5 2 Larval development times (d) for three Ulidiidae reared on different plant species in Belle Glade, FL. ............................................................................... 108 5 3 Comparison of pupal development times (d) of three species on various plant species in Belle Glade, FL. ............................................................................... 109 5 4 Comparison of number of pupae emerged of three species of ulidiids on various plant species in Belle Glade, FL. .......................................................... 110

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10 5 5 Percentage pupal survival (d) of ulidiids on various plants. .............................. 111 5 6 Ulidiidae species collected in fields from commodities/weeds in Florida. ......... 112 6 1 Range of body measurements (mm) on 1st and 2nd instar larvae of three species of picturewinged fly pests of corn. ...................................................... 137 6 2 Comparison of 3rd instar larval measurements among three species of picturewinged fly pests of corn. ....................................................................... 138 6 3 Comparison of creeping welt measurements (mm) among larvae of three species of picturewinged fly pests of corn. ...................................................... 141 6 4 Diagnostic characteri stics separating the immature stages of three species of picturewinged fly pests of corn. ....................................................................... 143 7 1 Analysis of variance for Ulidiidae developmental times on two food sources. .. 167 7 2 Developmental times (d) for three Ulidiidae species on corn ears in field trials conducted in March and December, 2009, and May 2010 in Belle Glade, FL. 168 7 3 Developmental times for three species of Ulidiidae on artificial diet at 26.5 1 .0 C, 14:10 (L: D) photoperiod and 55 70% RH. .......................................... 169 7 4 Developmental times (d) by stage for three species of Ulidiidae as reported in the literature. ..................................................................................................... 170 7 5 Analysis of variance of adult longevity. ............................................................. 171 7 6 Mean SEM adult longevity (in d) of three species of picturewinged flies reared on artificial diet and corn ears. .............................................................. 172 7 7 Mean SEM (range) percentage survival of immature stages of three species of corn infesting picture winged flies (20 cohorts of each species) on artificial diet. ...................................................................................................... 173 7 8 Reproductive parameters of three species of picturewinged flies on artific ial diet. ................................................................................................................... 174 7 9 Life table parameters of three species of picturewinged flies on artificial diet. 175 8 1 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by ear ages, June 2009. ........................................................................................ 197 8 2 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by 2 h time period, June 2009. .............................................................................. 198 8 3 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by ear age, May 2010. ........................................................................................... 199

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11 8 4 Mean SEM n umber of eggs and proportion of corn ears with ulidiid eggs by 2 h time period, May 2010. ............................................................................... 200 8 5 Fly counts of three ulidiid species caught in sweep nets from corn fields selected for daily pattern of oviposition experiment. ......................................... 200 8 6 ANOVA table of intra ear distribution of flies. ................................................... 201 8 7 Least squared mean S EM (range) number of adults of three Ulidiidae species reared from three segments of corn ears, 20092010 (n = 156). ......... 201 8 8 Frequency of corn ears with ulidiid species alone or in combinat ion with other species in two years of ear sampling. ............................................................... 201

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12 LIST OF FIGURES Figure page 3 1 Adults of four species of picturewinged flies ...................................................... 63 3 2 Distribution of Ulidiidae species infesting corn in Florida by county during the 20072008 surveys ............................................................................................. 64 4 1 Regression plot between mean total number of flies caught on sticky traps and proportion of infested corn ears in small and largescale corn felds. ........... 92 6 1 Scanning electron microscope views of eggs of three species ......................... 144 6 2 Scanning electron microscope views of larvae of three species ...................... 145 6 3 Light microscope views of larvae of three species ........................................... 146 6 4 Light microscope views of pupae ...................................................................... 147 7 1 Mean no. of eggs deposited by females of C. massyla on artificial diet summed over 3d period. .................................................................................. 176 7 2 Mean cumulative eggs deposited by females of C. massyla on artificial diet. .. 177 7 3 Mean no. of eggs deposited by females of E. eluta on artificial diet summed over 3 d period. ................................................................................................ 178 7 4 Mean cumulative eggs deposited by females of E. eluta on artificial diet. ........ 179 7 5 Mean no. of eggs deposited by females of E. stigmatias on artificial diet summed over 3d period. .................................................................................. 180 7 6 Mean cumulative eggs deposited by females of E. stigmatias on artificial diet. 181

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13 Abst ract of Dissertation Presented t o t he Graduate School o f t he University o f Florida i n Partial Fulfillment o f t he Requirements f or t he Degree of Doctor o f Philosophy MORPHOLOGY, BIOLOGY AND DISTRIBUTION OF CORNINFESTING ULIDIIDAE By Gaurav Goyal December 2010 Chair: Gregg S. Nuessly Major: Entomology and Nematology Corn silk fly, Euxesta stigmatias Loew is serious pest of sweet corn in Florida. Experiments were conducted to determine the pest status of three most common ulidiids, C haetopsis massyla (Walker) E uxesta eluta Loew and E stigmatias All three species were reared from both uninfested and infested ears indicating their primary as well as secondary mode of attack. More flies of all three species emerged from corn ears infested with S. frugiperda than uninfested corn ears indicating that ulidiid adults are attracted to damaged ears by the p resence of olfactory cues in ears damaged by Lepidoptera larvae, or that previously damaged corn ear s are more suitable for larval survival F ewer adults of E. stigmatias and C. massyla emerged from ears infested with E. eluta in nochoice tests, suggesting the presence of visual or olfactory cues that negatively affect the oviposition. Surveys were conducted throughout Florida to evaluate species richness and distribution of corninfesting Ulidiidae Adults were sampled using s weep net s and reared from fly larvaeinfested corn ear s collect ed from representative corn fields in 16 and 27 counties in 2007 and 2008, respectively. Euxesta eluta and C. massyla were found infesting field and sweet corn throughout Florida. Euxesta stigmatias was only found in Martin, Miami Dade, Okeechobee, Palm Beach and St. Lucie Counties on field

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14 and sweet corn. Euxesta a nnonae (F.) was found in sweet corn in Miami Dade, Okeechobee, and Palm Beach Counties, but field corn was not sampled in these counties. Euxesta eluta, E. stigmatias and C. massyla were collected from corn throughout the corn reproductive stage. Raising adults from fly larvaeinfested ears provided the best method for assessing rates of ear infestation and species richness. Spatial and temporal distributions of flies in corn fields were determined by sampling flies caught on sticky traps arranged in a certain pattern in small and large scale corn fields from corn at first silk appearance to corn harvesting. Four corn ears were sampled close to each trap location for presence of immatures and held for immature development to adults under laboratory condit ions to determine the number and species of adults obtained. Morisitas index showed the distribution of the flies to be aggregated in both small and large scale fields at most of the sampling dates. More flies were found on the sides of the field than i n the center in most of the large scale fields. More flies were found in field sides bordered by sugarcane, fallow or residences than sides bordered by corn. The proportion of infested corn ears to total number of sampled corn ears was found to be strong ly correlated with the flies caught on sticky traps. Field surveys and laboratory evaluations were conducted to evaluate crop and noncrop plants for their potential to act as developmental hosts and determine developmental and survivorship rates for the i mmatures of these flies. Several commodities and weeds (bell pepper, spiny amaranth, cattail, sugarcane, and johnsongrass) were found in field surveys to provide satisfactory food sources for successful development from eggs to adults In laboratory eval uations, all three species

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15 were able to complete development on alternative commercial crops and weedy species (bell pepper, cabbage, radish, papaya, hass avocado, sugarcane, little hogweed, habaero pepper, tomato, southern cattail, spiny amaranth, johnso ngrass). Morphology of the immature stages was examined for three species of Ulidiidae using light and scanning electron microscopy. Many egg, larval, and pupal characters were measured and described, but only a few traits in each stage could be used to separate the species due to considerable range overlap. Pores were restricted to the posterior end of C. massyla eggs, but distributed over the entire surface of E. eluta and E. stigmatias eggs. A distinct tooth on the ventral surface of the mouth hooks s eparates C massyla larvae from the other species Euxesta eluta and E stigmatias had more oral ridges than C massyla T he two Euxesta species could be separated based on the length of the 2nd and 6th creeping welts. The first row of spinules was disc ontinuous on the 4th 8th creeping welts in larval C. massyla while it was continuous in Euxesta species. The larval posterior spiracles were black on E. eluta, dark brown to black on E. stigmatias and brown on C. massyla. C haetopsis massyla pupae were reddish brown while E. eluta and E. stigmatias pupae were light brown to black. The posterior spiracular plates in C. massyla pupae were trapezoidal in shape while those in E. eluta and E. stigmatias were ovoid. Studies were conducted to determine and com pare biological parameters (i.e., adult longevity, developmental periods, and survival of different immature s tages ) of the three species of these flies on artificial diet and corn ears. Euxesta eluta lived longer, deposited more eggs and had shorter preoviposition period compared to other species. Flies of all three species developed faster in corn ears than in artificial diet. The

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16 development periods in corn ears in general were shortest in May followed by in March followed by December. Survival of immature stages was not significantly different among three species. The percentage pupal survival was greatest followed by egg survival and then larval survival. Field studies were conducted to determine the flies period of oviposition by examining sweet corn ears for eggs naturally deposited by Ulidiidae every 2 h periods of the day. Chaetopsis massyla deposited more eggs in young ears whereas E. stigmatias deposited more eggs in ears during and after peak silk extension. Chaetopsis massyla depos ited eggs throughout the day whereas E. eluta deposited more eggs and in more corn ears from 11001700 h than during other times of the day. Euxesta stigmatias deposited more eggs and in more ears from 11001500 h than during other times of the day. In s tudies to understand the intra ear resource competition among these flies, it was found that more larvae were in ear tips than middle and bottom segments This indicat es that flies may compete for their resources forcing the larvae to go deeper in the cor n ears. Moreover, differences in the frequency of ears with individual versus multiple species indicated the presence of behavioral interactions among these species in oviposition preference and development within the corn ears.

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17 CHAPTER 1 REVIEW OF LI TERATURE Introduction The picturewinged fly Euxesta stigmatias Loew (Diptera: Ulidiidae) has been reported feeding on a wide range of fruits, vegetables and field crops, and is an economic pest of maize ( Zea mays L.), especially sweet corn (App 1938). This insect is highly injurious to maize, with up to 100% damage in untreated fields reported by workers beginning in the 1940s (Bailey 1940, Walter and Wene 1951, Hayslip 1951, Seal and Jansson 1989, Evans and Zambrano 1991, Hentz and Nuessly 2004). Histor y Early records of E. stigmatias distribution indicated a range from Argentina in South America through Central America to s outh ern Florida and T exas in North America (App 1938 Hayslip 1951). Specific U.S. National Museum collection records for E. stigma tias cited by App (1938) included the West Indies (Puerto Rico, Cuba, Dominican Republic, Nassau, Virgin islands), British West Indies (Barbados, Dominica, St Vincent, Jamaica), Central America (Canal Zone, Panama), South America (Brazil, Bolivia, Paraguay Peru) and North America (Mexico, Florida). Their larvae were found infesting corn in Texas by Walter and Wene (1951). According to Daly and Buntin (2005), ulidiid larvae were recovered from corn in Georgia. The first published observation of the pest in Florida was in Dade County during February and March, 1936 by Barber (1939). Hayslip (1951) confirmed the pest status of this species in Florida. Damage Damage to corn plants primarily occurs by larval feeding in ears. The insect deposits its eggs pri marily on the styles (silks) present at the tips of ears. Eggs have

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18 also been found at the base of leaves and on the male inflorescence (tassels) (Seal and Jansson 1989). The larvae (maggots) feed on silks and on kernels (Seal and Jansson 1989, App 1938) App (1938) observed softening of ears after the larvae entered the cob. Infested ears are considered unmarketable. Ears with late damage restricted to silks after the kernels are set (i.e. past fertilization) can be marketed only if the ear tops are r emoved and the ears tray packed for marketing. This latter practice is the minor part of the market usually restricted to the early summer months. The market value of maize ears gets reduced unless the infestation is restricted to silks and tip of maize ears is removed during packaging. Larvae in ears disrupt pollination by clipping the silks (Bailey 1940). Life Cycle The fly can complete its life cycle in 18 to 37 d (Hentz and Nuessly 2004, Seal and Jansson 1993), completing approximately 20 generations per year (Bailey 1940). The egg, larval and pupal periods range from 1.4 to 4 d, 7.46 to 27 d and 5.6 to 9.2 d, re spectively (App 1938, Seal and Jansson 1989, Hentz and Nuessly 2004). Larvae complete their development in maize ears. The majority of late instar larvae leave the ears to pupate in the soil. Some larvae pupate near the opening of the silk channel on the ears (App 1938). Adults can survive for approximately 116 d under artificial conditions when provided with honey and water ( Hentz and Nues sly 2004). Chemical Control Management of the fly using insecticides has reached varying degrees of success. The use of chemicals is directed primarily towards the adults as the eggs are protected within the silk channel. Larvae are protected by husks an d pupae are protected under the soil or in ears. Sampling program and economic threshold are not currently

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19 available for determining the proper timing of control strategies. Chemical control of these flies has been evaluated since the 1940s. Nuessly and Hentz (2004) tested all labeled insecticides used for control of armyworm in maize against E. stigmatias adults. They found that all the tested insecticides except thiodicarb quickly killed or caused incapacitating sub lethal effects to >75% flies when kept in direct contact with insecticides. Insecticide residues on dipped leaves and on field treated plants were much less effective at killing adults compared with direct contact tests. Dried insecticide residues on leaves killed < 20% of adult flies 48 h after they were applied in the field. Pyrethroid (i.e., esfenvalerate, cyfluthrin and lambdacyhalothrin) residues induced greater levels of sub lethal effects (57 70%), and for longer periods than other types of insecticides. Evidence suggests that prolonged use of low rates of pyrethroids with associated sublethal effects may result in the development of resistance to these insecticides (Nuessly and Hentz 2004). Bailey (1940) conducted experiments to test the efficacy of combinations of pyrethrum extract and mineral oil for controlling the larvae of these flies. Better control (94%) of fly larvae was obtained by injecting two applications of a mixture of pyrethrum and mineral oil (1:5) into corn silks with eye droppers as compared to one applicati on (89%) and when pyrethrum and mineral oil were applied alone. Derris extract (Botanical insecticide extract from Derris eliptica Benth) proved to be ineffective in increasing the effectiveness of the pyrethrum: mineral oil mixture. Plant Resistance Plan t resistance to insects has also been exploited as one of the control measures against the flies. Damage by E. stigmatias larvae was reduced in two high maysin varieties of corn, Zapalote Chico 2451 and Zapalote Chico sh2 compared to standard

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20 sweet corns (Nuessly et al. 2007 ). Tightness of ear tips was reported to be one of the host resistance factors against attack by E. stigmatias by Scully et al. (2000). Resistance to an unknown species of Euxesta was identified in two sweet corn test hybrids developed in Brazil (Branco et al. 1994). Tight husks were also found to improve plant resistance to E. eluta (Evans and Zambrano 1991). Early workers examined cultural control strategies for this pest. App (1938) found that cutting of silks and squeezing ear ti ps had little effect on larvae. Covering the ear or constricting the husks gave better control of this insect than other methods. Use of wires, hog rings and paper bags also gave better control compared to tying strings around ear tips. Daly and Bunti n (2005) found fewer larvae and adults on transgenic corn expressing Cry 1Ab toxin from Bacillus thuringiensis var kurstaki (Bt) than on standard non GMO corn varieties. They explained this effect was due to less attractiveness of the plant for fly due to less damage caused by Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) Arce de Hamity (1986) found corn ear color of field corn to affect the damage caused by E. eluta in Jujuy, Argentina. Corn ears with colored grains suffered more damage by E. eluta in comparison with white colored grains with yellow, purplelilac and red suffering more damage than creamy colored grains. Biological Control Little is known about the biocontrol of these flies. Seal and Jansson (1989) reported that some unidentified earwigs (Dermaptera: Forficulidae, Labiidae), hemipterans and mites preyed upon E. stigmatias eggs. Van Zwaluvenberg (1917) observed a capsid bug (Hemiptera: Miridae) feeding on the eggs of this fly. Valicente (1986) reported Dettmeria euxestae Borgmeier (Hymenoptera: Eucoilidae) as a parasitoid of Euxesta eluta in the Sete Lagoas region, MG, Brazil.

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21 Additional Ulidiidae Species That Damage Corn in America South of the U nited States Additional Euxesta species are reported to attack corn in Central and South America. Euxesta major Wulp (Diptera: Ulidiidae) is reported to be a serious pest of seedling corn in Guatemala (Melhus and Harris 1949). Painter (1955) collected adults of E. sororcula (Wiedemann) (Diptera: Ulidiidae) at Antigua, Tiquisate, Progreso, Jutiapa in Guatemala and larvae of E. sororcula at Antigua (Guatemala) and adults of E. obliquestriata Hendel ( Diptera: Ulidiidae) at Antigua (Guatemala) on corn. Fras L (1978) reported E eluta and E. annonae to be the pests of corn. Chaetopsis aenea ( Wiedemann) (Diptera: Ulidiidae) has been found in the damaged stems of corn above ears in Ohio (Gossard 1919). Euxesta mazorca Steyskal (Diptera: Ulidiidae) has also been reported to attack corn (Huepee et al. 1986). Problems with Correct Species Identifi cation Misidentification of Euxesta species has been recurring problem in the literature. Walter and Wene (1951) mentioned in his paper App (1938) possibly confused the larva of Megaselia scalaris Loew (Diptera: Phoridae) with that of E. stigmatias sinc e his published figure shows a spindle shaped larva typical of M. scalaris instead of one with a very broad and blunt caudal end as in E. stigmatias The photograph on the front page in Hayslips 1951 paper is clearly that of E. eluta but it was described as E. stigmatias in the figure caption. Similarly, photographs of E. eluta described in the figure caption in Huepee et al. (1986) are not of E. eluta but of some other species. One reason for this misidentification can be the minute differences in w ing banding patterns and other adult characteristics.

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22 Identification of Immature Stages Little work has been published on identifying characteristics of the immature stages of pest picturewing fly species. Allen and Foote (1992) described the three larva l instars of C. massyla using various characteristics of the body including the anterior and posterior spiracles and the cephalopharyngeal skeleton. App (1938), Hayslip (1951) and Seal et al. (1995) described all the stages of E. stigmatias using general external morphological descriptions. Arce de Hamity (1986) provided morphological characteristics for immature stages of E. eluta Identification to species using characters of immature stages has been successful on other calypterate flies that feed on decomposing human remains. Sukantason et al. (2003) used relative thickness of the branches of posterior spiracular hairs and body pubescence to distinguish Chrysomya megacephala (F.) (Diptera: Calliphoridae) from C. rufifacies (Macquart) (Diptera: Calli phoridae). Several characteristics of anterior and posterior spiracles and mouth hooks were used to describe the morphology of all larval instars of Parasarcophaga dux (Thomson) (Diptera: Sarcophagidae) by Sukantason et al. (2003). Many characters, inclu ding the length and width of puparia, number of papillae in the anterior spiracle, position of posterior spiracles, shape of posterior spiracular discs, and intersegmental spines between prothorax and mesothorax were used to differentiate pupae of a blow f ly, Lucilia cuprina (Diptera: Calliphoridae) and a flesh fly, Liosarcophaga dux (Diptera: Sarcophagidae) (Sukantason et al. 2006). Similar characters were used to separate puparia of the housefly, Musca domestica L (Diptera: Muscidae) from those of Chryso mya megacephala (F.) (Siriwattanarungsee et al. 2005). Studies on morphological characteristics of the immature stages of these ulidiids could

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23 provide characters useful for species identification without the need for rearing them to adults from infested plant material. Biology and Ecology Host plants of these flies have been studied by certain workers. Adults and immature stages of these flies have been reported on various plants by several workers. App (1938) reported the adult flies on sugarcane ( Sacch arum officinarum L.), guava ( Psidium guajava L.), corn (Zea mays L.), Eugenia species, orange ( Citrus aurantium L.), and the stem of banana ( Musa species). Seal and Jansson (1989) collected different developmental stages of this fly from sorghum ( Sorghum bicolor Moench), tomato ( Lycopersicon esculentum Mill.) and potato ( Solanum tuberosum L.) from fields at Homestead, FL. Adult flies were also collected from volunteer potato ( Solanum tuberosum L.), sugarcane and some weed species (Seal and Jansson 1989). Franca and Vecchia (1986) reported E. stigmatias larvae feeding on carrot roots ( Daucus carota L.) in Brasilia, Brazil. Armstrong (1986) defined host status of a fruit fly as Any fruit or vegetable in which fruit fly oviposits under field conditions, th e eggs hatch into larvae, and the larvae acquire sufficient sustenance to form viable pupae from which adults eclose that are capable of reproduction. Thus, field collection of adults from a potential host cannot confirm the plant as a host. Kroening et al. (1989) reported that apple ( Ma lus domestica) fruits cannot be considered as host of the apple maggot, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) even though the adults of apple maggot were collected from apple orchards, as larvae and pupae were not collected from the apple fruits. To examine the plant as a host of an insect for quarantine purposes under laboratory conditions, plants are exposed to high populations of adults for them to deposit eggs on plants and allow the eggs to complete their life cycle till

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24 adults emerge. Spitter et al. (1984) reported survival of only five pupae out of 515,982 eggs deposited on lemons ( Citrus limon L., cv. Eureka and Lisbon) confirming that lemon should not be considered as host of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (D iptera: Tephritidae) Thus, host range of these flies and their biology on those hosts needs to be established to have better knowledge of their ecology and dynamics in nature. This information will also be helpful in understanding the various sources of infestation in nature which can lead t o development of new recommendations. Conflicting evidence exists regarding primary versus secondary nature of E. eluta and E. stigmatias in the present literature. E uxesta stigmatias was shown to be a primary pest by bagging ears immediately after the ap pearance of eggs by Van Zwaluvenberg (1917) in Puerto Rico, but the author could not provide enough evidence about the precautions taken to avoid any infestation of corn ears before bagging of ears. According to Seal et al. (1996), E. stigmatias was secon dary pest on several plants as it was found infesting plants that included sugarcane, guava, banana, orange, atemoya, orchid and potato only after they got decayed. Arce de Hamity (1986) observed E. eluta to be both primary as well as secondary pest of c orn in Jujuy, Argentina. In contrast, Fras (1981) found more E. eluta larvae on the ears damaged earlier by H. zea as compared to E. annonae that prefers undamaged ears. It also indicates the primary nature of E. annonae. Allen and Foote (1992) reported that C. massyla larvae were secondary invaders in decomposing stem tissue of cattail ( T ypha latifolia L.) previously damaged by moth larvae belonging to family Noctuidae. They also reported C. massyla larvae in Carex lacustris Willd. stems previously damaged by larvae of Epichlorops

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25 exilis (Coquillett) (Diptera: Chloropidae). Many Chaetopsi s species (Diptera: Ulidiidae) are saprophagous, some being found in damaged stems of corn (Gossard 1919). Knowing primary and secondary nature of an insect may be crucial for growers as well as researchers especially in case of fall armyworm or corn earworm infested ears that may not be of any use to grower making the fly control unnecessary. Knowledge on the spatial as well as temporal distribution of flies in a corn field will be helpful in developing sampling plans and economic thresholds. Spatial dist ribution of an insect in a field can be random, regular or aggregated. Distribution of flies in a field before and after the application of an insecticide will further help in attaining this objective. Sampling techniques can vary depending upon the dist ribution of flies in the field. Temporal and spatial distribution of insects has been studied by few workers. Boivin (1987) found two distinct periods of adult activity per year in case of carrot rust fly, Psila rosae (F.) (Diptera: Psilidae). The appar ent pupae of Gonometa postica Walker were found to be aggregated on larval host plants while the cryptic G. rufobrunnea pupae were aggregated on nonhost plants as reported by Veldtman (2007). Miliczky (2007) found that the density of Western flower thrips, Frankliniella occidentalis (Pergande) decreased with increasing distance into the orchard surrounded by native sagebrush steppe habitat. Moreover, t he regression analysis showed that the thrip density and damage were positively correlated. Studies on distribution of insects have been conducted using several techniques. Kozlov (2007) studied the change in distribution of archaic moth, Micropterix calthella (L.) (Lepidoptera: Micropterigidae), in St Petersburg, Russia visually as well as by extensive s weep netting. Dist ribution of olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae)

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26 within an orchard has been studied using McPhail traps by Dimou et al. ( 2003) Decante (2008) showed random spatial distribution of green leafhopper, Empoasca vitis (Goethe) (Hemiptera: Cicadellidae) using statistical and geostatical analysis from adult trappings and nymphal counts. Distribution of cotton jassid, Amrasca bigutulla bigutulla was found to be aggregated when population was high and random when population was low using various distribution parameters such as variance mean ratio, dispersion parameter (K), Lloyd's Index of mean crowding, patchiness index, Taylor's power law and Chi square test by Mohapatra (2007). Reay Jones (2007) studied the movem ent of Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae) through the Texas Rice Belt using pheromone baited traps. It has been demonstrated by Nuessly and Hentz (2004) that residues on dipped leaves and field treated plants with insectic ides like esfenvalerate, cyfluthrin, lambdacyhalothrin, chlorpyriphos, methomyl and thiodicarb were less effective at killing adults compared with direct contact tests. Also, flies seem to have the ability to recolonize a field following insecticide appl ication as was shown by Seal (2001), who found more than 19.9 maggots per ear after the application of chemical every 34 days. Knowledge on the movement pattern of flies into a field can have impact on decision of grower for spraying certain portions of field rather than spraying the entire field. This information will be helpful in developing sampling plans and economic thresholds for this insect. It seems that apart from the present distribution, these flies may be present in areas previously unknown t o inhabit them. Barber (1939) reported E. stigmatias in Miami Dade County, Florida. Uli di i ds have also been found attacking corn in Texas (Walter and Wene 1951) and Georgia (Daly and David 2005) implying the possibility of

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27 expanding range of flies. Adul t specimens of these flies at museum in Division of Plant Industry, Gainesville, FL show these flies to be collected from many areas of Florida in previous years, thus implying the possibility of intra state dispersal of flies. Fras L (1981) found that E eluta and E. annonae prefer different microhabitats i.e. one preferring mature ears and another less mature ears and thus implying the different time of finding the flies in field. Thus, understanding of population dynamics and spatial and temporal dist ribution of different species in various areas of Florida and adjoining states will be useful for scientists as well as growers as it may lead to need for development of area specific control measures. Research Objectives Pest status of picturewinged flie s Distribution of corninfesting picture winged flies in Florida Spatial and temporal distribution of corninfesting picture winged flies in corn fields Alternate hosts of corn infesting picturewinged flies Comparative morphology of the immature stages of three corninfesting Ulidiidae Developmental and life table studies of corninfesting picturewinged flies Ecological studies of corninfesting picturewinged flies

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28 CHAPTER 2 PEST STATUS OF PICTU RE WINGED FLIES Adults of four species of picturewinged fl ies, Euxesta annonae, E. eluta E. stigmatias and Chaetopsis massyla have been observed in corn fields at Everglades Research and Education Center (EREC), Belle Glade, Florida. Although E. stigmatias has been recognized as a primary pest of corn in many countries for over 75 years ( Van Zwaluvenberg 1917) the pest status of the other three species has not been formally examined. They may attack corn as primary pests or they may require prior infestation of corn ears by other insects before they can use t he crop as a food source (secondary pest). Scully et al. (2000) reported both E. stigmatias and S frugiperda larvae infest ing ears concurrently in south Florida. Nuessly et al. (2007) suggested that S podoptera frugiperda (J. E. Smith) (Lepidoptera: Noct uidae) larval tunnels through the silk channel might provide E. stigmatias easier access to the cob and kernels compared to ears without substantial silk channel damage. Fras L (1981) determined that E. eluta development was favored by previous activity of corn earworm larvae, Helicoverpa zea Boddie (Lepidoptera: Noctuidae). Ulidiid eggs frequently have been observed within the entry and exit holes of Lepidoptera larvae on corn ears (pers. obs. G. Goyal). The pest status (primary versus secondary pest) of these flies is important from a management standpoint. Control strategies are required to prevent primary pests from entering ears, while no treatment is required for secondary pests if primary pest damage is avoided, as the presence of prior infestati on is required for their feeding. The information may be helpful for future research on oviposition cues for these flies.

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29 Materials and Methods Choice and nochoice tests were conducted to evaluate the pest status of the flies. T he studies were conducted at EREC on C. massyla, E. eluta, and E. stigmatias The flies used for the first no choice test (2007) were collected from corn fields using sweep nets and kept in laboratory conditions for 814 d before setting up the experiment due to the unavailabilit y of flies reared in laboratory Flies used for the second nochoice test (2008) and both choice tests (2009 and 2010) were obtained from fly colonies maintained in laboratory conditions at the EREC described below Colony Maintenance Colonies of C. massyla, E. eluta and E. stigmatias were established and maintained in the laboratory to facilitate development studies. Colonies of each species were initiated from flies collected using sweep nets in corn fields in and around Belle Glade, Florida. Adults w ere maintained in Plexiglas and screen covered cages (0.45 x 0.45 x 0.62 m). Water was provided using dental wicks (15.2 1.3 cm, Richmond Dental Co., Charlotte, NC) placed in 50 ml Erlenmeyer flasks. Parafilm (Pechiney Plastic Packaging, Chicago, IL) was used to form a seal between the dental wick and the flask. Food was provided for adults by suspending cotton balls dipped in 50 to 60% honey water solution on the roof and sides of the cages and by painting thin streaks of honey on the roof of the cag es using a small syringe. Frequent introductions of wild flies of all the three species were made to boost the colony and remove any inbreeding depression. Flies used in the experiment had completed 36 generations before beginning the studies. Artificia l Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) diet (Product # F9393B, Bioserv, Frenchtown, NJ) served as the ovipositional substrate and larval food

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30 source for colonies of all three species. One liter of dried diet consisted of ground pinto bean (11 1.0 g), stabilized 30mesh wheat germ (50.0 g), torula yeast (34.0 g), ascorbic acid (3.0 g), phydroxybenzoic acid methyl ester (methyl paraben) (2.0 g), and sorbic acid (1.0 g). The larval diet was prepared by boiling 14.0 g powdered agar with 1500 ml w ater and then blending with 200.0 gm of the dry diet. Diet slants were prepared by pouring the liquefied diet into sterilized test tubes (2.5 cm dam 15 cm long) placed at a 45 angle to produce a larger surface area on the solidified diet for oviposition. After the diet cooled and solidified, the open ends of the diet tubes were covered with aluminum foil and the vials stored in plastic bags at 0C to reduce bacterial and fungal contamination. Diet tubes were removed from cold storage and allowed to reach room temperature before placement at the top of adult cages for oviposition. Diet tubes with eggs were removed every 12 d, plugged with cotton balls and held horizontally in trays. Larvae were allowed to complete development within the diet tubes. Larvae normally left the diet to pupate away from their food source. While many pupated within the cotton balls plugging the tubes, each tray was lined with a dry cotton sheet to provide a site for pupation of larvae that escaped from the diet tubes. The cotton balls and sheets with attached pupae were hung in the cages to allow adults to emerge directly into the colony. Adults and immatures were maintained at 26.5 1.0 C, 14:10 (L: D) photoperiod and 55 70% RH. No c hoice T est s Studies were conducted using potted plants in a greenhouse in December 2007 and using plants grown in a field in May 2008. Sweet corn ( cv. Obsession Seminis Vegetable Seeds, Inc., Saint Louis, MO ) was planted on 17 October 2007, in 18.9 L (5 gal) buckets filled with organic (Dania Muck) soil. Fertilizer based on the plant

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31 requirements was manually hand mixed with the soil in buckets a day before planting. The crop was hand seeded by sowing 3 seeds in each bucket at a depth of approximately 2.5 cm. The plants were later thinned to one per bucket on 5 November. Plants were irrigated as needed. The insecticide Praxis (gamma cyhalothrin, Dow Agro Sciences Indianapolis, IN) was used to protect the plants from Lepidoptera larvae until tassel emergence after which no insectic ide was applied. The plants were grown in a fanand padcooled greenhouse maintained within 3.5 C of the ambient temperature. Corn plants with ears at first silk appearance not previously damaged or infested by insects were moved together in the greenhouse and labeled in sets of three plants each. Eight replications were conducted for each species. The experiment was a split plot design with three main treatments and three sub treatments. The main treatments consisted of one uninfested ear, one ear pr eviously infested with E. eluta larvae, and one ear previously infested with S. frugiperda larvae of the three plants of a set Five 2nd instar larvae of E. eluta were placed within the silk channel of one ear per set with the aid of a fine camels hair brush (size 0). Second instar larvae from the E. eluta colony were easily recognized by the nonoverlapping ranges of body length and width. Newly formed 2nd instar larvae were collected using a brush, placed in 30ml diet cups half filled with H. zea die t, and transferred to the greenhouse to infest the corn ears. Three 3rd instar larvae of cornstrain S. frugiperda larvae were placed within the silk channel of one ear in each set. Larvae were reared from eggs obtained from a laboratory colony (Insect B ehavior and Biocontrol Research Unit, USDA, Gainesville, FL) maintained on H. zea artificial diet. Third instar larvae were recognized based on

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32 their body length, color and cuticle molts. The three corn ears in each set were then covered with pollination bags (6.25 2.5 21.25 cm) to protect the ears from infestation by S frugiperda or Ulidiidae. Bags were held tightly around the middle of the ear with a rubber band. The sub treatments were applied to sets of ears 7 d after application of the main tre atments. The sub treatments consisted of caging one fly species on each of the three ears in a set. Each of the three fly species was applied to a different set of three ears. Five pairs of flies were caged on each ear using 11 30 cm, 17 mesh bags (17 openings per linear 2.5 cm ) The open end of each bag was held tight around the ear using a binder clip to prevent flies escaping from the bag. A cotton ball dipped in 5060% honey solution was kept in the bag as food for flies. Adults in mesh bags wer e examined on alternate days and replaced with new adults as old ones died. Euxesta eluta pupae that developed from the main treatment were removed from ears and mesh bags 3 d after application of the sub treatment to avoid later confusion with immatures developing from the sub treatments. The sub treatments (flies and mesh bags) were removed after 10 d and recovered with pollination bags to protect the ears from additional infestation by other insects until the end of the experiment. Larvae that emerged from eggs deposited by flies in the sub treatment were allowed to continue to develop in the ears for 7 d after removing the sub treatments. Seventeen days after application of the sub treatments, the top third of each ear was removed using a knife and placed individually in 1.83 L Ziploc bag s. Two paper towels were added to each bag to reduce moisture accumulation. The e ars were held in a room maintained at 26.5 1.0 C, 14:10 (L : D ) photoperiod and 55 70% RH. To reduce the accumulation of

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33 moistur e and associated fungus, bags were left partially open, p aper towels were changed frequently, and the air was constantly circulated using box fans. Pupae were removed fr om the bags and placed on moistened filter paper (Whatman 3) in covered Petri dishes f or adult emergenc e The dishes were sealed with P arafilm to reduce moisture loss. Adults that emerged were counted at the end of the experiment. No choice field studies were conducted in May 2008 at the EREC. Sweet corn ( cv. Obsession ) was planted on 19 March 2008 in organic (Dania muck) soil and manage d according to local standards ( Ozores Hampton et al. 2010). Insecticides were used to protect the plants from Lepidoptera larvae until tassel emergence after which no insecticides were applied. The pr ocedures for applying and evaluating main and sub treatments in the field study were the same as for greenhouse studies except that only one pair of laboratory reared 515 dold adults were caged in each bag as sub treatments instead of five pairs used in the greenhouse trial. Nine replications were conducted for each species. Choice T est s Studies were conducted in sweet corn fields in December 2009 and May 2010 to evaluate the pest status and oviposition preference of the flies. Sweet corn was planted on 15 and 29 September 2009 (cv. Garrison, Rogers Brand, Syngenta Seed, Wilmington, DE ) and 15 February and 3 March 2010 ( cv. Obsession ) at EREC. Sets of three ears (at first silk appearance) adjacent to each other not previously damaged or infested by insects were covered with pollination bags (6.25 2.5 21.25 cm) as done in the nochoice tests and labeled. The main treatments (i.e., uninfested, E. eluta infested and S. frugiperda infested ears) were applied to ears similarly as in the nochoice tes ts. Each set of three plants were enclosed by a mesh cage (2.40 m x 0.90 m

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34 x 0.90 m, 8 openings per linear cm) with a full length zipper to provide easy access to the ears supported by polyvinyl chloride pipes set 0.4 m in the soil. Sub treatments were applied by releasing five pairs of 515 d old laboratory reared flies of a single species in each cage. The pollination bags were removed from ears before releasing the flies in cages. Food was provided for the flies by hanging two cotton balls dipped in a 5060% honey solution in each cage. Six replications of each species were conducted in the first week and six replications were conducted in the second week of December 2009. Seven replications of C. massyla and E. eluta and six replications of E. stig matias were conducted in first and second weeks of May 2010, respectively. Ears were re covered with pollination bags 10 d after exposing them to the sub treatments. The bags were removed after 7 d and ears harvested and held for adult emergence similarl y as for the nochoice tests. Statistical A nalysis Proc GLM (SAS institute 2008) was used to conduct an analysis of variance of the results of choice and nochoice tests to determine pest status. The main effects (ears uninfested or infested with either E eluta or S. frugiperda), sub treatments (three fly species), replicate (year) and their interactions were modeled in the nochoice test analysis. Numbers of adults that emerged per ear were used as dependent variables. The independent variables tested in the choice test model were the main effects (ears uninfested or infested with either E. eluta or S. frugiperda), sub treatments (three fly species), replicate and their interactions. The choice test was conducted twice in each of two years for a total of four replicates. The replicates were treated as random variables in the model. Numbers of adults that emerged per corn ear were used as dependent variables in the choice test model. The percent of adults that emerged per

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35 main effect ear treatment was calculated by dividing the number of adults that emerged per ear within a cage by the total number of adults that emerged from all the three corn ears within a cage and multiplied by 100. Percent adults were transformed using arcsine root transformation to improve the normality and homogeneity of variance, but retransformed for presentation purposes. Different numbers of ears were exposed to flies of different species. Least square means were used rather than arithmetic means due to unequal numbers of ears exposed per species. The Tukey's honestly significant difference (HSD) test (SAS institute 2008) was used for means separation with P = 0.05. Results and Discussion No choice T est s All three ulidiid species oviposited and were able to complete development to the adult stage in uninfested ears, as well as in ears already infested with S. frugiperda or E. eluta larvae. The number of adults that emerged per ear was significantly affected by f ly species, treatment, and year (replicate) (Table 2 1). The number of adults that emerged per ear was affected by fly species but the results depended on treatm ents. The number of adults that emerged per ear w as also affected by treatments, but the results depended on fly species More adults emerged per ear in 2008 ( least squares mean SEM range ; 24.2 0.84, 458) than 2007 (14.5 0.89, 040), but data were pooled over the two years for means comparison because the trends among treatments were the same for both years. Significantly more adults of all the thr ee fly species emerged from ears already infested with S. frugiperda than with E. eluta (Table 22 ). More E. eluta and E. stigmatias adults emerged from uninfested ears than from ears already infested by E. eluta More E. stigmatias adults than E. eluta and C. massyla

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36 adults emerged from uninfested ears and those already infested by S. frugiperda The mean number of adults of all three species that emerged per ear was 1.52x greater in ears already infested with S. frugiperda than with E. eluta Choice T est s All three fly species deposited eggs and completed development to the adult stage in uninfested ears and those already infested by S. frugiperda and E. eluta when given the choice among these main effect treatments. Fly species and treatment had sign ificant effects on the number of adults that emerged per ear (Table 2 3). The number of adults that emerged per ears w as also significantly different among fly species with different treatments. Data from the four replicates were pooled for mean comparis on of adults that emerged per ear because replicates were not a significant source of variation in the model. Significantly more E. eluta (least squares mean SEM, range; 23.1 2.6, 090) and E. stigmatias (19.5 1.8, 0 58) adults emerged per ear than C. massyla (8.0 1.0, 040). Results of the biology studies reported in the last chapter indicated that the species have different reproductive potentials in terms of the number of eggs deposited in their reproductive periods. Therefore, the percent of adults that emerged per cage from each ear were used to compare the main effect treatments and interaction means. The percent of adults that emerged per ear was significantly affected by treatment ( F = 63.67; df = 2, 216; P < 0.0001) The percent of adul ts that emerged per ear was also significantly affected by fly species but the results depended on treatments ( F = 6.53; df = 4, 216; P < 0.0001). A greater percentage of adults of all three fly species emerged from ears already infested with S. frugiper da than from uninfested ears or those already infested with E. eluta (Table 2 4). A greater

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37 percentage of E. eluta than the other two fly species emerged from ears already infested with S. frugiperda. All three species were reared from uninfested corn ear s in both choice and nochoice tests indicating that these flies attack corn as primary pests. All three species also oviposited and successfully completed development in ears already infested with S. frugiperda or E. eluta larvae. These results indicate that these three species can successfully use previously damaged ears as hosts. These results were in accordance with some of the previous studies done on the primary and secondary nature of these flies. Chaetopsis massyla were also reared from both uni nfested standard sweet corn and from Bacillus thuringiensis enhanced sweet corn in commercial and experimental fields in southern Florida (Goyal et al. 2010). The secondary mode of attack of C massyla has been reported on wetland monocots such as cattail Typha latifolia L. (Typhaceae: Typhales) damaged by moth larvae (Lepidoptera: Noctuidae), and stems of Carex lacustris Willd. (Cyperaceae: Cyperales) that were previously damaged by larvae of Epichlorops exilis (Coquillett) (Diptera: Chloropidae) (Allen and Foote 1992). Euxesta eluta was identified as a primary pest of corn by Arce de Hamity (1986) in Jujuy, Argentina. Both Arce de Hamity (1986) and Fras L (1981) in Chile reported that E. eluta completed development in ears damaged by other insects. E uxesta stigmatias has been recognized as a primary pest of sweet corn in Florida for over 70 years (Barber 1939, Haysl ip 1951, Seal and Jansson 1989). Euxesta stigmatias has been reported as a secondary pest of several fruits in southern Florida (Seal et al. 1996). While it is unknown which of these types of hosts (i.e., uninfested or infested with other insects) originally attracted these species to maize, the results of greater numbers

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38 and percentages of flies emerging from S. frugiperdainfested than un infested ears suggests that ulidiid adults are attracted to damaged ears. Fras L (1981) studies in Chile suggested that E. eluta also preferred Lepidopterainfested ears to uninfested ears. His first study found no E. eluta in uninfested ears, while 6 of 62 ears examined were found with both E. eluta and H zea larvae. Two ulidiid species were found in infested ears in his second study in which E. eluta comprised 86.3 % of the adults that emerged from H zea infested ears but only 49.6% of the adults t hat emerged from undamaged ears. Taken together, these results may indicate the presence of attractive olfactory cues in ears damaged by Lepidoptera larvae that results in significantly greater oviposition in infested ears compared to uninfested ears. It is also possible that survival of fly larvae was better in corn ears infested with S. frugiperda than in uninfested ears Conversely, the results of significantly fewer E. stigmatias and C. massyla adults emerging from ears already infested with E. eluta larvae compared to uninfested ears in the nochoice tests suggests the presence of visual or olfactory cues in fly larvaeinfested ears that negatively affects oviposition by the other two species. It is also possible that competition between the larvae of fly species inhibited their development resulting in fewer adults emerging from ears. Differences in the number of adults that emerged from ears between years in the nochoice tests may have resulted from differences in experimental design (greenhouse versus field trial), using fieldcaptured adults versus laboratory reared adults, and the numbers of adults caged on ears. The results from the earlier biology chapter indicated that egg deposition increases to its maximum around 1013 d after adult eclos ion and then decreases towards the end of their adult life. Fieldcaptured flies of unknown age

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39 classes were used in the 2007 nochoice tests. Therefore, even with the assumption of a uniform age distribution with the subsample of flies used in that experiment, field collected flies would likely produce fewer flies than flies of a known age from a laboratory colony. However, some laboratory reared insects may not perform as well as field collected individuals. Glas et al (2007) showed that field collec ted Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) produced more eggs on maize than laboratory reared individuals.

