University of Florida Thesis Or Dissertation

ASSESSING VIRULENCE OF Beauveria bassiana AGAINST HOUSE FLY (Musca domestica) IMMATURES AND ADULTS

ROXIE LOURENE WHITE

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

To my mother for her never-ending love and support

ACKNOWLEDGMENTS

Above all, I want to express great appreciation to my main advisor, Dr.

Christopher Geden for his support, knowledge, and guidance throughout this process.

His encouraging words matched with his eternal patience has made this learning adventure a manageable and pleasurable experience. His expert knowledge helped formulate the methods of my research and he answered my relentless questions in the laboratory. I am greatly appreciative of Dr. Geden for providing the funding necessary for me to further my education as well as employing me as a full-time technician at the

USDA. He has taught me many things about life both within and outside of the laboratory and for that, I am thankful.

I would also like to show great appreciation to my co-chair, Dr. Phillip Kaufman, for consistently guiding me, providing feedback and for pushing me to take things a step further than the minimally required. Through his weekly writing meetings, my writing has improved tremendously, while being able to edit scientific literature for other students. I am very thankful and appreciative of the lessons learned through Dr. Kaufman’s writing club. Although I may have tried to hide from Dr. Kaufman at one point or another, he has always had an open door and open lab to make me feel very welcome and part of his lab.

An extended thank you is due to Dr. Emma Weeks as she has served almost as an unofficial committee member. She has graciously reviewed all my writing and has provided tips, tricks and insights on things I may have overlooked. She has also provided a soft place when things got tough and remained encouraging while not pushing too hard during the sensitive times.

Tremendous thanks are extended to the members of the Geden lab. In particular,

Dana Johnson has been a very critical aspect of my success. She has taught me most of what I know about fungal culturing, sterile lab procedures and microbiology techniques. Dana has been a huge help in preparing fungal strains, isolating strains for pure cultures and assistance in bioassays. She has been a listening ear when things got tough, always encouraged me to “just keep swimming”, and helped voice some of the hesitations I was too afraid to speak about. Thanks are extended to Lindsey Granko,

Katie Carrol and Rachel Dillard for maintaining house fly colonies, keeping the laboratory in tip-top shape and their assistance in bioassays.

Great thanks are due to my friends, family, and colleagues for helping me in various ways throughout the last few years. I would like to thank my mother Rhonda

White for always encouraging me to become the best while pursuing my dreams. She has remained encouraging and supportive for the duration of my educational journey including the last two years of graduate school. Without her support, graduate school would not have been possible. Lastly, I would like to thank my four-legged children for picking me up daily with their endless wet kisses and wagging tails. Coming home to a happy family of animals was a very enriching part of my day during the stressful parts of graduate school.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

ABSTRACT ...... 11

CHAPTER

1 LITERATURE REVIEW OF FILTH FLIES AND ENTOMOPATHOGENIC FUNGI FOR HOUSE FLY MANAGEMENT ...... 13

Introduction ...... 13 House Fly Biology and Life History ...... 14 Medical, Veterinary and Economic Importance ...... 16 Need for IPM to Manage House Flies ...... 16 House Fly Parasitoids ...... 19 Entomopathogenic Fungi Overview ...... 22 Beauveria bassiana: Biology, History, and Development...... 25 Virulence of Beauveria bassiana and Metarhizium anisopliae against Adult House Flies ...... 27 Research Objectives ...... 29

2 EXPOSURE OF HOUSE FLY LARVAE TO Beauveria bassiana ...... 30

Introduction ...... 30 Materials and Methods...... 32 House Fly Rearing ...... 32 Beauveria bassiana used in Assays ...... 33 Larval Test 1: Assays with Dry L90 Conidia and BotaniGard® ...... 35 Larval Test 2: Effect of Host age and Dose on Response to Treatment with B. bassiana...... 35 Larval Test 3: Effect of Temperature and Host Age on Response to Treatment with B. bassiana...... 37 Larval Test 4: Effect of Medium Composition and Host Age on Response to Treatment with B. bassiana...... 38 Statistical Analysis ...... 39 Results ...... 40 Larval Test 1: Assays with Dry L90 Conidia and BotaniGard® ...... 40 Larval Test 2: Effect of Host age and Dose on Response to Treatment with B. bassiana...... 41

Larval Test 3: Effect of Temperature and Host Age on Response to Treatment with B. bassiana...... 42 Larval Test 4: Effect of Medium Composition and Host Age on Response to Treatment with B. bassiana...... 42 Discussion ...... 43

3 SELECTION FOR A FASTER-KILLING Beauveria bassiana STRAIN ...... 51

Introduction ...... 51 Materials and Methods...... 55 Sources of Flies and B. bassiana ...... 55 Selection for Early-dying Flies ...... 56 Comparing Virulence of Selected and Unselected B. bassiana strain NFH10.. 58 Statistical Analysis ...... 61 Results ...... 61 Discussion ...... 62

4 COMPARISON OF VIRULENCE OF Metarhizium anisopliae AND FOUR STRAINS OF Beauveria bassiana AGAINST ADULT HOUSE FLIES ...... 75

Introduction ...... 75 Materials and Methods...... 77 Sources, Isolation and Propagation of Fungal Strains ...... 78 Harvesting of Conidia and Preparation of Inocula for Testing ...... 81 Bioassay Method ...... 82 Statistical Analysis ...... 84 Results ...... 84 Discussion ...... 85

5 DISCUSSION OF FINDINGS AND FUTURE RESEARCH DIRECTIONS FOR USING Beauveria bassiana AGAINST HOUSE FLY (Musca domestica) IMMATURES AND ADULTS ...... 100

LIST OF REFERENCES ...... 105

BIOGRAPHICAL SKETCH ...... 119

LIST OF TABLES

Table page

2-1 Percent pupation and percent adult emergence of Musca domestica after 100 second instar larvae were exposure to Beauveria bassiana in the form of dry L90 conidia or an aqueous suspension of BotaniGard® ES...... 47

2-2 Mean (SE) percent pupation and percent adult emergence of Musca domestica after exposure to BotaniGard® ES (Beauveria bassiana strain GHA)...... 48

2-3 Effect of temperature and conidia dose on percent pupation and percent adult emergence after Musca domestica larvae were exposed to BotaniGard® ES (Beauveria bassiana strain GHA)...... 49

2-4 Effect of larval medium and dose of B. bassiana (BotaniGard®) on house fly (Musca domestica) pupation and emergence when conidia were added to medium 1 or 2 days after egg placement...... 50

3-1 Adult house fly LT50 and LT95 values at different concentrations for an unselected Beauveria bassiana (strain NFH10) and for a strain that was selected for 10 generations for faster mortality...... 72

3-2 Adult house fly LC50 and LC95 values post-exposure to an unselected Beauveria bassiana (strain NFH10) and for a strain that was selected for 10 generations for faster mortality...... 73

4-1 LT50 and LT95 values (in days) for female house flies exposed to a 0.1% CapSil® solution containing 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana (strains GHA, HF23, NFH10 and L90) or Metarhizium...... 95

4-2 LC50 and LC95 values expressed as the log function for female house flies exposed to 0.1% CapSil® containing 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana (strains GHA, HF23, NFH10 and L90) or...... 97

LIST OF FIGURES

Figure page

3-1 Knockdown device with CO2 tube inserted for anesthetizing adult house flies (Musca domestica L.) for experimental set up, A) assembled knockdown device used to anesthetize flies with CO2,...... 65

3-2 Wet harvesting Beauveria bassiana (Balsamo) Vuillemin using 0.1% CapSil®. Notice the left side of the plate has been scraped with the sterile plastic disposable spatula shown...... 65

3-3 Flask, funnel and squeeze bottle used for wet harvesting Beauveira bassiana (Balsamo) Vuillemin, A) glass wool placed inside glass funnel that is placed on top of 250 mL flask ...... 66

3-4 Beauveria bassiana (Balsamo) Vuillemin conidia collected in flask, while debris remains in glass wool inside funnel after wet harvest...... 67

3-5 Observation containers used to contain adult house flies (Musca domestica L.) in bioassays, A) observation container is made from 473 mL deli cups, food and water is provided at libitum...... 67

3-6 Mortality (%) of adult house flies, Musca domestica L., seven days after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of an unselected...... 68

3-7 Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 109 colony forming units of an unselected Beauveria bassiana (Balsamo) ...... 69

3-8 Mortality (%) of adult house flies, Musca domestica L., nine days after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of an unselected Beauveria bassiana ...... 70

3-9 Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 108 colony forming units of an unselected Beauveria bassiana...... 71

4-1 Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 109 colony forming units of Beauveria bassiana (Balsamo)...... 91

4-2 Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 108 colony forming units of Beauveria bassiana (Balsamo) ...... 92

4-3 Mortality (%) of adult house flies, Musca domestica L., seven days after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana ...... 93

4-4 Mortality (%) of adult house flies, Musca domestica L., nine days after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana ...... 94

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science

ASSESSING VIRULENCE OF Beauveria bassiana AGAINST HOUSE FLY (Musca domestica) IMMATURES AND ADULTS

Roxie Lourene White

December 2019

Chair: Christopher Geden Major: Entomology and Nematology

Resistance to chemical insecticides has fueled investigation of alternative control strategies for many insect species, including the house fly (Musca domestica L.).

Entomopathogenic fungi have shown promising results for managing house fly populations while being safe for humans and other animals. The overall objective was to analyze the effectiveness of Beauveria bassiana against house fly immatures and adults. Beauveria bassiana strains GHA and L90 were evaluated against larval house flies. Beauveria bassiana strains GHA, HF23, L90 NFH10 and Metarhizium anisopliae strain F52 were evaluated against adults. Another objective was to select for a faster adult house fly-killing strain (NFH10) using a fly-to-fly passage system.

Larval treatments largely were unsuccessful at providing effective control in that reductions in pupation and adult emergence rates were only significant when massive numbers of conidia were applied during a narrow window of susceptibility. Fly immatures were susceptible to B. bassiana only in larval medium when conidia were applied 1-2 days after deposition of eggs. Mid- to late-stage third instars were refractory to infection at any concentration. Contact exposure of adult flies to either B. bassiana or

M. anisopliae conidia (106–109 cfu/mL) caused significant mortality compared to the

controls. Based on LC and LT values, strains GHA and NFH10 were generally the most virulent for adult flies. Ten generations of selection for a faster killing sub-strain did not result in substantial change in virulence against adult flies. These findings demonstrate that management efforts using B. bassiana should focus on adult control.

CHAPTER 1 LITERATURE REVIEW OF FILTH FLIES AND ENTOMOPATHOGENIC FUNGI FOR HOUSE FLY MANAGEMENT

House flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.),

(Diptera: Muscidae) are major pests to both humans and livestock. These nuisance fly species belong to a group known as filth flies because of their attraction to decaying organic matter and fecal deposits, which provide resources as larval development sites.

House flies are a cosmopolitan species that thrive in diverse geographical locations and occur across the globe, except in Antarctica. House flies are pests in livestock facilities such as milk processing facilities, feedlot and meat processing facilities, poultry and swine operations, and equine farms (Malik et al. 2007).

Introduction

House flies pose medical and veterinary concern as they are capable of mechanically vectoring over 100 disease-causing organisms that affect both humans and other animals (Graczyk et al. 2001, Malik et al. 2007, Shah et al. 2016).

Economically damaging house fly and stable fly populations are a concern to farmers as unrestricted fly numbers decrease animal production and performance, which reduces profits (Catangui et al. 1997, Taylor et al. 2012). In addition, farmers potentially can face legal problems if fly populations migrate off farm to nearby establishments and cause annoyance and public health risks (Skoda and Thomas 1993).

Multiple methods are utilized to decrease fly populations such as biological, cultural, and chemical controls. Extensive use of Dichlorodiphenyltrichloroethane (DDT) to manage filth flies led to insecticide resistance (Varzandeh et al. 1954) that subsequently has shaped the way in which fly populations are managed today. The failure of chemical management alone has led to the widely adopted strategy of

combining management tactics together to suppress filth flies, known as integrated pest management (IPM). Using natural enemies to reduce filth fly populations without increasing chemical pressure is a component of IPM. Biological control can be carried out with naturally-occurring pupal parasitoids and entomopathogenic fungi, such as

Beauveria bassiana, both of which target house flies (Machtinger and Geden 2018,

Weeks et al. 2018).

House Fly Biology and Life History

Adult house flies are non-biting and have modified sponging mouthparts from which they regurgitate saliva onto solid foods creating a liquid diet of predigested material (West 1951, Graczyk et al. 2001). Adults are 6–8 mm in length with females typically longer than males, and each sex has four dark stripes on a grey thorax

(Chapman 1998, West 1951). Females have nine abdominal segments, of which only the first five are noticeable, while males only have eight segments and have a darkened tip to their abdomen (West 1951).

House flies exhibit complete metamorphosis with discrete egg, larval, pupal and adult life stages. Female adult house flies typically lay their eggs on moist manure and in livestock bedding (Shah et al. 2016). Flies can lay eggs as early as four days post emergence (Lysyk and Axtell 1987), and the eggs hatch into larvae within 24 hours. The larvae develop through three instars before pupating around 5 to 9 days post oviposition, depending on temperature (Lysyk and Axtell 1987). Time from pupal to adult stage is highly dependent on temperature and pupae can survive for an extended period if maintained at cooler temperatures, thus allowing emergence only once the temperature increases (Larsen and Thompson 1940, Lysyk and Axtell 1987). The typical life cycle in warm environments (30 °C) is 7 to 10 days for house flies and 14

days for stable flies (Larsen and Thompson 1940, Hinkle et al. 2001, Stafford 2008).

Female house flies can produce up to 1,000 eggs in her life time (LaBrecque et al.

1972), laying 120-150 eggs per batch (James 1947). The quantity of eggs each female can contribute, along with the short development time of the fly, means that populations can reach overwhelming numbers rapidly in the absence of control efforts.

The average adult longevity of house flies in the field is approximated at 10 to 14 days (Hogsette and Farkas 2000), but adults can survive for up to two months under laboratory conditions (West 1951). Warm summer months experience the highest fly numbers, with populations in North America remaining high through September and tapering off and reaching low numbers in the cooler months (LaBrecque et al. 1972).

House flies can travel considerable distances searching for food and oviposition substrates (Sacca 1963), with dispersal of 20 km from release sites being documented

(Bishopp and Laake 1921). The long-range dispersal flights of house flies results in the movement of flies between farms and dispersal from farms (Pickens et al. 1967), frequently resulting in high fly populations in residential areas surrounding concentrated animal feeding operations (Winpisinger et al. 2005) and posing potential legal issues to farmers (Thomas and Skoda 1993).

House flies are attracted to a variety of substrates containing decomposing organic matter. Immature house flies develop in a wide range of substrates that contain decaying organic waste. In addition to laying eggs on moist manure, straw and other livestock bedding (Schmidtmann 1988, Hogsette and Farks 2000, Machtinger et al.

2014), house flies will also lay their eggs on other attractive sites include decaying hay and vegetation, spilled feed (Meyer and Petersen 1983, Skoda and Thomas 1993),

stockpiled manure (Skoda et al. 1991, Lysyk 1993), and compost piles (Skoda and

Thomas 1993). Horse manure has been identified as a preferred substrate for house fly oviposition and development (Bishopp 1915, Coffey 1951). The release of ammonia from horse manure is what makes this substrate an attractive oviposition site (Yates et al. 1952), making fermenting fresh horse manure a favorite option for oviposition

(Ehmann 1997).

Medical, Veterinary and Economic Importance

House flies are known mechanical vectors of over 100 disease-causing organisms affecting both humans and animals (Malik et al. 2007, Khamesipour et al.

2018). Prevention, diagnosis and treatment of these diseases have an economic impact on the community. When flies leave an animal facility, they can arrive in nearby neighborhoods, schools and businesses. This movement of flies from farms to areas of urban development can cause legal problems to the farmer because of disease concerns and annoyance. Stable flies cause additional losses because their blood- feeding behavior can decrease livestock productivity and animal physical condition

(Taylor et al. 2012). Dislodging flies throughout the day takes energy away from livestock’s ability to rest and eat, and thus, can affect weight gain or milk production

(Catangui et al. 1997, Taylor et al. 2012). It is estimated that economic losses associated with stable flies are $2.2 billion per year (Taylor et al. 2012).

Need for IPM to Manage House Flies

Extensive use of chemical insecticides to control M. domestica has been a standard management practice for decades. Constant insecticide pressure has resulted in M. domestica developing resistance to new classes of insecticides. Resistance usually develops within the first few years of an active ingredient being released onto

the market (Yasutomi 1973; Plapp 1984; Scott et al. 1989, 2000, 2013; Kaufman et al.

2001a, Shah et al. 2015). Resistance to multiple active ingredients has created a need for alternative control methods that reduce house fly populations.

To control fly populations, farmers and ranchers should have a well-defined integrated pest management (IPM) plan. IPM programs aim to maintain pest populations below acceptable levels in an economical and efficient manner, while minimizing damage to human health and the environment. The foundation of an IPM program is based on identification of the pest insect, determining the economic threshold level, monitoring of pest populations, and utilization of multiple control methods, which includes preventive methods and selective use of insecticides. The beneficial aspect of IPM is the combination of cultural, mechanical, biological, and chemical control strategies for providing sustainable long-term pest management. One goal of IPM is to decrease the reliance on and minimize the use of insecticides

(Radcliffe et al. 2009). However, an IPM plan should preemptively identify which chemical products will be utilized and when the use of those chemicals is deemed necessary.

