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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.

Permanent Link: http://ufdc.ufl.edu/UFE0041391/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.
Physical Description: Book
Language: english
Creator: Diclaro, Joseph
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Joseph Diclaro.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Koehler, Philip G.
Electronic Access: INACCESSIBLE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041391:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041391/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.
Physical Description: Book
Language: english
Creator: Diclaro, Joseph
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Joseph Diclaro.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Koehler, Philip G.
Electronic Access: INACCESSIBLE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041391:00001


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1 BEHAVIOR RESPONSE OF MU SCA DOMESTICA TO VISUAL STIMULI AND THE DEVELOPMENT OF A NOVEL FILTH FLY CONTROL DE VICE By JOSEPH WILLIAM DICLARO II A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Joseph William Diclaro II

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3 To my family, alway s keeping me focused and driven

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4 ACKNOWLEDGMENTS I would like to thank the United States Navy for granting me the opportunity to pursue a PHD in entomology under the Medical Service Corps Inservice Procurement Program: an experience I have found rewarding and challenging. CAPT (ret.) Gary Breeden, CAP T Stan C ope, CDR George Scheoler, CDR David Hoel, LCDR Craig Stoops, and LCDR Pete Obenauer provided exceptional support and irreplaceable guidance throughout this entire process All of which, I hope will continue throughout the remainder of my career. I would like to extend a special thanks to Dr. Gary Breeden who started me on the path of entomology and continued to guide me throughout my studies. He was always available for guidance along with pushes when I needed them. His leadership, advice, and example w ere part of the foundation that kept me focused and on track. I will always carry the deepest respect for him and will pay his generosity and support forward to the next person. I would like to acknowledge Dr. Phil Koehler who has been my academic advisor for these three years His guidance, mentorship, and above all friendship have made me a better person professionally and personally. Dr. Koehler always took time to discuss or review research projects and never was too busy to help with any aspects of his students life. His leadership is truly, what helped me be successful through my entire term at the University of Florida I would also like to thank Dr. Roberto Pereira who spent more time in front of the computer with me going over stats and writing than he may have cared too. Even at his busiest times, he would always stop what he was doing to help students with any aspects of their research. He had taught me more about my new chosen field than he

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5 realized and I am truly grateful. I hope to conti nue in emulating his methodology in approaching questions. Dr. Richard Patterson I would like to thank for his unknowingl y being the person who seemed to always arrive at the right time. H e always had time to talk with me and was never too busy to discuss any subject, which was always a welcomed distraction. I would like to thank Dr. Ellis Greiner who was able to step in as my external member. I was his last entomology student before he starts the lush retired life. Even though I did not get a chance to know him as much as I would have liked, h e opened my eyes on subjects I never knew I had an interest Mark Mitola made the fly program func tion every day over the last three years and without all of his dedicated and consistent work, the house fly colony would have died. He has helped more than any other has when it came to my research. He was always there to provide a helping hand and would often collect data when I was unavailable to do so. Without his help, this research could not have been completed. To my fraternal brother LT (FMF) Jeffrey C. Hertz we have been through many adventures and will surly see many more. Thank you for your friendship and continued support. You were there for some of the best and worse times of my life, but you were alwa ys there. The compass you gave me kept me in the right direction. So mote it be. Most of all I would like to thank my family and friends who have sacrificed their time and energy that contributed to my success. Thank you for all that you did and contin ue to do.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 LITERATURE REVIEW .......................................................................................... 16 Musca domestica Linnaeus .................................................................................... 16 Identification ..................................................................................................... 16 Eggs ................................................................................................................. 16 Larva ................................................................................................................ 17 Pupa ................................................................................................................. 17 Adult ................................................................................................................. 17 Insect Vision ........................................................................................................... 18 Structure and Function ..................................................................................... 19 Ommatidia .................................................................................................. 19 Ocelli .......................................................................................................... 20 Optic lobes ................................................................................................. 21 Vision ................................................................................................................ 21 Image formation ......................................................................................... 22 Resolution .................................................................................................. 22 Photoreceptors ........................................................................................... 23 House Fly Importance ............................................................................................. 24 Control: Baited Fly Traps ........................................................................................ 25 Using Visual Attraction for Control of House Flies .................................................. 28 3 RESPONSE OF MUSCA DOMESTICA L. TO VISUAL STIMULI ........................... 32 Introduction ............................................................................................................. 32 Materials and Methods ............................................................................................ 34 Results .................................................................................................................... 39 Discussion .............................................................................................................. 41

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7 4 FLY KILL CAUSED BY TOXICANTS IN WOOL YARN APPLIED TO A BLUE COLORED FLY CONTROL DEVICE ...................................................................... 50 Introduction ............................................................................................................. 50 Materials and Methods ............................................................................................ 51 Results .................................................................................................................... 59 Discussion .............................................................................................................. 61 5 FIELD EVALUATION OF A FLY ATTRACTANT SYSTEM WITH TOXICANT TREATED CORDS ................................................................................................. 71 Introduction ............................................................................................................. 71 Materials and Methods ............................................................................................ 72 Results an d Discussion ........................................................................................... 77 6 CONCLUSION ........................................................................................................ 84 APPENDIX A EFFECTIVENESS OF FLY TRAPS AND TREATED CORDS IN CATCHING AND KILLING HOUSE FLIES (DIPTERA: MUSCIDAE) ......................................... 87 Introduction ............................................................................................................. 87 Materials and Methods ............................................................................................ 87 Results .................................................................................................................... 89 Discussion .............................................................................................................. 90 B CONCISE DESCRIPTION OF THE INVENTION ................................................... 93 LIST OF REFERENCES ............................................................................................... 98 BIOGRAPHICAL SKETCH .......................................................................................... 104

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8 LIST OF TABLES Table page 3 1 Wavelengths (nm) and reflected intensity (cd/m2) of light from light sources on visual targets used in electroretinogram and twosided light tunnel experiments. ....................................................................................................... 45 3 2 Measured depolarization of female house fly equatorial region of right compound eye and median ocellus from white LED light source reflected off of plas tic visual target. ........................................................................................ 46 3 3 Comparison of house fly response to fabric visual targets and plastic visual targets used in twosided light tunnel with GE daylight fluorescent bulb. ........... 46 4 1 House fly mortality in choice laboratory cage study using twelve 10 cm pieces of black wool yarn distributed uniformly across ceiling of cage. .............. 64 5 1 Estimated percent fly control calculated per FAST TC location separated by fly species. .......................................................................................................... 80