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40 Table 2 1 ANOVA table of nochoice test for adults obtained per corn ear Source df F P Main effects treatments 2 23.13 < 0.0001 Sub treatments (fly species) 2 24.32 < 0.0001 Year (Replicate) 1 63.19 < 0.0001 Fly species Treatment 4 2.86 0.0261 Fly species Year 2 2.38 0.0965 Infestation Year 2 7.31 0.0010 Fly species Treatment Year 4 0.95 0.4353 ANOVA (PROC GLM, SAS Inst itute 2008) df, Error = 135. Table 2 2 Mean SEM number of adults that emerged per corn ear in no choice test s, 20072008 (n = 17 per fly species per main effects treatment ) Sub treatment Fly Species Main effects treatment E. eluta S. frugiperda Uni nfested C. massyla 12.9 1.84ef (2 23) 18.1 1.84cd (0 42) 15.7 1.84ef (4 40) E. eluta 12.5 1.84f (0 26) 21.1 1.84bc (0 42) 17.8 1.84cde (4 36) E. stigmatias 17.5 1.84cdef (6 29) 34.4 1.84a (13 58) 24.0 1.84b (12 35) Mean SEM followed by different letters are significantly different (Tukey, P > 0.05, SAS Institute 2008). Table 2 3 ANOVA table for number of adults that emerged per ear in choice test s. Source df F P Main effect t reatment s 2 47.91 < 0.0001 Sub treatments (f ly specie s ) 2 28.81 < 0.0001 Replicate 3 1.32 0.2674 Main effect sub treatment 4 15.96 < 0.0001 ANOVA (PROC GLM, SAS Institute 2008) df, Error = 216

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41 Table 2 4 Mean SEM (range) p ercent adults that from corn ears previously infested with E. eluta or E. sti gmatias or uninfested (n = 24 ears per treatment ) Sub treatment Fly species Main effects treatments E. eluta S. frugiperda Uninfested C. massyla 17.5 3.6d e (0 52.2) 57. 8 3.6 ab (0 100) 24.7 3.6c de (0 87.5) E. eluta 16.9 3.6d e (0 100) 68.1 3. 6 a (0 97.7) 14.9 3.6 e (0 43.8) E. stigmatias 22.0 3.8 de (0 40) 43.2 3.8 bc (0 100) 34.7 3.8 cd (0 100) Mean SEM followed by different letters are significantly different (Tukey, P > 0.05, SAS Institute 2008).

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42 CHAPTER 3 DISTRIB UTION OF CORN INF ESTING PICTURE WINGED FLIES IN FLORIDA There are 671 species of Ulidiidae worldwide, but less than ten species in two genera are known to damage corn (Allen & Foote 1992; Anonymous 2008c; Goyal et al. 2010; Van Zwaluvenberg 1917). Van Zwaluvenberg (1917) first reported the pest nature of E stigmatias (Figs. 3 1 g, h) in Puerto Rico where it damaged up to 100% of untreated corn. It was first discovered damaging corn in Miami, Florida in 1938 (Barber 1939) and north into central Florida by 1951 (Hayslip 1951). This species has become a serious pest of Florida sweet corn requiring multiple insecticide applications during the ear stage to maintain a marketable crop (Mossler 2008, Nuessly & Hentz 2004, Seal 1996, 2001, Seal & Jansson 1994) Sweet corn ( Zea mays L.) is an important crop in Florida with 22.8% of the total USA fresh market sweet corn production (Anonymous 2010a). Euxesta stigmatias also has been reported infesting sweet corn in Georgia (Daly & Buntin 2005), Texas ( Walter & Wene 1951 ), Californ ia ( Fisher 1996) Antigua (Painter 1955), and Brazil (Franca & Vecchia 1986) The insect deposits its eggs primarily on silks (styles) in the tip s of ear s. The larvae feed on silks, kernels and cobs. Bailey (1940) observed pollination disruption due to larval feeding on silks. Larvae enter through the soft pericarp of milk stage kernels to completely consume the developing embryo and endosperm (Seal & Janss o n 1989). App (1938) observed larval feeding on cobs followed by mold development resulting in si gnificant reduction in market value Several other ulidiid species are known maize pests in the Caribbean and in the Americas south of Texas ( Arce de Hamity 1986, Barbosa et al. 1986, Chittenden 1911, Diaz 1982, Evans & Zambrano 1991, Gossard 1919, Painter 1955, Wyckhuys & ONeil

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43 2007) but only one other species is currently recognized as a pest in the USA Chaetopsis massyla (Walker) (Figs. 3 1 a, b) was recently determined to be a primary pest of sweet corn in Florida (Goyal et al. 20 10 ). Evidence sugg esting the possibility of a dditional picturewinged species attacking corn in Florida include a picture of E eluta (Figs. 3 1 e, f) on the cover of Hayslips (1951) paper entitled Corn silk fly control on sweet corn misidentified as E. stigmatias Exam ination of the U lidiid ae collection at the Division of Plant Industry in Gainesville, F lorida revealed that E. eluta and E. annonae (F.) (Diptera: Ulidiidae) (Figs. 3 1 c, d) have been collected in several Florida counties since at least 1948, but these sp ecimens were not labeled as being collected or reared from corn. These later two species are recognized pests of corn in South America ( Diaz 1982; Fras L 1978). Therefore, it is possible that additional Ulidiidae species are feeding on corn in Florida. The objective of this study was to evaluate species richness and distribution of corn infesting ulidiids throughout Florida. Materials and Methods C orn grown throughout Florida was sampled for ulidiid species. Extension personnel and researchers from all 67 Florida counties provided information on corn types and growing season needed to s elect representative fields. C orn fields were visited with the assistance of extension agents. One to two c orn fields were sampled for Ulidiidae in each of sixteen coun ties from July through O ctober 2007 (Table 3 2). One to 4 corn fields were sampled for Ulidiidae in each of 27 counties during February through June 2008 (Table 33 ), including 10 counties visited in 2007 Adult ulidiids can be elusive and difficult to reliably observe and collect. They frequently avoid direct sunlight and walk or fly away from the direct line of sight of workers approaching them within fields. They are more easily collected from the tassels

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44 and upper leaves in the hour after sunrise and before sunset, but it was not possible to sample all fields at these times. Adults can also be killed after ovipositing on a plant host before they are sampled, particularly within crops that are frequently treated with insecticides, such as sweet corn. Therefore, fields were sampled for both adults and immatures to determine whether the plants served as developmental hosts for ulidiid species and to determine the viability of using adult collection records for determining ear infestation. Preference was given to sampling corn that was between the silking and dough stages because both the adult and immature stages of flies can best be collected during the first three weeks of corn reproduction. Neither adults nor immatures in ears were found in fields s ampled before silking in Lake County (sweet corn) in 2007 and in Jefferson (field corn) and Walton (sweet corn) Counties in 2008, therefore; data from these three fields were not included in the results. Sweet corn fields were preferred over field corn for sampling because the flies cause less damage in field corn than in sweet corn (Scully et al. 2000). Corn type (i.e., f ield, sweet, Bt enhanced, and standard corn) and variety number of days before or after first silk appearance, and locations of the fi eld were recorded. Visual observations were taken for the presence of ulidiid adults. Flies were collected from corn fields using a sweep net (37.5 cm diameter). The sample size was adjusted depending on the estimated field siz e A minimum of 3 and a ma ximum of 9 pairs of rows were sampled in each field. In fields 4 ha, 3 pairs of corn rows were selected for sampling: 1 pair f rom each side of the field and 1 pair in the middle of the field. In fields > 4 ha, 9 pairs of rows were selected for sampling: 1 on each side of the field, 1 in the middle of the field, and 6 pairs of rows randomly selected

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45 from between the field margins S weep net s ampling for flies was done while walking the length of the field swinging the net 100 times between two rows in each pair of selected rows. Flies were preserved in 70% ethyl al cohol for later identification and counting Identified U lidiidae specimens housed at the Division of Plant Industry, Gainesville, FL and keys of Euxesta ( Ahlmark & Steck, unpublished, Curran 1928, 1934 and 1935) and Chaetopsis (G. Steyskal unpublished) were used to confirm identities. C orn ears were examined for the presence of fly larvae in the same fields sampled with sweep nets. Ears found to contain larvae were collected and held for adult emergence to confirm species infestation. The number of ear s s ampled per field was adjusted depending on the number of planted rows in each field. Fifty six ears were examined in fields with < 90 rows and 88 ears were examined in fields with > 90 rows. In a field with < 90 rows, 10 groups of 4 plants each were r andomly selected for ear inspection. In a field with 90 rows, ears were examined in every 10th row starting from the first row and continuing to the other side of the field (total of 10 rows) In a field with > 90 rows, 6 rows were sampled from each side of the field (each sampled row separated by 10 rows), and 6 additional rows were randomly selected and sampled in the middle of the field. One ear on each of f our plants in the middle of each selected row was examined for fl y larvae (40 and 72 ears per f ield for < 90 and > 90 rows, respectively) An additional 4 plants in each corner of the field were examined for fly larvaeinfested ears (16 ears per field). The top third of each infested ear was removed using a knife and placed individually in Ziploc bag s (1.83 L S.C. Johnson & Son, Inc., Racine, WI). Two paper towels were added to each bag to reduce moisture

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46 accumulation. Bags were stored in portable coolers in the field and during transportation back to the laboratory. Fly larvae infested e ars in Ziploc bags were held in an air conditioned room maintained at 26.0 1C and L 14: D 10 h photoperiod to collect pupae and adults for identification. To reduce the accumulation of moisture and associated fungus, bags with corn were left partially open, p aper towels were changed frequently, and the air was constantly circulated using box fans. Corn ears collected on March 6, 2007 were placed collectively in 3.78 L Ziploc bag s and then transferred to plastic containers with mesh tops. Pupae were removed fr o m the bags and plastic containers and placed on moistened filter paper (Whatman 3, Whatman International Ltd, Maidstone, England) in covered Petri dishes for adult emergenc e The dishes were sealed with P arafilm (Pechiney Plastic Packaging, Chicago, IL) to reduce moisture loss. Adults that emerged were preserved in 70% ethyl alcohol for later identi fication and counting as above. Statistical Analysis The results were tested using analysis of variance to examine the effects of sample technique, corn type (field and sweet), corn ear age at sample time (17, 8 14, 1521 d) and sample year on the mean numbers of each species collected (Proc GLM, Version 9.0; SAS Institute 2008) Year was used as a random variable in the model. The mean number of flies sweep netted per pair of rows used in the data analysis was calculated for each field by dividing the total number of flies caught in sweep nets by the number of pairs of rows sampled in that field. The mean number of flies per infested ear was calculated for each field by dividing the total number of flies reared from infested corn ears by the number of infested ears in each field. Different numbers of ears and plant rows were sampled in each field and more fields were sampled in 2008

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47 than in 2007; therefore the results were presented as least square means rather than arithmetic means of flies caught per row and reared per corn ear. Results and Discussion The mean number of ulidiid adults caught in sweep nets was significantly affected by fly species, corn ty pe, survey year (Table 3 1). The mean number of ulidiid adults caught in sweep nets was also significantly affected by fly species but the results depended on year. Significantly more E. eluta (least squares mean SEM; 3.80 0.63) and C massyla (3.62 0.63) were caught in sweep nets per row than E. stigmatias (1.33 0.63) and E. annonae (0.14 0.63). More adults were caught per row using sweep nets in sweet corn (2.95 0.34) than in field corn (1.49 0.49). Sweep net counts per row were greater in 2007 (3.89 0.51) than in 2008 (0.55 0.32). The mean number of adults emerged per ear were significantly affected by fly species, corn type and survey year (Table 3 1). Significantly more E. eluta (least squares mean SEM ; 1.41 0.30) were reared from each corn ear than E. stigmatias (0.40 0.30) and E. annonae (0.05 0.30). The mean number of C. massyla per ear (0.82 0.30) was not significantly different than the other species. More adults were reared from each corn ear in 2007 (1.19 0.25) than in 2008 (0.15 0.15). Significantly more adults per ear were reared from sweet corn (1.02 0.16) than from field corn (0.33 0.24). Results for species by county and reared from fields were presented separately for 2007 (Table 3 2) and 2008 (Table 3 3) due to significant differences in mean counts between years. The correlation between adults caught in sweep nets and those reared from ears varied by species. Correlation coefficient were 0.79 ( P < 0.0001) for E. stigmatias 0.62

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48 ( P < 0.0001) for C. massyla 0.58 for ( P < 0.0001) E. annonae and 0.51 ( P < 0.51) for E. eluta. 2007 Field Survey Four Ulidiidae species were c aught in sweep nets and reared from fly larvaeinfested ears in Florida corn during the first s urve y year (Table 3 2) Chaetopsi s massyla was collected in more counties throughout the state than other species and was netted in 100% of the sampled fields. This was followed by E eluta that was netted (88% of sampled fields) in all except Lake and Lee Counties (Table 3 2 ). Euxesta annonae and E. stigmatias were netted from only three counties in central and southern Florida: Miami Dade, Okeech obee, and Palm Beach Counties. As a result of the more limited distribution, both E. annonae and E. stigmatias were netted in only 18% of fields sampled. The species netted and reared varied by corn type. Adults of E. annonae and E. stigmatias were netted only from sweet corn fields in Miami Dade, Okeechobee, and Palm Beach Counties but field corn was not sampled in these counties (Table 3 2 ). A dults of E. eluta and C. massyla were nett ed from both fiel d and sweet corn fields throughout the state Euxesta eluta and C. massyla were netted from 50 and 100% of field corn fields, respectively, while both species w ere netted from 100% of sweet c orn fields. The percentage of ulidiidinfested ears ranged from 5% in Escambia to 38% in Santa Rosa County (Table 3 2). Euxesta eluta and C. massyla were reared from ears collected from all but Lee, Lake and St. Johns Counties. These two species were rea red from infested ears in 82% of corn fields statewide. Euxesta annonae and E. stigmatias were reared only from corn ears collected from Miami Dade, Okeechobee, and Palm Beach C ounties amounting to only 18% of fields sampled Adults of E. eluta

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49 and C. m assyla were reared from both field and sweet corn ears E uxesta annonae and E. stigmatias emerged from sweet corn ears in fields from Miami Dade, Okeechobee, and Palm Beach Counties, but field corn fields were not sampled in these counties Euxesta eluta and C. massyla were each reared from 50% of field corn and 92% of sweet corn fields. Euxesta annonae and E. stigmatias were reared from 100% of the sweet corn fields in above mentioned Counties The a ge of sampled corn in 2007 ranged from 4 to 21 d after first silk appearance (Table 3 2) Euxesta annonae and E. stigmatias were sweep net ted from fields 8 to 21 d after first silk appearance, but no fields < 8 d after first silk appearance were sampled in counties infested with these two species. Euxesta eluta and C. massyla emerged from corn ears collected from fields 4 to 21 d after first silk appearance, while E. annonae and E. stigmatias from ears collected 8 to 21 d after first silk appearance (Table 3 4). Corn ears were not collected from fields < 8 d after first silk appearance in counties with E. annonae and E. stigmatias 2008 Field Survey The same four species were again collected in sweep nets and reared from fly larvae infested ears in Florida corn during the second study year (Table 3 3). U lid iid adults were netted in 23 of 27 counties sampled in 2008. No adult picturewinged flies were captured in corn in Dixie, Jackson, Sumter, Taylor or Volusia Counties. Chaetopsis massyla was collected from more counties than other species throughout the state and was netted in 66% of the fields sampled. Euxesta eluta was netted in 49% of the fields sampled. Chaetopsis massyla was the only species collected from corn in Alachua, Jefferson and Marion Counties, while E eluta was the lone species collected from corn in Okaloosa County. Euxesta stigmatias was netted only in Martin,

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50 Okeechobee, Palm Beach and St. Lucie Counties amounting to only 11% of the fields sampled. Euxesta annonae was not collected in sweep samples in 2008. Euxesta eluta, E. stigmat ias and C. massyla were netted from field and sweet corn fields (Table 3 3). Euxesta eluta were netted from 27 and 59%, and C. massyla from 40 and 78% of the field and sweet corn fields throughout the state, respectively. Euxesta stigmatias was caught fr om 100 and 67% of the field and sweet corn fields, respectively in Martin, Okeechobee, Palm Beach, and St. Lucie Counties. No E. annonae adults were netted in field or sweet corn fields. Ulidiid infested ears were found in 13 of 27 counties sampled (Table 3 3). The percentage of ulidiid infested ears ranged from 2% in Volusia to 39% in Lake County. Only E. eluta were reared from corn ears collected from Alachua, Jefferson and Walton Counties. Chaetopsis massyla was the only species reared from corn ears collected from Volusia County Euxesta eluta and C. massyla were reared from 32% and 28% of the corn fields sampled throughout the state. Euxesta eluta were reared from 20 and 38% and C. massyla from 13 and 34% of the field and sweet corn fields, respec tively throughout the state. Euxesta stigmatias was only reared from infested sweet corn ears in Martin and Palm Beach C ounties amounting to 6% of the total fields sampled. Adults of E. annonae were only reared from infested sweet corn ears collected fro m Palm Beach County amounting to approximately 2% of the total fields sampled. Field corn fields were not sampled in the counties where E. stigmatias and E. annonae were reared from ears. The age of corn ears in surveyed fields ranged from 1 to 21 d after first silk appearance (Table 3 3). More E. eluta and C. massyla were caught in sweep nets and

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51 reared from corn ears 15 to 21 d old compared to 0 to 14 d old. More E. stigmatias were caught in sweep nets and reared from corn ears in fields with 15 to 21 d old ears than in younger fields. In counties where E. annonae was found, it was only reared from fields sampled 15 to 21 d after first silk appearance The results of this twoyear study confirmed that several species of Ulidiidae flies were infesting c orn in Florida. Ulidiidae flies were found infesting both sweet and field corn fields across the Florida panhandle from Escambia to Nassau Counties and south through the peninsula to Miami Dade County. Flies were collected in sweep nets or reared from corn ears from 29 out of 33 sampled counties during the two survey years (Fig. 3 2). Flies were more common in the 2007 compared to 2008 surveys probably due to differences in sampling times. Corn fields in 2007 were largely sampled from August to October, except for Miami Dade County that was sampled in March. In contrast, surveys were conducted from February to June in 2008. The flies may be more common in midsummer through fall months in northern Florida. While more research has been conducted on E st igmatias than the other species, it was found to be much less common than C. massyla and E. eluta in this survey. Euxesta eluta and C. massyla were distributed in most fields sampled throughout the state in both years, while E. stigmatias and E. annonae w ere found in only several counties of southern Florida (Martin, Miami Dade, Okeechobee, Palm Beach, and St. Lucie) The distribution of alternate host plants and differences in acceptable temperature ranges for each species may explain some of the variation present in the distribution of ulidiids infesting corn in Florida. Euxesta eluta, E. stigmatias and C. massyla were collected from both field and sweet corn, while E. annonae was collected only from

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52 sweet corn fields. Sweet corn is mostly grown in sou thern Florida in comparison to northern Florida where field corn predominates (Anonymous 2008a ). However, E. stigmatias was not collected or reared from sweet corn fields in northern Florida. Fras L (1978) in Chile found that higher temperature and lower relative humidity led to greater numbers of E. annonae while the reverse led to greater numbers of E. eluta S ampling using both sweep nets and collecting infested corn ears gave a more complete picture of fly distribution in Florida corn fields than eit her sampling technique alone Low correlation values indicate that sweep netting is not an efficient method to estimate ulidiid species infesting corn ears. The relationship between sweep nets and fly species that emerged from infested ears accounted for > 60% of the variation for E. stigmatias and C. massyla but < 60% for E. eluta and E. annonae. There were also a few locations where flies were observed but not collected with sweep nets. These were the places where flies were uncommon (1 or 2 per site) and netting was not the best sampling technique for insects at low densities. Seal et al. (1996) found that E. stigmatias congregated on the top of plants in late evening. Fly species in our study may have been more active or more accessible with nets at times of the day when sampling was not possible. Therefore, sweep netting can be used to indicate the potential for ear infestation, but identifying adults reared from infested ears is currently the only method available for differentiating species wit hin ears. The external physical characteristics of the immature stages of Ulidiidae infesting Florida corn are currently being examined by the authors to determine the possibility of using eggs, larvae or pupae for the identification of flies infesting co rn.

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53 E uxesta eluta E. stigmatias and C. massyla were collected from corn throughout the corn reproductive stage. Adult E. annonae may be present in fields during the first week of silking, but only fields > 8 d after first silk appearance were sampled in counties where the species was found. In general, there was a tendency for greater infestation by all four species as sweet corn ears neared harvest and as field corn ears approached the dough stage. These flies have also been reared from other parts of the corn plant that effectively expands the potential host period on this crop. The authors have frequently reared E. eluta E. stigmatias and C. massyla from tassels and stems of corn plants. T his is the first report of E. annonae infesting corn in Florida and the USA This species was not common in any location and was always netted from fields and reared from ears along with other Ulidiidae species. Euxesta annonae was the least collected species in sweep nets and it was r eared from corn ears collected only from the southern end of the Florida peninsula (Fig. 3 2). Euxesta annonae is also reported as a pest of corn in Chile ( Fras L 1978). The authors have frequently observed E. annonae on Annona spp. (Magnoliales: Annonaceae) and Chinese long bean, V igna sesquipedalis (Fabales: Fabaceae) in southern Florida and reared E. annonae adults from field collected Annona spp. fruit (Magnoliales: Annonaceae) Plants of Annona spp. are recorded in several southern and central Florida counties (Brevard, Broward, Collier, De Soto, Glades, Hendry, Highlands, Indian River, Lee, Manatee, Martin, Miami Dade, Monroe, Palm Beach, and St. Lucie) (Wunderlin & Hansen 2008) where they may provide alternative food plants for this species. The authors have also reared this

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54 species from decaying corn stalks and from spiny amaranth, Amaranthus spinosus L. (Caryophyllales: Amaranthaceae) roots collected from the field at Belle Glade, Florida. E uxesta eluta was widely collected in this study from fields sampled throughout Florid a (Fig. 3 2). These flies were commonly observed in fields and as many as 62 were reared from an individual ear. While this is the first known record of E. eluta being a pest of corn in Florida and the USA, its image in Hayslip (1951) suggests that it was present in Florida corn fields > 50 yr ago, but incorrectly identified as E. stigmatias The wide distribution of E. eluta in Florida and its discovery on both sweet and field corn indicates this fly is a much greater threat to corn than E. stigmatias which is found in a much smaller portion of Florida. Euxesta eluta was recognized as infesting corn in Puerto Rico > 60 yr ago (Wolcott 1948) and has been recorded as an ear pest in Ecuador (Evans & Zambrano 1991), Chile ( Fras L 1978, Olalquiaga 1980), P eru (Diaz 1982), Argentina (Arce de Hamity 1986), and Brazil (Franca & Vecchia 1986). Euxesta eluta is a pest of loquat Eriobotrya japonica (Thumb.) Lindl. (Rosales: Rosaceae) in Alachua County, Florida (Anonymous 2008b). Loquat is grown as a dooryard plant and is distributed in several counties throughout the state (Wunderlin & Hansen 2008). Euxesta stigmatias was found in sweep net collections and reared from corn ears from southern and central Florida counties only (Fig. 3 2). Weather differences in southern and northern Florida may explain part of the variation in distribution of the species. Adult E. stigmatias have been reared from damaged or decayed inflorescences of sorghum, Sorghum bicolor ( L .) Moench (Cyperales: Poaceae), tomato fruit, Lycoper sicon esculentum L. (Solanales: Solanaceae) (Seal & Jansson 1989), and decaying carrot roots, Daucus carota L. (Apiales: Apiaceae) (Franca & Vecchia 1986).

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55 Chaetopsis massyla was caught in sweep nets and reared from corn ears in the majority of surveyed counties (Fig. 3 2). This fly was common in field and sweet corn fields throughout the year in southern Florida counties. The relative abundance and development range across corn types indicates this species is a much greater threat to Florida corn than pr eviously recognized. Its habit of feeding on a range of monocots may help explain its widespread distribution throughout Florida. Allen & Foote (1976) reported it to be a secondary invader of wetland monocots. Chaetopsis massyla has been reared from cat tail, Typha spp. (Typhales: Typhaceae) in California (Keiper et al. 2000) Typha spp. is found in most Florida counties except Flagler, Gadsden, Glades, Hernando, and Suwannee (Wunderlin & Hansen 2008). The authors made several personal observations of C massyla plant associations during the course of this statewide survey. Chaetopsis massyla was frequently observed by the authors on cattail plants on ditch banks and feeding on sugary exudates from sugarcane plants (a complex hybrid of Saccharum spp.) i n Belle Glade (Palm Beach County). Chaetopsis massyla adults were reared from sugarcane stems that were actively infested with the sugarcane borer, Diatraea saccharalis (F). (Lepidoptera: Crambidae) collected by the authors in November 2009 from sugarcane fields at Clewiston (Hendry County) and Sebring (Highlands County), Florida. Chaetopsis massyla was also successfully reared by the authors from otherwise healthy sugarcane stems exposed to colonies in the laboratory in which 0.5 cm diam holes were drill ed in billets to simulate emergence and frass evacuation holes produced by D saccharalis. Other plants from which C. massyla has been reared include hairy sedge, Carex lacustris Willd. (Cyperales: Cyperaceae) (Allen & Foote 1992), Narcissus spp. (Liliale s: Liliaceae) (Blanton 1938) and onions,

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56 Allium cepa L. (Asparagales: Alliaceae) (Merrill 1951). Carex spp. is found throughout the state while the distribution of Narcissus spp. is considered to be limited to Alachua, Calhoun, Escambia and Leon Counties (Wunderlin & Hansen 2008). Two additional Chaetopsis spp. have been reported feeding on corn, but neither was found in this two year survey of Florida corn fields. Large populations of fly larvae that were discovered in corn stalks within tunnels likely produced by European corn borer, Ostrinia nubilalis Hbner (Lepidoptera: Pyralidae) in Ohio were reared to adults and identified as Chaetopsis aenea (Wiedemann) by Gossard (1919). Larvae of C. fulvifrons (Macquart) were reared from within the tunnels of southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), in the Texas high plains (Knutson 1987). Langille (1975) reported that Chaetopsis spp. larvae were commonly associated with diapausing D. grandiosella within corn stalks in Missou ri and hypothesized that ulidiid larvae feed on the decaying stalks or microbial growth within the bored stalks. In conc lusion, four species of picturewing ed flies were found infest ing corn in Florida. Evidence presented herein is the first known documentation for E. annon a e and E. eluta as pests of corn in Florida and the USA. The four species were not uniformly distributed throughout Florida corn growing regions. E uxesta eluta and C. massyla were found infesting field and sweet corn throughout Florida. Euxesta stigmatias was only found infesting corn in Martin, Miami Dade, Okeechobee, Palm Beach, and St. Lucie Counties. Euxesta annonae (F.) was found in sweet corn in Miami Dade, Okeechobee, Palm Beach Counties, but field corn was not sampled in these counties. Euxesta eluta, E. stigmatias and C. massyla were collected from corn throughout the corn reproductive stage. Euxesta annonae was reared from 821 d old

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57 ears only, but fields with ears < 8 d old were not sampled in the counties where this speci es was found. The relative abundance of E. eluta and C. massyla in Florida field and sweet corn indicates the need for more research into their biology and ecology. The discovery of E. eluta and C. massyla attacking corn ears in many of the northernmost Florida counties suggests that further surveys of corn growing areas across the borders in to neighboring states is warranted to determine the extent of corn infesting picturewinged fly infestations in the southern U.S The statewide distribution of E. e luta and C. massyla in reproducing corn also suggests that additional studies should be conducted to evaluate additional food sources that support these species in the absence of corn.

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58 Table 3 1 A nalysis of variance for sweep net caught flies and corn r eared flies on species, corn type, age of corn and year Sweep net Corn ears Source df F P F P Species 3 8.12 < 0.0001 3.73 0.0120 Corn type 1 6.77 0.0099 6.53 0.0113 Age of corn 2 1.82 0.1638 1.09 0.3368 Year 1 33.17 < 0.0001 13.82 0.0003 Species corn type 3 0.68 0.5642 1.52 0.2091 Species age of corn 6 0.31 0.9304 0.67 0.6739 Species year 3 5.50 0.0011 2.00 0.1145 ANOVA (Proc GLM, SAS Institute 2008), denominator df = 236.

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59 Table 3 2. Ulidiidae species collected in fields or reared from infested ears in Florida, 2007. County field no. Corn type a Sample date Ear age (d) b No. rows sampled with sweep net Mean no. adults captured per 100 sweeps c No. ears sampled (no. infested) Mean no. adults emerged per ear (per infested ear) E. anno nae E. eluta E. stigmatias C. massyla E. annonae E. eluta E. stigmatias C. massyla Alachua Swt 16 Aug 15 21 3 0.0 1.8 0.0 4.2 56 (6) 0 0.9(8.3) 0 1.4 (13.5) Bradford Swt 17 Oct 15 21 9 0.0 0.9 0.0 2.9 88 (14) 0 0.7(4.4) 0 1.6 (9.9) Miami Dade Swt 6 Mar 1521 3 2.8 26.0 11.2 11.7 56 (16) 0.4(1.3) 17.5 (61.3) 5.6 (19.4) 5.9 (20.7) Escambia 1 Fld 2 Aug 8 14 3 0.0 11.5 0.0 3.8 56 (5) 0 0.2(2.2) 0 0.3 (3.2) Escambia 2 Bt swt 2 Aug 7 9 0.0 5.8 0.0 6.9 56 (3) 0 0.3(6.3) 0 0.6 (10.3) Gadsden Swt 17 Sep 7 3 0 .0 6.3 0.0 4.8 56 (6) 0 1.1(10.3) 0 1.0 (9.3) Holmes Swt 16 Oct 1521 3 0.0 3.7 0.0 2.3 56 (11) 0 0.6(3.2) 0 0.6 (2.8) Lake Fld 14 Sep 4 5 9 0.0 0.0 0.0 3.2 56 (0) 0 0 0 0.0 (0.0) Lee Fld 17 Oct 15 21 3 0.0 0.0 0.0 1.5 56 (0) 0 0 0 0.0 (0.0) Liberty F ld 13 Sep 15 21 3 0.0 2.3 0.0 1.8 56 (4) 0 0.3(4.3) 0 0.5 (7.3) Marion Swt 4 Sep 8 14 3 0.0 16.5 0.9 26.0 56 (11) 0 4.2(21.2) 0 7.1 (36.3) Okeechobee Swt 18 Sep 8 14 3 1.1 0.9 1.0 2.9 88 (6) 0.1(1.3) 0.6(8.3) 0.3(4.3) 0.8 (12.3) Palm Beach Swt 14 Nov 8 14 3 1.7 21.3 33.5 18.3 56 (17) 1.7(5.5) 5.8(19.1) 8.8(29.1) 1.8 (5.9) Santa Rosa Swt 3 Aug 8 14 3 0.0 3.7 0.0 8.3 56 (21) 0 4.5(12.1) 0 1.1 (3.0) St. Johns Swt 13 Sep 1521 3 0.0 10.7 0.0 11.3 56 (0) 0 0 0 0.0 (0.0) Suwannee Swt 13 Sep 15 21 3 0.0 1 1.5 0.0 2.7 56 (12) 0 2.8(13.2) 0 4.6 (21.3) Washington Swt 2 Aug 15 21 3 0.0 4.3 0.0 3.3 88 (16) 0 11.3(62.3) 0 3 (16.3) aCorn type: Fld = field corn; Swt = sweet corn; Bt swt = Bacillus thuringiensis enhanced sweet corn. bEstimated days after first sil k appearance at time of sampling. c0 = no flies were detected in sweep nets.