The economic threshold is defined by the population level at which management intervention should be initiated to prevent pest populations from reaching the economic injury level (Stern et al. 1959). Two important steps in a successful IPM program are identification and monitoring of the pests. It is important to be able to distinguish between house flies and other common fly species as economic injury levels and control strategies vary between species. Before fly control methods are utilized, population size must first be assessed. Spot cards, sticky tapes, baited traps, and direct

observations are accepted methods for trapping and monitoring house fly populations

(Williams 1973, Lysyk and Axtell 1986). Between the years 1911 and 1995, at least 14 baited trap types were used to monitor fly numbers. Although the goal of these traps is to contain and monitor fly populations, traps may actually facilitate immature fly development sites (Howard and Bishop 1924, Fenton and Bieberdorf 1936, Davidson

1962, Pickens 1995). Fly populations should be monitored prior to, during, and after management efforts are applied in order for success or failure to be determined.

Cultural control involves changing the environment in which fly larvae are developing including reduction of development sites by proper manure management, and prevention of contact between flies, humans, and other animals (Keiding 1986,

Machtinger et al. 2015). House flies are attracted to decaying organic matter and the reduction of these materials can help to reduce fly presence and proliferation potential.

Sanitation of facilities is time-consuming, labor-intensive, and tedious; however, daily removal of fly development sites can reduce adult house fly populations by two-thirds

(Pickens and Miller 1987). Farms with the best sanitation practices typically have the lowest house fly populations (Kaufman et al. 2005). Research has shown that modifying the environment through cultural control methods was the most important method for controlling stable flies (Greene 1993), yet this strategy is often neglected, partly due to a lack of time (Machtinger et al. 2012). Other reasons why cultural control strategies are underused include a shortage of labor and appropriate machinery, gap of education and lack of recognition of potential immature fly developmental sites, and environmental conditions that are out of human control. Mechanical control includes the application of energy consuming tactics such as manipulation of manure piles to make them

unsuitable as fly developmental sites, which can be as simple as covering or composting the manure or spreading it over a field to encourage drying (Machtinger et al. 2015). Installation of screens or air curtains to deny flies access to sensitive areas such as milk rooms and food service areas is another example of mechanical control.

Biological control methods involve a diverse array of approaches, which are perceived as safer options when compared to chemical insecticides (Howarth 1991).

The immature stages of house flies are susceptible to several natural enemies. Control strategies can exploit the susceptibility of immature house flies by incorporating predators, parasitoids, as well as fungal, bacterial and viral pathogens, all of which have been shown to be successful under certain conditions (Malik et al. 2007). However, each of the house fly biological control methods has limitations. Due to their sensitivity to biotic and abiotic factors, successful field application of biological control agents may be hindered (Barbosa 1998). An overview of the biological control agents of interest is presented below.

House Fly Parasitoids

Filth fly parasitoids are natural enemies of muscoid flies, which decrease the fly population without disturbing humans or other animals (Machtinger et al. 2015,

Machtinger and Geden 2018). Pupal parasitoids have been considered a viable biological control option to manage fly densities since they were successfully used to suppress fly populations over 40 years ago (Morgan et al. 1975). Several parasitoid species are available for purchase through commercial insectaries, and the selection of appropriate parasitoid species remains an inexact science.

Hymenopteran parasitoids used for filth fly management are members of the family Pteromalidae. Pteromalidae is a large family consisting of approximately 3,506

species (Noyes 2017). Members of Pteromalidae are diverse and many species play an integral part of IPM programs. The different parasitoid species used in fly management vary in their developmental time, foraging behavior, host preference, habitat choice, distance traveled to locate pupae and attack rate (Machtinger et al. 2015). As reviewed by Machtinger et al. (2015), there are six commercially available species including:

Muscidifurax raptor Girault and Sanders, Muscidifurax raptorellus Kogan and Legner,

Muscidifurax zaraptor Kogan and Legner, Spalangia cameroni Perkins, Spalangia endius Walker and Nasonia vitripennis (Walker).

The release of parasitoids to manage fly populations has been effective in many studies (Rutz and Axtell 1979, Morgan and Patterson 1990, Geden et al. 1992,

Petersen et al. 1992, Petersen and Cawthra 1995, Crespo et al. 1998, Geden and

Hogsette 2006). However, the suppression of fly populations is not consistent across all parasitoid release studies and some studies were unsuccessful at lowering fly populations (Meyer et al. 1990, Andress and Campbell 1994, Weinzierl and Jones 1998,

McKay and Galloway 1999, Kaufman et al. 2001b). Possible reasons for conflicting results in prior studies is partly attributed to the environmental factors that affect parasitoid abundance and distribution (Skovgård 2004). Likewise, the release of species not adapted to the climatic conditions in which the studies were conducted could result in poor parasitoid performance (Rutz and Axtell 1980, Petersen et al. 1983, Geden et al.

1992). Previous studies emphasized the importance of releasing house fly parasitoids that are climatically adapted to the environment in which they are released (Legner and

Olton 1971, Tingle and Mitchell 1975). Other possible reasons for contradictory results include site-specific environmental factors such as sensitivities to insecticides,

availability of hosts, microhabitat preferences, and lack of optimal timing and release methods (Petersen and Meyer 1985).

The life cycle of parasitoids is similar to filth flies in the aspect that they undergo complete metamorphosis which includes egg, larva, pupa and adult stages. In comparison to the dipteran species, parasitoid developmental time is varied among species and dependent on temperature (Lysyk 2004). The normal range of parasitoid development is 2 to 4 weeks, which is longer than the development time for flies

(Machtinger and Geden 2018). The longer developmental time of pupal parasitoids can be problematic when trying to manage the fly population with parasitoids alone

(Machtinger and Geden 2018). This is why a well-defined IPM plan is critical. The female parasitoid deposits venom and one (solitary species) or more (gregarious species) eggs into the fly puparium, and the developing parasitoid consumes the developing fly in order to survive, thus causing fly mortality (Machtinger et al. 2015).

Both the male and female parasitoids must consume the fluids of fly pupae to survive, providing another means of killing the host. Females feed on host pupa by drilling a hole through the puparium and feeding on the resulting pupal exudate of hemolymph. Males also feed on these exudates. Host-feeding by females adds an additional host mortality component that can be 30% of the mortality due to oviposition and parasitism.

There are several species of parasitoids and each have different qualities and thus the release of more than one species may improve efficacy (Machtinger and

Geden 2018). Two of the most commonly used parasitoid species are M. raptor and S. cameroni. Both are solitary species that oviposit a single egg into the fly puparium,

resulting in only one wasp offspring per fly pupa; however, the biology of the two species is quite different.

Parasitoids in the genus Spalangia are known for inhabiting and searching deep into substrates to locate and parasitize fly pupae, whereas Muscidifurax species concentrate their searching near the surface (Rueda and Axtell 1985, Geden 2002,

Floate and Gibson 2008). Under natural conditions, S. cameroni must contend with competition from other parasitoid species including M. raptor. Muscidifurax raptor has a shorter development time and higher fecundity than S. cameroni, giving it an advantage in interspecific interactions. Muscidifurax raptor also has an aggressive and highly mobile first larval stage that searches within the host puparium to kill any other parasitoids that are already present. Spalangia cameroni lack this aggressive characteristic and therefore are at a distinct disadvantage when they are forced to compete with M. raptor (Wylie 1972; Machtinger et al. 2015). This may account in part for the different depths in which they search a habitat for suitable hosts, with the two species concentrating their searching in different zones to avoid competition with each other.

Entomopathogenic Fungi Overview

Naturally-occurring entomopathogenic fungi are well known for their deleterious effects to many insect species (Butt et al. 2001). Qualities that make an entomopathogenic fungus successful include rapid germination, abundant sporulation, efficient discharge of conidia to transmit the infection, and increased insect mortality

(Roberts et al. 1991, Milner 1997). Other important determining characteristics of entomopathogenic fungi that must be considered before utilization as a biological control agent involve the selection of a highly virulent strain, the ability to easily and

economically mass produce the strain, and an extended shelf life of formulated products

(Roberts et al. 1991, Milner 1997). Additionally, the fungus must be noninfectious to humans, other vertebrates and nontarget invertebrates.

More than 700 species of entomopathogenic fungi have been identified (Samson et al. 1988). Fungi used in insect pest management include Entomophthora muscae

(Cohn) Fresen, Metarhizium anisopliae (Metchnikoff), and Beauveria bassiana

(Balsamo) Vuillemin. One of the most commonly used, B. bassiana, has been studied extensively with promising results for filth fly management (Roberts et al. 1991, Geden et al. 1992, Watson et al. 1995, Kaufman et al. 2005, Hasaballah et al. 2017). For the purpose of this research, B. bassiana was be the main pathogen of choice because it has been successfully isolated from naturally infected house flies in the wild (Steinkraus et al. 1990, Geden et al. 1995, Skovgaard and Steenberg 2002).

In addition to direct insect mortality, infection with fungal pathogens also causes a significant reduction in the reproductive fitness of the female house fly population

(Acharya et al. 2015). Infection with B. bassiana results in a 70–80% reduction in egg production and a 13–20% reduction in egg viability compared to non-infected female house flies of the same age (Acharya et al. 2015). Thus, the use of fungal pathogens may reduce house fly populations not only through direct insect mortality but also by suppressing their reproductive output.

In addition to inducing insect mortality with the potential of causing epizootics, entomopathogenic fungi play other important roles in nature. Entomopathogenic fungi are known to be plant endophytes, antagonists of plant pathogens, beneficial rhizosphere colonizers and plant growth promoters (Elliot et al. 2000, Vega et al. 2009,

Behie et al. 2012, Jaber and Salem 2014, Lacey et al. 2015). Jaber and Salem (2014), demonstrated that squash plants treated with endophytic B. bassiana had a significant reduction in the incidence and severity of zucchini yellow mosaic virus, providing protection against the virus in the squash plants. Thus, entomopathogenic fungi play multiple roles in nature, such as decreasing the pest insect population and protecting plants from diseases while promoting plant growth (Vega et al. 2009, Jaber and Salem

2014).

The fungal pathogen Entomophthora muscae is reported to be a species complex with several genetically distinct species that are difficult to distinguish morphologically (Keller 1987, Keller 2002, Gryganskyi et al. 2013). The forms that make up the species complex differ in characteristics such as size of conidia, the number and size of nuclei, and host affinity (Keller 1987, Keller et al. 1999). Another challenging feature of E. muscae is that this fungus can produce secondary and even tertiary conidia from conidia that may not contact a host on initial discharge, making determination of exact conidial concentrations difficult (Mullens 1986, Bellini et al.

1992). The most common host of E. muscae is the house fly (Kramer 1980, Keller et al.

1999). Exposure to E. muscae conidia typically resulted in adult house fly mortality in 4–

8 days (Mullens 1986, Mullens 1990, Geden et al. 1995, Pinnock and Mullens 2007,

Geden 2012). The transmission of E. muscae occurs through direct contact with new hosts during conidial discharge from a killed host, thus the successful development of this fungus is dependent on fly population densities and dissemination from infected cadavers to unexposed flies (Geden et al. 1995). Due to the difficulty in reproducing and

stockpiling viable E. muscae outside of house fly colonies, this fungus is not currently utilized as an augmentative biocontrol agent for fly management.

Metarhizium anisopliae has a wide host range and has been a research subject for management of various pest insects (Roberts and Hajek 1992, Lacey et al. 2015).

Although some strains of M. anisopliae have been proven virulent to house flies (Barson et al. 1994, Renn et al. 1999, Mishra et al. 2011, Acharya et al. 2015), there are no reports of isolating this fungus naturally from house flies.

Beauveria bassiana: Biology, History, and Development

Beauveria bassiana is a widely studied entomopathogenic fungus that has a worldwide distribution (Mascarin and Jaronski 2016). Although soil and infected insects are the primary sources for isolating B. bassiana, it has been isolated successfully from the interior and exterior surfaces of various plants (Lipa 1963, Meyling and Eilenberg

2006), including the bark of Ulmus spp. (elm trees) (Doberski and Tribe 1980) and

Carpinus caroliniana (ironwood, hop hornbeam) (Bills and Polishook 1991). Beauveria bassiana was first identified as an insect pathogen in 1834 by Agostino Bassi di Lodi when it was reported to infect silkworms, Bombyx mori L., with white muscardine disease (Glare and Milner 1991). Since the discovery and description of B. bassiana, it has been used as a biological control agent to manage a variety of insect pests (Uma

Devi et al. 2008). This fungus has been documented infecting ~700 insect species with widely differing life histories (MacLeod 1954, Lipa 1963, Glare and Milner 1991).

MacLeod (1954) reported that B. bassiana was isolated from 63 different insect species from different regions across Canada. Steinkraus et al. (1990) documented the first natural occurrence of B. bassiana infecting house flies.

The mode of infection of B. bassiana is through direct contact, requiring that the insect must first physically encounter the fungus. The infection process begins once the fungal conidia attach to the host cuticle (Barbarin et al. 2012, Hasaballah et al. 2017).

Insects may independently acquire conidial spores; however, efficient autodissemination has been documented as well. Barbarin et al. (2012) demonstrated that direct conidia exposure to only 50% of a bed bug (Cimex lectularius L.) population sharing close harborages resulted in bed bug mortality exceeding 95%.

Within six hours after application to house fly adults, the conidia of B. bassiana attach to the ommatidia and integument (Hasaballah et al. 2017, Mwamburi et al. 2018).

The conidia bind to the insect cuticle due to the cuticle surface hydrophobic proteins,

Hyd1 and Hyd2 (Ortiz-Urquiza and Keyhani 2013). Conidial attachment across the insect cuticle is not uniform and there is a difference in conidial-cuticular binding affinities depending on segmental regions of the insect. Areas with higher densities of conidial attachment include the intersegmental regions, the legs, the eyes, and the base of the setae, while areas with no setae were observed to contain lower densities of conidial attachment (Hasaballah et al. 2017, Mwamburi et al. 2018). There is an immediate and tight conidial adhesion on hydrophobic surfaces, whereas hydrophilic regions experienced weak conidial binding, allowing the conidia to easily be washed, brushed or groomed off (Holder and Keyhani 2005, Ortiz-Urquiza and Keyhani 2013).

Conidial germination occurs 12 to 48 hours after the conidia attach to the cuticle

(Mwamburi et al. 2018), and results in a stronger delayed adhesion of fungal conidia

(Holder and Keyhani 2005). Hydrolytic enzymes such as chitinases, proteases, and lipases promote germination, help degrade the insect cuticle and facilitate penetration

(Ortiz-Urquiza and Keyhani 2013). Beauveria bassiana has multiple specialized infection structures such as penetration pegs, which are produced during the germination phase (Ortiz-Urquiza and Keyhani 2013). Formation of penetration pegs occurs between 36–72 hours after initial exposure (Mwamburi et al. 2018). Following germination, mycelium penetrates the cuticle (Ortiz-Urquiza and Keyhani 2013). The primary penetration sites are located at the base of setae and follow a similar distribution pattern to high conidial attachment sites (Mwamburi et al. 2018). Eventually, the mycelium reaches the hemocoel, resulting in insect mortality.

Virulence of Beauveria bassiana and Metarhizium anisopliae against Adult House Flies

When adult house flies are treated with B. bassiana, the time to death is approximately 5–8 days, although mortality rates are affected by strain and method of delivery (Geden et al. 1995, Lecuona et al. 2005, Mwanburi et al. 2010). Exposure of adult house flies to B. bassiana at 1 x 109 conidia/mL concentration resulted in 100% mortality by day 9, with 75% mortality occurring by day 5 post-exposure (Geden et al.

1995, Mishra et al. 2011, Acharya et al. 2015). Geden et al. (1995) concluded that treating plywood boards with a dust formulation was more effective than a liquid suspension containing the same quantity of conidia. Exposing adult house flies to B. bassiana-treated food or water resulted in 90–99% mortality nine days after the start of treatment (Geden et al. 1995).

When adult house flies are treated with M. anisopliae the time to death is approximately 7 days, although mortality rates are affected by strain and method of delivery. For example, Renn et al. (1999) documented up to 100% adult house fly

motility 10 days after exposure to M. anisopliae bait containers, while Barson et al.

(1994) observed 100% mortality within 3 days with topical treatments.

Both B. bassiana and M. anisopliae have been researched extensively and compared for house fly control (Barson et al. 1994, Mishra et al. 2011, Acharya et al.

2015). Strain differences, suspension formulations, application methods, and different laboratory methods may account for different results in comparative studies.

Barson et al. (1994) evaluated six species of entomopathogenic fungi (including

B. bassiana) for house fly management and concluded that of the six, M. anisopliae was the most effective pathogen against adult house flies. This conclusion was reached because M. anisopliae provided the highest mortality, in the shortest time, at the lowest dose. These researchers documented 100% mortality of adult house flies 6 days after treatment with aqueous B. bassiana or M. anisopliae suspensions. They further evaluated the effects of suspending M. anisopliae conidia in various oils. Oil suspensions resulted in faster mortality compared to aqueous suspensions of this species. When M. anisopliae conidia were suspended in soybean, linseed, or cotton seed oil, 100% house fly mortality was observed in 3, 3, and 4 days, respectively. A limitation of their study was that B. bassiana was not evaluated in oil suspensions and the conclusion was made based on data collected from oil suspensions of M. anisopliae.

Mirshra et al. (2011) added separate spore suspensions of both B. bassiana and

M. anisopliae to house fly diet and reported 100% adult house fly mortality 4 days after treatment for both fungi. Although both fungi had the similar mortality results, based on the lethal concentration 50% and 99% mortality, LC50 and LC99 respectively, this study

concluded that M. anisopliae was a better control agent than B. bassiana in terms of adult mortality. Acharya et al. (2015) evaluated spray treatments containing oil suspensions of both B. bassiana and M. anisopliae. When treated with the same concentration (1 x 109 conidia/ mL) of B. bassiana and M. anisopliae, 100% mortality was documented within 8 and 16 days, respectively.

