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Development of an IPM program for the tropical sod webworm Herpetogramma phaeopteralis Guenee

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Title:
Development of an IPM program for the tropical sod webworm Herpetogramma phaeopteralis Guenee
Creator:
Tofangsazi, Nastaran
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
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english
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1 online resource (122 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
ARTHURS,STEVEN P
Committee Co-Chair:
MEAGHER,ROBERT LEO
Committee Members:
CHERRY,RONALD H
TRENHOLM,LAURIE E
Graduation Date:
12/19/2014

Subjects

Subjects / Keywords:
Infestation ( jstor )
Insecticides ( jstor )
Insects ( jstor )
Instars ( jstor )
Larvae ( jstor )
Moths ( jstor )
Pests ( jstor )
Roundworms ( jstor )
Species ( jstor )
Turf grasses ( jstor )
Entomology and Nematology -- Dissertations, Academic -- UF
sod -- tropical
City of Boca Raton ( local )
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
Entomology and Nematology thesis, Ph.D.

Notes

Abstract:
Tropical sod webworm, Herpetogramma phaeopteralis Guenee (Lepidoptera: Crambidae) is one of the most serious pests of warm season grasses in the Gulf Coast of the United States and the Caribbean islands. Despite the apparent economic importance of H. phaeopteralis, little information on biology and integrated pest management (IPM) programs for this species has been reported. In order to develop degree day models, I studied the developmental biology of this species over a range of temperature. The relationship between temperature and developmental rate of H. phaeopteralis were described mathematically. The Briere-1 model provided the best fit with estimated lower, upper, and optimum thresholds for total development of 14.9, 34.3, and 29.4 C, respectively. Current control recommendations for H. phaeopteralis are mainly application of above-ground chemical insecticides against larval stages. I estimated resistance baselines and concentration response of H. phaeopteralis to six insecticide classes. Chlorantraniliprole, the anthranilic diamide labeled for turf usage, was the most toxic compound tested (LC50 values of 4.5 ppm). In field tests, all compounds at label rates were effective (94% mortality of larvae exposed to fresh residues). However, a more rapid decline in activity of clothianidin (a neonicotinoid) and bifenthrin (a pyrethroid) was observed in field tests on St Augustinegrass compared with chlorantraniliprole. Demand for more environmentally friendly landscaping encourages the use of alternatives to chemical insecticides such as biopesticide. Commercially available entomopathogenic nematode products including Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophora, H. megidis and H. indica were tested against three different larval sizes of H. phaeopteralis. All tested entomopathogenic nematode were pathogenic to H. phaeopteralis larvae in the laboratory, but S. carpocapsae caused the highest mortality in the laboratory and greenhouse. Our data suggest that commercial formulations of S. carpocapsae can be a viable non-chemical option for H. phaeopteralis biocontrol. In order to provide a new and valuable monitoring tool to replace the crude monitoring tool currently used for this purposeI investigated pheromone compounds of this moth. In my results I identified several candidate pheromone compounds consisting of C14-acetate and C16- acetates with single or double bonds and an unsaturated hydrocarbon that elicit an electroantennogram (EAG) response in the laboratory. Thus, we hypothesized that these compounds might be sex pheromone of this species. Future studies should focus on confirmation of the structure of all compounds, and synthesis of any compounds that are not commercially available. This dissertation presents new information regarding the biology and biological and chemical management options of H. phaeopteralis. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: ARTHURS,STEVEN P.
Local:
Co-adviser: MEAGHER,ROBERT LEO.
Statement of Responsibility:
by Nastaran Tofangsazi.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Resource Identifier:
974372615 ( OCLC )
Classification:
LD1780 2014 ( lcc )

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DEVELOPMENT OF AN IPM PROGRAM FOR THE TROPICAL SOD WEBWORM H erpetogramma phaeopteralis G uenée By NASTARAN TOFANGSAZI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Nastaran Tofangsazi

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To my parents

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4 ACKNOWLEDGMENTS I would like to express the deepest appreciation to my committee chair and co chair, Professor Steven Arthurs and Professor Ron Cherry , for their attitude and invaluable advice throughout the course of this pro jec t from the initial planning of experiments through t o preparation of manuscripts . Without their guidance and help this dissertation would not have been possible. I would like to thank you for encouraging my research and for allowing me to grow as a research er . I am grateful to my other supervisory committee members Professor Robert Meagher and Professor Laurie Trenholm for their excellent guidance and suggestions on how to improve my work. I would like to acknowledge t he Center for Landscape Conserv ation and Ecology at University of Florida and Mid Florida Research & Education Center (MREC) for providing financial assistance for the duration of my Ph.D. research. I am thankful to The New Zealand Instit ute for Plant and Food Research and Professor Ash raf M. El Sayed and David Maxwell Suckling for allowing me to work in their laborator ies . I am grateful for the technical help of Robert Leckel , James Kerrigan . I would like to deeply appreciate Luis Aristizábal for supporting me as a lab assistant and as a friend. I appreciate the love and support that I received from my family and friends. I would like to thank all of them for making Florida a home away from home. I would like to thank my family for their long distance support. I appreciat e my parents, J amileh Mousavi and Akbar Tofangsazi and my sisters, Yasmin Tofangsazi and Ladan Tofangsazi. Their love provided my inspiration and was my driving force. I owe them everything and wish I could show them how much I love them.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURE S ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Importance of Turfgrasses ................................ ................................ ...................... 14 Insect Pests of Warm Season Turfgr asses ................................ ............................. 15 The Caterpillar Complex In Warm Season Turfgrass ................................ ............. 16 Web Worms ................................ ................................ ................................ ............ 17 Tropical Sod Webworm Taxonomy ................................ ................................ ......... 18 Tropical Sod Webworm Distribution And Life History ................................ ............. 18 Morphological Description ................................ ................................ ....................... 20 Hosts and Damage ................................ ................................ ................................ . 21 Integrated P est Management Of Tropical Sod Webworm ................................ ....... 22 Sampling ................................ ................................ ................................ .......... 22 Economic Threshold ................................ ................................ ......................... 22 Chemical Control ................................ ................................ .............................. 23 Microbial Co ntrol ................................ ................................ .............................. 24 Entomopathogenic Nematodes ................................ ................................ ........ 25 Cultural Control ................................ ................................ ................................ 26 Natural Enemies ................................ ................................ ............................... 27 Objectives ................................ ................................ ................................ ............... 28 Outline of Dissertation ................................ ................................ ............................. 29 2 THERMAL REQUIREMENTS AND DEVELOPMENT OF HERPETOGRAMMA PHAEOPTERALIS (LEPIDOPTERA: CRAMBIDAE: SPILOMELINAE) .................. 42 Background ................................ ................................ ................................ ............. 42 Materials And Methods ................................ ................................ ........................... 44 Insect Rearing ................................ ................................ ................................ .. 44 Diet Suitability ................................ ................................ ................................ ... 44 Temperature Dependent Development ................................ ............................ 45 Developmental Rate And Mathematical Models ................................ ............... 45 Results ................................ ................................ ................................ .................... 47

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6 Diet Suitability ................................ ................................ ................................ ... 47 Temperature Dependent Development ................................ ............................ 48 Developmental Rate and Mathematical Models ................................ ............... 48 Discussion ................................ ................................ ................................ .............. 49 Background ................................ ................................ ................................ ............. 61 Materials and Methods ................................ ................................ ............................ 63 Insects and insecticides ................................ ................................ .................... 63 Laboratory Bioassays ................................ ................................ ....................... 63 Field Studies ................................ ................................ ................................ ..... 65 Results ................................ ................................ ................................ .................... 67 Laboratory Bioassays ................................ ................................ ....................... 67 Residual Efficacy Under Field Conditions ................................ ......................... 67 Discussion ................................ ................................ ................................ .............. 68 3 EFFICACY OF COMMERCIAL FORMULATIONS OF ENTOMOPATHOGENIC NEMATODES AGAINST TROPICAL SOD WEBWORM ................................ ........ 78 Background ................................ ................................ ................................ ............. 78 Materials And Methods ................................ ................................ ........................... 80 Nematodes And Insects ................................ ................................ ................... 80 Laboratory Experiments ................................ ................................ ................... 80 Nemato de Reproduction ................................ ................................ ................... 81 Greenhouse Experiments ................................ ................................ ................. 81 Data Analysis ................................ ................................ ................................ ... 82 Results ................................ ................................ ................................ .................... 83 Laboratory Studies ................................ ................................ ........................... 83 Greenhouse Studies ................................ ................................ ......................... 83 Discussion ................................ ................................ ................................ .............. 84 4 ATTRACTION OF HERPETOGRAMMA PHAEOPTERALIS ADULTS TO FLORAL LURES AND PHERMONE CAND IDATES ................................ ............... 91 Background ................................ ................................ ................................ ............. 91 Materials and Methods ................................ ................................ ............................ 92 Attraction to Floral Lures ................................ ................................ .................. 92 Field Bioassays Using H. licarsisalis Pheromone Blends ................................ . 93 Shipping Protocol ................................ ................................ ............................. 93 Gas Chromatography (GC) ................................ ................................ .............. 94 Electroantennogram (EAG) ................................ ................................ .............. 94 GC E lectroantennographic Detection ................................ ............................... 95 Results ................................ ................................ ................................ .................... 95 Attraction Of Tropical Sod Webworm to Floral Lures ................................ ....... 95 Field Bioassays Using H. licarsisalis Pheromone Blends ................................ . 96 EAG Recording and GC EAD Detection ................................ ........................... 96 Discussion ................................ ................................ ................................ .............. 96 5 SUMMARY AND CONCLUSIONS ................................ ................................ ........ 108

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7 LIST OF REFERENCES ................................ ................................ ............................. 111 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 122

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8 LIST OF TABLES Table page 1 1 Significant turfgrass pests in Florida. ................................ ................................ .. 31 1 2 Current insecticides registered for use against turf caterpillars .......................... 32 2 1 Models evaluated ................................ ................................ ............................... 52 2 2 Duration (days) of larval, prepupal, and pupal development .............................. 53 2 3 Percentage stage survivorship and successful adult emergence ....................... 54 2 4 Developmental time (days) of immature stages of H. phaeopteralis at six ....................... 55 2 5 Larval, prepupal, and pupal survival (%) of H. phaeopteralis . ............................ 56 2 6 Estimates of linear regression parameters, minimum temperature threshold ..... 57 2 7 Estimated coefficients, thermal constants, and goodness of fit for linear ........... 58 3 1 Insecticides tested for control of H. phaeopterali . ................................ ............... 73 3 2 Toxicity of commercial insecticides to medium size ................................ ............ 74 3 3 Mean mortality of H. phaeopteralis exposed to aged insecticide residues ......... 75 3 4 Mean percentage mortality of H. phaeopteralis exposed to aged insecticide ..... 76 4 1 Percentage mortality of H. phaeopteralis ................................ ............................ 87 4 2 Lethal concentrations (LC 50 ) of five entomopat hogenic nematodes ................... 88 4 3 Effect of five species of entomopathogenic nematodes 89 5 1 Adult H. phaeopteralis collected after one week on green delta traps .............. 100 5 2 Adult H. phaeopteralis collected after one week on green deltatraps. .............. 101 5 3 Electroantennogram responses of male H. phaeopteralis moths ..................... 102

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9 LIST OF FIGURES Figure page 1 1 La rvae of tropical sod webworm ................................ ................................ ......... 33 1 2 Bluegrass webworm ................................ ................................ ........................... 34 1 3 Tropical sod webworm egg cluster laid on grass leaf sheath. ............................ 35 1 4 Tropical sod webworm newly emerged larva ................................ ...................... 36 1 5 Tropical sod webworm larval instars, pre pupae and pupa. ............................... 37 1 6 Pupa found in cocoon in St. Augustinegrass thatch. ................................ .......... 38 1 7 Adult tropical sod webworm resting in grass. ................................ ...................... 39 1 8 St. Augustinegrass residential lawn damaged by tropical sod webworm ............ 40 1 9 Window feeding caused by younger larval instars of tropical sod webworm. ..... 41 2 1 Abnormal and normal wing of H. phaeopteralis . ................................ ................. 59 2 2 Temperatur e dependent developmental rates of immature stages .................... 60 3 1 Cumulative percentage mortality, moribund and survivorship ............................ 77 4 1 Greenhouse tests showing mortality of H. phaeopteralis . ................................ ... 90 5 1 Deployment of delta shaped sticky traps baited ................................ ............... 103 5 2 Caged H. phaeopteralis virgin female i n top middle of trap .............................. 104 5 3 Response of H. phaeopteralis one day old male antennae .............................. 105 5 4 Coupled gas chromatogram electroantennograms of H. phaeopteralis male ... 106 5 5 H. phaeopteralis female chordotonal organ. ................................ ..................... 107

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10 LIST OF ABBREVIATIONS AA A cetic acid AI Active ingredient AIC Akaike information criterion ANOVA A nalysis of variance EAG Electroantennogram EFN E xtra floral nectaries EPNs Entomopathogenic nematodes ETa Evapotranspiration FQPA Food Quality Protection Act FRC Field recommended concentration GC EAD Gas Chromatogram Electroantennogram Detector GC MS Gas Chromatogram Mass Spectrometry IJ Infective juveniles IPM Integrated pest manag e ment K Thermal constant LC50 Median lethal concentration 3MeB 3 methyl 1 butanol (isoamyl alcohol) MREC Mid Florida Research and Education Center PAA P henylacetaldehyde PPM Parts per million R2 Coefficient of determination RH Relative humidity RSS residual sum of squares SEM Standard error mean

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11 SSE Sum of the squared error T Temperature T0 Lower thermal threshold TL Upper thermal thresholds Topt Optimum temperature TSW Tropical sod webworm USDA U.S. Department of Agriculture

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF AN IPM PROGRAM FOR THE TROPICAL SOD WEBWORM Herpetogramma phaeopteralis Guenée By Nastaran Tofangsazi December 2014 Chair: Steven P. Arthurs Major: Entomology and Nem atology Tropical sod webworm, Herpetogramma phaeopteralis Guenée (Lepidoptera: Crambidae) is one of the most serious pests of warm season grasses in the Gulf Coast of the United States and the Caribbean islands. Despite the apparent economic importance of H. phaeopteralis , little information on biology and integrated pest management (IPM) programs for this species has been reported. In order to develop degree day models, I studied the developmental biology of this species over a range of temperature. T he relationship between temperature and developmental rate of H. phaeopteralis were described mathematically. The Briere 1 model provided the best fit with estimated lower, upper, and optimum thresholds for total development of 14.9, ectively. Current control recommendations for H. phaeopteralis are mainly application of above ground chemical insecticides against larval stages . I estimated resistance baselines and concentration response of H. phaeopteralis to six insecticide classes. C usage, was the most toxic compound tested (LC 50 values of 4.5 ppm). In field tests, all residues). However , a more rapid decline in activity of clothianidin (a n eonicotinoid ) and

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13 bifenthrin (a pyrethroid) was observed in field tests on St Augustinegrass compared with chlorantraniliprole. Demand for more environmentally friendly landscaping encourages the use o f biopesticides as alternatives to chemical insecticides . C ommercially available entomopathogenic nematode products including Steinernema carpocapsae , S. feltiae , Heterorhabditis bacteriophora , H. megidis and H. indica were tested against three different larval sizes of H. phaeopteralis . All tested entomopathogenic nematode s were pathogenic to H. phaeopteralis larvae in the laboratory, but S. carpocapsae caused the highest mortality in the laboratory and greenhouse . Our data suggest that commercial formulations of S. carpocapsae can be a viable non chemical option for H. phaeopteralis biocontrol. In order to provide a new and available monitoring tool to replace the crude monitoring tool currently used for this purpose , I investigate d pheromone compounds of this moth. In my results I identified several candidate pheromone compounds consisting of C14 acetate and C16 acetates with single or double bonds and an unsaturated hydrocarbon that elicit an electroantennogram (EAG) response in the laboratory. Thus, we hypothesized that these compounds might be sex pheromones of this species . Future studies should focus on c onfirmation of the structure of all compounds, and synthesis of any compounds that are not commercially available . This diss ertation presents new information regarding the biology and biological and chemical management options of H. phaeopteralis .