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60 Table 3 3. Ulidiidae species collected in fields or reared from infested ears in Florida, 2008. County field no. Corn type a Sample date Ear age (d) b No. rows sampled with sweep net Mean no. adults captured per 100 sweeps c No. ears sampled (no. infested) Mean no. adults emerged per ear (per infested ear) E. annonae E. eluta E. stigmatias C. massyla E. annonae E. eluta E. stigmatias C. massyla Alachua Swt 4 Jun 7 3 0.0 0. 0 0.0 1.3 56 (8) 0 0.2(1.4) 0 0 Bradford 1 Swt 23 Jun 18 21 3 0.0 2.3 0.0 8.7 56 (15) 0 3.3(12.3) 0 1.1(4.3) Bradford 2 Swt 23 Jun 1014 3 0.0 3.3 0.0 5.7 56 (17) 0 10.2(33.5) 0 0.4(1.5) Columbia Swt 24 Jun 8 14 9 0.0 1.1 0.0 2.2 88 (2) 0 0.1(2.5) 0 0.05(2.0) Dixie Fld 4 Jun 2 3 9 0.0 0.0 0.0 0.0 88 (0) 0 0 0 0 Gilchrest 1 Swt 23 Jun 15 21 9 0.0 0.7 0.0 1.3 88 (6) 0 0.6(9.3) 0 0.8(11.3) Gilchrest 2 Bt fld 23 Jun 7 9 0.0 0.0 0.0 2.1 88 (4) 0 0.1(2.3) 0 0.2(5.3) Gilchrest 3 Bt fld 23 Jun 7 9 0.0 1. 8 0.0 0.8 88 (0) 0 0 0 0 Hamilton 1 Swt 24 Jun 7 3 0.0 1.3 0.0 1.3 56 (0) 0 0 0 0 Hamilton 1 Fld 24 Jun 14 3 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Hendry Swt 26 Feb 1521 9 0.0 4.6 0.0 1.4 88 (18) 0 2.7(13.3) 0 4.4(21.4) Holmes 1 Swt 26 Jun 21 3 0.0 8.3 0.0 0 .7 56 (0) 0 0 0 0 Holmes 2 Fld 26 Jun 21 9 0.0 2.1 0.0 2.9 88 (9) 0 0.4(3.4) 0 0.1(1.4) Jackson 1 Bt fld 5 Jun 2 3 9 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Jackson2 Bt fld 5 Jun 2 3 9 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Jefferson 1 Fld 25 Jun 8 14 9 0.0 0.0 0.0 0. 0 88 (0) 0 0 0 0 Jefferson 1 Swt 25 Jun 14 3 0.0 0.0 0.0 0.7 56 (10) 0 0.4(2.4) 0 0 Lafayette 1 Swt 24 Jun 14 3 0.0 1.0 0.0 1.3 56 (0) 0 0 0 0 Lafayette2 Swt 24 Jun 14 3 0.0 0.0 0.0 1.7 56 (0) 0 0 0 0 Lafayette 3 Fld 24 Jun 14 9 0.0 0.0 0.0 0.0 88 (0) 0 0 0 0 Lake 1 Swt 6 Jun 21 9 0.0 0.0 0.0 5.2 56 (0) 0 0 0 0 Lake 2 Swt 6 Jun 21 9 0.0 2.0 0.0 1.8 56 (22) 0 1.7(4.4) 0 4.1(10.4) Marion Bt fld 3 Jun 14 3 0.0 0.0 0.0 56 (0) 0 0 0 0 Martin 1 Swt 11Mar 7 3 0.0 3.0 1.0 1.3 56 (0) 0 0 0 0 Martin 2 Swt 11 Mar 1 2 3 0.0 0.0 0.0 0.0 56 (9) 0 0.4(2.3) 3.1(19.0) 0.5(3.3) Martin 3 Swt 11 Mar 1 2 9 0.0 0.0 0.0 0.9 88 (0) 0 0 0 0 Martin4 Swt 11 Mar 14 3 0.0 0.0 14.3 4.7 56 (8) 0 0 0.5(3.4) 0 Nassau 1 Swt 23 Jun 15 21 3 0.0 0.0 56 (0) 0 0 0 0 Nassau 2 Fld 23 Jun 15 21 9 0.0 0.0 0.0 0.0 88 (0) 0 0 0 0

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61 Table 3 3. C ontinued. County field no. Corn type 1 Sample date Ear age (d) 2 No. rows sampled with sweep net Mean no. adults captured per 100 sweeps 3 No. ears sampled (no. infested) Mean no. adults emer ged per ear (per infested ear) E. annonae E. eluta E. stigmatias C. massyla E. annonae E. eluta E. stigmatias C. massyla Okaloosa 1 Swt 5 Jun 5 3 0.0 2.0 0.0 0.0 56 (0) 0 0 0 0 Okaloosa 2 Swt 5 Jun 10 3 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Okeechobee S wt 19 Apr 14 9 0.0 3.6 7.2 1.9 88 (0) 0 0 0 0 Palm Beach Swt 10 Apr 15 21 3 0.0 11.3 20.7 12.7 56 (15) 0.9(3.2) 3.3(12.3) 1.1(4.3) 2.5(9.2) Santa Rosa 1 Bt swt 5 Jun 10 3 0.0 0.0 0.0 1.7 56 (16) 0 16.1(56.4) 0 0.4(1.4) Santa Rosa 2 Swt 5 Jun 14 3 0.0 0.0 56 (0) 0 0 0 0 Santa Rosa3 Bt swt 5 Jun 1521 3 0.0 2.3 0.0 56 (0) 0 0 0 0 St. Johns 1 Bt swt 6 Jun 8 14 3 0.0 6.3 0.0 56 (0) 0 0 0 0 St. Johns 2 Bt swt 6 Jun 14 3 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 St. Lucie Bt fld 29 May 14 9 0.0 0.0 4.1 5.7 56 (0) 0 0 0 0 Sumter Swt 6 Jun 1521 3 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Suwannee Fld 4 Jun 15 21 3 0.0 1.0 0.0 1.0 56 (0) 0 0 0 0 Taylor Fld 25 Jun 15 21 3 0.0 0.0 0.0 0.0 56 (0) 0 0 0 0 Volusia Swt 6 Jun 21 3 0.0 0.0 0.0 0.0 56 (1) 0 0 0 0.02(1.0) Wa lkulla Swt 25 Jun 14 3 0.0 2.0 0.0 56 (6) 0 0.3(3.2) 0 0.3(2.3) Walton Fld 26 Jun 14 9 0.0 1.8 0.0 0.0 88 (4) 0 0.1(2.3) 0 0 Walton Swt 26 Jun 15 21 3 0.0 0.0 2.0 56 (0) 0 0 0 0 Walton Swt 26 Jun 15 21 9 0.0 0.0 0.0 0.0 88 (0) 0 0 0 0 aCorn type: F ld = field corn; Swt = sweet corn; Bt swt = Bacillus thuringiensis enhanced sweet corn. bEstimated days after first silk appearance at time of sampling. c0 = no flies were detected in sweep nets; = fly species was observed only, not collected.

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62 Table 3 4. Percentage of fields with Ulidiidae species sweep netted or reared from infested ears by ear age. Species Sweep netted Reared from corn ears Ear age (d) Ear age (d) 0 to 7 d 8 to 14 d 15 to 21 d 0 to 7 d 8 to 14 d 15 to 21 d 2007 E. a nnonae 0 100 100 0 100 100 E. eluta 67 100 89 67 100 78 E. stigmatias 0 100 100 0 100 100 C. massyla 100 100 100 67 100 78 2008 E. annonae 0 0 0 0 0 100 E. eluta 36 42 64 27 32 35 E. stigmatias 33 100 100 33 33 100 C. massyla 54 68 71 18 21 4 1

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63 Figure 31. Adults of four species of picturewinged flies Chaetopsis massyla male (a) and female (b); Euxesta annonae male (c) and female (d); E. eluta male (e) and female (f); E. stigmatias male (g) and female (h)

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64 Figure 32. Distributio n of Ulidiidae species infesting corn in Florida by county during the 20072008 surveys. Symbols in figure represent species collected using sweep nets and reared from corn ears in each of the sampled (shaded) counties.

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65 CHAPTER 5 SPATIAL AN D TEMPORAL DI STRIBUTION OF CORN INFESTING PICTURE WINGED FLIES IN CORN FIELDS Current research has found four species of flies, C. massyla E. annonae E. eluta and E. stigmatias attacking corn in Florida. Insecticides of various chemistries are applied beginning at the silking stage of sweet corn to manage these flies. No published economic thresholds are known for any of the fly species. In the absence of economic thresholds and sampling plans, insecticides are sprayed as frequently as daily to kill the flies bef ore they can deposit eggs in the ears. Sampling by growers and corn scouts is done based on personal experience. Moreover, information on spatial and temporal dis tribution and movement of flies required to develop sampling plans and economic thresholds i s not available. Seal and Jansson (1989) reported that the E. stigmatias populations increase through movement of adults from neighboring fields, or by reproduction in the field itself. Mostly anecdotal information indicates that adults of these species q uickly reenter treated fields. Seal (2001) demonstrated that E. stigmatias re colonized insecticidetreated experimental fields by showing that > 19.9 maggots per ear were found at harvest after insecticide applications every 34 d. Seal et al. (1996) earlier found that E. stigmatias abundance increased with ear age up to 3 wk after tassel emergence. Information on the spatial distribution of flies in corn fields may help growers to direct their control measures towards the areas with flies rather than s praying entire corn fields. Similarly, knowledge on the temporal distribution of the flies may help growers to optimize insecticide sprays. Moreover, this information will be helpful in developing sampling plans and economic thresholds for these flies. Therefore, the current study

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66 was conducted to evaluate the spatial and temporal distribution of these ulidiid flies in corn fields. Materials and Methods The distribution of ulidiid species was examined in small experimental plots (1.8 ha) and in large scale commercial (4 16 ha) fields of sweet corn (Table 4 1). The small scale fields were sampled in May and December 2008 at the EREC. The largescale commercial corn fields were sampled in May 2008, May 2009 and May 2010 in Belle Glade, Pahokee and South Bay, Florida, respectively Sampling was initiated in corn fields at first silk appearance. Double sided, yellow sticky traps (7.5 cm x 12.5 cm, Great Lakes IPM, Inc., Vestaburg, MI) were used to sample adult ulidiid species. Sticky traps were chosen for this research over other available methods (e.g., sweep nets, visual observations) due to their advantage of continuous capture throughout the day during the 3wk periods. Preliminary trials with the manufactured cards determined that they caught flies f or up to 6 d before they needed to be replaced. The cards were replaced more frequently if the effective surface area of the traps was reduced by blowing soil, insects, bird feathers, or leaves. The traps were attached to 1.0 to 1.5 m bamboo sticks inser ted upright into the soil in the planted rows between corn plants or attached directly to corn stalks using a small or medium binder clip. The position of the trap was adjusted as necessary to maintain a height midway between the top of the ear and the tassel The sticky cards were examined during the afternoon hours daily or whenever reentry to insecticidetreated fields was allowed. Flies trapped on both sides of the sticky traps were identified to species and counted. Trap counts were recorded for each trap separately for later use in examining distribution in each field at each date. Rather than replacing the traps at

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67 each sample date, trapped flies were crushed on the card to avoid confusion with counting the same flies on the next sampling date. The numbers of traps placed in each field was based on field size and the number and length of rows in each field (Table 4 1). Traps were placed in a total of 5 11 rows in each field. Sweet corn in southern Florida is planted on 76.2 cm (30 in) centers. Traps were always placed in the first and last rows in each field with traps in an additional 39 rows between the two field edges. The distance between rows with traps varied from 2040 rows (1530 m) depending on the size of the field. Traps were plac ed on each end of the field and along the trap rows at 1530 m intervals resulting in 828 traps per row depending on the field length. Therefore, the traps were equidistant apart between and within trap rows across the entire field. The small and largescale fields were bordered by crops (sugarcane, corn) or barren fields or residence (Table 4 2). Corn ears were sampled for ulidiid larvae and pupae infestation at approximately 3 wk after first silk appear a nce Two corn ears on two plants on each side of the trap along the row were examined (i.e., four ears at each trap). The ears were examined for the presence of fly immatures by partially lifting the ends of the corn husk away from the silk channel to avoid the loss of larvae and pupae from ears. Ears found with immature stages were collected and placed in separate Ziploc bags (1.83 L). B ags were stored in portable coolers in the field and during transportation back to the laboratory. Fly larvae infested e ars in Ziploc bags were held in an air condit ioned room maintained at 26.0 1C and L 14: D 10 h photoperiod to collect pupae and adults for identification. To reduce the accumulation of moisture and associated fungus, bags with ears were left partially open, p aper towels were changed frequently, and the air was constantly circulated

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68 using box fans. Pupae were removed fr om the bags and placed on moistened filter paper in covered Petri dishes for adult emergenc e The dishes were sealed with P arafilm to reduce moisture loss. Adults that emerged were preserved in 70% ethyl alcohol for later identi fication and counting Identified specimens housed at the Division of Plant Industry, Gainesville, FL and keys of Euxesta ( Ahlmark & Steck, unpublished; Curran 1928, 1934 and 1935) and Chaetopsis (G. Steyskal unpublished) were used to confirm identities. Statistical Analysis The number of flies captured on traps varied considerably in each field during the 3 wk sampling period. The data were used to determine the distribution of ulidiid flies in each field at each date. Iwao (1968) found Morisitas index as superior in measuring aggregation tendency as it is not affected by the difference in mean density. Morisitas index has been used to determine the dispersion of the oriental fruit fly, D acus dorsalis Hen del and melon fly, Dacus cucurbitae Coquillett (Diptera: Tephritidae) on Kauai, Hawaii (Vargas et al. 1989). Therefore, I measure d the dispersion of flies at each sampling date using Morisitas index, I. Morisitas index was calculated as I = n i(xi1)/N(N 1) where n = number of samples, xi = number of flies on each trap, and N = total number of flies on all traps (Davis 1993). The value of I indicates the estimated dispersion: if I >1, then individuals are aggregated; if I = 1, then individuals are distributed randomly; if I < 1, then individuals are distributed uniformly. The level of aggregation increases with increasing values of I > 1 (Khaing et al. 2002). The Z test of significance was used to determine if the sampled population signific antly differed from random (Davis 1993).

PAGE 69

69 The trap counts were further analyzed to look for differences in overall patterns of fly density along the field edges compared to the center of the fields and to examine whether the types of surrounding fields had a significant effect on trap counts. Proc MIXED (SAS institute 2008) was used to conduct an overall analysis of variance of the results of trap fly counts. Field, fly species, date after first silk appearance (das), and their interactions were used as independent variables. The mean number of flies per sticky trap on the traps present on the outermost one row of traps on each of four sides of the field (edge) were compared to mean number of flies caught on rest of the traps in the field ( Proc GLM SAS I n stitute 2008) Data were analyzed separately by field due to differences in field size and type of surroundings that bordered the fields. Data on mean total number of flies of each species per trap were also compared among different types of borders for each field (Proc GLM, SAS Institute 2008). The middle trap row of fields with odd number of trap rows was not included in the data analysis when comparing different types of borders. The effect of mean total number of flies per sticky trap on the proporti on of infested e a rs was tested by linear regressions using the model y = bx + a, where y is proportion of infested ears, x is mean number of flies per sticky trap for a corn field, and a and b are the regression parameters obtained from the regression (Pro c REG SAS Institute 2008) The proportion of infested ears was calculated by dividing the number of infested ears in a field by the total number of sampled ears. Results and Discussion The number of flies caught per trap w as affected by field ( F = 78.60; df = 8, 18000; P < 0.0001), fly species ( F = 80.14; df = 2, 18000; P < 0.0001), and date after first silk appearance (das) (F = 28.81; df = 26, 18000; P < 0.0001) The number of flies

PAGE 70

70 caught per trap w as also affected by field but the results depended on fly species ( F = 97.38; df = 16, 18000; P < 0.0001), by field with results depending on das ( F = 27.24; df = 32, 18000; P < 0.0001), and by fly species with results depending on das ( F = 8.89; df = 52, 18000; P < 0.0001). As a result of the significant interactions within and among main effects, results are presented separately by field (smallscale and large scale fields) and by species All three species were captured by sticky traps in all but one of the fields intensively sampled with sticky traps during the threeyear study. Euxesta stigmatias was much less common in 2009 and 2010 than in 2008 in all fields and it was not captured in any trap placed in large scale field 3 in 2010. Flies differed in their mean counts and their distribution within f ields with the ear age. The distribution of flies between field center and field edge for any of the fields did not follow a single pattern at different sampling dates. Therefore, the number of flies caught was summed over the entire season to calculate the mean total number of flies per trap. Small Scale F ields Field 1. The mean number of flies in this field varied from 16 per sticky trap per sample date. The mean trap counts varied from 0 4 for C. massyla ( m ean SEM, 0.18 0.02), 0 5 for E. eluta ( 0.29 0.03) and 021 for E. stigmatias (1.67 0.09). The Morisitas index calculation showed that fly distribution was aggregated at 8 of the 10 sampling dates (Table 4 3 ). Flies were uniformly distributed at 6 d after first silk appearance (d as ) and randomly distributed at 7 das. The aggregation decreased and increased with sample dates with no apparent pattern relative to spray dates in this field. In evaluating the effect of distance into field on mean total fly counts, it was determined that the d istance into field affected the mean total number of flies per sticky

PAGE 71

71 trap for E. stigmatias ( F = 4.57 ; df = 1, 48; P = 0.0378) and species total ( F = 6.13; df = 1, 48 ; P = 0.0169) only and not for C. massyla ( F = 1.29 ; df = 1, 48; P = 0.2623) or E. elu ta ( F = 3.78; df = 1, 48; P = 0.0578 ) Significantly more E. stigmatias were caught on traps at the edge compared to in the center of field (Table 45). The species total followed the same pattern as that of E. stigmatias In determining the effect of ty pe of field border on mean number of flies it was found that there was no significant effect of type of border (sugarcane and fallow) on mean total number of C. massyla ( F = 0.05; df = 1, 42; P = 0.8203 ) E. eluta ( F = 0.44 ; df = 1, 42; P = 0.5098 ) E. st igmatias ( F = 0.48 ; df = 1, 42; P = 0.4931) and species total ( F = 0.61; df = 1, 42 ; P = 0.4391) per trap. Field 2. The mean number of flies in this field varied from 04 per sticky trap per sample date. The mean trap counts varied from 02 for C. massyl a (0.14 0.02), 011 for E. eluta (0.29 0.08), and 010 for E. stigmatias (1.30 0.08). The Morisitas index calculation showed that fly distribution was aggregated at 10 of 11 sampling dates (Table 43). Flies were aggregated at 4 and 5 das, but bec ame uniformly distributed at 6 das before the insecticide treatment later that day. The fly distribution again became aggregated at 7 das. The level of aggregation varied between 7 and 16 das showing no pattern with ear age or insecticide application. In determining the effect of distance into field on mean total number of flies it was found that the distance into field affected the mean total number of flies per sticky trap for E. stigmatias ( F = 8.34; df = 1, 38 ; P = 0.064) and species total ( F = 8.04 ; df = 1, 38; P = 0.0073) only and not for C. massyla ( F = 0.02 ; df = 1, 38; P = 0.8901 ) and E. eluta ( F = 3.68; df = 1, 38 ; P = 0.0625) Significantly more E. stigmatias were caught on traps

PAGE 72

72 at the edge of field compared to on rest of the traps (Table 4 5). The species total followed the pattern same as that of E. stigmatias In determining the effect of type of field border on mean fly counts, it was found that there was no significant effect of type of border (sugarcane and fallow) on mean total number of C. massyla ( F = 0.95 ; df = 1, 34; P = 0.3362 ) E. eluta ( F = 0.83 ; df = 1, 34; P = 0.3680 ) E. stigmatias ( F = 0.00; df = 1, 34 ; P = 0.9513) and species total ( F = 0.27 ; df = 1, 34; P = 0.6078) per trap. Field 3. The mean number of flies in this field varied from 04 per trap per sample date. The three ulidiids were not present at all the sampling dates in this field. No E. eluta were collected in traps at 17, 19, and 20 das and no E. stigmatias were collected in traps at 5, and 20 das. The mean t rap counts varied from 012 for C. massyla (0.62 0.07), 01 for E. eluta (0.02 0.01), and 03 for E. stigmatias (0.05 0.01). The Morisitas index calculation for flies showed that the fly distribution was aggregated except at 5 and 20 das (Table 43 ). The distribution was uniform at 5 das ( I = 0.83) before the spraying on 6 das. The distribution again became aggregated after 6 das ( I = 2.90) and reached the maximum level of aggregation at 19 das ( I = 7.14). The fly distribution switched to uniform at 20 das and then back to aggregated at 24 das. In determining the effect of distance into field on mean total number of flies it was found that the distance into field had no significant effect on mean total number of flies per sticky trap for C. massyla ( F = 0.01 ; df = 1, 48 ; P = 0.9104 ) E. eluta ( F = 2.83; df = 1, 48 ; P = 0.091) E. stigmatias ( F = 0.36; df = 1, 48 ; P = 0.5496) and species total ( F = 0.02 ; df = 1, 48; P = 0.8929)

PAGE 73

73 In determining the effect of type of field border on mean number of flies it was found that there was no significant effect of type of border (sugarcane and fallow) on mean total number of C. massyla ( F = 3.47; df = 1, 42; P = 0.0694 ) E. eluta ( F = 3.41 ; df = 1, 42; P = 0.0720 ) E. stigmatias ( F = 0.61 ; df = 1, 42; P = 0.4401) and species total ( F = 3.05; df = 1 42 ; P = 0.0878) per trap. Field 4. The mean number of flies in this field varied from 03 per sticky trap per sample date. Chaetopsis massyla and E. stigmatias were found in traps at all the sampling dates. Adults of E. eluta were caught in traps at 2 and 21 das only. The mean trap counts varied from 011 for C. massyla (1.07 0.01), 0 1 for E. eluta (0.01 0.01), and 04 for E. stigmatias (0.40 0.04). The Morisitas index calculation for each sample date showed that the flies distributed themsel ves in an aggregated manner throughout the sampling period (Table 43). In determining the effect of distance into field on mean total number of flies it was found that the distance into field had no significant effect on mean total number of flies per st icky trap for C. massyla ( F = 1.01 ; df = 1, 36 ; P = 0.3218 ) E. eluta ( F = 0.87; df = 1, 36 ; P = 0.3570) E. stigmatias ( F = 0.85; df = 1, 36 ; P = 0.3635) and species total ( F = 0.23; df = 1, 36 ; P = 0.6376) In determining the effect of type of field bor der on mean number of flies, it was found that there was no significant effect of type of border (sugarcane and fallow) on mean total number of C. massyla ( F = 1.67; df = 1, 32; P = 0.2055 ) E. eluta ( F = 0.36 ; df = 1, 32; P = 0.5502 ) E. stigmatias ( F = 0 .96 ; df = 1, 32; P = 0.3342) and species total ( F = 0.50; df = 1, 32 ; P = 0.4846) per trap.

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74 Large S cale F ields Field 1. The mean number of flies in the field varied from 01 per sticky trap. The mean trap counts varied from 04 for C. massyla (0.18 0.0 1), 0 4 for E. eluta (0.33 0.02), and 09 for E. stigmatias (0.45 0.02). A Morisitas index value > 1 at each sampling date indicated that the flies distributed themselves in an aggregated manner on all the sampling dates (Table 44). The field was t reated with insecticides seven times in the first two weeks of silking. The index increased from 1.86 at 5 das to 2.30 at 8 das after 2 continuous insecticide treatments at 6 and 7 das. The aggregation decreased at 11 das (after 2 days of spray), but peaked on 13 das after the sixth insecticide treatment. Morisitas index values indicated an aggregated distribution throughout the remaining sample dates, but they declined at each sampled date following the peak. In determining the effect of distance into field on mean total numbers of the three ulidiids, it was found that the distance into field affected the mean total number of flies per sticky trap for C. massyla ( F = 32.34; df = 1, 218; P < 0.0001) E. eluta ( F = 7.47 ; df = 1, 218; P = 0.0068 ) E. stigm atias ( F = 13.69; df = 1, 218 ; P = 0.0003) and species total ( F = 33.65 ; df = 1, 218 ; P < 0.0001 ) The number of all three species and total numbers of flies captured on traps were greater on the field edge than on traps in the remainder of the field (T able 45). In determining the effect of type of field border on mean fly counts, it was found that there was no significant effect of type of border (fallow, sugarcane and corn) on mean total number of C. massyla ( F = 0.69; df = 2, 205; P = 0.5044 ) and E. stigmatias ( F = 0.90; df = 2, 205 ; P = 0.4077) per trap. The type of border had significant effect on

PAGE 75

75 mean total number of E. eluta ( F = 5.16; df = 2, 205; P = 0.0065) and total flies ( F = 4.01 ; df = 2, 205 ; P = 0.0196 ) caught on traps. Euxesta eluta caught on the side s of the field bordered by fallow (0.43 0.04; 01.86) were significantly higher in numbers than those caught on the side of the field bordered by corn (0.24 0.04, 00.71). The total number of flies caught on the side of the field bordered by fallow (1.14 0.08, 0.143.43) w as significantly higher than those caught than on the side bordered by corn (0.81 0.08, 0.14 1.57). Field 2. The sticky traps were sampled in this field beyond the usual 21 das due to the delayed harvesting. Ulid iid trap counts in this field were lower than in the other fields sampled. The mean number of flies varied from 0.10.5 per sticky trap. Euxesta eluta was captured in the sticky traps at all the sampling dates, but the other two species were not found on the traps on several sample dates. Chaetopsis massyla was not caught at 9 or 13 das, and E stigmatias were not caught at 9, 25, 26, or 27 das. The mean number of flies varied from 02 for C. massyla (0.04 0.01), 0 4 for E. eluta (0.1 0.02), and 01 for E. stigmatias (0.04 0.01). Fly captures were generally lower at the first three sample dates before peaking at 13 and 21 das. The ulidiids were uniformly distributed during the first 9 das, but became aggregated as the fly trap counts increased fr om 12 to 21 das (Table 4 4 ). The Morisitas index values showed that the fly distribution remained aggregated until past usual 21 das. The distribution of flies at the last sampling date of 27 das could not be calculated due to presence of very low numbers of flies found in each sample. In determining the effect of distance into field on mean total fly counts of three species, it was found that the distance into field had no significant effect on the mean

PAGE 76

76 total number of flies per sticky trap for C. massyla ( F = 0 ; df = 1, 58; P = 1.000) E. eluta ( F = 0.54; df = 1, 58 ; P = 0.4671) E. stigmatias ( F = 2.65 ; df = 1, 58 ; P = 0.1090) and species total ( F = 0.06; df = 1, 58 ; P = 0.8119) In determining the effect of type of field border on mean fly counts, it was determined that there was no significant effect of type of border (residence, fallow and corn) on mean total number of C. massyla ( F = 0.76; df = 2, 49 ; P = 0.4751) and E. stigmatias ( F = 1.46; df = 2, 49 ; P = 0.2420) per trap. The type of border ha d significant effect on mean total E. eluta ( F = 4.31 ; df = 2, 49; P = 0.0189) and total number of flies ( F = 2.42; df = 2, 49 ; P = 0.0999) caught on traps. Significantly fewer E. eluta were caught in the sides of fields bordered by fallow (0.11 0.03, 0 0.63) and corn (0.05 0.02, 0 0.25) than by residences (0.29 0.16, 01.14). A s ignificantly higher total number of flies w as caught on traps on field side bordered by residences (0.33 0.17, 0 1.14) than by corn (0.13 0.02, 00.38). Field 3. The mean number of flies in this field varied from 0.10.5 per sticky trap per sample date. Both C. massyla and E. eluta were found at all the sampling dates, but E. stigmatias was not captured in any trap on any sample date. The mean trap counts varied from 0 for C. massyla 4 (0.20 0.02) to 03 for E. eluta (0.06 0.01). The calculation of Morisitas index showed that the flies distributed themselves in an aggregated manner at all the sampling dates (Table 44). The index was 1.10 at the first sample da te and peaked at 7.47 on 6 das (2 d after spraying). The index decreased from 6 to 10 das (3 d after spraying). The second peak in Morisitas index values was observed at 20 das after four continuous sprays of insecticides. The index decreased again at 25 das (3 d after spraying).

PAGE 77

77 In determining the effect of distance into field on mean total counts of three species of flies, it was found that the distance into field had no significant effect on the mean total number of flies per sticky trap for C. massyla ( F = 0.04; df = 1, 222; P = 0.8488) only. Mean total E. eluta ( F = 35.59 ; df = 1, 222; P < 0.0001 ) and species total ( F = 9.99; df = 1, 222 ; P = 0.0018) were affected by the distance into field. Significantly more E. eluta per trap were caught on traps at the field edge than on rest of the traps in the field (Table 45). The species total followed the pattern similar to that of E. eluta In determining the effect of type of field border on mean fly counts, it was found that there was no significant e ffect of type of border (corn and fallow) on mean total number of C. massyla ( F = 0.05; df = 1, 222 ; P = 0.8267) and species total ( F = 0.81 ; df = 1, 222; P = 0.3693 ) per trap. Mean total E. eluta ( F = 4.18; df = 1, 222; P = 0.0420) caught on traps were affected by the type of border and were greater in number on field side bordered by fallow (0.08 0.02, 01) than by corn (0.04 0.01, 00.4 ). Field 4. The mean number of flies in this field varied from 01 per sticky trap per sample date. The mean trap counts varied from 08 for C. massyla (0.32 0.03), 05 for E. eluta (0.25 0.03), and 03 for E. stigmatias (0.06 0.01). The Morisitas index showed that the flies distributed themselves in an aggregated manner at all sample dates (Table 44). The peak Morisitas index value was observed on 16 das which fell immediately after the spray on the same day. In determining the effect of distance into field on mean total fly counts of three species, it was found that the distance into field affected the m ean total number of flies per sticky trap for C. massyla ( F = 9.24 ; df = 1, 206 ; P = 0.0027) and E. eluta ( F = 22.16; df = 1, 206; P < 0.0001) only but not on E. stigmatias ( F = 3.24 ; df = 1, 206 ; P =

PAGE 78

78 0.0731) and species total ( F = 0.13; df = 1, 206 ; P = 0.7173 ) Significantly fewer C. massyla and more E. eluta were caught in traps on field edge than rest of the traps in the field (Table 45). In determining the effect of type of field border on mean fly counts, it was found that type of border (sugarcane and corn) had significant effect on mean total number of C. massyla ( F = 12.85; df = 1, 206; P = 0.0004) E. eluta ( F = 18.53; df = 1, 206; P < 0.0001) E. stigmatias ( F = 3.95; df = 1, 206 ; P = 0.0483) and species total ( F = 27.70; df = 1, 206; P < 0.00 01 ) per trap. The field side bordered by sugarcane caught more flies per trap ( C. massyla 0.51 0.07, 0 4, E. eluta 0.38 0.04, 02; E. stigmatias 0.09 0.02, 01; species total 0.98 0.09, 05.5) than field side bordered by corn ( C. massyla 0.21 0.03, 0 1.33; E. eluta 0.14 0.03, 01; E. stigmatias 0.04 0.01, 0 0.33; species total 0.40 0.04, 01.67). Field 5. The mean number of flies in this field varied from 0.31.9 per sticky trap per sample date. Euxesta stigmatias were caught at 13 and 14 das only. The mean trap counts varied from 09 for C. massyla (0.41 0.04), 0 9 for E. eluta (0.51 0.05), and 01 for E. stigmatias (0.004 0.003). The Morisitas index calculations showed that fly distribution was aggregated in 3 of 5 sample dates (13, 19, and 20 das) and uniform at 2 dates (10 and 14 das) (Table 44). The fly population went from uniform at 14 das to aggregated at 19 das peaking at 25 das with the greatest I value determined at any sample date in any field over the threeyear study. In determining the effect of distance into field on mean total fly counts of three species, it was found that The distance into field affected the mean total number of flies per sticky trap for E. eluta ( F = 93.06 ; df = 1, 98; P < 0.0001 ) and species total ( F =

PAGE 79

79 30.07; df = 1, 98 ; P < 0.0001 ) only but not on C. massyla ( F = 0.95; df = 1, 98; P = 0.3318) and E. stigmatias ( F = 3.69 ; df = 1, 98 ; P = 0.0577) Significantly more E. eluta were caught on traps on field edge than on rest of the traps in the field (Table 45). The species total followed the pattern similar to that of E. eluta In determining the effect of type of field border on mean fly counts, it was found that there was no significant effect of type of border (fallow, corn and sugarcane) on mean total number of E. eluta ( F = 1.85; df = 2, 97 ; P = 0.1630) and E. stigmatias ( F = 0.50 ; df = 2, 97; P = 0.6081) per trap. The type of border had significant effect on mean total C. massyla ( F = 7.04 ; df = 2, 97 ; P = 0.0014) and species total ( F = 7.06; df = 2, 97; P = 0.0014 ) caught on traps. Fewer flies were caught on traps on field side bordered by corn ( C. massyla 0.36 0.04, 0 1, species total 0.87 0.10, 02.8) and sugarcane ( C. massyla 0.25 0.05, 00.8; species total 0.61 0.10, 02) than field side bordered by fallow ( C. massyla 0.68 0.15, 02.6; species total 1.37 0.2, 0 3.8). Corn E ar I nfestation The proportion of corn ears infested with ulidii d larvae was different between small and large scale corn fields. Fly infested corn ears were found in all the small scale corn fields. The proportion of infested corn ears varied from 0.42 for field 1, 0.41 for field 2, 0.26 for field 3, and 0.31 for fi eld 4, in small scale category. Ulidiid infested ears were only found in large scale field 1. The proportion of infested ears to total sampled ears in field 1 and field 5 of large scale fields varied from 0.23 for field 1 and 0.03 for field 5. Significa nt linear relationship was indicated for the regressions of proportion of infested corn ears on mean total number of ulidiid adults caught per trap per season ( F = 53.89; df = 1, 7; P < 0.0001) (Fig. 4 1 ). This suggested that as mean flies caught per trap

PAGE 80

80 increased, the proportion of infested corn ears increased and vice versa. The equation y = 0.0227x 0.0045 accounted for 85% of variability in the linear relationship between the proportion of ulidiid infested ears and mean total fly counts. Comparatively very low infestation of corn ears was observed in four of five commercial corn fields. In comparison, higher infestation of corn ears was observed in all the small scale corn fields. The mean densities of the flies were lower in the large scale corn f ields (1.3 1.9 flies per sticky trap) in comparison to small scale corn fields (6.519.1 flies per sticky trap). The graph showed that the minimum mean total fly counts to cause infestation in corn ears lies between 1.94.6 per sticky trap. The Morisita s index for calculating the level of aggregation was found to be different among the corn fields and seasons. This can probably be explained by the variation in fly species complex and abundance of each species of flies in different years and seasons. Fl ies of at least one species were found at different sampling dates in each of the sampled fields. Flies of three species, C. massyla E. eluta and E. stigmatias were present in different densities in different fields. Adults of E. annonae were found only very rarely; therefore, they were not included in the studies. The study was conducted over a period of more than 2 years. The number of species found in a particular field and abundance of each species changed with years and seasons within a year. Dist ribution of flies was found to be aggregated in majority of the sampling dates in all the small scale and large scale fields. The distribution was random or uniform at 6, and 7 das in field 1, 6 das in field 2, 5, and 20 das in field 3 in small scale fiel ds. A few dates in large scale fields also found uniform or random distribution of flies at 4, 6, and

PAGE 81

81 9 das in field 2, and 10, and 14 das in field 5. The Morisitas index ranged from 1.027.14 to 1.107.47 for aggregated distributions of flies in small and large scale fields, respectively. The Morisitas index at 27 das in field 2 of large scale fields was not calculated due to zero value of denominator in the equation of Morisitas index. This was because only one adult of C. massyla was caught on the trap in the entire field. The Morisitas index was also found to be zero at few sampling dates (4, 6, and 9 das in field 2 of large scale fields). This happened due to presence of only one fly in many sticky traps on these sampling dates. The fly dist ribution became more aggregated after insecticide spraying from uniform or less aggregated before spraying at most of the sampling dates. This could possibly mean the flies coming in from outside of the field and settling in the field at certain locations of the field after the insecticide spray. Hurlbert (1990) reported that Morisitas index is independent of sample size; therefore, different number of samples in different fields should not make difference in examination of Morisitas index as a measure of aggregation in small scale and large scale fields. The Morisitas index value provided information on the type of distribution for the population in consideration, but not the location of aggregation in the field. The distribution of flies was aggregat ed at majority of the sampling dates in all the fields, but the aggregation was not seen when data were analyzed to compare the fly catch among field edge and center of the field at many sampling dates. The flies of all three species are very active flier s and move rapidly when approached. Therefore, integrated approach of examining the various sections of field along with using Morisitas index helped understand the distribution of flies better than anyone alone.

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82 The small scale and largescale fields di ffered in the abundance of different species of flies. More E. stigmatias were caught from small scale fields in April 2008 and more C. massyla were caught in small scale fields in December 2008. In comparison, largescale corn fields in 2009 and 2010 ha d more C. massyla or E. eluta than E. stigmatias Only one field in the large scale category (Field 1, sampled in April 2008) had more E. stigmatias than other species at majority of the sampling dates. This could probably be explained by differences in temperature, humidity etc. among the different seasons and years. Fras L (1978) found that E. eluta and E. annonae differ in their seasonal occurrence. He found that adults of E. eluta were higher in abundance in March compared to January and February due to higher humidity in March compared to earlier months, but E. annonae were fewer in February and March than January. App (1938) found the E. stigmatias infestation to be present throughout the year in different fields in Puerto Rico, but higher infest ation was found in the months of January, February, May, June and September as compared to October, November and December months. Hayslip (1951) found that damage by E. stigmatias may be expected both in fall and spring with slightly higher population occ urring in the fall and late spring. Barber (1939) reported that E. stigmatias adults increased in abundance as the season advanced each year and were more in number in the late March. Seal and Jansson (1989) found that a higher level of corn ear infestat ion by E. stigmatias was found in the month of April which decreased in May followed by June. The effect of distance into the field affected the different species differently and in different fields. Fewer differences were found between mean total number of flies caught on traps between field edge and center of field in small scale fields than large-

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83 scale fields. Distance into the field of small scale fields showed the effect on mean flies caught on traps in field 1, and field 2 only and not in field 3, and field 4. Moreover, differences between field edge and center in field 1 and field 2 were seen with respect to E. stigmatias only. This could probably be explained by the smaller overall density of C. massyla and E. eluta than E. stigmatias caught in traps in field 1, and field 2. Mean total E. stigmatias caught per trap were 49 times than E. eluta and C. massyla in field 1 and field 2. The mean total number of E. stigmatias caught in field 1 and field 2 were 432 times more than those caught in field 3 and field 4. Therefore, the flies were probably too few in some of these fields to cause any differences between field edge and center of field. Differences between mean flies per trap at field edge and center were observed in more fields and in mor e species in largescale fields than small scale fields. More E. eluta were found on traps on field sides than center in four out of five sampled fields. More C. massyla were found on field side than center in field 1, and in center than field side in fi eld 4. Adults of E. stigmatias were more on field side in field 1 only. No significant differences were seen between flies of any species caught between field side and center for field 2. This could probably be explained by the overall smaller density o f flies of all three species ( C. massyla 0.03, E. eluta 0.13, E. stigmatias 0.05) caught in this field compared to other largescale fields and sampling in December rather than sampling in April May. It is also possible that field size was too small (6 ha) for flies to show any significant differences between field side and center. In field 5, differences in mean total flies caught between field side and center were observed for E. eluta only. This could again be explained by lower mean C. massyla (0.44) and E.