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9 LIST OF FIGURES Figure page 3 1 Typical electroretinogram elicited from female house fly equatorial region of compound eye A) and median ocellus B) after 0.5 s stimulus of white reflected light. ..................................................................................................... 46 3 2 Mean behavioral response of house flies to fabric visual targets compared to black after 2 h in twosided light tunnels. ............................................................ 47 3 3 Mean behavioral response of house flies to fabric visual targets in two sided light tunnel; white vs. yellow, blue vs. yellow and blue vs. white. ........................ 47 3 4 Mean behavioral response of house flies to different colored lines on blue backgrounds in twosided light tunnels compared to solid black. ....................... 48 3 5 Mean physiological response of house flies from electroretinogram experiments with house fly behavioral response in twosided light tunnels. ..... 48 3 6 Spectral reflectance of sunlight from colored plastic visual targets. ................... 49 4 1 Schematic drawing of collapsible FAST TC. ...................................................... 65 4 2 Mortality of flies exposed to cords treated with different active ingredient. ........ 66 4 3 Mean number of dead house flies after 1, 3, 5, and 24 h exposure to wool cord or wool yarn treated with 2.5% imidacloprid fly bait. ................................... 68 4 4 Indoor and semi outdoor trap height comparisons over 24 h ............................. 69 5 1 Daily percent of dead house flies collected under FAST TC in relation to total dead flies collection (based on 44,598 total flies). .............................................. 82 5 2 Daily percent of dead scuttle flies collected under FAST TC in relation to total dead flies collection (based on 61,705 total flies). .............................................. 82 5 3 Daily percent of dead blow flies collected under FAST TC in relation to total dead flies collection (based on 3,136 total flies). ................................................ 83 A 1 Mortality of house flies at 1, 24, and 48 h after initial exposure to different fly trap treatments in field cages. ............................................................................. 92 A 2 Correlation between annoyance index and number of surviving flies observed in field cages. ...................................................................................................... 92 B 1 Cylindrical fly attractant system with toxicant treated cords. .............................. 95

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10 B 2 Fly attractant system with toxicant treated cords placed near fly infested garbage cans, which eliminated 3006 flies in 24 hours. ..................................... 96 B 3 Dead flies killed by the fly attractant system with toxicant treated cords near a house fly infested dumpster. ............................................................................ 97

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BEHAVIOR RESPONSE OF MUSCA DOMESTICA TO VISUAL STIMULI AND THE DEVELOPMENT OF A NOVEL FILTH FLY CONTROL DE VICE By Joseph William Diclaro II May 2010 Chair: Philip G. Koehler Major: Entomology and Nematology House flies see reflected sunlight from objects by both their compound eyes and ocelli and use this vi sual input for survival. Neurological responses elicited by reflected light from plastic colored visual targets of house flys compound eye and ocelli were measured using an electroretinogram. Light of the same intensity reflected from multiple plastic visual targets showed that house fly compound eye and ocellus were capable of color vision. The greatest neurological responses for the compound eyes and ocelli were to white, blue, and yellow. Behavioral responses of house flies to colored plastic and fabric visual targets compared to black were determined using a twosided light tunnel. The house fly attraction was White = Blue > Red > Green > Yellow. In a paired test blue visual target was more attractive than white. Yellow visual targets proved to be visual repellent for house flies. However, the addition of black lines, which simulated a harborage area, enhanced the attractiveness of the blue background. Combining visually attractive color patterns with wool insecticide treated cords led to the construction of a fly control device: FAST TC Comparisons of active ingredients, (deltamethrin, thiamethoxam, imidacloprid, indoxacarb, fipronil, and cyfluthrin) applied to

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12 FAST TC in field cages effectively reduced the house fly population by > 88% in 24 h compared to the 3% decrease of the untreated control Bioassay conducted with sticky cards hung simultaneously at three heights caught 50% of the house flies released in a lab oratory room at the lowest height, which d emonstrat ed that house flies tend to fly low. At all three of the tested heights the m odified version of the FAST TC was able to attract and kill ~90% of the house flies In field evaluations, 2.5% imidacloprid was applied to the FAST TC and was able to control two species of fly populations. The FAST TC achieved >70% control for Musca domestica population and 79% fly control of Megaselia scalaris Due to the FAST TC efficiency in field evaluations, the University of Florida submitted a patent application for this device.

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13 CHAPTER 1 INTRODUCTION The U.S. Department of Defense (DOD) initiated a program in 2004 for the development and testing of tools for control of pest and diseasecausing vectors called the Deployed War Fighter Protection (DWFP) program. The house fly is a mechanical vector of 10 0 known pathogens (Greenburg 1971) and a major pest/disease vector during natural disast ers and wartime. The purpose of the following research was to develop a new tool that effectively control s the house fly and other filth fly populations that can be useful for the DOD and as well as the civilian sector. House fly control is best achieved using integrated management strategies combining cultural, mechanical, biological, and chemical control methods (Kolbe 2004). Baited fly traps have been available in various designs since the early 1900s and are often used in combination with other methods for fly control (Pickens 1995). There is some debate whether baited traps fit into a fly management program best as monitoring rather than a control d evices. To reduce a fly population by 5090%, traps must remove 2458% of the house flies daily (Weidhaas and Haile 1978); however, traps alone have not been shown to reduce fly populations to acceptable levels (Smallegange 2004). A c hemical control met hod used in the past included the use of cords treated with insecticide. Cords treated with organophosphate and chlorinated hydrocarbon insecticides were introduced in 1947 and were found to provide good house fly control (Smith 1958). However, use of tr eated cords fell in popularity due to the ease of applying residual insecticide sprays directly to structures. In the 1990s there was a decrease in residual insecticide usage due to public opinion. Which spurred research to utilize modern low risk insect icides and new materials with the vintage idea of

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14 insecticide treated cords. In field conditions l ow concentrations of new insecticides, such as 0.1% fipronil and 1.2% indoxacarb impregnated in natural fiber cords were shown to effec tively kill flies (Her tz 2007) Initial experimentation sought to improve a baited fly trap with the addition of a inse cticide applied to a wool cord (Appendix A ). Baited fly traps have two main lures to catch flies: visual lure and chemical lure. The visual lure attracts fli es in from a distance, and when the fly is in close proximity to the trap, the chemical lure attracts the fly to enter the trap (Pickens 1995). The additional of the insecticide treated cord would eliminate the flies that are visually attracted to the trap even when they only contact the outside surface of the trap. Flying insects such as the house fly rely on their compound eyes to guide them to locate food and potential mates (Nation 2002). An u nderstanding of the physiological and behavioral responses of house flies to visual cues was used in the development of ef fective control device (Chapter 3 ). The use of electroretinogram s allowed for the measurement of house fly physiological responses to reflective color with the compound eyes and ocelli. Beh avioral response to reflective color was observed with the use of a two sided light tunnel, which was designed to compare attractiveness of colors and house fly behavior in a controlled environment. Once attractive colors were identified, a fly control dev ice was designed and constructed. This fly control device used attractive color combinations to lure flies in from a distance and then eliminated the flies with the use of insecticide treated cords. Considerations in development of this device were to make it practical for use (Chapter 4), including construction with low cost materials and effective insecticide s.

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15 To determine the ability of this device to control fly populations, extensive fieldtesting was conducted i n a variety of settings (Chapter 5). The goal of the devic e was to reduce fly populations by 50% in the first 24 h. In addition, this device was field evaluated on more than one species of filth fly.

