Distribution and Mechanisms of Insecticide Resistance and Isolation and Evaluation of Beauveria Bassiana in Haematobia I...

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Distribution and Mechanisms of Insecticide Resistance and Isolation and Evaluation of Beauveria Bassiana in Haematobia Irritans (L.)
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1 online resource (96 p.)
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english
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Holderman, Christopher J
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University of Florida
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Kaufman, Phillip Edward
Committee Members:
Geden, Chris J
Bloomquist, Jeffrey R

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Subjects / Keywords:
biological -- control -- fly -- horn -- insecticide -- resistance
Entomology and Nematology -- Dissertations, Academic -- UF
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Entomology and Nematology thesis, M.S.
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
Horn flies are a major pest of cattle in most of North America.  Insecticides are a primary method of control,with few effective alternatives. Beginning in Fall 2010 a study was initiated to evaluate the mechanisms of insecticide resistance in field-collected horn flies and to screen field collections for entomopathogenic fungi.  Horn flies were collected from three field sites; Labelle, Ona, and the Beef Teaching Unit, and were screened for insecticide resistance using contact bioassays.  Four assays were then used to characterize resistance mechanisms; polymerase chain reactions for genetic point mutations, acetylcholinesterase inhibition by diazoxon, cytochrome P450 and general esterase quantification assays.  Resistance to multiple insecticide classes was present in the Labelle population, which generated resistance of 66-fold and 15-fold for permethrin and diazinon, respectively.  Three strains of Beauveria bassiana,including one field isolate and two reference strains, were evaluated using three different application techniques.  Virulence varied with application technique, with the field isolate out-performing the reference strains in cornstarch formulations and the reference strains yielding superiority in filter paper assays.  Mortality did not reach over 41% for the most pathogenic strain (Florida isolate EN1) in the cornstarch formulation.  Multiple insecticide class resistance is of major concern due to the limited number of available chemistries available for fly control.  The future of horn fly control must involve additional insecticide chemistries and/ or alternative biological control methods if producers expect to manage this pest effectively.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Christopher J Holderman.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Kaufman, Phillip Edward.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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lcc - LD1780 2012
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UFE0044740:00001


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1 DISTRIBUTION AND MECHANISMS OF INSECTICIDE RESISTANCE AND ISOLATION AND EVALUATION OF BEAUVERIA BASSIANA IN HAEMATOBIA IRRITANS (L.) By CHRISTOPHER JOSEPH HOLDERMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Christopher Joseph Holderman

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3 To my family and friends w ithout their constant support I would not be who I am toda y

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4 ACKNOWLEDGMENTS I wish to thank the Entomology and Nematology Department for the teaching assistantship that I was awarded under the supportive guidance of Dr. Rebecca Baldwin I would like to thank Dr. Phillip Kaufman for funding my research and the Kaufman Lab for all of your support in my research; Mark Halverson, Lois Wood, Timothy Davis, Amanda Eiden, Cori e Singer Lucas Carnohan, and Dr. Emma Weeks. Additionally, I would like to thank Dr. Jeff r e y Bloomquist for allowing me to conduct the ma jority of my toxicological experiments in his laboratory; and the assistance of Fan Tong, Lacey Jenson, and Daniel Swale. Appreciation is also extended to Dr. Christopher Geden for helping me make sense of my fungal trials and the statistics that accompan y them. I wish to thank Dr. Jimmy Pitzer for fly shipments and ceaseless advice and Dr. Jennifer Gillett Kaufman for never ending edits and corrections to my manuscripts. Additionally, thanks to Dr. Jim A rends for providing a pure culture of fungus to be gin my work and Jesse Savell for working with me on collections at the UF Beef Teaching Unit I would also like to thank t he faculty and staff of the Entomology and Nematology D epartment for all of their help through my degree. Additionally I would like to thank the county agents and producers who allowed me the privilege of working with them for field collections.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 L IST OF TABLES ................................ ................................ ................................ ............. 7 LIST OF FIGURES ................................ ................................ ................................ ........... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 LITERATURE R EVIEW OF THE HORN FLY (Haematobia irritans L.) ....................... 11 Introduction and Economic Impact ................................ ................................ .............. 11 Distribution and Biology ................................ ................................ ............................ 13 Management Methods ................................ ................................ ................................ 14 Entomopathogenic Fungi ................................ ................................ ..................... 15 Horn Fly Genetic Manipulations and Determinations ................................ .............. 17 Host Vaccination ................................ ................................ ................................ 18 Horn Fly Predators and Parasitoids ................................ ................................ ....... 19 Other Non chemical Methods ................................ ................................ ............... 20 Inorganic and Natural Source Insecticides and Repellents ................................ ....... 20 Synthetic Insecticides ................................ ................................ .......................... 21 Insecticide Resistance Development and Evaluations ................................ .................... 23 Types of Resistance Mechanisms ................................ ................................ .......... 23 Emergence and Evaluation ................................ ................................ ................... 24 Pyrethroid resistance ................................ ................................ ..................... 25 Organophosphate resistance ................................ ................................ ........... 29 Research Objectives ................................ ................................ ................................ .. 31 2 TOXICOLOGICAL AND BIOCHEMICAL EVALUATIONS OF FLORIDA HORN FLY INSECTICIDE RESISTANCE ................................ ................................ ........... 32 Introduction ................................ ................................ ................................ .............. 32 Materials and Methods ................................ ................................ ............................... 33 Fly Rearing ................................ ................................ ................................ ........ 33 Contact Assay ................................ ................................ ................................ .... 34 Molecular and Enzymatic Assays ................................ ................................ ......... 36 Polymerase chain reaction ................................ ................................ ............. 36 Acetylcholinesterase inhibition assay ................................ .............................. 38 Cytochrome P450 monooxygenase assay. ................................ ....................... 39 Carboxylesterase quantification ................................ ................................ ..... 40 Statistics ................................ ................................ ................................ ................... 41 Results ................................ ................................ ................................ ..................... 43 Discussion ................................ ................................ ................................ ................ 45

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6 3 LABORATORY EVALUATION OF BEAUVERIA BASSIANA ISOLATE PATHOGENICITY AGAINST ADULT HORN FLIES ................................ ................ 58 Introduction ................................ ................................ ................................ .............. 58 Materials and Methods ................................ ................................ ............................... 60 Isolation and Viability ................................ ................................ ......................... 60 Fungal Strains and Colony Maintenance ................................ ................................ 61 Laboratory Bioassay ................................ ................................ ............................ 62 Conidia in Surfactant Solutions ................................ ................................ ............ 63 Conidia Impregnated on Fil ter Paper Contact Assay ................................ ............... 64 Conidia in Inert Carrier Formulations ................................ ................................ .... 64 Statistics ................................ ................................ ................................ ................... 65 Results ................................ ................................ ................................ ..................... 66 Discussion ................................ ................................ ................................ ................ 68 4 IMPLICATIONS AND FUTURE DIRECTIONS FOR HORN FLY RESEARCH ........... 81 APPENDIX A HORN FLY COLLECTIONS: NUMBERS, DATES, HERD SIZES AND TIMES .......... 85 LIST OF REFERENCES ................................ ................................ ................................ .. 86 B IOGRAPHICAL SKETCH ................................ ................................ ............................. 96

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7 LIST OF TABLES Table page 2 1 Sequences of polymerase chain reaction ( PCR ) primers used to identify kdr and G262A genotypes fro m field collected horn fly populations. ................................ ..... 52 2 2 cyfluthrin and diazinon on glass to horn fly adults collected from Florida beef cattle ranches. ................................ ............................... 53 2 3 Percentage genotype composition of laboratory strain and Florida field collected horn fly populations as expressed through a multiplex PCR. ................................ ...... 54 2 4 Mean 50% inhibition concentration (IC 50 ) of diazoxon in nM to horn fly head homogenates. ................................ ................................ ................................ ........ 55 2 5 Relative enzymatic activity (SEM) per gram of horn fly abdomen protein. .................. 56 3 1 Mortality of adult horn flies exposed by immersion into a Beauveria bassiana conidia dilution containing a 0.1% Tween 80 and water solution. ............................... 74 3 2 Mortality (corrected for controls) of adult horn flies exposed to Beauveria bassiana conidia dispersed into a 0.1% Tween 80 and water solution impregnated onto filter paper. ................................ ................................ ................................ ................... 75 3 3 Mean (SEM) m ortality of adult Haematobia irritans following exposure to Beauveria bassiana conidia impregnated filter paper discs. ................................ ....... 76 3 4 F values for mean percent mortality of adult Haematobia irritans ex posed to Beauveria bassiana conidia impregnated on filter paper in a 0.1% Tween 80 and water solution. ................................ ................................ ................................ ...... 77 3 5 Mortality (corrected for controls) of adult horn flies exposed to Beauveria bassian a conidia dispersed into a cornstarch formulation spread onto filter paper. ..................... 78 3 6 Mean (SEM) mortality of adult Haematobia irritans following exposure to Beauveria bassiana conidia in starch f ormulation. ................................ .................... 79 A 1 Horn Fly collections: numbers, dates, herd sizes and times. ................................ ....... 85

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8 LIST OF FIGURES Figure page 2 1 Typical gel photo showing polymerase chain reaction ( PCR ) amplification products. ... 57

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Parti al Fulfillment of the Requirements for the Degree of Master of Science DISTRIBUTION AND MECHANISMS OF INSECTICIDE RESISTANCE AND ISOLATION AND EVALUATION OF BEAUVERIA BASSIANA IN HAEMATOBIA IRRITANS (L.) By Christopher Joseph Holderman August 2012 Chai r: Phi l lip E. Kaufman Major: Entomology and Nematology Horn f l i es are a major pest of c attle in most of North America. Insecticides are a primary method of control, with few effective alternatives. Beginning in Fall 2010 a study was initiated to evaluat e the mechanisms of insecticide resistance in field collected horn flies and to screen field collectio ns for entomopathogenic fungi. Horn flies were collected from three field sites; Labelle, Ona, and the B eef T eaching U nit and were screened for insecti cide resistance using contact bioassays F our assays were then used to characterize resistance mechanisms; polymerase chain reactions for genetic point mutations, acetylcholinesterase inhibition by diazoxon, c ytochrome P450 and general esterase quantifica tion assays. Resistance to multiple insecticid e classes was present in the Labelle population which generated resistance of 66 fold and 15 fold for permethrin and di azinon respectively Three strains of Beauveria bassiana including one field isolate and two reference strains, were evaluated using three different application techniques. Virulence varied with application technique with the field isolate out per forming the reference strains in c orn starch formulations and the reference strains yielding superior ity in filter paper assays M ortality did not reach over 41% for the most pathogenic strain (Florida isolate EN1) in the c orn starch formulation

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10 Multiple insecticide class resistance is of major concern due to the limited number of available che mistries available for fly control. The future of horn fly control must involve additional insecticide chemistries and/ or alternative biological control methods if producers expect to manage this pest effectively

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11 CHAPTER 1 LITERATURE REVIEW OF THE HO RN FLY (HAEMATOBIA I RRITANS L.) Introduction and Economic Impact The horn fly, Haematobia irritans (Linnaeus), is a major pest of both beef and dairy cattle. The economic impact of this obligate, blood feeding fly has been evaluated numerous times. A stu dy comparing untreated controls to treatments with DDT estimated that the loss for Kansas in 1945 was 10 million dollars (Laake 1946). Laake (1946) also reported that feeding animals for harvest from June to September was impossible in some areas due to t he loss caused by heavy infestations of horn flies. In more contemporary studies, national loss estimates are extremely high, over 730 million dollars (reported in Byford et al. 1992 from Drummond et al. 1981), and 876 million dollars (Kunz et al. 1991) i n the United States, and approaching one billion dollars per year in North America (Cupp et al. 2004). The United States was reported to have over 30 million beef cattle inventoried in 2007 with a cash value of over 24 billion dollars (USDA NASS 2009a). In the 2007 Census of Agriculture, the state of Florida ranked 11 th nationally in beef cow inventory, with over 900,000 beef cattle (USDA NASS 2009b). Management of cattle and their pests is critical to Florida's and the United State's beef industry inclu ding the numerous individuals and families that are supported economically by the sale, distribution, and market end point of beef products. A ttempts to determine the impact of horn flies on cattle varied ; Schwinghammer et al. (1986a) suggested that this i nconsistency was due to the variability of the factors used (breed, age, size, environmental conditions, management practices, etc). Reports as early as Freeborn (1925) documented reduced milk production among dairy cattle exposed to horn flies. Beef cat tle weight has been shown to be affected by the irritation and biting of H. irritans with a gain, due to removal of the flies by insecticide treatment, of one half to two thirds of a pound per

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12 animal per day (Cheng 1958). Campbell (1976) investigated the effects of horn flies on weaning weights and showed a $4.75 return on every dollar in vested in insecticide treatment of calves. Quisenberry and Strohbehn (1984) demonstrated a significant positive increase in weaning weight with horn fly control, as well as a lack of compensatory gain after the end of the fly season in untreated control animals. Additionally, Kunz et al. (1984) found that although the benefit to cost ratio was not always statistically significant, the return on investment was at least do ubled for ev ery dollar spent on insecticide impregnated ear tags, when compared to untreated controls. Alternatively, Hogsette et al. (1991) showed that cattle (Angus or Brangus yearling heifers and cow calf pairs) could tolerate upwards of 200 horn flies per animal for 70 d without a significant change in condition scores. The majority of research has shown that horn flies have an impact on beef cattle, however this impact has been variable, difficult to measure consistently and may be reflected in the variability of test subjects (breed and age of cattle) and experimental setup (environmental conditions, horn fly levels, management practices etc.). Laboratory infestations of horn flies on beef steers (Angus x Hereford yearlings) by Schwinghammer et al. (1986a), showed higher blood cortisol levels, increased body temperature, heart rate, respiration rate, and increased activity; resulting in reduced nitrogen retention being significantly different between the high infestation level (n=500 horn flies per head) and the lower infestation level (n=100) or controls. Similarly, Schwinghammer et al. (1986b) found a significant increase in temperature, urine production, and nitrogen excretion with exposure to stable flies, Stomoxys calcitrans (L.). In a combine d infestation of both horn and stable flies Schwinghammer et al. (1987) determined a decrease in nitrogen retention for fly exposure concentrations (100 horn flies plus 25 stable flies, or 500 horn flies plus 50 stable flies per head) and an increase in he art rate, respiration, and temperature. Schwinghammer et al. (198 6 a,b)

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13 depicted physiological responses within normal ranges, however, many of these studies were completed in confined locations (stalls) or in paddocks where flies could move between treatm ent groups. This may have reduced the impact of the infestations and prevented the typical Distribution and Biology Haematobia irritans is native to Europe, Northern Africa, and Western Asia. The horn fly was introduced from Europe to North America with cattle in 1885 1886, possibly from France into Philadelphia (summarized in Butler and Okine 1999). The horn fly soon spread across the continent reaching Florida by 1891, Michigan in 1892 and Californi a by 1893 with introduction extends from southern Canada to Chile and Argentina (Barros et al. 2001, Cupp et al. 2004). In their native range, horn flies usually do n ot develop in great numbers, but in most introduced areas fly numbers can reach extreme levels of 10,000 20,000 flies on an individual animal (Bruce 1964). Adult flies take blood meals intermittently, up to 20 times each day (Bruce 1964, Harris and Miller 1969), and have been reported to live up to 8 weeks (Bruce 1964). The female mates once to several times while on the host and takes as few as two pre ovipositional days before producing viable eggs (Harris et al. 1968). Adult flies have been reported t o oviposit 3.5 d post ecosion and live for 6.6 d (Krafsur and Ernst 1983). Eggs are laid exclusively in fresh cattle manure. Individual female flies have been reported to produce 24 eggs per cycle, with 15 cycles during a lifetime (Bruce 1964), alternati vely, Krafsur and Ernst (1983) reported that females produced only 78 eggs per fly. No natural development of horn flies has been shown in manure other than what is typically produced by grass fed cattle (Greer and Butler 1973). Differences i n horn fly at traction to cattle has been observed with some breeds and colors of beef type animals. According to Bruce (1964), horn flies exhibit color preferences at low

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14 temperatures toward black and dark animals, but show no preference in hot weather. Schreiber and Campbell (1986) demonstrated a preference for black over red hair coat in white faced cattle. However, Tugwell et al. (1969) demonstrated that color is of little importance, but found that as Brahman breed influence increased, horn fly attractiveness dec reased, although other factors were involved. Testosterone injections in steers by Dobson et al. (1970) showed an attractiveness of horn flies for steers treated with their low dose, but a repulsion for those treated with their high dose; concluding that bulls are likely more attractive due to the secondary effects of testosterone. Horn flies have been shown to disperse over long distances, arriving at new herds 1.7 km from source herds wi thin six days of placement populations of cattle, or physical borders such as trees at the edges of pastures (Byford et al 1987). Gui llot et al. (1988) evaluated horn fly dispersal, with an emphasis on age grading, and found that males and females are equally likely to migrate, h owever, the majority of dispersing females had mated, but were pre vitellogenic. Many biological factors have been evaluated for control purposes yet most have not been developed into management methods. Management Methods Numerous techniques have been pr oposed for management of horn fly populations including: entomopathogenic fungi, horn fly genetic manipulations and determinations, host vaccination, predators and parasitoids, other non chemical control methods, inorganic and natural source insecticides a nd repellants, and synthetic insecticides. Most attempts at manage ment are effective to some degree but most do not achieve desired levels of control, or are in need of additional research. Horn fly control is attempted in most areas of the Americas. C urrent strategies still rely heavily on the use of pyrethroids and organophosphates against adult flies, typically as

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15 insecticide impregnated ear tags, pour on, or spray formulations. Avermectins are used primarily for their effect on internal cattle para sites, but due to their insecticidal activity can and have been used for horn fly control. IGR s in feed through minerals and salt supplements are not frequently utilized, but are available. Very little research has been conducted on biological controls, and no species are recommended for horn fly control in a pasture setting. Entomopathogenic Fungi A control method that previously has been evaluated includes the use of entomopathogenic fungi. Of the fungi that infect arthropod populations 700 species are known, yet only 10 are currently used or under evaluation for control purposes (Hajek and Leger 1994). Steenberg et al. (2001), in a survey of fungi that infected flies in agricultural settings, reported the following species of fungi isolated from h orn flies from Denmark: Beauveria bassiana (Balsamo) Vuillemin, Verticillium lecanii (Balsamo) Vuillemin, V. fusisporum W. Gams, and Furia americana (Thaxter) Humber. All fungi located in the survey were recovered at a very low prevalence. The use of pat hogenic fungi in veterinary entomology has been limited. Within the muscoid flies, H. irritans is an understudied insect with respect to the number of evaluated fungal species currently reported This list includes only four fungal species: Metarhizium a nisopliae (Metschnikoff) Sorokin B. bassiana Isaria fumosorosea (Wize) (Brown and Smith) (formerly Paecilomyces fumosoroseus) and Isaria farinosa (Holmskjold) Fries (formerly Paecilomyces farinosus) (Angel Sahagun et al. 2005, Lohmeyer and Miller 2006, Mochi et al. 2010a Zimmermann 2008 ). Among muscoid flies in general, Steinkraus et al. (1990) was the first to show the presence of B. bassiana infections in wild house flies ( Musca domestica L.) from New York dairies. Skovgard and Steenberg (2001) have shown the presence of B. bassiana in house flies, along with several other fungi. Watson et al. (1990) evaluated the efficacy of B.