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84 stigmatias (0.01) per trap caught compared to E. eluta (1.06) and smaller size of this field (4 ha). Differences were observed between small scale and largescale fields with respect to effect of field borders on mean fly counts in fields. T ype of border (fallow and sugarcane) did not show any effect on mean fly counts of any of the species in small scale fields. In largescale fields, flies caught were significantly less on sides bordered by corn compared to sugarcane/fallow/residence. Sim ultaneous spraying of adjacent corn fields may have affected the movement of flies from those corn fields into the main corn field. In comparison, sugarcane being sprayed less frequently may have acted as reservoir of flies and led to more flies in corn f ields bordered by sugarcane. Work has been done in the past on understanding the distribution of insects in various crops. Various indices have been used to measure the aggregation of populations. Vargas et al. (1989) used Morisitas index to determine the distribution of Oriental fruit fly and melon flies on Kauai, Hawaiian islands. Khaing et al. (2002) showed different amounts of aggregation from different values of Morisitas index in populations of Helicoverpa armigera on rainfed cotton and irrigat ed cotton. Reay Jones (2010) showed the significant distance from the field border effect on adults of stink bugs in South Carolina. Miliczky (2007) found that the density of w estern flower thrips, Frankliniella occidentalis (Pergande) decreased with increasing distance into the orchard surrounded by native sagebrush steppe habitat. The fly trap count was found to be a good indicator of the damage done to corn ears. The proportion of infested corn ears decreased with decrease in fly density until a

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85 poi nt is reached where no infested corn ears were found. This information can be helpful in developing economic thresholds for these flies in the future. In conclusion, the ulidiid flies were found to be aggregated in distribution in corn fields sampled in 20082010. The flies were found mostly at the sides of the fields rather than in the center of fields especially in largescale fields Field sides bordered by corn were found to have fewer flies than sides bordered by sugarcane/fallow/ residence. The fly infestation on corn ears was found to be strongly correlated with the flies caught on sticky traps; therefore, traps provide a good tool for determining the sampling plans and economic thresholds for these flies in future.

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86 Table 4 1. Small and largesca le sweet corn fields sampled for determining the spatial and temporal distribution of corninfesting ulidiids, 20082010 Field Location Size (ha) Year Distance of trap placement Total no. trap rows Trap distance (m) First date of sampling traps Date of col lection of infested ears Length wise Cross wise Small scale fields 1 Belle Glade 1.8 2008 6 May (2 DAS a ) 5 10 15 8 May 22 May 2 Belle Glade 1.8 2008 6 May (2 DAS) 5 8 15 8 May 23 May 3 Belle Glade 1.8 2008 1 Nov (0 DAS) 5 10 15 5 Nov 1 Dec 4 Belle Glade 1.8 2008 26 Oct (0 DAS) 5 8 15 27 Oct 24 Nov Large scale fields 1 Belle Glade 16 2008 8 May (4 DAS) 11 20 30 9 May 22 May 2 Pahokee 6 2009 19 Feb (1 DAS) 5 12 30 22 Feb 17 Mar 3 Belle Glade 14 2009 17 Apr (1 DAS) 8 28 30 19 Apr 11 May 4 So uth Bay 14 2009 16 Apr (3 DAS) 8 25 30 26 Apr 11 May 5 Belle Glade 4 2010 23 Apr (8 DAS) 10 10 15 25 Apr 5 May aDAS days after first silk appearance

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87 Table 4 2 Borders of small and largescale sweet corn fields, 20082010 Field North South East West Small scale fields 1 Sugarcane Sugarcane Fallow Sugarcane 2 Fallow Sugarcane Sugarcane Sugarcane 3 Sugarcane Sugarcane Sugarcane Fallow 4 Fallow Sugarcane Sugarcane Sugarcane Large scale fields 1 Corn Sugarcane Fallow Sugarcane 2 Residence a Fallow F allow Corn 3 Fallow Fallow Fallow Corn 4 Sugarcane Sugarcane Sugarcane Corn 5 Corn Corn Fallow Sugarcane aResidence Local houses and shopping market

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88 Table 4 3 Morisita s index ( I) for ulidiids in smallscale corn fields, 2008 Field N a Sampling date (das) b Insecticide spray (das) Mean SEM number of Ulidiidae per trap per date I c Field 1 50 4 3 0.86 0.13 1.05S 5 1.32 0.17 1.11S 6 2.06 0.18 0.89 S 7 1.42 0 .17 0.98NS 8 d 8 2.48 0.40 1.89S 9 2.52 0.28 1.24S 10 1.96 0.25 1.33S 11 1.34 0.18 1.20S 15 12, 15 5.78 0.64 1.43S 17 16 1.64 0.19 1.08S Field 2 40 4 3 2.13 0.29 1.28S 5 1.55 0.27 1.61S 6 e 6 2.00 0.22 0.99 NS 7 3.25 0.49 1.57S 8 3.65 0.34 1.08S 9 1.45 0.27 1.67S 10 0.80 0.19 2.10S 11 0.30 0.10 1.82S 14 12 1.03 0.17 1.17S 15 c 15 1.98 0.43 2.38S 17 16 0.93 0.18 1.38S Field 3 50 4 2 3.46 0.41 1.40S 5 0.3 2 0.08 0.83S 16 6 0.84 0.21 2.90S 17 0.26 0.10 4.49S 19 0.14 0.07 7.14S 20 0.14 0.05 0S 24 0.64 0.15 2.22S 28 0.48 0.12 1.81S 30 0.24 0.07 1.52S Field 4 40 1 3.16 0.48 1.62S 2 1.84 0.25 1.24S 3 1.08 0.17 1.02S 14 4, 7, 11 1.11 0.21 1.58S 21 2.76 0.34 1.28S 22 0.61 0.14 1.43S 24 0.47 0.13 1.83S 25 0.42 0.13 2.67S 28 1.89 0.33 1.69S

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89 Table 43 continued aN number of samples bdas days after first s ilk appearance cI Morisitas index: If < 1 (uniform population), = 1 (random population), > 1 (aggregated population), S (Population significantly different from random), NC (not calcul ated due to zero value of denominator in the equation of index) dField was s ampled after the insecticide treatment on the same day eField was sampled before the insecticide treatment on the same day.

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90 Table 4 4 Morisitas index ( I) for ulidiids in large scale corn fields 20082010 Field N a Sampling date ( das ) b Insecticide spray (das) Mean SEM I c Field 1 220 5 1, 3 0.88 0.08 1.86 8 6, 7 1.19 0.12 2.30 11 9 0.40 0.05 2.07 13 12 0.66 0.08 3.14 15 14 0.97 0.10 2.76 16 1.37 0.12 2.20 17 1.22 0.11 2.05 Field 2 60 4 2 0.18 0.05 0 6 0.08 0.04 0 9 7 0.05 0.03 0 13 11 0.29 0.09 3.14 21 14, 16, 19 0.45 0.11 2.51 25 22 0.14 0.05 1.67 26 0.15 0.05 1.67 27 0.02 0.02 NC Field 3 224 3 0.09 0.03 1.10 6 4 0.47 0.05 7.47 10 7 0.22 0.04 1.77 20 11, 13, 16, 17 0.40 0.04 3.05 25 22 0.13 0.02 1.14 Field 4 208 13 7, 9, 10 1.03 0.11 2.20 16 d 16 0.72 0.11 4.15 18 17 0.14 0.03 1.43 Field 5 100 10 5, 7, 9 0.28 0.05 0.99 13 1.94 0.23 1.85 14 0.73 0.11 0.55S 19 17 1.23 0.18 4.75 20 0.48 0.11 9.95 aN number of samples bdas days after first silk appearance cI Morisitas index: If < 1 (uniform population), = 1 (random population), > 1 (aggregated population), S (Population significantly different from random), NC (not calculated due to zero value of denominator in the equation of index) dField was s ampled after the insecticide treatment on the same day.

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91 Table 4 5 Mean SEM (range) flies per sticky trap for three Ulidiidae species in small scale and largescale fields, 20082010 Field C. massyla E. eluta E. stigmatias Species total Center Edge Center Edge Center E dge Center Edge Small scale fields 1 0.15 0.03 (0 0.5) 0.20 0.03 (0 0.5) 0.22 0.04 (0 2.5) 0.35 0.05 (0 1.3) 1.45 0.11 B (0.7 2.5) 1.88 0.17 A (0.8 4.9) 1.82 0.12 B (0.9 2.90) 2.43 0.21 A (1 6.7) 2 0.15 0.04 (0 0.55) 0.14 0.02 (0 0.36) 0 .17 0.03 (0 0.55) 0.38 0.09 (0 2.09) 1.05 0.08 B (0.45 1.73) 1.51 0.13 A (0.553.09) 1.37 0.11 B (0.82 2.27) 2.03 0.19 A (0.915.36) 3 0.67 0.09 (0 1.67) 0.65 0.12 (0 2.33) 0 0.03 0.01 (0 0.22) 0.04 0.01 (0 0.22) 0.06 0.03 (0 0.67) 0.71 0.09 (0.11 1.67) 0.74 0.13 (0 2.44) 4 1.17 0.10 (0.33 1.89) 0.98 0.15 (0.11 2.44) 0.01 0.01 (0 0.11) 0.01 0.01 (0 0.11) 0.36 0.06 (0 0.78) 0.43 0.06 (0 1.11) 1.54 0.12 (0.67 2.44) 1.43 0.18 (0.67 3.33) Large scale fields 1 0.13 0. 01 B (0 0.71) 0.32 0.04 A (0 1.29) 0.29 0.03 B (0 1.86) 0.61 0.06 A (0 1.86) 0.39 0.03 B (0 2.29) 0.61 0.06 A (0 1.86) 0.81 0.04 B (0 2.86) 1.35 0.1 A (0.143.43) 2 0.03 0.01 (0 0.13) 0.03 0.01 (0 0.25) 0.13 0.04 (0 1.14) 0.09 0.03 (0 0.63) 0.02 0.01 (0 0.13) 0.05 0.01 (0 0.25) 0.18 0.04 (0 1.14) 0.17 0.04 (0 0.75) 3 0.20 0.02 (0 1.2) 0.21 0.03 (0 0.8) 0.02 0.01 B (0 0.4) 0.14 0.03 A (0 1.0) 0 0 0.22 0.02 B (0 1.2) 0.35 0.04 A (0 1.8) 4 0.47 0.06 A (0 4) 0.20 0.04 B (0 1.5) 0.20 0.03 B (0 1.5) 0.47 0.06 A (0 2) 0.05 0.01 (0 1) 0.10 0.02 (0 1) 0.72 0.07 (0 5.5) 0.76 0.1 (0 4) 5 0.45 0.07 (0 2.6) 0.35 0.04 (0 1) 0.20 0.03 B (0 1.2) 1.06 0.11 A (0 2.2) 0 0.01 0.01 (0 0.2) 0.65 0.08 B (0 3.8) 1.43 0.13 A (0 2.8)

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92 Fig ure 4 1 Regression plot between mean total number of flies caught on sticky traps and proportion of infested corn ears in small and large scale corn felds.

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93 CHAPTER 5 ALTERNATE HOSTS OF CORNINFESTING PICTURE WINGED FLIES Corn is not available throughout the year for fly development, yet C. massyla and Euxesta spp. adults routinely appear in corn fields at the beginning of each season following cornfree periods Therefore, other plants in Florida are likely acting as food sources for maint enance and development of these flies in the absence of commercial corn. Several commodities in Florida can act as potential reservoirs for these flies. The larvae or adults of E. stigmatias have been collected from damaged or decayed sorghum ( Sorghum bi color Moench), tomato ( Lycopersicon esculentum Mill.) sugarcane ( Saccharum officinarum L.), guava ( Psidium guajava L.), banana ( Musa spp.), orange (C itrus aurantium L.), atemoya ( Annona squamosa l. x A cherimolia Miller), orchid (Dendrobium spp.), and po tato ( Solanum tuberosum L.) from the Homestead, Florida area (Seal and Jansson 1989, 1996). Florida ranks second in vegetable production, area harvested and value behind California (Anonymous 2010 a ). Many other monocot and dicot crops, weeds and native plants may also provide resources for E. stigmatias and the other ulidiid species in the absence of corn. Nothing is currently known of the alternative food sources in Florida for the other three ulidiid species discovered feeding on corn. Allen and Foote (1992) collected C. massyla larvae from decomposing cattail Typha latifolia L. (Typhaceae: Typhales) stems. Thus, there is potential for vegetables, fruits and weeds within and surrounding these fields to serve as reservoirs for these flies in Florida. Therefore, the present studies were conducted to determine the alternate hosts of the three most common corninfesting ulidiids in corn fields: E. eluta, E. stigmatias and C. massyla

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94 Materials and Methods Field surveys were conducted to evaluate crop and non crop plants commonly found in fields near maize growing areas for their potential to act as developmental hosts for E. eluta, E. stigmatias and C. massyla Laboratory studies were conducted to determine the developmental and survivorship rates for the immature stages of these flies reared on these alternative hosts. Laboratory E valuation Locally available fruits vegetables and weeds were evaluated for successful development to the adult stage for each fly species (Table 5 1) The selection of these commodities was based on their proximity to maize fields in southern Florida. Fully ripe v egetables and fruits to be tested as larval development hosts were purchased from local market s. Weeds used in the experiment were collected from fields at the EREC Each commodity and weed ( Table 5 1 ) was exposed to adults of each of the three species to acquire plant material naturally infested by direct oviposition by flies, rather than by artificial infestation using eggs from the colonies. Potential development hosts were placed in Plexiglas cages ( 15 cm 15 cm 15 cm) with ten pairs of a single fly species (5 15 d old). Adults of the three ulidiid species used for the experiment were reared on H. zea artificial diet using the method of Hentz and Nuessly (2004) as described above. The flies were provided with supplementary honey and water. Following a 24 h exposure period that began at 0900 hr, the plant material was removed from the cages and placed in plastic containers ( 15 high 11 cm dia m ) with a scree n top (9 cm2 area) lined with paper towels to allow development to the pupal stage. The plant parts and paper towels were checked for pupae daily after 7 d. The pupae were placed on moist ened filter p aper (Whatman 3) in P etri plates and held for adult

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95 em ergence. The plat es were sealed with P arafilm to reduce moisture loss and held under the same environmental conditions as the eggs and larvae. All phases of this experiment were conducted at 26.5 1.0 C, 14:10 (L: D) photoperiod, and 55 70% RH. Adul t emergence was recorded daily. Flies were preserved in 70% ethyl alcohol for later identification. Identifi cations were determined using identified specimens and keys as indicated above. The successful emergence of adults from these plant specimens was considered to be evidence of a potential host of that species. While most of the tested plant parts were not altered before exposure to flies, a few were manipulated before exposure to the flies in cages. Chaetopsis massyla has been reared from sugarcane billets cut from stalks naturally infested with larvae of the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae) (G. Nuessly, pers. comm.). However, preliminary laboratory trials determined that undamaged sugarcane stalks did not support development of these flies. Therefore, 0.5 cm diam holes were drilled 0.5 cm into the internodes of 7cm long sugarcane billets to mimic the damage of Lepidopteran larvae before exposing them to flies. The cut ends of billets were covered with P arafilm to re duce moisture loss. The main stems of weeds ( spiny amaranth, j ohnsongrass, l ittle hogweed, southern cattail) used in the experiments also were cut into 7 cm lengths and placed in cages. To expose more of the surface area to oviposition, the segments of w eeds, cattail, and sugarcane stems were placed upright inside the cage with one end touching the floor several cm away from a wall and the other touching one cage wall. Individual leaves of cabbage were separated from heads and placed on the cage floor for their evaluation.

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96 Observations were recorded on the length of the combined egg and larval stages, the number of pupae produced, the length of the pupal stage, and the number of adults emerged for each species on each tested plant species. Quantification of oviposition was initially attempted, but it was discontinued after it was determined that more adults emerged from hosts than the number of eggs originally observed. It probably occurred due to fly oviposition in cracks and crevices of the commodities Percentage pupal survival was calculated by dividing the number of adults that emerged by the number of pupae on each host. Field S tudies Adults of these corninfesting species have frequently been observed in sweet corn, fruiting vegetable, sugarcane f ields, and on several grass and broadleaved weeds. Therefore, field surveys were conducted to examine the occurrence of flies in different crops (Table 56) It is possible that flies move from corn fields to fields of other commodities or vice versa. T herefore, field surveys were conducted at the EREC and the Tropical Research and Education Center, Homestead, Florida and in commercial fields of fruits and vegetables in Palm Beach and Miami Dade Counties, where approximately 80% of the sweet corn produce d in Florida has historically been produced ( Anonymous 2008a ; A. Kirstein, Palm Beach Co. Coop. Ext. Economist, pers commun .). Damaged and lower quality products of some commodities grown in southern Florida are either left in fields or dumped in cull pi les to decay. This decaying plant matter could provide a reservoir for development of immature pest Ulidiidae species or a food source for adults of these flies. Therefore, samples were taken from cull piles of snap beans ( Phaseolus vulgaris L.) and radi shes ( Raphanus sativus L.) at several locations in western Palm Beach County. Samples for adults consisted of 100

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97 consecutive sweeps using sweep nets (37.5 cm diam) in commercial fields or visual observations as discussed below in small acreage plots. Sw eep net samples were taken along the borders and in the interior of fields. Sampling consisted of sweeping in five rows including two outside rows and three rows randomly selected in the field with 20 sweeps in each row. In sugarcane, sweep nets were used only along field borders due to the difficulty in sweeping inside the field. Insects collected in nets were placed in 70% ethyl alcohol and returned to the laboratory to identify Ulidiidae to species. Small experimental plots of a few commodities that i ncluded garden plots (25 100 m2) at the EREC or individual plants were also visually examined for fly occurrence (Table 56) Plants were first examined thoroughly for approximately 10 min per field site for adults. Flies observed were identified to species in the field and counted. A representative sample was also collected using vials or sweep nets where possible. Approximately 10 min also were spent checking fruits and fruiting vegetables on plants per field site along field borders and inside fiel ds for fly larvae. Infested produce was collected into Ziploc bags with paper towels and brought to laboratory to rear out adults for identification. To reduce the accumulation of moisture and associated fungus, the bags were kept partially opened p aper towels were changed frequently, and the air was constantly circulated using box fans. Pupae were removed fr om the bags and placed on moistened filter paper in covered Petri dishes sealed with Parafilm for adult emergenc e Adults that emerged were identi fied to species level, counted, and preserved in 70% ethyl alcohol. Statistical Analysis Proc GLM (SAS institute 2008) was used to conduct an analysis of variance of the results of the laboratory evaluations of alternate hosts. Fly species, plant species, and

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98 season were used as independent variables. The lengths of the egg plus larval and pupal stages, number of pupae, and percentage pupal survival were used as dependent variables in the model. The Tukey's honestly significant differenc e (HSD) test (SAS institute 2008) was used for post hoc means separation with P = 0.05. Results and Discussion Laboratory E valuation All three species deposited their eggs on all the plant species tested. Potato and carrot were the only plants tested on which none of the fly species successfully completed development. The length of the egg plus larval stages was significantly affected by plant host ( F = 1,007.16; df = 11, 954; P < 0.0001), fly species ( F = 884.80 ; df = 2, 954 ; P < 0.0001) The length of the egg plus larv al stages was also affected by the plant host with varying fly species ( F = 18.44; df = 22, 954 ; P < 0.0001). The season ( F = 0. 05 ; df = 2, 954 ; P = 0.9510), plant host s with varying season s ( F = 0.39 ; df = 22, 954 ; P = 0. 9949) and fly species with varyin g seasons ( F = 0.24 ; df = 4, 954 ; P = 0. 9147) did not have significant effect s on the length of egg plus larval developmental times Therefore, the data were pooled across seasons for means comparisons. The egg plus l arval development al time pooled across plant species was shortest for E. eluta followed by E. stigmatias then C. massyla (Table 5 2 ). The development times varied from 12.7 to 27.0 d for C. massyla to 10.1 to 19.9 d for E. eluta, and 11.5 to 25.0 d for E. stigmatias The egg plus larval developmental times for both C. massyla and E. stigmatias were shortest in bell pepper and longest in spiny amaranth. The egg plus larval developmental time for E. eluta was significantly shorter in bell pepper, cabbage and tomato and longer in spiny amaran th and cattail than the other tested plant parts

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99 The pupal developmental time was significantly affected by plant host ( F = 1 ,040.92 ; df = 11, 953 ; P < 0.0001), and fly species ( F = 117.61 ; df = 2, 953 ; P < 0.0001) The pupal development time was also af fected by fly species with varying plant host s ( F = 200.70 ; df = 22, 953 ; P < 0.0001) The season ( F = 0.94 ; df = 2, 953 ; P = 0. 3891), and plant host s with varying seasons ( F = 0.46; df = 22, 953; P = 0. 9849) and fly species with varying seasons ( F = 0.85 ; df = 4, 953 ; P = 0.4938) did not significant ly a ffect pupal developmental times Therefore, the data were pooled across seasons for means comparisons. The pupal development al time of C. massyla varied from 6 d in tomato to approximately 10 d in hogweed (Table 5 3 ) The length of pupal development for E. eluta ranged from approximately 5 d (tomato and papaya) to 12 d (cabbage and avocado). The pupal development time of E. stigmatias varied from approximately 6 d (tomato, bell pepper and papaya) to 12 d (avocado and spiny amaranth). No overall pattern of pupal development speed was observed with respect to the fly or plant species. The pupae of E. eluta developed faster than other species in six out of 12 plant species evaluated ( i.e., spiny amaranth, johnsongrass, little hogweed, papaya, sugarcane and tomato ). The pupae of C. massyla developed faster than other species in five of the evaluated plant species ( i.e., avocado, cabbage, c attail, habaero, and radish). The pupae of E. stigmatias developed faster than other species only in bell pepper. The number of pupae obtained per plant species w as significantly affected by fly species ( F = 142.55 ; df = 2, 954; P < 0.0001), and plant host ( F = 655.36 ; df = 11, 954; P < 0.0001) The number of pupae obtai ned per plant species was also affected by fly

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100 species with varying plant host s ( F = 12. 98 ; df = 22, 954; P < 0.0001). The s eason ( F = 0. 74 ; df = 2, 954; P = 0. 4756), fly species with varying seasons ( F = 0. 83 ; df = 4, 954; P = 0. 5082), and season s with v arying plant hosts ( F = 0.6 5 ; df = 22, 954; P = 0. 8904) did not have significant effect on the number of pupae. Therefore, d ata were pooled across seasons to compare the mean number of pupae of each species obtained in different plant species Significant ly fewer pupae of each of fly species were found associated with weedy species compared to plant species grown for commercial production (Table 5 4 ). The number of pupae of each fly species was greatest in bell pepper followed by the other plant species. The number of pupae varied from cattail, johnsongrass, and little hogweed) to 71 for C. massyla 107 for E. eluta and 82 for E. stigmatias in bell pepper. There were significant differences among fly species in the number of pupae that em erged from all tested plant species except habaero and little hogweed. Euxesta eluta produced more pupae than the other fly species in bell pepper, cabbage, cattail, papaya, radish, and tomato. P upal survival was significantly affected by plant host ( F = 18.64 ; df = 11, 954; P < 0.0001), but not by fly s pecies ( F = 1. 33; df = 2, 954; P = 0. 2651), season ( F = 2.21; df = 2, 954; P = 0. 1099), fly species with varying plant host s ( F = 1.21; df = 22, 954; P = 0. 2315), fly species with varying seasons ( F = 0. 39 ; df = 4, 954; P = 0. 8127), or seasons with varying plant host s ( F = 1.41; df = 22, 954; P = 0. 1011). Therefore, the data were pooled across seasons and fly species to compare the pupal survival among the hosts. The pupal survival was significantly great er in avocado, bell pepper, cabbage, papaya,

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101 and tomato than in spiny amaranth, johnsongrass, habaero, little hogweed and sugarcane (Table 5 5 ). Field E valuation Adults of the three fly species used for cage studies plus E. annonae were found in sampled f ields commodities, native plants and weeds (Table 5 6 ). Spiny amaranth and Annona trees harbored all four fly species. E uxesta annonae was the only fly species not found on johnsongrass, little hogweed, Santa Maria feverfew, cattail, cabbage, sorghum and sugarcane. More flies were found in snap bean within garden plots and experimental fields at EREC compared to commercial snap bean fields. Surveys of commercial bell pepper, mangoes, guava, cucumber, and squash fields at Homestead on 2 July 2008 did not yield any flies in sweep nets. Euxesta eluta and C. massyla adults were observed on banana fruits and stems at Fruit and Spice Park, Homestead, FL on 12 June 2010. No banana fruits were found infested with Ulidiids after searching for approximately 15 m in. Flies of all four species have been observed on many of the plant species evaluated at EREC at other random instances. It seems that proximity of a plant species to corn fields influenced the fly numbers that could be seen or sweep netted in plant sp ecies Comparatively more flies were found in weeds, and in other plant species (sugarcane, beans etc.) that surrounded corn or were close to corn fields rather than those fields far from corn. It is possible that flies may be avoiding the insecticide sprays being applied to corn by hiding in the crops nearby. In several instances, corn fields that were sprayed recently had low abundance of flies, but nearby weeds had high abundance of flies. Plant parts infested with ulidiid larvae were collected during field sampling and during several other accidental collections. Four johnsongrass plants were found

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102 infested with Ulidiidae larvae during routine sampling in a sweet corn field at EREC on 25 May 2009. These plants were returned to the lab from which a t otal of 5 C. massyla 20 E. eluta and 3 E. stigmatias ultimately emerged. Six additional ulidiid infested johnsongrass plants were located in another corn field at EREC on 5 May 2010 that yielded a total of 19 C. massyla and 33 E. eluta. Two decaying bel l peppers collected at the EREC garden on 16 June 2010 yielded a total of 14 E. eluta and 7 C. massyla No flies were reared from snap beans collected from a cull pile at South Bay, FL on 12 December 2008 nor were any ulidiids reared from radish samples collected from cull piles located near Belle Glade, Florida on 5 February 2009. Ulidiidae eggs were found between leaf sheaths on 1 of 58 cattail plants intensive sampled on 6 December 2009. The plant was returned to the lab where a total of 11 C. massyl a adults ultimately completed development within the decaying stalk. Adults of E. eluta and E. annonae were reared from a decayed spiny amaranth weed collected on a roadside on 9 July 2009. An intensive examination of spiny amaranth plants growing around corn fields at EREC on 21 May 2010 yielded another damaged plant with 3 fly eggs that developed into E. eluta adults in the laboratory. Most of the tested plant species were found to support the development of all three species of flies under laboratory c onditions (Table 5 1 ). No studies have been conducted in the past to study the development of these flies on alternate hosts under laboratory conditions. However, the plants that successfully served as hosts for the u lidiid flies in our studies match those found by others in the past. Huepee et al. (1986) and Koch and Waterhouse (2000) also determined that E. eluta used bell peppers as a host in Chile. In an unrelated study, Chaetopsis massyla adults were reared from

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103 sugarcane stems infested with D saccharalis collected in November 2009 from Clewiston (Hendry County) and Sebring (Highlands County), Florida and from sweet sorghum stems collected at the EREC in September 2010 (unpublished data, G. Nuessly). Sugarcane is produced on over 161K ha in southern Florida and the majority of the sweet corn acreage is surrounded by sugarcane. Keiper et al (2000) found C. massyla larvae and puparia in cattail plants in California. Allen and Foote (1992) also collected C. massyla larvae from decomposing cattail s tems previously damaged by n octui d (Lepidoptera) larvae, as well as from Carex lacustris Willd. (Cyperale s: Cyperaceae) stems previously damaged by Epichlorops exilis (Coquillett) (Diptera: Chloropidae) larvae in Ohio. Seal et al. (1996) collected larvae and adults of E. stigmatias from damaged or decayed sugarcane and sorghum ( Sorghum bicolor Moench). Several commodities not surveyed in the current study have been reported as developmental hosts of these flies in the literature. Chaetopsis massyla has been reared from onions, Allium cepa L. (Liliales: Liliaceae) in Michigan (Merrill 1951), and from decaying Narcissus bulbs (Liliales: Liliaceae) in New York (Blanton 1938). Illingworth (1929) recorded E. annonae as a minor pest of pineapple in Honolulu. S everin and Hartung (1912) reared E. annonae from bananas decaying around the flower scar. Euxesta eluta has been reported as a pest of loquat, Eriobotrya japonica (Thumb.) Lindl. (Rosales: Rosaceae) in Alachua County, Florida (Anonymous 2008b). Several pl ant species in the current study acted as food source for adults in the field conditions. Adults of all four species were observed or sweepnetted from beans, annona, and spiny amaranth plants, while adults of C. massyla E. eluta and E.

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104 stigmatias were observed in sorghum, sugarcane, little hogweed, Santa Maria feverfew, cattail, cabbage, johnsongrass (Table 5 6 ), guava, and banana in the current study. Adults of E. eluta were observed on eggplants (N. Larsen, pers. comm.). Surveys in the past reported the presence of E. stigmatias adults in sorghum, sugarcane, banana, guava, orange ( Citrus aurantium L.) (Sapindales: Rutaceae), atemoya, orchid, and potato in Florida (Seal et al. 1996), and C. massyla on beach grass at the edge of tide water in New York ( Blanton 1938) and on Melaleuca quinquenervia (Cav.) S.T. Blake at Weston and Fort Lauderdale, Florida (Costello et al. 2003). Some of the observations on sweep net caught adults and adults reared from infested fruits in the current study did not match those of previous studies. Decaying fruits of guava did not yield any ulidiids in the current study, whereas Seal et al (1996) reared adults of E. stigmatias from larvae in guavas grown in field. Fresh carrots and potatoes did not support the development o f flies in the current studies, but larvae of E. eluta and E. stigmatias were reared from decaying carrot roots in Brazil (Franca and Vecchia 1986), and E. stigmatias was reared from larvae found in decayed potatoes in southern Florida (Seal et al. 1996). The larval developmental time, in general increased in the following order from bell pepper, cabbage, tomato, papaya, avocado, radish, sugarcane, habaero, little hogweed, johnsongrass, cattail to spiny amaranth while number of pupae decreased in the same above order with few exceptions (Table 5 2 ). Pupal development time of each of the species did not follow a single order for all the commodities evaluated (Table 5 3 ). While experimenting with tomato fruits as an alternative hosts for olive fruit fly, B actrocera oleae (Rossi) (Diptera: Tephritidae), Navrozidis and Tzanakakis (2005)

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105 found that the resistance of a cultivars epicarp to penetration by probes affected the number of eggs received by a cultivar. Cultivars with epicarp less resistant to probing with needles were found to be more attractive to flies for oviposition. Moreover, the fewer the eggs deposited per fruit, the longer the time from oviposition to pupation was observed. Nutritional differences in different plant species tested could pro bably explain some of the variation seen in developmental times in my studies. Abdel Fattah et al. (1977) observed shorter larval and pupal development times of cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) in food commodities w ith high concentrations of carbohydrates, nitrogen and essential growth elements. The pupal survival was greater in avocado, bell pepper, cabbage, papaya and tomato than in habaero and in most of the weeds (johnsongrass, little hogweed, and spiny amaranth). Nutritional differences among weeds and other plant species may explain a part of this variation in pupal survival While radishes were found to support the development of three ulidiid species under laboratory conditions, no picturewinged flies emerg ed from samples collected from either radish or snap bean cull piles. Sorting the fruit by quality and throwing the damaged, decayed, lower quality fruits in the field during harvesting of certain commodities is a common practice among growers. Perhaps environmental conditions or competition for the food prevented u lidiid larvae from completing development in the samples. It is also possible that there were no fly larvae in the samples collected. But the successful completion of development of all three fly species in radish bulbs in the laboratory suggests that cull piles may serve as potential reservoirs of these flies when corn is not available.

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106 In conclusion, many commodities and weeds found in the vicinity of sweet corn fields in Florida provided sa tisfactory food sources for successful development from eggs to adults All three species were able to complete development on alternative commercial crops and weedy species under laboratory conditions. The plant species tested here are available in abun dance especially in Miami Dade and Palm Beach Counties, where most of Floridas sweet corn is grown. The presence of multiple host crops throughout the sweet corn production areas of Florida may help explain the occurrence of these flies immediately after prolonged absences of corn. T hree species ( C. massyla E. eluta and E. stigmatias ) completed their development on a wide range of locally grown as well as several weedy plant species under laboratory conditions. Moreover, several plant species, bell pepper, spiny amaranth, cattail, sugarcane, johnsongrass supported the development of flies under field conditions indicating the potential of these plants as reservoirs of flies when corn is absent.

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107 Table 5 1 Various commodities/weeds evaluated under laboratory conditions or surveyed in fields in Belle Glade and Homestead, FL. Order Family Common name Scientific name Plant part Caryophyllales Amaranthaceae Spiny amaranth Amaranthus spinosus L. Stem Magnoliales Annonaceae Annona Annona spp. L. Fruit A piales Apiaceae Carrot Daucus carota L. Root Asterales Asteraceae Santa Maria feverfew Parthenium hysterophorus L. Whole plant Capparales Brassicaceae Cabbage Brassica oleracea L. Leaf Capparales Brassicaceae Radish Raphanus sativus L. Root Violales Ca ricaceae Papaya Carica papaya L. Fruit Fabales Fabaceae Snap bean Phaseolus vulgaris L. Whole plant Laurales Lauraceae Hass avocado Persea americana Mill. Fruit Cyperales Poaceae Johnsongrass Sorghum halepense (L.) Pers. Stem, root Cyperales Poaceae So rghum Sorghum bicolor (L.) Moench Whole plant Cyperales Poaceae Sugarcane Saccharum officinarum L. Stem Caryophyllales Portulacaceae Little hogweed Portulaca oleracea L. Stem Solanales Solanaceae Habaero pepper Capsicum chinense Jacquin Fruit Solanale s Solanaceae Bell pepper Capsicum annum L. Fruit Solanales Solanaceae Tomato Solanum lycopersicum L. Fruit Solanales Solanaceae Potato Solanum tuberosum L. Stem tuber Typhales Typhaceae Southern cattail Typha spp. Stem

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108 Table 5 2 Larval development t imes (d) for three Ulidiidae reared on different plant species in Belle Glade, FL Commodity/ Weed Mean SEM (n; range) a C. massyla E. eluta E. stigmatias F P do Spiny amaranth 27.0 0.2Aa (26; 23 31) 19.9 0.2Ca (26; 17 22) 25.0 0.2Ba (26; 23 27) 154.54 < 0.0001 2, 75 Avocado 16.7 0.2Af (26; 15 18) 14.3 0.2Cd (26; 12 19) 15.2 0.2Be (26; 12 19) 31.33 < 0.0001 2, 75 Bell pepper 12.7 0.2Ah (26; 12 16) 10.1 0.2Cf (26; 8 12) 11.5 0.2Bh (26; 10 13) 48.50 < 0.0001 2, 75 Cabbage 14.9 0.2Ag (27; 13 17) 10.9 0.2Cf (27; 9 13) 14.0 0.2Bf (1739; 10 18) 146.83 < 0.0001 2, 78 Cattail 25.3 0.2Ab (28; 23 27) 19.1 0.2Ca (28; 17 21) 23.2 0.2Bb (28; 22 26) 236.90 < 0.0001 2, 81 Johnsongrass 21.4 0.2Ac (32; 18 24) 18.0 0.2Cb (32; 16 20) 19.0 0.2Bc (32; 17 21) 59.15 < 0.0001 2, 93 H abaero 19.4 0.2Ad (28; 17 21) 17.2 0.2Cb (27; 16 18) 18.7 0.2Bc (394; 17 21) 32.78 < 0.0001 2, 80 Papaya 15.5 0.2Ag (27; 13 17) 12.0 0.2Ce (27; 10 14) 12.7 0.2Bg (27; 11 15) 81.49 < 0.0001 2, 78 Little hogweed 21.0 0.2Ac (36; 19 23) 17.4 0.2Cb (36; 15 20) 18.9 0.2Bc (275; 16 22) 70.84 < 0.0001 2, 105 Radish 17.5 0.2Aef (31; 16 19) 15.4 0.2Cc (30; 13 17) 16.2 0.2Bd (31; 14 18) 51.67 < 0.0001 2, 89 Sugarcane 17.9 0.2Ae (25; 16 20) 15.7 0.2Cc (25; 14 18) 17.1 0.2Bd (25; 15 19) 34.68 < 0.0001 2, 72 Tomato 15.1 0.2Ag (28; 13 18) 11.0 0.2Cf (28; 10 14) 13.3 0.2Bfg (28; 11 16) 114.15 < 0.0001 2, 81 F 388.71 291.35 741.26 P < 0.0001 < 0.0001 < 0.0001 df 11, 328 11 326 11, 328 Mean SEM within a row followed by the same capital letter and within a column followed by the same small letter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute 2008) an = number of fruits/leaves/stems.