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14 CHAPTER 1 INTRODUCTION Importance o f Turfgrasses The production and sale of turfgrass is a thriving industry in the United States . T urfgrass is the largest irrigated crop in the United States and covers three times the land area of any other crop (Haydu et al. , 2006). In the United States , turfgrass covers an estimate d 40,475,861 acres in residential yards , athletic fields, golf courses, parks, commercial and institutional parks (Haydu et al. , 2008). Turfgrass has three important roles in human life: namely utility, beautification and recreation (Trenholm et al. , 2000). Utility function in cludes reducing soil erosion, glare, noise, air pollution and enhancing ground water recharge (Haydu et al. , 2006, 2008). Also, healthy lawns provide a pleasant environment for residents of suburban and recreational opportunities in urban landscapes for many centuries (Beard 1 973). In addition, recreational use of turfgrass is extensive throughout the world. Common sports activities played on turf include football, soccer and golf (Haydu et al. , 2006). Ornamental and beautification of urban areas has a positive effect on mental health and can increase property values (Behe et al. , 2005). Economics associated with turf industry is estimated to be $75 billion per year in U.S. (Haydu et al . , 200 8 ). The golf sector is the largest component of the turfgrass industry, accounting for a 44% share. The nearly 16,000 golf courses generated $33.2 billion in (gross) output impacts, contributed $20.6 B in value added or net income, and generated 483,649 jobs A summary of this chapter has been accepted in the Journal of Integrated Pest Management. Nastaran Tofangsazi, Ron. H. Cherry, Robert L. Meagher, and Steven P. Arthurs. Tropical Sod Webworm (Lepidoptera: Crambidae): a Pest of Warm Season Turfgrasses.

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15 nationwide. The lawn care goods retailing sector produced $9.1 Bn in output impacts, contributed $5.8 Bn in value added, and sustained 114,294 jobs. The lawncare services sector generated nearly $19.8 Bn in output impacts, $13.3 Bn in value added, and 295,841 jobs. Golf courses had $23.3 Bn in output impacts, $14.5 Bn in value added, and 361,690 jobs in U . S. (Haydu et al ., 2008). In Florida, turfgrass is grown in home lawns, parks, sports fields, cemeteries, rights of way and golf courses. To supply this need, turfgrass is grown on commercial sod farms and is marketed through many small a nd large specialist distributers and home and garden retail outlets throughout the state . Florida's sod industry comprised approximately 125 producers, producing an estimated 37 , 635 hectares and harvesting nearly 25 , 495 hectares in 2002 (Haydu et al. , 2008). St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze is adapted to both tropical and subtropical regions. It is native to coastal regions of the Mediterranean and the Gulf of Mexico ( Trenholm and Unruh 2004 ) . St. Augustinegrass can establish quickly in many soil types and various regions in Florida and other southern states in the continental United State ( Trenholm and Unruh 2004 ) . It is the most common turfgrass planted in home lawns in Florida (Trenholm et al. , 2000). Insect Pests o f Warm Season Turfgrasses Warm season grasses cultivated for use in the southern U.S. are subjected to various pests. Any organism causing damage to the functional or aesthetic value of turf can be considered as a pest. Significant arthropod pests include severa l species of caterpillars, mole crickets, chinch bugs, spittlebugs, scale insects, aphids, mites and the larval stage of several beetles (Table 1 1). Simple techniques to monitor for many of these pests are described by Hellman (1995). Imported fire ants, Solenopsis spp., can

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16 be a nuisance, although they can be beneficial by preying on herbivorous insects near the colony (Potter 1988). Most insect pests can be recognized by the type of damage they cause. Caterpillars feed on the leaf blade and stems, while mole crickets and more damaging larval stages of the beetles largely occur underground where they attack roots. The true bugs and mites suck plant sap, or individual cells, causing distortion, discoloration and die back. Cumulatively, pests listed in Table 1 1 have economic impact through direct damage and control costs (Potter 1998). The Caterpillar Complex I n Warm Season Turfgrass In the southern U.S., the fall armyworm, Spodoptera frugiperda ( J. E. Smith ) , striped grass , loopers Mocis spp., the fiery skipper, Hylephila phyleus ( Drury ) , and the tropical sod webworm , Herpetogramma phaeopteralis ( Guenée ) are the most common caterpillar species causing significant economic impact through direct damage and control costs (Potter 1998). Larva e of these caterpillars are equipped with sharp mandibles and damage lawns by chewing turfgrass leaf blades. Early damage is hard to notice but later damage creates a ragged appearance as larvae grow. These species are most damaging in newly established tu rf (Potter 1998). Tropical sod webworm larvae can be distinguished from other warm season turf caterpillars by their size and markings . Tropical sod webworm has four brown spots on each body segment, whereas mature fall armyworm larvae (1.5 inches, 38 mm) have an inverted light colored 'Y' on the head ( Brandenburg and Freeman 2012 ) . Mature striped grass loopers larvae (1.4 inches, 35 mm) have several narrow stripes on the head and backs ( Brandenburg and Freeman 2012 ) . Mature fiery skipper larvae (1 inch, 25 is constricted behind the black head ( Potter 1998 ) (Figure 1 1).

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17 Web Worms Various species of native sod feeding webworms in the family Crambidae (crambid snout nosed moths) occur across North America. Common species which seem to prefer cool season turfgrass include the bluegrass webworm, Parapediasia teterrellus (Zincken), distributed over the eastern United States from Massachusetts and Connecticut west into Colorado and through mid Texas and eastward . It is most abundan t in the limestone districts of Kentucky and Tennessee where Kentucky bluegrass Poa pratensis is dominant. The western lawn moth, Tehama bonifatella (Hults) is found west of the Rocky Mountains and along the Pacific Coast, western Canada into Alaska. Strip ed sod webworm or changeable grass veneer, Fissicrambus mutabilis (Clemens) are found from New York to Florida, west to Illinois to Texas and north to Ontario. The larger sod webworm, Pediasia trisecta (Walker) ranges from southern Canada south into North Carolina and west into Tennessee, Texas, New Mexico, Colorado, and up to Washington State. The tropical sod webworm Herpetogramma phaeopteralis ( Guenée ) is more common in warm season turfgrass and it is present from South Carolina to Florida, west to Texas , south through Central America and Caribbean (current distributions based on the Global Biodiversity Information Facility database http://gbif.org, 2014 04 29; North American Moth Photographers Group http://mothphotographersgroup.msstate.edu/MainMenu.shtml ; Brandenburg and Freeman 2012; Heppner 2003). As an easy diagnostic characteristic, H . phaeopteralis hold its wings out flat when at rest, whereas all other North American webworms partially fold their wings around their body (Figure 1 2 ). Currently little published information exists for many of these webworm species, although they comprise important pests of turf.

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18 Tropical Sod Webworm Taxonomy Nine species of Herpetogramma occur in North America. These small brownish moths are frequently collected at light traps ; h owever their variable shading led to many species being independently described multiple times. Tropical sod webworm was formerly classified in the family Pyralid ae. According to a recent systematic revision this species was moved to subfamily of Spilomelinae and family Crambidae (Solis 2010). Synonymies associated with H. phaeopteralis in the literature includes Botys phaeopteralis (Guenée), B. vecordalis (Guenée) , B. vestalis (Walker), B. additalis (Walker), B. plebejalis (Lederer), B. cellatalis (Walker), B. communalis (Snellen), B. intricatalis (Möschler); Acharana descripta (Warren) is designated as a new synonym of H. phaeopteralis (Solis 2010). Tropical So d Webworm Distribution a nd Life H istory The tropical sod webworm H. phaeopteralis occurs exclusively in warm season turfgrass. This species occurs from South Carolina to Florida and the Caribbean, and west to Texas, and south through Central America (Heppner 2003, Kerr 1955). However, a comprehensive survey for this pest has not been conducted in recent years. Businesses primarily impacted by this pest in the southeastern United States include sod production, lawn care services and golf courses and at hletic fields. These industries are well represented in the southeast, with economic impacts of over $3 billion in Florida alone (Haydu et al. , 2006) although the economic impact of tropical sod webworm has not been documented .

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19 Adult TSW communicate via s ex pheromones with virgin females lacking obvious calling posture and mating occurring 3 4 hours before sunrise (Meagher et al. , 2007). T ympanic and associated chordotonal organs have observed in moths which suggest ultrasonic courtship behavior. T he relea se of acoustic signals from males of species ha s not confirmed yet , as has been noted among other Crambidae (Nakano et al. , 2008; Takanashi et al. , 2010). Adults live for 10 14 days when provided 5% v/v honey water in greenhouse cages and likely nectar fee d on flowering plants. Sourakov (2008) noted this species feeding on extra floral nectaries (EFN) of passion vines, Passiflora incarnate (L.). The presence of both flowers and EFN on plants will support the flight ability, longevity and fecundity of the ad ult moth as well as some of its natural enemies (Rudgers 2004; Wäckers et al. , 2007). Adults have a high preference to reside in tall grass and shrubs during the day and are most active at dusk (Cherry and Wilson 2005) . Multiple generations may occur during a year (e.g. three or four generations in southern Florida) (Kerr 1955). In southern Florida, tropical sod webworm adults are present year round, with significantly higher numbers in the fall (September through November) (Cherry and Wilson 2005). P opulations decline over the winter and increase slightly beginning in the spring (March through May). In Central Florida , adults ha ve been observed from May through November. In north central Florida (Gainesville), the peak of flight activity was reported in October and November (Kerr 1955) and August October (John Capinera, personal communication ). Indications are that this species does not survive the winter in the northern part of the state (Kerr 1955). Based on the observed patterns of abundance, I

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20 hy pothesize that some seasonal migration of this species may occur from south into central and central into north Florida during the late spring and early summer. Despite the apparent economic importance of H. phaeopteralis , little information on biology and phenology of this species ha s been reported. Understanding the factors governing H. phaeopteralis development and implementing this knowledge into forecast models may facilitate pest management decisions. Morphological Description Eggs Adult females dep osit clusters of 10 to 35 creamy white eggs on the upper surface of grass blades. Eggs are flattened, overlapping and slightly oval in shape; 0.7 mm (length), 0.5 mm (width) and 0.1 mm (height). Eggs are pale white when laid and become brownish red as they mature (Figure 1 3). The duration of egg stage is variable , 2012). Larvae Young larvae are cream colored with two pairs of brown spots on each segment and a dark, yellowish brown head and when disturbed, may adopt a defensive C shape pose. The larvae become greener in late instars (Figure 1 4). Larval head capsules at maximum width measure 0.225, 0.344, 0.489, 0.676, 0.944 and 1.267 mm, respectively and mature larvae range from 0.7 1 inch (18 25 mm) (Figure 1 5). Pupae The reddish brown pupae are about 8.5 9.5 mm long and 2.1 2.9 mm wide. The pupae may be exposed or buried in thatch (Figure 1 6).

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21 Adults Moths of H. phaeopte ralis are dingy brown and their wing spread is about 0.8 inch (20 mm) (Figure 1 7 ). Adult males have six abdominal segments, whereas females have five (Kerr 1955). Hosts a nd Damage Tropical sod webworms prefer St. Augustinegrass, Stenotaphrum secundatum (Walter) Kuntze and bermudagrass Cynodon spp ( L. ) . Other major warm season turfgrass subject to annual infestation by H. phaeopteralis are centipedegrass Eremochloa ophiur oides ( Munro. Hack.), seashore paspalum Paspalum vaginitium ( Swartz), carpetgrass Axonopus spp., zoysiagrass Zoysia japonica ( Steudel) and bahiagrass Paspalum notatum ( Flugge) (Kerr 1955). Symptoms are dependent on the stage of infestation (Figure 1 8 ). T he first four grass blades, and so the injury they cause is often overlooked (Figure 1 9 ). Fifth and sixth instars can severely damage grass by chewing entire sectio ns off the leaf blade night and larvae hide in the thatch during the day. Caterpillars prefer dry and hot grass areas. Grass may recover if infestations are not too sever e, but extensive feeding damage causes yellowish and brown patches and often leads to the ingress of weeds ( Brandenburg and Freeman 2012 ). Most damage occurs during late fall when tropical sod webworm are most active and the rate of grass growth has declin ed so that damage is not repaired as readily.

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22 I ntegrated P est M anagement O f Tropical S od W ebworm Sampling early infestations ( Brandenburg and Freeman 2012 ) . Larvae can be found in infested sod by parting grass in the periphery of poorly growing turf areas and looking for chewing damage and greenish larval frass in the thatch layer. Larvae can be found curled i n a C shape near the soil surface. Larger larvae feed at night and leave silk trails as they move from one grass blade to another. These webs are easily seen in the morning if dew is present. Larvae can be agitated during the day using soap flushes (1 tabl espoon of dishwashing soap in a gallon of water applied in a 1 m 2 area of turf). Detection is enhanced by movement of larvae climbing up grass blades within 5 minutes ( Anonymous 2008 ) . Moths hiding in shrubs and bushes fly low to the ground when disturbed. Once in place, tapping the vegetation encourages moths to fly upwards into the nets. This initial presence of adults indicates that oviposition and later feeding damage may be expected. Adults are attracted to porch lights (personal observations) and can be captured using black light traps (Cherry and Wilson 2005). Light traps are useful for sampling and/or collecting adults. Disadvantages are cost and the presence of many other insects attracted to the light (Muirhead Thompson 1991). Sex pheromones that a llow specific capture of turf pests ( such as fall armyworm ) , are not currently available for sod webworms . Economic T hreshold The number of larvae that can cause economic damage depends on turf vigor, maintenance practice, other biotic and abiotic factors and the tolerance of turf damage

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23 of homeowners or turfgrass managers. On golf courses, damage in roughs is less noticeable tha n damage in fairways, and damage on tees and greens may not be tolerated at all. Aesthetic thresholds are thus variable, although a threshold of 5 10 webworm per square meter may warrant a control treatment in high quality turf in dry and sunny areas (Anon ymous 2008). Additional damage to turf may occur through mammals and birds digging holes in search of larvae. Chemical Control Insecticides are frequently used against damaging populations of tropical sod webworms. Insecticide application 10 to 12 days after observing flying moths may control smaller larvae that are generally easier to control than larger larvae (Watschke et al ., 1995). When the damaged area is small and early infestations are detected spot treatments may be applied. Monitoring is recomm ended to confirm the activity of insecticide treatments. Several products are registered for sod webworms for home owner and professional use (Table 1 2 ). With contact materials, irrigation should not be applied within 24 hours to allow the insects to cons ume the foliage and make contact with treated surfaces. Granular formulations may be less effective against sod webworms since insecticidal activity on the leaf blades may be insufficient. With systemic materials, post treatment irrigation should be applie d if rainfall is not expected with 24 hr to move the material to the root zone (Held and Potter 2012 ) . Other lawn chemicals may impact tropical sod webworm populations. T he D, 0.3% mecoprop, 0.090% dicamba, plus emulsifiers) have been reduced survival of small and medium sized

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24 tropical sod webworm larvae ( Cherry et al. , 2010 ) . Mortality was caused by a feeding response (starvation or toxicity via ingestion) rather than direct contact or volatilization. Research has shown that silicon can enhance plant resistance to some turf pests (Saigusa et al. , 1999). However, Korndorfer et al. , (2004) tested applications of calcium silicate to turfgrass, but did not observe any differences in subsequen t feeding and development of H. phaeopteralis compared with control plants. Reinert in 1973 and 1983 evaluated carbaryl, chlorpyrifos, bendiocarb and ethoprop against H. phaeopteralis larvae. However , to date these insecticides have been canceled or res tricted following the Food Quality Protection Act (FQPA) of 1996. Information is not available on toxicity of newer insecticides and formulations to control H. phaeopteralis larvae. Understanding residual properties of current insecticides under field cond itions might prevent unnecessary insecticide reapplication and associated costs. Microbial Control Hazards associated with the over use of insecticides in turf include insecticide resistance, negative impacts on beneficial and other nontarget species, and contamination of groundwater (Racke 2000; Potter 2005). These issues along with public concern have stimulated interest in application of microbial insecticides as part of integrated management of turfgrass insect pests (Grewal 1999; Racke 2000). Microbia l insecticides are generally specific to their target pest(s), considered nontoxic to humans and can be applied with standard pesticide equipment such as pressurized sprayers, mist blowers, and electrostatic sprayers. However, biopesticides have had limite d market success and sales of microbial insecticides constituted < 0.1% of the estimated $500 million U.S. turfgrass insecticide market in 1998 (Koppenhöfer 2007).