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16 bassiana in house fly control, developing the concept of what is currently used in a commercially available product, balEn Few studies have evaluated the efficacy of entomopathogenic fungi against horn flies. Angel Sahagun et al. (2005) evaluated the efficacy of several isolates of M. anisopliae, B. bassiana a nd I. fumosoroseus to horn fly eggs, larvae, pupa e and adult stages and the following is from their paper. Horn fly eggs were placed onto manure and a conidial suspension was applied; the treated eggs were found to have reduced adult emergen ce with some fungal strains yielding total mortality. Pupae were treated in a continuous exposure assay on filter paper, with some strains yielding over 50% mortality of adult horn flies. Adult horn flies were inoculated in a continuous exposure assay with a liquid s uspension of conidia which generated high mortality in some strains. Angel Sahagun et al. (2005) showed through their proof of concept study that horn flies can have fungal induced mortality, however, strain specificity was highly variable. Additionally, the methods of Angel Sahagun et al. (2005) utilized continuous exposure assays to evaluate adult control and the authors did not suggest a means of distributing the conidia to the dung pats in a pasture setting. Lohmeyer and Miller (2006) evaluated powder formulations of M. anisopliae (strain ESCI, Bioblast, EcoSc ience Corp., East Brunswick, NJ ) B. bassiana (strain GHA, Botanigard 22 WP, Emerald BioAgriculture, Lan sing, MI ), and I. fumosoroseus (strain ARSEF 3581, formulated in house with diatomaceous ear th) in an on host mimic, two hour contact exposure to adult horn flies using acrylic faux fur as the contact substrate. These authors generated a lethal time for 50% mortality, LT 50 of 2.7, 4.98, 7.97 and 9.42 days for B. bassiana M. anisopliae I. fumo soroseus and untreated controls, respectively. The powder formulations utilized could be

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17 readily adapted to current technologies used to control horn flies, i.e. dust bags ( Lohmeyer and Miller 2006) Mochi et al. (2010a) evaluated conidial suspensions of M. anisopliae (strains E9, IBCB425, and IBCB159) B. bassiana (strains JAB06, JAB07, and AM09), I. fumosoroseus (strains IBCB133 and CB75), and I. farinous (strains CG189 and CG195) against wild collected horn fly adults and pupae reared from eggs from th e field collected horn flies. Mochi et al. (2010a) reported significant mortality for pupae treated with M. anisopliae B. bassiana and I. farinous but not I. fumosoroseus Treated adults with M. anisopliae and B. bassiana had significant mortality, alt hough I. fumosoroseus and I. farinous were not as effective in generating adult mortality. In general, Mochi et al. (2010a) reported pathogenicity for the adult stage was greater at higher conidial concentrations. Mochi et al. (2010b) evaluated mortality of horn fly eggs following inoculation with M. anisopliae They reported no effect on egg hatch; however, mortality in larvae from these hatched eggs was observed. Mochi et al. (2010b) found no activity of B. bassiana on eggs or larvae, whereas I. farino us was found to increase the mortality of larvae and pupae that hatched from treated eggs (Mochi et al. 2010b). The authors suggest incorporating the active isolates into manure as the next step in generating a viable control strategy for the immature sta ges of horn flies, but did not suggest a method for such dispersal. Horn Fly Genetic Manipulations a nd Determinations A thorough understanding of the genetics of horn flies, their natural variability, and biology could lead to improvements in control tac tics. Eschle et al. (1973) demonstrated that horn flies could be suppressed by release of sterile, irradiated, males into an isolated population. In this evaluation, low numbers of flies arrived from nearby herds to the treated cattle herd, which was pre sumed to have prevented eradication in west Texas (Eschle et al. 1973).

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18 Methoprene, an insect growth regulator (JH analog), was used in conjunction with irradiated pupa, to nearly eradicate horn flies in an area on one of the Hawaiian Islands (Eschle et a l. 1977). In the failure of the Hawaii field trial, fly infested cattle were moved into the treatment area and methoprene in the cattle water supply dropped below effective levels. Intensive fly control for a short period of time has been shown to eradic ate flies in confined areas, however, neither the cost of such measures nor the feasibility of such an operation on a large scale were discussed. Expressed sequence tags (ESTs) have been used to isolate metabolic, proteinacious, insecticide resistance and other functional coding groups in horn flies (Guerrero et al. 2004). Guerrero et al. (2008) assembled a database of approximately 10,000 cDNA clones to aid in discovery of coding for metabolic resistance, novel antigen targets, and develop new targets for pesticide development. Guerrero et al. (2009) developed several candidate genes for development of specific conditional lethality tied to both sex and life stage. Torres et al. (2011) also generated a cDNA library with over 2000 EST s and suggested candi date antigens for development into vaccines. These genetic approaches have yet to yield any applicable products for horn fly control. Host V accination The development of a vaccine for ectoparasites has been considered for many species including the catt le grubs, cattle fever ticks, and horn flies ( Willadsen et al. 1995, Colwell 2010, Cupp et al. 2004). Such vaccines incorporate either parasite component proteins or parasite produced substances, which are used by the cattle to elicit an immune response t hat prevents or reduces feeding of the parasite. Cupp et al. (2004) demonstrated, both in a laboratory host (rabbit) and in cattle, an immune response following vaccination to a horn fly produced salivary protein, thrombostasin, using a recombinant peptide The reaction caused reduced feeding and a reduction in egg production of

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19 horn flies. Cupp et al. (2010) showed a similar result, however, the specific allele for the protein had multiple isoforms in the horn fly, thereby requiring that multiple isoform s of the protein be included for a vaccine to be effective in a field setting. Horn F ly P redators and P arasitoids Many types of arthropods are known to attack immature horn flies. These consist of generalist egg and larvae predators as well as pupal par asitoids. Geden et al. (2006) reported that several parasitoid wasps could parasitize horn flies in a laboratory setting, but rates were low compared to other hosts in some cases. These parasitoids included: Muscidufurax raptor Girualt and Sanders, Spala ngia cameroni Perkins, S. endius (Walker), S. nigroaenea Curtis, S. gemina Boucek (Hymenoptera: Pteromalidae), and Dirhinus himalayanus Masi (Hymenoptera: Chalcididae). In an ecological approach to evaluating immature horn fly predation, Hu and Frank (1996 ) found that the arthropod community caused a significant reduction (71.3%) in horn flies seeded on artificial dung pats in north central Florida. Solenopsis invicta Buren, the red imported fire ant, was reported to raise mortality to at least 93.9% (Hu a nd Frank 1996). In a laboratory setting, Hu and Frank (1997) evaluated the feeding rates of several Philonthus species (Coleoptera: Staphylinidae), which are predacious as adults and larvae. Due to the developmental lifespan of the Philonthus being longe r from egg to first instar than horn flies, the authors conclude d that beetle larvae had the great est impact on the later arriving dipterans (Hu and Frank 1997). Additionally, the authors surmised that the adult beetles did not provide adequate control of horn fly populations, as they were currently dispersed across Florida, and in some cases Holarctic in distribution, but provided a lack of fly control across their distribution This was attributed to availability of alternative food sources or presence of low beetle

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20 populations that are not maintained at adequate levels for horn fly control purposes (Hu and Frank 1997). Other N on chemical M ethods Other non chemical control methods exist for horn fly control. One such option is the Bruce trap that dates back to pre WWII (Bruce 1938). The trap is of simple construction, and relies on the behavior expressed by horn flies wherein they leave a host when it enter s a darkened building (Hall 1996). The trap is constructed so that cattle must walk through on a frequent basis, and yields a reduction in fly numbers (Bruce 1938). One major issue is that cattle must pass through the trap, which can be difficult and costly to realize in large open pastures, or areas where mineral or water sources are not in corrals or paddocks. Inorganic and Natural Source Insecticides and Repellents Numerous products have been applied to cattle as repellents and control treatments; however, many were toxic to cattle, ineffective, or too costly for practical use (Byford et al 1998 ). Vegetable derived oils, kerosene, sulfur compounds, cabrolic acid, coal tar, and pine tar are just a few examples of the historically applied products (Cory 1917 Bruce 1964). The first successful topical agents applied to cattle were pyrethrum and to bacco powders (Smith 1889 from Schmidt and Morgan 1978). Pyrethrum sprays and forced dusts were frequently used until 1944 when DDT and residual animal treatments resulted in substantial and extended H. irritans control (Bruce 1964). N,N diethyl m toluam ide with methoxychlor, and pyrethrins mixed with piperonyl butoxide ( PBO ) were shown by Cheng (195 8 ) to reduce flies and stimulate weight gain of one half to two thirds of a pound per animal per day, due to reduced fly numbers. A photoactive substance, phl oxine B has been evaluated against immature horn fly stadia (Filiberti et al. 2009). However photoactive substances may have little application because the inside of a dung pat, where the susceptible larval flies develop, does not typically reach the

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21 req uired luminosity (~4000 lux for larvae fed 0.1 mM phloxine B to generate 50% mortality) for the compounds to become lethal. Avermectins are a group of compounds produced by Streptomyces soil dwelling microbes (Jackson 1989). Avermectins interact with the likely by activating the GABA receptor causing flaccid paralysis and death (summarized in Jackson 1989). An avermectin was shown to inhibit deve lopment of horn flies and stable flies in cattle manure (Schmidt and Kunz 1980). Wit h subcutaneous injections of an avermectin, horn fly development in cattle manure collected from the injected animal was retarded for over 28 d (Schmidt 1983). Ivermectin has been shown to be effective in reducing populations of horn flies for approximate ly six weeks with pour on treatments of cattle (Marley et al. 1993). When pour on ivermectin was combined with diazinon impregnated ear tags, duration was additive when seasonal fly pressure was low (Lysyk and Colwell 1996). However, a major concern with the use of such products is residual activity and pasture fouling due to dung not being recycled into the soil (summarized in Jackson 1989, Fincher 1992). Synthetic I nsecticides After World War II, synthetic insecticides were heavily utilized for pest c ontrol. Laake (1946) reported that spraying or dipping cattle with DDT provided excellent horn fly control. Many classes of insecticides were used for horn fly control including: organochlorines, organophosphates, carbamates and pyrethroids (Sparks et al 1985). These compounds have been formulated into numerous products that were applied to animals as whole animal dips, sprays, oilers, dusts pour ons, spot on s and ear tags. E xposure of horn fly populations to these compounds has been extensive, with c ross resistance of pyrethroids typically linked to previous DDT exposure (summarized in Sparks et al 1985). The widespread utilization of insecticides as

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22 control measures generated excellent initial control, but a lack of alternative control methods has lead to an overreliance on these compounds, resulting in insect ic ide resistance issues. In the mid 1970 s, the development and rele ase of insecticide impregnated ear tags provided a n inexpensive, long term, and relatively easy option for horn fly population control (Li et al. 2009). The formulation of these same active ingredients into pour ons and spot on s occurred in the 1990 s as an alternative to ear tags. These products provided several weeks of control at a time when ear tag failures due to insecticid e resistance were increasingly reported (summarized in Guerrero et al. 2002) The number of new compounds introduce d began to decrease in the 1970 s while older materials were removed from use, exacerbating the overuse of some compounds and increasing se lection for insecticide resistant flies. This decline continues today due to numerous factors including increasing cost of new registrations, perceived or real environmental impact, increasing government regulations, lack of new products and perception of a lack of need for the cattle industry (Kaufman et al. 2001). Today, the most widely available insecticides for horn fly control are in the pyrethroid class, with a few remaining members of the organophosphate and carbamate groups (Kaufman et al. 2011). Pyrethroids and DDT act at a similar site on voltage sensitive sodium channels and effectively prolong channel closure, causing hyperactivity, tremors, and rigid paralysis and death of insects (summarized in Yu 2008). Pyrethroids are commonly used to con trol horn flies and two pyrethroid types are utilized in current control methods. Type I pyrethroids are typically esters of acids and alcohols (e.g. permethrin), where type II specifically contain an cyano 3 phenoxybenzyl alcohol (e.g. deltamethrin) (Yu 2008) Type I pyrethroids induce repetitive electrical discharges in neurons, whereas type II pyrethroids typically cause a stimulus dependent

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23 depolarization of the membrane potential, leading to los s of the ability to transmit an action potential (summarized in Yu 2008). Carbamate and organophosphate insecticides are inhibitors of acetylcholinesterase (AChE), a serine hydrolase necessary for regulating the synaptic action of the neurotransmitter acet ylcholine (ACh) within both insects and mammals. Anticholinesterase reacts with a serine residue that is located at the catalytic site found within the AChE gorge (summarized in Yu 2008). The carbamoylated or phosphorylated enzyme is incapable of hydroly zing AChE, yielding an increase in synaptic ACh concentration that produces excessive excitation and eventual death (Yu 2008) Carbamates are known to be reversible inhibitors of AChE with a half life of about 40 min, whereas organophosphates have a half life of days or weeks and are said to be irreversible inhibitors of AChE (Yu 2008) Insecticide Resistance Development and Evaluations Types of Resistance Mechanisms Insects have several methods of expressing metabolic resistance to insecticides, primary metabolism by cytochrome P450 monooxygenases and esterases to a more water soluble form and secondary metabolism such as glutathione conjugation, both of which are of major focus in detoxification of insecticides (Pa rk and Lee 2007). Cytochrome P 450 s in insects perform many t asks and their role is often up regulated to aid in detoxification of xenobiotics in insecticide resistant ins ects (Feyereisn 1999). Such up regulation of metabolic enzymes is one method of insecticide resistance in the horn fly as found in a highly pyrethroid resistant pop ulation (Sheppard and Joyce 1998 ). Chlorfenapyr, a metabolic de coupler, was metaboliz ed by up regulated cytochrome P 450 s in the resistant horn flies to the active form, and was negatively cross resistant because the susceptible horn flie s did not exhibit P 450 levels as high as the resistant horn flies (Sheppard and Joyce 1998). Esterases have not frequently been shown to

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24 have much activity in horn flies, although they were linked to some metabolic resistan ce by B ull et al. (1988) and elevated levels of transcript concentrations of an esterase was reported to be active against diazinon (Guerrero 2000). Barro s et al. (2001) linked up regulation of GST s to diazinon resistance in a Louisiana horn fly populatio n. Alternatively insects can develop mutations to the target sites of insecticides. Point mutations are known to occur in receptors or ion channels of the nervous system, which alter their interaction with insecticides, preventing or reducing the effects of insecticides (Yu 2008). These mutations prevent or reduce pyrethroid and DDT interacti on with the sodium channel and have been documented in several insects. Additionally, AChE can carry point mutations that confer resistance to a nticholinesterases b y changing the structure, folding, or active site conformation of the AChE (Yu 2008). Point mutations in AChE prevent the anticholinesterases from binding to the active site, but such mutations must allow normal hydrolysis of ACh in the insect. Emergence and Evaluation Insecticide resistance has emerged to many compounds when formulated as insecticide impregnated ear ta gs, beginning in the early 1980 s (Kunz and Schmidt 1985). Within two years of initial use, H. irritans developed resistance to the pyreth roid fenvalerate, that was used in insecticide impregnated ear tags (Sheppard 1984). Sheppard and Hinkle (1985) theorized that several factors aligned for rapid selection of insecticide resistant individuals; all flies could be treated (selection pressure ) for long time periods, the expressed active ingredient in ear tags drops slowly over the life of the tag, which allows for rapid selection of heterozygous and homozygous resistant individuals. Res ults were dramatic. A fter only four years of utilizing o ne active ingredient (fenvalerate) the percent control observed one day after a pplication was reduced from 100 to 59% (Sheppard and Hinkle 1985). The addition of synergists to block