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109 Table 5 3 Comparison of pupal development times (d) of three species on various plant species in Belle Glade, FL. Common plant name Mean SEM (n; range) a F P df C. massyla E. eluta E. stigmatias Spiny amaranth 9.3 0.1Bb (25; 711) 7.7 0.1Cd ( 26; 69) 11.5 0.1Aa (26; 10 13) 168.07 < 0.0001 2, 74 Avocado 9.2 0.1Ab (26; 810) 12.0 0.1Ca (26; 1113) 11.5 0.1Ba (26; 11 12) 31.33 < 0.0001 2, 75 Bell pepper 6.6 0.1Bf (26; 6 7) 8.0 0.1Ad (26; 7.9 8.2) 6.0 0.1Cg (26; 5.8 6.2) 1,333.2 5 < 0.0001 2, 75 Cabbage 8.6 0.1Ccd (27; 8 9) 11.7 0.1Aa (27; 11 12) 9.2 0.1Bc (27; 8.9 9.4) 1,810.6 0 < 0.0001 2, 78 Cattail 7.3 0.1Ce (28; 5 9) 10.5 0.1Ab (28; 9 12) 9.8 0.1Bb (28; 9 10) 169.24 < 0.0001 2, 81 Johnsongra ss 8.8 0.1Abc (32; 8 1 1) 5.7 0.1Bf (32; 4 7) 8.5 0.1Ad (32; 7 9) 184.11 < 0.0001 2, 93 H abaero 6.8 0.1Cf (28; 5.7 7.4) 7.8 0.1Bd (27; 7 9) 8.8 0.1Ad (28; 8 9) 154.97 < 0.0001 2, 80 Papaya 8.1 0.1Ad (27; 7.8 8.2) 5.1 0.1Cg (27; 5.0 5.3) 6.0 0.1Bg (27; 5.7 6.3) 5,810.8 7 < 0.0001 2, 78 Little hogweed 10.4 0.1Aa (36; 812) 10.0 0.1Ac (36; 912) 10.1 0.1Ab (36; 9 12) 3.09 0.0497 2, 706 Radish 6.5 0.1Cfg (31; 5 7) 6.6 0.1Be (30; 6 7) 7.4 0.1Ae (31; 7 8) 57.49 < 0.0001 2, 89 Sugarcane 7.4 0.1Ae (25; 6 8) 6.0 0.1Cf (25; 5 7) 6.8 0.1Bf (25; 6 7) 94.62 < 0.0001 2, 72 Tomato 6.0 0.1Ag (28; 5.7 6.4) 5.1 0.1Cg (28; 5.0 5.3) 5.8 0.1Bg (28; 5.5 6.2) 448.61 < 0.0001 2, 81 F 175.80 739.76 741.26 P < 0.0001 < 0.0001 < 0.0001 df 11, 327 11, 32 6 11, 328 Mean SEM within a row followed by the same capital letter and within a column followed by the same small letter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute 2008) an = number of fruits/leaves/stems

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110 T able 5 4 Comparison of number of pupae emerged of three species of ulidiids on various plant s pecies in Belle Glade, FL. Common plant name Mean SEM (n; range) a F P df C. massyla E. eluta E. stigmatias Spiny amaranth 2.9 2.0 b Bf (26; 1 6) 5.2 2.0Af (26; 3 7) 4.3 2.0Ag (26; 1 9) 14.88 < 0.0001 2, 75 Avocado 35.6 2.0Bcd (26; 14 89) 54.3 2.0Ad (26; 32 82) 46.8 2.0Ad (26; 27 70) 9.30 0.0002 2, 75 Bell pepper 71.0 2.0Ba (26; 45 96) 107.3 2.0Aa (26; 66 179) 81.7 2.0Ba (26; 49 105) 23. 79 < 0.0001 2, 75 Cabbage 60.7 2.0Bab (27; 41 75) 87.6 2.0Ab (27; 50 136) 64.4 2.0Bb (27; 49 84) 33.04 < 0.0001 2, 78 Cattail 7.1 2.0Bef (28; 3 12) 10.0 2.0Af (28; 4 14) 7.8 2.0Bfg (28; 3 14) 9.94 0.0001 2, 81 Johnsongrass 6.1 1.8Bef (32; 1 16) 10.4 1.8Af (32; 1 20) 8.5 1.8ABfg (32; 1 14) 8.04 0.0006 2, 93 H abaero 12.7 2.0Aef (28; 5 25) 15.2 2.0Aef (27; 8 26) 14.1 2.0Aefg (28; 7 20) 2.01 0.1409 2, 80 Papaya 41.9 2.0Cc (27; 18 61) 73.4 2.0Ac (27; 49 101) 51.4 2.0Bcd (28; 24 81) 45.62 < 0.0001 2, 78 Little hogweed 7.2 1.7Aef (36; 1 12) 7.8 1.7Af (36; 1 18) 7.6 1.7Afg (36; 1 17) 0.25 0.7768 2, 105 Radish 14.0 1.9Be (31; 3 31) 25.3 1.9Ae (30; 16 39) 16.0 1.9Bef (31; 7 24) 30.26 < 0.0001 2, 89 Sugarcane 28.6 2 .0Ad (25; 15 47) 25.4 2.0ABe (25; 17 34) 22.4 0.9Be (25; 12 30) 7.75 0.0009 2, 72 Tomato 54.7 2.0Bb (28; 26 78) 77.1 2.0Abc (28; 34 121) 59.5 2.0Bbc (28; 43 84) 15.44 < 0.0001 2, 81 F 170.56 234.51 298.14 P < 0.0001 < 0.0001 < 0.0001 df 11, 328 11, 326 11, 328 Mean SEM within a row followed by the same capital letter and within a column followed by the same small letter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute 2008) an = number of fruits/ leaves/stems

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111 Table 5 5 Percentage pupal survival (d) of ulidiids on various plant s Common plant name Mean SEM (n; range) a Spiny amaranth 86.7 1.3d (78; 0 100) Avocado 99.9 1.3a (78; 98 100) Bell pepper 99.9 1.3a (78; 98 100) Cabbage 99.9 1.3a (81; 95 100) Cattail 98.2 1.2ab (84; 80 100) Johnsongrass 88.5 1.2 cd (96; 11 100) H abaero 89.1 1.2cd (83; 52 100) Papaya 99.9 1.3a (81; 96 100) Little hogweed 89.8 1.1cd (108; 40 100) Radish 97.7 0.2ab (92; 68 100) Sugarcane 93. 7 1.3bc (75; 61 100) Tomato 99.9 1.2a (84; 97 100) F 19.01 P 11, 954 df < 0.0001 Mean SEM within a column followed by the same small letter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute 2008 ) an = number of f ruits/leaves/stems

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112 Table 5 6 Ulidiidae species collected in fields from commodities/weeds in Florida. Plant name Date Location Sweep net/visual observation a Sampling unit C. massyla E. annonae E. eluta E. stigmatias Annona 10 June 2008 Garden b P ahokee 10 min observ 0 25 13 18 Annona 11 June 2008 EREC c 10 min observ 3 8 2 6 Spiny amaranth 17 June 2008 EREC 100 sweeps 0 3 29 1 Spiny amaranth 18 Aug. 2009 EREC 100 sweeps 11 1 19 4 Snap beans 4 Nov. 2008 Clewiston 100 sweeps 0 0 0 0 Snap beans 6 June 2008 Garden, EREC 10 min observ 0 29 12 4 Snap beans 9 June 2008 Garden, EREC 10 min observ 0 14 11 6 Snap beans 5 Aug. 2008 Garden, EREC 10 min observ 6 1 10 8 Snap beans 7 Oct., 15 Oct. 2008 Garden, EREC 10 min observ 0 0 0 0 Snap beans 10 Dec. 2008 EREC 100 sweeps 0 0 0 0 Snap beans 2 Dec. 2009 EREC 100 sweeps 16 0 31 11 Snap beans 28 May 2010 EREC 100 sweeps 3 5 21 5 Cabbage 15 April 2009 EREC 10 min observ 3 0 7 2 Cattail 18 Aug. 2009 EREC 100 sweeps 5 0 1 0 Cattail 2 Dec. 2009 EREC 100 sweeps 14 0 3 5 Johnsongrass 25 May 2009 EREC 100 sweeps 19 0 43 12 Johnsongrass 5 May 2010 EREC 100 sweeps 31 0 59 2 Little hogweed 7 Nov. 2008 EREC 100 sweeps 4 0 2 6 Little hogweed 20 Oct. 2009 EREC 100 sweeps 19 0 36 11 Santa Maria feverfew 17 June 2008 EREC 100 sweeps 0 0 4 1 Santa Maria feverfew 3 Apr. 2009 Pahokee 100 sweeps 2 0 15 10

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113 Table 5 6 continued Plant name Date Location Sweep net/visual observation Sampling unit C. massyla E. annonae E. eluta E. stigmatias Sorghum 26 Sept. 200 9 EREC 100 sweeps 6 0 1 20 Sugarcane 17 Aug. 2009 EREC 100 sweeps 7 0 3 0 Sugarcane 18 Aug. 2009 EREC 100 sweeps 12 0 2 1 Sugarcane 5 May 2010 EREC 100 sweeps 61 0 40 13 aSweep net/visual observation: Total number of flies found in 100 sweep nets/10 mi n visual observation. bGarden refers to a small scale plot (range 25 100 m2) cEREC refers to Everglades Research and Education Center, Belle Glade, FL.

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114 CHAPTER 6 COMPARATIVE MORPHOLO GY OF THE IMMATURE S TAGES OF THREE CORNINFESTING ULIDIIDAE The picturewinged fly Euxesta stigmatias Loew (Diptera: Ulidiidae) feeds on a wide range of fruits, vegetables and field crops (Seal and Jansson 1989) and is an economic pest of maze ( Zea mays L.), especially sweet corn (App 1938). This insect is highly injurious to maze with up to 100% damage to ears in untreated fields reported by many workers beginning in the 1940s (Bailey 1940, Nuessly and Hentz 2004, Seal and Jansson 1989). Several additional ulidiid species are known maze pests in the Caribbean and in North, C entral and South America ( Arce de Hamity 1986, Barbosa 1986, Evans and Zambrano 1991, Painter 1955, Wyckhuys and ONeil 2007) Four species in two genera are currently recognized pests of maze in Florida: Euxesta annonae (F.), E. eluta Loew, E. sti gmatias Loew and Chaetopsis massyla (Walker). While the genus Chaetopsis is restricted to the New World, the genus Euxesta occurs in both the Old and New Worlds. Previously referred to as Otitidae, Ulidiidae is the family name currently accepted by dipterists and used in the Biosystematics Database of World Diptera (Thompson 2006). Euxesta is represented by 31 species north of Mexico (Steyskal 1965) and 69 species south of the United States (Steyskal 1968). Chaetopsis is represented by 10 species each north of Mexico (Steyskal 1965) and south of the United States (Steyskal 1968) with 4 species common to both regions. Euxesta annonae, E. eluta, E. stigmatias and C. massyla can frequently be observed on maze in the same field in southern Florida; therefor e, species identification is important. While the adults can be identified using wing and other body features (Ahlmark and Steck 2010 unpublished key, Curran 1935, Goyal et al. 2010b), no such distinguishing characteristics have been reported that can be used to differentiate the

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115 immature stages of these four economically important species. General size and color characteristics have been reported for some immature stages of E. annonae ( Fras L 1981), E. eluta (Arce de Hamity 1986, Fras L 1981), E. stigm atias (App 1938, Hayslip 1951, Seal et al. 1995) and C. massyla (Allen and Foote 1992), but these immatures were all reared using different food sources under different environmental conditions. These measurements and observations are inadequate to separa te the species, because body size may vary greatly depending on the quality and quantity of food available. The current studies were undertaken to describe the immature stages of the three most common ulidiid species infesting corn in Florida ( C. massyla E. eluta and E. stigmatias ) and to search for characters suitable for distinguishing these species without relying on the timeand spaceconsuming practice of rearing the larvae found in ears to the adult stage. Materials and Methods Eggs, larvae and pu pae for this project were obtained from laboratory colonies maintained as described in the chapter on Pest status Egg Eggs of each species for our comparative morphology studies were collected from the surface of the diet within test tubes attached to t he inside top surface of adult cages. To obtain eggs of the same age for all three species, diet tubes were placed in the cages of each of the species at the same time and then examined for eggs every 2 h. Newly deposited eggs were removed from the diet using a c amel s hair brush (size 0) and placed on filter paper within an airtight container. Older eggs were obtained for microscopic examination by subsampling eggs from the original cohort of 2 h old eggs

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116 every 2 hr for a total of 20 h. Eggs were stor ed at 20 C to kill the eggs in preparation for imaging. Eggs were placed on glass slides to measure the greatest length and width of eggs using light microscopy before preparing them for scanning electron microscopy (SEM). No differences in egg surface characteristics were observed among eggs aged 2 to 20 h old. Eggs were selected to standardize the procedure for eggs of all three species. To present the various egg surfaces for examination, frozen eggs were thawed to room temperature and then variously attached to sticky carbon paper on aluminum stubs and sputter coated with goldpalladium (3 min) within a Denton Vacuum Desk III, LLC (Denton Vacuum, LLC USA, 1259 North Church St. Bldg 3, Moorestown, New Jersey). Eggs were imaged using a JEOL JSM 5510 LV SEM (JEOL Ltd., 1 2, Musashino 3chome Akishima, Tokyo, Japan) in an accelerating voltage of 15 kV at working distances of 10 and 25 mm to look for additional surface features that were not apparent under light microscopy. Larva Larvae in each of the three instars were initially examined for distinguishing characteristics. The larvae in diet tubes were examined daily from the day of eclosion to pupation. A subsample of larvae was collected daily to measure body length and widths. Larvae were then cleared in 10% NaOH for 1215 h to allow accurate measurements of the cephalopharyngeal skeleton (CPS). First and second instars were easily recognized based on body length and width, and nonoverlapping sizes of their CPS (Table 6 1). Third instar larvae were subsequently chosen for detailed morphological descriptions presented here because their structures were larger and more easily manipulated and measured than the earlier instars. N ewly molt ed 3rd

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117 instars were separated from other instars and then placed in new diet tubes to continue develop ment for 3 d before they were removed from the diet and prepar ed for detailed examination and imaging Third instar larvae were prepared for examination under dissecting and compound light microscopy by clearing them in 10% NaOH. Different subsets of l arvae were used for examining the structures on the posterior segment, the dorsal and lateral body surfaces, and the ventral body surface. The posterior larval segment was cut in cross section to the body to view the posterior spiracular plate and posteri or spiracles. Other specimens were cut with iridectomy scissors longitudinally along the middorsal line to allow the cuticle to lie flat for careful examination of the ventral surface. Similarly, some other specimens were cut longitudinally on the midv entral line for examination of the dorsal surface. Care was taken to avoid any damage to the mouth parts. The cut larvae and posterior larval segments were then cleared for 1215 h in 10% NaOH and the remaining body tissues were removed from the larvae using a c amel s hair brush. After clearing, the sclerotized mouth parts were carefully removed from the cut larvae with the aid of very fine forceps and mounted laterally on a glass slide. The larval cuticles and posterior spiracular plates were mounted f lat on glass slides, covered with glycerin and glass cover slips, and labeled. An ocular micrometer was used to measure body parts large enough to be viewed with a compound light microscope. The external larval characteristics observed included: the great est length and width of 3rd instar larvae; total number of creeping welts; size, number of rows, and arrangement of spinules in individual creeping welts; shape, size, and position of the anal lobes with respect to the anal opening; and the

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118 arrangement of spinules around the anal lobes. Several parameters of the CPS were examined including: total length and width; lengths of the mandible, hypopharyngeal sclerite, parastomal bar, dorsal arch, dorsal bridge, and dorsal and ventral cornua; and the presence or absence of a distinct tooth on the mandibles (Fig s. 6 3a, b, c, d, e, and f). The larval mouth parts were photographed using a Syncroscopy Automontage System (Syncroscopy, 5108 Pegasus Court, Suite M, Frederick, Maryland) with JVC KY F70B digital camera (JVC Headquarters and East Coast Sales, 1700 Valley Road, Wayne, New Jersey) mounted on a DMLB compound microscope (Leica Microsystems Inc., 2345 Waukegan road, Bannockburn, Illinois). The head and thoracic sections of larvae were examined for the size and shape of the antennae, number of oral ridges, and the size and shape of papillae on the anterior spiracles. The caudal segment was examined to measure the length of posts supporting the posterior spiracles; the distance between the posts; the number and arrangement of spiracular slits; the size and position of the spiracular scar; and the number, arrangement and branching pattern of spiracular hairs. The trunks and tips were counted for all the posterior spiracular hairs. Ratios of measurements on related structures also were calculated for use as characteristics to differentiate the species and to avoid the biases caused by unusually small or large specimens. Some larvae were cross sectioned and mounted vertically to obtain SEM views of the anterior and posterior surface structures. The methods of Grodowitz et al. (1982) were followed to prepare the larval specimens for examination under SEM. Live 3rd instar larvae were rinsed 2 times in deionized water followed by sonication in mild detergent for 30 s. The larvae were again rinsed 2 times in deionized water and then

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119 placed for 1 min in super skipper solution (kerosene, 17parts; glacial acetic acid, 11parts; 95% ethyl alcohol, 50 parts; isobutyl alcohol, 17 parts; dioxane, 5 parts) (Elzinga 1981) fol lowed by two rinses in Carls solution (95% ethyl alcohol, 28 parts; 40% formaldehyde, 11 parts; glacial acetic acid, 4 parts; water, 57 parts) (Barbosa 1974). The larvae were then placed in Carls solution for 1215 h followed by a dehydration series in increasing concentrations of ethyl alcohol (15 min in each of 30, 50, 70, 80, 90, 95% and 3 times in 100%). The specimens were dried using a Samdri 780A Critical Point Dryer (Tousimis Research Corporation, P.O. Box 2189, Rockville, Maryland). The specimens were variously attached to sticky carbon paper on aluminum stubs to allow SEM views of both dorsal and ventral sides. Specimens were sputter coated with goldpalladium dorsally, ventrally and laterally (3 min each). Pupa Four day old pupae were removed from the colony and stored in 70% ethyl alcohol for examination. Pupae were examined under light microscopy using a dissecting microscope. The anterior and posterior surfaces were observed by placing pupae upright in small depressions in styrofoam block s. The greatest length and width of pupae, the number and position of papillae in anterior spiracles, the position of posterior spir ac les, and the shape of posterior spiracular plates were observed and described (Figs. 6 4a, b, c, and d). The pupae were photographed with JVC 3 CCD camera (JVC Headquarters and East Coast Sales, 1700 Valley Road, Wayne, New Jersey) and Z16 APO lens (Leica Microsystems Inc., 2345 Waukegan Rd. Bannockburn, I llinois ). Voucher specimens for each species were deposited in the F lorida State Collection of Arthropods at the Division of Plant Industry (DPI), Florida Department of

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120 Agriculture and Consumer Services (FDACS), Gainesville, Florida. Morphological terminology used in this manuscript follows Teskey (1981). Terminology of the maxillary palp and dorsolateral group follows ChuWang and Axtell (1972). Statistical Analysis One way analysis of variance (PROC GLM, SAS Institute 2008) was used to determine whether the measurements and ratios of measurements were affected by specie s (fixed variable). Replicates were used as random effects. The Tukey's honestly significant difference (HSD) test (SAS institute 2008) was used for means separation with P = 0.05. Descriptions Chaetopsis massyla Egg. White, elongateovoid to somewhat s pindle shaped; posterior end smoothly rounded while tapering from micropylar end; widest in middle (Fig. 6 1a); length 0.68 0.01 (mean SEM) (range 0.60 0.75) mm; width 0.15 0.003 (0.12 0.18) mm; length: width 4.61 0.09 (3.55 5.71) (n = 36); diameter of collar surrounding micropyle 0.02 0.0005 (0.02 0.03) mm (n = 36); hatching line (hl) starting from the micropylar end and spanning approximately 20 25% of the egg length (Fig. 6 1b); micropyle situated in a shallow pit at the anterior end of the egg; chorion appearing to be smooth under a light microscope, but reticulated with elevated ridges under SEM; entire egg surface covered mostly with elongate hexagons, occasionally irregular squares, pentagonal, and hexagonal cells; cell length reduces towards both ends of eggs and ranges from 14 40 m for anterior, 20 42 m for middle and 16 34 m for posterior cells (based on randomly selected cells from 100 m long sections in the anterior, middle and posterior regions of eggs); distinct por es 1.95 0.09 (1.05 2.92) m diam (n

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121 = 36) where present at the vertices of polygons on the chorion surface at the posterior end of eggs (Fig. 6 1c). Larva. General structure. Creamy white to yellow in color elongate in shape with a pointed head, and br oad, rounded posterior end ; third instar length 8.23 0.09 (7.25 9.25) mm; width 1.17 0.04 (0.45 1.50) mm; length:width 7.40 0.50 (5.17 18.33) (n = 24) (Table 6 2). Head. Antennomaxillary complex (Fig. 6 2a, d): ce phalic segment bilobed with an antennal and maxillary sensory complex at the end of each lobe (Fig. 6 2a); antenna 2segmented, distal segment apically hemispherical; antennal length approximately 1.5x greater than width (Table 6 2); maxillary complex with 3 papilla sensillae (p1, p2, p3) and 2 knob sensillae (k1, k2); dorsolateral group bearing 2 additional well developed papilla sensillae (p, m) much closer to palp than to antennae (Fig. 6 2d). Oral ridges (Fig. 6 2a): 23 0.4 (19 27) oral ridges (or) (n = 29) below the antennomaxil lary lobes; number varies between the two sides of the same larva; finely dentate with individual processes pointed and curved inward towards the mouth cavity; a few of the dentate processes in broken groups near the ends of the oral ridges close to the an tennomaxillary complex; two medial oral lobes (mol) on the inside of the mandibles; labial lobe (lal) at posterior of mouth cavity. Cephalopharyngeal skeleton (CPS) (Figs. 6 3a, d): sclerotized structure consisting of three major parts : mandibles (m), hypopharyngeal sclerite (hps), and t entoropharyngeal sclerite (tps) (Fig. 6 3a and Table 6 2); two mandibles (Fig. 6 3d) with elongate, heavily sclerotized basal half and lightly sclerotized distal half comprising sharp sickle shaped mouth hooks that are dista lly grooved ventrally, a distinct tooth (at)

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122 ventrally at about the midpoint of the mouth hook just posterior to the end of the groove (Fig. 6 3d); hps lying between the mandibles and tps with posterior portion of hps overlapping the anterior margin of tps two longitudinal bars articulated broadly with the mandibles anteriorly and with the tps posteriorly (Fig. 6 3a); posterior portion of the hps lies interior to the anterior ventral cornua; two bars of the hps joined on the ventral side by a lightly scler otized bar forming an H shape when viewed ventrally; a pair of slender parastomal bars fused to the hps for half their length posteriorly then tapering and diverging over the anterior half; the labial sclerite and the epipharyngeal sclerite lie below hps towards its anterior end; tps comprised mainly of dorsal cornu and ventral cornu, upper and lower arms of U shaped sclerites on ei ther side of the pharynx; ventral cornua fused on each side with the pharynx; dorsal cornua at their anterior end joined by a dorsal bridge. Thorax. Three thoracic segments clearly visible; no creeping welts on any of the thoracic segments; 2530 discontinuous rows of spinules (sr) encircle the anterior one third of the first thoracic segment (Fig. 6 2a). Anterior spiracles: str ucture projects laterally on prothorax like a fan from middle part of segment (Figs. 6 2e, 6 3g); spiracular openings (so) in depressions at the ends of almost equal length finger like papillae (pa) projecting from a common base; papillae in a single row i n the shape of a fan, vary in number (810) between sides of the same larva; papillae increase in width towards their distal end; only the papillae and a small portion of the main body of the spiracle normally seen outside the larval body under SEM while t he remainder hidden within the body; length of spiracle (asl) about equal to

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123 its width (asw) (Table 6 2); papillae length (pl) about 1.5 2x greater than their width (pw). Abdomen. Eight abdominal segments clearly visible. Locomotory structures: creeping welts (Fig. 6 2i, j, k and Table 6 3 ) present on the ventral s urface of anterior margins of all eight abdominal segments (i.e., CW1 to CW8) ; welts comprise transverse swollen ridges bearing multiple rows (R) of spinules which were all pointed, curved and symmetrical to asymmetrical; width of CW8 narrower than other welts; CW3, CW4, and CW6 wider than CW7 and CW8; CW7 wider than CW8 ; CW4 and CW5 longer than CW1 to CW3; welt width to length ratio greatest for CW1 followed by CW2 that was followed by CW4 to C W8. CW1 and CW8 with 5 rows of spinules (i.e., R1 to R5) ; CW2 usually with 6 rows, but occasionally with 5 rows; CW3 to CW7 with 6 rows of spinules Spinules in R1 of each welt anteriorly oriented while posteriorly oriented in following rows; CW1 with al l the spinules of almost same size; spinule groups in R4 of all welts except CW1 are angled to body midline and thus appear to radiate from the body with angle increasing laterally. CW1: R1 to R4 discontinuous, R5 continuous. CW2 to CW8: R1, R2, R4, and R5 discontinuous; R3 and R6 continuous, rarely discontinuous; spinules in R4 broader and in distinct groups on raised ridges with 49 spinules in each group; spinule size largest in R4 reducing in size in rows anterior and posterior to R4. CW2 to CW7: spi nules of R4 and R5 overlap spinule groups of the same row and join spinules between different rows at various points. CW8: spinules in R4 merge with those of R5 when the latter are discontinuous. Posterior spiracles (Fig. 6 3j and Table 6 2): present on elevated sclerotized cylindrical posts on the last abdominal segment and facing posteriorly; spiracular plate

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124 bearing a circular ecdysial scar and 3 elongateoval shaped spiracular openings arranged at right angles; ecdysial scar situated at the medial marg in of the spiracular plate; moderately developed rimae (rm) and numerous trabeculae (tr) on the spiracular openings; four groups of spiracular hairs arising from the spiracular plate between the ecdysial scar and the spiracular openings; spiracular hairs m ore than half the length of the spiracular posts; SP 1(first on left from ecdysial scar) comprising 3 4 trunks and 21 28 tips, basal width 6 16 m (n = 23 specimens); SP 2 (second on left from ecdysial scar) comprising 2 4 trunks and 14 35 tips, basal width 8 18 m; SP3 (third on left from ecdysial scar) comprising 2 4 trunks and 14 28 tips, basal width 4 14 m; SP4 (fourth on left from ecdysial scar) comprising 3 4 trunks and 20 28 tips, basal width 6 19 m. Anal complex : paired w rinkled and grooved anal lobes on either side of the anus (Fig. 6 2l and Table 6 2) present ventrally on anterior portion of last abdominal segment; anal lobes surrounded by 1 or occasionally 2 rows of spinules overlapping each other at several places and a few groups of spinules scattered around the anal lobes more laterally; spinules pointed and curved and almost always posteriorly oriented, except a few of the spinules anterior to anal lobes oriented anteriorly in few specimens; a robust anal hook with 6 9 sharp points at the posterior end of the anus. Pupa. Length 4.95 0.06 (4.38 5.75) mm; maximum width at 3rd abdominal segment 1.58 0.01 (1.35 1.75) mm (n = 39) (Fig. 6 4d); reddish to reddish brown in color; barrel shaped, narrowing sharply towards the anterior end while broadly towards the posterior end; notable cuticle shrinkage at the anterior end; anterior spiracle visible with only papillae and small portion of stalk protruding as in third instar; all creeping

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125 welts clearly visible similar to third instar; anal lobes reddish brown to black in color; spinules around anal lobes clearly visible; posterior spiracular plate surrounded by groves forming trapezoidal shape (Fig. 6 4a); the posts supporting posterior spiracles brown in color; poster ior spiracles dark black in color with spiracular hairs apparent. Euxesta eluta Egg. Color and shape of eggs similar to those of C. massyla ; l ength 0.72 0.01 (0.62 0.80) mm; width 0.14 0.001 (0.12 0.14) mm; length:width 5.27 0.06 (4.56 6.11) ( n = 33); diameter of collar surrounding the micropyle 0.02 0.0003 (0.02 0.03) mm (n = 33) (Fig s. 6 1d, e). The remainder of the description is the same as for eggs of C. massyla except for the following: length of polygonal cells on chorion surface r ange from 12 36 m for anterior, 24 35m for middle, and 20 37m for posterior cells; chorion covered with two sizes of pores over entire surface when observed under SEM; minute pores 0.89 0.04 (0.72 1.27) m diam (n = 20) widely distributed across the surface, but do not cover the seam of the hatching line (hl); distinct pores 2.56 0.07 (1.91 3.00) m diam (n = 33) present at the vertices of polygons at posterior end of eggs (Fig. 6 1f). Larva. General structure. Third instar larvae of E. e luta are the same as in C. massyla larvae, except for the following: length 7.52 0.09 (6.39 8.63) mm; width 1.18 0.01 (1.10 1.38) mm, length to width ratio 6.38 0.10 (5.17 18.33) (n=23) (Table 6 2) Head. Antennomaxillary complex (Fig. 6 2b) : same as in C. massyla except that the antennal length is approximately 1.18x greater than the width (Table 6 2).

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126 Oral ridges : 34 0.6 (30 42) oral ridges (or) (n = 25) present below the antennomaxillary lobes (Fig. 6 2b); remainder of description is the same as for C. massyla Cephalopharyngeal skeleton (CPS) : same as that of C. massyla except that no distinct tooth as seen on mandibles of C. massyla (Fig s. 6 3b, e) (Table 6 2, 6 4) Thorax. Three thoracic segments visible; no creeping welts on any of the thoracic segments; 27 30 discontinuous rows of spinules (sr) encircle the anterior third of the first thoracic segment (Fig. 6 2b). Anterior spiracles (Figs. 6 2e, 6 3h): length (asi) about 1.23x greater than width (asw) (Table 6 2); 7 10 finger l ike papillae in a single row ; papillae length (pl) about 1.4x greater than width (pw) (Table 6 2); remainder of the description is the same as that of C. massyla Abdomen. Eight abdominal segments are clearly visible. Locomotory structures: c reeping welts on the ventral side of the anterior margins of all eight abdominal segments (Table 6 3); welts comprise transverse swollen ridges bearing rows of spinules which are all pointed, curved and symmetrical to asymmetrical CW3 to CW5 wider than CW1, CW2, CW7 and CW8; CW4 to CW6 longer than CW 1 CW3 and CW8 ; CW1 shorter than other welts; welt width to length ratio greatest for CW1 followed by CW2 and CW3 and then by CW6 to CW8 Spinule groups in R4 of all welts except CW1 angled to body midline and thus appear to radiate from the body with the angle increasing laterally. CW1 and CW8 have 5 rows of spinules; CW2 to CW7 have 6 rows of spinules; CW1, majority of spinules were of equal size; CW2 to CW8, spinule size largest on R4 and in distinct groups on raised ridges with 3 8 spinules in

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127 each group and reducing in size in rows anterior and posterior to R4. R1 spinules on CW1 and CW2 with anterior orientation (occasionally with posterior orientation); R1 spinules on CW3 to CW7 with anterior orientation, but R 2 to R6 with posterior orientation, R4 spinules angled to body midline and thus pointing opposite to each other. CW1 to CW3: R1 and R2 usually continuous (rarely discontinuous), R3 and R4 discontinuous. CW2 and CW3: R5 and R6 continuous. CW4 to CW8: R2, R4, R5 discontinuous while R1, R3 and R6 continuous. CW2 to CW7: spinules of R4 and R5 overlap spinule groups of the same row and join spinules between different rows at various points. CW8: spinules in R4 merged with those of R5 when the latter were di scontinuous. Posterior spiracles (Fig. 6 3k and Table 6 2): SP 1 comprising 2 3 trunks and 19 25 tips. basal width 5 15 m (14 specimens); SP 2 comprising 2 3 trunks and 14 26 tips, basal width 6 17 m; SP3 comprising 2 4 trunks and 12 21 tips, basal width 6 18 m: SP 4 comprising 2 4 trunks and 18 29 tips; basal width 6 21 m. Remainder of description is the same as in C. massyla. Anal complex: the description is the same as that of C. massyla (Fig. 6 2k and Table 6 2). Pupa. Length 4.25 0.06 (3.75 4.75) mm; maximum width at 3rd abdominal segment 1.42 0.03 (1.25 1.70) mm (n = 34) (Figs. 6 4b, d). Light brown (but not reddish) to black in color. The remainder of description is the same as in pupae of C. massyla except for the following: posterior spiracular plate surrounded by groves forming ovoid shape; posts supporting posterior spiracles black in color.

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128 Euxesta stigmatias Egg. Color and shape of eggs similar to those of C. massyla ; length 0.76 0.01 (0.69 0.83) mm; width 0.15 0.004 (0.12 0.19) mm; length:width 5.20 0.12 (4.05 6.69) (n = 39); (Figs. 6 1g, h, i); diameter of collar surrounding the micropyle is 0.02 0.0002 mm (n = 39). The remainder of the description is the same as for eggs of C. massyla and E. eluta except for the following: length of polygonal cells on chorion surface range from 12 44 m for anterior, 24 38 m for middle, and 15 37 m for posterior cells; chorion covered with two sizes of pores over entire surface when observed under SEM; minute pores 0.63 0.06 (0.16 0.81) m diam (n = 14) widely distributed across the surface, but do not cover the seam of hatching line (hl); distinct pores 2.84 0.07 (2.353.52) m diam (n = 39) present at vertices of polygons at posterior end of eggs (Fig. 6 1i). Larva. General structure. Third instar larvae the same as in C. massyla except the following: length, 6.24 0.14 (5.25 7.75) mm and width 0.95 0.03 (0.60 1.15) mm, length: width 6.64 0.24 (5.83 11.67) (n = 24) (Table 6 2) Antennomaxillary complex : same as in C. massyla (Fig. 6 2c and Table 6 2) Oral ridges: 35 0.6 (31 40) oral ridges (or) (n = 22) present below the antennomaxillary lobes (Fig. 6 2c ). Remainder of the description is the same as for C. massyla. Cephal opharyngeal skeleton (CPS) : same as that of C. massyla except no distinct tooth as seen on mandibles of C. massyla (Fig s. 6 3c, f and Table 6 2). Thorax. Three thoracic segments clearly visible; n o creeping welts on any of the thoracic segments ; 27 29 discontinuous rows of spinules (sr) encircle the anterior third of the first thoracic segment (Fig. 6 2c)

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129 Anterior spiracles: same as for C. massyla except the following (Figs. 6 2g, h, 6 3i); spiracle (asl ) length about 1.3x width (asw) (Table 6 2 ) ; 7 10 finger like papillae in a single row ; p apillae about 2x longer than wide (Table 6 2 ). Abdomen. Eight abdominal segments are clearly visible. Locomotory structures: Creeping welts on the ventral side of anterior margins of all 8 abdominal segments (Tabl e 6 3); welts comprise transverse swollen ridges bearing rows of spinules which were all pointed, curved and symmetrical to asymmetrical. CW1, CW7, and CW8 narrower than CW4 ; CW3 to CW7 longer than CW1, CW2 and CW8; width to length ratio significantly greater for CW1 followed by CW2 followed by CW3 to CW8 The size, shape, number of rows, angle, grouping and the orientation of spinules on the rows were the same as for E. eluta CW1 and CW2: R1, R2, R4 and R5 discontinuous (the later two rarely continuous ) R3 and R6 (CW2) continuous ; CW3 to CW8 : R2, R4, and R5 discontinuous while R1, R3 and R6 continuous (R1 rarely discontinuous) Posterior spiracles ( Fig. 6 3l): SP1 comprising 2 4 trunks and 22 31 tips basal width 5 14 m (14 specimens) ; SP 2 com prising 2 4 trunks and 11 25 tips basal width 5 15 m ; SP3 comprising 2 4 trunks and 13 27 tips basal width 5 15 m ; SP4 comprising 2 4 trunks and 15 29 tips basal width 6 18 m. The remainder of the description is the same as in C. massyla (Table 6 2). Anal complex (Fig. 6 2l): same as for C. massyla and E. eluta except that the anal hook has 5 8 points (Table 6 2). Pupa. Length 3.92 0.05 (3.40 4.75) mm; maximum width at 3rd abdominal segment of 1.36 0.02 (1.05 1.60) mm (n = 33) (Figs. 6 4c, d). Light brown (but not

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130 reddish) to black in color. Description is the same as that of pupae of C. massyla except the following: posterior spiracular plate surrounded by groves forming an ovoid shape; posts supporting posterior spir acles black in color. Diagnoses Egg The only characteristic found t o differentiate eggs of the two genera wa s the size and distribution of pores. All three species had pores at the vertices of polygons on the egg surface but only E. eluta and E. stigmatias also had pores over the entire egg surface (Table 6 4). Significant differences were determined for several additional egg characteristics but overlapping ranges prevent them form being diagnostic for species separation. The diameter of the pores at the vertices of polygons at the posterior end of eggs varied significantly among species ( F = 33.68, df = 2, 105; P < 0.0001) and was greatest in E. stigmatias and smallest in C. massyla Egg length ( F = 38.95, df = 2, 105; P < 0.0001) width ( F = 7.78, df = 2, 105; P = 0.0007) and length to width ratio ( F = 14.58, df = 2, 105; P < 0.0001) varied significantly among species Euxesta stigmatias eggs were the longest and C. massyla eggs were the shortest. Eggs of E. eluta were narrower than the other two species. The length to width ratio wa s smaller in C. massyla than the other two species No consistent differences in egg shape or color were found. No difference in egg collar diameter was found among the three species ( F = 1.68, df = 2, 105; P = 0.19 06) Larva The body length of first instar larvae varied significantly among species (Table 6 1). The first instars of C. massyla and E. eluta were significantly longer than those of E. stigmatias Body width and CPS length were not significantly differe nt among species.

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131 Due to considerable overlap in ranges of body measurements, no body measurements of first and second instar larvae were suitable for separating the three species. Several characteristics were found in the current studies that could be us ed to distinguish third instar larvae among the species. These characteristics were the presence or absence of a distinct tooth on the mandibles, number of oral ridges, differences in the continuity of the first row of spinules (R1) in creeping welts, dif ference in length of a creeping welt and color of the posterior spiracles (Table 6 4). The mandibles have a distinct tooth in all C. massyla specimens examined while no distinct tooth was visible on E. eluta and E. stigmatias mandibles. The number of or al ridges varied significantly among species ( F = 166.97, df = 2, 73; P < 0.0001) The mean number and range of oral ridges was less in Chaetopsis massyla than in either of the Euxesta species. The R1 in creeping welts wa s discontinuous in CW2 to CW8 of C. massyla but continuous in CW4 to CW8 of E. eluta and CW3 to CW8 of E. stigmatias The width, length, and width:length ratio of creeping welts varied significantly among species (Table 6 3); however, CW2 was the only welt with mean separation and nono verlapping ranges suitable for distinguishing E. eluta from E. stigmatias The posterior spiracles in E. eluta remained dark black in color after the 12 15 h 10% NaOH treatment while they had weakened to light brown in C. massyla and dark brown to black i n E. stigmatias (Figs. 6 3 j, k, l ). The various parts of the posterior spiracular plate can be seen after clearing with 10% NaOH on C. massyla but they are indistinguishable in E. eluta and difficult to differentiate in E. stigmatias Several other larv al characteristics were found with significant differences among the three species, but they could not be used to distinguish the species due to their

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132 overlapping ranges. The mean length and width of third instar E. stigmatias larvae were significantly le ss than the two other species (Table 6 2). The mean length of C. massyla larvae was significantly longer than E. eluta, but the mean widths of the two species were not significantly different. No significant difference was found in mean larval width:leng th ratios among the three species. Mean antennal length, width and their ratio did not vary significantly among the three species. The mean overall length of the cephalopharyngeal skeleton (CPS) was longer in C. massyla than in the Euxesta species. Mandible segment d was longer in C. massyla than the other species, while mandible segment a was longer in E. stigmatias than the other species. Mandible b was longer in C. massyla than E. stigmatias None of the ratios of various mandible lengths was consis tently largest or smallest in any one species. The dental sclerite hps overlap of tps over hps dorsal cornu and notch lengths were all significantly greater in C. massyla than in E. eluta and E. stigmatias Mean ventral cornu and parastomal bar lengths were significantly greater in C. massyla than in E. stigmatias which were greater than in E. eluta The dorsal bridge length was significantly shorter in C. massyla than in the other species. Other CPS parameters were not found to be different among spec ies. The mean length and length:width of the anterior spiracles were significantly greater in C. massyla than in the two other species. The width of the anterior spiracles was greater in E. stigmatias than in C. massyla The length and length:width of pa pillae on the anterior spiracles of E. stigmatias were significantly greater than on E. eluta The mean width of CW1 to CW7 was greater in C. massyla and E. eluta than in E. stigmatias The majority of E. stigmatias welts were narrower than the other species. Significant differences in the width:length ratio were only observed in CW2 and CW4.