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25 Entomopathogenic Nematodes Entomopathogenic nematodes are soft bodied, non segmented roundwo rms that are obligate or sometimes facultative parasites of insects ( Kaya and Gaugler 1993 ) . Entomopathogenic nematodes occur naturally in soil environments and locate their host in response to carbon dioxide, vibration, and other chemical cues (Grewal et al. , 2005). Species in two families (Heterorhabditidae and Steinernematidae) have been effectively used as biological insecticides in pest management programs (Kaya and Gaugler 1993). The infective juvenile stage (IJ) is the only free living stage of entom opathogenic nematodes. The juvenile stage penetrates the host insect via the spiracles, mouth, anus, or in some species through intersegmental membranes of the cuticle, and then enters into the hemocoel (Bedding and Molyneux 1982). Both Heterorhabditis and Steinernema are mutualistically associated with bacteria of the genera Photorhabdus and Xenorhabdus , respectively (Kaya and Gaugler 1993). The juvenile stage release cells of their symbiotic bacteria from their intestines into the hemocoel. The bacteria m ultiply in the insect hemolymph, and the infected host usually dies within 24 to 48 hours. After the death of the host, nematodes continue to feed on the host tissue. The nematodes develop through four juvenile stages to the adult, and then reproduce. Depe nding on the available resources, one or more generations may occur within the host cadaver, and a large number of juveniles are eventually released into the environment to infect other hosts and continue their life cycle (Kaya and Gaugler 1993). Entomopat hogenic nematodes use two search strategies: ambushers or cruisers (Grewal et al. , 1994). Ambushers such as S. carpocapsae have an energy conserving approach and lie in wait to attack mobile insects (nictitating) in the upper soil. Cruisers like S. glaseri and H. bacteriophora are highly active and generally subterranean,

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26 moving significant distances using volatile cues and other methods to find their host underground. Therefore, they are effective against less mobile pests such as white grubs (Scarab beetl es). Some nematode species, such as S. feltiae and S. riobrave , use an intermediate foraging strategy (combination of ambush and cruiser type) to find their host. Entomopathogenic nematodes have been effective in pest management programs for other lepidop teran species that attack various grasses ( Gonz á lez Ram írez et al. , 2000; Medeiros et al. , 2000; Negrisoli et al. , 2010). I therefore hypothesized that EPNs might offer an alternative tool to manage H. phaeopteralis in turf. Cultural C ontrol Maintenance practices can influence turfgrass susceptibility to insect pests including tropical sod webworm. Excessive fertilization, improper watering and mowing may result in a layer of thatch, i.e. accumulated dead and living stems located above the soi l surface (Trenholm and Unruh 2004). It is therefore important to avoid conditions that favor thatch build up and to dethatch to reduce insect habitat and minimize pesticide applications (Potter 1998). Close mowing stresses grass and encourages webworm dam age (Trenholm and Unruh 2004) . In Florida, recommendations are that g rass should be mowed to a recommended height of 3.5 4 inches (9 10 cm) for St. Augustinegrass, 2.5 3 inches (6 8 cm) for dwarf St. Augustinegrass, 3 4 inches (8 10 cm) for bahiagrass, 1 3 inches (3 8 cm) for zoysiagrass, 0.75 1.5 inches (2 4 cm) for bermudagrass and 1.5 2 inches (4 5 cm) for centipedegrass (Trenholm and Unruh 2004). It might be helpful to remove grass clipping while adults are present since this would remove webworm eggs which can be determined by observing blue gray color of grass blades and visible

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27 footprints on the grass. Irrigation systems should be calibrated to deliver 0.75 inch (2 cm) of water with frequen cy timed for local water restrictions. Using pest resistant turfgrass varieties is one potential tool for managing turf pests. T urfgrass breeding programs usually place emphasis on aesthetic traits and abiotic factors, such as drought, heat, cold and othe r physiological stresses, rather than developing cultivars with pest resistance (Reinert et al. , 2009). Nevertheless, Reinert and Busey (1983) reported that fewer larvae completed development and less damage 289922 C. dactylon ). T he reported as less preferred for adult oviposition and larval feeding damage compared with other cultivars (Reinert et al., 2004) . TSW populations on an individual site, such practices will not reduce TSW populati ons on regional scale unless these cultivars become widely marketed and used by the sod industry. Natural Enemies Beneficial arthropods observed attacking tropical sod webworm include several generalist predators, i.e. spiders, ants , i.e. Lasius neoniger ( Emery ) and Solenopsis molesta ( Say), lady beetles, big eyed bugs ( Geocoris spp.), syrphid flies, ground and tiger beetles, rove beetles and a variety of parasitoids, mostly Trichogramma , braconid, encyrtid and scelionid wasps and an ichneumonid wasp ( Horogenes sp.) (Kerr 1955). I observed the egg parasitoid Trichogramma fuentesi (Torre) , parasitizing > 80% of tropical sod webworm eggs in my colony. Preserving natural enemies by using low -

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28 toxicity insecticides may help limit outbreaks of this pest . However, the impact of natural enemies on tropical sod webworm has not been documented. Insecticide applications against insect pest populations in yards and landscapes can harm beneficial insects and insectivorous birds and ultimately cause more severe insect pest outbreaks in their subsequent generations (Hostetler 2002). Preserving natural enemies by using low toxicity insecticides may help limit outbreaks of TSW. Beneficial insect populations can theoretically be increased by providing flowering plan ts throughout the season as pollen and nectar sources and by manipulating natural enemy activity through landscape design (Braman et al. , 2002). Increasing vertical layering in the yards and landscapes by planting a variety of plants with different sizes a nd heights will provide more cover and feeding opportunities for beneficial insects. However, the use of such strategies would first require knowledge of specific natural enemies that are important locally, as well as the selection specific plants that the y are adapted to. Objectives 1. To determine artificial diets and thermal requirements for development of H. phaeopteralis . 2. To determine d ose response relationship, toxicity and residual activity of insecticides to control Herpetogramma phaeopteralis (Lepidoptera: Crambidae) in St. Augustinegrass . 3. To determine efficacy of commercial formulations of entomopathogenic nematodes against tropical sod webworm, H. phaeopteralis . 4. To identify the sex pheromone of H. phaeopteralis to use as a monitoring tool in an IPM program.

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29 Outline o f Dissertation Herpetogramma phaeopteralis is an important turfgrass pest in Florida and there is a need for pest management program for this species . This dissertation presents original research on important aspects of biology of H. phaeopteralis along with different management tactics to control this pest. Currently, some basic information regarding the biology of this pest has not been well documented. Temperature is a critical abiotic factor influ encing the biology of all pest species (Huffaker et al. , 1999). Thus, in Chapter 2, I studied the development of H. phaeopteralis in the laboratory to develop a degree day model to describe its temperature dependent development and identify its upper and l ower thermal limits. Insecticides provide an important management option for controlling a wide range of turfpests, including H. phaeopteralis . In C hapter 3 , the resistance baselines and lethal activity of six different insecticides representing six major classes of insecticides were estimated. I also compared r esidual efficacy of several registered insecticides to control H. phaeopteralis under field conditions . In C hapter 4 , I investigated the use of an alternative to current insecticides, i.e. entomopathogenic nematodes (EPN) , as potential tools to manage the damaging larval stages of H. phaeopteralis . In this C hapter, efficacy of commercially available EPN products in the United States against different larval sizes of H. phaeopteralis was evaluated under laboratory and greenhouse conditions. Understanding the seasonal biology of this species is critical for timing pesticide or bio insecticide applications and for developing a successful monitoring program. Hence , in C hapter 5 I attempt to i dentify the sex pheromone of H. phaeopteralis .

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30 Synthetic lures composed of the pheromone components can be used to monitor populations and determine seasonal activity of insect pest s .

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31 Table 1 1. Significant turfgrass pests in Florida. Type Common and scientific name Order and family Caterpillars Tropical sod webworm, Herpetogramma phaeopteralis Lepidoptera: Crambidae Fall armyworm, Spodoptera frugiperda Lepidoptera: Noctuidae Cutworms Lepidoptera: Noctuidae Mole crickets Tawny, southern, and short winged; Scapteriscus spp. Orthoptera: Gryllotalpidae Beetles Small weevil called billbugs, e.g. Sphenophorus venatus Coleoptera: Curculionidae Masked chafer, Cyclocephala borealis Arrow, May/June beetles, Phyllophaga spp., Green June beetle, Cotinus nitida Coleoptera: Scarabaeidae Southern chinch bug, Blissus insularis Hemiptera: Blissidae Two lined spittlebug, Prosapia bicincta Hemiptera: Cercopidae Soft and armored scale insects Hemiptera: Coccoidea Aphids Schizaphis graminum (Rondani) Hemiptera: Aphididae Mites Bermudagrass mite, Eriophyes cynodoniensis Acarina: Eriophyidae Ants Imported fire ants, Solenopsis spp. Hymenoptera: Formicidae

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32 Table 1 2. Current insecticides registered for use against turf caterpillars in North America. Active ingredient Chemical subgroup IRAC class Trade name(s) Home owner products Carbaryl Carbamate 1A Sevin Bifenthrin Pyrethroid 3A Talstar, Onyx Ortho Bug B Gon Max Insect Killer for Lawns Cyfluthrin Pyrethroid 3A Tempo Bayer Advanced Complete Insect Killer Deltamethrin Pyrethroid 3A DeltaGard Hi Yield Turf Ranger Insect Control Granules Lambda/gamma cyhalothrin Pyrethroid 3A Scimitar, Demand Triazicide Insect Killer for Lawns Clothianidin Neonicotinoid 4A Arena, Aloft Imidacloprid Neonicotinoid 4A Merit Hi Yield Grub Free Zone III, Bayer Advanced Complete Insect Killer Spinosad Spinosyns 5 Conserve Natural Guard Spinosad Landscape and Garden Insecticide Bacillus thuringiensis subsp. aizawai and kurstaki Bacterial derived 11B Dipel Safer® Brand Caterpillar Killer II with B.t. Halofenozide Diacylhydrazine 18 Mach 2 Hi Yield Kill A Grub Indoxacarb Indoxacarb 22A Provaunt Chlorantraniliprole Anthranilic Diamide 28 Acelepryn, GrubEx Azadirachtin Azadirachtin UN Azatrol, Neemix Steinernema carpocapsae Microbial Millenium S . feltiae Microbial NemAttack Heterorhabditis bacteriophora Microbial B Green H . indica Microbial Beauveria bassiana Microbial Botanigard 1 Insecticide Resistance Action Committee.

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33 Figure 1 1. A) Larvae of tropical sod webworm . B) fall armyworm . C) striped grass and D) loopers fiery skipper. Credit: A) James Kerrigan, University of Florida. Credit: Anonymous, B) Nastaran Tofangsazi, C) Bernardo Navarrete, D) Amy Goodman. A B C D

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34 Figure 1 2 . A) Bluegrass webworm . B) larger sod webworm . C) striped sod webworm . D) western lawn moth and E) sod webworm wings horizontally at rest . Credit: A) Tom Murray . B) Tom Murray . C) Anita Gould . D) Jim Moore . D) and E) James Kerrigan . D E

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35 Figure 1 3 . Tropical sod webworm egg cluster laid on grass leaf sheath. Credit: Nastaran Tofangsazi.

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36 Figure 1 4 . A) Tropical sod webworm newly emerged larva, lighter in color . B) mature greener larva resting, C shape , in grass blades. Credit: A) Luis F. Aristizábal , B) Nastaran Tofangsazi . A B

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37 Figure 1 5 . Tropical sod webworm larval instars, pre pupae and pupa. Credit: James Kerrigan.

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38 Figure 1 6 . Pupa found in cocoon in St. Augustinegrass thatch. Credit: Steven Arthurs.

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39 Figure 1 7 . Adult tropical sod webworm resting in grass. Credit: James Kerrigan.

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40 Figure 1 8 . St. Augustinegrass residential lawn damaged by tropical sod webworm. Credit: Steven Arthurs.

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41 Figure 1 9 . Window feeding caused by younger larval instars of tropical sod webworm. Credit: Steven Arthurs.

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42 CHAPTER 2 THERMAL REQUIREMENTS AND DEVELOPMENT OF HERPETOGRAMMA PHAEOPTERALIS (LEPIDOPTERA: CRAMBIDAE : SPILOMELINAE) Background Turfgrass (sod) production is an important industry in the United States, and Florida is one of the main sod producing states cultivating an estimated 37,635 ha and harvesting over 25,495 ha annually (Trenholm and Unruh 2004, Hay du et al. , 2006). The tropical sod webworm, Herpetogramma phaeopteralis Guenée (Lepidoptera:Crambidae: Spilomelinae) is a destructive pest of warm season turfgrasses in Florida, infesting centipedegrass Eremochloa ophiuroides ( Munro Hackel), bermuda grass C ynodon spp. (L) , carpetgrass ( Axonopus spp.), bahiagrass Paspalum notatum ( Flugge), and St. Augustinegrass Stenotaphrum secundatum (Walter Kuntze) (Reinert et al. , 2009). This pest is especially destructive on newly established sod, lawns, athletic fi elds, and golf courses (Kerr 1955, Buss and Meagher 2006). Herpetogramma phaeopteralis has been previously recorded from Florida, Georgia, Louisiana, Texas, Hawaii, Mississippi, Alabama, and the Caribbean islands (Kerr 1955); however, a comprehensive survey for this pest has not been conducted in recent years. St. Augustinegrass, the most common turfgrass planted in home lawns in Florida (Trenholm and Unruh 2004) can be severely defoliated by H. phaeopteralis . The side of the grass blade, and their injury is often overlooked (Buss and Meagher 2006). The fifth and sixth instars are defoliators, and produce silk and frass (Kerr 1955). Larval feeding damage reduces turfgrass This chapter has been published as Tofangsazi N., Buss E.A., Meagher R.L., Mascarin G.M. and S.P. Arthurs. 2012. Thermal requirements and development of Herpetogramma phaeopteralis (Lepidoptera 1580.

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43 aesthetics, vigor, photosynthesis, and densi ty. Peak adult emergence is from October to November in southern Florida (Cherry and Wilson 2005). Adult H. phaeopteralis may benefit from the presence of nectar sources (Sourakov 2008). Despite the apparent economic importance of H. phaeopteralis , little information on integrated pest management (IPM) programs has been reported. Several insecticides may be used to control this pest, but appropriate timing, risks of resistance and nontarget impacts need to be considered (Reinert 1983). Varying leve ls of resistance to H. phaeopteralis have been identified among certain cultivars or hybrids of bermuda grass (Reinert and Busey 1983), zoysiagrass, and St. Augustinegrass (Reinert and Engelke, 2001, Reinert et al. , 2009). Korndorfer et al. , (2004) tested applications of calcium silicate to turfgrass for antibiosis, but did not observe any differences in subsequent feeding and development of H. phaeopteralis compared with control plants. It is essential to create laboratory colonies to study the behavior, l ife history, and feeding habits for this pest. No arti ficial diets have been published for H. phaeopteralis , so our first objective was to evaluate the suitability of various commercial diets. In addition, because insect growth and development rates are cl osely tied to environmental temperatures (Huffaker et al. , 1999), understanding the factors governing H. phaeopteralis development and implementing this knowledge into forecast models may facilitate pest management decisions. Therefore, our second objectiv e was to study thermal requirements of H. phaeopteralis using linear and nonlinear models.

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44 Materials A nd Methods Insect Rearing A colony of H. phaeopteralis was established on St. Augustinegrass Palmetto from adults collected from north, central, and south Florida. Larvae were reared in a greenhouse on 15 cm pots of St. Augustinegrass inside rearing cages (60 × 60× 60 cm) covered with nylon mesh fi tted with sleeves for access. Additional pots of grass were provided as needed. To obtain cohorts, newly emerged adults were collected and released into an oviposition cage (26 × 26 × 26 cm) with St. Augustinegrass blades as oviposition sites. Growth chamber conditions were at 25 C, 70% relative humidity (RH), and a photoperiod of 14:10 (L:D) h. Adults were pr ovided with a 10% honey solution in a snap cap vial with extruding dental wicks. Diet Suitability Five artificial diets were evaluated:1) a corn based diet, S.W. Corn Borer F0635 (dry mix) and F0717 (vitamin mix); 2) a soy wheat germ diet, general purpose diet for Lepidoptera (F9772); 3) a corn cob wheat germ base diet, European corn borer ( Ostrinia nubilalis Hübner ) diet (F9478B); 4) a corn cob soy flour base diet, for the sugarcane borer, Diatraea saccharalis (F.), U.S. Department of Agriculture (USDA) f ormula (F9775B), (diets #1 4 were obtained through Bio Serve, Frenchtown, NJ); and 5) pinto bean diet (Guy et al. 1985; diet ingredients purchased at several locations and prepared at USDA ARS CMAVE, Gainesville, FL). Diets were prepared according to their directions and dispensed immediately. Agar was added to autoclaved water and boiled for 1 min. Dry mixes and vitamins were added to the agar solution and blended for 2 min. The host plant St. Augustinegrass Palmetto was used as a control.