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16 CHAPTER 2 LITERATURE REVIEW Musca domestica Linnaeus Musca domestica, or house fly, is one of approximately 4,200 species out of 190 genera in the Muscidae family (Moon 2009). It is a synanthropic fly, commonly found in agricultural environments and unsanitary conditions, is often viewed as a nuisance and embarrassment. Breeding s ites include but are not limited to garbage, manure, rotting plants, carrion, and other decaying organic matter. In nature, house flies serve a vital function in recycling animal manure and refuse by replacing nutrients back into the soil (Hedges 1998). However, due to its close interaction with humans and propensity to harbor diseasecausing pathogens the house fly has been labeled as one of the most dangerous insect s known to man (West 1951). Identification The house fly is classified in the class Hexa poda, order Diptera, the fourth largest order suborder Brachycera, infraorder Muscomorpha (Cyclorrhapha), and family Muscidae (Triplehorn and Johnson 2005). Eggs House fly eggs are white in color measuring 1 mm in length and wider in diameter on the posterior end than on the anterior end (West 1951). The overall shape of egg being bananalike with both ends bluntly rounded. Eggs are laid in large clusters of 120150 eggs. Female house flies are capable of producing up to six batches of eggs in thei r lifetime, depending on environmental factors.

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17 Larva House fly larva are known as maggots and have three instars (Moon 2009). Maggots have no antennae, eyes, or appendages. The body is tapered to a point at the anterior end, while the posterior end is w ider and rounded. Strong mouth hooks, made of sclerotized mouthparts, are the main component of the cephalopharyngeal skeleton on the anterior end of the larvae. On the posterior end, there is a pair of black spiracular plates, used for gas exchange, sha ped like two half circles (D shaped) back to back. Pupa The house fly, being a holometabolous insect, undergoes pupation when the third instar larvae contracts and hardens its integument to form a puparium. This process is completed within a 6h period ( West 1951). The puparium is reddish brown in color and is 6.3 mm in length. It has a cylindrical shape with bluntly rounded ends. As in the larval instars, two button shaped spiracles are located on the posterior end that are left over from larval devel opment but are no longer utilized for gas exchange. Between the fifth and six body segments, there is a pair of spiracles on the dorsal side used for gas exchange. Adult Adult house flies are dull gray in color and about 6 to 9 mm in length (Moon 2009). The top of their thorax has four black longitudinal stripes with wings that are longer than the abdomen. The wings have key identification characteristics in their venation; the first anal vein is present but does not reach the wing margin, and there is a sharp upward bend in the fourth longitudinal wing vein, m1 (Kolbe 2004).

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18 Male and female house flies can be differentiated by the abdomen, both sexes have modifications in the last six segments (West 1951). The male house fly has a genital pouch that con tains an aedeagus. The female house flys abdomen terminates into a telescopic ovipositor. The head has two large compound eyes with three ocelli at the dorsal midline. Male house flies compound eyes are holoptic and female compound eyes are dichoptic (T riplehorn and Johnson 2005). The antenna has a scape, pedicel, and an arista. The house fly, a nonbiting fly, has sponging mouthparts consisting of a proboscis that has a basal rostrum that articulates with a haustellum, and terminates at the labellum (Moon 2009). An important defining characteristic of the house fly, as well as other muscid flies, is the enlarged labella that are made up of two lobes with a mesal surface of pseudotracheae. Ps e udotracheae assist in the digestion and scraping of food by acting as a salivasoaked sponge. Also located within this structure are dentate prestomal teeth that assist in the scraping and liquefying of food. Insect Vision Visual systems in insects help relay external information to their brain to help guide behavior, such as flying and walking. In flying insects such as Diptera, the eyes serve as a way to detect movement on a vertical and horizontal plane. A visual system in most insects consists of two compound eyes and a set of ocelli. The compound eyes are structured with ommatidia which can range in number from insect species to species. In dragonflies, approximately 10,000 ommatidia can be found, whereas a Drosophila fly may only have 800 ommatidia (Chapman 2006). Not all insects have this type of visual system; more primitive insects and some larva may only have simple eye structures or other types of sensory organs.

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19 Structure and Function Ommatidia The primary func tion of the ommatidium is to allow light into the compound eye which is transformed by sensory cells into electrical energy to form an image. Light enters the ommatidium by first entering a transparent cuticle also known as the cornea or lens (Nation 2002). Directly below the cornea is a cone that helps direct the light to photosensitive cells called photoreceptors. The photoreceptors are long, slender receptor neurons known as the retinular cells. These cells are considered to be the main receptors for the ommatidia because their axons are directly linked to the optic lobe of the brain with no gaps (synapses) (Chapman 2006). Although ommatidia in many species contain similar functional cells, like the retinular cells, there is variation in structure among insect species. There are two main variations within the ommatidia: photopic and scotoptic (Nation 2002). Photopic ommatidia are common with insects that are active during the day, such as house flies, and have multiple rhabdomere that extend through the retinular cell from the cone to the retinular cell axon. The light that enters a photopic compound eye is focused in the ommatidium after the cone and is not permitted to leak into other ommatidia by the presence of shielding cells or pigment cells. Pigment cells surround the retinular cells and the proximal angles of the cone bl ock light from spilling to other surrounding ommatidia. Scotoptic eyes are common in insects such as moths that are mostly active at night. The structural difference between the scotoptic and photopic ommatidia is that, after the cone in the scotoptic ommatidium, there is a crystalline structure or tract that extends to the retinular cell. Within the retinular cell, fused rhabdomere extend from the

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20 crystalline tract to the r etinular cell axon. This crystalline structure is clear and surrounded by pigment cells. However, unlike the photopic ommatidia that have little or no movement in their pigment cells, the scotoptic pigment cells contract and move their pigment distally uncovering the crystalline tract, which allows light to be shared between ommatidia and stimulate their rhabdomere as well. In dim or dark light conditions, this phenomenon gives nocturnal insects the ability to see (Nation 2002). Ocelli Generally, insects that have compound eyes also have a set of dorsal ocelli or simple eyes. Usually arranged as an inverted triangle in a set of three (Chapman 2006), the ocelli have some of t he same functional components as compound eyes. However, unlike the compound eye the ocelli retinular cell axon is synaptically connected to the ocellar interneuron, leading to either the protocerebrum of the brain or to ganglia on the central nervous system (Mizunami 1994). These ocellar interneurons are classified as large fibers and small fibers (Chapman 2006). The function of the ocelli is unclear but it is speculated that the configuration of the small fibers that lead to multiple areas of the brain may be able to form a crude image; whereas the large fibers that provide connec tions from the ocelli to larger ganglia (subesophageal, prothoracic, or meso and metathoracic) may stimulate responses when there is a change in light intensity. Due to the large fibers having a direct pathway to the ganglia, there is speculation that the occelli may affect insect behavior because of the rapid transmission of ocellar stimulation to other areas of the insects body (Mizunami 1994).