Research Objectives

The overall objective of this MS research project was to explore the use of entomopathogenic fungi as a management tool for house flies. The specific objectives were:

1. To determine whether Beauveria bassiana has potential for use against fly larvae

2. To select for a faster adult house fly-killing Beauveria bassiana strain

3. To compare the virulence of four Beauveria bassiana strains and a Metarhizium anisopliae strain against adult house flies

CHAPTER 2 EXPOSURE OF HOUSE FLY LARVAE TO Beauveria bassiana

Introduction

The house fly, Musca domestica L. (Diptera: Muscidae) has developed high levels of resistance to many classes of insecticides, which has created the need for alternative pest management strategies (Scott et al. 2013). Modern integrated pest management (IPM) plans include the use of cultural, mechanical, biological, and chemical control methods to maintain target populations below acceptable threshold levels (Stern et al. 1959). Biological control may include the use of hymenopteran parasitoids and/or entomopathogenic fungi such as Beauveria bassiana (Balsamo)

Vuillemin (reviewed in Machtinger and Geden 2018 and Weeks et al. 2018, respectively).

Beauveria bassiana has been recovered from many naturally infected insects, including the house fly (Lipa 1963, Steinkraus 1990, Geden et al. 1995). Adult house fly mortality typically occurs 5–7 days after treatment with B. bassiana (Barson et al. 1994,

Geden et al. 1995, Watson et al. 1995, Mwamburi et al. 2010, Mishra et al. 2011, 2013).

This latency period allows for the fly to continue to be a nuisance to humans and other animals, transmit disease-causing pathogens, reproduce, and contribute to the next house fly generation before it succumbs to infection (Lysyk and Axtell 1987, Malik et al.

2007). If this fungus could successfully suppress immature populations of house flies by causing larval mortality or by preventing adult emergence, the overall adult house fly population would be reduced. The application of entomopathogenic fungi formulations to favorable larval rearing sites may also serve as an oviposition deterrent, thus decreasing the number of eggs laid in treated larval habitats (Machtinger et al. 2016).

Previous research on the efficacy of B. bassiana against house fly larvae has produced mixed results. Some studies have yielded promising results, with larval mortality greater than 70% (Steinkraus et al. 1990, Sharififard et al. 2011). Other studies have demonstrated marginal results with 30–70% larval mortality (Barson et al. 1994,

Watson et al. 1995, Mishra et al. 2011, 2013). Furthermore, low (<30%) larval mortality has been reported (Mwamburi et al. 2010), while others were unable to infect house fly larvae with B. bassiana (Geden et al. 1995, Lecuona et al. 2005). Factors that may contribute to inconsistencies among studies include variations in strain, isolate virulence

(Roberts and Yendol 1971, Lecuona et al. 2005), fungal culturing method, conidial dose or concentration, age, feeding status of the larval host, bioassay exposure method, properties of larval rearing medium, and differences in susceptibility among fly strains

(Geden et al. 1995, Lecuona et al. 2005).

Although there are discrepancies in the results of studies examining the use of B. bassiana as a fly larvicide, a common theme is that the conidial concentration required to induce house fly larval mortality is significantly higher than the concentration required to cause adult mortality (Barson et al. 1994, Watson et al. 1995, Mwamburi et al. 2010,

Sharififard et al. 2011, Mishra et al. 2011, Machtinger et al. 2016). The cuticle acts as a protective barrier against foreign substances and is known to thicken with each successful molt, thus making older larvae less susceptible to infection (Boucias and

Pendland 1998, Mwamburi et al. 2010). These properties of the insect cuticle can alter successful infection with entomopathogenic fungi. Moreover, the near-constant movement of the larvae through larval habitats provides ample opportunity for conidia to be dislodged from larval cuticle before penetration has begun. One of the objectives of

this study was to examine the effectiveness of B. bassiana when applied as a larvicide under a range of test conditions to determine which conditions might favor use of this pathogen as a management tool.

Combining biological control methods, such as using pupal parasitoids and B. bassiana within the same system may have an additive effect by decreasing the house fly population more than when the practices are utilized separately. Optimization of control using these two methods simultaneously would be beneficial, however, the fate of parasitoid eggs deposited in B. bassiana infected pupal hosts is unknown. A second objective of this study was to determine whether application of B. bassiana against house fly larvae would result in production of large numbers of infected pupae that could have an adverse effect on pupal parasitoids.

Materials and Methods

House Fly Rearing

House flies used in tests were obtained from a colony that has been maintained for several decades by United States Department of Agriculture (USDA) Center for

Medical, Agriculture and Veterinary Entomology (CMAVE) in Gainesville, Florida. The flies are housed in wire mesh cages (46 x 37 x 37 cm). The cage opening is covered with 25.4 cm diameter cloth sleeves (Alba non-sterile stockinette #ABH86912

Rockwood, Tennessee) cut into approximately 50 cm long pieces. Sleeves are secured into place on the cages by rubber spline cords and fastened closed with a hair clip.

Adult flies are fed a diet consisting of a 4:4:1 ratio (by volume) of dried non-fat milk powder, sugar, and dried egg yolk. Water is provided in 3.8 L plastic buckets with foam packing “peanuts”. The foam peanuts, which float, provide flies with a dry resting site

while they drink water. House fly cages are kept in rearing chambers that are maintained under constant light at 23–25°C and approximately 80% RH.

Rearing medium is prepared by placing 355 g (500 cm3) of Calf-Manna® (Manna

Pro Products LLC, Chesterfield, MO), 1,500 grams (6,500 cm3) of wheat bran (Siemer

Milling Company, Teutopolis, IL) and 3,750 mL water into rearing trays (55 x 43 x 8 cm) and mixing thoroughly by hand. An oviposition ball is made by placing 30–50 grams of spent rearing medium, obtained from week-old larval rearing trays, into a water- moistened 15 x 15 cm black cloth, which is folded to form an oviposition “pillow”. The pillow is placed in a foam cup (Dart® Food Container Squat Foam 8 oz #8SJ32 Mason,

MI) and placed inside a cage containing 2-week-old house fly adults. Females typically oviposit within 1–3 hours after placement of the oviposition pillow. Once deposited, eggs are rinsed off the pillow into a container using tap water and gently shaken with water to allow the eggs to sink to the bottom of the container. Using a transfer pipette, eggs are transferred to graduated centrifuge tubes to a volume of 2.0–2.5 mL (20,000–25,000 eggs) of settled eggs for each rearing tray. Eggs in the centrifuge tubes are shaken and deposited on the surface of the larval rearing medium. The rearing tray containing fresh medium and eggs is covered with a king-size pillowcase, secured with a rubber band and kept in a larval rearing chamber maintained at 27–30°C and approximately 80% RH under constant light until the pupae are sclerotized at 6–7 days. Pupae are separated from the rearing medium by water floatation and then placed in a forced-air blower for drying.

Beauveria bassiana used in Assays

For the larval assays conducted in this objective, the L90 and GHA B. bassiana strains were used. The L90 strain was isolated from house fly cadavers collected on a

dairy farm in upstate New York in 1990 and is known to be virulent to house flies

(Geden et al. 1995). The commercial product BotaniGard® ES (BioWorks Inc., Victor,

NY) was the source of strain GHA to be tested. BotaniGard® was provided by a representative of the company (Dr. Daniel Peck), who stated that the product was fresh from the production line.

Strain L90 was cultured on Sabouraud dextrose yeast agar (SDY) (2.0% glucose,

1.0% peptone, 0.5% yeast extract, pH 7.0) in an incubator set at 26°C in complete darkness for 7 days or until heavily sporulated. Once adequate sporulation had occurred, the plates were dried in a room with a dehumidifier kept at 25°C and 30–60%

RH for another week. After drying, the conidia were scraped from the agar with a sterile plastic disposable spatula. Dry conidia were placed in sterile 20-mL glass scintillation vials (WheatonTM, DWK Co., Millville, NJ) and stored at 4C for up until 4 weeks before use in experiments. All glassware i.e. vials, bottles, flask, funnel were sterile upon purchase or sterilized by an autoclave at 121°C for 45 minutes.

Viability of stored conidia was confirmed before using a given vial of spores by suspending 10 mg of conidia in 100 mL Luria-Bertani (LB) broth (LB broth, Fisher

BioReagents, Pittsburgh, PA) and placing on a shaker table at 25°C. After 24 hours, samples of the broth were placed on microscope slides and examined to determine the percentage of conidia with germination tubes. Germination rates (viability) during these tests were consistently in the range of 90–99%.

For the L90 strain, bioassays were conducted using weights of conidia as a measure of dosage. Based on extensive data collected on this strain, 1 mg of conidia is known to contain approximately 108 spores when cultures are grown and harvested

using our lab protocol (Johnson et al. 2018, Burgess et al. 2018). In tests with the commercially formulated GHA strain (BotaniGard®), the conidia concentration on the manufacturer’s label (2 x 1010 conidia/ mL), was used to calculate test concentrations.

Larval Test 1: Assays with Dry L90 Conidia and BotaniGard®

This experiment compared effects of dry conidia from the L90 strain with the commercial product BotaniGard®. For the dry treatments, conidia were weighed into batches of 10, 25, and 50 mg and mixed with 5 g of freshly prepared house fly rearing medium in sterile 60 mL clear glass bottles (height 5 cm, diameter 5 cm) (FisherbrandTM

Fisher Scientific Hampton, NH #02911763). For the BotaniGard® treatments, 2 mL of suspensions containing either 4 x 109, 2 x 1010, or 4 x 1010 were stirred into the medium.

Groups of 100, 2-day old larvae (2nd instars) were added to the medium. The bottles were covered with cotton muslin secured with a rubber band and held at 25°C. An additional 5 grams of freshly prepared house fly medium was added to each bottle, including the controls, 48 hours after the start of the experiment as supplemental food.

Pupae were removed by hand, counted, and held at 25°C for fly emergence. This experiment was performed three times with three cups per treatment condition.

Larval Test 2: Effect of Host age and Dose on Response to Treatment with B. bassiana.

For the experiments that follow, a larval medium was prepared with less water to compensate for the subsequent addition of aqueous suspensions of conidia. For this medium, the amounts of wheat bran and Calf-Manna® remained the same as before, while only 2,500 mL of water was added instead of 3,750 mL used in the standard medium. In the sections that follow, this is referred to as “reduced-water” medium.

For tests on the effects of larval age and amounts of B. bassiana conidia applied, reduced water medium (200 g) was added to 473 mL clear deli cups (Waddington North

America, Chelmsford, MA #WNAAPCTR16). A small pocket on the surface of the medium, in the center of the medium was created by pressing a finger approximately 3 cm into the medium. Eggs were collected from gravid house flies and measured in graduated centrifuge tubes. Six cm3 of settled eggs were combined with 114 mL of tap water to create a 1:20 egg suspension, then the mixture was stirred using a magnetic stirrer. One mL of this suspension containing approximately 500 eggs was deposited in the pocket on the medium and lightly covered with medium to prevent egg desiccation.

For each experiment, three 1 mL samples of the egg solution were deposited on black cloth and counted using 10 X magnification to determine the actual number eggs used for each batch of egg suspensions. The average of the latter egg number, rather than the target value of 500 eggs, was used to calculate percent pupation and adult emergence.

For the high concentration in this experiment, I used 20 mL of undiluted

BotaniGard® (total, 4 x 1011 conidia). This high concentration was serially diluted to make suspensions containing 4 x 1010, 4 x 109, and 4 x 108 conidia in 20 mL. A set volume (20 mL) of tap water or the given concentration was added to the reduced water medium on either day 0 (eggs), 1 (1st and 2nd instars), 2 (2nd instars), or 3 (3rd instars) after egg deposition. For the treatment on day 0, the solution (or water for the control) was mixed into the medium prior to the addition of eggs. For day 1, day 2, and day 3 treatments, the suspension was added to the experiment cups, then gently mixed using

a glass rod so as to not cause mechanically-induced mortality in the developing fly larvae.

Cups were covered with cloth lids and held in an incubator at 25°C until pupation.

Once pupariation was complete, pupae were separated from medium using water floatation. The pupae were dried, counted, placed in clean containers, covered and placed back in the incubator for adult eclosion. Once the adult flies died, the numbers of adults were recorded. This experiment was repeated three times with three cups per treatment.

Larval Test 3: Effect of Temperature and Host Age on Response to Treatment with B. bassiana.

To determine potential effects of temperature on fly larval susceptibility to B. bassiana, cups with medium and larvae were treated with BotaniGard® and held at one of two different temperatures within the typical fly larval development range. Reduced water medium (50 g) was added to 163 mL plastic cups. Groups of 50 house fly eggs were counted on black cloth using 10X magnification, then the cloth (2 x 3 cm) with the eggs was placed in the cups containing medium with the egg side gently pressed into the medium. Cups were covered and subsequently held at either 25°C. On day 1 or day

2 after egg placement, depending on treatment condition, cups were removed from the

25°C incubator and treated with a 5 mL volume of either 0 (water-controls), 1010 or 109 conidia and the solution was gently stirred into the mixture using a glad rod. Following treatment, the cups were then held at either 22°C or 32°C to finish development. After pupariation, the pupae were separated with water floatation, counted, dried, placed back in the cups within the designated rearing incubator and held for adult eclosion.

Treatments varied in day of exposure, concentration applied, and temperature in which

they were held. This experiment was repeated three times with three cups per concentration, per treatment day at each set temperature.

Larval Test 4: Effect of Medium Composition and Host Age on Response to Treatment with B. bassiana.

Calf-Manna®, an ingredient in the USDA-CMAVE standard fly larval medium, contains propionic acid (https://www.mannapro.com/products/cattle/cattle), which has antifungal properties at sufficiently high concentrations. To determine whether this could affect the ability of B. bassiana to infect larvae, two different media were prepared and tested. The standard, Calf-Manna®-containing larval house fly diet medium was used as a baseline following our reduced-water protocol, as previously described. A second diet was prepared by combining 355 grams (500 cm3) of GRO ‘N WIN™ ration balancer horse feed (Buckeye Nutrition, Dalton, OH) with 1,500 grams of wheat bran and 2500 mL of water. The ingredient list for GRO ‘N WIN™ does not include propionic acid or any known antifungal agents (https://www.buckeyenutrition.com/products/gro-n- win.aspx).

Plastic cups (473 mL) were filled with 200 grams of the above medium. A house fly egg suspension was made with eggs as previously described and approximately 500 eggs were added to each of the cups. The cups were covered with cloth, secured by lids with a hole cut out of the center for ventilation and held at 25°C. Cups were treated with BotaniGard® either 1 or 2 days after egg deposition, depending on treatment condition, by adding 20 mL of solution containing either 4 x 1011 or 4 x 1010 conidia to the medium and mixing using a glass rod. Control cups were treated with an additional

20 mL of tap water on day 0 before the addition of eggs. After pupation, the medium and pupae were separated by water floatation, dried, counted, placed in clean

containers and held at 25°C until adult eclosion. Treatments varied in medium type and day of exposure. This experiment was performed three times with three cups per treatment.

Statistical Analysis

Percent pupation and percent adult emergence were the dependent variables for all of the experiments. Data were analyzed using the General Linear Model Procedure

(Proc GLM) of SAS® software, Version 9.4 of the SAS System for Windows, Copyright

© 2002-2012, SAS Institute Inc., Cary, NC. The replication main effect and its interactions with other main effects were included in the model during an initial run. If the interaction term involving replication had been significant, ANOVA F’s for other main effects were to be re-calculated by dividing main effect mean squares by the mean square of the interaction (Sokal and Rohlf 1981). The latter step sometimes was necessary because the SAS default is to construct F-values for all treatments effects using the error mean square as the denominator. In practice, none of the experiments presented here required this adjustment in the construction of F statistics, and analyses were re-run excluding the interaction terms involving replication as a model effect.

Data from Larval Test 1 (dry L90 conidia and liquid BotaniGard® solution) were analyzed by separate one-way ANOVAs for the two fungal sources using dose as the main effect, and differences among treatment means evaluated by Tukey’s method at

P=0.05 by using the Means/Tukey Statement in Proc GLM. Data from Larval Test 2

(effect of host age and dose) were initially analyzed by two-way ANOVA using host age, dose and host age x dose as model effects, then separate one-way ANOVAs were performed for each day of fungal treatment using dose as the main effect; treatment means within days of treatment were examined by Tukey’s procedure as before. For

Larval Test 3 (effect of temperature and dose), data were analyzed by separate two- way ANOVAs for each day using temperature, dose and temperature x dose as model effects. For Larval Test 4 (effect of larval medium and dose), data were analyzed by separate two-way ANOVAs for each day using larval medium, dose and larval medium x dose as model effects.

Results

Larval Test 1: Assays with Dry L90 Conidia and BotaniGard®

Treatments with dry conidia of the L90 strain of B. bassiana significantly reduced pupation success (F=12.26, df =2,34, P=0.0002) and adult emergence (F=16.64, df

=2,34, P<0.0001) (Table 2-1). Treatments with BotaniGard® also reduced pupation success (F=18.17, df =2,34, P<0.0001) and adult emergence (F=39.92, df =2,34,

P<0.0001).

When dry L90 conidia were used, fewer larvae pupated at the high dose of approximately 5 x 109 conidia (48.6%) than in untreated fly developmental medium

(70.9%). Fewer adults emerged from the three L90 treatments as compared to the untreated control (64.4%), and the lowest emergence (42.4%) was observed in developmental medium treated with the high dose of 5 x 109 conidia.

When liquid BotaniGard® solutions were used, significantly fewer larvae pupated and fewer adult flies emerged from B. bassiana-treated medium compared with the untreated controls (Table 2-1). Percent pupation in untreated developmental medium was 70.0% compared with 48.4, 36.7 and 25.4% in medium treated with B. bassiana- containing BotaniGard® at 4 x 109, 2 x 1010 and 4 x 1010 conidia, respectively. Similarly, adult emergence from untreated medium was 64.4% compared with 40.0, 25.7, and

14.3% in medium treated with this B. bassiana-containing formulation at 4 x 109, 2 x

1010 and 4 x 1010 conidia, respectively.