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133 Spinule size and orientation (anterior/posterior) did not vary in any welt among the three species. The mean length of the posts supporting the posterior spiracles was significantly greater in C. massyla than in the other species, while the distance between the two posterior spiracles was significantly greater in E. eluta than in the other species (Table 6 2). Rima thickness ( rm ) in C. massyla was nearly 2x as wide as in E. stigmatias Rima thickness could not be accurately measured for E. eluta using a light microscope due to the poor clearing of the posterior spiracles using 10% NaOH. The mean width and length:width of the anal complex were significantly larger for E. eluta than E. stigmatias followed by C. massyla The mean length of the anal hook was significantly shorter in C. massyla than in E. eluta The mean length of the anal complex did not differ significantly among the three species; however, the mean length of the anus in E. stigmatias was significantly shorter than in C. massyla Pupa The overall color and the shape of the posterior spiracular plate were the only pupal characteristics that were distinctive enough for use in separating the three spec ies. The pupae of C. massyla were reddish in color compared to the light brown to black pupae of E. eluta and E. stigmatias (Table 6 4 d ). The posterior spiracular plate in C. massyla pupae was trapezoidal while those of E. eluta and E. stigmatias were ovoid, lacking the hard angles observed in C. massyla (Fig. 6 4 a, b, c ) Some pupal characteristics were found with significant differences among the three species, but they could not be used to distinguish the species due to their overlapping ranges. Th e mean pupal length, width and their ratio were significantly greater in C. massyla than in E. eluta which were greater than in E. stigmatias

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134 Discussion The above descriptions of morphological characters for the immature stages of C. massyla E. eluta and E. stigmatias add to the physical descriptions for these species in the literature. The descriptions and measurements published by previous workers generally fall within the ranges determined in the present study, with some notable exceptions. The differences in diet among the studies may help explain some of the significant differences reported. Most of the comparative studies were conducted on immatures that were field collected or otherwise reared from plants compared to the artificial diets used in our study. Allen and Foote (1992) described eggs of C. massyla reared on decayed cattail and lettuce leaves as white, semi polished, spindle shaped with striated chorion, 0.86 0.94 mm long (significantly longer than in the current study) and 0.15 0. 18 mm wide. Euxesta eluta and E. stigmatias eggs in our study were within the range of size described in other studies. Fras L (1981) described E. eluta eggs found in sweet corn in Chile as white in color, 0.7 mm long and 0.15 mm wide. Arce de Hamity ( 1986) reported that eggs of E. eluta collected in Jujuy, Argentina reared on artificial diet were cylindrical, dull white in color with a thin and sculpturous chorion, 0.73 0.03 mm long and 0.12 0.002 mm wide. App (1938) found eggs of sweet cornrea red E. stigmatias to be minute, elongate and whitish. Seal et al. (1995) found E. stigmatias eggs from field collected sweet corn ears to be creamy white, slender, 0.85 0.004 mm long (slightly longer than those in our study), and 0.16 0.001 mm long. Haylsip (1951) reported E. stigmatias eggs in sweet corn to be white, slender and 0.78 mm long. Our measurements of third instar larval characters for these three species closely resemble those made by previous workers. Allen and Foote (1992) descri bed 3rd instar

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135 larvae of C. massyla as white with transparent integument, 4.50 9.90 mm long; 0.57 1.28 mm wide; CPS length 0.99 1.10 mm; anterior spiracles with 9 12 finger like marginal papillae. Arce de Hamity (1986) reported 3rd instar larvae o f E. eluta to be 7.55 0.73 mm long, 1.18 0.33 mm wide, with anterior spiracles 0.09 0.008 mm long and 0.15 mm wide They also found the ratio of the hypostomal (other name for hps) to pharyngeal sclerites (other name for tps) as 1:4 in comparison to as the value 1:3.87 [hps/ventral cornu (tps)] found in our study. Fras L (1981) described E. eluta mandibles as black posteriorly and brown anteriorly. Seal et al. (1995) reported E. stigmatias larvae as 5.39 0.04 mm long and white in color with a gl ossy cuticle that turns yellow as larvae mature. The measurements of E. stigmatias larvae length reported by App (1938) were 9.38 mm, nearly 33% longer than those observed in our study. Published observations of pupal characteristics were nearly identical to those found in our study. Allen and Foote (1992) described pupae of C. massyla as reddish brown, 3.06 5.50 mm long and 0.96 1.63 mm wide. Arce de Hamity (1986) found the length and width of E. eluta pupae to be 4.25 0.42 mm and 1.44 0.15 mm, respectively. Seal et al. (1995) found pupae of E. stigmatias to be barrel shaped, reddish brown in color, 3.91 0.02 mm long and 1.37 0.01 mm wide. Only a few characteristics for each developmental stage were found in our study that could be used to s eparate the immature stages of C. massyla E. eluta and E. stigmatias without rearing them to the adult stage. While the means of most of the characteristics were significantly different among species, their overlapping ranges made them unsuitable for sep arating the species. Moreover, some of the

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136 characteristics found to separate the species (egg surface, larval mandibles, larval creeping welts, larval posterior spiracles, and pupal posterior spiracular plate) require the use of compound and scanning elec tron microscopes which limits their usefulness to field scouts, growers or others working in field conditions. However, pupal color and shape of posterior spiracular plates in pupae can be used to separate the pupae of two genera ( Chaetopsis and Euxesta) in the field using a hand lens.

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137 Table 6 1. Range of body measurements (mm) on 1st and 2nd instar larvae of three species of picturewinged fly pests of corn Larval body measurements Mean SEM (n; range) Instar C. massyla E. eluta E. stigmatias F d f P Body length 1st 1.83 0.09A (12; 1.20 2.20) 1.78 0.10A (13; 1.00 2.10) 1.4 0.14B (9; 0.90 2.00) 4.30 2, 31 0.0225 2nd 4.25 0.20 (15; 2.60 4.90) 3.72 0.27 (10; 2.40 5.00) 3.52 0.29 (12; 2.20 4.80) 2.58 2, 34 0.0905 Body width 1st 0.19 0.01 (12; 0.15 0.25) 0.20 0.01 (13; 0.15 0.22) 0.18 0.01 (9; 0.12 0.22) 1.43 2, 31 0.2555 2nd 0.40 0.02 (15; 0.30 0.50) 0.36 0.03 (10; 0.25 0.50) 0.39 0.02 (12; 0.25 0.45) 1.27 2, 34 0.2926 Cephalopharyngeal skeleton length 1st 0.26 0.01 (12 ; 0.22 0.30) 0.28 0.01 (13; 0.20 0.32) 0.26 0.01 (9; 0.20 0.30) 1.49 2, 31 0.2407 2nd 0.56 0.01 (15; 0.50 0.60) 0.55 0.02 (10; 0.45 0.65) 0.52 0.02 (12; 0.45 0.60) 2.60 2, 34 0.0893 ANOVA (PROC GLM, SAS Institute 2008) Means followed by different letters within a row are significantly different (Tukey, P > 0.05, SAS Institute 2008).

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138 Table 6 2. Comparison of 3rd instar larval measurements among three species of picturewinged fly pests of corn Larval measurementsa Mean SEM (n; range) C. m assyla E. eluta E. stigmatias Larval Length (L) (mm) 8.23 0.0A (24; 7.25 9.25) 7.52 0.09B (23; 6.39 8.63) 6.24 0.14C (24; 5.25 7.75) Width (W) (mm) 1.17 0.04A (24; 0.45 1.50) 1.18 0.01A (23; 1.10 1.38) 0.95 0.03B (24; 0.60 1. 15) L:W 7.40 0.50A (24; 5.17 18.33) 6.38 0.10A (23; 5.50 7.22) 6.64 0.24A (24; 5.83 11.67) Antenna Length (al) 15.29 1.20A (12; 11.00 22.50) 14.69 1.99A (10; 8.80 22.50) 15.29 1.34A (12; 11.00 22.50) Width (aw) 10.67 0.33A (12; 9.00 12.50) 12.19 1.13A (10; 8.80 20.00) 12.38 0.49A (12; 11.00 15.00) al:aw 1.46 0.13A (12; 1.00 2.25) 1.18 0.09A (12; 0.80 1.80) 1.22 0.07A (12; 1.00 1.80) Anterior spiracle Length (asl) 115.88 3.15A (15; 92.40 132.00) 105.93 2.02B (23; 88.00 125.00) 103.57 2.84B (15; 81.40 122.50) Anterior spiracle Width (asw) 120.36 3.42B (15; 99.00 145.00) 130.36 3.35AB (22; 110.00 175.00) 135.66 4.98A (16; 99.00 185.00) asl:asw 0.97 0.03A (15; 0.81 1. 11) 0.82 0.02B (22; 0.66 0.96) 0.77 0.03B (15; 0.54 1.11) Basal width (tw) 53.11 1.93A (16; 33.00 66.00) 50.27 1.46A (24; 39.60 62.50) 50.94 1.88A (17; 32.50 67.50) Papilla length (pa) 21.19 0.62AB (14; 17.50 25.00) 19.57 0.66B (19; 15.00 25.00) 24.03 1.46A (12; 17.50 32.50) Papilla width (pw) 13.24 0.58A (14; 10.00 17.60) 14.13 0.72A (19; 10.00 20.00) 12.82 0.32A (14; 11.00 15.40) pa:pw 1.63 0.07AB (14; 1.33 2.00) 1.44 0.08B (19; 1.00 2.25) 1.85 0.1 3A (12; 1.33 2.60) Posterior spiracle Post diam 140.86 2.88A (24; 121.00 175.00) 140.71 3.22A (23; 106.80 162.50) 148.33 4.91A (21; 120.00 187.50) Post length 119.53 3.42A (16; 87.50 137.50) 104.17 3.69B (21; 62.50 125.00) 98.21 3.14B (21; 75.00 137.50) Distance between posts 115.47 4.11B (23; 87.50 160.00) 144.39 4.08A (23; 112.50 187.50) 106.67 3.82B (21; 75.00 130.00) Ecdysial scar width (es) 20.66 0.67A (26; 15.40 28.00) 23.12 1.63A (17; 15.50 35.00) 21.39 1.08A (21; 15.40 27.50) Rima thickness (rm) 4.63 0.07A (18; 4.40 5.00) c 2.34 0.03B (20; 2.20 2.50) Opening length (sol) 31.12 1.45A (12; 25.00 42.00) 32.10 0.72A (17; 28.00 40.00) 30.35 0.59A (17; 27.00 37.00)

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139 Table 6 2. continued. Larval measurements a Mean SEM (n; range) C. massyla E. eluta E. stigmatias Posterior spiracle Opening width (sow) 22.19 0.73A (18; 17.50 28.60) c 18.36 0.62A (14; 15.00 22.00) Cephalopharyngeal skeleton (CPS) CPS length (mm) 0.99 0.01A (19; 0.88 1.09) 0.87 0.01B (21; 0.75 0.97) 0.90 0.01B (21; 0.73 0.99) Mandible segments a 58.61 1.54B (16; 49.50 70.00) 53.75 1.19B (18; 49.50 62.50) 65.32 1.70A (17; 57.50 85.00) b 52.94 1.58A (16; 35.00 63.00) 48.43 0.95AB (23; 37.50 57.50 ) 44.74 1.65B (18; 30.00 55.00) c 100.35 1.91A (16; 80.00 110.00) 95.76 2.68A (23; 66.00 117.50) 104.04 3.51A (18; 80.00 149.60) d 20.88 1.57A (16; 10.00 30.00) 15.53 0.95B (23; 7.50 25.00) 15.43 1.69B (18; 6.60 30.00) e 84.52 1.82A (16; 67.50 92.40) 88.43 1.75A (23; 75.00 107.50) 89.19 1.28A (17; 77.50 105.00) Mandible segment ratios a to b 3.80 0.15A (16; 3.00 5.71) 3.94 0.09A (23; 3.39 5.00) 2.69 0.30B (16; 1.12 4.35) a to c 1.98 0.03A (16; 1.73 2.25) 2.00 0.05A (23; 1.67 2.73) 1.93 0.06A (17; 1.28 2.38) a to e 2.36 0.07A (16; 2.05 2.96) 2.14 0.04B (23; 1.75 2.67) 2.24 0.03AB (17; 2.00 2.48) Dental sclerite length (ds) 54.27 1.66A (2 3; 39.60 66.00) 44.74 1.30B (28; 31.50 60.00) 45.29 1.12B (25; 37.40 55.00) hypopharyngeal sclerite (hps) length 176.29 4.61A (20; 151.30 250.00) 146.98 3.69B (90.00 175.00) 156.51 3.16B (23; 117.00 187.50) Cephalopharyngeal skeleto n Parastomal bar length (pb) 184.58 3.56A (19; 162.00 225.00) 154.43 2.95C (21; 126.00 187.50) 171.20 3.34B (23; 150.00 212.50) Overlap of tps over hps 58.61 1.54A (16; 49.50 70.00) 53.75 1.19B (18; 49.50 62.50) 65.32 1.70B (17; 5 7.50 85.00) Dorsal arch length (da) 129.85 3.90A (17; 99.00 170.00) 128.12 3.08A (19; 97.90 150.00) 118.13 4.61A (16; 70.00 135.00) Dorsal bridge length (db) 77.82 2.24B (17; 63.00 99.00) 89.69 2.25A (21; 60.00 106.80) 87.89 1.39A (22; 70.00 100.00) Dorsal cornu length (dc) (mm) 0.61 0.01A (29; 0.51 0.74) 0.54 0.01B (35; 0.45 0.65) 0.51 0.01 B (33; 0.45 0.56) Notch length (n) (mm) 0.28 0.01A (17; 0.25 0.34) 0.24 0.01B (17; 0.20 0.30) 0.25 0.01B (18; 0.12 0.28) dc:n 0.47 0.01A (17; 0.40 0.52) 0.46 0.01A (17; 0.40 0.54) 0.50 0.02A (18; 0.24 0.57) Ventral cornu length (vc) (mm) 0.70 0.01A (31; 0.58 0.77) 0.57 0.01C (28; 0.49 0.63) 0.61 0.001B (34; 0.35 0.69)

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140 Table 6 2. continued. Larval measurements a Mean SEM (n; range) C. massyla E. eluta E. stigmatias Anal complex Anal organ width (aow) 0.45 0.01C (21; 0.39 0.51) 0.60 0.02A (24; 0.48 0.69) 0.52 0.02B (24; 0.39 0.63) Anal organ length (aol) 0.28 0.01A (21; 0.24 0.32) 0.30 0.01A (24; 0.26 0.35) 0.29 0.01A (24; 0.23 0.39) aow:aol 1.61 0.02C (21; 1.49 1.70) 2.01 0.05A (24; 1.71 2.41) 1.79 0.04B (24; 1.55 2.20) Anus length 0.16 0.00A (21; 0.13 0.18) 0.15 0.00AB (24; 0.13 0.17) 0. 14 0.00B (24; 0.10 0.17) Anal hook 31.24 1.16B (21; 23.20 38.40) 39.60 0.85A (24; 36.40 45.00) 35.51 2.38AB (24; 21.00 56.00) Means followed by different letters within a row are significantly different (Tukey, P > 0.05, S AS Institute 200 8). aAll the parameters except ratios are in m unless specified. brm and sow not measured for E. eluta due to poor visibility of posterior spiracle under compound microscope.

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141 Table 6 3. Comparison of creeping welt measurements (mm) among larvae of three species of picturewinged fly pests of corn. Welt no. Parameter a Mean SEM (n; range) C. massyla E. eluta E. stigmatias 1 Width (W) 0.80 0.03Abc (18; 0.58 0.98) 0.75 0.02Ac (24; 0.53 0.94) 0.65 0.02Bb (18; 0.52 0.80) Length (L) 0.14 0. 01Ad (18; 0.09 0.28) 0.13 0.01ABd (21; 0.09 0.22) 0.10 0.01Bc (18; 0.07 0.14) W:L 6.41 0.45Aa (18; 2.84 9.10) 6.28 0.37Aa (21; 3.36 8.66) 6.80 0.31Aa (18; 4.32 8.80) 2 Width (W) 0.86 0.03Aabc (18; 0.62 1.02) 0.83 0.02Abc (24; 0.67 1.02) 0.71 0.03Bab (15; 0.53 0.90) Length (L) 0.18 0.01Bcd (18; 0.12 0.23) 0.20 0.00Ac (21; 0.17 0.23) 0.14 0.01Cb (15; 0.12 0.16) W:L 4.88 0.14Ab (18; 3.56 5.66) 4.28 0.12Bb (21; 3.35 5.45) 5.04 0.14Ab (15; 4.03 5.84 ) 3 Width (W) 0.93 0.03Aab (15; 0.761.20) 0.97 0.02Aa (24; 0.77 1.16) 0.74 0.02Bab (18; 0.61 0.89) Length (L) 0.22 0.01ABbc (15; 0.17 0.30) 0.23 0.01Ab (21; 0.17 0.29) 0.19 0.00Ba (18; 0.17 0.22) W:L 4.45 0.22Abc (15; 2.67 5 .45) 4.36 0.18Ab (21;3.23 6.16) 3.90 0.07Ac (18; 3.36 4.50) 4 Width (W) 0.96 0.03Aa (12; 0.80 1.16) 0.98 0.02Aa (24; 0.76 1.20) 0.77 0.02Ba (18; 0.60 0.95) Length (L) 0.28 0.02Aa (12; 0.21 0.37) 0.26 0.00Aa (21;0.23 0.29) 0.2 1 0.01Ba (18;0.18 0.24) W:L 3.46 0.13Bcd (12; 2.76 4.22) 3.82 0.08Abc (21;3.22 4.64) 3.72 0.08ABc (18; 3.09 4.39) 5 Width (W) 0.91 0.03Aabc (12; 0.71 1.11) 0.97 0.02Aa (24;0.76 1.17) 0.73 0.02Bab (18; 0.59 0.89) Length (L) 0.28 0.02Aa (12; 0.19 0.37) 0.26 0.01Aa (21; 0.19 0.29) 0.21 0.01Ba (18; 0.19 0.26) W:L 3.35 0.20Acd (12; 2.38 4.56) 3.78 0.13Abc (21; 2.91 5.19) 3.53 0.08Ac (18; 2.77 4.11) 6 Width (W) 0.94 0.03Aa (12; 0.73 1.13) 0.91 0.03 Aab (24; 0.71 1.16) 0.72 0.02Bab (18; 0.59 0.92) Length (L) 0.25 0.01Aab (12; 0.19 0.33) 0.27 0.00Aa (24; 0.26 0.29) 0.20 0.01Ba (18; 0.15 0.24) W:L 3.79 0.21Abcd (12; 2.91 4.98) 3.46 0.09Ac (24; 2.67 4.18) 3.57 0.12Ac (18; 2.89 4.79)

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142 Table 6 3. continued. Welt Parameter Mean SEM (n; range) C. massyla E. eluta E. stigmatias 7 Width (W) 0.79 0.02Ac (12; 0.67 0.93) 0.83 0.02Abc (24; 0.67 1.05) 0.65 0.02Bb (18; 0.52 0.83) Length (L) 0.24 0.02Aabc (12; 0.15 0.32) 0.25 0.00Aab (24; 0.21 0.28) 0.19 0.01Ba (18; 0.10 0.26) W:L 3.53 0.26Acd (12; 2.36 5.08) 3.38 0.07Ac (24; 2.77 3.95) 3.67 0.25Ac (18; 2.57 6.10) 8 Width (W) 0.65 0.03Bd (12; 0.49 0.82) 0.78 0.02Ac (18; 0.62 0.94 ) 0.55 0.03Cc (15; 0.37 0.76) Length (L) 0.22 0.01Aabc (12; 0.14 0.28) 0.23 0.01Ab (18; 0.17 0.28) 0.15 0.01Bb (15; 0.08 0.21) W:L 3.06 0.26Ad (12; 2.04 4.64) 3.32 0.14Ac (18; 2.42 4.31) 3.82 0.35Ac (15; 2.34 6.40) Means wi thin a row comparing species followed by different capital letters, and within a column comparing parameters between welts of the same species followed by different small letters are significantly different (Tukey, P > 0.05, SAS Institute 2008).

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143 Table 6 4. Diagnostic characteristics separating the immature stages of three species of picturewinged fly pests of corn. Stage Characteristic C. massyla E. eluta E. stigmatias Egg Surface structure pores present at vertices of polygons only pores present across entire surface pores present across entire surface Larva Posterior spiracle light brown black dark brown Distinct tooth on mandible present absent absent Oral ridges 23 0.4 (range: 19 27) 34 0.6 (range: 30 42) 35 0.6 (range: 31 40) Creeping we lts Length mean and range for CW2 and CW6 longer than E. stigmatias mean and range for CW2 and CW6 shorter than E. eluta Continuous/ Discontinuous spinules in row 1 dis continuous in CW2 to CW8 spinules in row 1 continuous in CW4 to CW8 spinules in row 1 continuous in CW3 to CW8 Pupa Color reddish to reddish brown light brown to black light brown black Shape of posterior spiracular plate trapezoidal oval oval

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144 Figure 6 1. Scanning electron microscope views of eggs of three species, Chaetopsis massyla (a, b, c), Euxesta eluta (d, e, f) and Euxesta stigmatias (g, h, i); habitus (a, d, g), anterior end (b, e, h) and posterior end (c, f, i); hl, hatching line.

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145 Figure 6 2. Scanning electron microscope views of larvae of three species Chae topsis massyla (a, d, g, h, i, j), Euxesta eluta (b, e, k) and Euxesta stigmatias (c, f, l); face (a, b, c), anterior spiracle (d, e, f), creeping welts 2nd (g), 7th (h), 8th (i), anal complex (j, k, l); ah, anal hook, al, anal lobe, aol, anal complex leng th, aow, anal complex width, ant, antenna, k1, knob sensilla 1, k2, knob sensilla 2, lal, labial lobe, m, modified papilla sensilla 1 of dorsolateral group, mol, median oral lobe, mxp, maxillary palp, or, oral ridges, p, modified papilla sensilla 2 of dors olateral group, pa, papillae, p1, papilla sensilla 1, p2, papilla sensilla 2, p3, papilla sensilla 3, so, spiracular opening, sr, spinule row.

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146 Figure 6 3. Light microscope views of larvae of three species Chaetopsis massyla (a, d, g, j), Euxesta elut a (b, e, h, k) and Euxesta stigmatias (c, f, i, l); Cephalopharyngeal skeleton (a, b, c), mandibles (d, e, f), anterior spiracle (g, h, i), posterior spiracle (j, k, l), asl, anterior spiracle length, asw, anterior spiracle apical width, cwl, length of creeping welt, cww, width of creeping welt, da, dorsal arch, db, dorsal bridge, dc, dorsal cornu, dt, distinct tooth es, ecdysial scar, hps, hypopharyngeal sclerite, m, mandible, ma, total mandible length a, mb, mandible length b, mc, mandible base length c, md, mandible length d, me, mouth hook length e, pb, parastomal bar, pl, papilla length, pw, papilla width, rm, rima, sph, spiracular hair, tps, tentoropharyngeal sclerite, tr, trabeculae, tw, anterior spiracle basal width, vc, ventral cornu.

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147 Figure 6 4. Light microscope views of pupae Posterior spiracular plate of Chaetopsis massyla (a), Euxesta eluta (b), Euxesta stigmatias (c); habitus view of three species; Ee, Euxesta eluta, Es, Euxesta stigmatias Cm, Chaetopsis massyl a; psp, posterior spiracular plate.

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148 CHAPTER 7 DEVELOPMENTAL AND LI FE TABLE STUDIES OF COR N INFESTING PICTURE WINGED FLIES Current s tudies have identified four species of picturewinged flies, Euxesta annonae (F.) E. eluta Loew, E. stigmatias Loew, and Chaetopsis massyla (Walker) th at attack corn in Florida. Information on developmental time and survival of each instar is limited for each species. Several studies have been published with this information on E. stigmatias in Florida (Hentz and Nuessly 2004, Seal et al. 1995), Brazil (Franca and Vecchia 1986), Puerto Rico (App 1938), on E. eluta in Jujuy, Argentina (Arce de Hamity 1986), E. eluta and E. annonae in Chile (Fras L 1978), and C. massyla in Ohio (Allen and Foote 1992). However, these studies were conducted using different food sources and no studies have compared the developmental times and survival of these species at one location and reared on a single food source. Moreover, information is unavailable on adult longevity and other life table parameters required to determine the reproductive potential of flies. A few studies have been conducted on number of eggs deposited daily throughout the life cycle for C. massyla and E. eluta (Allen and Foote 1992, Fras L 1978). However, similar information is lacking for other s pecies. The purpose of the present studies was to determine and compare biological parameters (i.e., adult longevity, developmental periods, and survival of different immature stages, and life table parameters) of the three most important corninfesting u lidiids in Florida: C. massyla E. eluta and E. stigmatias Materials and Methods Studies to determine the length of egg, larval and pupal stages for E. eluta, E. stigmatias and C. massyla were conducted within corn ears in the field and on artificial

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149 die t in the laboratory. Tests were begun using eggs collected from laboratory colonies maintained as described earlier in the chapter on Pest status Developmental Times On corn ears The development of Ulidiidae larvae in corn ears was studied by placing 20egg cohorts into ears on plants grown in the field. Field studies were conducted in two main corn growing seasons: spring (March 2009, and May 2010) and fall (December 2009). Sweet corn (cv. Garrison, Rogers Brand, Syngenta Seed, Wilmington, DE) wa s planted 15 Jan uary and 29 September 2009 and on 15 February 2010 (cv. Obsession Seminis Vegetable Seeds, Inc., Saint Louis, MO) at the EREC Belle Glade, Florida and managed according to local standards ( Ozores Hampton et al. 2010). Insecticides were used to protect the plants from Lepidoptera larvae until tassel emergence after which no insecticides were applied. Corn ears (35 d after first silk appearance) not previously damaged or infested by insects were covered with a pollination bag (6.25 2.5 21.25 cm, Lawson Bags, Northfield, IL) the day before the beginning of each study to protect the ears from infestation by Spodoptera frugiperda (J.E. Smith), Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) or Ulidiidae. Newly deposited eggs (< 4 h old) of each species produced by flies in the colonies were collected from the larval diet and placed in plastic cups for transfer to the field. Later that same day, 20 eggs of a single species were placed between the silk and husk of pre selected ears using a camels hair brush (size 0) (March 2009 15 ears, December 2009 20 ears, May 2010 20 ears) Ears were then recovered with the pollination bags in the March 2009 study or with a 67 mesh cloth bag (30 11 cm, 67 openings per 10 cm) in the Dec 2009 and May 2010 studies to prevent infestation of the

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150 corn silks and tips by additional insects. Bags were held tightly around the middle of the ear with a rubber band. Ears and bags were checked daily after 7 d for the presence of pupae until the last lar va pupated. Pupae were found within the silk channel, on dried silks outside the channel, and trapped within the bags. The time from the day eggs were placed in corn ears until larvae pupated was defined as the egg larval developmental time. Most of the larvae pupated in the bags rather than in corn ears. Pupae were collected and transported to the laboratory to complete development to prevent their desiccation and death under field conditions. Larvae that leave ears normally pupate in the soil or beneath surface leaf trash where they are less likely to experience the higher temperatures and lower humidity of the plant canopy. Pupae were placed on moistened filter paper within Petri plates and transported to the laboratory to monitor pupal development. Parafilm (Pechiney Plastic Packaging, Chicago, IL) was used to seal the plates to reduce moisture loss and pathogen infestation. Each dish held pupae that emerged from a single ear on a single day. The Petri plates were placed under the same conditions as those described above for colony maintenance. Adult emergence was recorded daily for each dish. The time from collection of the pupae to adult eclosure was defined as the pupal developmental time. A fter the adult emergence, the flies were examined using a hand lens to determine the sex of the dead adults. Fifteen corn ears were selected for the study in the March 2009 study and 20 corn ears each were set up in the December 2009 and May 2010 studies. The total developmental time was defined as the s um of the egg larval and pupal developmental times.

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151 On artificial diet Developmental times of C. massyla E. eluta and E. stigmatias were also evaluated on artificial diet at constant temperature in the laboratory. Studies were conducted in January 2009 and August 2009 under the same temperature, photoperiod, and relative humidity as stated above for colony maintenance. The egg developmental time of each species was determined by holding 20egg c ohorts in Petri plates until larvae eclosed. Eggs ( adult cages and placed inside the lid of Petri plates using a moistened camels hair brush. The bottom half of the Petri plates were fitted with moist ened orangecolored paper over moistened filter paper (Whatman 3, Whatman International Ltd, Maidstone, England) to increase the visibility and viability of eggs. Petri plates were sealed with Parafilm to reduce moisture loss and fungal contamination. E ggs were checked every 2 h for larval emergence with the help of a hand lens of up to 10 X magnification The larval developmental time for each species was determined by placing old larvae that emerged from eggs in Petri plates into diet cups (clear, 30 ml creamer plastic cup, Bio Serv Frenchtown, NJ) half filled with the same artificial diet used in colony maintenance. Larvae of a single species that emerged from eggs on the same day were kept in the same cup. A cotton ball was placed above the diet in each cup to provide a dry location for larvae to pupate. Each cup was placed within a 185ml cylindrical plastic vial which was then covered wit h a paper towel held in place with a rubber band to prevent larval escape. The cotton and diet were monitored for pupae every 24 h beginning 7 d after setting up each experimental unit until all the larvae pupated. The pupal developmental time and sex of resulting adults was determined as

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152 above for larvae that developed within corn ears. The total developmental time was calculated by summing the egg, larval and pupal developmental times. Adult Longevity Adult longevity was studied by caging newly emerged adults of each species individually in Petri plates and observing them daily until they died. A single sheet of filter paper placed in the bottom of each plate was moistened with deionized water every 2 3 d and replaced weekly. A 5060% honey solution w as provided as adult food on a cotton ball inserted into a 1.5 cm diam hole drilled in each plate lid. The honey solution was replenished daily and cotton balls replaced weekly. Longevity was studied for flies reared from sweet corn ears collected from a field at EREC Belle Glade, Florida in May and Dec 2009. The corn field was planted with sweet corn (cv. Garrison, Rogers Brand, Syngenta Seed, Wilmington, DE). Additional tests were conducted to evaluate the longevity of flies reared on corn earworm artificial diet (see above) in the laboratory in January and August 2009. Petri plates with adults were maintained under the same conditions as those described above for colony maintenance. Current studies with colony maintenance revealed that the Helicov erpa zea diet normally used was not a preferred ovipositional medium for all three species. Several other alternatives were investigated for use in determining life table parameters before settling on a diet made from whole kernel corn that provided both an attractive ovipositional medium and a suitable larval diet for all three ulidiid species tested. The diet was prepared using canned whole corn kernel obtained from a local market. The whole kernels were drained, rinsed and then hand dried using paper towels to remove all free moisture. The diet was prepared by adding 6.0 g powdered agar to 150 ml

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153 boiling water and then blending the mixture with 300.0 gm of the prepared whole kernel corn for 12 min. The liquid diet was poured into glass test tubes for oviposition and diet cups (30 ml clear plastic cups, Bioserv, Frenchtown, NJ) for use in survival and oviposition studies. After the diet cooled and solidified, the cups were covered with lids (30 ml over cap snap, Product # 9053, Bioserv, Frenchtown, N J) and stored at 0 C to reduce bacterial and fungal contamination. Diet cups were removed from cold storage and allowed to reach room temperature before using them for the studies. Survival Survival of egg, larval and pupal stages was studied under labor atory conditions in August 2009. Cohorts of 20 eggs corn kernel artificial diet in the adult cages and placed inside the lid of Petri plates using a moistened camels hair brush. The bottom half of the Petri plates were fitted with moistened orangecolored paper over moistened filter paper (Whatman 3) to increase the visibility and viability of eggs. Petri plates were sealed with Parafilm to reduce moisture loss and fungal contamination. Eggs of indiv idual species were held in Petri plates until larval emergence and then reared on artificial diet made of whole corn kernels. Petri plates were maintained under the same conditions as those described above for colony maintenance. Eggs were checked for larval emergence every 2 h with the help of a hand lens beginning 15 h after placement in plates. Emerged larvae were transferred to 30ml diet cups 1/3 filled with the whole kernel corn diet. The larvae that emerged from a cohort of eggs were transferred together to a single diet cup. A cotton ball was placed above the diet in each cup to provide a dry location for larvae to pupate. Each cup was placed within a 185ml cylindrical plastic vial which was then covered with a paper towel held in place with a rubber band to prevent larval escape. Twenty

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154 replications were conducted for each of the three species with 20egg cohorts per cup in the majority of replicates. One of the replicates for E. stigmatias consisted of 15 eggs and one of the replicates of C massyla consisted of 16 instead of 20 eggs. Percentage survival was calculated for each stage and each container by dividing the number from each stage that successfully reached the following stage by the number of immatures in the previous stage. Repro ductive Periods Reproductive periods (preoviposition, oviposition and post oviposition) and egg production were studied by providing male: female pairs with an oviposition source and monitoring them daily until their death. Studies were conducted from April through October 2009 on adults reared from a whole kernel diet under laboratory conditions. An experimental unit consisting of a 30ml plastic cup 1/3 filled with the whole kernel corn diet covered with a 50ml cylindrical plastic vial to provide extr a room for the adult flies above the diet. A hole was drilled in the bottom of the 50ml vial to insert a dental wick moistened with a 5060 % honey solution as food for adults. The 50ml vials were narrower than the 30ml diet cups so that when inverted over the diet cups the rim of the 50ml vials nested inside the rims of the diet cups trapping the adults over the diet. It was observed that females preferred to deposit eggs within cracks in the diet or between the diet and the sides of the cups. Ther efore, forceps were used to create a gap between the solidified diet and the wall of the diet cups prior to exposure to pairs of the flies. Condensation on the walls of cups was removed with paper towels prior to exposure to the flies to reduce flies getti ng trapped in the free moisture. Single male: female pairs of newly emerged flies ( monitored daily for their mortality Dead males were replaced as needed to maintain

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155 pairs. The diet cups were replaced daily until the female died. The cups were checked usi ng a hand lens to record daily oviposition. The number of days from first exposure to the first day of oviposition was defined as the preoviposition period. The oviposition period was the number of days between the first and last day of oviposition. The number of days from the last day of oviposition to the day the female died was defined as the post oviposition period. The diet cups with eggs were placed in separate cylindrical vials (185 ml) as indicated above for measuring the number and sex of adult s emerged. The vials were kept in the same environmental conditions as used for rearing the flies. The vials were examined for adults completing development every 3 d beginning 10 d after egg deposition until all the adults emerged. Fungal growth on the diet or cups was removed using paper towels. Life Table Parameters All the life table parameters were calculated using data for each original cohort of eggs. Net reproduction rate ( Ro = xmx), and the mean length of a generation ( T = xmx/ xmx) were calculated with lx = probability of being alive at age x, and mx = the mean number of female offspring produced in a unit of time x (Birch 1948). Values of lx used in calculation of net r eproduction rate, mean length of a generation, intrinsic rate of increase, stable age distribution were obtained from developmental periods of egg, larval and pupa stages on corn earworm artificial diet. The intrinsic rate of increase ( r ) was solved through iteration from the equation 7 rxlxmx = 1 097 (Birch 1948) The stable age distribution (erx/ rxlx) was calculated for each stage and for each species (Carey 1982).

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156 Statistical Analysis Proc GLM (SAS institute 2008) was used to conduct an analysis of variance of the results of developmental and life table studies. For analysis of developmental times and adult longevity, dependent variables tested were egg, larval, and total developmental times and adult longevity. Independent variables (species, season, and sex) and their interactions were tested in the model for their effect on the developmental times. Species, season, sex, food and their interactions were used as independent variables in the model for adult longevity. Season was used only in an interaction term with food sour ce in the model for adult longevity, because both food sources were not tested in all seasons. For analysis of survival, developmental stage and species were used as independent variables and percentage survival was used as the dependent variable. For analysis of reproductive periods, the number of eggs deposited, and preoviposition, oviposition and post oviposition periods were used as dependent variables and species was used as the independent variable. The cumulative number of eggs deposited per fema le of an individual species was plotted against days of oviposition. For analysis of life table parameters, intrinsic rate of increase ( r ), net reproduction rate ( Ro) and mean generation time ( T ), were compared among species. The Tukey's honestly signif icant difference test (SAS institute 2008) was used for means separation ( P = 0.05) when analysis of variance identified a source of variance as significant at P < 0.05. Results and Discussion Developmental Times On corn ears The length of egglarval, pupal and total development was significantly affected (P < 0.05) by species and season (Table 7 1). The length of egg larval and pupal development time was also affected by species but the results

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157 depended on season. The sex of the resulting adults and it s interaction with species was found to have no significant effect on development. Therefore, results were pooled across sex to compare developmental times of each species by season (Table 7 2). Times for egg larval, pupal and total development were not affected by species in the March 2009 field trial, but species did affect the development times in the Dec 2009 field trial. The egg larval developmental time was shortest in E. stigmatias followed by C. massyla and then by E. eluta. The pupal stage was shortest in C. massyla followed by that of E. stigmatias and E. eluta The total developmental time was shorter in E. stigmatias and C. massyla than in E. eluta The pupal and total developmental times were also affected by species in the May 2010 field trial. The pupal developmental time was shortest in E. stigmatias followed by that of C. massyla and then by E. eluta Total developmental time was shorter in C. massyla and E. stigmatias than in E. eluta The seasons had a significant effect on developmental times for each species reared in the field in corn ears (Table 7 2). Egg larval and total developmental times of C. massyla were shortest in the May 2010 study and longest in the December 2009 study. The C. massyla pupal developmental time was sig nificantly shorter in May 2010 than in the other two studies. The shortest egg larval and total development times for E. eluta were in May 2010 followed by March 2009 and then by December 2009. The pupal stage in E. eluta was shortest in March 2009 followed by May 2010 and then December 2009. The egg larval, pupal and the total developmental times were all shortest for E. stigmatias in the May 2010 field trial and longest in the Dec 2009 field trial.