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25 resistance mechanisms has done little to restore efficacy in severely aff ected populations. Currently, Argentina, Brazil, and most of North America have been shown to contain horn fly populations resistant to pyrethroids (Li et al. 2009). Insecticide resistance in the horn fly has been studied utilizing many different approac hes for insecticide resistance evaluation. Methods used previously in evaluation of adult horn flies includes: insecticide residues on muslin cloth (field test kits) (Schmidt et al. 1985, Kunz et al. 1991), insecticide residues on glass (Sheppard 1984, Sh eppard and Marchiondo 1987, Hinkle et al. 1989), insecticide residues on filter paper (Sheppard and Hinkle 1987, Sheppard and Joyce 1992, 1998, Sheppard 1995, Sheppard and Torres 1998, Barros et al. 2001, Li et al. 2003, Barros et al. 2007, Li et al. 2007, Li et al. 2009, Oyarzun et al. 2011) and topical assays (Bull et al. 1988, Li et al. 2007). Additionally, formulated insecticides were evaluated on filter papers by Kaufman et al. (1999). Pyrethroid r esistance Quisenberry et al. (1984) reported horn f ly resistance to permethrin and fenvalerate in field collections from Louisiana; likely due to previous utilization of DDT, which conferred cross resistance to pyrethroids. Schmidt et al. (1985) first reported that horn flies in areas of Florida were no l onger being controlled by pyrethroids. Further investigations depicted less than two and greater than four fold resistance ratios to fenvalerate and permethrin, respectively, possibly linking the resistance to the then new utilization of insecticide impre gnated ear tags (Schmidt et al. 1985). Byford et al. (1985) measured high resistance ratios for several Louisiana sites fo r several pyrethroids. These resistance ratio s were not reduced completely with synergists, as 35 fold resistance only synergized 2. 2 and 3.5 fold, with S,S,S Tributyl phosphorotrithioate ( DEF ) and PBO respectively, suggesting a non metabolic resistance mechanism (Byford et al 1985). Kunz and Schmidt (1985) reported widespread pyrethroid resistance in 10 states from Florida to

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26 Calif ornia. Sheppard (1987) demonstrated insecticide impregnated ear tags selecting for pyrethroid (fenvalerate) resistance faster than sprays in field populations. Byford et al. (1998) reported that in a laboratory setting resistance could be generated in a p opulation with continuous selection, but was delayed with mixtures or rotation of permethrin, diazinon and ivermectin. In field settings, rotation of pyrethroid and organophosphates every two years resulted in reduced pyrethroid resistance, but yearly rot ation did little to affect resistance (Byford et al. 1998). However, a mosaic strategy, applied in a field setting, yielded no change in resistance over three years (Byford et al. 1998). In a laboratory setting, horn fly strain s with permethrin resistance quickly dropped low levels when horn flies were not under continued insecticide selection pressure (Kunz 1991). Alternatively, a rapid increase in resistance was found when horn flies were selected with permethrin, possibly indicating a trade off between resistance mechanisms and fitness costs (Kunz 1991). Kaufman et al. (1999) documented resistance to permethrin in all samples of horn flies ( resistance ratios 4 to 517) collected from Wyoming. Barros et al. (2007) documented heavy pyrethroid resistance ( resistance ratios 27.6 91.3 for cypermethrin) in horn flies collected from Brazil, where the primary control method included pyrethroid sprays (mostly cypermethrin and deltamethrin). Cross resistance between insecticide classes ha s been reported in numero us arthropod species, including Spodoptera littoralis (Boisd.), Culex quinquefasciatus Say, and Musca domestica L. typically between similar modes of action such as carbamates and organophosphates or pyrethroids and DDT (summarized in Yu 2008). Byford e t al. (1985) documented little cross resistance with carbamate (bendicarb) and organophosphate (dioxathion, stirofos, and sulprofos) insecticides to pyrethoid resistant horn flies. Additionally, lower

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27 synergism ratios were observed than what was expected if resistance was only due to metabolic factors (Byford et al. 1985). Sheppard and Marchiondo (1987) reported that pyrethroid resistant horn flies were more susceptible to diazinon in laboratory and field trials. Bull et al. (1988) selected for permethri n resistant horn flies and subsequently generated cross resistance to other pyrethroids ( f envalerate etc. ) and DDT, but did not find cross resistance to the organop h osphates (stirofos or cumaphos). PBO and to a lesser extent DEF only partially synergized permethrin to resistant horn flies, suggesting a target site insensitivity (Bull et al. 1988). Several mutations are responsible for conferring resistance to pyrethroid insecticides (summarized in Sparks et al. 1985). Guerrero et al. (1997) reported on t hree different amino acid point mutation substitutions found in a population of Louisiana horn flies. Two of the mutations were expressed only in laboratory induced pyrethroid resistant ( kdr resistant ( superkdr ) populations of H. i rritans However, the third mutation was expressed in both a resistant and a susceptible laboratory population, suggesting that it had no role in pyrethroid resistance expression. A leucine to phenylalanine substitution was reported to be associated with kdr (knockdown resistance) and a methionine to threonine substitution was associated with a superkdr (Guerrero et al. 1997). Two sodium channel mutations of single nucleotide substitutions, kdr and superkdr that occur in the S6 transmembrane segment of d omain II and S4 S5 loop of domain II, respectively, have been linked to pyrethroid resistance in the house fly (Smith et al. 1997, Lee et al. 1999) and horn fly (Guerrero et al. 1998). Jamroz et al. (1998) postulated that the low frequencies of kdr and su perkdr mutations may be due to a high fitness cost, as expressed in the allelic frequencies found, and further reported that superkdr was never found in the absence of a kdr mutation.

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28 Guerrero et al. (1998) demonstrated the possibility of developing a Poly merase Chain Reaction (PCR) amplification of specific alleles (PASA) assay for rapid screening of one of the kdr associated point mutations. Their results showed the capability for a PASA assay to describe point mutations; however, it was limited in the s cope of application, only recognizing one point mutation. Jamroz et al. (1998) determined allelic frequencies for kdr and superkdr mutations in several Texas field populations and two laboratory colonies They showed that wild colonies had low er allelic mutation frequencies than laboratory colonies with similar resistance ratios. Suggesting insecticide resistance is not entirely due to mutations and postulating that resistance point mutations likely carried a high fitness cost in the field. Guerrero et a l. (199 7 ) also illustrated the dramatic suppression of pyrethroid resistance of the superkdr colony with addition of the synergist piperonyl butoxide (PBO). LC 50 s were suppressed with addition of PBO from 1810 to 27 ug/cm 2 where the resistant colony was only reduced 2.7 to 2.0 ug/cm 2 This showed that multiple resistance mechanisms can be present with the kdr point mutation. The Jamroz et al. (1998) PCR method determined homo or heterozygosity of the alleles of interest. Guerrero et al. (2002) alt ernated pyrethroid and organophosphate insecticide impregnated ear tags yearly in a pyrethroid resistant field population; however, this rotation did not reduce kdr levels in the samples, and the population became uncontrolled by either insecticide class. Li et al. (2003) demonstrated permethrin resistance and presence of kdr alleles in wild caught horn flies from Mexico and Texas. Foil et al. (2005) evaluated resistance and presence or absence of kdr and superkdr determining that kdr provides protection from pyrethroids and superkdr only enhances this protection. Li et al. (2009) described a population of horn flies from Texas that had increased kdr and superkdr frequencies and elevated metabolic enzymes which were partially suppressed by PBO. These re searchers

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29 illustrated the difficulty of lowering pyrethroid resistance genetic allele frequencies from a population on ce they reached very high, near fixed levels. Guerrero and Barros (2006) reported that Brazilian horn fly populations were predominated by metabolic resista nce rather than point mutations. However in a highly resistant population elevated esterase levels and kdr were present but in most populations surveyed esterase levels and kdr did not play a significant role in the resis tance to pyreth roids. Sabatini et al. (2009) screened horn fly populations in Brazil, and found that individual ranch management programs likely determine prevalence of kdr with insecticide use, rather than a metapopulation having a significant genetic effect; however, kdr was found to be widely dispersed. Conversely Oyarzun et al. (2011) test ed field populations from Chile 16 years after horn fly introduction, and located resistant genotypes ( kdr ); both in insecticide managed herds and cattle that had not received in secticide treatments for five years prior to the study. Additionally, kdr has been identified in a subspecies the buffalo fly, Haematobia irritans exigua De Meijere in Queensland, Australia by Rothwell et al. (2011) suggesting that kdr is widespread in d istribution Oremus et al. (2006) treated three pyrethroid resistant populations of horn flies with mid season doramect in pour on treatments of cattle and yielded five weeks of control post treatment. In a field location where kdr was presumed to not be genetically fixed at a high rate in the population, some reduction in kdr genetics was observed. However, the authors believe that at the other two sites the kdr alleles had become fixed and the treatments therefore were not successful in reducing the f requency of kdr (Oremus et al. 2006). Organophosphate resistance Horn flies were documented in eight of twenty five populations in Wyoming as being resistant to diazinon (Kaufman et al. 1999). Adult horn fly resistance to an organophosphate (OP) has been reported wherein a field population is no longer controlled by diazinon containing

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30 ear t napthyl acetate was significantly higher in OP resistant field populations, suggesting an in crease in GST s and a me tabolic means for OP resistance (Barros et al. 2001). B arros et al. (2001 ) reported in a nine year study where efficacy of ear tag treatments, impregnated with diazinon, was reduced from greater than 20 weeks to less than one week. Organophosphate resistance in horn flies as generated by Barros et al. (200 1 ) documented a n increase in esterase activity and illustrated an increase in diazinon cross resistance to other organophosphates. However, resistance ratios to diazinon decreased from each Fall to the following Spring when there was no selection with diazinon, suggesti ng a fitness cost associated with the diazinon resistance mechanism (Barros et al. 200 1 than one class of insecticide for horn fly control (Barros et al. 200 1 ). The rol e of esterases in the metabolism of diazinon was evaluated by Li et al. ( 2007 ), and depicted a biphasic effect of PBO. The authors suggest that at high PBO concentrations, the PBO prevents metabolic bio activation, at medium concentrations there was no ef fect, and at low concentrations PBO effectively synergized diazinon by facilitating penetration or inhibited detoxification (Li et al. 200 7) Barros et al. (2007) reported low levels of resistance to diazinon in horn flies from Brazil ( resistance ratio 1.1), suggesting that resistance has not yet evolved to this organophosphate i nsecticide in South America In an attempt to lo cate a point mutation, diazinon resistant horn flies were used to generate two esterase cDNA libraries; however this approach failed to locate a poi nt mutation in the acetylcholin esterase gene. Transcript concentration was higher i n diazinon resistant horn flies than susceptible flies (Guerrero 2000). Temeyer et al. (2008) generated an AChE cDNA library that likely coded for di azinon insensitivity (G262A) in horn flies, and tested several molecular

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31 techniques for determining the presence of this point mutation in field resistant horn flies. This le d Foil et al (2010) to develop a multiplex PCR method combining the G262A mutati on with the kdr mutation. This single molecular technique allowed for rapid assessment of genetic diversity and genotyping of individual flies for kdr and G262A mutations Research Objectives Research on the horn fly in published literature is extensiv e; however there are some major gaps in the current understanding of insecticide resistance. Many studies were conducted during the 1980s to 1990 s on pyrethroid resistance and more recently from the 2000 s to today on organophosphate resistance in the horn fly, yet few studies have evaluate d both types of resistance and possible interaction s in field populations. Florida is unique in regards to the longer horn fly season than that observed in most of the US, but little has been published on conditions in t his state Biological control development has been under studied with respect to the horn fly especially when compared to livestock pests such as the house fly, due in large part to the quick development and ephemeral developmental locations. Conceptual papers have been published on the ability of Beauveria to induce mortality of adult horn flie s, but few studies have examined extensively different formulations and doses. My obj ectives in this research included : 1. Determine the level and relati ve presence of insecticide resistance including the modes of resistance in field populations of horn flies from across Florida 2. Survey for potential fungal pathogens to the horn fly, specifically Beauveria bassiana Test formulations and effective conce ntrations of B. bassiana conidia to reduce horn fly populations and determine its efficacy as a possible horn fly biological control agent.

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32 CHAPTER 2 T O XICOLOGICAL AND BIOCHEMICAL EVAL UATIONS OF FLORIDA HORN FLY INSECTICIDE RESISTAN CE Introduction Hor n flies are one of the most damaging pests of cattle in the United States. Each year they inflict over 876 million dollars in losses to U.S. cattle producers (Kunz et al. 1991). Horn flies are typically controlled by use of synthetic insecticides applied to animal hosts (summarized by Li et al. 2009). A common method of insecticide applic ation is the use of insecticide impregnated ear tags. Resistance to active ingredients formulated in ear tags developed after the initial widespread us e of ear tags in the early 1980 s and has continued to the present (Kunz and Schmidt 1985). Resistance, likely metabolic up regulation of detoxification enzymes, was first reported to the pyrethroid fenvalerate, and evolved into cross resistance to numerous other pyrethroi ds and DDT, and more recently into point mutations in the sodium channel (Byford et al. 1985, Sheppard and Hinkle 1985, Guerrero et al. 1997). Initially, synergists were added to formulations, but this method of resistance mitigation is ineffective in pop ulations where point mutations, such as knockdown resistance ( kdr ), are at high frequencies. Byford et al. (1985) documented populations of horn flies that were not controlled by the addition of the synergists S,S,S Tributyl phosphorotrithioate ( DEF ) and piperonyl butoxide ( PBO ) Bull et al. (1988) also documented what appeared to be target site insensitivity for pyrethroid resistant, laboratory selected horn flies. Guerrero et al. (1997) reported what is now known as kdr and superkdr mutations in the s odium channel that confer insensitivity to pyrethroids, and are homologous to similar mutations in Musca domestica L. and Lucilia cuprina (Wiedemann). The superkdr genotype has yet to be found in the absence of the kdr mutation (Jamroz et al. 1998). A po lymerase chain reaction

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33 (PCR) assay for identification of these genotypes has been developed by Jamroz et al. (1998). Li et al. (2009) described the difficulty in controlling horn fly populations with fixed or high levels of kdr This difficulty in manag ement also was found in Chilean horn fly populations by Oyarzun et al. (2011), where insecticide treatments had been discontinued for five years yet kdr remained prevalent in the population. Barros et al. (2001 ) provided evidence of individual organophos phate (OP) active ingredient resistance and cro ss resistance to non applied OP s. Resistance to diazinon was found in Wyoming by Kaufman et al. (1999). Temeyer et al. (2008) described a mut ation, G262A, in acetylcholine sterase (AChE) for horn flies that i s thought to cause reduced sensitivity of AChE to bind to organophosphates. Foil et al. (2010) developed a multiplex PCR method that allowed for identification of both the G262A mutation and the kdr mutation. The authors evaluated several horn fly popula tions and found in some populations, cases of shared resistant alleles, with individual horn flies exhibiting both the G262A and the kdr mutations. The objectives of this study were to collect field strains of H. irritans and determine 1) insecticide susce ptibility in treated jar assays against live flies, 2) genetic prevalence of the AChE G262A and the kdr point mutations in field populations, and 3) determine levels of metabolic detoxifiers, genera l esterases and cytochrome P450 s, in field populations. M aterials and Methods Fly Rearing An insecticide susceptible colony of horn flies was obtained from New Mexico State University that had originated from the USDA, ARS, KBLIRL in Kerrville, Texas and served as the strain to compare field collected flies. T his strain has been in colony since 1961 (Guerrero et al. 1997) and after acquisition was reared at the University of Florida Veterinary Entomology Laboratory. A second fly strain was acquired through the same

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34 source a ggs were collected daily from adults held in cages of approximately 0.283m 3 Damp paper towels paced und er cages (app. 1 cm) were used to collect eggs; each day eggs were washed off of the towels and rinsed with tap water into a small beaker. Eggs were pipetted off the bottom of the beaker and placed onto rearing media. Larvae were reared using a method mod ified from Geden et al. (2006). Briefly, 1 mL eggs were placed on a media mixture of fresh collected, frozen solid, then thawed bovine feces (2 L) and (1 L) hydrated peanut hull pellet mixture (1 L peanut pellets and 1.25 L tap water) (Bio Plus Inc. Ashbu rn GA). Manure was collected off the ground after passing from mature cows at the University of Florida Beef Teaching Unit that had not been treated with any insecticide or anti parasitic treatments for the previous 60 days. Under these conditions larval flies pupated around day five to seven and were separated from the feces peanut hull mixture by water flotation between days seven to nine. Extracted pupae were dried under slow airflow in a forced air chamber. Pupae were placed into cages where adults typically eclosed three to five days later. Adults were fed daily with citrated (12 g L 1 sodium citrate) bovine blood on cotton pads. Bovine blood was collected twice monthly from a local slaughterhouse and stored at 4C in the laboratory until use. Ad ults and larvae were reared in a 12:12 L:D photoperiod at room temperature. Contact Assay Horn fly exposure to treated glass surfaces for evaluation of susceptibility to an insecticide was first described by Sheppard and Hinkle (1986), and was modified in this study to use glass jars (Pitzer et al. 2010). Technical grade insecticides used in treated jar assays were permethrin (47.6% cis : 50.4% trans ) beta cyfluthrin (99.5%), and diazinon (99.5%) and were