Differences in the range of doses used for the L90 and BotaniGard® treatments do not allow direct statistical comparison between effects of the two sources of B. bassiana. However, both percent pupation and percent adult emergence were similar in treatments with 5 x 109 L90 conidia (48.6 and 42.4%, respectively) and in treatments with a similar quantity (4 x 109) of conidia in the BotaniGard® product (48.4 and 40.0% for percent pupation and adult emergence, respectively).

Larval Test 2: Effect of Host age and Dose on Response to Treatment with B. bassiana.

Conidial dose (F=31.15, df=4,159, P<0.0001), host age (F=14.34, df=4,159,

P<0.0001), and the dose*age interaction (F=14.34, df=4,159, P<0.0001) all had a significant effect on larval pupation success. Similarly, conidial dose (F=35.65, df=4,159, P<0.0001), host age (F=8.57, df=4,159, P<0.0001), and the dose*age interaction (F=5.43, df=4,159, P<0.0001) all had an effect on adult fly emergence.

When BotaniGard® was added to developmental medium on day 0 or day 1 after egg deposition, there were no significant differences in percent pupation or percent adult emergence in the cups treated with 4 x 108, 4 x 109, or 4 x 1010 conidia when compared to the control (Table 2-2). However, significantly fewer larvae pupated and fewer adults emerged in medium treated with the high dose of 4x1011 when compared to the control (F=2.7, df=3, 39, P=0.0425). The treatment effect was particularly prominent at this concentration when conidia were applied 1 day after egg deposition, when only

24.3% pupation and 17.2% adult emergence were observed compared to 83.8 and

76.7% in the controls, respectively. In contrast, when conidia were applied 2 or 3 days

after egg deposition, the effects of B. bassiana application on pupation and adult fly emergence were either small or non-existent (Table 2-2).

Larval Test 3: Effect of Temperature and Host Age on Response to Treatment with B. bassiana.

The effect of temperature on B. bassiana treatment is presented in Table 2-3.

The temperature at which larvae were reared had no effect (P>0.05) on percent pupation or adult emergence in B. bassiana-treated medium regardless of whether conidia were added to medium on days 1 or 2 after egg deposition. Similarly, the temperature*dose interaction had no effect on pupation or adult emergence (P>0.05) regardless of whether conidia were added to rearing medium on days 1 or 2 after egg deposition. Slightly fewer larvae pupated and adult flies emerged from B. bassiana- treated medium, but only when conidia were applied 2 days after egg deposition.

Larval Test 4: Effect of Medium Composition and Host Age on Response to Treatment with B. bassiana.

Somewhat higher rates of pupation and adult fly emergence were observed when larvae were reared in medium that included the GRO ‘N WIN™ pelleted horse feed than in the standard USDA diet that includes Calf-Manna® pellets, but this was only true when the water or BotaniGard® solutions were added 2 days after egg placement (Table

2-4). Treatment with B. bassiana reduced both pupation and adult emergence success when conidia were added on either days 1 or 2 after egg placement. Fewer than 15% of the initially deposited eggs produced adult flies when rearing medium was treated with the high rate of 1011 conidia compared with 47.6-64.1% among larvae reared on untreated medium. The lack of a significant (P<0.05) diet x dose interaction term indicated that the effect of B. bassiana treatment was unaffected by the type of larval medium.

Discussion

Natural infections of adult house flies were first reported by Steinkraus et al.

(1990) in flies collected from New York dairy farms. In that publication, the authors reported that 73% of mature third instar larvae that were exposed to 2 x 109 conidia

(strain HF88) died as pupae and produced mycoses. However, when the same authors revisited the topic of larval susceptibility, they were unable to kill or infect mature fly larvae in media treated with 107 conidia/cm3 (2 x 109 total in 200 cm3) (Geden et al.

1995). Mwamburi et al. (2010) were unable to infect mature (7-day-old) larvae after dipping them in aqueous conidial suspensions, and Barson et al. (1994) observed no significant mortality when mature larvae were dipped in 0.01% Tween solutions containing up to 108 conidia/mL. Similarly, Lecuona et al. (2005) tested 17 strains of B. bassiana by dipped mature third instar larvae in 0.01% Tween solutions containing 108 conidia/mL and never observed mortality that was higher than in the controls. In preliminary experiments not presented here, I was unable to kill or infect mature larvae by direct contact with conidia of the L90 strain, regardless of concentration or whether conidia were dry or in an aqueous suspension. With the exception of the initial report by

Steinkraus et al. (1990), there is no evidence for successful B. bassiana infection of mature house fly larvae.

Other studies have reported mixed results regarding use of B. bassiana for younger fly larvae. In one report, 50-94% mortality was observed when “medium-sized” fly larvae were dipped for 10 seconds in B. bassiana suspensions containing 108 conidia/mL in a 0.01% Tween solution (Sharififard et al. 2011). However, the authors did not provide data on mortality of control larvae that were treated with the Tween surfactant alone. In preliminary results not presented here, I observed unacceptably

high mortality among control larvae that were treated with another surfactant (CapSil®).

The cause of death appeared to be due to the surfactant entering the spiracles of the larvae, traveling through the trachea and preventing normal respiration.

Sharififard et al. (2011) reported substantial larval mortality when larvae were placed in 50 grams of larval medium treated with 5 x 1010 to 5 x 1012 conidia. These assay results are somewhat similar to the results reported in Table 2-2 of this study.

After adjusting for differences in the volume of media and conidia doses used in the two studies, the results of the two studies are in fairly close agreement. I observed 24% pupation when young larvae were treated with 4 x 1011 conidia per 200 grams of media, whereas Sharifiard et al. (2011) observed 55-78% larval mortality at a nearly equivalent conidial concentration. Results in Table 2-2 also demonstrate that there is a narrow window of fly larval age at which B. bassiana treatments are effective. I only observed substantial larval mortality when treatments were applied 24 hours after egg deposition.

This narrow window may account for some of the discrepancies in the published literature on larval susceptibility to this pathogen.

Mishra et al. (2011) applied 1 mL of conidia suspensions to larval developmental medium in Petri dishes containing second instar larvae and observed the highest larval mortality (97%) at the 109 conidia/mL concentration. These researchers also documented high mortality (79%) at the 107 conidia/mL concentration, which was a considerably lower concentration than those used in my study. A shortfall of this study was that the amount of developmental medium present in each Petri dish was not stated. The challenge with comparing this study to the current study is that a dilution of conidia per weight or volume of developmental media could not be calculated. In

addition, the number of insects tested was low, with 10 in each Petri dish resulting in a total of 30 per concentration. Moreover, the strain of B. bassiana used in that study was not named or characterized. In a subsequent paper, the same authors reported 55% larval mortality when sugar-encapsulated conidia were applied to fly larvae in rearing medium (Mishra et al. 2013). When B. bassiana from the L90 strain was applied to a cow manure/sawdust substrate containing second instar larvae, Watson et al. (1995) observed about 50% mortality at the high rate of 1010 conidia/cm3, or about 1012 conidia in about 100 cm3 of medium. The latter authors noted that the conidial doses needed to have any effect on larvae were 100 times greater than amounts that provided high levels of mortality against adult flies.

Why are house fly larvae so much more refractory to infection than adults? One factor may be that there are fewer optimal sites for conidial adhesion. Hasaballah et al.

(2017) applied B. bassiana to the abdomens of adult house flies and examined the distribution of conidia on the flies 12 hours after exposure. Conidia with germination tubes were unevenly distributed and were most prevalent on “protected” sites that are difficult to groom, especially at the bases of setae and between the ommatidia of the eyes. With the exception of the spiracles, house fly larvae are comparatively lacking in protected structures near which conidia can adhere. The location of conidial adhesion and penetration in larval house flies has yet to be determined. A second factor is undoubtedly the fact that larvae are moving through moist substrates in ways that provide near-constant opportunities for mechanical removal of conidia before germination can occur. Finally, the physical properties of house fly larval cuticle may be less conducive to adhesion than other insect hosts. Conidial adhesion is favored by

hydrophobic, nonpolar surfaces (Holder and Keyhani 2005). The elastic cuticle of muscoid fly larvae is more polar (less hydrophobic) than the hardened cuticle of adult insects (Hillerton and Vincent 1983), making it somewhat more difficult for conidia to attach and remain adhered.

The results of the present study demonstrate that although it is possible to use B. bassiana as a biological larvicide, house fly larvae are only susceptible to such treatments for a relatively brief time during development. Currently it is unknown why there is an age effect on larvicidal success, but it may be that the insect is more susceptible immediately after molting before the new larval cuticle has fully hardened.

Moreover, the quantities of conidia required to exert even modest levels of control are so great that the cost of such treatments would be prohibitively high. The effects of such massive quantities of conidia on beneficial insects such as parasitoids and predators also would need to be documented. One objective of this study was to determine whether B. bassiana use against fly immatures would result in accumulations of infected pupae that could have adverse effects on pupal parasitoids. Results presented here and in previously published work indicate that large numbers of infected pupae are unlikely to accumulate following a B. bassiana treatment, at least with currently available strains and application methods. For the metrics of this study, the numbers of interest were the percent pupation and the percent adult emergence and thus larval and pupal cadavers were not held for further testing to examine if they produced conidia blooms. Although

B. bassiana has potential as a fly management tool, the most appropriate application of this pathogen is through the use of infective baits and as treatments of surfaces where flies typically alight and rest.

Table 2-1. Percent pupation and percent adult emergence of Musca domestica L. after 100 second instar larvae were exposure to Beauveria bassiana (Balsamo) Vuillemin in the form of dry L90 conidia or an aqueous suspension of BotaniGard® ES. The conidia were mixed into 5g of larval rearing medium and an additional 5 g of fresh medium was added 48 hours after initial exposure. Source of Dose (no. conidia) Mean (SE) 1 Mean (SE) 1 conidia % pupation % adult emergence

L90 0 (control) 70.9 ( 6.00) a 64.4 ( 7.33) a L90 (10 mg) ca. 1 x 109 55.1 (12.32) bc 51.1 (11.54) b L90 (25 mg) ca. 2.5 x 109 62.1 ( 9.83) ab 52.7 ( 9.91) b L90 (50 mg) ca. 5 x 109 48.6 (12.95) c 42.4 (11.14) c

ANOVA F (P)2 10.26 (0.0002) 16.64 (<0.0001)

BotaniGard® 0 (control) 70.9 ( 6.00) a 64.4 ( 7.33) a BotaniGard® 4 x 109 48.4 (14.29) b 40.0 (11.72) b BotaniGard® 2 x 1010 36.7 (13.90) bc 25.7 (10.67) c BotaniGard® 4 x 1010 25.4 (12.85) c 14.3 ( 7.73) c

ANOVA F (P)2 18.17 (<0.0001) 39.92 (<0.0001) 1 Means within columns under the same source of B. bassiana (L90, BotaniGard®) followed by the same letter are not significantly different at P=0.05 (Tukey’s HSD). 2 df, 2,34

Table 2-2. Mean (SE) percent pupation and percent adult emergence of Musca domestica L. after exposure to BotaniGard® ES (Beauveria bassiana (Balsamo) Vuillemin strain GHA). Approximately 500 eggs were added to 200 g of larval rearing medium and the cups were treated with BotaniGard® either on day 0, 1, 2 or 3 after the eggs were added. Dose1 % pupation or adult emergence of flies when exposed to B. bassiana on day after egg deposition:

Day 0 Day 1 Day 2 Day 3 (eggs) (1st and 2nd instars) (2nd instars) (3rd instars)

Mean (SE) % pupation2 0 81.4 (2.9) a 83.8 (3.2) a 83.5 (3.0) ab 88.0 (5.3) a 4x108 80.5 (2.9) a 87.1 (3.5) a 92.5 (3.3) a 87.6 (3.9) a 4x109 81.0 (3.0) a 81.6 (4.5) a 96.6 (3.0) a 81.7(4.9) a 4x1010 80.5 (2.5) a 76.3 (6.8) a 89.3 (3.2) a 87.0 (3.4) a 4x1011 65.6 (5.1) b 24.3 (8.0) b 73.2 (4.4) b 71.9 (4.8) a

ANOVA F (P)3 4.0 (0.0081) 27.3 (<0.0001) 7.2 (0.0003) 2.7 (0.0425)

Mean (SE) % adult emergence2 0 73.3 (2.9) a 76.7 (3.6) a 75.6(3.0) a 81.5 (6.1) a 4x108 70.6 (3.2) a 80.3 (3.8) a 80.7 (2.9) a 73.3 (3.3) ab 4x109 67.0 (2.1) a 69.4 (4.9) a 79.0 (4.3) a 62.9 (4.6) b 4x1010 71.4 (3.2) a 67.9 (7.5) a 79.6 (3.8) a 73.5 (4.7) ab 4x1011 50.0 (4.3) b 17.2 (7.5) b 60.5 (3.7) b 60.1 (5.2) b

ANOVA F (P)3 8.5 (<0.0001) 25.3 (<0.0001) 7.5 (0.0001) 3.6 (0.0133) 1 Total number of conidia applied in 20 mL of water-diluted BotaniGard® (BioWorks Inc., Victor, NY), water was used in the controls with 0 conidia. Means within columns under measured variable (% pupation, % adult emergence) followed by the same letter are not significantly different at P=0.05 (Tukey’s HSD). 2 df, 3,39

Table 2-3. Effect of temperature and conidia dose on percent pupation and percent adult emergence after Musca domestica L. larvae were exposed to BotaniGard® ES (Beauveria bassiana strain GHA) and subsequently held at either 22°C or 32°C. Temperature Dose1 Mean (SE) % pupation2 Mean (SE) % adult emergence2 (oC) Day 1 Day 2 Day 1 Day 2

22 0 67.6 (4.1) 78.7 (2.8) 60.9 (4.3) 71.1 (4.0) 32 0 72.9 (3.3) 80.7 (2.8) 60.9 (2.9) 69.8 (2.4)

22 109 61.3 (8.7) 71.8 (4.2) 56.4 (8.1) 68.2 (4.0) 32 109 76.4 (3.3) 72.2 (2.8) 65.8 (4.0) 62.2 (4.3)

22 1010 68.7 (3.6) 68.2 (3.9) 64.7 (3.5) 59.3 (3.6) 32 1010 68.0 (5.2) 72.0 (3.0) 52.9 (6.5) 60.9 (3.3)

ANOVA F (P) Temp (df 1, 46) 3.8 (0.056) 0.8 (0.366) 0.1 (0.814) 0.8 (0.371) Dose (df 2, 46) 0.1 (0.895) 6.6 (0.003) 0.2 (0.830) 7.8 (0.001) Temp x Dose 1.9 (0.167) 0.2 (0.836) 3.2 (0.052) 1.1 (0.353) (df 2, 46) 1 Total number of conidia applied in 5 mL of water-diluted BotaniGard® ES. Water was used in the controls (dose 0). 2 B. bassiana conidia were added either 24 or 48 h after egg placement. Larvae on day 1 after egg placement were 1st and early 2nd instars; larvae on day 2 were mid-sized 2nd instars. 50 eggs were placed in 50 g of larval developmental medium and held at 25°C prior to treatment.

Table 2-4. Effect of larval medium and dose of B. bassiana (Balsamo) Vuillemin (BotaniGard®) on house fly (Musca domestica L.) pupation and emergence when conidia were added to medium 1 or 2 days after egg placement. Diet1 Dose2 Mean (SE) % pupation3 Mean (SE) % adult emergence3 Day 1 Day 2 Day 1 Day 2

Calf-Manna®2 0 52.2 (6.1) 52.2 (6.1) 47.6 (5.5) 47.6 (5.5) GRO ‘N WIN™ 0 68.9 (9.7) 68.9 (9.7) 61.1 (8.4) 64.1 (2.6)

Calf-Manna® 1010 58.7 (2.2) 57.9 (6.2) 53.4 (2.1) 52.4 (5.8) GRO ‘N WIN™ 1010 68.0 (2.7) 71.9 (6.5) 58.2 (2.5) 63.8 (5.7)

Calf-Manna® 1011 28.5 (10.8) 27.1 (8.6) 12.4 (6.4) 8.2 (4.0) GRO ‘N WIN™ 1011 15.8 (10.0) 35.6 (5.4) 6.9 (4.7) 14.1 (4.0)

ANOVA F (P) Diet (df 1,35) 0.52 (0.4761) 4.8 (0.0375) 0.9 (0.3405) 4.5 (0.0408) Dose (df 2,35) 19.0 (<0.0001) 12.3 (<0.0001) 47.4 (<0.0001) 39.7 (<0.0001) Diet x Dose 2.1 (0.1403) 0.2 (0.8522) 1.6 (0.2260) 0.2 (0.2065) (df 2,35) 1 Larval medium was prepared by combining 1.5 kg wheat bran, 2.5 L water, and 355 grams of either Calf-Manna® (Manna Pro Products LLC, Chesterfield, MO) or GRO ‘N WIN™ (Buckeye Nutrition, Dalton, OH) pelleted livestock feed. 2 Total number of conidia applied in 20 mL of water-diluted BotaniGard® ES (BioWorks Inc., Victor, NY); water was used in the controls with 0 conidia. 3 B. bassiana conidia were added on either day 1 or 2 after egg placement. Larvae on day 1 after egg placement were 1st and early 2nd instars; larvae on day 2 were mid-sized 2nd instars. 500 house fly eggs were added to 200 g of developmental medium.

CHAPTER 3 SELECTION FOR A FASTER-KILLING Beauveria bassiana STRAIN

Introduction

The house fly (Musca domestica L.) is an important pest insect in livestock production facilities and nearby residential neighborhoods. High fly populations can result in an increased likelihood of transmission of human and animal disease-causing pathogens (Malik et al. 2007, Shah et al. 2016). Along with stable flies, severe house fly pressure can have negative impacts on food animal production, resulting in animal discomfort and economic losses (Catangui et al. 1997, Taylor et al. 2012).