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21 Optic lobes The brain optic lobes, which receive direct information from the compound eyes and are connected t o the ocelli by the small fibers interneurons, are located in the protocerebrum. The protocerebrum has integrative centers that are thought to contain olfactory input from the antennae. The combination of the protocerebrum, deutocerebrum, and tritocerebrum make the structure that is referred to as the brain or supraesophageal ganglion (Nation 2002). Fukushi (1975) demonstrated that house flies process visual stimulus by using monochromic colored lights. This author used conditioning by exposing a hous e fly to a colored light and allowing the fly to feed on a sucrose solution. After the flies were conditioned, 70% of them responded positively by lowering their proboscis when exposed to the light, whereas the control group had only 10% positively respond ing This clearly demonstrated that the visual information the brain receives is processed for learning, and that visual stimulus is important in the learning process. Vision Insects, especially flying insects, rely on their compound eyes to guide them by environmental elements to locate food, prey or potential mates (Tobin and Stoffolano 1973). High intensity of light can cause saturation in the photoreceptors and can limit vision. Adaptations in various insects have been made to regulate the amount of light that reaches the photoreceptors. Goldsmith (1965) suggested that there might be different types of color receptors in the compound eyes of flies, possibly even leakage in the pigment cells with the color red. Goldsmith showed that red light passed through the accessory pigment cells and the light still reached the receptors when it should not have. This experiment suggests

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22 that there may be information missing on how house flies form images Wehrhahn (1984) showed that house flies with blinded c ompound eyes would show a positive phototaxis and edge fixation when only receiving visual stimulus through the ocelli. This may suggest that the ocelli play a larger role in visual stimulus. Image formation The way an insect constructs an image is by focusing light by the use of dioptric structures or light guides. As mentioned earlier, light passes through the cornea in to the ommatidia. The eyes of flies, for example the horse fly, have noticeable bands that run across the compound eyes. These bands are darker across when the ommatidia have a thicker cornea. This is an adaptation to the eyes of a horse fly that enables it to see a host in an open field (Nation 2002). The cornea acts as polarizing sunglasses do for humans. Depending on the insect the cornea filters light and only lets certain wavelengths of light into the next structure, the cone. The cone helps to further focus the light to the retinular cells. In some higher O rder s of insects, such as Coleoptera and Diptera, there is a clear, hard secretion from the semper cells. Semper cells make up the cone helping in the focusing of light by the means of refraction (Chapman 2006). This secreted substance is in higher abundance in insects that have photopic ommatidia and has a gelatinous t exture unlike the scotoptic ommatidia which is more crystalline in formation. Resolution The quality in the refraction of light by the shape of the cone and the size and shape of the rhabdom will determine the degree of resolution of the image formation (C hapman 2006). If the cone is shorter and more rounded, as it is in scotoptic ommatidia, then there is less refraction, which is due to shielding cells. However, if the

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23 interommatidial angles are small, meaning the angles between adjacent ommatidia, then the resolution is high. If the rhabdom is not twisted and has a large diameter, then the resolution is also greater than with a smaller twisted rhabdom. Some predatory insects, like the mantids, have large rhabdom towards the front of their eyes that hel p s in the detection of prey movement Photoreceptors Once light has been focused in the cone of the ommatidia, it enters the photoreceptors, which stimulate nerves directly linked to the optic lobe. Rhabdomere within these photoreceptors are made up of approximately 60,000 microvilli (Zuker 1995). The photons that are received by the eye cause an intercellular reaction in the microvilla called phototransduction. Phototransduction occurs when the light that enters the eye is converted from a light signal to an electrical signal for the nervous system via a biochemical reaction (Zuker 1995). The microvilla contain a light receptor molecule, rhodopsin, made of protein called opsin, retinal, and an aldehyde of vitamin A (Chapman 2006). Rhodopsin is covalently bonded to chromophere, a light sensitive pigment. The manner in which the chromophere reacts to photons depends on the colors of light that can be attenuated. If the pigments are greensensitive and UV sensitive, they respond to light that falls into the green and UV spectrum (Chapman 2006). When the photon contacts this light sensitive pigment, there is a change in the configuration of the chromophore structure. This changes the rhodopsin into its active form metarhodopsin. Metarhodopsin is the molecule from the receptor that activates the G protein within the cell membrane. After the metarhodopsin activates the G protein, it is reverted back into its inactive state. Metarhodopsin deactivation is accomplished by absorption of the

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24 same wavelength of photon that activated it. This process reverts metarhodopsin to rhodopsin, making the cell ready for light stimulation again. Zuker (1995) presented strong evidence that extracellular calcium is used to regulate the activation and deactivation of rhod opsin. When extracellular calcium is not present, there is a slow response in the activation of rhodopsin, but if there is an overabundance, the reaction is accelerated. The G protein causes activation of a phospholipase by binding to a protein, most likely arrestin, which creates two intercellular messengers, inosital trisphosphate and diacylglycerol, from the catalyzed breakdown of a phospholipid membrane (Zuker 1995). These intercellular messengers signal the opening of light activated ion channels t o create a depolarizing receptor potential to the axon. The messenger cascade amplifies the energy and causes an action potential to lead to a response of post synaptic cells (Chapman 2006). House Fly Importance The house fly is distributed worldwide and is considered a mechanical vector capable of harboring 100 known diseasecausing pathogens to man (Greenburg 1973). Mechanical vectoring of pathogens by this insect occurs when hairs on the flys body or legs pick up microorganisms and are physically car ried to food or surfaces associated with humans and animals. Ability of vectoring can also occur when house flies ingest pathogens and pass them via vomitus or fecal waste (West 1951). The capacity of this fly to vector disease is increased because it i s commonly associated with human habitation and primarily breeds in manure and other decaying organic material. Once contaminated house flies disseminate bacteria, such as Escherichia coli (Toshinori et al. 2000). The house fly is also known to vector some

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25 viruses, such as the Poliovirus (Greenburg 1973). Under some circumstances, houseflies can help spread protozoan diseases and spirochete infections (West 1951). Fly borne diseases have been documented throughout American history extending back to the Civil War. Control: Baited Fly Traps House fly populations can be minimized by simple sanitary procedures, however any break in the process that leaves any type of breeding material can lead to a serious fly infestation. It is estimated that approximately 250,000 cubic feet of flies would exist if one pair of house flies mated and every progeny lived to reproduce over one summer (West 1951). Lawrence Pickens, who worked with filth fly traps over a thirty year period, is internationally known as the expert on fly traps. According to Pickens (1995), traps are underutilized because attractants used in the traps cause unfavorable odors and are aesthetically displeasing. Traps require weekly upkeep, such as the unpleasant chore of re baiting and empting the c aptured, rotting flies. There were fourteen different types of baited traps in use as nontoxic methods of controlling flies in 2006 (Welch 2006). M ost commonly attractants placed in the traps consist of decomposing organic material that release gases t hat attract flies. The best fly trap design tested by Pickens (1995) had the following elements: vertical bright white panels which form sharp corners and edges; a cone with a steep sixty degrees slope and a small exit hole ~1 cm; a greater than 25 cm diam eter base placed 1.2 cm above the bait; 10 to 15 cm deep plastic pan that had fly entry points cut into two of its sides; and fly entry points made so flies can not look direct ly out of the trap once they were inside.