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45 To study instar specific survivorship and development, newly emerged larvae were placed individually in 30 ml plastic cups prefilled with 5 ml of diet. Equivalent cups prefilled with 5 ml of water agar containing leaves of St. Augustinegrass blades served as controls. Th ere were 50 replicate larvae per diet. Artificial diets were replaced every 2 wk, whereas St. Augustinegrass was replaced daily. Survival and larval development were monitored every 24 h. Exuviae indicated that the larvae molted and nonfeeding shortened la rvae indicated the prepupal stage. This study was conducted in a growth chamber at 25 C, 70% RH, and a photoperiod of 14:10 (L:D) h. Temperature Dependent Development Day old neonates were transferred singly to 30 ml plastic cups containing water agar and St. Augustinegrass and were reared at six constant temperatures (15, 20, 25, Larval, prepupal, and pupal survivorship and development were determined as previously describ ed. In addition, 100 eggs (within 24 h of oviposition) were also incubated at each assigned temperature. Eggs were checked daily for eclosion. Developmental Rate A nd Mathematical Models The developmental rate (reciprocal of developmental time in days, 1/d) was measured for each immature stage exposed to different temperatures. Two linear (common and second order polynomial) and three nonlinear (Briere 1, Briere 2, and Lactin 2) models were used to explain the relationship between temperature and H. phaeopte ralis development (Table 2 1). Thermal requirements were determined for all immature stages (eggs, first to sixth instar larvae, prepupae, and pupae) and overall immature stages by regressing developmental rates against temperature. The parameters of inter est were lower (T 0 ),

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46 and upper thresholds (T L ), optimum temperature (T opt ), and thermal constant (K). These models were selected based on previous fi ndings (Briere and Pracros 1998, Briere et al. , 1999, Aghdam et al. , 2009, Sandhu et al. , 2010). The Briere 1, Briere 2 and Lactin 2 models provide estimates of all thermal parameters, except for thermal constant (K), whereas the linear model estimates only T 0 and K. T 0 estimate developmental thresholds and other empirical constants, we used iterative analysis at six temperatures (15 35 C) for eggs and five temperatures (15 32.5 C) for the remaining stages, because H. phaeopteralis failed to complete development at the highest temperature. The optimum temperature for the Briere 1 and Briere 2 models was calculat ed using the expression: Where the constant m equals two (Briere 1) or is estimated by the iterative analysis (Briere 2) (Briere et al. , 1999). For the second order polynomial model, T 0 a nd T L were obtained by solving the roots of the equation, while the T opt came from the first derivative of dR(T)/dT = 0. For nonlinear models, developmental rate curves were fitted based on the Levenberg Marquardt algorithm using the PROC NLIN command (Stati stical Analysis System [SAS] Institute 2008). For the prepupal stage in Briere 2, we used the Newton Raphson algorithm because the parameters were not highly correlated (>0.8 or< 0.8). For Lactin 2, parameter estimates were derived by simulation method where developmental rate (1/d) was 0 for T 0 and T L (identifying upper and lower thresholds intersecting the x axis), whereas T opt was obtained when developmental rate was maximized (Jandricic et al. , 2010). Linear models were fitted using PROC REG ( SAS Institute 2008).

PAGE 47

47 Models were validated through R 2 (coefficient of determination), RSS (residual sum of squares) and AIC (Akaike information criterion) (Aghdam et al. , 2009, Sandhu et al. , 2010). The RSS was derived from analysis of variance (ANOVA), wh ere lower values indicate a better fit. Better models have lower AIC values, which was computed as AIC = nln(SSE/n) + 2p, where n is the number of observations, p is the number of parameters in the model, and SSE is the sum of the squared error (Sandhu et al. , 2010). Results Diet Suitability Larval diets significantly affected the duration of development for all immature stages (Table 2 2). H. phaeopteralis did not survive beyond the first instar on the pinto bean diet. Only larvae reared on St. Augustineg rass and soy wheat germ diets successfully developed to pupae. However, the adults that emerged from soy wheat germ diets had aberrant wing pattern (Fig ure 2 1). Total development time and individual instars of H. phaeopteralis developed significantly faster on St. Augustinegrass (41.0 ± 0.9 d; instar range 3.2 10.9 d) compared with soy wheat germ diet (69.5± 2.9 d; range 6.5 20.7 d). Development was also significantly slower on the sugarcane borer, S. W. Corn borer and European corn borer diets, and none of the individuals successfully pupated. Total survival to the adult stage was 83.6% on St. Augustinegrass and 10.0% on the soy wheat germ diet (Table 2 3). The lowest survivorship on the soy wheat germ diet was recorded at the pupal stage (30.7%) and onl y 50.0% of these moths successfully emerged from the pupal case. Larvae reared on the S. W. corn borer diet, sugarcane borer diet and European corn borer diet failed to survive after the prepupal, sixth and third instars, respectively.

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48 Temperature Dependen t Development Herpetogramma phaeopteralis successfully completed its life cycle between 20 temperatures (Table 2 4). The number of days required for eggs to hatch ranged from 29.6 d at 15 C to 3.2 d at 30 C. For larvae, the first instar required the longest time to develop at all temperatures. The pupal stage required the longest development period, ranging from 5.3 d at 30 C to 11.4 d at 20 C. Longevity significantly decreased from 47.8 d at 20 C to 21.1 d at 30 C and then increased to 32.6 d at 32.5 C. Larvae failed to complete development at 15 and 35 C during the first and fifth instar, respectively (Table 2 5). The cumulative survivorship was highest at 30 C, followed by 25, 20, and 32.5 C. Developmental Rate a nd Mathematical Models The lower developmental thresholds (T 0 ) and degree days values (K) were estimated by the common linear model over the linear response range (excluding 32.5°C) (Table 2 6). The estimated lower temperat ure thresholds varied from 6.9°C (1st instar) to 15.3°C (fourth instar). The R 2 values for Briere 1, Briere 2, 2nd order polynomial and Lactin 2 for total development were 0.9, 0.4, 0.9 and 0.9, respectively (Table 2 7). The polynomial 2nd order model atta ined a good fit for the data considering the small value of AIC for eggs ( 50.6), larval stage ( 40.3), pre pupae ( 35.3), pupae ( 33.6) and the total development ( 44.5), but the upper developmental threshold for immature stages (42.1 65.9°C) were higher than observed in the laboratory (Table 2 7). The Briere 1 and Lactin 2 models provided a good fit for immature stages and total development. Overall we consider the Briere 1 model superior by providing a closer

PAGE 49

49 match to values observed in the laboratory. T he predicted value of the developmental ra te as a function of temperature is presented (Figure 2 2). Discussion This is the first attempt to describe the development of H. phaeopteralis mathematically. Previously, Kerr (1955) described the development of H. phaeopteralis as having seven larval instars that need 25 observed six instars requiring 22 d at 25 C and a prepupal stage (5 d) before pupation. The T 0 , T opt and T L estimated by the Briere 1 model for all immatu r e stages and total development better re fl ected the measured parameters than the Briere 2, Lactin 2, and polynomial models. The polynomial and Lactin 2 models overestimated an d underestimated the lethal threshold (47.5 and The estimate d R 2 values by Briere 2 were low (0.4). Sandhu et al. , (2010) reported that the Briere 1 model provided the best fit for the lesser cornstalk borer, Elasmopalpus lignosellus Zeller (Lepidoptera: Pyralidae), stating that T 0 , T opt and T L for total developmen t of immature stages were 9.3, 31.4, and 37.9 °C, respectively. Although these thresholds are within the results of this study for T 0 (14.9), T opt (29.4) and T L (34.2) estimated by Briere 1, differences in the host plant and insects will result in differen t thermal adaptations (Bergant and Trdan 2006). Kerr et al ., (1955) noted that adults became inactive at 14°C. Our study demonstrated that the lower thermal threshold for total development is 13.1°C using the linear model and between 12.2 and 15.0°C using the nonlinear models. Jensen and Cameron (2004) concluded that the lower developmental threshold for the grass webworm, Herpetogramma licarsisalis (Walker) falls somewhere between 10.8 and 15°C. The optimum temperature for H. licarsisalis was reported at o r slightly above 31°C based on the limited observations of Tashiro (1976), which is close to the

PAGE 50

50 observed (30°C) and estimated optimum temperature for egg to adult development by our nonlinear models (29.4 32.9°C). Jensen and Cameron (2004) studied the dev elopmental time of H. licarsisalis and found that larvae fed kikuyu grass, Pennisetum clandestinum ( Hochst. ex Chiov ) , required 25.0 d at 25°C, 43.4 d at 20°C, 50.4 d at 18°C, and 81.4 d at 15°C to complete the development from egg to adult. These developmental times appear faster than H. phaeopteralis at 25 and 20°C on St. Augustinegrass (41.0 and 47.8 d, respectively). In our experiment , survivorship declined with decreasing temperature, a trend also reported with H. licarsisalis (Jensen and Cameron 2004). It appears that H. licarsisalis that successfully developed at 15°C have a lower developmental threshold compared with H. phaeopteralis (our study), which may result in different ecological adaptations or host plant associations. To facilitate our research, we evaluated several existing lepidopteran artificial diets for their usefulness in rearing H. phaeopteralis . N one of the tested diets were suitable because of high mortality and slower developmental time. Failure to survive on these diets could be because of absence of important nutrients or feeding stimulants (secondary metabolic compounds). Insects often develop better on plant mate rials than on artificial diets (Dosdall and Ulmer 2004, Shen et al. , 2006). The diets based on corn starch were not suitable for rearing H. phaeopteralis ; similar results were observed in studies with Lymantria xylina (Shen et al. , 200 6). Our findings suggest that the soy wheat germ diet, with the highest protein content, was the most suitable compared with other tested diets, in that some larvae successfully pupated and eclosed. Previous studies have also reported that protein source i s critical component of insect diets (Schoonhoven et al. , 1998, Cohen 2003). For example, Spodoptera exigua ( Hübner ) and

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51 Helicoverpa zea ( Boddie) reared on synthetic diets comprising casein (animal protein) had superior growth compared with those fed soybea n powder (vegetable protein) (Duffey et al. , 1986). However, emerged adults from soy wheat germ diet had morphological abnormalities on the wings or scales. Malnourishment leading to wing deformities can be caused by variety of diet related deficiencies, i ncluding various lipids, amino acids, and vitamins as well as mineral deficiency (Cohen 2003). Little is known about identifying nutritional deficiency in insects by observing a specific set of symptoms (Cohen 2003). Lack of some fatty acids such as linole ic and linolenic acids cause wing deformity in cabbage Trichoplusia ni (Hübner) (Grau and Terriere 1971). The diet composition should be adjusted to improve survival and development of H. phaeopteralis in future studies. Our findings provide important inf ormation on the developmental biology and thermal characteristics of H. phaeopteralis . This information, in conjunction with other ecological data such as fecundity, intrinsic rate of increase, and mortality will be useful for predicting the potential dist ribution of this species and conducting pest management strategies such as timing pesticide applications, scheduling sampling intervals, and forecasting occurrence of different life stages in turfgrass.

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52 Table 2 1. Models evaluated to describe the relationship between temp erature and developmental rate for immature H. phaeopteralis . Model Paramet er Equation Reference Linear (common) 2 Y = a + bX Roy et al. , (2002) Briere 1 3 aT (T T 0 ) × sqrt (T L T) Briere et al. , (1999) Briere 2 4 aT (T T 0 ) × (T L T) 1/m Briere et al. , (1999) Polynomial (2 nd order) 3 aT 2 + bT + c Lamb et al. , (1984) Lactin 2 4 L [(T L Lactin et al. , (1995) In linear, a and b were constants and T0 = a/b and degree days were estimated through K = 1/b; where T is rearing temp (°C), T0 is lower threshold, TL is upper threshold, and Topt is optimum temperature. In Lactin optimal and upper threshold a

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53 Table 2 2. Duration (days) of larval, prepupal, and pupal development of H. phaeopteralis Diet 1 st instar 2 nd instar 3 rd instar 4 th insta r 5 th instar 6 th insta r Prepupa Pupa Total St. Augustine grass 4.0d 3.7c 3.2c 3.8c 3.9b 3.5b 3.6b 10.9b 41.0 b Soy wheat germ 6.5c 8.1b 8.6b 8.5b 8.8a 7.5a 8.0a 20.7a 69.5 a Sugarcan e borer 8.7b 13.0a 14.1a 12.2a 10.7a 8.0a European corn borer 10.3a 11.4a 18.0a S. W. corn borer 10.4a 10.8a 10.4a b 10.0b 9.6a 7.4a 9.0a F 118.1 54.2 44.2 67.3 49.0 46.5 71.2 137.5 44.2 Df 4, 106 4, 92 4, 80 3, 71 3, 63 3, 52 2, 45 1, 34 1, 44 Means within a column followed by the same letter are not significantly different (P , indicates no survival at that stage. Sample size was 50 larvae per diet.

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54 Table 2 3 . Percentage stage survivorship and successful adult emergence (eclosion) of H. phaeopteralis reared on five artif Augustinegrass at 25°C. Diet 1st instar 2nd instar 3rd instar 4th instar 5th instar 6th insta r Prepupa Pupa Total St. Augustin egrass 97.9 100 100 97.91 100 95.7 97.7 93.1 83.6 Soy wheat germ 100 100 90.0 94.4 100 82.3 92.8 30.7 10.0 Sugarca ne borer 95.0 84.2 93.7 46.6 57.1 50.0 0 0 0 Europea n corn borer 95.0 47.3 11.1 0 0 0 0 0 0 S. W. corn borer 90.0 94.4 94.1 87.5 71.4 50.0 20.0 0 0 Pinto bean 0 0 0 0 0 0 0 0 0 Sample size was 50 larvae per diet.

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55 Table 2 4 . Developmental time (days) of immature stages of H. phaeopteralis at six Temp. (°C) Egg 1 st insta r 2 nd instar 3 rd instar 4 th insta r 5 th insta r 6 th insta r Prepup a Pupa Total 15 29.6 a 20 6.5b 4.7a 4.3a 3.9a 4.0a 4.2a 4.4a 5.6a 11.4 a 47.8 a 25 4.1c 4.1b 3.8b 3.2b 3.8a b 3.9a b 3.6b 4.6b 10.9 a 41.0 b 30 3.2d 2.7c 2.0d 2.0d 1.5c 1.8d 2.4d 3.7c 5.3b 21.1 d 32.5 3.4e 3.4b 2.6c 2.5cd 3.2b 3.0c 2.9c 4.3cb 6.3b 32.6 c 35 3.4e 4.2b 2.6c 2.6 c 3.7a b 3.4c b F 60.9 46.5 111.0 48.0 63.3 72.3 101. 0 34.9 122. 7 121. 6 Df 5, 35 4, 155 4, 145 4, 142 4, 135 4, 125 3, 115 3, 113 3,10 9 3, 107 Means within a column followed by the same letter are not significantly different (P 0.05;

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56 Table 2 5 . Larval, prepupal, and pupal survival (%) of H. phaeopteralis reared at six Temperature (°C) 1 st instar 2 nd instar 3 rd instar 4 th instar 5 th instar 6 th instar Prepupa Pupa Total 15 2.5 0 0 0 0 0 0 0 0 20 92.1 94.2 100 100 96.9 96.8 96.7 100 78.9 25 97.9 100 100 97.9 100 95.7 97.7 93.1 83.6 30 97.5 94.8 100 100 97.2 100 97.2 100 87.5 32.5 82.5 90.9 80.0 87.5 95.1 90.0 88.8 93.7 37.5 35 92.5 83.3 83.3 76.0 36.8 0 0 0 0 Sample size was 50 larvae per temp erature .