Although many chemical control strategies have provided effective fly management, the widespread application and possible misuse of insecticides has created situations that exacerbate the challenges of managing this pest. House flies are notorious for developing insecticide resistance through enhanced mechanisms that allow them to detoxify chemical insecticides (Kaufman et al. 2001a, Scott et al. 2013).

Insecticide resistance can develop within the first two years of an active ingredient being released on the market (Shah et al. 2015).

Biological control agents (BCAs) have been an attractive alternative to traditional chemical insecticides due to increased insecticide resistance emergence and consumer preferences shifting to include all-natural products. BCAs are thought to be a safe alternative to chemical insecticides and generally can be used in organic production systems (Howarth 1991). Common BCAs include predators, parasitoids, and multiple entomopathogens including bacteria, viruses and fungi. In order to be competitive with chemical insecticides, BCAs need to be effective, fast-acting, and cost-effective.

The strains of entomopathogenic fungi used in integrated pest management programs should be matched with the pest insect and the environmental conditions in which it will be utilized (Eilenberg and Hokkanen 2006). The fungal entomopathogen

Beauveria bassiana (Balsamo) Vuillemin has been isolated from naturally infected house flies in the wild (Steinkraus et al. 1990, Geden et al. 1995, Skovgaard and

Steenberg 2002) and has been proven to have strong potential for house fly management (Kaufman et al. 2005, Hasaballah et al. 2017, Johnson et al. 2018). A challenge associated with the use of B. bassiana for house fly management is that high fly mortality does not occur until 6–7 days after exposure (Geden et al. 1995). This is a challenge to consumers who demand quicker results, but also because house flies are sexually mature and capable of successful reproduction by day five post-ecolosion

(Lysyk and Axtell 1987, Malik et al. 2007).

There are several possible approaches for developing faster-killing entomopathogenic fungi. One is to screen a variety of strains to identify those with superior virulence (Mwamburi et al. 2010, Sharififard et al. 2011), and this is the focus of

Chapter 4. Another approach is to attempt to synergize the fungal pathogen by combining it with a second pathogen, especially bacteria (Wraight and Ramos 2005,

Johnson et al. 2018). The use of modern molecular biological methods to produce genetically modified (GM) strains with higher virulence is now possible and provides another approach (Karabörklü et al. 2018), but general public disfavor with GM products may hamper the release and widespread use of such strains (Scott et al. 2016). Novel mutants can be created from established strains by use of ultraviolet radiation, a

method that can rapidly produce an abundance of new isolates to be evaluated (Zhao et al. 2016).

In contrast, conventional selection of established strains for faster kill rates could result in superior products that could be introduced rapidly into the growing biopesticide marketplace (Arthurs and Dara 2018). It has long been known that the virulence of entomopathogenic fungi decreases with successive passage on artificial medium, whereas virulence can increase with passage through susceptible hosts (Steinhaus

1949, Müller-Kögler 1965, Hall 1980, Tanada and Kaya 1993). Steinhaus (1949) suggested that insect pathogens may increase in virulence during passage through insects in the buildup to an epizootic wave.

Butt et al. (2006) reviewed the literature on the effects of continued in vitro culture of entomopathogenic fungi and concluded that virulence in attenuated strains often was restored after passage through an insect host. This may require more than a single passage through insect hosts. For example, Hall (1980) investigated the effects of repeated sub-culturing on agar and passage of Verticillium lecanii (Zimmerman) (now known as Lecanicillium lecanii) through an insect host on pathogenicity, morphology and growth rates of the fungus. He documented that when V. lecanii was passed through an insect host (Chrysanthemum aphid, Macrosiphoniella sanborni Gillette), and not through artificial medium, pathogenicity did not differ significantly from the parental strain. However, repeated culturing on agar resulted in gross colonial changes and those changes were still evident after a single host insect passage, suggesting that further insect passages were needed to return the fungus to its former morphological form.

Surprisingly few studies have been conducted to improve the virulence of entomopathogenic fungi by deliberate selection. Some have shown promising results with selecting for a more virulent strain while others have had less success. Daoust and

Roberts (1982) reported a substantial increase in virulence for two strains of

Metarhizium anisopliae (Metchnikoff) after only one passage through Culex pipiens L. larvae; however, the enhanced virulence was not always stable after several subcultures on agar. Additionally, not all of the M. anisopliae isolates tested generated the same virulence increase, with some isolates remaining unchanged while others even experienced a decrease in virulence. They concluded that the passage of fungal strains through mosquito larvae proved effective in increasing the virulence of M. anisopliae.

Not all attempts to select for “improved” entomopathogenic fungi have been successful. Ignoffo et al. (1982) did not document a significant decrease or increase in the virulence of Nomuraea rileyi (Farl.) after 12 passages in larvae of Trichoplusia ni

(Hübner). Also, Holderman et al. (2017) documented no changes in virulence after seven selective passages of B. bassiana through the horn fly, Haematobia irritans (L.).

Although the literature provides mixed results on the success of selecting for more virulent entomopathogenic fungi, it has been proven that repeated in vitro sub- culturing decreases virulence and that passage through an insect host can help maintain and restore virulence. Objective 2 efforts focused on the selection for a faster adult house fly-killing Beauveria bassiana strain. The goal of this Objective was to determine whether B. bassiana can be selected to provide a more virulent or faster- killing strain for use against house flies.

Materials and Methods

Sources of Flies and B. bassiana

House flies were reared as previously described in Chapter 2. Approximately 500 live adult house flies were removed from a cage 2–5 days post emergence using a modified vacuum with an insect collection tube. To remove the flies from the collection tube, flies were briefly anesthetized using CO₂. To keep the flies anesthetized for sex determination and counting, a custom-built, dual-chamber CO2 “knockdown” device was used (Figure 3-1A). The bottom chamber of the device is formed by cutting away the upper walls of a 2-liter container so that only the bottom 10 cm remains, forming a shallow, circular dish with a diameter of just over 15 cm. A 2 cm hole is cut into the side of the bottom chamber to allow insertion of vinyl tubing to deliver CO2 (Figure 3-1B). The upper chamber is made of a large (15 cm) plastic Petri dish lid with numerous small perforations to allow CO₂ to flow through the holes without the flies passing through the holes (Figure 3-1C). The upper chamber is tightly nested within the bottom chamber and the CO₂ delivery tube is inserted into the hole in the bottom chamber. The CO2 is pushed up through the holes in the upper chamber by the flow from the tank and forms a blanket of CO2 over the surface of the upper chamber.

The North Florida Holsteins (NFH10) strain of B. bassiana was used for this selection experiment using a fly-to-fly passage system. The NFH10 strain was isolated in 2010 from house flies which were collected from a dairy located in Gilchrist County,

Florida. As this strain was originally isolated from a Florida dairy, it is expected to be adapted to Florida conditions and the dairy environment.

This fungal strain had been maintained for the first eight years after collection by occasional fly-to-fly passage interspersed with storage of spore-laden fly cadavers at

4°C for up to one year. Prior to these experiments, conidia were removed from a batch of cadavers stored at 4°C and transferred to Petri dishes with Sabouraud dextrose yeast agar (SDY) (2.0% glucose, 1.0% peptone, 0.5% yeast extract, pH 7.0) for isolation and subsequent propagation. These plates formed the basis on which fungal cultures were maintained. Plates were grown inverted in a 26°C and 80–99% RH incubator in complete darkness for 7 days or until a dense carpet of conidia was visible on the surface of the plates. Once adequate sporulation had occurred, the plates were dried at

25°C and 30–60% RH for seven days. After drying, the conidia were scraped from the agar with a sterile plastic disposable spatula, placed in a sterile 20 mL borosilicate glass scintillation vial (Wheaton, Millville, NJ) and stored in a refrigerator at 4°C until use. All glassware i.e. vials, bottles, flasks, and funnels were sterile upon purchase or sterilized by an autoclave at 121°C for 45 minutes.

Selection for Early-dying Flies

For the first selection experiment (generation 1), 50 mg of dried NFH10 conidia

(aproxmaitly 5 x 109 spores) previously stored at 4°C was placed in a glass bottle i.e., a sterile 60 mL clear, straight-sided, round glass bottle (FisherbrandTM Fisher Scientific

Hampton, NH #02911763). Approximately 150 initially anaestisized female flies were placed in the glass bottle containing the conidia for five minutes.

A clean 24.5 x 24.5 x 24.5 cm mesh BugDormTM (MegaView Science, Taiwan

#4F2222) cage was prepared with adult house fly food, (4:4:1 ratio [by volume] of dried non-fat milk powder, sugar, and dried egg yolk). Water was provided in a 59 mL plastic cup (Dart Solo, Mason, MI #PL200n) with two slits cut in the lid to form a cross with a cotton dental wick (Absorbal Co.,Wheat Ridge, CO ) inserted to act as a wick to deliver water. The cage was labelled with the date and NFH10 generation number. After the

flies were exposed for five minutes, the glass bottle with condidia was placed in the cage and the lid removed.

Dead flies were removed from the cage daily, counted, and placed on sterile, water-moistened 90 mm diameter grade 2 filter paper (WhatmanTM #1002090 GE

Healthcare Maidstone, England) placed in the lid of a Petri dish (Fisher Scientific

Hampton, NH) to promote conidial sporulation. Dishes with fly cadavers were covered with the lids, labeled with the date and the number of days since exposure to conidia, and placed in an incubator at 26°C and 80–99% RH in complete darkness for 7 days.

Sterile Deionized (DI) water was added to the filter papers approximately every other day to maintain moisture that encouraged conidia sporulation in infected flies. After one week in the incubator, the Petri dishes containing the cadavers were vented and moved into a drying room containing a dehumidifier that kept the room at 30–60% RH at 25°C for another 7–14 days.

Ten of the earliest-dying (day 5) cadavers from generation 1 were removed from the Petri dish and placed in a glass bottle and crushed with a sterile glass rod. Two hundred CO2 anesthetized female flies (2–5 days post emergence) were placed in the glass bottle containing the crushed cadavers. The lid was loosely secured, and the bottle was gently shaken for 10 seconds to mix the anesthetized flies with the crushed cadavers and conidia. The live female flies were held in the glass bottle with the crushed cadavers for five minutes before releasing them into a clean BugDormTM. The glass bottle containing the conidia and cadavers was removed on day 4 (96 hours after placement).

Dead flies were removed from the cage and counted daily, starting at one-hour post treatment. Cadavers were placed on water-moistened filter paper in Petri dishes and held in an incubator set at 26°C in complete darkness for sporulation. Sterile DI water was added to the filter papers approximately every other day to maintain moisture. After one week in the incubator, dishes containing the cadavers were moved to the drying room. Sporulated cadavers were dried for 7–14 days before being used for the next generation of selection, remaining cadavers were placed in the refrigerator for long term storage. This procedure was repeated until 10 rounds of selection were completed.

Comparing Virulence of Selected and Unselected B. bassiana strain NFH10

To compare the new selected strain of NFH10 with the unselected founder strain a concentration-response experiment was conduted. This required re-isolating B. bassiana from the cadavers and culturing on medium. To do this, three of the earliest- dying cadavers (day 5) from generation 10 were placed in 1.5 mL microcentrifuge tubes and 1 mL of sterile 0.1% CapSil® was added. The tube was vortexed to remove conidia from the cadavers and to suspend the conidia in the liquid. Once the cadavers appeared clear of conidia, 100 μL of the vortexed liquid was placed on SDY agar and spread with a sterile disposable plastic L-shaped cell spreader (Fisher Scientific

Hampton, NH #14-665-231). Similarly, conidia were re-isolated from three cadavers from the preselection “generation-0” flies which died on day 5. SDY plates were labeled and placed in an incubator set at 26°C and held in complete darkness for seven days.

One week after inoculating the agar, the SDY plates were harvested using a wet harvesting method (Mishra et al. 2013). This method was used instead of dry harvesting because wet harvesting is more efficient, does not require a long drying period before

harvest, and results in suspensions that have less mycelium and other debris. The B. bassiana wet harvest method includes flooding the fungal plate with approximately 15 mL sterile 0.1% CapSil® and scraping the surface of the agar with a plastic disposable spatula (Figure 3-2). A sterile 250 mL Erlenmeyer flask was prepared with a sterile glass funnel resting on the top. A piece of glass wool approximately 70 mm in length was cut from the roll and unfolded to arrange a square (approximately 70 x 70 x 2 mm) to fit inside the funnel (Figure 3-3A). Once placed inside the funnel, the wool was premoistened by rinsing with 0.1% CapSil® and discarding the liquid collected in the flask (Figure 3-3B). The premoistening step was needed to improve the flow of the suspension through the glass wool. Once the wool was premoistened, the plate containing the wet conidia was carefully poured into the funnel and thoroughly rinsed into the funnel with a squeeze bottle containing 0.1% CapSil® (Figure 3-4).

The collected B. bassiana conidia suspension in the flask, henceforth referred to as the stock solution, was vortexed for 30 sec to homogenize the mixture before samples were taken for conidia counting. Conidial counts were conducted using an automatic cell counter (Cellometer® X2; Nexcelom Bioscience LLC, Lawrence, MA).

After the conidial concentration (conidia/mL) was determined for the stock solution, the stock solution was adjusted to obtain the desired 1x109 spores/mL concentration. The stock solution of the highest concentration of B. bassiana (1x109 spores/mL) was serially diluted to make the 1x108, 1x107 and 1x106 spores/mL concentrations in

CapSil®.

Concentration-response experiments were performed by exposing adult house flies to wet WhatmanTM #2 filter paper. Plastic disposable 100 mm diameter Petri dishes

(Fisher Scientific Hampton, NH) were prepared by placing a filter paper inside the lid of the dish with the outer surface of the bottom of dish was labeled with treatment conditions. Three replicates were included for each treatment: CapSil® 0.1% negative control and 1x106, 1x107, 1x108, and 1x109 B. bassiana spores/mL concentrations.

Working from the negative control then from the lowest to highest B. bassiana concentration, 1 mL of the solution was applied evenly on the filter paper with a pipette.

House flies used for this experiment were removed from cages 2–5 days post emergence and sorted using the knockdown device as previously described (Figure 3-

1). Twenty-five female adult flies were placed on the recently treated and wet filter paper, the dish was closed and lightly shaken to encourage conidia to fly contact. The flies were held in the Petri dishes for an exposure period of two hours. After two hours, the flies were anesthetized briefly using CO₂ and transferred to observation containers

(Figure 3-5A). These observation containers were 473 mL clear deli cups (Waddington

North America (WNA) Chelmsford, MA #WNAAPCTR16,) with fly food provided in a 30 mL plastic cup (Dart Solo, Mason, MI #PLN100). Water was provided in a 59 mL plastic cup as described previously. The food and water cups were taped to the bottom of the observation containers to help prevent the cups from tipping and spilling. The observation containers were covered with a 14 x 14 cm piece of mesh Tulle fabric and secured with deli cup lids that had a 6 x 6 cm square removed from the center (Figure

3-5B). Observation containers were held in an incubator at 25°C for the duration of the experiment (9 days).

The entire concentration-response (selected vs. unselected) experiment was repeated three times with three treatment replications per repetition using different fly cohorts and fungi batches.

Statistical Analysis

For the fly-to-fly passage experiment, the LT50 or time to 50% mortality was calculated for selected and unselected B. bassiana with Probit analysis using SAS® software, Version 9.4 of the SAS System for Windows, Copyright © 2002-2012, SAS

Institute Inc., Cary, NC. Differences in LT50 values were evaluated by examining non- overlapping 95% confidence intervals.

For the concentration response experiments the LC50, or the conidial concentration that kills 50% of the population, was determined separately for critical time points post infection, days 4─9. In addition, the LT50 for each concentration was determined for the selected and unselected strain using the Probit Procedure in SAS.

Differences in LT50 values were evaluated by examining non-overlapping 95% confidence intervals.

Results

A dose-response relationship for mortality was observed for both the unselected and selected strains of NFH10. At the highest concentration of 1 x 109 cfu, 100% mortality was observed seven days after exposure (Figure 3-6, Figure 3-7). Both strains resulted in greater than 92% mortality nine days after exposure at the 1 x 108 cfu concentration (Figure 3-8–Figure 3-9).

The time to 50% and 95% mortality (LT50 and LT95) was determined for each concentration (Table 3-1). Mortality rates were similar for both the unselected and

8 selected strains. The LT50 values at the 1 x 10 concentration were 5.1 and 4.8 days for

the unselected and selected strains, respectively. The LT50 values at the highest concentration of 1 x 109 cfu were and 3.9 and 3.7 days for the unselected and selected strains, respectively. The only significant difference (P<0.0001) between the strains was

7 at the 1 x 10 cfu concentration, where the LT50 was 8.1 and 6.5 days for the unselected

7 and selected strains, respectively. Although the LT95 at the same concentration (1 x 10 cfu) was about 2.5 days shorter for the selected strain, there was a slight overlap in the confidence intervals of this estimates for the two strains. Thus, there was not a

7 significant difference in the LT95 values at the 1 x 10 cfu concentration.

Lethal concentration (LC) values were determined at critical time points post exposure (days 4-9) and are expressed as the log function (Table 3-2). The LC50 for day

7 was 7.0 and 6.9 for the unselected and selected strains, respectively. Similarly, the

LC50 for day 9 was 6.6 and 6.5 for the unselected and selected strains.

Based on overlapping 95% confidence intervals for both the LC and LT values, there was little to no evidence of improved virulence after 10 generations of selection for

7 early dying flies. The only exception to this was the LT50 at the 1 x 10 cfu concentration, where the selected flies died somewhat faster than the unselected flies

(Table 3-1). This trend, however, was not significant for the LT95 values at the same concentration.

Discussion

Beauveria bassiana has substantial potential for management of adult house flies. However, the amount of time required to induce mortality using this entomopathogenic fungus may be an obstacle to its wide-spread use in the successful control of house flies. Laboratory selection for faster killing strains is one way to improve the virulence of selected strains.