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158 Developmental times varied significantly among seasons for the three species when reared on corn ears. The difference in egg larval developmental times among the seasons was probably the result of temperature differences. Average temperatures during the the studies were 22 C in March (6 33), 25 C in May ( range: 1834), and 16 C in December ( 1 31) (Anonymous 2010 b ). However, pupal developmental times also varied by season, even though they were returned to the laboratory for the majority of their development. This result suggests that conditions experienced by developing larvae carry over to affect pupal developmental times. On artificial diet Developmental times also varied significantly by species when the larval stage was reared on artificial diet at constant temperatures in the laboratory (Table 7 1). The season (replicate), and sex of the resulting adults, did not have significant effect on development The pupal development time was significantly affected by species but the results depended on season. The data were pooled across sex and replica tes for means comparison. The fastest developmental times varied by species for each stage (Table 7 3). While E. stigmatias had the shortest egg stage, E. eluta larvae developed faster than the other species in the diet, and the pupal stage was significa ntly faster in C. massyla than the other species. Total developmental times followed the trend for the larval stage with the fastest development in E. eluta All three species developed more quickly within corn ears compared to artificial diet. Chaetopsi s massyla E. stigmatias and E. eluta larvae developed in diet in 22, 19 and 13 d, respectively, but developed in approximately 11 d in corn ears in the May 2010 study. The pupae of all three species completed development to the adult stage on corn ears f aster than on artificial diet. Total developmental time for all three species

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159 was also faster for flies reared on corn ears in May 2010 study compared to those reared on artificial diet. Seal and Jansson (1993) reported a decrease in larval developmental time for E. stigmatias when reared on artificial diet (25 d) compared to corn ears (22 d). These differences in developmental times between two food sources may be due to nutritional differences between the two food sources, such as nutritional deficienc ies in the diet. Chang et al (2000) found that omission of any one of the growth factors from an artificial diet for Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) prolonged developmental time. Conversely, some studies were found in the literature where larvae developed faster in artificial diet than in corn ears. Euxesta stigmatias larvae were found to develop faster in a corn earworm artificial diet (13.3 1.8 d) (Hentz and Nuessly 2004) than on corn ears (22 d) (Seal and Jansson 1993). Differ ence in larval developmental time of E. stigmatias between our study and that by Hentz and Nuessly (2004) remains unexplained. There was less difference in length of development among species when reared on corn ears compared to artificial diet. Egg larva l developmental time did not vary among species when reared on corn ears in the March and May studies, and there was only 1.5 d difference in development among species in December. In contrast, there was a difference in larval development of 3 d between E eluta and E. stigmatias and 6 d between E. stigmatias and C. massyla Pupal and total developmental times in corn ears were not significantly different among species in March 2009. In contrast, pupal and total developmental times were significantly dif ferent among species when reared on artificial diet.

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160 Differences observed between developmental times found in the current study and some of the studies in the past can partially be explained by diet and temperature differences (Table 7 4). The shorter eg g developmental time for C. massyla in our study compared to that of Allen and Foote (1992) may be due to the higher temperature used in our studies. While our results found no significant difference in egg development time between sexes, Seal and Jansson (1993) showed males developing several hours faster than females. In our study, C. massyla had a wider range of larval developmental times (i.e., 10 30 d in corn ears, 1432 d in artificial diet) compared to studies done on decaying cattail leaves (1223 d; Allen and Foote 1992) (Table 7 4). Fras L (1978) also showed that E. eluta larvae developed faster at higher temperatures than low temperatures (Table 7 4). Euxesta stigmatias completed larval development in 8 d on corn ears in studies by App (1938) conducted in Mayagez, Puerto Rico, but temperature data was not provided. Our finding that there was no difference in male and female larval developmental times supports the results of Seal and Jansson (1993). Pupae of C. massyla developed in 6.9 d on artificial diet in our studies and 7.0 d in studies by Allen and Foote (1992). No significant difference was found in male and female pupal developmental times of C. massyla in current study; however female C. massyla pupae (810 d) took a day longer to d evelop than male pupae (79 d) in the Allen and Foote (1992) study. Euxesta eluta pupae developed in 8 d at 26.5 1.0 C in the current study and in 10 d at 25 C in studies by Fras L (1978). Euxesta stigmatias pupae developed in 5 and 6 d in May (26 C) and March (23 C), respectively in the current study which were close to the results of Seal and Jansson (1993) (7 d). The pupal developmental time of E. stigmatias reared on artificial diet in our study (8 d) was

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161 close to that of Hentz and Nuessly (9 d). Our findings of faster developmental times with increases in ambient temperature support the results of Vargas et al. (1996) who found that egg, larval and pupal development of Bactrocera cucurbitae (Coquillett), B. dorsalis (Hendel), B. latifrons (He ndel), and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) decreased with increasing temperatures from 16 to 32 C. Adult Longevity Longevity in Petri plates provided with honey was significantly ( P by species, sex, and food (Table 7 5). The season food interaction was not significant; therefore, adult longevity was determined using all the data across both replicates by sex and food source (corn ears and corn earworm artificial diet) for each species (Table 7 6). None of the other interactions were significant sources of variation in the model (Table 7 1). Males and females of E. eluta lived longer than the other two species and C. massyla adults died sooner than the other two species when reared from either corn ears or artificial diet (Table 7 6). Male and female E. stigmatias and C. massyla and female E. eluta reared from corn ears lived significantly longer than those reared on artificial diet. Females of all three species l ived longer than males when reared on either food source. Female C. massyla lived significantly longer than males when reared from corn ears ( F = 13.70; df = 1, 75; P = 0.0004) and on artificial diet ( F = 24.89; df = 1, 77; P < 0.0001). Euxesta eluta fem ales lived significantly longer than males on corn ears ( F = 7.86; df = 1, 77; P = 0.0064) as well as artificial diet ( F = 5.64; df = 1, 82; P = 0.0198). Euxesta stigmatias females lived significantly longer than males on both corn ears ( F = 21.46; df = 1, 77; P < 0.0001) and artificial diet ( F = 7.13; df = 1, 79; P = 0.0092).

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162 Adult longevity was dependent on species, sex and food source. The results of these studies were similar to those of previous studies. Adult C. massyla reared on corn earworm artif icial diet in our study lived 1.52x longer than those reared on decaying cattail and lettuce under laboratory conditions (mean 25 d, range 1562 d; Allen and Foote 1992). Female C. massyla captured in the field in Kent, Ohio lived longer (4045 d) than m ales (3036 d) (Allen and Foote 1992). Male and female E. eluta reared on a wheat germ based diet in our study lived an average of > 6x longer than those reared on a cornmeal based diet (12 2 d) in studies by Arce de Hamity (1986) started from flies col lected in Jujuy, Chile. Fras L (1978) in Chile found that 11.8% of field collected E. eluta females died within 20 days and 38.2% died within 40 d. Flies are easily damaged by nets during the collection process, and these adults also probably included a mixed age distribution that further contributed to the shorter life spans of roughly 50% of the captured adults compared to our studies using lab colonies. In our current study, no E. eluta adults died within 20 d while only 2.6% adults died in the firs t 40 d. Both sexes of E. stigmatias in our study that were reared from a wheat germ based diet lived > 1.5x longer than those reared on a pinto beanbased diet (Seal et al. 1995). Laboratory studies by Seal et al. (1995) determined that E. stigmatias lon gevity increased from maximum of 10.0 d without water to mean longevity of 15.2 d with water to 23.4 d when provided sugar cubes, honey, corn ears or pieces of sugarcane stalks. Hentz and Nuessly (2004) demonstrated that providing honey to E. stigmatias a dults under laboratory conditions increased their mean longevity to 90 d for males and 116 d for females from < 5 d when provided with only water. They also

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1 63 showed that females lived longer than males when both were provided with water only, honey, or hon ey plus torula yeast. Survival Percentage survival during development in the whole kernel corn diet was significantly affected by species ( F = 3.52; df = 2, 171; P = 0.0319) and developmental stage ( F = 26.82; df = 3, 171; P < 0.0001). The survival was not affected by species when developmental stages varied ( F = 0.77; df = 4, 171; P = 0.5454). The percentage survival for E. eluta (95.4) was greater than C. massyla (92.6) but not E. stigmatias (93.3). The percentage pupal survival (97.4) was greatest fol lowed by egg survival (94.5) followed by larval survival (89.4). When compared within each stage, survival was not significantly different among species (Table 7 7). When compared within a species, stage was found to have significant effect on survival i n C. massyla ( F = 8.86; df = 2, 57; P = 0.0004), E. eluta ( F = 8.08; df = 2, 57; P = 0.0008) and E. stigmatias ( F = 11.20; df = 2, 57; P < 0.0001). Both percentage egg and pupal survivals of C. massyla and E. stigmatias were greater than percentage larval survival. Percentage pupal survival was greater than larval survival in E. eluta Greater survival was seen in the current study compared to previous studies. Fras L (1981) found larval and pupal survival of E. eluta to be 32 and 71%, respectively in c omparison to 92, and 99%, respectively in the current study. Seal and Jansson (1989) reported egg, larval and pupal survivals of E. stigmatias as 90.0, 54.5, and 80.0% respectively in comparison to 94.0, 88.4, and 97.4% in the current study. The difference in survival could be related to different diet mediums used in different studies.

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164 Reproductive Periods Species was found to have significant effect on the number of eggs deposited by a female throughout the reproductive period (Table 7 8) and the length of the pre oviposition period. Euxesta eluta and E. stigmatias deposited approximately double the mean number of eggs deposited by C. massyla Euxesta eluta displayed the shortest pre oviposition period followed by E. stigmatias and C. massyla Mean dai ly oviposition data varied significantly from day to day. Flies normally deposited eggs for a day or two and then did not deposit eggs for a day or two before starting the cycle again. Therefore, mean oviposition data were summed over a 3d period for co mparison purposes. Oviposition data was also presented cumulatively over the life span of each species. Egg deposition in C. massyla started a day (mean 10 d) after adult emergence and peaked on the 16th d (12 eggs per d) before tapering off (Fig. 7 1) A second degree polynomial equation fitted to the data described 29 % of the variation. The third and fourth degree polynomial equations fitted to the data described greater amount of variation than second degree polynomial equation, but showed no biolo gical meaning. The mean cumulative number of eggs produced by C. massyla was 191 in 63 d (Fig. 7 2). Half of the eggs were deposited in the first 19 d. Chaetopsis massyla lived an average of 8 d following the end of the oviposition period. Egg depositi on in E. eluta started 2 d (mean 3 d) after adult emergence and peaked on the 11th d (24 eggs per d) before tapering off (Fig. 7 3). Approximately 77% of the variation in the 3d sums of average oviposition was explained by the fourth degree polynomial equation. The mean cumulative number of eggs deposited by E. eluta was 521 at the age of 67 d (Fig. 7 4). Euxesta eluta deposited half of their eggs in the first 20 days. On an average, females lived for 72 d, but stopped depositing eggs at 67 d after adult

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165 eclosion. Egg deposition in E. stigmatias started 4 d (mean 8 d) after adult emergence and peaked on the 11th d (19 eggs per d) before tapering off (Fig. 7 5). Ninety two percent of the variation in the 3d oviposition results was again explained by a fourth degree polynomial equation. The mean cumulative number of eggs deposited by E. stigmatias was 245 eggs in a 40 d reproductive period (Fig. 7 6). They deposited half of their total eggs in the first 17 d. Females lived for a mean of 50 d, but stopped producing eggs after 40 days. Previous studies on the reproductive periods of E. eluta and C. massyla are available for comparison. Allen and Foote (1992) found fecundity of C. massyla reared on decaying cattail leaves ranged from 150 to 398 in c ontrast to 4 to 801 eggs obtained in the current study. They determined the preoviposition period to be 3 d in comparison to 10 d in the current study. Fras L (1978) found that the preoviposition period for E. eluta to be 11.5 12.8 d at 16 C and 6. 4 1.9 days at 25 C. The mean cumulative number of eggs deposited by a female at 25 C were in close proximity to eggs deposited by E. eluta females in this study. Life Table Parameters Life table parameters for the three species are presented in Table 7 9. Of the three species, Euxesta eluta had the greatest net reproductive rate (110), the least mean generation length (36 d) and the greatest intrinsic rate of increase (0.139 per female per day). Chaetopsis massyla had smallest net reproductive rate ( 45), the greatest mean generation length (46 d), but the smallest intrinsic rate of increase (0.084). The net reproductive rate ( R0) revealed that C. massyla population would be able to multiply 45x in a mean generation time of 46 d. The stable age distr ibution showed that the natural population of C. massyla would consist of approximately 93%

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166 immatures and only 7% adults. Larvae made up the majority of immatures population followed by eggs and pupae. The R0 for E. eluta revealed that E. eluta populatio n would be able to multiply 110x in a mean generation time of 36 d. The stable age distribution showed that a natural population of E. eluta would consist of approximately 91% immatures with only 9% adults. My results indicated that a population of E. st igmatias would be able to multiply 80x in a mean generation time of 44 d. The stable age distribution showed that a natural population of E. stigmatias would have approximately 95% immatures and only 5% adults. Based on the stable age distribution calculations for the three species, the majority of individuals in a population would be in the immature stages (especially larval stage) (Table 7 9). The larval stage constituted 67 to 78%, while the adult stage constituted of only 3 to 7 % of the total populat ion across species. No comparative studies were located that calculated the life table parameters of these flies. The overwhelming proportion of each species consisting of the larval stage suggest that destroying the crop immediately after harvest may be an effective strategy for bringing the population under control. Overall, E. eluta developed faster, lived longer and produced more eggs compared to other species in laboratory conditions. I also found E. eluta as pest of more serious nature than other s pecies throughout Florida after conducting surveys in corn fields. Food (artificial diet and corn ears) was found to affect the developmental periods, and adult longevity in laboratory experiments. Differences in these biological parameters between artif icial diet and corn ears will have to be considered while extrapolating the studies to field conditions.

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167 Table 7 1. Analysis of variance for Ulidiidae developmental times on two food sources Developmental time Source df Egg larval Pupal Total developm ental time Reared on corn ears Species 2, 985 F = 12.44; P < 0.0001 F = 60.83; P < 0.0001 F = 40.90; P < 0.0001 Season 2, 985 F = 4,659.99; P < 0.0001 F = 242.81; P < 0.0001 F = 5,175.50; P < 0.0001 Sex 1, 985 F = 0.37; P = 0.5451 F = 0.05; P = 0.8 175 F = 0.46; P = 0.4982 Species sex 2, 985 F = 1.86; P = 0.1559 F = 4.00; P = 0.0186 F = 0.48; P = 0.6199 Species season 4, 985 F = 6.55; P < 0.0001 F = 51.31; P < 0.0001 F = 1.24; P = 0.2914 Reared on artificial diet Species 2, 602 F = 373.49; P < 0.0001 F = 37.34; P < 0.0001 F = 292.89; P < 0.0001 Season 1, 602 F = 0.02; P = 0.8945 F = 0.25; P = 0.6145 F = 0.00; P = 0.9508 Sex 1, 602 F = 0.00; P = 0.9490 F = 1.21; P = 0.2709 F = 0.24; P = 0.6234 Species sex 2, 602 F = 0.37; P = 0.6936 F = 0.04; P = 0.9629 F = 0.41 ; P = 0.6626 Species season 2, 602 F = 0.19; P = 0.8244 F = 4.03; P = 0.0183 F = 1.27; P = 0.2803 ANOVA (PROC GLM, SAS Institute 2008)

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168 Table 7 2. Developmental times (d) for three Ulidiidae species on corn ears in field trials conducted in March and December, 2009, and May 2010 in Belle Glade, FL. Stage Mean SEM (n; range) Season C. massyla E. eluta E. stigmatias Egg larval Mar. 20.4 0.4Ba (74; 14 27) 21.7 0.5Ba (59; 14 28) 20.4 0.5Ba (48; 13 27) Dec. 26.9 0.1Ab (127; 24 30) 27.5 0.2Aa (113; 23 31) 26.0 0.2Ac (106; 24 31) May 11.9 0.1Ca (146; 10 15) 12.0 0.1Ca (221; 9 16) 12.2 0.1Ca (103; 10 14) Pupal Mar. 6.4 0.1Aa (74; 5 8) 6.2 0.1Ca (59; 5 8) 6.3 0.1Ba (48; 5 8) Dec. 6.3 0.1Ab (127; 5 9) 7.7 0.1Aa (113; 5 9) 7.6 0.1Aa (106; 4 10) May 5.5 0.1Bb (146; 3 8) 6.7 0.1Ba (221; 3 8) 5.0 0.1Cc (103; 3 7) Total development Mar. 26.8 0.4Ba (74; 20 34) 27.9 0.5Ba (59; 20 35) 26.6 0.6Ba (48; 19 33) Dec. 33.2 0.2Ab (127; 29 38) 35.2 0.2Aa (113; 29 40) 33.6 0.2Ab (106; 31 39) May 17.3 0.1Cb (146; 15 18) 18.7 0.1Ca (221; 15 22) 17.2 0.2Cb (103; 14 20) Means within a column for each developmental stage followed by the same capit al letter and within a row followed by the same small letter are not significantly different (Tukey, P > 0.05, SAS Institute 2008)

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169 Table 7 3. Developmental times for three species of Ulidiidae on artificial diet at 26.5 1.0 C, 14:10 (L: D) photoperiod and 55 70% RH. Stage (unit of time) Mean SEM development time (n; range) C. massyla E. eluta E. stigmatias Egg (h) 42.0 0.2* A (240; 38 51) 32.5 0.2 B (330; 26 40) 27.8 0.1 C (200; 26 32) Larva (d) 21.8 0.4 A (218; 13 36) 12.7 0.2 C ( 305; 9 23) 18.7 0.2 B (182 13 27) Pupal (d) 6.9 0.1 C (180; 4 13) 8.1 0.1 A (271; 5 12) 7.5 0.1 B (160; 5 10) Total developmental (d) 30.2 0.4 A (180; 2143) 21.6 0.2 C (271; 1731) 27.4 0.3 B (160; 2036) Mean SEM within a row followed by the same letter are not significantly different (Tukey, P > 0.05, SAS Institute 2008) .

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170 Table 7 4. Developmental times (d) by stage for three species of Ulidiidae as reported in the literature. Species and temperature Mean SEM Total developm ent Food s ource Reference Egg Larva Pupa C. massyla 23.5 1 C 2.0 3.0 12.0 23.0 7.0 10.0 Decaying cattail leaves Allen and Foote (1992) E. eluta 16.0 C 31.8 6.8 a 55.8 5.1 Nutrina, Agar agar Fras L (1978) 25.0 C 18.8 3.2 2 8.3 3.7 Nutrina, Agar agar Fras L (1978) E. stigmatias 20.0 C 3.0 0.2 30.1 0.3 13.3 0.1 46.4 Corn earworm artificial diet Seal and Jansson (1993) 25.0 C 1.7 0.7 25.2 0.2 10.6 0.2 37.5 Corn earworm artificial diet Seal and Jansson (1993) 30.0 C 1.4 0.3 21.6 0.2 5.2 0.2 28.2 Corn earworm artificial diet Seal and Jansson (1993) 24.0 C 2.8 0.1 25.4 26.9 7.0 Seed corn Seal and Jansson (1993) 24.0 C 3.1 0.1 25.4 26.9 7.0 Seed corn Seal and Jansson (1993) 24.0 C 2.6 0.1 20.6 22.7 7.0 Sweet corn Seal and Jansson (1993) 24.0 C 2.9 0.1 20.6 22.7 7.0 Sweet corn Seal and Jansson (1993) 26.5 C 1.5 2.0 13.3 1.8 9.2 0.4 Corn earworm artificial diet Hentz and Nuessly (2004)

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171 Table 7 5. Analysis of variance of adult longevity Source df a F P Species 2 441.15 < 0.0001 Sex 1 49.61 < 0.0001 Season (rep) 1 0.81 0.3688 Food 1 67.92 < 0.0001 Season (rep) Food 1 0.98 0.3773 Species Sex 2 0.07 0.7848 Species food 2 1.11 0.3300 Sex food 1 0.18 0.6696 Species sex food 2 0.26 0.7708 ANOVA (PROC GLM, SAS Institute 2008) aError term, df = 465

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172 Table 7 6. Mean SEM adult longevity (in d) of three species of picturewinged fl ies reared on artificial diet and corn ears. Mean SEM longevity (d) ( n; range) Sex Food C. massyla E. eluta E. stigmatias F P df Male Corn ears 36.5 1.4Ac (43; 10 45) 86.4 3.1Aa (40; 25 115) 51.9 1.5Ab (41; 13 65) 146.40 < 0.0001 2, 121 Artificial diet 23.5 1.1Bc (38; 11 39) 76.6 4.6Aa (46; 8 124) 39.0 2. 2Bb (39; 7 62) 73.07 < 0.0001 2, 120 F 52.44 2.98 22.72 P < 0.0001 0.0877 < 0.0001 df 1, 79 1, 84 1, 78 Female Corn ears 44.9 1.9Ac (34; 19 58) 99.6 3.4Aa (39; 30 129) 64.3 2.3Ab (38; 25 80) 102.29 < 0.0001 2, 108 Artificial diet 3 1.8 1.2Bc (41; 16 52) 90.3 3.2Ba (38; 19 114) 47.2 2.1Bb (42; 8 66) 176.72 < 0.0001 2, 118 F 36.27 3.77 31.44 P < 0.0001 0.0560 < 0.0001 df 1, 73 1, 75 1, 78 F df, and P values represent ANOVA of species and diet by s ex (PROC GLM, SA S Institute 2008). Mean SEM for each sex within a column followed by a different capital letter and within a row followed by a different small letter are significantly different (Tukey, P > 0.05, SAS Institute 200 8 )

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173 Table 7 7. Mean SEM (range) perc entage survival of immature stages of three species of corninfesting picturewinged flies (20 cohorts of each species) on artificial diet Species Egg Larvae Pupae C. massyla 94.5 1.5 a (75 100) 87.4 1.5b (68 95) 95.8 1.5a (83 100) E. eluta 95.0 1.5ab (80 100) 92.3 1.2b (84 100) 98.9 0.5a (93 100) E. stigmatias 94.0 1.2ab (80 100) 88.4 1.6b (76 100) 97.4 1.2a (79 100) F 0.12 3.11 1.79 P 0.8849 0.0521 0.1763 df 2, 57 2, 57 2, 57 Mean SEM within a row followed by the same small le tter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute)

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174 Table 7 8. Reproductive parameters of three species of picturewinged flies on artificial diet Species Mean SEM (Range) No. eggs deposited Pre oviposition period (d) Oviposition period (d) Post oviposition period (d) C. massyla (n = 23) 121.7 39.4b (4 801) 10.2 2.0a (1 42) 20.6 3.0 (0 58) 2.6 0.9 (0 16) E. eluta (n = 42) 263.2 31.3a (18 678) 3.0 0.2b (2 7) 20.5 2.9 (1 64) 2.6 0.5 (0 14) E. sti gmatias (n = 19) 217.3 29.4ab (6 474) 8.1 1.0a (4 20) 22.4 2.1 (1 36) 3.0 1.0 (0 15) F 4.35 11.17 0.10 0.07 P 0.0160 < 0.0001 0.9059 0.9363 df 2, 81 2, 81 2, 81 2, 81 Mean SEM within a column followed by the same small letter are not significantly different (Tukey, P > 0.05); ANOVA (Proc GLM; SAS Institute)

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175 Table 7 9. Life table parameters of three species of picturewinged flies on artificial diet Parameter Mean SEM (range) C. massyla E. eluta E. stigmatias Net Reproduction rate ( R 0 ) a 45.2 17.1 (12.369.8) 110.0 28.9 (32.6167.4) 79.5 10.5 (69.089.9) Mean length of generation ( T )b (d) 46.2 2.5 (41.549.9) 36.1 2.4 (29.041.3) 43.6 0.7 (42.944.2) Intrinsic rate of increase ( r ) c 0.084 0.01 (0.06 0.10) 0.139 0.01 (0. 12 0.15) 0.106 0.00 (0.10 0.11) Stable age distribution d (%) Egg 17.1 2.3 (12.6 20.3) 13.0 0.6 (11.8 14.5) 10.8 0.15 (10.6 10.9) Larva 70.0 1.9 (66.2 72.3) 68.6 3.3 (55.8 73.5) 77.9 0.2 (77.7 78.1) Pupa 5.7 1.2 (4.2 8.1) 9.7 1.1 (6.5 12.4) 6.7 0.3 (6.4 6.9) Adult 7.1 3.0 (3.2 13.1) 3.1 0.3 (2.2 4.1) 4.8 0.2 (4.6 4.9) aRo = xmx; lx = probability at birth of being alive at age x, mx = the mean number of female offspring produced in a unit of time x bT = xmx/ xmx cr from equation rxlxmx = 1 cStable age distribution = erx/ rxlx

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176 Fig ure 7 1. Mean no. of eggs deposited by females of C. massyla on artificial diet summed over 3d period.

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177 Figure 7 2. Mean cumulative eggs deposited by females of C. massyla on artificial diet

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178 Fig ure 7 3. Mean no. of eggs deposited by females of E. eluta on artificial diet summed over 3d period.

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179 Fig ure 7 4. Mean cumulative eggs deposited by females of E. eluta on artificial diet

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180 Fig ure 7 5. Mean no. of eggs deposited by females of E. stigmatias on artificial diet summed over 3d period.

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181 Fig ure 7 6. Mean cumula tive eggs deposited by females of E. stigmatias on artificial diet

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182 CHAPTER 8 ECOLOGICAL STUDIES O F CORNINFESTING PICTURE WINGED FLIES In southern Florida, insecticides are applied to control the adult stage of E. stigmatias starting as early as tass el emergence and continuing until the silks have dried (Scully et al. 2000). The immature stages are protected within the ear and in the soil. The insecticides are frequently applied early in the morning before the wind reaches 24 kph (15 mph). However, little information is available on the pattern of oviposition with respect to the time of the day and age of the ears. Euxesta stigmatias was reported to deposit most of their eggs between 1100 and 1300 hr (Seal and Jansson 1993). Information on the act ive period of the day for oviposition by the other species may help improve the timing of control strategies. It is possible that different species are partitioning the resource to reduce competition, resulting in more larvae deeper in the ear when multipl e species are present. Corn ears are currently sampled for fly damage by opening the tips of husks and examining the silks and kernels for presence of immatures or damage. Sampling infested ears by examining just the ear tips may be i neffective if flies are present only in the middle and bottom portions of th e ear. More information on the ecological aspects of these flies may help in the formulation of better management practices for these flies. Therefore, studies were conducted to determine and compare the various ecological parameters (daily pattern of oviposition and intra ear distribution) of the three most common corn infesting ulidiids in Florida corn fields: E. eluta E. stigmatias and C. massyla

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183 Materials and Methods Daily Pattern of Oviposition The study was conducted in the field by examining sweet corn ears for eggs naturally deposited by Ulidiidae over 4 wk from first silk appearance to 7 d beyond normal maturity. Sweet corn fields with visual presence of adult E. eluta E. stigmatias and C. massyla were selected for this experiment. The first study was conducted in June 2009 on sweet corn planted 19 March 2009 ( cv. Heavenly Syngenta Seeds). The study was repeated in May 2010 on sweet corn planted 15 February 2010 (cv. Obsession). Sweet corn fields were planted at the EREC in organic (Dania muck) soil and manage d according to local standards ( Ozores Hampton et al. 2010). Thirty six and forty plants with ears at first silk appearance were randomly selected from fields on 1 June 2009 an d on 4 May 2010, respectively Only ears without Lepidoptera damage were chosen for these experiments. The s ilks of each ear were cleaned of existing fly eggs using a camels hair brush (size 0) and then covered with a pollination bag (6.25 2.5 21.25 cm) to protect the ears from infestation by S frugiperda H. zea and Ulidiidae infestation. The ears were divided into two subsets of 18 ears in 2009 and two subsets of 20 ears in 2010. To examine oviposition patterns of the three species observed in t he fields, the pollination bags were removed to allow natural oviposition in subsets of ears for alternating 2h periods. The first subset of ears was exposed beginning at 0700 h and then covered at 0900 h (EST) The second subset of ears was exposed beg inning at 0900 h and then covered at 1100 h. The first subset of ears was examined for eggs during the period that the second subset of ears w as exposed to oviposition. The process of exposing and then examining alternating groups of ears was repeated until 1900 h. All ears were covered with pollination bags from 1900 h until

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184 0700 h on the next sampling day. Before exposing the corn ears on the next sampling day, they were examined for any natural infestation of Ulidiidae or Lepidoptera larvae. Ears th at had become naturally infested by these insects were replaced by another ear of similar age for the remainder of the experiment. Sampling was conducted up to twice per week depending on weather conditions for 4 wk. Sampling was continued past the norma l 21 d maturity of sweet corn to test the attractiveness of these mature ears for oviposition. The eggs found deposited on each corn ear during a 2h period were collected into separate 30 ml plastic cups containing H. zea artificial diet using a camels hair brush and transported to the laboratory. A cotton ball was placed above the diet in each cup to provide a dry location for larvae to pupate. The diet cups with eggs were placed singly into individual 185ml cylindrical vials and then covered with pap er towels held in place with rubber bands to prevent larvae from escaping when they left the diet to pupate. The vials with diet cups were held under laboratory conditions at 26.5 1.0 C, 14:10 (L: D) photoperiod, and 55 70% RH. Adults that emerged fr om pupae were removed from the vials every 3 d until all the adults had emerged. Flies that emerged in each vial were counted and identified to species using a hand lens and keys of Euxesta ( Ahlmark & Steck, unpublished, Curran 1928, 1934 and 1935) and Ch aetopsis spp. (G. Steyskal unpublished) together with identified u lidiid specimens housed at the Division Plant Industry, Gainesville, FL. Eggs of different ulidiid species cannot be differentiated. Therefore, numbers of eggs/species were estimated from the number of adults emerg ing after the eggs hatched and the flies were reared to maturity

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185 The flies in the sweet corn fields were sampled using sweep nets to correlate the fly abundance in the field with the eggs obtained on corn ears. Sweep netting was done at the beginning of the experiment at the time of first silk appearance on 2 June 2009 and 5 May 2010. Samples were taken from five corn rows that included one at each end of the field and three randomly selected in the middle of the field. Ten sw eeps were made in each row and flies were collected and brought in laboratory to identify to species level. Intra E ar D istribution The intra ear distribution of larvae was studied on ulidiidinfested sweet corn ears collected 3 wk after silk initiation. I nfestation was confirmed by pulling back the tips of husks just enough to determine the presence of Ulidiidae larvae. Ears were collected from four sweet corn fields planted at the EREC during 2009 and 2010. Forty ears were collected on 18 June 2009 from a sweet corn field planted 19 March 2009 (Field 1: cv. Heavenly). Twenty four and 38 ears were collected on 20 May 2010 from two sweet corn fields planted 15 February 2010 (Field 2 and Field 3: cv. Obsession). Fifty four ears were collected on 25 June 2010 from a sweet corn field planted 1 April 2010 (Field 4: cv. Passion, Seminis Vegetable Seeds). Collected ears were held in the shade befo re transferring them within 2 h to the laboratory. The ears were cut with a sharp knife into three approximately equal segments (i.e., tip, middle, and bottom thirds). Each segment was placed in a separate 1.83 L Ziploc bag with paper towels to reduce the accumulation of free moisture. The ear segments were maintained at same environmental conditions used in t he previous experiment to allow larvae to complete development and pupate within the bag. Pupae were collected from the ears, bags and paper towels every 2 3 d and transferred onto moistened filter paper within Petri

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186 plates. Parafilm was used to seal t he dishes to reduce moisture loss and pathogen infestation. Adults that emerged were counted at the end of the experiment. The flies were identified to species and counted to determine the distribution across the ear segments at harvest time. Statistical Analysis Daily pattern of oviposition. The effect of time of day on oviposition by each fly species was evaluated using repeated measures analysis of variance (PROC MIXED, SAS Institute 2008). Fly species, time of day, and their interaction were used as fixed variables in the model. The number of eggs deposited per infested ear in each 2h period and the proportion of corn ears infested during each 2h period were tested separately as the dependent variables. The individual ears used in the experiment were used as the repeated variable due to repeated examinations of the same ear through time on multiple days. The betweenwithin method was used by the SAS software to calculate the degrees of freedom. The effect of ear age on oviposition by each fly species was again evaluated using repeated measures analysis of variance (PROC MIXED, SAS Institute 2008). The number of eggs deposited per 2h period were summed across the day to produce number of eggs per day per ear. Fly species, ear age in days, and their interactions were used as fixed effects in the model. The individual ears used in the experiment were used as the repeated variable due to repeated examinations of the same ear through time on multiple days. The number of eggs deposited per corn ear in one day and the proportion of corn ears infested by an individual species were tested as the dependent variables. The proportion of infested corn ears was calculated by dividing the number of ears that received eggs of an individual species by the total sampled ears

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187 per day. Least square means were used to compare numbers and proportions for treatments due to the covariance structure of the model (different number and ages of ears conducted at different seasons). The LSMeans and the PDIFF commands wer e used to perform paired t test comparisons of the least square means. Intra ear distribution. Proc Mixed (SAS institute 2008) was used to conduct an analysis of variance of the results of intra ear distribution due to the presence of both fixed and random effects. Dependent variables tested were the number of adults that emerged per ear segment. Independent variables tested were fly species, ear segment and their interactions. Year and fields were used as random variables initially in the model, but variance associated with year was estimated to be zero and it was removed from the random statement. SAS calculated the degrees of freedom using the containment method. The LSMEANS statement was used to generate least square means and their standard errors and the PDIFF command was used to perform the paired t test comparison of the least square means. Analysis of variance was also conducted to evaluate whether ear segments from which more than one fly species emerged had a significant fly species effect. The numbers of corn ears with presence of individual species alone or in combination with other species were compared with the adults caught in sweep nets in both 2009 and 2010. Results and Discussion Daily Pattern of Oviposition There was a significant difference in the naturally occurring species composition in experimental fields at the EREC in 2009 and 2010. Chaetopsis massyla adults were uncommon in 2009 and E. stigmatias were rarely observed in 2010. Therefore, the results of experiments to examin e the daily pattern of oviposition were presented and

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188 analyzed separately for each year. No eggs of any species were observed on corn ears on the 1st or 2nd d after first silk appearance in both study years; therefore, analysis was begun with oviposition on the 3rd d after first silk appearance. June 2009 The results were first examined using the number and proportion of eggs deposited per day during the 3wk sweet corn ear maturation period up to the normal harvest date. The number of eggs deposited per day were significantly ( P > 0.05) affected by fly species ( F = 7.42 ; df = 2, 34; P = 0.0021) ear age ( F = 6.12; df = 7, 119; P < 0.0001) The number of eggs deposited per day w as also affected by fly species with varying ear ages ( F = 5.34 ; df = 14, 238 ; P < 0.0001 ) Ear age significantly affected the number of C. massyla and E. stigmatias eggs deposited by day (Table 8 1 ). Chaetopsis massyla deposited < 0.3 eggs/ear/day, but significantly fewer were produced during the last week of ear development for sweet corn than during the first 2 wk when the silks were still growing. Euxesta eluta deposited 5.38.0 eggs per day throughout the normal 3 wk sweet corn ear development period. Egg deposition by E. stigmatias peaked at 10 d after first silk appearanc e and then decreased > 85% by 13 d after first silk appearance. While no oviposition by C. massyla was detected beyond 16 d after first silk appearance, both Euxesta spp. continued to deposit eggs throughout the 3 wk period. The proportion of the corn ear s infested per day were affected by fly species ( F = 107.55; df = 2, 34 ; P < 0.0001) and ear age ( F = 4.71 ; df = 7, 119 ; P = 0.0001) The proportion of corn ears infested per day w as also affected by the fly species but the results depended on ear age ( F = 4.56 ; df = 14, 238 ; P < 0.0001) The significant effect of fly species was the result of significant differences in oviposition by E. stigmatias

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189 among ear ages with most oviposition occurring early in ear life (Table 8 1 ). Euxesta stigmatias deposited eggs in 88% and 94% of sampled ears at 8 and 10 d after first silk. No corn ears were infested with eggs of E. stigmatias past the regular harvesting age of ears (21 d after first silk appearance). The proportions of ears in which E. eluta deposited eggs were relatively constant throughout the 3wk post silk emergence period. Chaetopsis massyla deposited eggs in The results were next evaluated for differences among 2h periods sampled throughout the 3wk ear period. The mean number of eggs deposited per ear per 2h period of the day did dot differ among t he fly species ( F = 2.43; df = 2, 34 ; P = 0.1034) nor among time of the day ( F = 0.50; df = 5, 85 ; P = 0.7757) Also, there was no difference in the oviposition pattern of fly species among time periods ( F = 0.79 ; df = 10, 170; P = 0.6346) However, a separate analysis of oviposition by species determined that E. stigmatias oviposition was significantly affected by time of day (Table 8 2 ). Euxesta stigmatias ovipositio n peaked from 1100 to 1500 h. There was no difference in oviposition between the periods of 0700 to 1100 h and 1500 to 1900 h. The proportion of corn ears infested in 2h time period differed among fly species ( F = 99.66 ; df = 2, 34; P < 0.0149) and time of day ( F = 10.19; df = 5, 85 ; P < 0.0001 ) The proportion of corn ears infested in 2h time period was also affected by the fly species but the results depended on time of day ( F = 4.57; df = 10, 170 ; P < 0.0001 ) The time of day affected the proportion of ears with E. eluta and E. stigmatias eggs (Table 8 2 ). Euxesta eluta deposited eggs on fewer ears from 07001100 h (35%) than during the other time periods (1719%). Euxesta stigmatias deposited eggs on a greater proportion of ears from 11001400 h (30%) than during the other time periods