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26 The differences in fly traps developed over the years usually involve a variation in size and shape of the fly entry port, which usually leads to a cone that allows the house fly to enter but not exit. Luring flies to a trap is usually achieved by a visual cue for the fly to see the trap f rom a distance and then odorous bait that causes the fly to enter the trap. The most important physical characteristics of a fly traps bait are: the ability to overcome prevailing air temperature, an odor plume that is recognized by house flies as a food source or oviposition source, and the ability to stand out over other odors in the same area (Pickens 1995). Baits are placed in three categories: sugars and their fermentation products, proteins, and animal excretions. Along with trap design Pickens (19 95) suggest ed that fly traps work best with proper trap placement, meaning 25 to 46 cm from the ground where full sunlight will reach the trap in the morning and late afternoon so the top of the trap will be fully illuminated. The trap also should be plac ed as close as possible to natural fly attractants, such as breeding or feeding areas. If there is a need for more than one trap, they should be spaced no more than 18 m apart. Studies conducted on house fly activity show peak hours of activity between 9 :00 a.m. to 4:00 p.m. Ninety percent of houseflies studied preferred to rest ing areas inside houses (Keiding 1965). On nights when the temperatures were above 20oC and the house had screened doors and windows 80% of the house flies rested on the ground outside or in animal shelters. Jug traps in the rafters of poultry houses caught a greater num ber of flies during hours of 7:00 a.m., 4:00 p.m., 7:00 p.m., and 10:00 p.m. (Burg and Axtell 1984). For sampling methods, fly traps can be made from 1 gal translucent milk jugs with four 5 cm diameter entry holes equally spread around the circumference and on the

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27 upper third of the jug (Burg and Axtell 1984). Using a standard yeast based bait more house flies were caught near the rafters in a narrow poultry house away from organic material, while in a residential highrise building fly traps located at the same level of organic material caught more flies than on the rafters or walkways. Two key attractants that have been used to lure house flies to a trap are olfactory and visual stimuli. Visual stimulus is the most important in directing fly behavior (Tobin and Stoffolano 1973). After a house fly has been visually stimulated to move towards a trap, it is important to have chemical or olfactory stimuli, to draw the fly inside the trap. One of the most commonly used chemical stimuli in fly traps is the closerange sexual stimulant, (Z) 9 tricosene (Schal et al 2001). Results by White et al (2007) question whether (Z) 9 tricosene is truly an effective method of increasing a fly bait s attractiveness in large areas because it attracted flies at a very limited distance and was not that effective in outdoor environments. New fly control methods are entering the market. One of the newest devices, the Fly Sentry (Mix 2007), exposes enough fly bait to cover a large area, but also protects the bait from the sun, wind, and rain. Another trap, which seems to be a promising choice for fly surveillance, is the sheltered Quick Strike trap. This device preserves flies intact in dry, odorless conditions (Geden 2005). Use of fly traps continue to decline despite studies showing an increase in house fly resistance to pesticides. House flies have been documented as resistant to pyrethrins, pyrethroids, organophosphates, ca rbamates, fiproles, insect growth regulators, avermectins, and organochlorines (White et al. 2007). In 1991, Bayer CropScience developed MaxF orce Fly Spot Bait, a water dispersible, sugar based

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28 bait formulation that contains 10% of imidacloprid. When i ngested this product is effective in controlling pest insects like cockroaches, fleas, cri ckets, termites, and flies. This chloronicotinyl class compound has shown promising results agai nst synthetic pyrethroidresistant house flies. Imidacloprid has a show the ability to reduce fly populations quickly (Pospischil et al 2005) In experiments conducted by Hertz ( 2007) imidacloprid sprayable bait applied to wool cords caused a high er mortality rate in house flies than methomyl granular fly bait in the first three hours. However, over 24 hours, the methomyl granular fly bait caused an overall higher mortality rate than imidacloprid. In experiments performed by White et al (2007), imidacloprid had noticeable effec tiveness in 30 min after initial exposure and after two hours most of the flies were nonresponsive. However, despite significant knockdown in fly populations, some flies recovered from the initial dose. Scientist s debate whether fly traps are monitoring or control devices (Geden 2006). To reduce a fly population by 5090%, a single trap would have to remove ~2458% of the houseflies daily (Weidhaas and Haile 1978); however, traps alone have not been shown to reduce fly populations to acceptable levels (Sm allegange 2004). Using Visual Attraction for Control of House Flies Understanding the physiology of insect vision can help in the development of effective control devices for house flies. There is uncertainty and disagreement when it comes to spectral r eflectance of colors and their visual attractants for house flies (Geden 2006). The ideal trap has been referred to as being bright white in color (Pickens 1995), which disagrees with other studies where yellow traps caught more flies than white traps (Burg and Axtell 1984).

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29 The physiology of the photoreceptors, suggest s that surfaces with strong contrast ing colors should have greater visual attractiveness. However, no studies have demonstrated this to be true. However, when house flies are given a choice of shapes to land on, they tend to land on the edges over the center of the shape (Pickens 1995). House flies also show preferences for shapes that were more complex. In addition, beadedtop traps catch more flies than traps with flat tops, and that cubical traps always outperform cylindrical trap s. House fly traps typically use a visual component as a long distance attractant, such as colors in specific wavelengths, lights, or patterns. Once the fly is in close proximity of the trap, a chemical at tractant may be used to lure the house fly into the trap. A visual stimulus may be the most important attractant for most insects (Tobin and Stoffolano 1973). Since house flies are strong flyers and have large compound eyes, it is speculated that visual cues are used to search for potential feeding and breeding sites over long distances (West 1951). However, according to Keiding (1965), house fly activities are not determined by visual cues but are random and explorative. Garret (1965) observed a clear differenc e in house fly color preference, with greater numbers resting on primrose, green, tan, and white. This suggests that visual stimulus guides the house fly behavior. Electroretinogram studi es of the house flys eye show sensitivity to UV light ran g ing from 340 nm to 370 nm, and blue gre en light ranging from 480 nm to 510 nm ( Bellingham 1994). The UV spectral range used in light traps for house flies and other flying insects, also is used in paints or fabrics used in baited traps. This transforms t he entire trap into a visual cue that may be similar to an environmental element that would

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30 naturally attract house flies. B lack or blue UV light s are c ommonly used with these types of traps (Hedges 1998). Other colors may not stimulate the photoreceptors of the ommatidia. House flies exposed to green or red UV light show negative phototaxis behavior (Bellingham 1994). Aubuchon (2006) used several different types of light sources as visual attractant s and showed that previous exposure to di fferent kinds of light did not influence h ouse fly attractiveness to light traps. In an experiment performed by Geden (2006), a known visual attractant for house flies and stable flies, alsynite, was compared to a known attract that for tsetse fly, blue f abric. The blue fabric had a maximum reflectance of 466 nm in sunlight and the alsynite was described as translucent, and reflected UV spectrum. More house flies were captured with alsynite traps than traps with blue fabric as the visual attractant. How ever, the blue fabric outperformed the white and black fabric traps. In addition, when the visual attractiveness of alsynite and blue fabric were combined, the trap captured as many or more than the alsynite alone (Geden 2006). Blue fabric visual targets have also shown promising results for capturing stable flies (Foil and Younger 2006). Pickens (1995) points out that the color used with a fly trap is dependent on the weather. White and yellow colors are preferred by house flies in cooler weather, whereas red, black, and blue are preferred in hotter weather. This may show that the atmospheric temperature contributes to the manipulation of the pigment cells in the ommatidia causing them to absorb colors of different wavelengths depending on the exte rnal temperature of the insect, possibly an adaptation that associates some colors to be attractive in hot and cold conditions.