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57 Table 2 6 . Estimates of linear regression parameters, min imum temp erature threshold (T 0 ), and thermal constant (K). Stage T 0 T u (degree days) Linear equation R 2 AIC Egg 10.1 62.9 0.9 20.8 1 instar 6.9 66.2 0.9 19. 3 2nd instar 12.3 38.2 0.8 14.4 3rd instar 10.5 40.3 0.9 16.2 4th instar 15.3 24.9 0.8 10.7 5th instar 13.9 32.3 0.8 12.5 6th instar 9.1 51.9 0.9 19.4 7th instar 13.1 106.4 0.9 29.7 Pupa 12.0 109.9 0.8 19.5 Eggs to adult 13.1 370.4 0.8 28.3

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58 Table 2 7 . Estimated coefficients, thermal constants, and goodness of fit for linear and nonlinear models of H. phaeopteralis Model Parameter Eggs Larvae Prepupae Pupae Total development Briere 1 A 0.0002 0.00008 0.0003 0.0001 0.00004 T 0 11.6 14.6 13.5 13.9 14.9 T L 38.2 34.4 34.4 37.3 34.2 T opt 32.0 29.4 29.3 31.7 29.4 R 2 0.9 0.9 0.9 0.9 0.9 RSS 0.0009 0.0004 0.003 0.002 0.0002 AIC 46.44 41.6 30.7 33.4 46.1 Briere 2 A 0.000005 0.00014 0.0003 0.0003 0.00009 M 0.6 9.2 1.1 37.2 52.0 T 0 13.4 12.6 18.7 11.5 12.2 T L 50.1 32.5 32.5 32.5 32.5 T opt 31.8 31.3 26.7 32.2 32.3 R 2 0.9 0.8 0.08 0.05 0.4 RSS 0.00028 0.0003 0.1 0.002 0.00008 AIC 51.8 41.2 12.2 32.1 47.2 Polynomial 2nd order A 0.0009 0.0003 0.001 0.0003 0.0001 B 0.06 0.02 0.08 0.02 0.009 T 0 14.1 14.9 14.9 14.7 15.0 T L 51.7 46.0 42.2 65.9 47. 5 T opt 32.9 30.5 28.5 40.3 31.3 R 2 0.9 0.9 0.7 0.9 0.9 RSS 0.0005 0.0005 0.001 0.002 0.0002 AIC 50.6 40.3 35.3 33.6 44.5 Lactin 2 0.033 0.005 0.045 0.008 0.002 14.7 0.1 11.3 0.2 0.2 Lambda 1.2 1.1 1.3 1.2 1.0 T 0 13.5 14.8 14.3 14.5 14.9 T L 43.1 32.7 33.5 32.6 32.5 T opt 32.0 32.0 26.0 31.5 32.0 R 2 0.9 0.9 0.9 0.9 0.9 RSS 0.0003 0.0003 0.0034 0.0013 0.00007 AIC 51.50 40.64 28.49 33.27 48.03

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59 Figure 2 1. A) Abnormal . B) normal wing of H. phaeopteralis . Photo A by Chris Fooshee . P hoto B by Lyle Buss.

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60 Figure 2 2 . Temperature dependent developmental rates of immature stages described by the Briere 1 model .

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61 CHAPTER 3 DOSE RESPONSE RELATIONSHI PS AND RESIDUAL ACTI VITY OF INSECTICIDES TO CONTROL HERPETOGRAMMA PHAEOP TERALIS (LEPIDOPTERA: CRAMBIDAE) IN ST. AU GUSTINEGRASS Background St. Augustinegrass, Stenotaphrum secundatum (Walter) Kuntze, and bermudagrass, Cynodon spp. (L.) , are the most widely used turfgrasses in Florida lawns and golf courses, respectively (Trenholm and Unruh 2004). Tropical sod webworm, Herpetogramma phaeopteralis (Guenée) is a serious pest of both of these grasses (Kerr 1955). All other major warm se ason turfgrass species, including centipedegrass ( Eremochloa ophiuroides { Munro. Hack. } ) , seashore paspalum ( Paspalum vaginitium { Swartz } ), zoysiagrass ( Zoysia japonica { Steud } ), bahiagrass ( Paspalum notatum { Flugge} ) are also subject to annual infestation by H. phaeopteralis (Reinert 1983). Herpetogramma phaeopteralis occurs from South Carolina to Florida, west to Texas in North America, the Caribbean and south through Central America (Brandenburg and Freeman 2012; H eppner 2003). In southern Florida, H. phaeopteralis adults are active year round, with significantly higher numbers in fall (September through November). Populations decline over the winter and increase slightly beginning in spring (March through May) (Che rry and Wilson 2005). In more northern regions of Florida (Gainesville), peak of flight activity was reported in October and November (Kerr 1955). Heavily infested sites include established sod, lawns, athletic fields and golf courses. Females lay eggs on , 2012). Young This chapter is accepted for a publication titled as ose response relationships and residual activity of insecticides to control Herpetogramma p haeopteralis (Lepidoptera: Cra mbidae) in St. Augustinegrass the Journal of Economic E ntomology.

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62 larvae (first through fourth instars) feed on adaxial side of grass blades and their injury is often overlooked (Kerr 1955). Older larvae (fifth and sixth instars) remove en tire grass blades causing brownish mown patches that allow weed ingress (Tofangsazi et al., 2012) . Lawn caterpillars including H. phaeopteralis larvae have traditionally been managed with broad spectrum insecticides; those used historically on Florida law ns include carbaryl, chlorpyrifos, diazinon, ethoprop, methomyl, trichlorfon, primiphos ethyl, isazofos, isofenphos, fonofos and toxaphene (Reinert 1983). Reinert in 1973 and 1983 evaluated carbaryl, chlorpyrifos, bendiocarb and ethoprop against H. phaeopt eralis larvae. However , to date these insecticides have been canceled or restricted following the Food Quality Protection Act (FQPA) of 1996. Information is not available on toxicity of newer insecticides and formulations to control H. phaeopteralis larvae . Insects are known for their ability to develop resistance to insecticides. Currently, twelve turfgrass pests have developed insecticide resistance in the USA, although fall armyworm, Spodoptera frugiperda (J.E. Smith), is the only lepidopteran turf pest with documented resistance to organophosphate, carbamate and pyrethroid compounds (Yu 1991, 1992). The ability of H. phaeopteralis to develop resistance is of concern due to its multiple generations per year and overlapping life stages, especially in Flori da where lawn and sod farms are treated with insecticides multiple times a year (e.g. 6 to 12 annually per conversations with sod farm managers and landscapers). It is thus important to plan and implement insecticide resistance management strategies for co ntrolling this pest before field control failure is encountered.

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63 Resistance monitoring programs require establishment of resistance baselines and survey ing for statistically significant shifts in lethal concentrations values (LC 50 ). These are normally est ablished through laboratory bioassays which should be initiated when frequency of resistant individual s are low or before a product is widely used to develop historical reference values (Cook et al. , 2004, Hardke et al. , 2011). Additionally, environmental factors such as UV light, temperature, rainfall, plant metabolism and microorganisms influence patterns and rates of insecticidal degradation under field environments (de Urzedo et al. , 2007, Hulbert et al. , 2011). Understanding residual properties of curr ent insecticides under field conditions might prevent unnecessary insecticide reapplication and associated costs. Thus, objectives of this study were to estimate resistance baselines and lethal activity range of insecticide classes, and determine relative effectiveness of a field aged sub set of these insecticides against H. phaeopteralis larvae. Materials a nd Methods Insects a nd insecticides Medium sized (third and fourth instar) H. phaeopteralis used in studies were obtained from a colony maintained since 2011 on potted St. Augustinegrass cv. New grass was provided every few days as needed and emerging moths moved to new cages and fed 5% v/v honey water. Formulated insecticidal compounds were obtained from commercial sources (Table 3 1). Laboratory B ioassays

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64 Initial bioassays were conducted to select insecticide concentrations that cause 10 to 99% mortality to estimate median lethal concentrations (LC 50 ) as suggested by Robertson et al. , (1984). A stock solution of each formulated insecticide was prepared at a concentration that reflected field recommended concentration (FRC), except for acephate (1/10th of FRC used as a stock solution) (Table 4 1). Subsequently, aliquots were taken from each stock solution and diluted with distilled water to prepare five concentrations of clothianidin (164.9, 82.5, 41.2, 20.6, 10.3 ppm), chlorantraniliprole (35.9, 17.9, 8.9, 4.5 , 2.2 ppm), B. thuringiensis (1327, 663, 333, 166, 83 ppm), bifenthrin (706.5, 353.2, 176.2, 88.3, 44.1 ppm), spinosad (97.7, 48.8, 24.4, 12.2, 6.1 ppm) and acephate (37.9, 7.6, 3.8, 2.5, 1.9 ppm) that were used to establish concentration response curves. For each tested insecticide, five hundred milliliters per serial dilution (plus 0.5ml/L of Tween 80% for contact insecticides B. thuringiensis , spinosad and Augustinegrass . Applications were made with a DeVries Research spray booth (Hollandale, MN) fitted with fan nozzle, calibrated to deliver the equivalent of 2,037 l/ha (218 gal./A) at a pressure of 207 kPa. After 24 hours, St. Augustinegrass stolons containing fresh sho ots were cut from the pots and placed individually into Petri dishes (8.5 cm diameter) containing 6 ml water agar covered with filter paper to maintain humidity. Five replicates were set up for each treatment with four medium sized H. phaeopteralis placed inside each Petri dish. All Petri dishes were kept in an incubator at 25±1°C, 70% RH and a photoperiod of 14:10 (L: D). Dead individuals (defined as no response to prodding) and moribund individuals (defined by uncontrolled twitching and other abnormal mov ements) were

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65 reported after 72 hours. Initial experiments indicated that moribund larvae after this time did not recover from insecticide exposure. Thus, moribund individuals were considered dead for analyses. The bioassay was replicated three times (i.e. 60 larvae per treatment concentration). Larval mortality was pooled for a given concentrations and subjected to analysis of variance (ANOVA; Proc glm) and Proc probit analysis on Log 10 concentrations to estimate LC 50 and LC 90 values. Significant difference s were based on non overlapping 95% confidence intervals. Field S tudies Experiments were conducted to measure residual control of insecticides from three different classes (chlorantraniliprole, clothianidin and bifenthrin) against medium sized (third and fourth instar) H. phaeopteralis . Experiments were conducted on 'Floratam' St. Augustinegrass plots maintained at the Mid Florida Research and Education Center , Apopka, FL . Individual grass plots were approximately 8 m 2 in surface area fitted with four pop up sprinklers in each corner and two in the middle of each long size at 105 at 241.3 kilopascal (kPa) for even irrigation. All plots were mowed, watered, and fertilized equally. Mowing was done before starting experiments at 10 cm height. Sta Green 12 2 8 fertilizer (Purcell Industries, Sylacauga, AL) was applied once at the rate of 1.04kg/ 92.9 sq. m (2.3 lb/1000 sq. ft) prior to fall experiments and two weeks following spring regrowth in April and end of May. Irrigation was cumulative reference evapotranspiration (ET o ) exceeded 19 mm, calculated from an adjacent weather station (Campbell Scientific Inc., Logon, UT) (Allen et al., 1989). Rainfall occurring between irrigation events was subtracted from the running ET o total.

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66 The amount of rainfall and solar radiation during experiments were reported from Florida Automated Weather Network http://fawn.ifas.ufl.edu/. The experiment was a repeated measures design. Plots were replicated f ive times. Each plot was assigned four subplots (1m × 1m) and treatments (3 insecticides plus controls) were randomized within each block. Buffer rows (100 cm) separated each subplot. Insecticide treatments were applied within label rate for clothianidin ( 7.0 kg per ha or 10 oz per A), chlorantraniliprole (146 ml per ha or 2 fl.oz per A) and bifenthrin (573 ml per ha or 7.8 fl.oz per A). Applications were made using a CO 2 back pack sprayer (R&D Sprayers, Opelousas, LA, USA) calibrated to deliver 5 gallon pe r 1,000 square feet (i.e. 2,037 l per ha or 218 gal. per A). Larvae for the bioassay were placed in field cages consisting of metal cylinders (17.8 cm diam. by 15.2 cm height) inserted into the ground in each subplot. Each week, fifteen third and fourth in star H. phaeopteralis were released into each metal cylinder (20 microplots) , which were covered with nylon mesh on top to prevent escape. Each metal cylinder was destructively sampled and larval survivorship was recorded by the end of each week. Experimen ts continued for 5 weeks under similar procedures. Plots were mowed prior to treatments but not during evaluations. Studies were conducted in the fall of 2013 (October 21 to November 24) and repeated the following spring (April 28 through June 2). On each occasion, a two factor (treatments and time) analysis was used to compare larval mortality using the GLIMMIX procedure (PROC GLIMMIX; SAS Institute 2013). Again, moribund individuals were considered as dead. Larval mortality percentage was arcsine transfor med to normalize the proportion data.

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67 Results Laboratory B ioassays Concentration that killed half of the H. phaeopteralis larvae ( LC 50 ppm ) was calculated as a measure of toxicity for each tested insecticide (Table 3 2). Dose response calculations indicated order of inherent toxicity was chlorantraniliprole > B. thuringiensis . The LC 90 values ranged from 30.6 ppm for chlorantraniliprole to 3101 ppm for bifenthrin. The L C 90 value of bifenthrin was greater than other tested compounds. Compared with recommended label concentrations, the LC 90 values derived in our study were lower for all tested insecticides except for B. thuringiensis and bifenthrin, suggesting that neither material was effective in the laboratory test. There was a significant two way interaction between treatment and concentration (F=1.84, df =15, P=0.03) indicating a variable larval response to different insecticides. The cumulative proportion data represe nting dead, moribund and live larvae after 72 h exposure to different insecticide concentrations revealed shallower concentration response for chlorantraniliprole and steepest for spinosad and clothianidin over the tested range (Fig ure 3 1, Table 3 2). Res idual E fficacy U nder F ield C onditions In residual efficacy field assays, all insecticide treated plots initially had significantly fewer H. phaeopteralis surviving compared to control plots during spring and fall experiments (Tables 3 3 and 3 4). All comp ounds at label rates were effective based on number of live recovered larvae was relatively high, possibly in part because not all larvae could be reliably recovered from field cages after one week. However, larval mortality was clearly affected by insecticide field aging, depending on material,

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68 with a more rapid decline in the activity of clothianidin and bifenthrin compared with chlorantraniliprole. Clothianidin had no s tatistically detectable activity after 4 weeks in the spring and the fall, and bifenthrin had no detectable activity after 3 weeks in the spring and fall. However , mortality) compared with other tr eatments throughout the 5 week study period. A generalized linear mixed model (GLIMMIX) showed that overall larval mortality was affected by insecticide treatment (F=376.3, df=3, P<.0001), time (F=58.1, df=4, P<.0001) and a treatment and time interaction ( F=10.3, df=12, P<.0001) as well as season effect (fall or spring) (F=35.4, df=1, P<.0001). Higher total rainfall, maximum daytime temperature and solar radiation (noon) were observed in spring, compared with the fall test, (i.e. 15.5 cm, 32.0°C and 723 w/m 2 versus 5.0 cm, 27.2°C and 506 w/m2). Discussion All tested compounds showed potential for controlling H. phaeopteralis . However , the most toxic compound tested. It had the lowest LC 50 values (4.5 ppm) in the laboratory and longest residual activity in the field. Chlorantraniliprole stimulates ryanodine receptors causing lethal paralysis in sensitive species (Cordova et al. , 2006). In addition to grass feeding caterp illars, chlorantraniliprole also controls turf damaging scarab grubs, as well as billbug and other weevil larvae, and invasive crane flies (Held and Potter 2012). Hardke et al. , (2011) evaluated chlorantraniliprole for baseline dose mortality responses for third instar fall armyworm, Spodoptera frugiperda (J. E. Smith). Chlorantraniliprole provided the least LC 50 values (0.068 ppm) compared to older insecticide s like spinosad (0.557 ppm) and the pyrethroid lambda cyhalothrin (5.27 ppm). The relatively lower values of Hardke et al. (2011) compared with the present

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69 study may reflect the different formulations of insecticides and methodologies used in their studies. For example, their insecticide was incorporated into an artificial diet, which may have increase d exposure and affected the LC 50 values accordingly. The neonicotinoid clothianidin has become widely used for control of caterpillars and other turf pests in Florida and elsewhere, due to its broad spectrum systemic activity and low application rate (Elb ert et al. , 2008, Silcox and Vittum 2008). We note that the susceptibility of H. phaeopteralis to clothianidin here (LC 50 = 46.6 ppm) was lower compared to previous reports for the western chinch bug, Blissus occiduus (Barber), a phloem feeding insect, when exposed to systemically acquired clothianidin on individual buffalograss plants (LC 50 = 16.6 ppm) (Stamm et al. , 2011). Stamm et al. , (2011) also noted differences in toxicity between several neonicotinoids against different life stages. Thiamethoxam was up to five fold more toxic to adult B. occiduus than clothianidin or imidacloprid in bioassays, although the opposite trend was noted for nymphs. It was also noted that the pyrethroid bifenthrin was significantly mor e toxic to B. occiduus in contact bioassays (i.e. exposed to dried residues on glass) compared with any of the neonicotinoid insecticides (Stamm et al. , 2011). It is not known why bifenthrin was less effective against H. phaeopteralis in our laboratory tes ts, although we speculate that larvae may have avoided feeding on the treated plant surfaces. Microbials historically constitute a tiny percentage (< 0.1%) of insecticides used on turfgrass in the United States (Grewal 1999). The bacterium Bacillus thuring iensis var. kurstaki (Btk) is labeled for turfgrass caterpillars but tends not to be used due to short activity (deactivation with sunlight), narrow pest spectrum, and weak activity against later instars (Held and Potter 2012). In our study, Btk had relati vely low activity