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190 (1 2%). The proportion of the ears infested with E. stigmatias eggs did not vary significantly between the periods of 07001100 h and 17001900 h. May 2010. The number of eggs deposited per day were significantly ( P > 0.05) affected by fly species ( F = 4.70; df = 7, 133 ; P = 0.0149) ear age ( F = 13. 12; df = 7, 133; P < 0.0001) The number of eggs deposited per day were also significantly ( P > 0.05) affected by the fly species but the results depended on ear age ( F = 2.22; df = 14, 266; P = 0.0074). Ear age significantly affected the number of eggs deposited by day for all 3 species (Table 8 3 ). Chaetopsis massyla deposited fewer eggs during the last week of ear development (01.9 eggs per day) for sweet corn than during the first week (6.0 9.5 eggs per day). Euxesta eluta deposited fewer eggs during 2022 d of ear development (0.52.7 eggs per day) than during the first two weeks (7.212.9). Euxesta stigmatias oviposition peaked at 17 d after first silk appeara nce and remained the same until 20 d after first silk appearance. Euxesta stigmatias deposited fewer eggs before and after 1720 d after first silk appearance. The proportion of ears infested by eggs was affected by fly species ( F = 14.83; df = 2, 38 ; P < 0.0001) ear age ( F = 7.07 ; df = 7, 133 ; P < 0.0001) The proportion of ears infested by eggs was also affected by the fly species but the results depended on ear age ( F = 12.39; df = 14, 266 ; P < 0.0001) Ear age affected the oviposition by all three f ly species among ear ages (Table 8 3 ). Chaetopsis massyla deposited a greater proportion of eggs during the first 2 wk (3080%) after first silk appearance than during the 3rd wk after first silk appearance. The proportion of ears with E. eluta eggs peak ed at 11 d (60%) after first silk appearance and then decreased in older age ears. Euxesta

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191 stigmatias deposited a greater proportion of eggs during the 3rd week (3545%) after first silk appearance than during the first 2 wk after the first silk appearanc e The number of eggs deposited per ear per 2 h period of the day varied among t he fly species ( F = 4.23; df = 2, 38 ; P = 0.0219) and fly species at different time s of day ( F = 2.09; df = 10, 190; P = 0.0269) The number of egg deposited per ear did not v ary with different t ime s of the ( F = 0.74; df = 5, 95; P = 0.5986) Euxesta eluta deposited more eggs (12.4 2.7) than C. massyla (10.2 2.7) and E. stigmatias (10.3 2.8). A separate analysis of oviposition by species determined that E. eluta oviposi tion was significantly affected by time of day (Table 84 ). Euxesta eluta deposited fewer eggs from 0700 1100 h and from 17001900 h than from 11001300 h and from 15001700 h. Chaetopsis massyla deposited 6.410.3 eggs per 2h time period throughout the day. The proportion of ears infested in a 2h time period varied among fly species ( F = 18.37; df = 2, 38 ; P < 0.0001 ) time of day ( F = 7.63; df = 5, 95 ; P < 0.0001) and fly species with different time s of day ( F = 3.28; df = 10, 190 ; P < 0.0001) The p roportion of ears deposited on by E. eluta and E. stigmatias was not uniform throughout the day. Euxesta eluta deposited eggs on a greater proportion of ears from 11001700 h (10 20%) than during the other time periods (13%). Euxesta stigmatias deposited eggs on a greater proportion of ears after 1100 h (24%) than before 1100 h (appr ox imately 0%). An egg free period was observed at the beginning and end of the reproductive period of corn in both years of the study Flies did not deposit eggs on corn ears at 1 or 2 d after first silk appearance in both the years. App (1938) found the silks i nfested with E. stigmatias eggs an average of 1.12 d after silk initiation. It is possible that flies respond differently to changes in the chemical composition of silks with time. Moreover,

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192 daily fly density was not recorded, which could have affected the number of eggs deposited on corn ears at different days from the beginning to the end of the reproductive period of corn. Information is also lacking on response of different species to different chemical composition of silks as they age. The day when the flies stopped depositing eggs was different in each year of my study. Some trends could be observed from results of both the years on effect of ear age on number of eggs deposited. Chaetopsis massyla deposited more eggs at the beginning of the reproductive period of corn, with egg numbers declining as the corn ears aged. The results on the proportion of corn ears infested with E. eluta eggs correspond with egg oviposition frequency on corn ears in 2010. However, no trend emerged for mean number of E. eluta eggs deposited, or proportion of ears with E. eluta eggs in 2009. In contrast to the other two species, E. stigmatias deposited more eggs towards the middl e and end of the reproductive period in 2009 and 2010, respectively. The results on proportion of the corn ears with E. stigmatias eggs closely followed the trend exhibited by those on mean number of E. stigmatias eggs in both the sampling years. Seal and Jansson (1989) found that more E. stigmatias eggs were deposited on fresh young ears than old corn ears. The t hree species differed in their time of oviposition. No difference was observed in C massyla oviposition among any 2h time periods of the day Results of 2010 showed that more E. eluta eggs were deposited and in more corn ears from 11001700 h than early in the morning or late in the evening. This trend was also shown by the proportion of ears with E. eluta eggs in 2009. The results of sampling in 2009 indicated that more E. stigmatias eggs were deposited and in more ears from 1100 1500 h than

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193 other times of the day. No trend could be seen for E. stigmatias eggs during the day in 2010, probably due to overall lower density of this species (Table 8 5 ) and fewer eggs deposited in 2010 than in 2009. The results on the oviposition pattern of E. stigmatias match the results of Seal and Jansson (1989) who found that E. stigmatias deposited more eggs at 1300 hr compared to other times of the day. Differences were observed between the two study years in number of eggs deposited by the three species. Eggs of primarily E. eluta and E. stigmatias were obtained in 2009, while eggs of C. massyla and E. eluta were obtained in 2010. Euxesta stigmati as eggs were obtained in 2010 only in older ears. Differences in mean numbers of eggs deposited by the three species between the studies may be explained to some extent by the fly counts of three species caught in sweep nets in respective years (Table 8 5 ). Seven times more female E. stigmatias were caught in sweep nets in 2009 (56) than in 2010 (6). Euxesta eluta and C. massyla females were approximately 2 and 5 times more common in 2010 than in 2009, respectively Intra Ear Distribution Chaetopsis mass yla E. eluta and E. stigmatias were reared from each ear segment indicating that these flies can utilize the entire corn ear for their development. However, t he total number of adults that emerged per segment of the ear differed significantly ( P < 0.05) among fly species and also when ear segments varied (Table 8 6 ) More flies emerged from the ear tip (l east squared mean SEM; 8.55 1.00) followed by the middle (1.96 1.00) and the bottom (1.37 1.00) segments. More E. stigmatias emerged per ear segment (6.35 1.00) than E. eluta (5.06 1.00) followed by C. massyla (0.47 1.00). Significantly more E. eluta and E. stigmatias emerged from the tip followed by the middle and bottom segments (Table 8 7 ). No significant

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194 difference was observed in the mean number of C. massyla adults that emerged among ear segments. Significantly more E. eluta and E. stigmatias emerged from ear tips than C. massyla In the middle segment of the ear, significantly more E. stigmatias emerged than C. massyla There was no significant difference in the number of flies of each species that emerged from the bottom segment. Our findings are in agreement with the results of Seal and Jansson (1989) who found greater densit ies of E. stigmatias larvae and pupae in the tip segm ent of the ear compared to middle and bottom ear segments. It is possible that larvae move deeper into the ears to reduce intra and inter species competition for food and space within the ears. There were significant differences in the distribution of fli es in the ear sections (Table 8 8 ). Ears with both E. eluta and E. stigmatias were much more common than the other combinations, including ears infested with only one of the two species. It is possible that the presence of adults or immatures of one of these species on corn ears affected the behavior of other species to deposit their eggs in the same corn ears. Results from the choice and nochoice tests indicated that fewer E. eluta and E. stigmatias adults emerged from ears previously infested with E. eluta larvae than those infested by S. frugiperda larvae. It is possible that both species preferred to deposit eggs in ears previously infested with any ulidiid species than in uninfested ears when no Lepidopterainfested ears were available. The findi ng that corn ears with all three species were more common than ears with only C. massyla or other combinations of C massyla with other species suggests that ear infestation by Euxesta spp. may affect oviposition behavior or larval survival of C. massyla w ithin corn ears. No ears were

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195 found with only C. massyla larvae in either year. While uninfested ears served as suitable oviposition and development hosts for C. massyla in the choice and nochoice tests described above, the results of this study suggest s that they may prefer ears previously infested with other insects than uninfested ears. It is also possible that C. massyla responds to potential hosts in some density dependent manner. C hoice test studies earlier showed that five C. massyla females in a cage were enough to result in oviposition and development in uninfested, as well as in previously infested ears. Only 2 and 11 C. massyla females were caught in 50 sweeps in 2009 and 2010, respectively (Table 8 5 ). C haetopsis massyla at low densities m ay prefer to oviposit in ears with previous infestation compared to uninfested ears. The reduction of corn ears with E. stigmatias alone from 8 in 2009 to 1 in 2010 probably reflected the reduction in adult densities as assessed by using sweep nets (56 fe males in 2009 and 6 females in 2010). In conclusion, it was found that Chaetopsis massyla deposited more eggs in young ears ( when silks were actively growing) while E. stigmatias deposited more eggs in ears during and after peak silk extension. The three species differed with respect to the times of the day for oviposition. Chaetopsis massyla deposited eggs throughout the day while Euxesta eluta deposited more eggs and in more corn ears from 11001700 h than during other times of the day. Euxesta stigmatias deposited more eggs and in more ears from 11001500 h than during other times of the day. The flies of these species utilize d the whole corn ear and not just any one part of ear for their nutrition. The finding that more larvae were in ear tips than middle and bottom segments showed that flies may compete for their resources forcing the larvae to go deeper in the corn ears. Moreover, differences in the frequency of ears with individual versus multiple species

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196 indicated the presence of behavioral int eractions among these species in oviposition preference and development within the corn ears.

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197 Table 8 1 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by ear ages, June 2009. Ear age (d) Numbers of e ggs /infested ear/day Proport ion of infested ears C. massyla E. eluta E. stigmatias C. massyla E. eluta E. stigmatias 3 0.2 0.04A 6.6 1.8 6.2 2.6C 0 a 0.44 0.1 0.72 0.1ABC 8 0.2 0.04A 8.0 1.9 13.8 2.6B 0.06 0.03 0.50 0.1 0.88 0.1A 10 0.2 0.04A 6.4 1.9 31. 1 2.6A 0.06 0.03 0.66 0.1 0.94 0.1 A 13 0.1 0.04A 7.1 1.9 3.7 2.6C 0 0.72 0.1 0.50 0.1CD 16 0.04 0.04B 6.3 1.9 4.2 2.6C 0 0.44 0.1 0.33 0.1D 19 0C 6.7 1.9 5.4 2.6C 0 0.44 0.1 0.78 0.1AB 21 0C 5.8 1.9 4.5 2.6C 0 0 .50 0.1 0.55 0.1BCD 24 0C 5.3 1.9 3.1 2.6C 0 0.56 0.1 0E F 7.23 0.74 13.39 0.87 0.79 10.74 P < 0.0001 0.6376 < 0.0001 0.5313 0.5945 < 0.0001 df 7, 119 7, 119 7, 119 7, 119 7, 119 7, 119 Mean SEM within a column followed by the same capital letter are not significantly different ( P > 0.05; PDIFF paired t tests); (Proc Mixed; SAS Institute) aProportion of infested ears was calculated as 0 or a negative value by SAS analysis due to very low value of actual number.

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198 Table 8 2 Mean SEM n umber of eggs and proportion of corn ears with ulidiid eggs by 2 h time period, June 2009. Oviposition time period (d) Numbers of e ggs /infested ear/2 h time period Proportion of infested ears C. massyla E. eluta E. stigmatias C. massyla E. eluta E. stigm atias 0700 0900 h 0 .03 0.09 6.8 3.8 11.6 4.9B 0 0.05 0.03B 0.09 0.03C 0900 1100 h 0.01 0.09 8.9 3.7 10.5 4.9B 0 a 0.03 0.03B 0.1 0.03BC 1100 1300 h 0 .04 0.09 11.2 3.7 29.8 4.9A 0 0.17 0.03A 0.3 0.03A 1300 1500 h 0.2 0.09 11.1 3.7 30.3 4.9A 0.01 0.004 0.19 0.03A 0.3 0.03A 1500 1700 h 0 .05 0.09 12.2 3.7 19.0 4.9AB 0 0.17 0.03A 0.2 0.03B 1700 1900 h 0.2 0.09 15.4 3.7 12.9 4.9B 0.01 0.004 0.17 0.03A 0.1 0.03BC F 1.18 0.09 0.67 3.54 0.80 6. 64 6.10 P 0.3255 0.6503 0.0059 0.5527 < 0.0001 < 0.0001 df 5, 85 5, 85 5, 85 5, 85 5, 85 5, 85 Mean SEM within a column followed by the same capital letter are not significantly different ( P > 0.05; PDIFF paired t tests); (Proc Mixed; SAS Institute) aProportion of infested ears was calculated as 0 or a negative value SAS analysis due to very low value of actual number.

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199 Table 8 3 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by ear age, May 2010. Ear age (d) Numbers of e ggs /infested ear/day Proportion of infested ears C. massyla E. eluta E. stigmatias C. massyla E. eluta E. stigmatias 3 9.5 1.2A 12.9 2.5A 0.11 0.3BC 0.80 0.1A 0.40 0.1ABC 0B 5 6.0 1.2B 8.2 2.5B 0.2 0.3BC 0.50 0.1BC 0.45 0.1AB 0B 9 4.8 1.2BC 7.2 2.5B 0.3 0.3BC 0.60 0.1AB 0.20 0.1CD 0B 11 3.4 1.2CD 7.2 2.5B 0.5 0.3BC 0 a D 0.60 0.1A 0B 14 3.1 1.2CD 5.1 2.5BC 0.8 0.3B 0.30 0.1C 0.30 0.1BC 0B 17 1.9 1.3DE 4.6 2.6BC 1.6 0.3A 0D 0.30 0.1BC 0.35 0.1 A 20 0.9 1.3DE 2.7 2.6CD 1.3 0.3A 0D 0D 0.45 0.1A 22 0E 0.5 2.6D 0.09 0.3C 0D 0D 0B F 8.53 4.90 8.00 17.92 5.05 11.29 P < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 df 7, 133 7, 133 7, 133 7, 133 7, 133 7, 133 Mean SEM within a column followed by the same capital letter are not significantly different ( P > 0.05; PDIFF paired t tests); (Proc Mixed; SAS Institute) aProportion of infested ears was calculated as 0 or a negative value by SAS analysis due to very low value of actual number.

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200 Table 8 4 Mean SEM number of eggs and proportion of corn ears with ulidiid eggs by 2 h time period, May 2010. Oviposition time period (d) Numbers of e ggs /infested ear/2 h time period Proportion of infested ears C. massyla E. eluta E. stigma tias C. massyla E. eluta E. stigmatias 0700 0900 h 8.3 2.7 6.7 4.1C 0.05 0.7 0.04 0.02 0.007 0.02B 0 a B 0900 1100 h 10.3 2.7 9.4 4.0C 0.3 0.7 0.06 0.02 0.02 0.02B 0B 1100 1300 h 8.6 2.7 18.6 4.0AB 1.7 0.7 0.07 0.02 0.1 0.02A 0.04 0.01A 1300 1500 h 9.1 2.7 14.5 4.1BC 1.1 0.7 0.07 0.02 0.1 0.02A 0.04 0.01A 1500 1700 h 7.7 2.7 21.6 4.1A 1.6 0.7 0.09 0.02 0.2 0.02A 0.02 0.01A 1700 1900 h 6.4 2.7 7.3 4.0C 0.8 0.7 0.09 0.02 0.03 0.02B 0.02 0 .01A F 0.40 4.16 0.86 0.78 8.57 2.75 P 0.8479 0.0018 0.5134 0.5674 < 0.0001 0.0231 df 5, 95 5, 95 5, 95 5, 95 5, 95 5, 95 Mean SEM within a column followed by the same capital letter are not significantly different ( P > 0.05; PDIFF paired t tests); ( Proc Mixed; SAS Institute) aProportion of infested ears was calculated as 0 or a negative value by SAS analysis due to very low value of actual number. Table 8 5 Fly counts of three ulidiid species caught in sweep nets from corn fields selected for daily pattern of oviposition experiment 2009 a 2010 b Species Male Female Male Female C. massyla 7 2 21 11 E. eluta 31 14 39 24 E. stigmatias 25 56 8 6 aAdults caught in 50 sweeps on 2 June 2009. bA dults caught in 50 sweeps on 5 May 2010.

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201 Table 8 6 ANOVA table of intra ear distribution of flies Source df F P Fly species 2 56.95 < 0.0001 Ear segment 2 94.56 < 0.0001 Fly species*ear segment 4 24.51 < 0.0001 ANOVA (PROC MIXED, SAS Institute 2008) error df = 1392 Table 87 Least squared mean SEM (range) number of adults of three Ulidiidae species reared from three segments of corn ears, 20092010 (n = 156) Ear segment (n = 156) a Species Tip Middle Bottom C. massyla 0.39 1.16 C (0 10) 0.46 1.16 C (0 12) 0.55 1.16 C (0 8) E. eluta 11.9 1.16 A (0 90) 2.05 1.16 BC (0 53) 1.20 1.16 C (0 18) E. stigmatias 13.3 1.16 A (0 97) 3.37 1.16 B (0 60) 2.36 1.16 BC (0 57) Means followed by the same letter are not significantly different (t test, P > 0.05, SAS Institute 2008) an = number of cor n ears Table 8 8 Frequency of corn ears with ulidiid species alone or in combination with other species in two years of ear sampling Corn ears with natural infestations of species Year 2009 Year 2010 C. massyla 0 0 E. eluta 5 3 E. stigmatias 8 1 C massyla, E. eluta 4 3 C. massyla, E. stigmatias 0 0 E. eluta, E. stigmatias 27 67 C. massyla, E. eluta, E. stigmatias 16 19

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202 CHAPTER 9 SUMMARY Euxesta stigmatias is one of several serious primary pest s of sweet corn in Florida among other insect pe sts. Adults of other Ulidiidae species E. eluta, E. stigmatias and C. massyla have been caught in sweep nets and suction traps at Belle Glade, FL but i nformation is lacking on the pest status of these species in Florida. Choice and nochoice tests in the green house and in the field were conducted to determine the pest status ( p rimary versus secondary mode of attack) of these species. Infested corn ears (infested with S. frugiperda or E. eluta ) and uninfested corn ears acted as main treatments. The c orn ears were exposed to each of the three fly species, E. eluta E. stigmatias and C. massyla in both choice and nochoice tests for 10 d. E ars infested with larvae were harvested and held for adult emergence. All three species were reared from both uninfested and infested ears in both choice and nochoice tests indicating their primary and secondary modes of attack More flies of all three species emerged from corn ears that were infested with S. frugiperda than uninfested c orn ears Presence of olfactory cues or easier access to larval tunneling in ears damaged by Lepidoptera larvae may explain the difference in number of adults emerged between S. frugiperda infested and uninfested ears. The finding that fewer adults of E. stigmatias and C. massyla emerged from ears infested with E. eluta in no choice tests suggests that visual or olfactory cues associated with infestation by this fly may negatively affect oviposition. Studies in the future may be directed towards finding oviposition cues that could be exploited in better managing the flies. While three species were confirmed as primary pests of sweet corn in these trials, only one species has been reported feeding on corn in Florida. Surveys were

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203 conducted throughout Florida to evaluate species ric hness and distribution of corninfesting Ulidiidae Adults were sampled using s weep net s and reared from fly larvaeinfested corn ear s collect ed from representative corn fields in 16 and 27 counties in 2007 and 2008, respectively. Four U lidiid ae species, C. massyla E. annonae, E. eluta and E. stigmatias were found in corn fields using both sampling techniques. Evidence presented herein is the first known documentation for E. annonae and E. eluta as pests of corn in Florida and the USA. The four species were not uniformly distributed throughout Florida corn growing regions. Euxesta eluta and C. massyla were found infesting field and sweet corn throughout Florida. Euxesta stigmatias was only found in Martin, Miami Dade, Okeechobee, Palm Beach and St. L ucie Counties on field and sweet corn. Euxesta annonae (F.) was found in sweet corn in Miami Dade, Okeechobee, and Palm Beach Counties, but field corn was not sampled in these counties. Euxesta eluta, E. stigmatias and C. massyla were sweep netted and reared from corn ears throughout the corn reproductive stage. The relative abundance of E. eluta and C. massyla in Florida field and sweet corn indicates the need for more research into their biology and ecology. Raising adults from fly larvaeinfested ear s provided the best method for assessing rates of ear infestation and species richness. Sweep netting did not provide reliable estimates to identify ulidiid species infestation. The discovery of E. eluta and C. massyla attacking corn ears in many of the northernmost Florida counties suggests that further surveys of corn growing areas across the borders into neighboring states is warranted to determine the extent of corn infesting picturewinged fly infestations in the southern U.S. The statewide distribution of E. eluta and C. massyla in reproducing corn also suggests that additional studies should be

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204 conducted to evaluate additional food sources that support these species in the absence of corn. In order to develop sampling plans and economic thresholds for these flies, it becomes important to understand the spatial and temporal distribution of the flies in corn fields. Yellow sticky traps were used to sample flies in corn fields ranging from 1.8 ha to 16 ha. At harvest, ears near the traps were examin ed for fly larvae and infested ears were collected and held to determine identity of the flies. The distribution of the adults was determined to be aggregated in both small and large scale fields at most of the sampling dates. More flies were found on th e edges than in the center of most of the sampled fields. More flies were found on field sides bordered by sugarcane/fallow/residence than those bordered by corn for most of the fields sampled. The proportion of infested corn ears was found to be strongl y correlated with t he season totals of flies caught per sticky t rap The information on number of flies to be observed on sticky traps to cause damage to corn ears can be used to determine the time of insecticide application. Sweet corn is not available t hroughout the year for fly development, yet adults of these species routinely appear at the beginning of each season following cornfree periods suggesting other plants are acting as food sources for maintenance and development of these flies. Field surveys were conducted to evaluate crop and noncrop plants commonly found in fields near maize growing areas for their potential to act as developmental hosts for three species of these flies. Sweep netting and visual observations were conducted to sample the plant species for adults. Infested plant parts were brought in laboratory and held to rear out the flies. Laboratory studies were

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205 conducted by exposing the different commodities to three fly species to determine the developmental and survivorship rates for the immature stages of these flies reared on alternative hosts. M any commodities and weeds (bell pepper, spiny amaranth, cattail, sugarcane, and johnsongrass) found in field surveys provided satisfactory food sources for successful development from eg gs to adults In la boratory evaluation, a ll three species complete d development on alternative commercial crops and weedy species (bell pepper, cabbage, radish, papaya, hass avocado, sugarcane, little hogweed, habaero pepper, tomato, southern cattail, spiny amaranth, johnsongrass) The larval development time increased in the following order from bell pepper, cabbage, tomato, papaya, avocado, radish, sugarcane, habaero, little hogweed, johnsong rass, cattail to spiny amaranth. The number of pupae decreased in the same above order with few exceptions. The plant species tested here are available in abundance especially in Miami Dade and Palm Beach Counties, where most of Floridas sweet corn is grown. The presence of multiple host crops throughout the sw eet corn production areas of Florida may help explain the occurrence of these flies immediately after prolonged absences of corn. Information is available to identify the adults of these species, but no information is available on identification of the imm atures. The ability to distinguish their immature stages would be useful for biological studies where their distributions overlap. Morphology of the immature stages of three species of these flies, C massyla E eluta and E stigmatias was examined S everal characteristics of eggs ( rd instar larvae (4 d old), and pupae (4 d old) were examined using light and scanning electron microscopy. Egg length, width and their ratio, diameter of pores, collar diameter were

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206 found to be significantly different among the three species, but these could not be used to distinguish the species due to the overlapping ranges. The only characteristics that can be used to distinguish the eggs of species are the distribution of pores on the surface of eggs. Pores were restricted to the posterior end of C. massyla eggs, but distributed over the entire surface of E. eluta and E. stigmatias eggs. Similarly, characteristics of larvae including body length, width, their ratio, cephalopharyngeal skeleton parameters, antennal length, width and their ratio, length, width and their ratio of anterior spiracles, and various parameters of posterior spiracles were found to be significantly different among three species, but could not be used to distinguish the species due t o the overlapping ranges. Only a few larval characteristics were found that can be used to distinguish the three species. A distinct tooth on the ventral surface of the mouth hooks separates C massyla larvae from the other species Euxesta eluta and E stigmatias had more oral ridges than C massyla T he two Euxesta species could be separated based on the length of the 2nd and 6th creeping welts. The first row of spinules was discontinuous on the 4th 8th creeping welts in larval C. massyla while it was continuous in Euxesta species. The larval posterior spiracles were black on E. eluta, dark brown to black on E. stigmatias and brown on C. massyla. The mean pupal length, width, and their ratio were significantly different among the species but again can not be used to distinguish the species due to the overlapping ranges. C haetopsis massyla pupae were reddish brown while E. eluta and E. stigmatias pupae were light brown to black. The posterior spiracular plates in C. massyla pupae were trapezoidal in shape while those in E. eluta and E. stigmatias were ovoid. The characteristics found to separate the species (egg surface, larval mandibles, larval creeping welts, larval

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207 posterior spiracles, and pupal posterior spiracular plate) require the use of com pound and scanning electron microscopes which limits their usefulness to field scouts, growers or others working in field conditions. However, pupal color and shape of posterior spiracular plates in pupae can be used to separate the pupae of two genera ( C haetopsis and Euxesta ) in the field using a hand lens. Information on development and reproductive periods of the flies can help in better timing of management tactics being used currently. Studies were conducted to determine and compare biological parameters (i.e., adult longevity, developmental periods, and survival of different immature stages, and life table parameters) of the three species of these flies: C. massyla E. eluta and E. stigmatias The development periods and adult longevity of these fli es were studied on both artificial diet in laboratory conditions and corn ears in field conditions. The survival and life table parameters of these flies were studied on artificial diet. Food (artificial diet and corn ears) was found to affect the developmental periods, and adult longevity in laboratory experiments. Flies of all three species developed faster in corn ears than in artificial diet. The development periods in corn ears in general were shortest in May (25 C) followed by in March (22 C) fo llowed by December (16 C) The larval and total development for E eluta in artificial diet was faster than other species. Both male and females of E. eluta lived longer than the other two species and C. massyla adults died sooner than the other two spe cies when reared from either corn ears or artificial diet. The adults lived longer when reared on corn ears than when reared on artificial diet. Females lived longer than males for all three species. Survival of immature stages was not significantly dif ferent among the three species. The percentage pupal survival (97.4) was greatest followed

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208 by egg survival (94.5) followed by larval survival (89.4). Euxesta eluta deposited significantly greater number of eggs (263) than C. massyla (122) and exhibited s horter pre oviposition period (3 d) compared to that of C. massyla (10 d) and E. stigmatias (8 d). The three species deposited eggs for an oviposition period of 20 d and stopped producing the eggs at an average of 3 d before dying. The intrinsic rate of increase was found to be 0.084 for C. massyla 0.139 for E. eluta and 0.106 for E. stigmatias Differences in these biological parameters between artificial diet and corn ears will have to be considered while extrapolating the studies to field conditions. Information on time of the day for oviposition by flies may help improve the timing of control strategies. Therefore, studies were conducted in corn fields by examining sweet corn ears for eggs naturally deposited by Ulidiidae over 4 wk from first silk appearance to 7 d beyond normal maturity. The corn ears were examined every 2h after exposing them to 2h periods of the day from 0700 1900 h. The eggs deposited at each 2h period were reared to adults under laboratory conditions. Chaetopsis massyla and E. eluta deposited more eggs in young ears (3 d after first silk appearance) when silks were actively growing, while E. stigmatias deposited more eggs in ears during and after peak silk extension (10 d after first silk appearance and later) The three species differed with respect to the times of the day for oviposition. Chaetopsis massyla deposited eggs throughout the day while Euxesta eluta deposited more eggs and in more corn ears from 11001700 h than during other times of the day. Euxesta stigma tias deposited more eggs and in more ears from 11001500 h than during other times of the day. The flies of these species utilized the whole corn ear and not just any one part of ear for their nutrition. The finding that more larvae were in ear tips than

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209 middle and bottom segments suggests that flies may compete for their resources forcing the larvae to go deeper in the corn ears. Moreover, differences in the frequency of ears with individual versus multiple species indicated the presence of behavioral i nteractions among these species in oviposition preference and dev elopment within the corn ears. Overall, four species of flies were found attacking corn rather than just one species, E. stigmatias thought in the past to attack corn. The finding that flies attacked corn in entire Florida suggests the presence of these flies attacking corn further north of Florida. Presence of various plant species acting as reservoirs and developmental hosts of these flies near corn fields suggests areawide pest management programs considering the population dynamics of flies on corn and alternate hosts. Information on presence of more flies on edges of fields would help growers in saving the money spent on insecticides by spraying them on edges only and at times needed r ather than in entire field and everyday to every other day. Identification of flies using immatures would help growers and researchers in identifying the species without rearing them to adult stage. Information on various biological parameters including developmental periods, survival and daily pattern of egg deposition in corn ears would help growers in timing the management options being used. Interaction of different species of flies within the corn ears leads into more research on behavioral interact ions among these flies.

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210 LIST OF REFERENCES Abdel Fattah, M. I., Y. S. Salem, and M. I. AbdelMegeed. 1977. Effect of larval diet on the development and fecundity of the cotton leafworm, Spodoptera littoralis (Boisd.). Z. Angew. Entomol. 84: 311315. Foote, B. A. 1976. Biology of picturedwing flies (Diptera: Otitidae). Dept. Entomol. Anniv. Pub. 8: 5159. Allen, E. J., and B. A. Foote. 1992. Biology and immature stages of Chaetopsis massyla (Diptera: Otitidae), a secondary invader of herbaceous s tems of wetland monocots. Proc. Entomol. Soc. Wash. 94: 320328. Anonymous. 2008a. Florida Statistical Abstract. University of Florida, Gainesville, FL. Anonymous 2008b. Florida Cooperative Agricultural Pest Survey Program. Quarterly report No. 220 08. Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL. Anonymous 2008c. Biosystematic Database of World Diptera. Available at http://www.sel.barc.usda.gov/diptera/names/Status/bdwdstat.htm (verified 1 0 Nov ember 2010). Anonymous. 2010a. USDA vegetables 2008 summary, January 2009. Available at http://usda.mannlib.cornell.edu/usda/current/VegeSumm/VegeSumm 0128 2009.pdf (verified 10 Nov 2010). Anonymous. 2010b Florida Automated Weather Network. University of Florida, Gainesville, FL. Available at http://fawn.ifas.ufl.edu/ ( verified 10 Nov ember 2010). App, B. A. 1938. Euxesta stigmatias Loew, an otitid fly infesting ear corn in Puerto Rico. J. Agric. Univ. P. R. 22: 181 188. Arce de Hamity, M. G. 1986. Biologia de Euxesta eluta (Dip: Ulididae) comportamiento en el ataque y putrefaccion de las espigas de maize. Acta Zool. L illoana 38: 119127. Armstrong, J W 1986. Pest organism response to potential quarantine treatments. Proceedings, 1985 ASEAN PLANTI Regional Conference on quarantine support for Agricultural Development. ASEAN Plant Quarantine Center and Training Institute, Serdang, Malaysia. 1: 2530. Bailey, W. K. 1940. Experiments in controlling corn ear pests in Puerto Rico. P. R. Exp Stn ., circular no. 23, USDA, Mayaguez, P. R. Barber, G. W. 1939. Injury to sweet corn by Euxesta stigmatias Loew in So uthern Florida. J. Econ. Entomol. 32: 879880.

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211 Barbosa, P. 1974. Manual of basic techniques in insect histology. Autumn Publisher, Massachusetts. Barbosa, P., A. E. Segarra Carmona, and W. ColonGuasp. 1986. Eumecosomyia nubila (Wiedemann), a new oti tid in Puerto Rico, with notes on the habits of the dipteran species complex of corn. J. Agric. Univ. P. R. 70: 155 156. Birch, L. C. 1948. The intrinsic rate of natural increase of an insect population. J. Anim. Ecol. 17: 1526. Blanton, F. S. 1938. Some dipterous insects reared from narcissus bulbs. J. Econ. Entomol. 31: 113116. Boivin, G. 1987. Seasonal occurrence and geographical distribution of the carrot rust fly (Diptera: Psilidae) in Quebec. Environ. Entomol. 16: 503506. Branco, M. C., G. L. Villas Boas, F. J. B. Reifschneider, and I. Cruz. 1994. Evaluation of resistance to Helicoverpa zea (Lepidoptera: Noctuidae) (Boddie) and Euxesta sp. (Diptera: Otitidae) in lines of Sweet corn. An n Soc. Entomol. Brazil. 23: 137140. Carey, J. R. 1982. Demography and population dynamics of the Mediterranean fruit fly. Ecol. Model. 16: 125150. Chang, C. L., R. Kurashima, and C. Albrecht. 2000. Effect of l imiting concentrations of growth factors in mass rearing diets for Ceratitis capitata larva e (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 93: 898903. Chittenden, F. H. 1895. An ortalid fly injuring growing cereals Insect life 7: 252354. Chu Wang, I. W., and R. C. Axtell. 1972. Fine structure of the terminal organ of the house fly lar va Musca domestica. Z. Zellforsch. 127: 287305. Costello, S. L., P. D. Pratt, M. B. Rayamajhi, and T. D. Center. 2003. Arthropods associated with aboveground portions of the invasive tree, Melaleuca quinquenervia, in south Florida, USA. Fla. Entomol. 86: 300322. Curran, C. H. 1928. Insects of Porto Rico and the Virgin Islands: Diptera or twowinged flies. New York Acad. Sci. Scientific Survey of Porto Rico and the Virgin Islands 11: 1118. Curran C. H. 1934. The families and g enera of North American Diptera. American Museum of Natural History, New York, N.Y. 512pp. Curran, C. H. 1935. New American Diptera. Am. Mus. Novit. 812: 124. Daly, T., and D. G. Buntin. 2005. Effect of Bacillus thuringiensis corn for lepidopteran control of nontarget arthropods. Environ. Entomol. 34: 12921301.

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214 Melhus, I. E., and H. M. Harris. 1949. A new insect pest of maize in Central America. Iowa Agric. Exp. Stn Res. Bull. 371: 603611. Merrill, L. S., Jr. 1951. Diptera reared from Michigan onions growing from seeds. J. Econ. Entomol. 14: 10151015. Miliczky, E., S. D. Cockfield, E. H. Bee rs, and D. R. Horton. 2007. Spatial patterns of western flower thrips (Thysanoptera: Thripidae) in apple orchards and associated fruit damage. J. Entomol. Soc. B C. 104: 25 33. Mohapatra, L. N. 2007. Spatial distribution of cotton jassid Amrasca bigutulla bigutulla Ishida. Indian J. Entomol. 69: 4650. Mossler, M. A. 2008. Crop profile for sweet corn in Florida. http://edis.ifas.ufl.edu/pi034 ( verified 10 Nov ember 2010) Navrozidis, E. I., and M. E. Tzanakakis. 2005. Tomato fruits as an alternative host for a laboratory strain of the olive fruit fly Bactrocera oleae. Phytoparasitica 33: 225236. Nuessly, G. S., and M. G. Hentz. 2004. Contact and leaf residue activity of insecticides against the sweet corn pest Euxesta stigmatias (Diptera: Otitidae). J. Econ. Entomol. 97: 496502. Nuessly, G. S., B. T. Scully, M. G. Heintz, R. Beiriger, M. E. Snook, and N. W. Widstrom. 2007. Resistance to Spodoptera frugiperda (Lepidoptera: Noctuida e) and Euxesta stigmatias (Diptera: Otitidae) in sweet corn derived from exogenous and endogenous genetic systems. J. Econ. Entomol. 100: 18871895. Olalquiaga, G. F. 1980. Aspectos fitosanitarios de la isla de pascua. Rev. Chil. Entomol. 10: 101102. Ozores Hampton, M., W.M. Stall, S.M. Olson, S.E. Webb, S.A. Smith, and R.N. Raid 2010. Sweet corn production in Florida. HS 737, University of Florida. Available at http://edis.ifas.ufl.edu/cv135 ( verified 10 Nov ember 2010). Painter, R. H. 1955. Insects on corn and teosinte in Guatemala. J. Econ. Entomol. 48: 3642. Reay Jones, F.P.F., L. T. Wilson, M.O. Way, T. E. Reagan, and C. E. Carlton. 2007. Movement of Mexican rice borer (Lepidoptera: Crambidae) through the Texas Rice Belt. J. Econ. Entomol. 100: 5460. Reay Jones, F.P.F. 2010. Spatial and temporal patterns of stink bugs (Hemiptera: Pentatomidae) in wheat. Environ. Entomol. 39: 944955. SAS Institute. 2008. PROC users manual, version 9th ed SAS Institute, Cary, NC.

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217 Wolcott, G. N. 1948. The insects of Puerto Rico. J. Agric. Univ. P. R. 32: 417 748. Wunderlin, R. P., and B. F. Hansen. 1996. Atlas of Florida Vascular Plants. Institute for Systematic Botany, University of South Florida, Tampa. Wyckhuys, K. A. G., an d R. J. O'Neil. 2007. Local agroecological knowledge and its relationship to farmers' pest management decision making in rural Honduras. Agric. Hum. Values 24: 307321.

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218 BIOGRAPHICAL SKETCH Gaurav Goyal was originally from Sangrur (Punjab), India. He received his b achelors d egree in agriculture with honors in plant protection from the Department of Entomology, Punjab Agricultural University, Ludhiana, India in 1999 with merit scholarship. He decided to pursue graduate education in entomology due to i nterest in insects. He obtained his m asters d egree in entomology from the same institute in 2005 with A.S. Sohi Memorial scholarship for academic achievements His masters research focused on cultural control of maize stem borer. He worked as a researc h fellow in ICAR project on pesticide residues in the same department for four months In August 2006, he enrolled at the University of Florida to pursue a Doctor of Philosophy degree in Entomology under the supervision of Dr. Gregg Nuessly. His research focused on the biology, morphology and distribution of corn silk flies serious pests of sweet corn He received several research and travel grants from the Department of Entomology and Nematology, The University of Florida, and from several scientific so cieties. He presented many talks and posters at annual and branch meetings of the Florida Entomological Society, Entomological Society of America, Florida State Horticultural Society, Caribbean Food Crops Society, Graduate Student Council Interdisciplinar y Research Conference Forum, and National Science Foundation Research Day He was also a member of the Gamma Sigma Delta honor society of agriculture. He won various scholarships and awards (10) at state, regional and national scientific meetings, as well as in college and at university in Punjab Agricultural University, India and University of Florida. He volunteered at several occasions some of which included serving as representative for the Entomology and Nematology Depar t ment at the University of Florida Graduate Student Council as judge

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219 of science fairs at middle school s, moderator of oral presentations at scientific meetings etc He was awarded best international student award for outstanding academic achievements from college of agricultural and life sciences, University of Florida in 2010.