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35 obtained from ChemService Inc. (West Chester, PA). All insecticides were serially diluted in acetone and one mL of solution was placed in each glass jar. The acetone carrier was evaporated prior to use by horizontal placement of the jars on an unheated hotdog rollers to allow for an even distribution of the insecticide on the sides of jars (Pitzer et al. 2010). Jars were dried for 24 hours and were approximately 60 mL in volume with an internal surface area of 67.87 cm 2 Between nine and ten dilutions of insecticide concentrations were generated betwee n 0.000003 and 30 ng/cm 2 for use in the contact assay. Three technical replicates of each dose were performed for each collection site. Adult flies were collected from the following seven Florida locations: four private commercial beef herds near Gainesvi lle, Kissimmee, Labelle, and Clewistion; and three University of Florida properties, the Range Cattle Research and Education Center (Ona), Beef Teaching Unit (BTU), and Beef Research Unit (BRU) (Appendix A) A beef cattle insecticide use history was obtai ned for each collection ranch to determine probable resistance selection. Field collected horn flies were obtained by sweep netting from the backs and bellies of mature cows and bulls at ranches described previously. Flies were placed into cages of appro ximately 0.283 m 3 and held in the shade or inside an air conditioned truck. Mixed sex and mixed age horn flies were removed from the holding cage, knocked down with CO 2 placed in groups of about 15 into glass jars previously treated with insecticides and examined for recovery. Horn flies that did not recover from knockdown were excluded from calculations as they were likely killed by handling. Horn flies were monitored for insecticide induced mortality two and four hours post placement into jars. The ho rn flies were considered dead if they were not able to walk or fly at the two time intervals. Controls consisted of glass jars in which 1 ml of acetone without the addition of insecticide was added to jars and allowed to

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36 evaporate as described previously. Excess flies from each collection site were removed from the cage and frozen alive at 70C for further analysis or retained for use in Chapter 3 Molecular and Enzymatic Assays Horn flies collected from Florida ranches were taken to the Veterinary Ent omology Laboratory at the University of Florida. Adult horn flies were placed into 100 mL plastic sample centrifuge tubes and frozen alive at 70 C until dissection. Frozen flies were placed onto a glass Petri dish pre chilled on ice and dissected into their three major body regions using forceps and a razor blade. Heads were retained for acetylcholinesterase (AChE) inhibition assays. The thorax was retained for PCR analysis. Abdomens were retained for general esterase (EST) activity assays or cytochr ome P450 (P450) assays. Heads and abdomens were placed separately in 1.5 mL microcentrifuge tubes and transferred to the Emerging Pathogens Institute, University of Florida, on ice, where they were stored at 80 C until the assays were performed. Labora tory strains were removed from the colony and treated in the same manner as the field collected flies. P olymerase chain reaction A DNA isolation method adapted from Guerrero et al. (2001) and a polymerase chain reaction ( PCR ) protocol adapted from Jamroz e t al. (1998) was used to determine presence and prevalence of known genetic point mutations that confer pyrethroid and organophosphate resistance in field collected horn flies captured from Florida ranches. Horn flies from collection sites were utilized f or examination in the PCR assay. At least 23 flies from each collection site, but not used in jar assays, were analyzed by PCR. Modifying the protocol outlined in Guerrero et al. (2001), DNA was extracted from thoracic regions of horn flies. Thoraces we re pulverized using pre chilled disposable pestles into 100 L of sample buffer (100 mM Tris, pH 8.3, 500 mM KCl) within 1.5 mL pre chilled microcentrofuge tubes that were kept on ice. Grinding of

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37 the fly thoraces continued until fully broken apart. Tube s containing ground thoraces were boiled for 3 min in a water bath. Tubes were centrifuged for 5 min at 15,000 x g. One microliter of the supernatant was used in the PCR assay. One microliter of DNA supernatant solution was placed into thin walled micro centrifuge tubes with 14 mM Tris (hydroxy methy) aminomethane hydrochloride pH of 8.3, 70 mM KCl, 0.15 mM dNTP, 4.5 mM MgCl 2 AmpliTaq DNA primers (Table 2 1) were used at 0.4 pmol per reaction. Amplification was completed in a Bio Rad DNA Engine Peltier Thermal Cycler (Foster City, CA) programmed for 96C for 2 min followed by 40 cycles of denaturation at 94C for 60 s, annealing at 62C for 1 min, and extension at 72C for 1 min, with a final extension at 72C for 7 min. Products were run on 4.0% agaro se TBE gel and visualized with ethidium bromide dye under ultraviolet light illumination and photographed on a BioDoc it Imaging System (Upland, CA). If imaging did not visualize DNA properly, the process was repeated using another aliquot of DNA in anoth er PCR; this process was repeated until the third PCR. If by the third PCR no product was observed on the gel, the sample was considered contaminated or unusable. These contaminated samples were not retained in evaluating the genotyping of the population s. The protocol utilizes two separate reactions, one that identifies susceptible genes and a second that identifies resistant genes at the kdr and the G262A alleles. Each reaction had the ability to generate three bands; GAPDH 154 bp, kdr 285 bp, and G262 A 116 bp (Figure 2 1). GAPDH, glyceraldehyde 3 phosphate dehydrogenase, is a constitutively expressed housekeeping gene and used as a positive control in each reaction to document that fly DNA was present, the diagnostic bands kdr and G262A indicated eit her a susceptible or resistant gene

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38 present in each fly (Guerrero et al. 2001). To ensure each reaction set was free of contaminates a negative control was performed, where the PCR reagents were placed without horn fly DNA in thin walled microcentrifuge tubes, processed and amplified as previously discussed. Results containing bands in this negative control were excluded from analysis. Positive controls were used in each preparation of PCR reagents and consisted of two horn flies, one from the Kerrville fly colony and another from the KDR fly colony, ensuring that the reaction generated bands visualized were correct. At the kdr allele, the Kerrville strain produced a susceptible band and the KDR strain generated a resistant band. If the PCR reaction ge nerated a positive control band (GAPDH) but did not generate a diagnostic band in either reaction the PCR was repeated for that sample. If no band was present the second time that fly was removed from the calculations for the allele of interest genotypi ng. A cetylcholinesterase inhibition assay The assay utilized to determine enzyme inhibition potencies was modified from Ellman et al. (1961) as outlined in Carlier et al. (2008). Briefly, acetylcholinesterase (AChE) hydrolyzed acetylthiocholine (ATCh) to dithio bis (2 nitrobenzoic acid) (DTNB) forming a yellow product. Absorbance was measured at 405 nm with the use of a DYNEX Triad spectrophotometer (DYNEX Technologies, Chantilly, VA, USA) for a 10 min cycle. Five frozen horn fly heads, from samples previously described, were placed into 1 mL of sample buffer, prechilled on ice in a glass tissue homogenizer. Sample buffer contained 0.1 M Na 2 HPO 4 buffer (pH 7.8, Fisher Scientific, Pittsburg, PA) 0.3% Triton X 100 ( Fisher Scientific ), and Bovine Serum Albumin (1 mg/ml, Fisher Scientific ). The horn fly heads were homogenized for approximately 5 seconds, or until the sample was homogenized thoroughly, with the use of an electric motor driven glass tissue homogenizer. This homogenate was placed

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39 into a microcentrifuge tube and centrifuged for 5 min at 5,000 x g and 4C. The supernatant of this homogenate was utilized in the assay as the enzyme source. An anticholinesterase reagent, diazoxon ( ChemSe rvice Inc.) was dissolved in dimethyl sulfoxide ( ChemService Inc. ) to a concentration of 10 mM and was further diluted (10x) in a serial manner with DMSO to a working concentration of 10 2 to 10 7 M. These working concentrations were then individually dil uted (100x) into buffer (0.1 M Na 2 HPO 4 pH = 7.8). Twenty L of diazoxon in buffer was added in triplicate in a 96 well microplate (Fisher Scientific). Final concentration of the anticholinesterase compound was 10 5 to 10 10 M. One hundred fifty L of 0 .1 M Na 2 HPO 4 (pH 7.8) buffer was added to each well. Ten L of enzyme source, described above, was placed in each sample well in triplicate. Ellman reagents, ATCh (0.4 mM final concentration) and DTNB (0.3 mM final concentration) were prepared fresh for each assay by combining 500 L of ATCh (1.0 M) and 500 L of DTNB (1.0 M) with 3 mL of 0.1 M Na 2 HPO 4 (pH 7.8) buffer. The microplate was incubated for 10 mi n at room temperature on a shaking vortexer Ellman reagents alone as a solution hydrolysis contro l were incubated on the bottom row of the microplate. After incubation, 20 L of the Ellman reagents were added to each well. Plates were then read on the microplate reader at an absorbance of 405 nm for 10 min. Included in each plate were wells contain ing only buffer with 0.1% DMSO, for blanks, and buffer with 0.1% DMSO and enzyme for end of the assay measurements. Cytochrome P 450 monooxygenase assay. Previously frozen horn fly abdomens were placed individually in a glass tissue homogenizer with 0.5 mL of sample buffer containing 0.1 M sodium phosphate buffer (pH 8, Fisher Scientific). The horn flies were homogenized for approximately 5 sec, or until the sample was homogenized thoroughly, with the use of an electric motor driven glass tissue homogeni zer.

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40 This homogenate was placed into a microcentrifuge tube and centrifuged for 20 min at 12,000 x g at 4C. The supernatant of this homogenate was utilized in the assay as the enzyme source. The assay to determine P450 activity utilized the 1 step Slow TMB Substrate Solution Kit ( 3,3, 5,5 tetramethylbenzidine (TMB), 1 mg/mL, with 3% hydrogen peroxide)(Fisher Scientific Fair Lawn, NJ ). When TMB oxidizes in solution a blue color is generated, and the absorbance is read on a spectrophotometer at 620 nM In a 96 well microplate 100 L of sample buffer was added to each well. Additionally, 20 L of enzyme source and 80 L of Slow TMB Substrate Solution Kit were added to each well. Each enzyme source was added in triplicate. The microplate was incubate d at room temperature for one hour and read on DYNEX Triad spectrophotometer (DYNEX Technologies, Chantilly, VA, USA) at 620 nM. One enzyme homogenate was considered one replicate with at least 5 replicates preformed from each horn fly collection site. Ca rboxylesterase quantification Horn fly abdomens were placed individually in 0.5 mL of sample buffer containing 0.1 M sodium phosphate buffer (pH 8, Fisher Scientific Fair Lawn, NJ ). The horn flies were homogenized for approximately 5 sec, or until the s ample was homogenized thoroughly, with the use of an electric motor driven glass tissue homogenizer. This homogenate was placed into a microcentrifuge tube and centrifuged for 20 min at 12,000 x g at 4C. The supernatant of this homogenate was utilized i n the assay as the enzyme source. The assay utilized to determine general esterase activity was modified from Grant et al. (1989). In this assay, NapA) conjugated to Fast Blue RR salt and acted as the enzyme substrate. The enzyme source, described above, or bovine serum albumin as a known standard (BSA, Sigma) was added to the substrate. In the ensuing reaction, the enzyme se parated the conjugated substrate into its constituents and the Fast Blue RR salt became colored

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41 in solution. Absorbance was measured at 450 nm with the use of a DYNEX Triad spectrophotometer. The level of absorbance was compared to a known standard, BSA, and indicated the relative level of carboxylesterase activity in the flies. In a 96 well microplate 150 L of sample buffer and 30 L of substrate was added to each well. Substrate was generated fresh for each assay by mixing 1 mg of Fast Blue RR salt i n 5 mL NapA) dissolved in DMSO. Twenty L of the enzyme source, described above, was added in triplicate to the plate. The plate was read immediately by the spectrophotometer One enzyme homog enate was considered one replicate and at least 5 replicates were performed from each horn fly collection site. Protein levels were quantified for each enzyme source by utilizing the method described in Bradford et al. (1976). Ten mg of Croomassie Brillia nt Blue G (Sigma Aldrich) dye was added to five mL of ethanol and dissolved. To this solution 8 mL of 10% HCl and 37 mL of deionized tap water. Under these acidic conditions, the dye forms a stable complex with amino acid residues of proteins thereby all owing protein quantification using a comparison to a standard. Bovine serum albumin ( BSA Sigma Aldrich ) stock was used as a standard by dissolving 1 mg into 0.15 M NaCl solution. BSA stock was diluted serially into 0.15 M NaCl solution, generating 8 con centrations. The absorbance of BSA was read on a DYNEX Triad spectrophotometer at 595 nm. The absorbance of BSA allowed a standard curve to be generated from absorbance values and known protein concentrations. The standard curve of BSA protein was compa red to each fly sample, which allowed for the conversion of sample activity to a per fly abdomen unit of carboxylesterase activity. Statistics Mixed sex, 2 5 day old insecticide susceptible Kerrville strain horn flies were used to generate a concentratio n response line for all insecticides. Insecticide applied to each jar was

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42 converted into grams per area. Bioassay data from each horn fly collection were pooled and analyzed by probit analysis (Finne y 1971) using PoloPlus ( LeOra Software, Petaluma, CA, U SA ) (Robertson et al. 2003) for each insecticide to generate Lethal Concentration (LC) values for resistance ratio calculations. Abbott's transformation (Abbott 1925) for control mortality correction was applied within PoloPlus. Resistance ratios were gen erated by dividing the LC 50 values obtained for horn flies from each field collection site by the LC 50 value obtained for the Kerrville strain. Ellman assay absorbance net change was calculated for each horn fly population or strain and analyzed by nonlin ear regression to the following formula: Y = Bottom + (Top Bottom) / (1 + 10 ^((LogEC 50 x)*Hillslope)); where the variable Bottom is the Y value at the bottom plateau, Top is the Y value at the top plateau, x = the logarithm of the concentration and Y = the response ( GraphPad Software, San Diego, CA, USA). The data were adjusted for back ground activity using the absor bance values obtained from reagent only blanks. Dose response curves were generated with GraphPad Prism and were utilized to determine I C 50 values (concentration to inhibit 50% of the enzyme), 95% confidence limits, Hillslope values, and r 2 values. One enzyme source (5 fly heads per homogenate) and one dilution of anticholinesterase compound were considered one replicate, with at least th ree replicates preformed per field collection site. All enzyme assays were performed in the linear range of the enzyme function; this ensured that the measured absorbencies were indicative of a zero order enzyme reaction. Reactions that did not meet zero order assumptions were not included in results. Absorbance data from replicates of flies at each field collection site and the two laboratory strains (Kerrville and KDR) were placed into GraphPad InStat ( GraphPad Software, San Diego, CA, USA) to calculat e means and

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43 standard deviation between sites and to test for significant differences in means (ANOVA) between sites. Average probable carboxylesterase activity and P450 absorbance values per fly (technical replicates) were compared by averaging technical r eplicates and an ANOVA was used to test differences between means of collection sites. The BSA standard curve generated a linear response that was used to back calculate the amount of protein in each sample. The protein quantification was used to determi ne enzyme activity per unit, per abdomen, and was converted to per gram of abdomen activity. Results Adequate numbers of flies from numerous collection sites were not available to run the glass jar assay, therefore, only horn flies from Labelle, BTU, a nd Ona sites were compared under insecticide resistance evaluations. Horn fly resistance to several insecticides was evaluated by mortality response to insecticide treated glass jars assessed four hours post exposure (Table 2 2). The Kerrville laboratory reference horn fly colony exhibited a LC 50 of 0.023 g/cm 2 for permethrin compared to 0.023 g/cm 2 for the pyrethroid resistant KDR strain. Permethrin LC 50 s of 1.53, 0.13, 0.068 g/cm 2 generated resistance ratios of 66.6, 5.65, and 3.0 with Labelle, BTU, and Ona collected horn flies, respectively. cyfluthrin generated a LC 50 of 0.0067 g/cm 2 for the Kerrville lab strain and a LC 50 of 0.53 g/cm 2 for the KDR lab strain. Comparatively, horn flies from the BTU and Ona ranches generated LC 50 s of 0.01, a nd <0.001 g/cm 2 respectively. These flies expressed resistance ratios of 79 for KDR, 1.4 for BTU, and <0.15 for Ona. Horn flies collected from the Labelle ranch did not generate homogenous data and therefore could not be properly analyzed. However, ex posure to the highest concentration (0.14 g/cm 2 cyfluthrin generated only 64% mortality.