Daoust and Roberts (1982) reported a substantial increase in virulence for two strains of Metarhizium anisopliae (Metchnikoff) after only one passage through Culex pipiens L. larvae; however, the enhanced virulence was not always stable after several subcultures on agar. Additionally, not all of the M. anisopliae isolates tested experienced the same increase in virulence, with some isolates remaining unchanged while others experienced a decrease in virulence. They concluded that the passage of fungal strains through mosquito larvae proved effective in increasing the virulence of M. anisopliae.

Other evidence for desirable trait selection in fungal pathogens comes from efforts to select for tolerance to fungicides that are commonly used in agricultural applications. Shapiro-Ilan et al. (2002) were successful in selecting for fungicide resistance (dodine, fenbuconazole and triphenyltin) in the GHA strain of B. bassiana and found that the resistance trait was stable after the selection pressure was removed.

They also found that the selected strain was as virulent as the parental GHA strain against larvae of the pecan weevil, Curculio caryae (Horn). However, in a subsequent study, these authors found that selection for fungicide resistance in B. bassiana and M. brunneum Petch had negative effects on other desirable traits such as germination success and virulence for wax moth larvae, Galleria mellonella (L.) (Shapiro-Ilan et al.

2011).

We found that it became increasingly difficult to obtain sufficient numbers of cadavers with adequate conidial blooms to continue the selection in the later generations. Cadavers obtained from generations 8 onward were characterized by sparse conidiation that made it difficult to assemble enough cadavers with sufficient

spore loads to continue the selection. These observations suggest attenuation of the selected strain as described by Sapiro-Ilan et al. (2011).

Nahar et al. (2008) documented an increase in the levels of virulence factors after M. anisopliae was passed through Helicoverpa armigera Hübner, resulting in a

th th lower LC50. After the 20 and 40 subculture on artificial medium, the LC50 values were higher than the first subculture, confirming that repeated passage through agar decreases virulence. Similarly, Adames et al. (2011) observed reductions in the LC50 of

M. anisopliae after successive passages through the cattle fever tick Rhipicephalus microplus (Canestrini).

Although it may seem counter-intuitive, the current study suggests that continuous passage through the insect host does not necessarily increase virulence.

Some unknown combination of passage through an insect host as well as artificial media may be required for stabilization of improved virulence of the selected strain.

Evidence from studies with other insect hosts indicates that selection for

“improved” entomopathogenic fungi does not necessarily result in the desired outcome.

Ignoffo et al. (1982) did not document a significant decrease or increase in the virulence of Nomuraea rileyi (Farl.) after 12 passages in larvae of Trichoplusia ni (Hübner). Also,

Holderman et al. (2017) documented no changes in virulence after seven selective passages of B. bassiana through the horn fly, Haematobia irritans (L.). Although the literature provides mixed results on the success of selecting for more virulent entomopathogenic fungi, it has been documented that repeated in vitro sub-culturing decreases virulence and that passage through an insect host can help maintain and restore virulence.

Figure 3-1. Knockdown device with CO2 tube inserted for anesthetizing adult house flies (Musca domestica L.) for experimental set up, A) assembled knockdown device used to anesthetize flies with CO2, B) shallow circular dish used as the bottom chamber, C) upper chamber made of a 15 cm Petri dish with holes for CO2 flow.

Figure 3-2. Wet harvesting Beauveria bassiana (Balsamo) Vuillemin using 0.1% CapSil®. Notice the left side of the plate has been scraped with the sterile plastic disposable spatula shown.

A B

Figure 3-3. Flask, funnel and squeeze bottle used for wet harvesting Beauveira bassiana (Balsamo) Vuillemin, A) glass wool placed inside glass funnel that is placed on top of 250 mL flask, B) glass wool after it has been moistened with 0.1% CapSil® in squeeze bottle.

Figure 3-4. Beauveria bassiana (Balsamo) Vuillemin conidia collected in flask, while debris remains in glass wool inside funnel after wet harvest.

Figure 3-5. Observation containers used to contain adult house flies (Musca domestica L.) in bioassays, A) observation container is made from 473 mL deli cups, food and water is provided at libitum, B) observation container covered with mesh Tulle fabric and secured with lid.

100

Mortality Mortality (%) 20

0 6 7 8 9 Log concentation in cfu/mL Unselected Selected

Figure 3-6. Mortality (%) of adult house flies, Musca domestica L., seven days after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and a sub-strain that was selected for 10 generations for faster mortality.

100

40 Mortality Mortality (%) 20

0 1 2 3 4 5 6 7 8 9 Time (Days) Unselected Selected

Figure 3-7. Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 109 colony forming units of an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and a sub-strain that was selected for 10 generations for faster mortality.

100

Mortality Mortality (%) 20

0 6 7 8 9 Log concentation in cfu/mL Unselected Selected

Figure 3-8. Mortality (%) of adult house flies, Musca domestica L., nine days after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and a sub-strain that was selected for 10 generations for faster mortality.

100

40 Mortality Mortality (%) 20

0 1 2 3 4 5 6 7 8 9 Time (Days) Unselected Selected

Figure 3-9. Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to a solution containing 0.1% CapSil® and 1 x 108 colony forming units of an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and a sub-strain that was selected for 10 generations for faster mortality.

Table 3-1. Adult house fly (Musca domestica L.) LT50 and LT95 values at different concentrations for an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and for a strain that was selected for 10 generations for faster mortality. 1 2 Concentration Selection Slope (SE) LT50 (95% CI) LT95 (95% CI) Chi-square P

CFU status

106 Unselected 0.243 (0.018) 10.54 (9.95–11.30) a 17.32 (15.93–19.15) a 190.5 <0.0001

Selected 0.185 (0.015) 11.47 (10.63– 12.61) a 20.34 (18.31–23.16) a 144.7 <0.0001

107 Unselected 0.325 (0.026) 8.11 (7.64–8.73) a 13.18 (12.02–14.87) a 152.2 <0.0001

Selected 0.387 (0.037) 6.54 (6.06–7.08) b 10.79 (9.79–12.30) a 111.1 <0.0001

108 Unselected 0.479 (0.048) 5.09 (4.59–5.59) a 8.52 (7.74–9.70) a 100.2 <0.0001

Selected 0.534 (0.062) 4.81 (4.23–5.36) a 7.88 (7.08–9.18) a 73.1 <0.0001

109 Unselected 0.928 (0.122) 3.91(3.51–4.32) a 5.68 (5.13–6.62) a 57.4 <0.0001

Selected 0.963 (0.078) 3.70 (3.47–3.92) a 5.40 (5.06–5.88) a 153.7 <0.0001

The total number of female house flies exposed at each strain and concentrated was 225. 1 Number of viable conidia (Colony Forming Units) applied in one mL of 0.1% CapSil® solution to 9-cm diameter filter paper disks; 25 female flies were confined in Petri dishes with treated filter papers for a two-hour exposure. 2 Lethal Concentration 95% Confidence Intervals (CI) within the columns were not significantly different due to a lack of non-overlapping CI indicated by (a)

Table 3-2. Adult house fly (Musca domestica L.) LC50 and LC95 values post-exposure to an unselected Beauveria bassiana (Balsamo) Vuillemin (strain NFH10) and for a strain that was selected for 10 generations for faster mortality. 1 2 Day Selection Slope (SE) LC50 (95% CI) LC95 (95% CI) Chi-square P status

4 Unselected 0.7303 (0.1276) 8.8944 (8.1883–11.7351) a 11.1468 (9.7224–20.5575) a 32.76 <0.0001

Selected 0.6993 (0.1261) 8.7713 (8.0115–11.9452) a 11.1234 (9.6301–22.1390) a 30.77 <0.0001

5 Unselected 0.9353 (0.1186) 7.7799 (7.1960–8.4690) a 9.5386 (8.7443–11.9287) a 62.19 <0.0001

Selected 0.9056 (0.0843) 7.5974 (7.1819–8.0354) a 9.4138 (8.7812–10.7640) a 115.27 <0.0001

6 Unselected 1.0294 (0.1630) 7.3300 (6.4502 –8.1833) a 8.9279 (8.1081–12.4883) a 39.89 <0.0001

Selected 1.0154 (0.1047) 7.1591 (6.6852–7.5944) a 8.7789 (8.1924–10.1213) a 94.01 <0.0001

7 Unselected 1.0313 (0.1504) 7.0022 (6.2043–7.6501) a 8.5967 (7.8686–11.2445) a 47.04 <0.0001

Selected 1.0755 (0.0629) 6.8788 (6.7767–6.9769) a 8.4082 (8.2386–8.6146) a 292.26 <0.0001

Table 3-2. Continued 1 2 Day Selection Slope (SE) LC50 (95% CI) LC95 (95% CI) Chi-square P status

8 Unselected 0.9578 (0.1798) 6.7453 (4.9428–7.6131) a 8.4626 (7.6006–14.8467) a 28.36 <0.0001

Selected 1.0099 (0.1054) 6.6697 (6.1547–7.0525) a 8.2985 (7.7656–9.5220) a 91.80 <0.0001

9 Unselected 0.9252 (0.1649) 6.5926 (4.8818 –7.3204) a 8.3705 (7.5537–13.2827) a 31.48 <0.0001

Selected 0.9919 (0.0662) 6.5012 (6.3767–6.6134) a 8.1594 (7.9773–8.3869) a 224.67 <0.0001

1 Day post exposure; 25 female flies were confined in Petri dishes with 9-cm diameter filter paper disks treated with one mL of Beauveria bassiana containing 1 x 106 through 1 x 109 colony forming units suspended in 0.1% CapSil® for a two-hour exposure. The total number of female flies exposed per strain and per concentration was 225. 2 LC values represent the log of the number of conidia applied to 9-cm diameter filter paper dish.

CHAPTER 4 COMPARISON OF VIRULENCE OF Metarhizium anisopliae AND FOUR STRAINS OF Beauveria bassiana AGAINST ADULT HOUSE FLIES

Introduction

House flies, Musca domestica L. (Diptera: Muscidae), are worldwide synanthropic and zoophilic pests. High populations are common in food animal production systems where there are ample decaying organic substrates for larval development. In addition to house flies being a nuisance, they are capable of transmitting over 100 disease-causing organisms that affect humans and other animals

(Malik et al. 2007, Khamesipour et al. 2018). Alternatives to chemical insecticides are needed for fly management as house flies have developed resistance to many active ingredients and they develop resistance to new chemicals within a short time after introduction (Kaufman et al. 2001a, Scott et al. 2013, Shah et al. 2015). Environmental concerns and the growing market for organically produced animal products have also played a role in the exploration of alternatives to chemical insecticides.

One of the most promising alternatives to chemical insecticides is the use of naturally-occurring entomopathogenic fungi. Although there are over 700 known species of entomopathogenic fungi (Samson et al. 1988), Beauveria bassiana (Balsamo)

Vuillemin and Metarhizium anisopliae/brunneum (Metchnikoff)/Petch have shown particular promise for house fly management (Kaufman et al. 2005, Mishra et al. 2011,

Acharya et al. 2015, Hasaballah et al. 2017, Johnson et al. 2018). A limitation of these fungi is the time required to cause high adult house fly mortality, which is typically 6–7 days post exposure (Geden et al. 1995, Mishra et al. 2011). This long latency period is an impediment to the adoption of fungi for fly management because it allows flies to develop at least one batch of eggs before succumbing to infection.

Currently the only commercial entomopathogenic fungal product registered for use against house flies in the United States is “balEnce" (Terragena Franklin, NC), which contains B. bassiana strain HF23. This strain was originally isolated from Florida house flies. There are also two widely used products whose labels mention a wide array of horticultural pests but do not include house flies. One is BotainGard ESTM (BioWorks

Inc., Victor, NY) which contains the GHA strain of B. bassiana stain. The other is

Met52TM (Novozymes BioAg, Durham, NC), whose label states that it contains the F52 strain of M. anisopliae, although this strain is currently viewed as belonging to M. brunneum Petch (Bischoff et al. 2009). All three commercial products are known to cause infections in adult house flies (Weeks et al. 2017).

Hundreds of strains of B. bassiana have been isolated from a wide range of host species, and these strains can differ markedly in their growth properties and virulence for different target pests (Roberts and Yendol 1971, Imoulan et al. 2017). Several studies have been conducted to evaluate different strains B. bassiana and M. anisopliae/brunneum against adult house flies (Barson et al. 1994, Geden et al. 1995,

Renn et al. 1999, Lecuona et al. 2005, Mwamburi et al. 2010, Mishra et al. 2011,

Sharififard et al. 2011).

Some of the studies cited above were conducted outside the US, using fungal isolates collected from the home country of the investigators. Because regulatory restrictions have made it increasingly difficult to import and release exotic biocontrol agents (Hajek et al. 2016), the search for superior pathogens for fly management in the

U.S. needs to concentrate on domestically-sourced material. Because strains collected from particular hosts and their environments may be somewhat adapted to those

circumstances (Hajek and St. Leger 1994), I decided to examine three strains of B. bassiana that were originally isolated from house flies collected on dairy farms (HF23,

L90, and NFH10). Of those strains, only HF23 has been incorporated into an EPA- registered product. For comparison I also evaluated the B. bassiana strain GHA and the

M. anisopliae/ brunneum strain F52 that are found in the commercial products

BotaniGard® and Met52TM, respectively. Although the latter two did not originate from house flies or from animal production systems, extending their use to include house fly management would only require data to expand the labels of EPA-registered products.

The objective of this study was to compare the virulence of four Beauveria bassiana strains and a Metarhizium anisopliae strain against adult house flies.

Materials and Methods

House fly rearing was carried out as previously described in Chapter 2. Adult house flies were fed a 4:4:1 ratio (by volume) of dried non-fat milk powder, sugar, and dried egg yolk. Water was provided in 3.8 L plastic buckets with foam packing

“peanuts”. House fly cages were kept in rearing chambers that are maintained at 23–

25°C approximately 80% RH under constant light. Rearing medium was prepared by placing 355 g (500 cm3) of Calf Manna® (Manna Pro Products LLC, Chesterfield, MO),

1,500 g (6,500 cm3) of wheat bran (Siemer Milling Company, Teutopolis, IL) and 3,750 mL water into rearing trays (55 x 43 x 8 cm) and mixed thoroughly by hand. House fly eggs were collected from a cage containing 2-week-old house flies. Eggs 2.0–2.5 mL

(20,000–25,000 eggs) were deposited on the surface of the larval rearing medium.

Larval trays were held in the larval rearing chamber maintained at 27–30°C approximately 80% RH under constant light. Once pupae were sclerotized at 6–7 days,

they were separated from the rearing medium by water floatation and then placed in a forced-air blower for drying.

The following strains of B. bassiana were investigated: L90, NFH10, HF23, and

GHA. In addition to the four B. bassiana strains compared, one strain of Metarhizium anisopliae sensu lato, F52, was included in the comparison. In the years since its original characterization this strain of M. anisopliae has been placed within the species

M. brunneum, but for historical reasons it is still often referred to as “M. anisopliae sensu lato” (Bischoff et al. 2009), and this designation was used here.

Due to the hydrophobic properties of B. bassiana, a wetting agent is required for adequate dispersion of conidial cells. For this purpose, CapSil® (Aquatrols, Paulsboro,

NJ) was diluted in sterile DI water to obtain a 0.1% CapSil® solution. Previous studies by Johnson et al. (2018) determined that CapSil® is compatible with B. bassiana. The

0.1% CapSil® solution was used as a diluent/carrier for all aspects of these experiments, including making new fungal plates, harvesting plates, and for diluting solutions.

Sources, Isolation and Propagation of Fungal Strains

In 1990, the L90 strain of B. bassiana was isolated from house fly cadavers on a dairy farm located in New York, and it since has been proven to be virulent to house flies (Geden et al. 1995, Johnson et al. 2018). In 2010, the NFH10 strain was isolated from house fly cadavers collected from a dairy located in Gilchrist county, Florida. Since original establishment of the NFH10 strain, to the author’s knowledge, this strain has not been investigated for virulence against house flies or other arthropods. Conidia of HF23 strain, used in the commercial product under the trade name “balEnce"TM (Terragena

Franklin, NC), were provided by the company that manufactures fungi for the

formulation (JABB of the Carolinas, Pine Level, NC). The conidia were provided in dry form and as a live culture on a barley medium.

Isolates of B. bassiana strain GHA and M. anisopliae strain F52 were cultured from fly cadavers that had been exposed to the commercial products BotaniGard® ES

(BioWorks, Victor, NY) and Met52TM (Novozymes BioAg, Durham, NC), respectively. To do this, live flies were exposed to various dilutions of the commercial products using the treated filter paper method described below in detail. Once mortality occurred, dead flies were placed on 90 mm diameter disks of grade 2 filter paper (WhatmanTM GE #1002090

Healthcare Maidstone, UK) moistened with sterile deionized water that were placed in plastic disposable 100 mm diameter Petri dishes (Fisher Scientific Hampton, NH). Petri dishes containing cadavers were placed within the fungal growth chamber (PercivalTM

I36VL, Perry, IA ) at 26°C and 80–99% RH and remoistened with sterile deionized (DI) water as needed to maintain moisture on the filter paper. After seven days, the cadavers were removed from the filter paper and four heavily sporulated cadavers were placed within a 1.5 mL microcentrifuge tube. Then, 1 mL of 0.1% CapSil® was added and vortexed for two minutes. Once the cadavers appeared to be washed clean of conidia, 100 μL of the conidial-containing CapSil® solution was applied to previously prepared plates containing Sabouraud dextrose yeast (SDY) (2.0% glucose, 1.0% peptone, 0.5% yeast extract, pH 7.0) agar in a Petri dish and spread with a plastic disposable L-shaped cell spreader (Fisher Scientific Hampton, NH #14-665-231). The

Petri dishes were placed within the fungal growth chamber as mentioned above for seven days.