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70 against mid sized larvae of H. phaeopteralis (LC 50 342 ppm) when applied at recommended field rates. Relatively better activity was observed with spinosad (LC 50 31.1 ppm) , another microbial derivative produced by fermentation of the soil bacterium Saccharopolyspora spinosa . Spinosad products are labeled for turf and effective against grass feeding caterpillars and some other turf pests, but also have short residual activity and use rates (Gosselin et al. , 2009, Held and Potter 2012). Sever al entomopathogenic nematodes are registered for turf pests, including caterpillars and white grubs. Tofangsazi et al. , (2014) reported that a commercial formulation of Steinernema carpocapsae was as effective as clothianidin against large larvae of H. pha eopteralis in greenhouse tests. Nevertheless, due to their cost and relative difficulty of use compared with chemicals, it is argued that the foreseeable use of microbial control products in turf will be restricted to niche markets, such as organic lawn ca re, school grounds and other sensitive areas (Held and Potter 2012). Longer residual activity of turf insecticides might reduce application frequency needed to maintain seasonal pest populations. Reinert (1983) screened thirty insecticides for controlling mixed populations of sod webworms, Herpetogramma spp. on 'Tifway' and 'Tifgreen' bermudagrass, Cynodon spp. They reported that fonofos and chlorpyrifos provided partial residual control of the second generation, but experimental plots become reinfested 3 o r 4 weeks post application. Both fonofos and chlorpyrifos (organophosphates) are classified as restricted insecticides for use on turf (Brandenburg and Freeman 2012). In our field study, only chlorantraniliprole provided residual control that might extend to the second generation of H. phaeopteralis if used early in the season. This result is consistent with findings of Held and Potter (2012) ,

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71 who noted that chlorantraniliprole has longer residual activity against caterpillars compared to neonicotinoids. En vironmental conditions affect the persistence of pesticides (Wackett 2007). Contact insecticides including pyrethroids may be more liable to leaching and photodegradation through rainfall and UV exposure compared with systemic materials which are taken up by the roots, stem and leaves. Future research using high performance liquid chromatography would be useful to quantify the persistent nature of different insecticide residue in the turf ecosystem. Turf is habitat for a variety of non target arthropods in cluding natural enemies, decomposers and pollinators (Bixby Brosi and Potter 2012, Peck 2009 and Larson et al. , 2013). Conserving beneficial species by selecting active ingredients that are less toxic against non target organisms is needed for integrated t urf pest management. Larson et al. , (2014) indicated that label rate of clothianidin may intoxicate a range of beneficial species, including the ground beetle Harpalus pennsylvanicus (DeGeer), black cutworm parasitoid Copidosoma bakeri (Howard), and bumble bees Bombus impatiens (Cresson), foraging on treated white clover in weedy turf. By contrast, chlorantraniliprole had no apparent adverse effects on any of the beneficial species. Moreover, chlorantraniliprole appears to have a synergistic or additive effe ct (similar to some neonicotinoids) with beneficial entomopathogenic nematodes used to control white grubs (Coleoptera: Scarabaeidae) in turfgrass (Koppenhöfer and Fuzy 2008). Since many turf managers practice multiple targeting insecticide application s to control foliage and root feeding turf pest s simultaneously (Held and Potter 2012), a combination of chlorantraniliprole with other low risk insecticides including microbial materials, offers

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72 promise for control of caterpillars such as H. phaeopterali , alo ng with grubs and other important pests of turfgrass.

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73 Table 3 1. Insecticide s tested for control of H. phaeopterali . Chemical Class Active Ingredient Trade Name Rate/ha (A) a Company Neonicotinoids Clothianidin Arena 50 WDG 672 g (9.6 oz) Valent USA Corp., Walnut Creek, CA Ryanodine receptor modulator Chlorantraniliprole Acelepryn® 140 ml (2 fl.oz) DuPont Professional Products, Wilmington, DE Organophosphate Acephate Acephate 75 SP 1.49 kg (1.33 lbs) Valent U.S.A. Corp., Walnut Creek, CA Pyrethroid Bifenthrin Talstar P Professional 573 ml (7.84 fl.oz) FMC Corp., Princeton, NJ Spinosyn Spinosad Conserve SC 795 ml (10.9 fl.oz) Dow AgroSciences, LLC, Indianapolis, IN Biological insecticide Bacillus thuringiensis subsp. kurstaki, DiPel®DF 1.12 kg (1 lb) Valent BioSciences Corporation, Libertyville, IL a Within range recommended for turf caterpillars .

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74 Table 3 2. Toxicity of commercial insecticides to medium size (3rd and 4th instar) H. phaeopteralis after 72 h. exposure. Insecticide n Intercept ± SE LC 50 a (CI b ) LC 90 a (CI b ) FR C c Clothianidin 60 0.1 ± 0.3 46.6 (37.1 57.9) 251.2 (172.5 442.6) 330 Chlorantraniliprole 60 0.04 ± 0.2 4.5 (3.3 5.8) 30.6 (21.3 53.9) 72 B. thuringiensis 60 0.2 ± 0.5 342.0 (262.6 447.6) 3041 (1795 7366) 550 Acephate 60 0.03 ± 1.8 8.6 (6.9 10.9) 40.4 (27.2 72.3) 731 Spinosad 60 0.12 ± 0.3 31.1 (26.3 37.2) 114.1 (85.9 170.5) 390 Bifenthrin 60 0.16 ± 0.2 282.7 (202.3 444.4) 3101 (1,479 11344) 281 a LC 50 , LC 90 = ppm product b CI = 95% confidence interval c Field recommended concentration= ppm product

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75 Table 3 3. Mean (±SEM) percentage mortality of H. phaeopteralis exposed to aged insecticide residues in field plots of St. Augustinegrass (spring experiment). Treatments Weeks after treatment 1 2 3 4 5 Clothianidin 97.3 ± 0.1 a a A b 97.3 ± 1.6 aA 70.6 ± 3.4 bB 69.3 ± 2.7 bB 53.3 ± 4.7 bB Chlorantraniliprole 100 ± 0.0 aA 98.6 ± 1.3 aA 97.3 ± 1.6 aA 97.3 ± 1.6 aA 84 ± 4.5 aB Bifenthrin 94.6 ± 0.4 aA 81.3 ±0.7 bB 66.6 ± 0.3 bBC 61.3 ± 0.4bcCD 42 ± 3.7 bD Control 50.6 ± 4.9 bA 40.0 ± 6.6 cA 46.6 ± 3.0 cA 46.6 ± 4.7cA 32 ± 4.4 bA a Column means followed by the same lower case letter are not significantly different, b Row means followed by the same capital letter are not significantly different (Proc glm,

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76 Table 3 4. Mean (±SEM) percentage mortality of H. phaeopteralis exposed to aged insecticide residues in field plots of St. Augustinegrass (fall experiment). Treatments Weeks after treatment 1 2 3 4 5 Clothianidin 97.3±1.6a a A b 93.3±0.2 aA 58.6±0.1b B 64±0.09bB 45.3±0.1bB Chlorantranilipr ole 97.3±1.7aA 100±0aA 90.6±0.2a A 94.6±0.1aA 94.6±0.2aB Bifenthrin 97.3±1.6aA 89.3±0.2 bB 69.3±0.3b BC 46.6±0.2bcC D 52±0.2bD Control 29.3 ± 8.0bA 48 ± 5.7 cA 18.6 ± 7.7cA 42.6 ± 4.5cA 32 ± 7.7bA a Column means followed by the same lower case letter are not significantly different, b Row means followed by the same capital letter are not significantly different (Proc glm, HSD).

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77 Figure 3 1. Cumulative percentage mortality, moribund and survivorship of 3rd and 4th instar H. phaeopteralis after 72 h exposure to different concentrations of tested insecticides.

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78 CHAPTER 4 EFFICACY OF COMMERCIAL FORMULATIONS OF ENTOMOPATHOGENIC NEMATODES AGAINST TROPICAL SOD WEBWORM Background Tropical sod webworm, Herpetogramma phaeopteralis Guenée, (Lepidoptera: Crambidae) is an economically injurious pest of warm season turfgrass in the southeastern United States and Caribbean islands (Kerr 1955; Meagher et al. , 2007). In North America, H. phaeopteralis has been recorded from Florida, Georgia, Louisiana, Texas, Hawaii, Mississippi, and Alabama (Kerr 1955; Meagher et al. , 2007). Infested s ites include lawns, athletic fields and golf courses (Vittum et al. , 1999). In southern Florida, the peak of adult emergence is from September to November (Cherry and Wilson 2005). After mating, females deposit clusters of 6 15 eggs on the grass blades and , 2012). Fifth and sixth instar H. phaeopteralis are the most harmful as the larvae chew whole sections of leaf and produce silk along the grass blades (Kerr 1955). Herpetogramma phaeopteralis larvae feed on all warm season turfgrasses affecting aesthetics, vigor, photosynthesis and density (Vittum et al. , 1999). Current control recommendations for H. phaeopteralis are mainly application of above ground chemical insecticides against larval stag es (Brandenburg and Freeman 2012). Turfgrass is an important industry in the southeast, comprising 1.6 million ha in Florida alone and generating revenue impacts of over $6 billion in 2007 (Hodges and Stevens 2010). Hazards associated with the over use of insecticides in turf include This chapter is accepted for a publication titled as efficacy of commercial formulations of entomopathogenic nematodes against tropical sod webworm, Herpetogramma phaeopteralis (Lepido ptera: Crambidae) in Journal of Applied Entomology.

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79 insecticide resistance, negative impacts on beneficial and other non target species, and contamination of groundwater (Racke 2000; Potter 2005). These issues along with public concern have stimulated interest in integrated mana gement of turfgrass insect pests (Grewal 1999; Racke 2000). Purchasing and releasing natural enemies for pest suppression is a fast developing tactic in augmentation biocontrol programs (Hajek 2004). Entomopathogenic nematodes (EPNs) in families of Heteror habditidae and Steinernematidae can be used in a rotational program with insecticides to reduce insect resistance (Grewal 1999). In addition to reduction in insecticide resistance and usage, EPNs are non toxic to humans, can be applied with standard pestic ide equipment and have some potential to recycle in pest populations (Potter 2005). Entomopathogenic nematodes have been effective in pest management programs for other lepidopteran species that attack various grasses (González Ramírez et al. , 2000; Medeir os et al. , 2000; Negrisoli et al. , 2010). We therefore hypothesized that EPNs might offer an alternative tool to manage H. phaeopteralis in turf. Susceptibility of lepidopteran larvae to EPN can be affected by several biotic and abiotic factors. Among the most important biotic factors are host developmental stages and nematode species or formulations (Koppenhöfer and Fuzy 200 5 ). However, the susceptibility of H. phaeopteralis to products containing different EPN species has not been reported. The objectives of this study were to compare the efficacy of available EPN products in the United States against different larval sizes of H. phaeopteralis under laboratory and greenhouse conditions. We included clothianidin, a neonicotinoid registered for webworm contr ol, as an insecticidal control for comparison.

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80 Materials A nd Methods Nematodes A nd Insects Products containing Steinernema carpocapsae , S. feltiae , and Heterorhabditis bacteriophora (Becker Underwood, Ames, IA and Biobest, Lafayette, CO), H. megidis (Bec ker Underwood) and H. indica (Southeastern Insectaries, Perry, GA) were obtained from commercial sources. All species were not available from all sources. Nematodes nema tode viability (> 90%) was confirmed by observing shape and mobility of 100 infective juveniles (IJ) at 100X magnification. Larvae of H. phaeopteralis came from a cm) at U niversity of Florida, Mid Florida Research and Education Center, Apopka, FL. Laboratory E xperiments All EPN species were tested against three different larval sizes of H. phaeopteralis . Small were first and second (1 5 mm) , medium were third and fourth (6 18 mm) , and large fifth and sixth instar larvae (18 25 mm) . Nematode IJ were diluted to different concentrations (10, 100, 1000 and 5000 IJ/mL) and applied in 1 ml of water to a filter paper in a 9 cm Petri dish using a pipette; controls received only water. Four larvae of one size were placed in each Petri dish with excised St. Augustinegrass leaves for food. Each Petri dish was one replicate and five replicates were set up for each nematode species, concentration, and larval size (i.e.75 treatments in total). The experiment was conducted three times each from different shipments from each company. Mortality was recorded daily over three days. Dead larvae were defined as those not able to move and with discoloration of the body. All Petri dishes were ma intained in a growth chamber at 25°C with photoperiod of 14: 10 (L:D) and 70% RH.

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81 Nematode Reproduction Entomopathogenic nematode reproduction was assessed using White traps (White 1927). Sixteen large size cadavers that were exposed to 5000 IJ/ml were col lected from the mortality bioassay. Four White traps containing four cadavers each were set up for each EPN species. The experiment was repeated two times. Once IJs started emerging from H. phaeopteralis cadavers, IJs were counted twice a week over three w eeks. The number of emerged IJs was estimated by taking an average of five counts of a 1cm 2 area of nematode solution in each White trap. Greenhouse Experiments Based on mortality obtained in laboratory studies, S. carpocapsae Becker Underwoo St. Augustinegrass (20.3 cm diameter) filled with a Fafard Growing Mix 2 (Canadian Sphagnum Peat Moss (75%), perlite, vermiculite, starter nutrients, wetting agent and dolomitic limestone) with 5.5 to 6.5 pH range were used. Pots were fertilized with Scotts Liquid Turf Builder (29 2 3) at the rate of 0.5kg/46.4 sq. m prior to the beginning of experiments once and watered two or three times weekly during experiment. Fifteen small, medium, or large (defined above) larvae of H. phaeopteralis were put in each experimental pot a day before S. carpocapsae or clothianidin application. Separate pots were used for each size of larva. Pots were caged individually by enclosing the upper portion of each pot in 60 × 60 cm nylon mesh supported by wires and braided elastic. Experimental pots (uncovered) were sprayed in a DeVries Research spray booth (Hollandale, MN) fitted with fan nozzle (TeeJet 8002E with filter removed) and calibrated to deliver the equi valent of 2500 L application rate per ha at a pressure of 206.8 kPa. Treatments consisted of S. carpocapsae at low rates 10 5 (IJ/liter) and 10 6 (label rate for

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82 turf), label rate of clothianidin (680 g per ha) and sterile water as control. Pots were irrigat ed prior and after S. carpocapsae or clothianidin application to deliver 0.5 and 0.1 inches of water as pre and post irrigation with a rainfall simulator. Soil samples were collected after spraying and soil moisture contents were estimated by gravimetric m ethod (Black 1965). Pots were arranged in a completely randomized block design in the greenhouse and emerging tropical sod webworm adults were counted daily for two weeks after initial moth emergence. There were five pots per treatment and experiments cond ucted on three separate occasions. Environmental conditions inside pots were monitored with HOBO temperature/relative humidity data loggers (Onset Computer Corp., Pocasset, MA). Data Analysis In laboratory experiments, larval mortality was pooled for each nematode concentration at each larval size for a given species and subjected to analysis of at alpha=0.05. Lethal median concentrations (LC 50 ) were estimated for the three larval sizes of H. phaeopteralis infected with five different nematode species by using Proc probit analysis on Log 10 dose (SAS 2008). For greenhouse experiments, a two way d etermine significance between means at alpha=0.05 if significant F test was detected (SAS 2008). Since there was no overall treatment × time interaction in the greenhouse trial (F 4, 126 = 1.4, P = 0.2), data were pooled across tests prior to analysis. Arcs ine transformations were used to normalize percentages values (larval mortality) for analysis.