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44 The Kerrville strain horn flies generated a LC 50 of 0.020 g/cm 2 under diazinon exposure, while the KDR strain exhibited 100% mortality at 0.029 g/cm 2 Horn flies collected from t he Labelle ranch expressed approximately only 20% mortality at the highest concentration, 15 g/cm 2 with an estimated resistance ratio of >150 The BTU collected horn flies generated a LC 50 of 0.06 g/cm 2 and a resistance ratio of 3.0. Horn flies at the Ona collection site did n ot generate homogenous data; therefore a LC 50 value was not calculated. A summary of PCR results is presented in Table 2 3. The Kerrville laboratory susceptible strain was 100% susceptible at the alleles evaluated. The KDR strain was largely heterozygo us, SR, at the kdr allele, with only 12.5% presenting as homozygous resistant, RR. Both strains were 100% susceptible at the G262A allele. The Labelle sample generated a 31.7% heterozygous and 45.12% homozygous resistant profile at the kdr allele and a 12 .94% heterozygous and 8.23% homozygous resistant expression at the G262A allele. The Ona collection generated 83.33% SR and 5.56% RR at kdr ; and 11.76% RR resistant for the G262A allele. Flies collected from the BTU showed 13.33 and 33.33% kdr SR and RR frequencies; with 7.14% SR at the G262A allele. No inhibition concentration differences were found between horn fly populations evaluated in Ellman assays (Table 2 4). Kerrville strain horn flies generated an IC 50 value of 7.5 nM for diazoxon, while all other strains were arithmetically slightly lower, but not significantly different, having overlapping 95% confidence intervals. Relative levels of enzyme activity are shown in Table 2 5. Activity was converted to activity per gram of abdomen and was gener ally higher in field collections over the susceptible Kerrville strain. Protein quantified per sample was divided by absorbance measured, and

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45 converted to per gram units. There were no statistical differences found between collection site for either cyto chrome P 450 activity or general esterase activity. Discussion Horn fly resistance was evaluated from several ranches across Florida. During the sampling period there were several confounding factors that limited access to cattle for fly collections. We based Cooperative Extension educators in counties that had high cattle production in the most recent (2007) Survey of Agriculture ( USDA NASS 2009a). Our aim was to collect horn flies from at least 10 coun ties, ranging from more temperate Northern areas to more sub tropical production areas in the South of Florida. Unfortunately, I only was allowed access to a few (4) private herds and University properties (3). Numerous flies were required for completion of contact assays, genetic, and metabolic screenings (~2000); but often these collection sites pro vided fly collections that were low and the needed fly numbers were not reached at several locations. Although the results of this research di rectly impact many stakeholders, this message was difficult to disseminate effectively leading to a lack of producer accessibility. Although multiple collections were attempted, due to statistical analysis requirements, the results presented herein are limited to those where adequate fly collections were obtained. Initially my research goal was to collect and use F1 horn flies in topical assays for insecticide susceptibility evaluations ; however, extreme difficulty in rearing field collected insects negated this goal. Instead, I evaluated insecticide resistance using residues on treated glass jars with field collected horn flies Several collection sites were excluded from the insecticide susceptibility evaluations due to low horn fly collection numbers when farms were visited and animal access was granted. The resultant three collection sites from which enough

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46 flies were collected to determine LC 50 values and subsequently resistance ratios were Ona, BTU, and Labelle. Historically, the pyrethoid fenvalerate was intense ly studied for its actions against horn flies; however the fe nvalerate EPA registrations have been canceled ( Bradbury 2008), negating its applicability to horn fly resistance research. Therefore we utilized the most commonly applied pyrethroids currentl cyfluthin. An additional benefit to utilizing these two compounds is that they are type 1 and type 2 pyrethroids, respectively, providing differing activity (Yu 2008). The Kerrville strain generated an LC 50 value of 0.023 g/cm 2 for permethrin and 0.0067 g/cm 2 cyfluthin and permethrin LC 50 values have not previously been published using our exposure method. The LC 50 value generated for the Kerrville strain of 0.020 g/cm 2 for diazinon is lower than that pres ented by Sheppard and Marchiondo (1987), who documented a mean of 2.0 g/cm 2 in the Kerrville strain. However they used a 2 hr exposure time and dried the glass for 48 hours before exposure. The difference between these two values may be due to metabolis m of diazinon to the active form that must occur in vivo or due to the longer post application drying time. A dditionally the Kerrville colony could have increased in diazinon susceptibility in the over 20 years since these studies were conducted. Byford et al. (1985) in exposing adult horn flies to permethrin residues on filter paper showed the toxicity of permethrin to be similar to bendiocarb (a carbamate), with other organophosphates examined being less toxic than permethrin. Byford et al. (1985) did cyfluthirn or diazinon, negating any direct comparisons to the evaluations presented here. Horn flies collected from the Ona ranch produced the lowest pyrethroid resistance ratios, cyfluthrin, that were ob served in this study (Table 2 2) Low

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47 resistance ratios have been documented in susceptible hou se flies by Kaufman et al. (2001 ), and the low ratios present in the horn fly collections may only represent natural variation in susceptible strains. However, t he kdr allele showed a n 83.3% heterozygous frequency for this ranch (Table 2 3). In light of the high susceptibility observed, one would have expected a much more homozygous susceptible genotype to have been found. The predominance of resistant alleles embedded in the heterozygote genotypes suggests that the heterozygote may be recessive or not continuously expressed in this population, and with continued insecticide pressure this horn fly population is predisposed to developing into a homozygous resist ant type. This high genetic frequency is lower than a population described by Foil et al. (2010) that contained 100% RR kdr horn flies evaluated in a sample (n=40). T herefore our results are in line with published literature for this genotype. Resistanc e ratios for pyrethroids examined were not presented in the Foil et al. (2010) evaluation. In the Soderlund (2008) review of pyrethroids and kdr in house flies kdr is genetically recessive; this recessive nature could explain the high frequency seen of t he heterozygotes, yet low resistance ratios in the jar assays. The horn flies collected from the BTU site generated slightly elevated resistance ratios comparaed to the Kerrville strain (Table 2 2 ). The resistance ratio of 5.7 1.4, and 3 for permethrin, cyfluthrin and diazinon respectively, were low in comparison to the Labelle collection site. Genetically the frequency was moderate with 33% of flies tested being kdr homozygous resistant, and supports the explanation for elevated pyrethroid resistance in the exposure assay. The proposed AChE mutation was also present in the PCR results with 11.76% homozygous resistant, and may partially explain the resistance ratio of 3.0 for diazinon. Although in the AChE inhibition assay, there was no difference fou nd between horn fly populations across collection sites. The point mutation described by Temeyer et al. (2008) was

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48 derived from a low level resistant population, and only confers a low level of resistance (Foil et al. 2010), as such may indicate multiple mechanisms for OP resistance in these horn flies. Foil et al. (2010) found populatio n samples ranged from 0 28.57% resistant G262A alleles, with an overall average of 14.34%. The authors evaluated discriminating doses of diazinon and found that the likel ihood of survival to the discriminating dose was increased if the G262A mutation was present. By far the most insecticide resistant horn fly population sampled was at the Labelle site. Here producers had extre mely high fly numbers on cattle, and these flies expressed significant resistance ratios; 66.6 for permethrin and >14.7 for diazinon (Table 2 2). The cattle that horn flies were collected from had previously been treated with i nsecticide impregnated ear tags. B oth diazinon and permethrin + PBO t ags were removed from the cattle two we eks before fly collection. P rior to that date insecticide use history was unavailable due to the cattle and property being sold to new owners. A worrisome combination of resistance to pyrethroids and organophosphat cyfluthrin generated data that was not suitable for PoloPlus analysis, but did show only 64% m ortality at the highest dose of 0.14 g/cm 2 Sheppard and Marchiondo (1987) found what was presumed to be negative cross resistance between pyrethroid resistant horn flies that expressed increased susceptibility to diazinon. This does not appear to be the case with the Labelle strain. Cross resistance has been documented in horn flies within the pyrethroi d insecticide class (Byford et al. 1985 Quisenberry et al. 1984 Barros et al 2007 Sheppard and Joyce 1992). To my knowledge no studies have compared horn fly resistance mechanisms in multiple classes. However, several studies have presented data showin g laboratory or field populations with insecticide resistance efficacy data across multiple insecticide classes. Bull et al. (1988)

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49 documented a pyrethroid resistant horn fly strain with low resistance to tetrachlorvinphos (2.1 fold) and c o umaphos (1.8 fo ld). Byford et al. (1985) documented a horn fly population that exhibited resistance to multiple ins ecticide classes with low level carbamate and organophosphate resistance observed. Byford et al. (1998) documented through laboratory selections with per methrin and diazinon that resistant populations of horn flies could develop in rotational systems to insecticides in multiple classes. Conversely, Sheppard and Marchiondo (1987) reported that diazin on was more toxic to pyrethroid resistant populations tha n pyrethroid susceptible populations Shared mechanisms of resistance to multiple classes of insecticide s in one horn fly population have not been reported. However, cross resistance to multiple insecticide s having different mode s of action has been descr ibed in other insects. I n house flies increa sed mixed function oxidases and decreased cuticular penetration generated resistance to abamectin in pyrethroid resistant strains, and in the German cockroach, Blattella germanica (L.) pyrethroid resistant popu lations had cross resistance to imidacloprid, which was not synergized with the addition of PBO (Scott 1989 Wei et al. 2001). It is unlikely that insecticide resistance to multiple insecticide classes that have different target sites would be a genetic p oint mutation at the same loci. I n the current study, due to the inherent differences between a sodium channel and AChE target site s multiple resistance mechanisms or increased metabolic factors are the two most likely mechanisms that conferred the obser ved resistance in the Labelle population. In the Labelle sampling the results of the PCR assay illustrated a high (45%) homozygous resistant kdr genotype, which likely conferred the high permethrin resistance observed in the contact assay. Additionall y, the AChE mutation was present at a low rate, which may confer some resistance capabilities to diazinon, as was observed in the contact assay.

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50 Additional replicates of the AChE inhibition assay could reduce the variance and possibly generate significant differences between populations (Table 2 4). The activity measured for P450 s and general esterases was arithmetically higher with the Labelle horn flies, yet not statistically different from the other strains due to high variability in the measurements ( Table 2 5). However, activity was indirectly measured in the assay; the active P450 responsible for metabolism of the xenobiotics may have a change in catalytic activity or not have been produced in high enough quantities to be realized in the assay (Scot t 198 9). A dditional replicat es could help determine if P450 s were upregulated. Moreover identification of the P450 responsible for detoxification or sequestration of the xenobiotic, the catalytic activity, and the amount of the specific P450 should be d etermined. Following this procedure the specific P450 s impact of the metabolic detoxification and thus insecticide resistance in the horn fly could be elucidated. Identification of a field population of horn flies that exhibited insecticide resistance to multiple insecticide classes would be a major concern to cattle producers. Such an event indicates that what Byford et al. (1998) documented through laboratory selections has come to fruition in field populations. Producers already are limited in the choices of insecticides that are available for horn fly control. Multiple insecticide class resistance is clear evidence that insecticides using new modes of action, application techniques, or management methods that achieve economic control, but do not p ressure horn fly populations to the extent of currently used ear tag technology, should be considered in the development of new insecticide chemistries and their utilization in future control programs. A comprehensive comparative analysis of the previousl y utilized insecticides using multiple exposure methods should be conducted for future studies. This would allow for the direct comparisons to potentially exhausted target sites, and chemistries that are no longer

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51 available, whereas the literature current ly consists of many outdated and no longer registered chemistries. Organophosphate resistance was low (1.4 fold) in the horn fly populations that were used to develop the AChE G262A PCR (Tenmeyer et al. 2008). The high diazinon resistance found in the La belle sample, coupled with the PCR results of a moderate percentage of positive G262A horn flies, indicates that this point mutation may be a contributing factor to diazinon resistance in this population. However this was not confirmed with the inhibition assay utilizing the active form of diazinon, diazoxon, against horn fly AChE. Future work should include conducting PCR to identify AChE genotype in each population grouping these flies by SS, SR and RR and determining if there are differences in inhib it ion by diazoxon to AChE associated with these three horn fly genotypes. This could help determine the level of resistance conferred by the heterozygote and homozygote resistant point mutations in AChE, and determine how serious this mutation is for futu re control utilizing diazinon. Additional replicates should be conducted on the enzyme assays to help determine if there is a true separation in the levels of activity for P 450 s and general esterases in Florida horn flies. These replicates could help det ermine if there is an up regulation of metabolic activity or if resistance is caused by an increase in catalytic activity. Knowing the types of resistance present in field populations can help producers know how best to handle populations that are no long er sensitive to insecticides, and guide the direction of future insecticide development.

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52 Table 2 1. Sequences of polymerase chain reaction ( PCR ) primers used to identify kdr and G262A genotypes from field collected horn fly populations Primer ID U83871 U83873 U83871, U83873 466160 466160 466160 GAPDH fragment obtained by reverse transcription PCR using degenerate primers, not published in GenBank (Table adapted from Foil, L. D., F. Guerrero, and K. G. Bendele. 2010. Detection of target site resistan ce to pyrethroids and organophosphates in the horn fly using multiplex polymerase chain reaction J. Med. Entomol. 47: 855 861. )

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53 Table 2 cyfluthrin and diazinon on glass to horn fly adults collected from Florida beef cattle ranches. LC 50 ug/cm 2 (95% CI) {Slope} Resistance Ratio 1 Insecticide Kerrville KDR Labelle BTU Ona KDR Labelle BTU Ona Permeth rin 0.023 ( 0.016 0.032 ) {2.47} 0.023 (0.0038 0.13) { 0.65 } 1.53 (1.01 2.6) { 1.29 } 0.13 (0.055 0.19) { 1.36 } 0.068 (0.013 0.36) { 0.85 } 1 .0 66.6 5.65 3 .0 cyfluthrin 0.0067 (0.0044 0.0096) { 1.55 } 0.53 (0 .13 1.31 ) { 0.99 } (ND) { ND } 0.01 ( 0.0013 0.13 ) { 0. 79 } <0.001 (ND) { ND } 79.0 1.4 <0.15 Diazinon 0.020 (0.14 0.029) {2.45} <0.029* (ND) { ND } >0.295 (ND) { 0.158 } 0.06 (0.046 0.27) { 1.33 } ND (ND) { ND } <1.4* >14.7 3 .0 ND Laboratory strains = Kerrville (susceptible); KDR (pyrethroid resistant). Field strains = Labelle, BTU and Ona. Kerrville n=900, other fly strains n=300. 1 Resistance ratio at LC 50 (i.e. LC 50 resistant strain/LC 50 Kerrville strain). ND not determined due to low fly collection numbers or problems in statistical analysis. Numbers aft er < or > indicate that the tested values did not generate a LC 50 value that was reliable, did not conform to heterogeneity, or statistical requirements for probit analysis; this indicates that the LC 50 was out of the range tested or data was not homogenou s enough to plot a reliable line. 100% mortality was achieved at a dose of 0.029 g/cm 2 at 0 .14 g/cm 2 generated 64% mortality.

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54 Table 2 3. Percentage genotype composition of laboratory strain and Florida field collected horn fly populations as exp ressed through a multiplex PCR kdr G262A Sample n SS SR RR n SS SR RR Kerrville 9 100 0 0 9 100 0 0 KDR 16 31.25 56.25 12.5 16 100 0 0 Labelle 82 23.17 31.70 45.12 85 78.82 12.94 8.23 Ona 18 11.11 83.33 5.56 17 88.24 0 11.76 BTU 15 53.33 13. 33 33.33 14 92.86 7.14 0 kdr and G262A are the point mutations referring to knockdown resistance and insensitive AChE in the horn fly (Foil et al 2010) Percent totals may not equal 100% due to rounding. n = number of flies evaluated that generated bands of the appropriate length in either the susceptible or resistant reactions

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55 Table 2 4. Mean 50% inhibition concentration (IC 50 ) of diazoxon in nM to horn fly head homogenates Fly Source n Mean (95% CI) ANOVA Kerrville 4 7.5 (5.9 9.0) F= 2.15 df = 4, 18 P=0.101 KDR 4 5.7 (1.8 9.7) Labelle 3 4.5 (1.0 7.9) Ona 3 5.3 (4.0 6.5) BTU 5 5.6 (4.5 6.7) Kerrville and KDR are a laboratory susceptible and pyrethroid resistant horn fly strain. Remaining strains were collected from Florida cattle ranches. Each replicate contained 5 fly heads and was repeated n times. All hillslopes were 0.99 or above.

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56 Table 2 5. Relative enzymatic activity (SEM) per gram of horn fly abdomen protein Collection Cytochrome P 450 General Esterase Kerrville 29.1 ( 8.9) 13 .5 (5.4) Labelle 50.5 (8.22) 40.6 (10.3) Ona 23.3 (10.1 23.0 (4.5) BTU 32.1 (18.4) 32.3 (6.8) ANOVA 0.93 2.69 F Value P Value 0.44 0.08 Individual horn fly abdomen protein concentration was used to adjust activity to a per gram basis. Five flies were averaged for each collection site. An ANOVA was performed on the results and no significant differences were found. df =3,16.

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57 Figure 2 1. Typical gel photo showing polymerase chain reaction ( PCR ) amplification products. Photo was cropped for clarity and adjusted in MS Powerpoint. L represents 100 bp ladder used to confirm band length. Lanes marked 1 and 2 are the susceptible reaction; Lanes marked 3 and 4 are the resistant reaction. Lane 1 and 3 contain PCR products containing DNA from t he KDR control, while Lane 2 and 4 contain PCR products containing DNA from the from the Kerrville control. Primers and loading dye produce the nonspecific bands present below the 100 bp band.