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31 When reviewing the Pickens (1995) analysis of the best trap design, it appears that the most important factor of the trap is its visual attributes. The described ideal trap has to have edges and be a color that reflects a wavelength that is attractive to flies. The trap has to be in a location where it is fully illuminated by the sun for most of the day. Pickens also mentioned t hat if there is a need for more tha n one trap, they should be space d at specific intervals

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32 CHAPTER 3 RESPONSE OF MUSCA DOMESTICA L. TO VISUAL STIMULI Introduction V isual stimulus is the most important attractant for inse cts (Tobin and Stoffolano 1 973), especially house flies since over half of their head is comprised of two large compo und eyes accompanied by a cluster of three simple eyes (ocelli). House flies rely on sunlight reflected from objects as visual input for survival. This gives flies the ability to see objects in their environment to avoid obstacles while flying, finding food, finding harborage, and being able to recognize resting areas. Light enters the compound eyes or ocelli and stimulates photosensitive cells that trigger a process called phototransduction, which converts light to electrical signals for the nervous system, sending signals to the insects optic lobe for interpretation (Zuker 1995). Signals received by the optic lobe result in fly behavior, like attractancy or repell ency. It is unknown how insect compound eyes physiologically form images other than interpreting intensity of light, motion, and plane of polarization (Hocking 1964). Color vision is the ability to distinguish differences in wavelengths of color and not t he intensity of light (Hilbert 1992). Color vision is thought to be associated with compound eyes; whereas the ocelli are believed only to perceive differences of light intensity and are not capable of detailed image formation (Mizunami 1994). However ~2 00 photoreceptor cells are within a house fly ocellus produce positive phototaxis when the compound eyes are blinded (Wehrhahn 1984). Typically, electroretinogram studies use a direct light source, such as a xenon bulb, to generate a narrow range wavelength to measure neural responses of insect eyes. Although electroretinogram studies with direct light sources show ed sensitivity of

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33 house fly compound eyes to UV light ( ranging from 340 to 370 nm ) and bluegreen light (ranging from 480 to 510 nm ) (Bellingham 1994), t here is disagreement on the effect of reflected colors and thei r visual at tractants on house fly behavior (Geden 2006). Light reflected from an object generates a wide range of wavelengths (Table 31) that will consist of various parts of the light sources color spectrum and filtering other parts (Hewitt 1998). Studies with specific fly trap colors have shown a direct correlation between visual sensitivity and fly trap efficiency (Agee and Patterson 1983). Musca flies are known to h ave bluegreen visual pigments in their compound eyes with sensitivity to ~440 to 540 nm (Salcedo et al. 1999). B lue fabric targets with the maximum reflectance of 466 nm have been shown to be more visually attractive to house flies in comparison to white and black fabric targets (Geden 2006) This same type of blue visual target has also shown promising results for capturing stable flies (Foil and Younger 2006). Previous observations demonstrated a clear difference in color preference for house flies which suggests that visual stimuli guide house fly behavior (Garret 1965). House fly behavior has shown an attraction to hanging cords and similar objects in field conditions as resting locations (Fehn 1958). Other studies have shown that house flies hav e a tendency to follow edges while foraging suggesting that the contrast of colors at the edge of lines may contribute to visual attractancy (Conlon and Bell 1991). The objectives of these studies were to: 1) measure house fly physiological response to re flective color using electroretinograms of both compound eyes and ocelli; 2) compare house fly behavioral attraction to reflective colors; 3) compare the effect of colored lines on attractiveness of the blue background.

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34 Materials and Methods Insects. Musc a domestica (Linnaeus) used for these experiments were from the University of Florida Horse Teaching Unit and reared at the University of Florida, Gainesville, FL. Room conditions for all developmental stages were maintained at 26+ 1 C and 55% RH with a 12:12 (L:D) photoperiod. Adult house flies were reared by placing eggs in a basin containing freshly mixed larval medium (1.5 liters of tap water, 250 ml Calf Manna pellets, Manna Pro Corp., St. Louis, MO, 15 ml methyl paraben, and 3 liters of wheat bran) Larvae were allowed one week to pupate. Pupae were collected by floating the basins content in tap water. Pupae were allowed to dry and were placed in a scree ned cage along with granulated sugar, powdered milk, and water ad libitum Adult house flies, 3 5 days old, were collected from screened cages by aspiration using a hand vacuum with a modified crevice tool. Electroretinogram (ERG) Equipment similar to and techniques modified from Bellingham (1994) were used to measure depolarization of nerves in compound eyes and ocelli exposed to reflected light. Equipment consisted of two ERG micromanipulators (EAG COMBI 10X, Syntech, Hilversum, NL) with glass sheathed tungsten electrodes to record depolarization of compound eyes and ocelli. Electrodes we re chemically sharpened by etching with p otassium hydroxide. Recording software used to measure depolarization was ElectroAntennography Version 2.4 (Syntech, Hilversum, NL). To reduce external electrointerference, ERG equipment was enclosed in a faraday cage, which was completely draped with black theater cloth allowing no light to enter cage. Internal cage conditions were 24+ 1oC and 45% RH. Visual target and light source Visual targets used for ERG were constr ucted from twin walled rigid plastic sheets (20 cm x 25 cm; White C201L, Blue C505L, Black

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35 C208L, Yellow C402T, Green C406T, and Red C404T, Coroplast, ThyssenKrupp Material NA, Inc. AIN division Madison Heights, MI) (Bere sford and Sutcliffe 2006) Visua l targets were placed 25 cm in front of the house flies head and arranged perpendicular to the floor. Light source was four warm white LED lights (RL5WWW7035 2940K/700 mcd/ 35o, Super Bright LEDs Inc. St Louis, MO) mounted on imprinted circ uit board and fitted with switch. Light source was powered by a 6V battery (BSL0955 6V 10Ah rechargeable battery, Universal Power Group Inc. Carrollton, TX) and hel d in place by threeprong clamp 30 cm from visual target. A black piece of Coroplast was placed under the light source to block any light other than that reflected from the visual target from reaching the test insect. All replicates were c onducted in complete darkness. Wavelength of reflective light from visual targets was measured using USB2000 Spectrom eter ( Ocean Optics, Dunedin, FL) with visual target s in place and spectrometer sensor secured to test insect (Table 3 1) Five independent readings were conducted for 1 minute each Light i ntensity of reflected light from visual targets was measured with a HOBO Light Intensity logger (Onset, Bourne, MA) in the same manner with HOBO device in place of test insect. ERG procedure Seventy f emale house fli es (40 for compound eyes and 30 for ocelli) were used for this experiment and were anesthetized with ice for 30 minutes. Each fly thorax was encased with wax and adhered to a glass slide, ensur ing no visual obstruction of the compound eyes or ocelli. The slide mounted fly was clamped in place with the head facing the visual target. Electrodes were inserted into the appropriate eye using a dissecting microscope for precise placement. For compound eye bioassay, the