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83 Results Laboratory S tudies All tested nematode species were pathogenic to H. phaeopteralis , with a clear concentration/mortality response for all species (F 3,120 = 236.6, P < 0.0001) (Table 4 1). Percentage mortality caused by each EPN species differed significantly especially at higher concentrations, demonstrated by a significant nematode species × concentration interaction (F 12,120 = 3.4, P < 0.001). Overall, H . phaeopteralis larvae were most susceptible to S. carpocapsae 76.3% mortality of H. phaeopteralis at 5000 IJs/ml. Based on LC 50 concentration and their confidence limits, nematodes were equally effectiv e against different larval sizes, the one exception being H. indica which had higher LC 50 for small larvae (Table 4 2). Overall, lethal concentrations were lowest for S. carpocapsae at all larval size being significantly lower than H. megidis and H. bacter iophora . In vivo IJs reproduction varied among EPN species (Table 4 3). The mean number of IJs produced per White trap was highest by larvae infected by H. bacteriophora . The number of IJs emerged from a H. bacteriophora White trap (9448 IJs) was approximately 2.4 times greater than for H. indica (3895 IJs) which produced the least IJs. Greenhouse S tudies In greenhouse experiments, clothianidin and the low and high rate of S. carpocapsae (10 5 and 10 6 IJ/liter) caused sig nificant mortality of all larval sizes compared to untreated control (Fig ure 4 1). The effect of treatment (F 3,168 = 501, P < 0.0001), host size ( F 2,168 = 7.0, P < 0.001) and their interaction (F 6,168 = 3.6, P < 0.005) were significant larval mortality fac tors. The high rate of S. carpocapsae caused

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84 significantly higher mortality than the low rate at all larval sizes. S. carpocapsae at 10 6 IJ/liter was as effective as clothianidin against large larvae, but not as effective as clothianidin against younger la rvae size, which may explain the treatment × host interaction. In each experiment, soil moisture was estimated immediately after spraying by taking three soil cores from each pots and the soil moisture range fell between 80% to 92% w/w. In greenhouse exper iments, environmental conditions averaged over the 81.5 % RH (range 48.8 to 100% RH). Discussion This study is the first report of susceptibility of any Herpetogramma spe cies to entomopathogenic nematodes. Our results showed that all larval size of H. phaeopteralis were susceptible to all tested EPN species, but S. carpocapsae appeared to be the most effective based on the products tested. Our results are similar to a prev ious study which evaluated the susceptibility of the armyworm, Pseudaletia unipuncta (Haworth), to five strains of entomopathogenic nematodes in the laboratory and found that S. carpocapsae A48 was most virulent compared to H. bacteriophora and S. glaseri Az26 (Medeiros et al. , 2000). Additional studies conducted in golf course fairways in New Jersey, USA, compared commercial EPN products including H. bacteriophora , S. carpocapsae , S. feltiae , and S. riobrave against the black cutworm , Agrotis ipsilon ( Huf nagel ) , and reported S. carpocapsae was the best species (Ebssa and Koppenhöfer 2011). There are some reports of EPN tested against species of stem boring Crambidae. Caroli et al. , (1996) reported that IJ penetration rates of Ostrinia nubilalis ( Hübner ) we re significantly higher for S. carpocapsae and S. feltiae compared with H. bacteriophora HP88 and NJ strains. More recently, Bellini and

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85 Dolinski (2012) reported that foliar applications of H. baujard LPP7 and S. carpocapsae on sugarcane stalks were equall y effective to control the sugarcane borer, Diatraea saccharalis ( F .) . The affinity of S. carpocapsae to these above ground grass feeding lepidopteran larvae may be related to its ambusher searching behavior which is adapted to control pests in upper soil profile (Moyle and Kaya 1981). In contrast, low virulence of S. carpocapsae against Diaprepes abbreviates (L.) larvae was reported (Shapiro and McCoy 2000). The low virulence of S. carpocapsae against D. abbreviates , a root feeder weevil, is likely due to the larvae being below ground. It is known that host age can affect virulence of entomopathogenic nematodes (Shapiro et al. , 1999; Lee et al. , 2002). Our greenhouse studies suggested that larger webworms were more susceptible compared with small and mediu m size larvae to S. carpocapsae inside turf pots. Other studies document that age susceptibility of insect hosts to Steinernema spp. is somewhat species specific (Fuxa et al. , 1988; Medeiros et al. , 2000). Another aspect of our study was to compare S. car pocapsae to a commonly used turf insecticide. Overall, clothianidin provided the best H. phaeopteralis control in greenhouse experiments. This finding is consistent with field trials that indicated insecticides among several classes (bifenthrin, chlorantra niliprole, thiamethoxam, and time released imidacloprid tablets) provided the best white grub control compared with H. bacteriophora and S. carpocapsae treatments in a conifer production (Liesch and Williamson 2010). Although clothianidin caused the highest larval mortality, greenhouse experiments also revealed that a single application of the label rate of S. carpocapsae was as effective as the insectic idal standard against large larvae, and also effective

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86 against small and medium size larvae. Considering the evidence of synergistic interaction between neonictionoid insecticides and EPN for control of several pests (Koppenhöfer et al. , 2000, 2002), the c ombination of clothianidin and S. carpocapsae could be tested in future studies to determine what interaction (e.g. synergism, additive, and antagonist) might occur. If clothianidin interacts synergistically with S. carpocapsae , this could reduce amount of insecticides applied in turfgrass. Steinernema carpocapsae (Millenium® or Carpocapsae System) is a good option for H. phaeopteralis biocontrol based on our data and corroborated the history of success of using S. carpocapsae to control grass feeding cate rpillars in the fields (Ebssa and Koppenhöfer 2011). Steinernema carpocapsae has also been reported virulent to other destructive turfgrass pests including annual bluegrass weevil ( Listronotus maculicollis Dietz), army worms ( Spodoptera exigua Hübner), bil lbugs ( Sphenophorus spp.), and black cutworm ( A. ipsilon ) (Shapiro Ilan et al. , 2002; Ansari et al. , 2007; McGraw and Koppenhöfer 2008). However, a challenging aspect of using EPN is that they are living organisms and are sensitive to UV light, high temper atures and drying conditions. The nematode efficacy can be enhanced by keeping the treated area wet for several hours post application and applying during early morning or evening hours (Grewal et al ., 2005). Thus, further studies are necessary to determin e how effective S. carpocapae will be against H. phaeopteralis and other warm season turf pests under different field conditions.

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87 Table 4 1 . Percentage mortality of H . phaeopteralis exposed to five concentrations of infective juveniles of five differe nt EPN species after 72 h in laboratory tests. Concentration (IJs/ml) Nematode species 0 10 100 1000 5000 S . carpocapsae 3.4Da 39.3 Ca 75.9 Ba 98.5 Aa 96.9 ABa S . feltiae 1.0 Da 29.9 Cab 55.1 Bb 84.4 Ab 91.4 Ab H . indica 3.4Ea 32.4 Dab 49.4 Cb 72.1 Bbc 86.4 Abc H . bacteriophora 2.2 Ea 24.0 Db 45.5 Cb 70.8 Bbc 88.6 Abc H . megidis 0.7 Da 28.5 Cab 44.4BCb 58.7 ABc 76.3 Ac Significant differences between concentrations for a given EPN species indicated with different capital letters in each row. Significant differences between species for a given concentration indicated with different small letters in each column. Means are pooled

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88 Table 4 2 . Lethal concentrations (LC 50 ) of five entomopathogenic nematodes (IJ/ml) applied against three larval sizes of H . phaeopteralis , after 72 h. Nematodes species Larval size S . carpocapsae S . feltiae H . indica H . bacteriophora H . megidis Small 28 (15 45) 72 (38 123) 387 (157 1056) 270 (126 577) 554 (210 1925) Medium 19 (11 29) 55 (27 96) 55 (26 100) 88 (46 152) 138 (47 333) Large 19 (10 33) 39 (13 83) 38 (11 85) 116 (58 210) 134 (55 283) different at (alpha= 0.05).

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89 Table 4 3. Effect of five species of entomopathogenic nematodes on IJs production in each White trap. Species Total IJs produced Range S . carpocapsae 7,366 b (2,099 14,185) S . feltiae 8,020 ab (2,837 18,156) H . indica 3,895 c (1,475 7,943) H . bacteriophora 9,448 a (4,709 17,022) H . megidis 6,556 b (1,248 15,887) Each white trap consists of four large size larvae of H . phaeopteralis infected with 5000 IJ/ml.

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90 Figure 4 1. Greenhouse tests showing mortality of H . phaeopteralis following treatments with S . carpocapsae , clothianidin and controls.

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91 CHAPTER 5 ATTRACTION OF HERPETOGRAMMA PHAEOPTERALIS ADULTS TO FLORAL LURES AND PHERMONE CANDIDATES Background Tropical sod webworm (TSW), Herpetogramma phaeopteralis (Guenée) (Lepidoptera: Crambidae), is a serious pest of turf grasses in the southern United States , C entral America and Hawaii (Tashiro 1976, Meagher et al. , 2007). This pest is of concern to turf production and sod farmers and may need multiple insecticide application to manage infestations in Florida ( Brandenburg and Freeman 2012. ). Monitoring traps can be used to determine the seasonal activity and distribution of H. phaeopteralis , which may improve control operations for this pest . Currently, however, only crude methods such as sweep nets or light traps are available for monitoring the flight activity of this species (Cherry an d Wilson 2005). Our goal was to develop trap baited with pheromone and /or floral lures as a monitor ing tool for H. phaeopteralis . Floral lures are an attractant for many nectar feeding moths. Phenylacetaldehyde ( PAA) , along with other chemicals, has been isolated from many flowering plants and shrubs including Zea mays (L.) (Cantelo and Jacobson 1978), Araujia sericofera (Brothero) (Cantelo and Jacobson 1979), Abelia grandiflora (André) (Haynes et al. , 1991), Cestrum nocturnum (L.) (Heath et al., 1992), and Gaura spp. (Shaver et al. , 1998). Floral lures ha ve been identified as attractant s for a number of species of Lepidoptera, including a number of the members of the Noctuidae subfamily Plusiinae ( Landolt et al., 201 2). The use of semiochemicals from plants acting as kairomones for monitoring and controlling insect pests would have the advantage of capturing both sexes (Avilla et al. , 2003).

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92 To date, the sex pheromone attractant of more than 41 crambid moth species ha s been identified (Gibb et al. , 2007, Peng et al. , 2012). Gibb et al. , (2007) identified a blend of (Z) 11 hexadecen 1 yl acetate (Z11 16:Ac) and (11Z,13E) hexadecadien 1 yl acetate (Z11,E13 16:Ac) as the major pheromone components of a related species, H. licarsisalis (Lepidoptera: Crambidae). Herpetogramma licarsisalis attacks pasture and ornamental turf throughout tropical and subtropical regions including central Africa, Sierra, Egypt, Saudi Arabia, India, Japan, Southeast Asia, Hawaii and northern Aust ralia (Tashiro 1976, Gibb et al. , 2007). However, lures with these chemicals have not been tested in North America for their attractiveness to H. phaeopteralis . Thus, my objective was to evaluate floral lures and pheromone blends attractive to H. licarsisals [based on Gibb et al. , (2007)] for their attractiveness to H. phaeopteralis . I also aimed to identify the sex pheromone of H. phaeopteralis in order to provide a synthetic attractant for monitoring and possibly enabling new control tactic s for this pest. The pheromone work was done in cooperation with Dr. Ashraf El Sayed from the New Zealand Institute of Plant and Food Research Ltd. Materials a nd Methods Attraction to F loral L ures The experiments were conducted at grassy areas of the Mid Flori da REC in Apopka and farther south at the Everglades Research and Education Center in Belle Glade in 2011. Different botanical lures listed in table 5 1 (Aldrich Chemical Co) and unbaited controls were used as treatments. 0.5 ml of each chemical were dispensed separately in hollow polyethylene stoppers (Kimble, Vineland, New Jersey) and placed in the middle of each green delta traps (Pherocon IIID, Trécé Inc., Adair, OK.) and were attached to PVC poles at a height of 1 m and 20 m spacing (Fig 5 1 ). The re were 5

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93 replicates for each treatment which were arranged in randomized block design. Blocks were separated by at least 50 m. After one week traps were collected and the number of adult H. phaeopteralis counted. To confirm the presence of H. phaeopterali s in our blocks, five sweeps with a sweep net (38 cm diameter) were taken per block. Each sweep consisted of a 180° sweep with each forward step. Field B ioassays U sing H. licarsisalis P heromone B lends The attraction of H. phaeopteralis males to the (Z) 11 hexadecen 1 yl acetate (Z11 16:Ac) and (11Z,13E) hexadecadien 1 yl acetate (Z11,E13 16:Ac) which are the two major sex pheromone components of the related species, H. licarsisalis and the corresponding alcohol, (11Z,13E) hexadecadien 1 ol (Z11,E13 16:OH) and six various combinations of the three sex pheromone candidate compounds were investigated with field populations of H. phaeopteralis . Virgin females were included as positive control. Studies were done on St . Augustinegrass cv. Floratam at the Everg lades Research and Education Center in Belle Glade, Florida, USA during the flight period of H. phaeopteralis ( September November ) . Green delta traps (Pherocon IIID, Trécé Inc., Adair, OK) were used and attached to PVC poles at a height of 1 m and 20 m sp acing. Treatments dissolved in acetone were loaded onto red rubber septa (Thomas Scientific Inc., Philadelphia, PA, USA).There were 5 replicates for each treatment and treatments were arranged in a randomized block design. Traps were collected after one we ek and total number of adults H. phaeopteralis counted. Shipping P rotocol Larvae of H. phaeopteralis were inside a greenhouse at University of Florida, Mid Florida Research and Education Center, Apopka, FL. Pupae were collected and shipped under permit to import live

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94 animals under Animal Imports and Exports Group, MAF Biosecurity Wellington, New Zealand. A pproximately 100 300 pupae were shipped to the Plant and Food Research Institute of Lincoln, New Zealand several times . H. phaeopteralis pupae were emerged individually in a s were sexed after emergence. One day old males were used in EAG recording and female s were used for pheromone gland extractions. G as C hromatography (GC) T he pheromone glands of 50 48 hr old female H. phaeopteralis were extracted hexane for 5 minutes. . gland extracts were derivatized, with either 4 methyl 1,2,4 tria zoline 3,5 dione (MTAD; Sigma Aldrich, NSW, Australia) or dimethyldisulfide (DMDS; Merck, Darmstadt, Germany) . Based on quantitative analysis using GC mass spectrometry (MS) several candidate pheromone compounds were identified . Electroantennogram (EAG) To determine the double bond positions and geometry of identified compounds from GC MS detected compounds in the gland extract of H. phaeopteralis , individual moths were tested for e lectroantennogram ( EAG ) response using a Syntech High Resistance EAG Probe, T ype ID 02 Signal Interface Box and Type IDAC 02 Intelligent Data Acquisition Controller (Syntech, Hilversum, Netherlands) and Syntech electroantennogram software. Laboratory experiments consist ed of recording electrophysiological activity of 1) five diene sex pheromone candidates, 2) f ive different (Z) isomers of monoene sex pheromone candidates, 3) f ive different (E) isomers of monoene sex pheromone

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95 candidates, 4) f our different alcohol compounds, 5) c omparison between Z11,E13 16:Ac and Z11 16:Ac and 6 ) c omparison between Z11 16:Ac and E11 16:Ac (Table 5 2). Stimuli were loaded on pieces of filter papers (20×5 10µg) and inserted into a Pasteur pipette. One or two antennal segments excised from the both ends of the antennae were inserted into saline fill ed glass electrodes with a micromanipulator (Syntech, Hilversum, Netherlands) . ANOVA was carried out to compare stimulus (treatments) GC E lectroantennographic D etection A n excised male antenna was set up between two glass electrodes, containing A 10 mm silver wire was inserted on e ach glass electrode to connect the preparation to the recording unit. Fifty pheromone gland extr acts analyzed by a Varian 3800 GC were coupled to an EAD Recordin g Unit (Syntech, Hilversum, The Netherlands). Extracts were run on DB 5 (30 m×0.25 mm Wax columns (30 film; Agilent) with 1:1 split outlets. Helium was used as the temperatures were 220° and 300°C, respectively, and the GC temperature program was 80°C for 1 min, 10°C Results Attraction O f T ropical S od W ebworm t o F loral L ures None of the floral lures were attractive to capture H. phaeopteralis at both locations (Table 5 1 ). Treatments did not significantly affect trap catch (F 4, 20 = 0.86, P = 0.57). Mean number of adult H. phaeopteralis collected in single sweep net catch in