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58 CHAPTER 3 LABORATORY EVALUATIO N OF BEAUVERIA BASSIANA ISO LATE PATHOGENICITY AGAINST ADULT HORN F LIES Introduction Integrated pest management (IPM) focuses on reducing pest populations with numerous tactics. Horn flies, Haematobia irritans (L.), are controlled on pastured beef cattle usually by only one control tactic, insecticides ( summarized by Li et al. 2009). Horn fly larvae develop in fresh undisturbed cattle manure (Bruce 1964) and long lasting population control methods are limited to insecticide impregnated ear tags that kill adults or insect growth regu lators (IGR) administered in feed through mineral supplements that target immatures. Insecticide impregnated ear tags have been implicated in numerous cases of insecticide resistance, and IGRs and systemic avermectins have generated concern with dung recy cling due to their perceived impact on beneficial and other non target dung inhabiting species (Fincher 1992). There have been few published research articles on the use of entomopathogenic fungi as an alternative management tool for horn fly populations (Angel Sahagun et al. 2005, Lohmeyer and Miller 2006, Mochi et al. 2010a,b). Such a tactic would allow for expansion of producer options in managing this pestiferous fly. Steenberg et al. (2001) noted the following species infecting horn flies in Denmark: Beauveria bassiana (Balsamo) Vuillemin, Verticillium lecanii (Balsamo) Vuillemin, V. fusisporum Gams, and Furia americana (Thaxter) Humber. These represent the only published field collected strains of fungi that infect horn flies. Within other muscid f lies several fungi have been isolated from field collections, but only one has been formulated and registered as a commercially B. bassiana ) for house fly control in poultry facilities (Andersen 2006).

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59 Of the few studies evalua ting entomopathogenic fungi in horn flies, a majority of the focus has been upon the immature stages; eggs, larvae, and pupae; with little attention given to infecting adult flies. Angel Sahagun et al. (2005) were the first to show artificial infection of horn flies with fungi, and did so with several isolates of Metarhizium anisopliae (Metschnikoff) Sorokin B. bassiana and Isaria fumosorosea (Wize) (Brown and Smith) (formerly Paecilomyces fumosoroseus) This proof of concept study used manure inoculati ons to infect eggs and larvae with conidia, as well as continuous exposure assays for pupae and adults. However a considerable challenge in reliance on an immature exposure paradigm is the delivery of conidia to immature flies that are widely dispersed ac ross pastures in ephemeral dung pats. Formulated fungal strains developed and sold for biological control of other insect species were evaluated by Lohmeyer and Miller (2006) and included M. anisopliae (strain ESCI, Bioblast, EcoScience Corp., East Brunswi ck, NJ) and B. bassiana (strain GHA, Botanigard 22 WP, Emerald BioAgriculture, Lansing MI, proprietary formulation). Additionally, Lohmeyer and Miller (2006) developed and tested an I. fumosorosea (strain ARSEF 3581) formulation with diatomaceous earth. Lohmeyer and Miller (2006) reported a lethal time for 50% mortality, LT 50 of 2.70, 4.98, 7.97, and 9.42 days for B. bassiana M. anisopliae I. fumosorosea and untreated controls, respectively, after a two hour contact exposure that simulated host treatm ents by using acrylic faux fur. Mochi et al. (2010a,b) evaluated conidia suspensions in an aqueous solution against field collected horn fly eggs, larvae, pupae and adults Although results varied between isolates of M. anisopliae (strains E9, IBCB425, an d IBCB159) B. bassiana (strains JAB06, JAB07, and AM09), I. fumosorosea (strains IBCB133 and CB75), and Isaria farinosa (Holmskjold) Fries

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60 (formerly Paecilomyces farinosus) (strains CG189 and CG195) virulence also varied with life stage, ranging from low infectivity in one stage to no infectivity in another life stage. Management of the horn fly using insecticides has led to considerable resistance. Producers are left with few options, opening the door for innovative control tactics. The use of fungal pathogens is one underutilized option that could provide acceptable control of this pest, however, considerable hurdles exist, including the location and isolation of a horn fly specific, pathogenic fungal strain and comparative studies that document effic acy. Therefore, the objectives of this study were to 1) evaluate presence of patent infections of entomopathogenic fungi in horn flies collected from Florida ranches, and 2) to compare fungal virulence against horn flies using a field collected horn fly B bassiana strain and two commercially available fungal strains under laboratory conditions. Materials and Methods Isolation and V iability Horn flies that were captured from Florida ranches and not used in insecticide resistance bioassays described in Cha pter 2 were retained until d eath to screen for infection by entomopathogenic fungi. Adult flies were sweep netted from the backs and bellies of mature cows and bulls at each collection site, placed into a cage and transferred to the Veterinary Entomology Laboratory at the University of Florida. Adult flies were collected from the following seven Florida locations: four private commercial beef herds near Gainesville (~250 flies), Kissimmee (~300 flies), Labelle (~5000 flies), and Clewistion (~500 flies); a nd three University of Florida properties, the Range Cattle Research and Education Center (Ona) (~300 flies), Beef Teaching Unit (BTU) (~300 flies), and Beef Research Unit (BRU) (~400 flies) a table with collection information is presented in Appendix A. The flies were surface sterilized by washing in 95% ethanol for 15 s, then placed onto dry paper towels and the ethanol was

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61 allowed to evaporate. The fly cadavers were then placed in groups according to collection sites in humidity saturated chambers to observe for flies that had fungal infections. The humidity chamber consisted of a plastic chamber lined with paper towels or filter papers, dampened with non sterile deionized water. The plastic container was sealed for several days while awaiting sporul ation. The flies were placed on the substrate, either paper towels or filter paper, so that they were not touching each other. Beauveria bassiana infections manifested around day four as an off white hyphal growth covering part of all of the fly body. T his hyphal growth formed conidia, which were subsequently isolated with a glass point and plated onto SDAY plates as modified from Geden et al. (1995). The end of a heated glass pasture pipette was pulled out to a very small diameter, resulting in a steri lized glass point for isolations. Subsequent growth of fungi was maintained in culture on SDAY media and was inoculated by means of a sterilized bacteriological loop that was used to gather conidia from the surface of mature plates. At each trial, to ens ure that fungal conidia were viable, germination rates of conidia were measured on SDAY plates. Each assay utilized germination rates of at least 95%, by counting at least 100 conidia per plate, under a compound microscope. Germination was enumerated bet ween 12 and 24 hours post inoculation, wherein germinated conidia appeared swollen and had germ tube hyphae emerging from individual conidia. Non germinated conidia, as well as those conidia that had swollen but had not yet extended hyphal structures, wer e considered non viable. Fungal Strains and Colony Maintenance The EN1 fungal strain collected from Florida horn flies (strain submitted for ARSEF numbering), GHA (ARSEF 6444) and the HF23 strain (ARSEF 7940), a commercially available strain used for hou se fly control, wer e evaluated for horn fly infectivity and mortality The EN1 strain was isolated from field collected horn flies captured near Gainesville, FL. Following isolation, the strain was maintained as discussed below. The GHA strain was isola ted from

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62 BotaniGuard 22 WP ( Laverlam International Corporation, Butte, MT, USA; isolated from product by E. N. I. Weeks), and maintained in culture in our laboratory as described below. The HF23 strain was received in pure culture from JABB of the Caroli nas (Pine Level, NC), and maintained in culture as described below. Fungal culture media, Sabouraud Dextrose Agar with yeast extract (SDAY), was prepared from Sabouraud Dextrose Broth (Difco, Sparks, MD) prepared as per instructions with 30 gL 1 agar (Fis her Scientific, Fair Lawn, NJ) and 5 gL 1 technical yeast extract added (Difco, Sparks, MD)(Goettel and Inglis 1997). This was autoclaved at 121 C for 20 min, after slight cooling, ~20 mL of media was poured onto each plate. Petri plates (50 x 10 mm) we re used for isolation of strains, while larger 90 x 10 mm plates, were used for culturing of fungus to provide a source of conidia for bioassays; all plates were obtained from Fisher Scientific (Fair Lawn, NJ). The number of passes through artificial medi a was recorded for the all field strains in culture. Fungi were not passed through insect hosts, and were all maintained on SDAY media isolat ed by strain in plastic chambers Fungus was grown on SDAY plates at 25 ( 7) C, a light: dark cycle of 12:12 h, and 50% relative humidity. Petri plates were inoculated with a sterile loop, using standard laboratory procedures. Under these conditions, fungal conidia were produced beginning on day seven with completion by day 30. Once conidia completed development on SDAY plates, they were harvested using a bacteriological loop to scrape the surface of the media, to gather conidia. After harvest this conidia containing powder was used immediately in any bioassays. Fresh conidia powder was generated for use in al l bioassays. Laboratory Bioassay Adult horn flies Kerrville strain, obtained from a long established colony of insecticide susceptible horn flies (described in Chapter 2), were treated with conidial suspensions in an

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63 attempt to determine the concentration (LC 50 ), time (LT 50 ) for 50% mortality, and the differences in strain virulence between concentrations and application techniques. Three inoculation techniques were utilized; in solution with surfactant, contact on dried filter paper, and in an inert carr ier. All horn flies used were between one and three days post eclosion. Conidia in S urfactant S olutions Conidial suspensions were generated by emulsifying the previously harvested conidial powder into a 0.1% Tween 80 (Fisher Scientific, Fair Lawn, NJ) an d water solution to generate concentrations as high as 1.0x10 9 conidia / mL. Tween 80 was added as a surfactant to aid in conidial suspension in a deionized water solution. Conidia suspensions were emulsified by vortexing in a test tube until all visible particulates were homogenized. The initial conidial suspension concentration was determined on an improved Neubaurer hemocytomer, and adjusted to desired concentrations using 0.1% Tween 80 and deionized water solution. The control treatments consisted o f a 0.1% Tween solution with no conidia and an additional treatment where the adult flies were knocked down with CO 2 and held without exposure to solutions to establish base line mortality. For each B. bassiana strain, 10 adult horn flies, 1 3 days post ec losion, were treated in groups. Flies were immobilized by CO 2 placed in a glass test tube containing 5 mL of a conidia solution or control solution, vortexed for 1 s, and strained in an 18 x 14 mesh steel window screen sieve, over a beaker, which collect ed the solution. Flies were gathered from the collection strainer with sterile forceps and placed into holding chambers. Exposure chambers were constructed from 90 x 10 mm Petri plates (Fisher Scientific, Fair Lawn, NJ), with 2.54 cm holes drilled in dis h lids and covered by screen (18 x 14 fiberglass mesh) which was secured with hot melt glue, and contained a 90 mm P5 filter paper (Fisher Scientific, Fair Lawn, NJ). The filt er paper was included to absorb excess water or feces.

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64 The horn flies were fed daily with citrated bovine blood soaked cotton balls placed over the screened hole. Mortality was measured 1 to 2 h post treatment and daily for seven days. Flies that were unable to walk or fly were considered dead. Each fungal strain assay was technic ally replicated 3 times for each fungal concentration with three true replicates, providing 90 horn flies per strain and concentration. Conidia I mpregnated on F ilter P aper C ontact A ssay Contact assay filter papers (90 mm, P5, Fisher Scientific, Fair Lawn, NJ) were treated on aluminum foil with conidia by placing 1 mL of the conidia solutions, as previously described, on each disc, and distributed in a circular motion with the pipetto r. Filter papers were allowed to dry for 1 h under a biological hood befor e use; after this time the treated filter papers were no longer visibly damp. For each B. bassiana strain, ~ 10 adult horn flies, 1 to 2 days post eclosion, were exposed in groups. Flies were immobilized by CO 2 and placed into a previously described expo sure chamber that each contained a treated filter paper disc. Contact time for the assay was 2 h. After the contact time had elapsed, the flies were immobilized by CO 2 and the treated filter paper disc was replaced with an untreated disc. Horn flies were fed daily with citrated bovine blood soaked cotton balls over the screened hole. Mortality was measured 1 to 2 h post treatment and daily for seven days. Horn flies that were unable to walk or fly were considered dead. Each fungal strain assay was tech nically replicated 3 times for each fungal concentration and 3 to 4 true replicates were performed, providing between 90 and 120 horn flies for each strain and concentration combination. Conidia in I nert C arrier F ormulations In this assay a small amount of conidia powder was first weighed and dissolved in 0.1% Tween 80 in deionized water, then the concentration of conidia in a known volume of solution was determined on an improved Neubaurer hemocytomer. Total conidia was then estimated by

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65 converting coni dia in the sample to a per gram of conidia containing harvested material. This allowed a calculation of conidia per gram of harvested material The conidia containing harvested material was diluted into the inert carrier, cornstarch (Great Value TM Walma rt, Bentonville, AR, USA), and was serially diluted in cornstarch to 1.0 x 10 7 conidia per g. One tenth of a gram of each concentration, 1.0 x 10 9 1.0 x 10 8 1.0 x 10 7 was applied to a filter paper disc (90 mm, P5, Fisher Scientific, Fair Lawn, NJ) that had been placed into the previously described chamber. Conidia and cornstarch mixtures were distributed as evenly as possible across the filter paper disc by lightly tapping the sides of the exposure chamber. Horn flies were immobilized by CO 2 and 10 adu lt flies were placed into each chamber. Following a 2 h exposure, the horn flies were immobilized by CO 2 and the filter paper and conidia containing dust was replaced with an unused filter paper. Horn flies were fed daily with citrated bovine blood soake d cotton balls over the screened hole. Mortality was measured 1 to 2 h post treatment and daily for seven days. Horn flies that were unable to walk or fly were considered dead. Each fungal strain assay was technically replicated 3 times for each fungal concentration and 3 to 4 true replicates were performed, providing between 90 and 120 horn flies for each strain and concentration combination. Statistics All replicates were aggregated and analyzed in JMP Pro 9.0.2 (SAS institute, Cary, NC, USA ) (JMP 200 7 ). Means and SEM standard error of the mean, were calculated using the distribution function. A one way Analysis of Variance (ANOVA) was performed on mortality data within each post exposure day, by comparing concentrations within strains and strains w ithin concentrations. A multiple factor ANOVA was conducted in SAS 9.2 (SAS institute, Cary, NC, USA) (SAS institute 2004) to test for strain, dose a nd strain x dose interactions. A dditionally orthogonal contrasts were applied to determine differences b etween the EN1 and

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66 two commercial strains, with a second evaluation between the two commercial strains. A dose. Bioassay data were pooled and transformed by pr obit analysis (Finnely 1971) using the Simple Probit add in within JMP Pro 9 ( SAS Institute Inc, Cary, NC, USA ) (JMP 2007 ) for each exposure method in an attempt to generate Lethal Concentration (LC) values for resistance ratio calculations. Abbott's tra nsformation (Abbott 1925) for control mortality correction was applied and regressed within JMP LT 50 values were generated by an inverse prediction of the mortality model generated for a repeated measures model in JMP using the analysis factors: day, d ose, strain, dose x strain, day x dose, and strain x strain. Results In over 7,000 horn flies sampled, only one fly yielded an isolate of B. bassiana EN1 (strain submitted for ARSEF numbering). This fly was collected from a beef cattle ranch near Gaine sville, FL on 24 November 2010 and was one of 250 horn flies examined for infection. No other fungal pathogens were observed among these 7,000 flies. In all immersion tests using a 0.1% Tween 80 and deionized water solution, Tween water control mortality was very high early in the experiment (Table 3 1). By post treatment day 3, 73.5% of control treatment flies were dead. In some replicates control treatment mortality reached 100% by day 3. Given the high control mortality, no statistical differences between strains and control groups could be detected. Control mortality was 23.6% at day seven for the assays where filter paper was treated filter paper assay s are presented (Table 3 2). Using this application technique, EN1 was not virulent generating negative adjusted values for all concentrations. St rain GHA was slightly

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67 virulent but did not follow a concentration response; wherein the middle concentrati on generated slightly higher mortality (12.7%) than the high or low dose (10.0 and 10.2%). Mortality in the JABB strain was greatest at the lowest concentration (12.2%). In all cases adjusted mortality was low at all doses on day seven and ranged from 0. 0 12.7%. Aggregated and analyzed by post treatment day, horn fly mortality following B. bassiana exposure to conidia on filter papers are presented in Table 3 3, with an accompanying set of F values (Table 3 4). For the overall model, strain was signific antly different between EN1, JABB, and GHA; while concentration and the interaction between concentration and strain were not significant (Table 3 4). Arithmetically EN1 mortality was considerably lower than GHA and JABB (Table 3 3). The F values present ed document that at post treatment days 4 through 7, GHA and JABB preformed similarly, with no differences found between them, whereas mortality in the EN1 was significantly lower (Table 3 4). Corrected mortality data for cornstarch formulations are prese nted in Table 3 5. At post treatment day 7, the EN1 strain showed increasing mortality with increased exposure concentration, from 3.5, 22.4, and 40.9% corrected mortality for concentrations ranging from 1 x 10 7 to 1 x 10 9 conidia per gram, respectively. Low mortality was observed with the GHA strain, and did not exceed 4% on day 7. The JABB strain resulted in low mortality at the lower two concentrations at post treatment day 7, but 18.73% mortality was observed at the highest concentration. Results of the treatment of horn flies with conidia dispersed into cornstarch are presented in Table 3 6, with associated F values presented in Table 3 7. Mean mortality of the most virulent strain, EN1, reached 58%, however, control mortality was 29.0% at day 7. No significant differences were found between concentrations, strains or their interaction. The only

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68 strain that followed a concentration response by day 7 post treatment was EN1, with increasing mortality as concentration increased (Table 3 6). Mortalit y observed with the remaining two strains, GHA and JABB, was variable and did not respond with increased mortality by increased concentration exposure. No statistical differences were observed between the EN1 strain and the two commercial strains or betwe en the two commercial strains using orthogonal contrasts. L C 50 values for the number of conidia required to infect 50% of adult horn flies on a given day were not established under our experimental conditions. Because corrected mortality did not exceed 50 % by day 7, reliable L C 50 or LT 50 values could not be generated. Discussion While evaluating the presence of entomopathogenic fungi in horn flies in Florida, I successfully discovered and cultured one isolate from a field infected fly. This isolate repre sents only the second report of fungi isolated from field collected horn flies; the first report is from Steenberg et al. (2001). Although my rate of discovery was lower than what was reported by Steenberg et al. (2001), their collection protocols and goa ls were different. Steenberg et al. (2001) collected live flies, but also searched extensively in agriculture barns and surrounding areas for fly cadavers, and reported a total of 70 infected flies (multiple species) out of over 6,000 incubated flies and cadavers. The aim of the current research was only on one host species, and the isolation methods may have excluded development of any fungi that had recently attached but not yet infected the fly hosts as field collected horn flies did not survive long o ff the cattle host. Furthermore, the biology of the horn fly likely interfered with collection of fungal strains. Horn flies are exclusively associated with their pastured cattle hosts and, other than dung pats, cannot be collected at off animal location s. Such a restriction prevented the acquisition of fly cadavers from a centralized location.