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36 m easuring electrode was inserted into the equatorial region of the right compound eye (Bellingham 1994) and the differential elect rode w as inserted into the fly s abdomen. For ocelli bioassay, the measuring electrode was inserted, just puncturing in the cuticle of the median ocellus and the differential elect rode was inserted into the fly s abdomen. After both electrodes were in place t he house fly was allowed time to recover in both bioassays. Each fly was randomly presented with every color (black, white, red, green, yellow, and blue) visual target, allowing a oneminute recovery between each 0.5 s exposure of light source on the vis ual targets (Figure 3 1) The ocelli ERGs were conducted separately from compound eye bioassays. Both experiments were run in a block design with each insect serving as a block, and being exposed to all colors Data analysis for ERG experiments. Data for the ERG consisted of measured neural responses measured in mV for the compound eyes and ocelli and averaged for each visual target. Oneway analysis of variance was performed and m eans were separated using Tukey test (P = 0.05; SAS 2001). Two Sided Light Tunnel (TSLT) TSLT was constructed with two 42.6 L capacity heavy duty ice chest (The Hercules ice chest No. 5345; LifeLike Products Inc., Baltimore, MD ). A circular hole (10 cm diam.) was cut in one of the side walls of the ice chest centered at 16 cm from the top, 17 cm from the bottom of the ice chest, and 29 cm from the sides of the ice chest. Each ice chest then was fitted with a black pipe flange (ABS DMV Schedule 40; NIBCO Inc, Elkhart, IN) secured in place with hot glue. Each ice chest contained a 45.7 cm fluorescent light fixture (Portfolio 18 under cabinet fluorescent light, model# GL9718T8 BK 1, Good Earth Lighting, Inc., Wheeling, IL)

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37 centered directly under the black flange. The fluorescent light fixture was fitted with a GE daylight fluorescent bulb (GE daylight F15 457 mm, F15T8/D/TP, General Electric Company, Cleveland, OH) as used by Shields (1989). A square hole (3 cm side) was cut in the lower corner of the side wall of each ice chest to allow the power cord from the light fixture to exit the ice chest. The a rea around the power cord was sealed with modeling clay (Marblex Self Hardening clay, American Art Clay Co., Inc., Indianapolis, Indiana) to prevent house flies from escaping. Two ice chests were connected by two 30.5 cm black PVC pipes (TrueFit System 3300 3 SCH 40 COEX ABS cellular core DMV pipe Charlotte Pipe and Foundry Company, Charlotte) which connect in the center to a T connector (NIBCO, IN ). The T connector was fitted with a cleanout adapter (NIBCO IN) with a modified plug (NIBCO, IN) so that a capped plastic vial (50 dram Crystal capped plastic vial; Thornton Plastics, Salt Lake City, UT) could snap into the cleanout adapter and be removed when necessary. The snapcap vial was modified wi th a rem ovable tab that prevented flies from leaving the vial until the tab wa s pulled out. Also the snap cap vial was lined with wire mesh that allowed the house flies to crawl up the sides of the vial to enter the TSLT. Similar wire mesh was also inserted in the modified plug and extended from the tab in the vial to the top of the cleanout adapter. This allow ed the house flies to climb from the vial to the T connector and enter the TSLT to make a choice between the ice chests with attractive visual target s. V isual targets Fabric visual targets were constructed from Nautolex skipper fabric (20cm x 25cm, OMNOVA Solutions Inc., Fairlawn, OH ) and plastic visual targets were constructed from twinwalled rigid plastic sheets (20cm x 25cm, Coroplast, ThyssenKrupp Material NA, Inc. AIN division Madison Heights, MI). Both fabric and

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38 plastic visual targets (Table 31) consisted of the colors: yellow, red, green, black, blue, and white. Visual targets were mounted to the center of ice chest opposite to the pipe flan ge Only the visual target s on each side could be seen from within the piping of the TSLT. For experiments with lined visual targets, lines were added on blue visual targets by attaching 6 mm wide strips of skipper cloth spaced 1 cm apart and attached ver tically on solid colored background. Vertical stripes were placed so that four lines could be seen when looking t hrough TSLT from T connector. Wavelength of reflective light from visual targets was measured with a USB2000 Spectrometer (Ocean Optics, D unedin, FL). The spectrometer sensor was secured in place with black cardboard in a centered orientation of T connector facing the visual target. Five readings were taken over one minute for each visual target (Table 3 1) Intensity of reflected light f rom visual targets was measured with a HOBO Light Intensity logger (Onset, Bourne, MA) in the same manner TSLT procedure All TSLT experiments were conducted at the Urban Entomology lab at the University of Florida with ambient temperature at 26.6o C. House flies ( 50 males and 50 females ) were anesthetized with ice and placed in modified snap cap vial. The snap cap vial was attached to the TSLT and the house flies were given 1 h to recover before removing the tab that allowed the house flies to enter the TSLT. Experiments were conducted in a Latin square design and after fly release each replicate ran 2 h. Once the allotted time was reached, the pipes were disconnected from the ice chests and all pipes and chest holes were immediately capped. Th e flies within ice chests and pipes were knocked down with CO2 and

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39 counted. House flies that flew into the ice chests were considered responsive to the reflective color, whereas the flies that remained in the connecting pipe were considered nonresponsive. F our TSLT experiments were conducted. In the first two experiments color targets were compared with black targets. Color targets included white, red, yellow, blue, and green, which were mounted in the ice chest opposite to black in the TSLT Two experi ments were conducted: the first one with all fabric visual targets and the second one with all plastic visual targets For the third experiment, t he most attractive plastic visual target s from the first e xperiments (blue vs. white) were compared with leas t attractive plastic target (yellow) The fourth experiment, color lines (red, yellow, white, black, and no lines ) were added to the blue target previously selected as the preferred color and again compared with black. Data analysis for TSLT experiments. House flies were collected and counted from capped pipes and ice chests. Total house fly response was calculated by subtracting nonresponsive flies (in piping) from total number of flies released in the TSLT. House flies collected from ice chest with vi sual target were divided by the total house fly response to get a percentage of house flies responding to individual visual targets. H ouse fly response to visual targets data were arcsinesquareroot transformed before analysis of variance and means were separated using Tukey s test (P = 0.05; SAS 2001). Results ERG Measurements of the depolarization of the nerves in the compound eyes showed that hous e flies had the greatest stimulus from the white visual target (Table 3 2 ). Compound eye ERG responses to visual targets were as follows: White = Blue >

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40 Yellow > Red > Green > Black. The compound eye had similar low neural responses in perceiving green and black visual targets. The measurements recorded from the ocellar response showed a significant ly greater in the color vision to white, blue, and yellow Ocelli ERG responses to visual targets had the following relationship: White > Blue > Yellow = Red > Green = Black. The green visual target had no significant differences to t he black visual target in responses from the ocellus. TSLT. In the experiment comparing attraction / repellency of colors when compared to black using the fabric visual targets (Figure 3 2 ) more flies were attracted to the white, blue, and red visual tar gets, White = Blue > Red > Green > Yellow. In addition, house fly attraction to the green and red visual targets was not significantly different. The yellow visual target was repellent having more flies in the ice chest that contained the black visual target than on the side with the yellow visual target. Replacing visual targets with plastic visual targets used in the ERG experiments, the house fly behavior was similar to that observed with fabric visual targets (Table 3 3 ) White and blue visual target s were the most attractive targets and had no difference in their house fly attraction, White = Blue > Red > Green > Yellow. However, different from the use of the fabric targets, the attraction of the red visual target was significantly lower than that f or white and blue targets. The yellow visual target was significantly different from all other colors tested and as with the fabric visual targets was repellen t Due to the repellency of the yellow visual target a larger percent of flies were attracted to blue and white visual targets when ran separately against yellow visual target tha n when compared to the black visual target (Figure 3 3) In addition, blue