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96 each experimental block at Everglades Research and Education Center were vary between 1.2 ± 0.2 to 3 ± 0.5 moths in block 1 5. Field B ioassays U sing H. licarsisalis P heromone B lends None of the H. licarsisalis p heromone b lends were attractive for capturing H. phaeopteralis adult males ( F 4 , 20 = 0.86 , P = 0.57) (Table 5 2 ) . Here we only presented the number of adult H. phaeopteralis collected after one week using major sex pheromone candidates of H. licarsisalis including Z11 16:Ac , Z11,E13 16:Ac and Z11,E13 16:OH and unbaited and positive control ( caged virgin female ) . EAG Recording and GC EAD Detection The electrophysiological act ivity of male antennae was significantly affected by different treatments (Figure 5 3 ). The highest response (mv) was observed by Z11 , E13 16 : AC (199 mv) compare d with other tested diene treatments in all experimental males (F 4,20 =20.8, P<.0001) (Table 5 3 , Figure 5 3 , A ) . Z11 16:AC elicited the highest response (123.6 mv) from all of tested Z and E monoene isomers (F 9 ,40 = 23.1, P<.0001) (Figure 5 3 , B and C ). None of tested alcohol compounds elicited significant antennal response compared with the solvent c ontrol (F 4,20 = 1.47, P = 0.25) (Figure 5 3, D ) . Z11,E13 16:Ac were compared to Z11 16:Ac and Z11,E13 16:Ac response was significantly higher (249.6 ± 20.3) compared with Z11 16:Ac (118.4 ± 5.8) (F 2,12 =62.4, P<.0001) (Figure 5 3, E ). In GC EAD assays, male H. phaeopteralis antennae responded repeatedly to Z11,E13 16:Ac in pheromone gland extracts (Figure 5 4 ). Discussion None of the tested floral lures were attractive for capturing H. phaeopteralis adults. However, we noticed a large number of adult bibionid fl ies , Plecia nearctica (Hardy) . Because the tested floral lures did not significantly affect traps catch , it is

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97 assumed that H. phaeopteralis adults were not able to perceive acetic acid, 3 methyl 1 butanol (isoamyl alcohol) and phenylacetaldehyde at the rate tested (0.5 ml of each chemical per stopper) . M ain sex pheromone candidate compounds of H. phaeopteralis were very similar to the chemical structure of the sex pheromone of H. licarsisalis . Both species produced 14 and 16 carbon acetate compounds with single or double bonds ( Gibb et al., 2007) . However , in the field bioassays, none of the H. licarsisalis pheromone blends were attractive for capturing H. phaeopteralis . After further experiments using gas chromatography (GC) it was clear that the can didate pheromone compounds of H. phaeopteralis were slightly different from H. licarsisalis with regard to the geometry and the position of the double bonds and that could explain why the H. licarsisalis pheromone did not capture H. phaeopteralis males. Tw o pheromone candidate s, Z11,E13 16:Ac and Z11 16:Ac , were identified in pheromone gland extract of H. phaeopteralis female moth by using GC EAD. Both Z11,E13 16:Ac and Z11 16:Ac elicit ed a strong antennal response using the EAG technique in the laboratory. The average a ntennal response for Z11,E13 16:Ac was highest and was 1.7 times greater than Z11 16:Ac . Thus, I hypothesized that Z11,E13 16:Ac and Z11 16:Ac are major and minor compounds of sex pheromone s of this species, respectively. However, the structure of an unsaturated hydrocarbon compound in the sex pheromone gland remains unknown , which likely play s a role in sex pheromone blend . A series of field experiments were conducted at the Everglades REC during 2011 2013 using Z11, E13 16: Ac as a s ingle component or in combination with Z11 16:

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98 Ac to attract male moths. The treatments represented different combinations of different isomers and ratio of these compounds . However, H. phaeopteralis moths were not attracted to lures loaded with Z11, E13 1 6:Ac and Z11 16:Ac and their combinations at different ratio s under field conditions. T his information is not reported since the exact treatments were not disclosed by our collaborator due to potential patent violations . It should also be noted that the l ures loaded with synthetic Z11, E13 16:Ac and Z11 16:Ac were not purified and may have included alcohol contaminants which prevented them from working as anticipated . Identifying pheromones, especially sex pheromones, is a challenging prospect. The major pheromone components used by male H. phaeopteralis to recognize female has been isolated and partially characterized in this study. It is proven that m oths use a variety of communication cues in the moments before mating ( Simmons and Conner 1996) . In some moths the courtship is simple and apparently involves only the release of a female sex pheromone and the attraction of a male. In other moths such as Utetheisa ornatrix (L.) (Lepidoptera: Arctiidae), a chemical conversation takes place, with the female ini tiating the conversation and the male replying with a courtship pheromone released from scent disseminating structures called coremata (Conner et al., 1981). In other moth s such as Cycnia tenera ( Hübner ) (Lepidoptera: Arctiidae) , the chemical cues have bee n joined by high frequency acoustic cues ( Conner 1987) . In Syntomeida epilais ( Walker ) (Lepidoptera: Arctiidae) sound production by both sexes is required for successful mating, and localization of the female by the male is mediated solely by acoustic cues (Sanderford and Conner,1990, 1995). T ympanic and associated chordotonal organs are present in H. phaeopteralis which suggest ultrasonic courtship

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99 behavior (Figure 5 4) . However, I have not confirmed the release of acoustic signals from this species, as ha s been noted among other Crambidae (Nakano et al., 2008; Takanashi et al., 2010). Presence of female chordotonal organs suggest that H. phaeopteralis may require acoustic cues for successful mating behavior and might also partially explain why Z11, E13 16 :Ac and Z11 16:Ac were not effective for capturing H. phaeopteralis in my field tests . Future studies should focus on c onfirmation of the structure of all compounds and synthesis of all pheromone compounds with a high degree of purity. Additionally, a ser ies of field experiments with different ratios and combinations of the identified compounds are essential to fully identify the sex pheromone blend . Future studies should also include investigation s of the acoustic communicative signals in the courtship be havior of H. phaeopteralis .

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100 T able 5 1 . A dult H. phaeopteralis (mean ± SE ) collected after one week on green delta traps baited with tested floral lures . Lure 1 Apopka Belle Glade AA + 3MeB 0 ± 0 a 0 ± 0a PAA 0 ± 0 a 0 ± 0 a myrcene 0 ± 0 a 1.2 ± 0.5 a PAA + methyl salicylate 0 ± 0 a 0.2 ± 0.2 a Unbaited 0 ± 0 a 0.0 ± 0 a 1 Abbreviations are as follows: AA = acetic acid, 3MeB = 3 methyl 1 butanol (isoamyl

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101 Table 5 2 . Adult H. phaeopteralis (mean ± SE ) collected after one week on green deltatraps . Treatment # H. phaeopteralis Z11 16:Ac 0.2 ± 0. 2 b Z11,E13 16:Ac 0.2 ± 0. 2 b Z11,E13 16:OH 0.2 ± 0. 2 b U nbaited 0.2 ± 0. 2 b C aged virgin female 20.2 ± 7.0 a tests) .

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102 Table 5 3 . Electroantennogram responses of male H. phaeopteralis moths to its sex pheromone candidate s . Treatment Electroantennogram response (± SE) (mV) Treatment Electroantennogram response (± SE) (mV) Diene Compounds Alcohol compounds Z9,Z11 16:Ac 85.4 ± 6.5 E9 16:OH 42.8 ± 4.9 Z,E 9,11 16:Ac 52.6 ± 3.6 E10 16:OH 50.8 ± 6.4 E9,E11 16:Ac 91.8 ± 6.3 E11 16:OH 49.2 ± 7.1 Z11,E13 16:Ac 199 ± 6.2 Z11 16:OH 55.6 ± 6.3 E11,Z13 16Ac 75 ± 8.4 (E) isomers of monoene Compounds (Z) isomers of monoene Compounds E9 16:Ac 44.2 ± 4.7 Z9 16:Ac 60.4 ± 7.1 E10 16:Ac 58.4 ± 3.3 Z10 16:Ac 51 ± 4.5 E11 16:Ac 89.6 ± 5.3 Z11 16:Ac 123.6 ± 12.1 E12 16 :Ac 60.2 ± 4.8 Z12 16:Ac 54± 6.3 E13 16:Ac 3.6 ± 8.1 Z13 16:Ac 52 ± 5.1 Ac; acetate, OH; alcohol.

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103 Figure 5 1 . A ) Deployment of delta shaped sticky traps baited with phenylacetaldehyde (PAA). B ) Captures of low number of H. phaeopteralis adults (note two TSW moths on top and bottom) but higher number of lovebugs, P. nearctica .

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104 Figure 5 2 . A) Caged H. phaeopteralis virgin female in top middle of trap . B) Captures of high number of H. phaeopteralis males. A B

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105 Figure 5 3 . Response of H. phaeopteralis one day old male antennae (mv) to different regioisomers of sex pheromone candidates of H. phaeopteralis . A B C D E F

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106 Figure 5 4 . Coupled gas chromatogram electroantennograms of H. phaeopteralis male antennae responding to Z11 , E13 16 : AC in pheromone gland extracts.

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107 Figure 5 5 . H. phaeopteralis female chordotonal organ .

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108 CHAPTER 6 SUMMARY AND CONCLUSIONS Tropical sod webworm, Herpetogramma phaeopteralis Guenée, (Lepidoptera: Crambidae) is one of the most serious insect of warm season grasses in the Gulf Coast of the United States , the Caribbean I slands and C entral America . This insect cause s economic dam age to turfgrass grown in athletic fields, golf courses, home lawns and recreational parks . Adult moths do not cause any type of damage to turfgrass but their larval stages feed on turfgrass and reduce turfgrass aesthetic value. According to one major sod farmer in central Florida, this species is the number one pest he has experienced in recent years . This dissertation raises awareness about this important species and prospects for improved integrated management. Achievements from my research include collating the existing fragmented literature on this pest regarding its taxonomy, description of stages, biology, host plants, potential for economic damage, scouting procedures, management and control options ( C hapter 1) . Monitoring is one of the most fundamental yet the most often neglected activity in an IPM program. Predicting when and where damaging pest infestations occur is among the greatest hurdles to reduc e insecticide usage on turf (Held and Potter 2012) . Thu s, the development al biology of this s pecies was described mathematically to develop degree day model ( C hapter 2) . S uch an approach is needed to better develop predictive models for the distribution and seasonal biology of this species throughout its geographical range and help turf managers to make informed decisions for control of H. phaeopteralis . Trap development can be used for monitoring population, mating distribution and trap out strategies. I investigated attraction of H. phaeopteralis to flor al

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109 lures and attempt ed to identify sex pheromone of H. phaeopteralis ( C hapter 5). Further work in this area is needed. Insect control on lawns, golf courses, and sport fields are insecticide driven and rely on preventive and/or curative insecticidal contr ol (Held and Potter 2012). Although resistance has not yet been documented for this species, it is likely to occur. To plan and implement insecticide resistant management strategies, resistance baseline for H. phaeopteralis to insecticides from six classes were established in C hapter 3. Results from field experiments indicated chlorantraniliprole provided longer residual control compare to clothianidin and bifenthrin. Chlorantraniliprole application could be rotated with older products such as clothianidin and bifenthrin to facilitate resistance management. Another approach is to combine two active ingredients for a broader spectrum of preventive control (Held and Potter 2012). Such products may work faster or slower depend on the interaction ( additive, syne rgist or antagonist between chemistries) if any. Further studies are necessary to study effect s of combined active ingredients on beneficial species on the risk of hastening insecticide resistance. Further I investigated the potential for insecticide alter natives. One such alternative is the use of microbial insecticides including entomopathogenic nematode as biological insecticides. I screened commercially available entomopathogenic nematode products against H. phaeopteralis and found that all larval stages of H. phaeopteralis were susceptible to beneficial entomopathogenic nematodes. Steinernema carpocapsae (Millenium® or Carpocapsae System) provided a promising alternative control option for H. phaeopteralis biocontrol (C hapter 4) . Entomopathogenic n ematodes may serve as a safe alternative to pesticides to control H. phaeopteralis but further field studies are

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110 warranted to confirm effectiveness under different environmental scenarios. While EPN might be effective for control of caterpillars , in some s ituations may not provide enough control . In previous studies the insecticide imidacloprid and the entomopathogenic nematode Heterorhabditis bacteriophora Poinar interacted synergistically against third instars of the masked chafers Cyclocephala hirta LeCo nte and C. pasadenae Casey (Coleoptera:Scarabaeidae) ( Koppenhöfer and Kaya 1998 ). Thus, Synergistic combinations of nematodes and neonicotinoid insecticides could be used as strategy that permits cost effective use of nematodes. There is no single solution to manage this pest ; however , application of best management practices in IPM programs should minimize producer costs and insecticide inputs in residential settings. In Florida, current control methods are primarily limited to applications of conventional insecticides. Future research is necessary to establish reliable economic thresholds under different scenarios and evaluate the impact of alternative controls including mechanical, cultural and biological strategies for management of this important pest. Herpetogramma phaeopteralis does not apparently survive the winter in north and C entral Florida . Some L epidopteran species such as fall armyworm (Lepidoptera: Noctuidae) use annual long distance migration as an adaptation method to extend their geograph ical range into areas that cannot support permanent population ( Nagoshi et al., 2012). I speculate that seasonal migration of this species may occur from south into central and central into north Florida during the late spring and early summer. The elucida tion of seasonal migration of this species is also needed.

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111 LIST OF REFERENCES Aghdam, H. R., Y. Fathipour, G. Radjabi, and M. Rezapanah. 2009. Temperature dependent development and temperature thresholds of codling moth (Lepidoptera: Tortricidae) in Ira n. Environ. Entomol. 38: 885 895. 662. Anonymous. 2008. Tropical Sod Webworm. Syngenta Turfgrass Encyclopedia. http://www3.hcs.ohio state.edu/turfwiki/index.php/Main_Page (5 June 2014). Ansari , M. A., L. Waeyenberge, and M. Moens. 2007. Natural occurrence of Steinernema carpocapsae , Weis er, 1955 (Rhabditida: Steinernematidae) in Belgian turf and its virulence to Spodoptera exigua (Lepidoptera: Noctuidae). Russ. J. Nematol. 15: 21 24. Avilla, J., D. Casado, N. Varela, D. Bosch, and M. Riba. 2003.Electrophysiological response of codling moth ( Cydia pomonella ) adults to semiochemicals. IOBC Bull. 26: 1 7. Beard, J. B. 1973. Turfgrass: Science and culture. Prentice Hall Upper Saddle River, NJ. Bedding, R., and A. Molyneux. 1982. Penetration of insect cuticle by infective juveniles of Hetero rhabditis spp. (Heterorhabditidae: Nematoda). Nematologica. 28: 354 359. Behe, B., J. Hardy, S. Barton, J. Brooker, T. Fernandez, C. Hall, J. Hicks, R. Hinson, P. Knight, and R. McNiel. 2005. Landscape plant material, size, and design sophistication increa se perceived home value. J. Environ. Hort. 23: 127 133. Bellini, L. L., and C. Dolinski. 2012. Foliar application of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) for the control of Diatraea saccharalis in greenhouse. Ciê nc. Agr. 33: 997 1003. Bergant, K., and S. Trdan. 2006. How reliable are thermal constants for insect development when estimated from laboratory experiment?. Entomol. Exp. Appl. 120: 251 256. Bixby Brosi, A.J., and D. A. Potter. 2012. Beneficial and innoc uous invertebrates in turf. In: Brandenburg RL, Prater CA (eds) Handbook of turfgrass insect pests. Entomological Society of America, Lanham, pp 87 93. Black , C. A. 1965. Methods of Soil Analysis: Part I Physical and mineralogical properties. American Soci ety of Agronomy, Madison, Wisconsin, USA.

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122 BIOGRAPHICAL SKETCH Nastaran Tofangsazi was born in Isfahan, Iran. She completed her undergraduate at Department of Plant Protection, College of Agriculture , Isfahan University of Technology (IUT) , one of the most prest igious universities in Iran . She earned her M. S. degree from Iran's oldest modern university , Department of Entomology and Plant Pathology, College of Agriculture , University of Tehran (UT), Tehran . She joined Dr. Arthurs research program in the fall of 2010 to study toward a doctorate degree in the Department of Entomology and Nematology . Her area of interest enc ompasses insect pest management, biological control, insect behavioral ecology, and e valuation of insect pathogens as microbial pesticides . H er research was focused on IPM program for tropical sod webworm . This dissertation is the culmination of the studies she conducted during 2011 201 4 as part of her Ph.D. degree . Funding College of Agricultural and Life Sciences . She presented more than fourteen oral and poster presentations and she earned several award s during he r Ph.D. program.