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69 The life expectancy of horn flies reported in literature is somewhat variable; Krafsur and Ernst (1983) reported that oviposition took place, on average, 3.5 d po st eclosion and adult flies only lived for 6.6 days on average, while life spans of 28 days to 8 weeks have been reported (summarized in Butler and Okine 1999). If the female flies contribute to the next generation early in their adult life, an entomopath ogenic fungus may not have enough time to be effective; although if they live longer an entomopathogenic fungus could be effective in reducing the adult population. However, if a highly virulent strain were to be developed, provided that it acted faster t han normal oviposition rates, an entomopathogenic fungus could be an effective addition to an IPM program. A better understanding of the life history of field populations is required before this can be confirmed with any certainty. The fungal strains in this study were not passed through a horn fly host to increase pathogenicity, as others have done. Passing a fungal strain through a host has been shown to change the characteristics of the fungi, by reducing LT 50 values for I. farinose (Hayden et al. 1 99 2). Instead inherent virulence of the fungi was evaluated, without any attempt at increa sing or selecting for virulent strains. Some isolates have been shown to be highly virulent to the hosts from which they were isolated, but this is not always the ca se. Several isolates from divergent hosts and hosts of interest have shown va riability in their virulence without host passages to other insects (Feng and Johnson 1990, Khachatourians 1992). Watson et al. (1990) showed that the same strains were differen t in their efficacy against house flies and stable flies, which could partially explain the differences observed between our strains. The bioassay results showed high control mortality following horn fly immersion into a Tween and water solution. This tec hnique has been used with other insects to apply entomopathogenic fungi (Khachatourians 1992, Nielsen et al. 2005), but not previously with

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70 horn flies. The treated flies had access to a dry filter paper inside of the exposure chamber, which helped remove excess solution from the flies. Some of the flies were observed to sporulate in the laboratory after such an exposure, although effects of the adjuvant may have had greater effects than the fungal agent. Water alone is an impractical carrier for B. bassi ana conidia, due to the hydrophobic nature of conidia. Water only solutions were unable to generate the conidial suspensions required for our assays with the conidia floating on the surface of the water. I hypothesize that the treatment of immersion into the Tween solution removed a protective wax coating on the horn flies cuticle or disrupted the cellular layers of the trachea and tracheoles within the respiratory system. The resultant observations of high control mortality indicated immersion in a 0.1% Tween 80 and deionized water solution is not an adequate method to evaluate pathogenicity of conidia against horn flies under o ur laboratory conditions. Because Tween is a surfactant it may act like an insecticidal soap toward the horn fly. Insecticidal soaps have been known to aid in the control of insects and have been shown by Szumlas (2002) to be active against the German cockroach, Blattella germanica (L.). Excluding the immersion technique for inoculation of horn flies our control mortality was si milar to what was seen by Lohmeyer and Miller (2006) (33.5% day 7), which utilized a horn fly colony with shared genetic stock. However, high control mortality is not always the case with horn flies, as Mochi et al. (2010b) reported lower (12.2% at day 15 ) control mortality. Each of our experiments had adequate controls, with either a Tween or starch only treatment, and a CO 2 only control to access mortality due to handling and CO 2 knockdown. In both Tween and cornstarch exposure methods, control mortal ity (Tween or cornstarch only) was very similar to the handling methods control (CO 2 knockdown). When maintained in colony, the Kerrville

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71 susceptible flies laid eggs around day 4; egg production peaked at day 5 and trailed off for 3 4 additional days. Th e field collected flies by Mochi et al. (2010 a ) were fed fresh blood twice daily and may have had a greater fitness than our laboratory colony. A range of concentrations as presented here was expected to elicit a concentration response line, but this was not the case in most experiments. When conidia were presented on filter papers, the commercial strains out performed the field collected strain, but never resulted s well in our case because it is important to note the actual percent mortality caused by the treatments; and prevents inflation due to the control mortalities. The cornstarch exposure method generated greater mortality, but also used higher doses. Also apparent in this method was a reversal of virulence, wherein the EN1 strain outperformed the commercial strains. In the cornstarch exposure the EN1 strain was the only strain to elicit a concentration response, with higher mortality at higher doses. Howe ver, for the cornstarch exposure method there were no differences between fungal strains. This result suggests that the EN1 strain has promise as a tool for horn fly management. The comparable stra ins were from commercial products that have been selected for production qualities over many years and are used in commercial application s That the EN1 strain performed equally to the commercial strains without selections, suggests that it may be good candidate for further development. Following multiple pass ages through horn flies, this strain could be selected to increase its virulence to its original host. That it performed poorly in the Tween containing studies suggests that the surfactant may have influenced its survival or capability to infect flies. R egardless, further development should focus on using a different adjuvant or no adjuvant practices for this specific strain.

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72 Beauveria bassiana was shown by Lohmeyer and Miller (2006) to generate 100% mortality of horn flies at day 7 post treatment. Howe ver, Lohmeyer and Miller (2006) utilized a Beauveria strain that was dispersed into a wettable powder formulation and applied without dilution, which may have resulted in an increased virulence. The formulation is a commercially available product, contain ing 9.09 x 10 8 conidia per gram of product. When concidering different volumes used in applying conidia, their application rate was over 4.5 times our highest application rate. Previous research studies examined a 12 hour spray inoculation at one concent ration (Mochi et al. 2010 a ), a continuous exposure assay at one conce ntration (Angel Sahagun et al. 2005), and a formulated product (Lohmeyer and Miller 2006). The variety of strains, concentrations, and application methods among these studies prevents me aningful comparisons. Establishment of an LC 50 or LT 50 at a specific concentration would provide a basis for evaluating strain and formulation differences. Unfortunately, our data did not meet the requirements for such a determination. Further work woul d be necessary with other application methods that could be mo re effective against this pest. The current work is unique in that it is the first evaluation of a horn fly Beauveria isolate examined against horn flies. Although most entomopathogenic fungi t end to be generalists some isolates can be host specific (summarized in Tanada and Kaya 1993). The search for highly pathogenic isolates should continue for incorporation into future control programs targeting important arthropod pests. Additionally, a confounding factor may be the impact of culturing fungal strains. Samsinakova and Kalalova (1982) showed the generation of spontaneous mutants from a parent colony that had greater or less virulence. Furthermore, they discuss the possible reduction of vi rulence in strains that are repeatedly sub cultured. Repeate d sub culturing without losing virulence and genetic isolation are a requirement for production of

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73 an isolate for production purposes. Further work should focus on isolating highly pathogenic st rains as well as working toward increasing the specificity of strains in culture. This work should be done in conjunction with efforts to develop formulations and application methods that recognize the unique challenge of delivering conidia to the horn fl y target. Integrated pest management seeks to continually develop and utilize novel methods for pest control; and horn flies have been seemingly overlooked in development of biological control agents. Due to their biology, ecology, pasture dwelling beha vior, and ability to develop insecticide resistant populations, alternative methods should continue to be investigated and developed to provide for future control of the horn fly.

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74 Table 3 1. Mortality of adult horn flies exposed by immersion into a Beau veria bassiana conidia dilution containing a 0.1% Tween 80 and water solution. Strain Concentration* Mean percent mortality on day post treatment 1 2 3 4 5 Control 0 2.0 33.0 73.5 GHA 0.1 0.0 15.0 68.4 2 0.0 17.5 75.8 4 14.8 23.1 91. 7 6 0.0 23.9 77.2 EN1 1 0.0 1.7 5.0 17.9 60.0 3 10.2 11.9 18.9 29.6 26.1 5 1.7 3.2 6.9 18.3 33.0 7 0.0 0.0 6.5 25.8 87.2 Concentration = 1.0 x 10 x conidia per mL of solution, where X is the value presented. Data not collected Standa rd errors are not included in this table; data are presented as a point of reference to illustrate high control mortality with this inoculation technique.

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75 Table 3 2. Mortality (corrected for controls) of adult horn flies exposed to Beauveria bassiana c oni dia dispersed into a 0.1% Tween 80 and water solutio n impregnated onto filter paper. Mean percent corrected mortality on day post treatment Concentration (conidia mL 1 ) Strain 4 5 6 7 1 x 10 5 EN1 0.0 0.0 0.0 0.0 GHA 2.4 4.9 5.1 10.2 JABB 9.9 1 2.2 12.0 12.2 1 x 10 6 EN1 0.0 0.0 0.0 0.0 GHA 1.8 6.1 5.4 12.7 JABB 10.9 10.9 8.6 9.8 1 x 10 7 EN1 0.0 0.0 0.0 0.0 GHA 7.5 8.7 9.6 10.0 JABB 6.4 8.5 9.1 8.1 One ml solution added to a 90 mm filter paper, subsequently dried with a 2 hr fly exposure. Corrected mortality generating negative values is presented as 0.0 (Abbott 1925). Tween 80 water control mortality was 17.4, 19.6, 21.7, and 23.6% for days 4, 5, 6, and 7, respectively. EN1 is a Florida collected, horn fly originated isola te, GHA (ARSEF 6444) and the JABB (HF23 strain, ARSEF 7940) are commercially available strains.

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76 Table 3 3. Mean (SEM) mortality of adult Haematobia irritans following exposure to Beauveria bassiana conidia impregnated filter paper discs Post T reatm ent Day Concentration (conidia mL 1 ) Strain 4 5 6 7 1 x 10 5 EN1 6.9 (2.0) 7.7 (2.0) 11.0 (2.5) 13.6 (2.7) GHA 19.4 (5.1) 23.5 (4.8) 25.7 (4.9) 31.4 (4.8) JABB 25.6 (4.5) 29.4 (5.1) 31.1 (5.1) 32.9 (5.3) 1 x 10 6 EN1 3.6 (2.3) 3.6 (2.3) 3.6 ( 2.3) 5.4 (2.9) GHA 18.9 (4.1) 24.5 (4.0) 25.9 (4.8) 33.3 (5.0) JABB 26.4 (6.4) 28.4 (7.4) 28.4 (7.4) 31.1 (7.1) 1 x 10 7 EN1 7.7 (1.9) 9.5 (9.8) 13.0 (2.7) 15.3 (2.7) GHA 23.5 (3.6) 26.5 (4.1) 29.2 (4.4) 31.2 (4.5) JABB 22.7 (4.7) 26.4 (5.2) 28.8 (5.3) 29.8 (5.2) EN1 is a field collected horn fly B. bassiana strain, GHA (ARSEF 6444) and the JABB (HF23 strain, ARSEF 7940) are commercially available strains.

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77 Table 3 4. F values for mean percent mortality of adult Haematobia irritans exposed to Beauveria bassiana conidia impregnated on filter paper in a 0.1% Tween 80 and water solution. Post Treatment Day Model effect 4 5 6 7 Concentration 0.10 0.11 0.52 0.19 Strain 13.54** 15.77** 13.61** 15.07** Concentration x Strain 0.35 0.24 0.28 0. 40 Contrasts EN1 vs GHA + JABB 25.90** 31.03** 26.96** 30.02** GHA vs JABB 1.48 0.74 0.41 0.03 **=p<0.01 EN1 is a field collected horn fly B. bassiana strain, GHA (ARSEF 6444) and the JABB (HF23 strain, ARSEF 7940) are commercially avail able strains.

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78 Table 3 5. Mortality (corrected for controls) of adult horn flies exposed to Beauveria bassiana conidia dispersed into a cornstarch formulation spread onto filter paper Mean percent corrected mortality on day post treatment Concent ration (conidia g 1 ) Strain 4 5 6 7 1 x 10 7 EN1 8.49 5.25 0.96 3.52 GHA 0.00 0.00 0.00 0.00 JABB 4.31 0.92 0.00 0.00 1 x 10 8 EN1 2.53 0.00 5.35 22.39 GHA 3.17 2.36 4.25 3.24 JABB 0.00 0.00 0.00 0.00 1 x 10 9 EN1 1.52 26.51 40.33 40.85 GHA 0.00 0.00 0.14 1.41 JABB 0.00 0.00 8.09 18.73 Conidia formulations were placed in 0.1 g quantities onto filter paper with a 2 hour exposure, mortality was assessed daily. Corrected mortality generatin g negative values is presented as 0.0. Control mortality for cornstarch without conidia was 21.1, 23.8, 27.1, and 29.0% for days 4, 5, 6, and 7, respectively. EN1 is a Florida collected, horn fly originated isolate, GHA (ARSEF 6444) and the JABB (HF23 st rain, ARSEF 7940) are commercially available strains.

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79 Table 3 6. Mean (SEM) mortality of adult Haematobia irritans following exposure to Beauveria bassiana conidia in starch formulation Post T reatment Day Concentration (conidia g 1 ) Strain 4 5 6 7 1 x 10 7 EN1 27.8 (12.7) 27.8 (12.7) 27.8 (12.7) 31.5 (11.5) GHA 15.3 (6.4) 16.5 (6.4) 20.1 (5.3) 21.3 (5.7) JABB 24.5 (7.4) 24.5 (7.4) 27.0 (7.3) 27.8 (7.6) 1 x 10 8 EN1 23.0 (7.8) 23.1 (7.8) 31.0 (9.8) 44.9 (16.0) GHA 23.6 (9.8) 25.6 ( 9.7) 30.2 (9.0) 31.3 (8.6) JABB 12.0 (4.1) 18.6 (5.3) 18.6 (5.3) 23.6 (6.2) 1 x 10 9 EN1 22.3 (9.1) 44.0 (16.8) 56.5 (19.5) 58.0 (18.9) GHA 18.7 (4.1) 21.0 (4.6) 27.2 (3.1) 30.0 (4.0) JABB 7.8 (3.4) 23.5 (10.0) 33.0 (12.1) 42.3 (13.9) EN1 is a field collected horn fly B. bassiana strain, GHA (ARSEF 6444) and the JABB (HF23 strain, ARSEF 7940) are commercially available strains.

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80 Table 3 7. F values for mean percent mortality of adult Haematobia irritans exposed to Beauveria bassiana conid ia in cornstarch formulations. Post Treatment Day Model effect 4 5 6 7 Concentration 0.54 0.57 1.88 1.92 Strain 1.20 1.08 1.47 2.03 Concentration x Strain 0.75 0.61 0.81 0.45 Contrasts EN1 vs GHA + JABB 1.76 2.13 2.94 GHA vs J ABB 0.62 0.03 0.00 0.23 value of 0.0543 EN1 is a field collected horn fly B. bassiana strain, GHA (ARSEF 6444) and the JABB (HF23 strain, ARSEF 7940) are commercially available strains.

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81 CHAPTER 4 I MPLICATIONS AND FUTU RE DIRECTIONS FOR HO RN FLY RES EARCH Control of the horn fly in pastured cattle has been achieved since WWII largely with the use of synthetic insecticides. This sole control method, coupled with horn fly biology, has helped select for insecticide tolerant and resistant populations. T here has been little work done to search for alternative control methods or the development an integrated pest management ( IPM ) program for horn flies. Insecticide resistance in the horn fly has been tied to the use of insecticide impregnated ear tags, fo llowing the emergence of this control technique in the early 1980 s (Kunz and Schmidt 1985). During the subsequent years, research was focused upon pyrethroids and identification of fi eld resistant horn fly populations (Byford et al. 1985, Sheppard and Hin kle 1985, Bull et al. 1988). Limited research was conducted on the association of non ear tag insecticide application techniques and non pyrethroid insecti ci des and their impacts on resistance selection (Sheppard 1987, Byford et al. 1998). Synergists eff ectively blocked metabolic detoxification of insecticides, yet as early as Byford et al. (1985), the addition of S,S,S Tributyl phosphorotrithioate ( DEF ) and piperonyl butoxide ( PBO ) were found to have limited effect on horn fly mortality. Bull et al. (19 88) also documented what appeared to be target site insensitivity for pyrethroids, a resistance mechanism that does not respond to synergist addition. From these pyrethroid resistance studies, kdr was characterized in horn flies (Guerrero et al. 1997, Jam roz et al. 1998); as such this research concurs with previous findings that the RR kdr genotype can be present in high numbers among horn fly populations (Foil et al. 2010). Organophosphate resistance levels reported in the literature typically have been low (Barros et al. 2001, Kaufman et al. 1999, Barros et al. 2007), and often presented as field control failures or reduced weeks of control (Barros et al. 2000). Rotational systems, utilizing multiple

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82 insecticide classes alternat ively have been impleme nted for insecticide use in horn fly control, when the pyrethroids began to lose efficacy (Kaufman 2011). E fforts have been conducted to determine the mode of action among resistant horn fly populatio ns. A promising acetylcholine sterase (AChE) mutation i n horn flies was described by Temeyer et al. (2008). This mutation, along with the characterization of the kdr mutation, was developed into a multiplex PCR by Foil et al. (2010). This technique was utilized in our research to identify the genetics of ind ividual horn flies at two point mutations tied to resistance, kdr and a mutation in the AChE (G262A). Coupled with contact bioassays, these data provided needed insights into the presence and development of multiple class insecticide resistance. If produ cers are to understand which treatments of insecticides are going to best control fly populations, future research should include measuring resistance to commonly utilized insecticides and determining not just if, but how, horn flies are resistant. This a pproach was utilized for three fly populations in Florida, wherein an organophosphate and pyrethroid target site insensitivity mutation did not fully explain the resistance levels observed among the field populations in live fly bioassays. Additional res istance mechanisms are possible; elevated but not significantly different, metabolic enzymes levels were found in this study. Identification of the metabolic factor that confers resistance should be more thoroughly evaluated in future research (Scott 198 9). However, complicating this process is the high off host mortality exhibited in the horn fly. Horn fly resistance has been evaluated numerous times in the past ; however selection pressures have likely changed, and resistance presence and intensity ne ed re evaluation at regular intervals. Additionally, insecticide integration into an IPM program likely will be required, yet success will be determined by individual horn fly genetics and inherent susceptibility to varied insecticides.