Stock solutions of the fungal isolates were stored at -80°C at United States

Department of Agriculture (USDA) Center for Medical, Agriculture and Veterinary

Entomology (CMAVE) in Gainesville, Florida. Stock solutions were prepared by placing approximately 10 mg of conidia and100 mL Luria-Bertani (LB) broth (LB broth, Fisher

BioReagents, Pittsburgh, PA) within a 250 mL Erlenmeyer flask and placing on a shaker table. Every third day, a conidia count was taken using an automatic cell counter

(Cellometer® X2; Nexcelom Bioscience LLC, Lawrence, MA). On the third day when conidia counts were adequate to make new plates, a 100 μL sample was taken and placed on each SDY plate and spread with an L-shaped cell spreader. The plates were placed within an incubator at 26°C and 80–99% RH to confirm adequate sporulation and to test for contamination. Once the solution was deemed free of contaminates based on the condition of the newly cultured plates, and the conidia count was sufficiently high (approximately 3–5 days preparation), 500 μL of the fungal LB solution and 500 μL 50% glycerol (50% water) solution were combined, yielding an overall 25% glycerol stock solution. The solution was labeled with the strain and date and stored in sterile plastic centrifuge tubes at -80°C. These stocks were used to prepare new plates for use in bioassays. Plates made from stock solutions were sub-cultured a maximum of two times to minimize the risk of potential changes in virulence over time by repeated sub-culturing.

New plates were prepared by mixing the stock solution with a sterile pipet tip and then rinsing the tip in 100 μL of 0.1% CapSil® within a 1.5 mL microcentrifuge tube. The conidia that adhered to the pipet tip provided sufficient quantities of conidia to prepare plates. The conidia-containing solution in the microcentrifuge tube was vortexed, and

the contents of the tube (approximately 100 μL) was applied to a Petri dish containing

SDY agar and spread with an L-shaped cell spreader. All fungal plates were grown inverted within the fungal growth chamber in complete darkness at 26°C and 80–99%

RH for seven days.

Harvesting of Conidia and Preparation of Inocula for Testing

Seven to ten days after the SDY plates were inoculated with fungi, they were harvested using a wet harvest method (Mishra et al. 2013). This method was preferred over a dry harvesting method as the wet harvesting method does not require an additional seven to ten days for drying. Another major advantage of using the wet harvesting method over the conventional dry harvesting method is that the wet harvest suspensions have less mycelium and other debris, making the overall solution cleaner, allowing for more accurate readings using the Cellometer®.

Prior to fungal harvest, the biosafety cabinet was prepared for wet harvesting B. bassiana by first sterilizing with the ultraviolet light for five minutes. The fume hood was then sprayed with alcohol and wiped down to kill any remaining contaminants. A sterile

250 mL Erlenmeyer flask was placed in the cabinet and a sterile glass funnel was placed on the top of the flask. A piece of glass wool (approximately 70 x 70 x 2 mm) was placed inside the funnel at its base and premoistened with sterile 0.1% CapSil®.

Pre-moistening the glass wool facilitated the passage of conidia. All glass lab equipment was sterilized upon purchase or sterilized by an autoclave at 121°C for 45 minutes.

Wet-harvesting of conidia was conducted by flooding a fungal plate with 15 mL of

0.1% CapSil® and carefully scraping the surface of the agar with a plastic disposable spatula. Once the agar appeared clean of conidia, the suspension was carefully poured over the funnel and through the glass wool. The plate containing the remaining agar

was rinsed with an additional 15 mL of 0.1% CapSil®. Finally, the glass wool was rinsed with 15 mL of 0.1% CapSil®, carefully folded within the funnel and lightly squeezed to remove remaining conidia. This process resulted in approximately 50 mL of wet harvested conidia suspension.

The collected conidia suspension in the flask was vortexed for 30 seconds to homogenize the mixture before a sample was taken for determining the conidia concentration using an automatic cell counter (Cellometer® X2; Nexcelom Bioscience

LLC, Lawrence, MA), as described in Chapter 3. After the conidial concentration

(conidia/mL) was determined for the stock solution, it was adjusted to prepare the high concentration of 1x109 spores/mL. Adjustments to the concentration were made by either adding additional 0.1% CapSil® to the suspension (if counts were too high) or by centrifuging (Centrifuge 5810, Eppendorf, Hauppauge, NY) conidia (2,500 rpm for 9 minutes) to form a pellet and then resuspending in smaller volumes of 0.1% CapSil®, if counts were too low. The high concentration of 1 x 109 spores/mL was serially diluted in

0.1% CapSil® to prepare concentrations of 1 x 108, 1 x 107 and 1 x 106 spores/mL.

Bioassay Method

A modified vacuum with an insect collection tube was used to remove two-to-five- day old adult house flies from a cage. The flies were removed from the collection tube by briefly anesthetizing them with CO₂. To keep the flies anesthetized for the setup of the experiment, they were placed in the upper chamber of a custom-built, dual-chamber

CO2 “knockdown” device (Figure 3-1) as described in Chapter 3. Briefly, this is a dual- chambered device consisting of an upper chamber with perforations on the bottom nestled into a lower chamber through which CO2 passes from a compressed-gas supply.

Plastic disposable 100 mm diameter Petri dishes (Fisher Scientific Hampton, NH) were prepared by labeling the bottom of the dish with the fungal strain being tested, and the date, concentration, and replicate number (1–3). A 9 mm Whatman #2 filter paper disk was placed in the lid of the dish. Working with one solution at a time and starting with 0.1% CapSil® negative control, then from the lowest to the highest fungal concentration, 1 mL of each concentration was applied evenly to the filter paper. While the paper was still damp, 25 female flies were placed on the paper and the dish was covered with the dish bottom. The dish was lightly shaken to disperse the flies across the paper and to encourage uniform coverage of conidia across the batch of flies. Flies were exposed to the treated filter paper for two hours.

After the two-hour exposure, the flies were briefly anesthetized with CO2 and moved to observation containers (Figure 3-5A). These observation containers were made from 473-mL clear plastic cups (Waddington North America (WNA) Chelmsford,

MA #WNAAPCTR16). Adult fly food was provided in a 30 mL plastic cup (Dart Solo,

Mason, MI #PLN100) and water was provided in a 59 mL plastic cup that was covered with a lid with two slits cut into it to form a cross into which a dental wick (Absorbal

Co.,Wheat Ridge, CO) was inserted. The food and water cups were secured to the bottom of the observation containers with tape to help prevent the cups from tipping and spilling. The observation containers were covered with a 14 x 14 cm piece of mesh Tulle fabric and secured with plastic cup lids that had a 6 x 6 cm square removed from the center (Figure 3-5B). Observation containers were held in an incubator at 25°C and

80% RH for the duration of the experiment. Beginning one hour after the start of the experiment (day 0), mortality was counted each day for an additional nine days (day 9).

Statistical Analysis

For each of the five fungal strains tested the LC50, or the conidial concentration that kills 50% of the population, was determined separately for critical time points post infection; days 4─9. In addition, the LT50, or the time until 50% of the population had died, for each strain and concentration was determined with Probit analysis using SAS® software, Version 9.4 of the SAS System for Windows, Copyright © 2002-2012, SAS

Institute Inc., Cary, NC. Differences in LT50 values were evaluated by examining non- overlapping 95% confidence intervals.

Results

Dose-response relationships for mortality were observed for all five of the investigated strains. Six days after exposure to the high concentration of 1 x 109 cfu, the four B. bassiana strains resulted in 92–97% mortality while strain F52 resulted in 73% mortality (Figure 4-1). At the 1 x 108 cfu concentration B. bassiana strains GHA, HF23, and NFH10 resulted in nearly 100% mortality eight days after exposure, while strain L90 resulted in 80% morality and the M. anisopliae F52 strain resulted in 50% mortality

(Figure 4-2). Seven days after exposure to the same concentration the four B. bassiana strains resulted in greater than 96% mortality, while the M. anisopliae F52 strain generated 77% mortality (Figure 4-3). Nine days after exposure to the high concentration the four B. bassiana strains resulted in 98-100% mortality and the M. anisopliae strain generated 89% mortality (Figure 4-4). Nine days after exposure to the low concentration of 1 x 106 cfu the five strains generated 15-23% mortality (Figure 4-4).

The time to 50% and 90% mortality (LT50 and LT95) was determined for each of the five strains at four concentrations as shown in Table 4-1. The LT50 at the high concentration of 1 x 109 cfu ranged from 3.8 to 5.2 days for all five strains (Table 4-1).

Beauveria bassiana strains GHA, NFH10, and L90 were more virulent than M. anisopliae strain F52, while B. bassiana strain HF23 was not significantly different from

8 either the M. anisopliae or the other B. bassiana strains. LT50 values of the 1 x 10 cfu concentration ranged from 4.9 to 7.8 days (Table 4-1). The B. bassiana strains were not significantly different from one another, however the M. anisopliae strain F52 was

7 significantly less virulent at this concentration. The LT50s for the 1 x 10 cfu concentration ranged from 7.6 to 9.4 days (Table 4-1). Based on non-overlapping confidence intervals, B. bassiana strains GHA and L90 were significantly more virulent than the M. anisopliae strain F52. Beauveria bassiana strains HF23 and NFH10 were not found to be statistically different from GHA, L90, or F52 at this concentration.

Lethal concentration (LC) values (expressed as the log of the concentration of cfu’s) were determined at selected time points post exposure and are presented in

Table 4-2 (days 4–9). The LC50 for day 7 ranged from 7.1 to 8.0 (Table 4.2) Beauveria bassiana strains GHA and NFH10 were significantly more virulent than strain HF23 and

M. anisopliae, while strain L90 was not found to be significantly different from any of the others (Table 4.2). The LC50 for day 9 ranged from 6.7 to 7.4 for the five strains (Table

4-2). When compared on day 9, B. bassiana strains GHA and NFH10 were significantly more virulent than HF23; strains L90 and F52 were not significantly different from the

GHA, L90 nor HF23 (Table 4.2).

Discussion

Beauveria bassiana and M. anisopliae have exhibited potential for management of adult house flies. However, a limitation with using these entomopathogenic fungi is the time required to induce mortality in the insect host. One of the objectives of this

study was to determine which fungal stains isolated from house fly hosts were the most virulent for house flies and which induced a faster time to death.

Previous studies have indicated that there is wide variation in the virulence of different B. bassiana strains against house flies. Geden et al. (1995) compared 14 strains of B. bassiana isolated from house flies on New York dairy farms. One of the most virulent strains, L90, has been used in subsequent studies (Watson et al. 1995,

Burgess et al. 2018, Johnson et al. 2018). Lecuona et al. (2005) compared 17 strains of

B. bassiana for their potential as biological control agents of the house fly and found that three, collected from the sugarcane borer Diatraea saccharalis Fab. in Argentina, warranted further study for house fly management. In a comparison of 34 South African strains of B. bassiana, several were observed to cause 100% mortality of adult house flies in as little as four days and all 34 strains resulted in 100% morality within six days after exposure to 106 conidia/mL (Mwamburi et al. 2010).

In some instances, both B. bassiana and Metarhizium spp. have been evaluated in the same study. In a comparison of 10 Iranian fungal isolates of both species, the

437C strain of M. anisopliae was the most virulent against adult house flies (Sharififard et al. 2011). Mishra et al. (2011) concluded that M. anisopliae was superior to B. bassiana in terms of adult house fly mortality. They documented 100% house fly mortality four days after exposure for both species. However, comparison of the LC and

LT values suggest that M. anisopliae required less conidia as indicated by lower LC value and a faster kill as indicated by a lower LT value. Similarly, Barson et al. (1994) observed that M. anisopliae was significantly more virulent for house fly adults than five other species of entomopathogenic fungi, including B. bassiana. Both M. anisopliae and

B. bassiana caused 100% adult house fly mortality 6 days after exposure at the 1 x 105 conidia/fly dose, while M. anisopliae caused 94% mortality at the 1 x 104 conidia/ fly dose when B. bassiana caused only 54% mortality (Barson et al. 1994). In contrast,

Acharya et al. (2015) found that M. anisopliae was less virulent than B. bassiana, requiring 16 and 8 days, respectively, to cause 100% adult fly mortality. Although there were differences in the number of days required to kill the host, both fungal species caused more than 90% reduction in fecundity and a slight (13-20%) reduction in egg viability. Thus, the authors suggest that both fungi reduced lifetime reproductive output

(Acharya et al. 2015). A large-cage study in the UK demonstrated that incorporation of

M. anisopliae conidia into a food bait was effective for controlling adult house flies

(Renn et al. 1999). Weeks et al. (2017) compared several commercial formulations of fungal pathogens for adult house flies and found that BotaniGard® containing B. bassiana strain GHA and Met52TM containing M. anisopliae strain F52 had similar efficacy and that both were superior to balEnce containing B. bassiana strain HF23.

In the current study, the B. bassiana strains evaluated were generally more virulent than the F52 strain of M. anisopliae, renamed as M. brunneum, isolated from the commercial product Met52TM. This was most apparent at the high concentration of 1

9 x10 cfu/mL, where the LT95 for F52 was 2.5-3.5 days longer than any of the B. bassiana strains (Table 4-1). To my knowledge, no natural infections of house flies with

Metarhizium spp. have been reported from the US. Further surveys of natural mycoses in field populations of adult house flies may reveal additional strains that warrant investigation in future studies.

One of my goals was to determine whether B. bassiana strains that had originated from naturally infected house flies would be more virulent than the GHA strain, which was originally isolated from corn rootworm larvae (Diabrotica spp.,

Coleoptera: Chrysomelidae). Anderson et al. (2011) found some fungal isolates to be less virulent than others for adult house flies and suggested that isolates may adapt to their current host and lose plasticity for affecting other arthropods. Therefore, the search for entomopathogenic fungi to be used as biological control against flies should include isolates from the target insect and system (Steinkraus et al. 1991). Evidence for the effectiveness of this approach was provided by Leland et al. (2005), with Lygus bugs

(Heteroptera: Miridae) as the target pest. Several strains that the authors isolated from naturally infected Lygus spp. were more virulent than the GHA strain for this target pest and performed better under the hot conditions (32°C) where the insects are often found in the field (Leland et al. 2005). In contrast, Holderman et al. (2017) found that EN1 B. bassiana strain isolated from a horn fly, Haematobia irritans (L.), (Diptera: Muscidae) was less virulent for this target pest than either the GHA or HF23 strains.

Differences among the B. bassiana strains were modest and did not indicate that the three house fly origin strains (HF23, NFH10, and L90) had higher virulence overall than strain GHA (Tables 4-2 and 4-3). Indeed, one of the house fly strains (HF23) had significantly lower virulence than the others, at least on some days, indicated by higher

LC95 values and at some concentrations, indicated by higher LT95 values. In the current study, based on LC and LT values, the most effective fly-derived strain, was NFH10.

However, the differences were generally small, only marginally significant and may not have biological significance. These small differences probably do not warrant the high

cost that would be required to develop NFH10 as an EPA-approved product. Additional surveys are needed to identify strains with unambiguously higher virulence for flies than have been identified to date.

It is also worth noting that the efficacy and practicality of a entomopathogen strain is affected by considerations beyond virulence under controlled experimental conditions in the laboratory. These include production characteristics such as conidial yields under mass-cultivation conditions, storage stability, and temperature tolerances.

Further research would be required to determine whether any new strain of fungal pathogen strain, regardless of innate virulence, can meet the requirements for large- scale or commercial production.

Commercial strains HF23 and GHA have been mass produced on artificial media for decades, and L90 was first cultured almost 30 years ago. In contrast, strain NFH10 has been produced on a much smaller scale and had gone through only 6 passages, all in house fly hosts, before this study was conducted. Continued passage through artificial media has been shown to attenuate B. bassiana virulence (Schaerffenberg

1964, Butt et al. 2006). In the current study, perhaps strains L90 and HF23 exhibited lower virulence than NFH10 due to the amount of time they have been maintained on artificial media.

In addition to exploring for strains that are innately superior for the target insect and the environmental conditions in which it will be utilized, other methods can be used to increase the virulence of strains that have already been discovered. One of the potential ways to improve virulence is through the selection for faster-killing subcultures using a fly-to-fly passage system; although it is uncertain whether improved virulence

factors would be maintained when the strains are returned to artificial media for commercial-scale production. The attempt to select for a faster killing sub strain of B. bassiana was the objective of Chapter 3; however I was unable to demonstrate a lower time-to-kill with the NFH10 strain. Future work should examine genetically modifying entomopathogenic fungi to increase virulence and generate faster killing strains.

Genetic improvement of naturally superior strains could result in strains capable of inducing greater than 90% mortality as early as day 3, which would be prior to first oviposition in female adults. The development of such fast-killing B. bassiana, whether by discovery, selection, or genetic manipulation, would be a major advance that would greatly improve fly management options, particularly for organic production systems, and make this pathogen competitive with chemical insecticides for use in conventional fly management programs as well.

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20 Mortality (%) Mortality (%) 0 1 2 3 4 5 6 7 8 9 Time (Days) GHA HF23 NFH10 L90 F52

Figure 4-1. Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 109 colony forming units of Beauveria bassiana (Balsamo) Vuillemin strains GHA, HF23, NFH10 and L90 or Metarizium anisopliae (Metchnikoff) strain F52.

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40 Mortality Mortality (%) 20

0 1 2 3 4 5 6 7 8 9 Time (Days) GHA HF23 NFH10 L90 F52

Figure 4-2. Daily mortality (%) of female house flies, Musca domestica L., after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 108 colony forming units of Beauveria bassiana (Balsamo) Vuillemin strains GHA, HF23, NFH10 and L90 or Metarizium anisopliae (Metchnikoff) strain F52.