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83 This was observed by Oyarzun et al. (2011), who found pyrethroid resistant fly populations where no insecticide treatments had been applied for the previous 5 years. One potential future component of an IPM program was evaluated herein, utilization of Beauveria bassiana a s a biological control. Entomopathogenic fungi are used infrequently in veterinary entomology due to several f actors. First, application is challenging. T he life stage must be treated easily and must be susceptible to the fungus, and fungal treatments m ust persist in the environment. Indoors, persistence is not an issue, where the commercial formulation, Several aspects of the horn fly life history lend themselves to use of an entomopathogenic fungus; adult flies largely remain on the animal, leaving only intermittently and to oviposit. Steenberg et al. (2001) was the first to survey entomopathogenic fungi in horn flies, with this work representing only the second sur vey, although it was more limited in scope th an the Danish survey The Florida strain that was isolated proved to be pathogenic at high concentrations in cornstarch formulations, with greater virulence than the two commercial strains evaluated. Impregnat ing conidia onto filter paper discs at lower concentrations, the commercial strains, was not above 50%, and strain selections or host passage may increase strain vi rulence. If Beauveria is to be developed into an effective control tactic, it must cause mortality or suppress oviposition of the adult flies prior to their first oviposition cycle. Population dynamics of the horn fly have not been evaluated in widesprea d areas and knowledge of their basic ecology is lacking in the literature. Bruce (1964) reported lifespans up to 8 weeks, Harris et al. (1968) reported as few as two pre ovipositional days before egg production, and Krafsur and Ernst (1983) reported ovipo sition to occur 3.5 days post eclosion, and lifespan to culminate at 6.6

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84 days. Due to the varied nature of these reports, horn fly ecology should be evaluated before any field studies are to be conducted against horn flies Provided that fungal applicati ons reduce the lifespan of the fly or suppress oviposition, all adults could be treated easily using a dust formulation, as is commonly done with certain insecticides (Kaufman et al. 2011). Additional selection experiments on pathogenic fungal strains sho uld be conducted in an attempt to reduce the LT 50 to a level that would reduce the reproductive potential of horn flies. Lohmeyer and Miller (2006) generated LT 50 values as low as 2.7 days for Beauveria which is lower than what this study generated. How ever Lohmeyer and Miller (2006) utilized commercial formulations 4.5 times our greatest concentration (corrected for volume differences between studies). Adult horn flies are easily accessible, whereas larval stadia are in ephemeral dung pats. A focus u pon adult fly fungal applications may be a better tactic than inoculation of manure in feed through systems. This is particularly important should the fungus interrupt dung recycling, as is seen following the use of ivermectin (Schmidt 1983), if the funga l feed through has any effects upon the rest of the dung community. The future of horn fly control must include alternative treatment strategies. This could include limited use of insecticide impregnated ear tags, with other application techniques or d evelopment of effective biological controls. Horn fly resistance management programs should be focused upon preventing resistance selection, rather than resorting to mitigation of resistant flies. The number of available insecticide classes and their use for fly control need to be conserved, additionally, more active ingredients should be considered for addition to livestock fly control programs.

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85 APPENDIX A HORN FLY COLLECTIONS : NUMBERS, DATES, HE RD SIZES AND TIMES Table A 1. Horn Fly collections: numbers dates, herd sizes and times. Location Date Appx. No. Horn Flies Screened for fungi Heard Size* Appx. Time of Collection Gainesville 11 24 10 250 Medium 3:00 pm Kissimmee 0 6 28 11 300 Small 8:00 am Labelle 0 7 0 8 1 1 5000 Large 7:30 am Clewiston 0 7 26 11 500 Medium 9:00 am Ona 0 8 18 11 300 Large 1:00 pm BTU 10 18 11 300 Medium 10:00 am BRU 0 8 0 3 11 400 Large 9:00 am Small 1 20 Medium 20 75, large 75+ reproductive female bovines per ranch

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86 LIST OF REFERENCES Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265 267. Andersen, J. L. 2006. Pesticide Product; Registration Applications. (http://www.epa.gov/fedrgstr/EPA PEST/2006/February/Day 15/p2160.htm) Angel Sahagun, C. A., R. Lezama G utierrez, J. Molina Ochoa, E. Galindo Velasco, M. Lopez Edwards, O. Rebolledo Dominguez, C. Cruz Vazquez, W. P. Reyes Velazquez, S. R. Skoda, and J. E. Foster. 2005. Susceptibility of biological stages of the horn fly, Haematobia irritans to entomopatho genic fungi (Hyphomycetes). J. Insect Sci. 5: 50 (http://insectscience.org/5.50) Barros, A. T. M., A. Gomes, and W. W. Koller. 2007. Insecticide susceptibility of horn flies Haematobia irritans (Diptera: Muscidae), in the state of Mato Grosso Do Sul Brazil. Brazil. J. Parasitol. 16: 145 151. Barros, A. T. M., J. Ottea, D. Sanson, and L. D. Foil. 2001. Horn fly (Diptera: Muscidae) resistance to organophosphate insecticides. Vet. Parasitol. 96: 243 256. Bradford, M. M. 1976. A rapid and sensi tive method for the quantification of microgram quantities utilizing the principle of protein dye binding. Anal. Biochem. 72: 248 254. Bruce, W. G. 1938. A practical trap for the control of horn flies on cattle. J. Kans. Entomol. Soc. 11: 88 93. Bru ce, W. G. 1964. The history and biology of the horn fly, Haematobia irritans (Linnaeus); with comments on control. N C Exp. Sta t Tech. Bull. 157 : 1 33 Bradbury, S. 2008. Environmental Protection Agency: Fenvalerate; Product cancellation order, p p. 39294 39296. In Federal Register, Wed. Jul. 9, Vol. 73, No. 132. US Government Printing Office, Washington, D.C. Bull, D. L., R. L. Harris, and N. W. Pryor. 1988. The contribution of metabolism to pyrethroid and DDT resistance in the horn fly (Dipte ra: Muscidae). J. Econ. Entomol. 81: 449 458. Butler, J. F., and J. S. Okine. 1999. The horn fly, Haematobia irritans (L.): Review of programs on natural history and control, pp. 625 646. In J. F. Burger (ed.) Contributions to the knowledge of Diptera : a collection of articles on Diptera commemorating the life and work of Graham B. Fairchild. Associa ted Publishers, Gainesville, FL Byford, R. L. A. B. Broce, J. A. Lockwood, S. M. Smith, D. G. Morrison, and C. P. Bagley. 1987. Horn fly (Diptera: Mu scidae) dispersal among cattle herds. J. Econ. Entomol. 80: 421 426.

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89 Greer, N. I., and J. F. Butler. 1973. Comparisons of horn fly development in manure of five animal species. Fla. Entomol. 56: 197 199. Guerrero, F. D. 2000. Cloning of a horn fly cDNA, encoding an esterase whose transcript concentration is elevated in diazinon resistant flies. Insect Biochem. Molec. Biol. 30: 1107 1115. Guerrero, F. D., and A. T. M. Barros. 2006. Role of kdr and esterase mediated metabolism in pyrethroid resistant populations of Haema tobia irritans irritans (Diptera: Muscidae). J. Med. Entomol. 43: 896 901. Guerrero, F. D., M. W. Alison, Jr., D. M. Kammlah, and L. D. Foil. 2002. Use of polymerase chain reaction to investigate the dynamics of pyrethroid resistance in Haematobia ir ritans irritans (Diptera: Muscidae). J. Med. Entomol. 39: 747 754. Guerrero, F. D., R. B. Davey, and R. J. Miller. 2001. Use of an allele specific polymerase chain reaction to genotype pyrethroid resistant strains of Boophilus microplus (Acari: Ixodi dae). J. Med. Entomol. 38: 44 50. Guerrero, F. D., R. C. Jamroz, D. Kammlah, and S. E. Kunz. 1997. Toxicological and molecular characterization of pyrethroid resistant horn flies, Haematobia irritans : Identification of kdr and super kdr point mutations Insect Biochem. Molec. Biol. 27: 745 755. Guerrero, F. D., S. E. Dowd, V. M. Nene, and L. D. Foil. 2008. Expressed cDNAs from embryonic and larval stages of the horn fly (Diptera: Muscidae). J. Med. Entomol. 45: 686 692. Guerrero, F. D., S. E. Dow d, Y. Sun, L. Saldivar, G. B. Wiley, S. L. Macmil, F. Najar, B. A. Roe, and L. D. Foil. 2009. Microarray analysis of female and larval specfic gene expression in the horn fly (Diptera: Muscidae). J. Med. Entomol. 46: 257 270. Guerrero, F. D., S. E. K unz, and D. Kammlah. 1998. Screening of Haematobia irritans (Diptera: Muscidae) populations for pyrethroid resistance associated sodium channel gene mutations by using a polymerase chain reaction assay. J. Med. Entomol. 35: 710 715. Guerrero, F. D., T. J. Lysyk, and L. Kalischuk Tymensen. 2004. Expressed sequence tags and new gene coding regions from the horn fly. Southwest. Entomol. 29: 193 208. Guillot, F. S., J. A. Miller, and S. E. Kunz. 198 8. The physiological age of female horn flies (Dipt era: Muscidae) emigrating from a natural population. J. Econ. Entomol. 81: 555 561. Hajek, A. E., and R. J. St. Leger. 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39: 293 322. Hall, R. D. 1996. Walk Through Tra p to Control Horn Flies on Cattle. (http://extension.missouri.edu/p/G1195#performance)

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90 Harris, R. L., and J. A. Miller. 1969. A technique for studying the feeding habits of the horn fly. J. Econ. Entomol. 62: 279 280. Harris, R. L., E. D. Frazar, and C. D. Schmidt. 1968. Notes on the mating habits of the horn fly. J. Econ. Entomol. 68: 1639 1640. Hayden, T. P., M. J. Bidochka, and G. G. Khachatourians. 1992. Entomopathogenicity of several fungi toward the English grain aphid (Homoptera: Aphidida e) and enhancement of virulence with host passage of Paecilomyces farinosus. J. Econ. Entomol. 85: 58 64. Hinkle, N. C., D. C. Sheppard, K. Bondari, and J. F. Butler. 1989. Effect of temperature on toxicity of three pyrethroids to horn flies. Med. Vet Entomol. 3: 435 439 Hogsette, J. A., D. L. Prichard, and J. P. Ruff. 1991. Economic effects of horn fly (Diptera: Muscideae) populations on beef cattle exposed to three pesticide regimes. J. Econ. Entomol. 84: 1270 1274. Hu, F. Y. and J. H. Fran k. 1996. Effect of the arthropod community on survivorship of immature Haematobia irritans (Diptera: Muscidae) in north central Florida. Fla Entomol. 79: 497 503. Hu, F. Y. and J. H. Frank. 1997. Predation on the horn fly (Diptera: Muscidae) by fi ve species of Philonthus (Coleoptera: Staphylinidae). Environ. Entomol. 26: 1240 1246. Humber, R. A. 2011. Collection of entomopathogenic fungal cultures. In ARSEF Catalog of Strains. (http://www.ars.usda.gov/SP2UserFiles/Place/19070510/ Catalog %20 Jan2011.pdf). Jackson, H. C. 1989. Ivermectin as a systemic insecticide. Parasitol. Today. 5: 146 156. Jamroz, R. C., F. D. Guerrero, D. M. Kammlah, and S. E. Kunz. 1998. Role of the kdr and super kdr sodium channel mutations in pyrethroid resistan ce: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies ( Haematobia irritans ). Insect Biochem. Molec. Biol. 28: 1031 1037. JMP 2007. JMP Statistics and Graphics Guid e, SAS Institute Inc., Cary, NC Kau fman, P. E, P. G. Koehler, and J. F. Butler. 2011. External parasites on beef cattle. Electronic Data Information Source, document ENY 274 (IG130). University of Florida, Gainesville, FL. Kaufman, P. E., J. E. Lloyd, R. Kumar, and T. J. Lysyk. 1999. Horn fly susceptibility to diazinon, fenthion, and permethrin at selected elevations in Wyoming. J. Agric. Urban Entomol. 16: 141 157. Kaufman, P. E., J. G. Scott, and D. A. Rutz. 2001 Monitoring insecticide resistance in house flies (Diptera: Mu scidae ) from New York dairies. Pest Manag. Sci. 57: 514 521.

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91 Khachatourians, G. G. 1992. Virulence of five Beauveria strains, Paecilomyces farinosus and Verticillium lecanii against the migratory grasshopper, Melanoplus sanguinipes J. Inv. Pathol. 59: 2 12 214. Krafsur, E. S., and C. M. Ernst. 1983. Physiological age composition and reproductive biology of hron fly populations, Haematobia irritans irritans (Diptera: Muscidae), in Iowa, USA. J. Med. Entomol. 20: 664 669. Kunz, S. E. 1991. Dynamics of permethrin resistance in a colony of horn flies (Diptera: Muscidae). J. Med. Entomol. 28: 63 66. Kunz, S. E., and C. D. Schmidt. 1985. The pyrethroid resistance problem in the horn fly (Diptera: Muscidae). J. Agri. Entomol. 2: 358 363. Kunz, S. E. J. A. Miller, P. L. Sims, and D. C. Meyerhoeffer. 1984. Economics of controlling horn flies (Diptera: Muscidae) in range cattle management. J. Econ. Entomol. 77: 657 660. Kunz, S. E., K. D. Murrel, G. Lambert, L. F. James, and C. E. Terrill. 1991. Estimated losses of livestock to pests pp. 69 98 In Pimentel, D. ( e d.), CRC Handbook of Pest Management in Agriculture, Vol. I. CRC Press, Boca Raton, FL Laake, E. W. 1946. DDT for the control of the horn fly in Kansas. J. Econ. Entomol. 39: 65 68 Lee, S. H., T. J. Smith, D. C. Knipple, and D. M. Soderlund. 1999. Mutations in the house fly Vssc1 sodium channel gene associated with super kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes. Insect. Biochem. Molec. Biol. 29: 185 194. Li, A. Y., F. D. Guerrero, and J. H. Pruett. 2007. Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Musc idae). Pest. Bioch. Phys. 87: 147 155. Li, A. Y., F. D. Guerrero, C. A. Garcia, and J. E. George. 2003. Survey of resistance to permethrin and diazinon and the use of a multiplex polymerase chain reaction assay to detect resistance alleles in the horn fly, Haematobia irritans irritans (L.). J. Med. Entomol. 40: 942 949. Li, A. Y., K. H. Lohmeyer, and J. A. Miller. 2009. Dynamics and mechanisms of permethrin resistance in a field population of the horn fly, Haematobia irritans irritans. Insect Sci. 16: 175 184. Lohmeyer, K. H., and J. A. Miller. 2006. Pathogenicity of three formulations of entomopathogenic fungi for control of adult Haematobia irritans (Diptera: Muscidae). J. Econ. Entomol. 99: 1943 1947.

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96 BIOGRAPHICAL SKETCH Chris Holderman grew up in Garnett, Kansas and graduated from Iola High S chool in May of 2006. During this time his interest in agriculture developed working on a family farm, which had a production cow calf operation. During high school he was active in the National FFA Organization which ignited his interest in entomology After graduation he went to Kansas State University (KSU) and majored in a nimal s cience and i ndustry, concurrently minoring in e ntomology. While at KSU he joined the laboratory of Dr. Ludek Zurek working on several projects during his undergraduate tra ining. Chris was offered a teaching assistantship under the direction of Dr. Rebecca Baldwin at the University of Florida where he began his training for his M aster of S cience degree in the f all 2010 semester. While at UF his research project was super vised by Dr. Phillip Kaufman. His are to become a research entomologist working with emphasis on livestock pests. He is attending UF for a doctorate beginni ng f all 2012. His research will be supervised by Dr. Phillip Kaufman.