100 80 60 40

Mortality (%) Mortality (%) 20 0 6 7 8 9 Log concentration cfu/mL GHA HF23 NFH10 L90 F52

Figure 4-3. Mortality (%) of adult house flies, Musca domestica L., seven days after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana (Balsamo) Vuillemin strains GHA, HF23, NFH10 and L90 or Metarhizium anisopliae (Metchnikoff) strain F52.

100 80 60 40

Mortality (%) Mortality (%) 20 0 6 7 8 9 Log concentration cfu/mL GHA HF23 NFH10 L90 F52

Figure 4-4. Mortality (%) of adult house flies, Musca domestica L., nine days after forced contact exposure to 1 mL of a solution containing 0.1% CapSil® and 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana (Balsamo) Vuillemin strains GHA, HF23, NFH10 and L90 or Metarizium anisopliae (Metchnikoff) strain F52.

® 6 Table 4-1. LT50 and LT95 values (in days) for female house flies exposed to a 0.1% CapSil solution containing 1 x 10 through 1 x 109 colony forming units of Beauveria bassiana (Balsamo) Vuillemin (strains GHA, HF23, NFH10 and L90) or Metarhizium anisopliae (Metchnikoff) (strain F52). 1 Concentration Strain Slope (SE) LT50 (95% CI) LT95 (95% CI) Chi- cfu/mL square2

106 GHA 0.2047 (0.0365) 12.96 (11.45–16.02) a 20.99 (17.44–28.34) a 31.38

HF23 0.2554 (0.0452) 13.17 (11.68–16.18) a 19.61 (16.49–26.02) a 31.86

NFH10 0.2507 (0.0362) 11.64 (10.65–13.36) a 18.20 (15.80–22.47) a 48.02

L90 0.1803 (0.0188) 13.55 (12.25–15.48) a 22.67 (19.86–26.90) a 92.02

F52 0.2163 (0.0208) 12.55 (11.53–14.01) a 20.15 (17.97–23.34) a 107.71

107 GHA 0.3143 (0.0319) 7.77 (7.25–8.47) a 13.00 (11.59–15.36) ab 96.88

HF23 0.2829 (0.0226) 8.39 (7.88–9.05) ab 14.20 (12.86–16.17) ab 156.18

NFH10 0.4132 (0.0701) 7.87 (7.15–9.10) ab 11.85 (10.18–16.29) ab 34.77

L90 0.3406 (0.0305) 7.60 (7.12–8.22) a 12.43 (11.27–14.17) a 124.52

F52 0.2658 (0.0168) 9.42 (9.00–9.94) b 15.61 (14.56–16.95) b 249.26

Table 4-1. Continued 1 Concentration Strain Slope (SE) LT50 (95% CI) LT95 (95% CI) Chi- cfu/mL square2

108 GHA 0.5841 (0.0884) 4.90 (4.19–5.57) a 7.72 (6.79–9.61) ab 46.69

HF23 0.4342 (0.0681) 5.86 (4.95–6.55) a 9.65 (8.48–12.36) bc 40.69 NFH10 0.7154 (0.0593) 5.14 (4.81–5.47) a 7.44 (6.87–8.35) a 145.67

L90 0.4223 (0.0325) 5.85 (5.46–6.25) a 9.74 (9.01–10.76) b 168.76

F52 0.3174 (0.0326) 7.83 (7.25–8.62) b 13.02 (11.60– 15.29) c 94.57

109 GHA 0.8430 (0.1116) 3.81 (3.30–4.23) a 5.76 (5.14–6.95) a 57.07

HF23 0.8764 (0.1634) 4.52 (3.79–5.20) ab 6.40 (5.61–8.26) a 28.79

NFH10 0.9718 (0.0442) 4.27 (4.17–4.37) a 5.96 (5.80–6.16) a 482.75

L90 0.7284 (0.0754) 4.29 (3.90–4.67) a 6.55 (5.99–7.38) a 93.23

F52 0.4178 (0.0351) 5.19 (4.75–5.63) b 9.13 (8.36–10.22) b 141.47

1 Number of viable conidia applied in one mL of 0.1% CapSil® solution to 9-cm diameter filter paper disks; 25 female flies were confined in Petri dishes with treated filter papers for a two-hour exposure. 2The P-value was <0.0001 for all accounts The total number of female house flies exposed was 225 in all the above categories. Strain GHA was isolated from the commercial product BotaniGard®, strain HF23 was provided from the commercial producers of “balEnce", strain F52 was isolated from Met52. Data were analyzed using Statistical Analysis System (SAS version 9.4). values within a column and concentration followed by the same letter are not significant different due to non-overlapping 95% confidence intervals.

® Table 4-2. LC50 and LC95 values expressed as the log function for female house flies exposed to 0.1% CapSil containing 1 x 106 through 1 x 109 colony forming units of Beauveria bassiana (Balsamo) Vuillemin (strains GHA, HF23, NFH10 and L90) or Metarhizium anisopliae (Metchnikoff) (strain F52). 1 2 2 Day post- Strain Slope (SE) LC50 (95% CI) LC95 (95% CI) Chi- exposure square3

4 GHA 0.8261 (0.0579) 8.60 (8.46–8.75) a 10.59 (10.26–11.01) a 203.28

HF23 0.4881 (0.0599) 10.24 (9.77–10.98) c 13.61 (12.52–15.39) b 66.49

NFH10 0.6826 (0.1082) 8.19 (7.46–9.62) ab 10.60 (9.33–16.76) ab 39.83

L90 0.5707 (0.0508) 9.17 (8.92–9.49) b 12.05 (11.42–12.94) b 126.16

F52 0.4568 (0.0554) 10.24 (9.76–10.99) c 13.84 (12.70–15.69) b 67.89

5 GHA 1.0247 (0.0949) 7.71 (7.31–8.12) a 9.32 (8.75–10.51) ab 116.48

HF23 0.9206 (0.1104) 8.19 (7.71–8.88) ab 9.98 (9.17–12.29) ab 69.58

NFH10 0.9137 (0.0512) 7.70 (7.59–7.81) a 9.50 (9.29–9.76) a 318.63

L90 0.7509 (0.1023) 8.22 (7.63–9.25) ab 10.41 (9.34–14.22) ab 53.92

F52 0.5996 (0.1032) 8.92 (8.15–12.12) b 11.66 (9.96–22.47) b 33.74

6 GHA 1.0647 (0.0571) 7.33 (7.24–7.43) a 8.88 (8.71–9.08) a 347.94

Table 4-2. Continued 1 2 2 Day post- Strain Slope (SE) LC50 (95% CI) LC95 (95% CI) Chi- exposure square3

6 HF23 1.0473 (0.0569) 7.56 (7.47–7.66) b 9.14 (8.96–9.35) a 338.31

NFH10 1.0930 (0.0950) 7.42 (7.06–7.79) abc 8.93 (8.42–9.92) ab 132.36

L90 0.8308 (0.0921) 7.47 (6.93–8.00) abc 9.45 (8.70–11.36) ab 81.43

F52 0.6366 (0.0806) 8.23 (7.67–9.212) c 10.81 (9.63–14.60) b 62.36

7 GHA 1.0881 (0.0606) 7.05 (6.95–7.14) a 8.56 (8.39–8.76) ab 322.65

HF23 1.0897 (0.0587) 7.38 (7.29–7.48) b 8.89 (8.73–9.09) a 344.56

NFH10 1.1557 (0.0648) 7.01 (6.92–7.11) a 8.44 (8.28–8.63) b 317.82

L90 0.9300 (0.0529) 7.19 (7.09–7.30) ab 8.96 (8.77–9.20) a 309.41

F52 0.5895 (0.0903) 7.98(7.24–9.26) b 10.77 (9.40–16.96) c 42.62

8 GHA 1.1448 (0.0660) 6.90 (6.80–6.99) a 8.33 (8.17–8.53) a 301.27

HF23 1.0948 (0.0603) 7.16 (7.06–7.26) b 8.66 (8.50–8.86) a 329.36

NFH10 1.0701 (0.0631) 6.84 (6.73–6.934) a 8.37 (8.20–8.58) a 287.59

Table 4-2. Continued 1 2 2 Day post- Strain Slope (SE) LC50 (95% CI) LC95 (95% CI) Chi- exposure square3

8 L90 0.9599 (0.0559) 6.98 (6.87–7.09) ab 8.70 (8.51–8.92) ab 294.90

F52 0.6467 (0.1091) 7.64 (6.65–8.87) ab 10.18 (8.92–17.35) b 35.14

9 GHA 1.1888 (0.0714) 6.76 (6.66–6.85) a 8.15 (7.99–8.34) a 277.29

HF23 1.0651 (0.0606) 6.97 (6.87–7.07) b 8.51 (8.34–8.72) a 308.98

NFH10 1.0892 (0.0668) 6.71 (6.60–6.81) a 8.22 (8.05–8.42) a 266.07

L90 1.0196 (0.1311) 6.82 (6.13–7.33) ab 8.44 (7.80–10.31) ab 60.48

F52 0.6555 (0.1023) 7.43 (6.46–8.34) ab 9.94 (8.80–15.1457) b 41.04

1 Day post exposure; 25 female flies were confined in Petri dishes with 9-cm diameter filter paper disks treated with Beauveria bassiana or Metarizium anisopliae suspended in 0.1% CapSil® for a two-hour exposure. 2 LC values represent the log of the number of conidia applied to 9-cm diameter filter paper disks. 3 The P- value was <0.0001 for all accounts. The total number of female house flies exposed was 225 in all the above categories. Strain GHA was isolated from the commercial product BotaniGard®, strain HF23 was provided from the commercial producers of “balEnce", strain F52 was isolated from Met52. Data were analyzed using Statistical Analysis System (SAS version 9.4). values within a column and concentration followed by the same letter are not significant different due to non-overlapping 95% confidence intervals.

CHAPTER 5 DISCUSSION OF FINDINGS AND FUTURE RESEARCH DIRECTIONS FOR USING Beauveria bassiana AGAINST HOUSE FLY (Musca domestica) IMMATURES AND ADULTS

House flies, Musca domestica L., are non-biting synanthropic pests found all over the world except Antarctica. They pose medical and veterinary concern as they are potential vectors for over 100 disease-causing organisms that affect both humans and other animals (Shah et al. 2016). Insecticide resistance paired with growing environmental concern has fueled the investigation of alternative management strategies (Scott et al. 2013). The use of biological control methods has been an area of focus as it utilizes natural enemies of the pest insect such as predators, parasitoids and pathogens. Although there are a few exceptions, resistance to biological control agents develops slowly and is not likely to lead to outright failure as with most insecticides. The use of biological control agents has both advantages and disadvantages and a better understanding of their direct potential is critical to their successful use.

The most wildly-studied entomopathogenic fungi for filth fly management,

Beauveria bassiana (Balsamo) Vuillemin, has shown promising results (Kaufman et al.

2005, Hasaballah et al. 2017). Asexual spores of this fungus, also known as conidia, must first attach to the cuticle of the host insect (Hasaballah et al. 2017). Upon rehydration, the conidia germinate and form specialized structures that aid in penetrating the host insect (Ortiz-Urquiza and Keyhani 2013). Once the conidia penetrate the exoskeleton, the pathogen proliferates within the hemocoel, utilizes host nutrients and ultimately results in insect death. In addition to causing direct insect mortality, it has been documented that B. bassiana also reduces fecundity (Acharya et al. 2015). The primary limitation to using B. bassiana is the time required to induce

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mortality, which can take up to seven days post exposure. In addition, it is unknown if B. bassiana is compatible with beneficial insects that also target the house fly host. The use of multiple-compatible biological control agents in a single system would have an additive effect on the management of adult house flies.

The ultimate goal of my MS research was to identify superior strains of B. bassiana for various life stages of the house fly. In addition, I examined a Metarizium anisopliae (Metchnikoff) strain against adult house flies. Similar research comparing B. bassiana and M. anisopliae strains against adult house flies has been conducted

(Barson et al. 1994, Acharya et al. 2015, Weeks et al. 2017). Unlike other studies, one of the goals of my work was to answer the following question: “Are strains of B. bassiana originally isolated from house flies more virulent against the house fly than strains isolated from other insect hosts?”

The original intent of my MS project was to find a reliable way to infect house fly larvae, to produce mass quantities of infected pupae, and to examine the susceptibility of hymenopteran pupal parasitoids to infected hosts. Through multiple larval assays was unsuccessful at reaching this goal, including the four assays presented in this thesis. Not included in the thesis are results of eight other experiments that did not produce infected pupae and were discontinued following one or two replications. The original objective of examining the compatibility of B. bassiana with pupal parasitoids was abandoned and larval assays were reevaluated in an effort to define experimental parameters that favored infection of fly immatures. Future research is needed on evaluating the virulence of B. bassiana and M. anisopliae against pupal parasitoids.

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In Chapter 2, two strains of B. bassiana were evaluated against larval house flies. Strain L90 was originally isolated from house fly cadavers and is not currently commercially available. Strain GHA was originally isolated from corn rootworm larvae

(Diabrotica spp., Coleoptera: Chrysomelidae) and was represented by the commercial product BotaniGard® ES (BioWorks Inc., Victor, NY). The results of this study indicate that there is a narrow (1- to 2-day) window of larval susceptibility, making infection with

B. bassiana challenging. Moreover, treatment effects were only seen when rates of conidial application were massive and economically unfeasible. Steinkraus et al. (1990) documented high pupal mortality when larvae were exposed as third instars. However,

Mwamburi et al. (2010) and Barson et al. (1994) were unable to infect larvae using a dipping method. The narrow window of susceptibly presented in the current study may be the reason why there are differences in the literature. Other factors that affect susceptibly include the strain, exposure method and fitness of the fungal culture. In addition, cuticular protective properties have been proposed as a potential impediment to successful larval infection (Holder and Keyhani 2005, Ortiz-Urquiza and Keyhani

2013). As such, it is hypothesized that the time of molting is a factor affecting susceptibility. The relationship between host age and susceptibility may be a major factor in which house fly larvae are resistant to infection to B. bassiana, however future research is needed to understand why.

The focus of Chapter 3 was to select for a faster adult house fly-killing strain of B. bassiana. For this experiment, I chose to use strain NFH10 which was isolated from a house fly cadaver on a dairy farm. A fly-to-fly fungal rearing system was utilized to select for improved virulence and time-to-death using the earliest dying flies to start

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each subsequent generation. This selection was conducted for 10 generations, after which selected and unselected strains were grown on SDY agar media prior to use in forced-contact exposure assays. After 10 generations of selection, there was not a significant difference in the unselected and selected strain in terms of percent mortality,

Lethal Time (LT) or Lethal Concentration (LC). However, there was a slight decrease

(non-significant) in the LT values and a stabilization of values after the selection. Future work could examine if a longer selection period would result in a significant difference of virulence and stabilization of the increased virulence over time. In addition, the strain I used for these tests may have lacked virulence alleles for faster host death and that there was insufficient genetic “raw material” on which the selection could act.

Unlike the larval assays, the experiments in Chapter 4 involving adult house flies were largely successful. In addition to strains L90, GHA, and NFH10, strains HF23 (B. bassiana) and F52 (M. anisopliae) were evaluated against adult house flies. Prior to testing, commercial strains were isolated from their products and all strains were grown on SDY agar. The B. bassiana strains resulted in the highest mortality while M. anisopliae strain F52 generated significantly less mortality. Based on LC and LT values, strains GHA and NFH10 were consistently identified as the superior strains.

Each experiment presented above were conducted to improve producer options for reductions in house fly populations using entomopathogenic fungi. Although the original objective of examining the compatibility of infected pupae with pupal parasitoids was not done, this research resulted in other useful findings. Successful larval infection has been narrowed to a short window of susceptibility. Future work is needed to answer questions about the refractory nature of house fly larvae to B. bassiana infection.

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Assessing the virulence of M. anisopliae against house fly larvae may provide useful results as suggested by Mishra et al. (2011).

My studies demonstrated the virulence of B. bassiana and M. anisopliae against house fly adults using a forced contact exposure method. Field assays are needed to determine if the superior strains and concentrations presented here are successful in agricultural settings. My studies demonstrated that strains isolated from house flies are not necessarily more virulent against the house fly host. An ongoing question is to determine if strains are better adapted to certain geographical or environmental conditions. As such, are strains isolated from cattle farms better suited for use on cattle farms compared to strains isolated from horticulture settings? Future research is also needed to decrease the latency period of B. bassiana to generate faster mortality.

Genetic modification of the fungi may have the greatest potential to improve virulence, but only if the regulatory climate changes in ways that allow field use of genetically- modified pathogens. Overall, my research confirms the potential utility of B. bassiana for house fly management, but leaves a number of unanswered questions on which future work can be built to improve potential practical application of this important biological control agent.

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BIOGRAPHICAL SKETCH

Roxie White was born in Port St. Lucie Florida to Thomas and Rhonda White.

From an early age, her affinity for animals inspired her dreams of becoming a veterinarian. During her youth, Roxie began riding and training horses and was involved in the local 4H where she raised two show steers. The more she learned about agriculture, the more she chased the lifelong passion of helping livestock. Throughout grade school, Roxie always excelled at math and sciences and even won the science fair in elementary school.

In 2012 Roxie graduated from Bayside High School in Palm Bay, Florida with her high school diploma as well as her Associate of Arts degree. In the spring of 2013 Roxie started her education at the University of Florida in Gainesville, FL. as an animal science major. During her undergraduate career, Roxie took challenging prerequisites required for veterinary school. In the summer of 2015, she began working at the USDA under Dr. Christopher Geden as a biological science technician and found herself doing entomological research. Roxie received her bachelor’s from the University of Florida in spring 2016.

After graduating with her bachelor’s, Roxie landed a full-time position at the

USDA where she performed experiments with biological control methods for house fly management. Her passion for science and helping livestock led her to a enroll in graduate school in 2017 under Dr. Christopher Geden to further her knowledge of medical and veterinary entomology. She finished her Master of Science in December

2019 from the University of Florida and continues to work at the USDA in hopes of expanding her research career.

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