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41 visual target ran against wh ite visual target, isolated the blue visual target as the most attractive target used in the TSLT. The addition of a lined pattern using vertical colored lines in TSLT experiment showed that black lines significantly enhanced the blue visual target s attractiveness (Figure 3 4). Adding red or white lines to the blue tar gets were no more attractive than where no lines were added. However, the addition of yellow lines on the blue visual target caused repellency. Discussion The neurological responses measured with the ERG experiments showed that the reflected light from the color targets elicited a neurological stimulus to the house fly compound eye and ocellus. The reflected light from all visual targets used had the same light intensity to the tested insect, suggesting that the neurological responses measured with the ERG were generated by the specific colors of visual targets. This is defined as color vision. The ERG showed that the house fly compound eye had high neural responses to the reflected light from white, blue, and yellow targets. White (~419738 nm) and blue (~423574 nm) reflected wavelengths that included known visual sensitivity wavelength ranges for house flies, 450510 nm (Agee and Patterson 1983, Bellingham 1994). The yellow visual target (~510586 nm) gave similar physiological responses as the blue visual target, which was surprising because yellows reflectance started at ~510 nm. Musca flies have bluegreen visual pigments in their compound eyes that have sensitivity to wavelengths 440 to 540 nm. This fact may explain why yellow targets generate d a higher response with the compound eyes (Salcedo et al. 1999). However,

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42 the lower neural response generated by the green visual target (462 571 nm) was not significantly different from black target. In experiments w h ere flies have had their compound ey es removed and are only using their ocelli for sight, they have a tendency to follow edges (Wehrhahn 1984). This demonstrates that the ocelli are able to form images. V isual pigments in fly ocelli have been shown to have a maximum absorption of light of 425 nm (Kirschfeld et al. 1988) which corresponds to white and blue visual targets Typically, the ocelli are seen as having high speed neurons that only detect changes i n light intensity (Chapman 2006). However, the light intensity of reflected light u sed for this experiment was the same for all the targets, suggesting that the tested ocellus was detecting the colors of visual targets. Therefore, the conclusion was drawn that the ocelli are capable of color vision. The TSLT allowed observation of the h o use flys behavioral response to colors in a controlled laboratory environment. When white, blue, yellow, green, and red were compared to black, more house flies were attracted to the colors white and blue than any other color. The color that repelled h ouse flies was yellow. In the past, laboratory experiments have shown that more house flies landed on yellow paper than other colors tested ( Awati and Swaminath 1920) Field observations using painted plastic jug traps demonstrated that yellow jugs caught more house flies (Burg and Axtell 1984). Other observations using colored pieces of cardboard found that black was most attractive in the laboratory, while yellow was most attractive outdoors (Nava 1967, Hecht et al. 1968). Confirmation of yellow as a repellant color in the TSLT showed that house flies were clearly more visually attracted to the white and blue, with a ~70% behavioral attraction, compared to ~20% attraction to yellow.

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43 A comparison of the two most attractive visual targets in the TSLT isolated blue (~60%) as more attractive than white (~25%). This is due to white having a wide range of wavelengths (Figure 3 6) that includes wavelengths peaking in the attractive blue range, but also in the repellen t yellow range. It has been argued that at mospheric conditions may contribute to the attraction of house flies to specific colors: white or yellow were preferred in cool er weather, whereas red, black, and blue were the preferred colors in warmer weather (Pickens 1995) Other studies conclude that house fly color preference is independent of temperature (Muniz 1967). The measured internal temperature before and after each TSLT replicate was 26+ 2o C I f attraction to a color is temperature dependent the n response to yellow and white targets should have caused the same behavioral attraction. It has been suggested that blue is attractive to tsetse fly and other flies because it is perceived as shaded resting area (Steverding and Troscianko 2003). In the TSLT study the blue visual target s were more attractive to house flies with the addition of black lines, which could be contributed to the scototaxis tendency of house flies (Hecht 1970). The house flies perceive the blue visual target as a potential resting area and the black lines as cracks or cre vices that the y can use as harborage. In other Diptera studies, similar shades of blue, which reflected in specific color bands of bluegreen and red have been shown to be effective as visual attractant s (Green and Flint 1986). Sunbrella blue fabric (pe ak wavelength 466 nm) was fieldevaluated as a visual attractant in substitution of blue cotton fabric which is a commonly used material in an effective tsetse fly trap due to demonstrated effectiveness of attracting flies in North America (Mihok et al. 2 006, Geden 2006). These results

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44 indicate that the material used as visual targets is not an essential factor in determining attraction to the flies, but the wavelengths of the target s reflect ed light is critical. The visual information from the compound eyes and the ocelli is integrated to work together in providing information to the optic lobe that directs fly behavior in response to reflected light from objects. The studies here have shown that the ocelli are capable of color vision and contr ibute more to house fly vision than originally thought. Comparison of the behavioral and physiological responses (Figure 35) shows a direct correlation of house fly attractiveness to visual targets and the intensity of neurological response. However, th e yellow visual target represents an exception, because it was repellant to house flies in the TSLT, but triggered relatively high neurological response. Y ellow stimulates the same amount of neural responses as blue, but the neurological response triggers an alternative behavioral res ponse. The triggered behavior response u nder the conditions of the TSLT causes house flies to be repelled by the yellow visual target s. Previous studies that have concluded that yellow was visually attractive to house flies may have been using yellow colors that were reflecting additional attractive wavelengths (Burg and Axtell 1984, Pickens 1995). From these experiments, there is no question that the compound eyes and ocelli have the capability of color vision. Each visua l target tested generated specific neurological responses, which was interpreted by the house fl y brain into behavior response of either attraction or repellency. Future studies with similar experiments should investigate various adult house fly responses at different life stages to determine if colors generate different neurological and behavior responses depending on if the house fly has mated, fed, or of a certain age.

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45 Table 31. W avelength s (nm) and reflected intensity (cd/m2) of light from light sources on visual targets used in electroretinogram and twosided light tunnel experiments. Light Source Material Color Peak(nm) ~ Reflected Intensity (cd/m2) Used with electroretinogram Warm white LED lights Plastic Blue 460 423574 11.8 Warm white LED lights Plastic White 500 419738 11.8 Warm white LED lights Plastic Yellow 564 510586 11.8 Warm white LED lights Plastic Green 543 462571 11.8 Warm white LED lights Plastic Red 603 588702 11.8 Warm white LED lights Plastic Black ** ** 11.8 Used with two sided light tunnel GE daylight fluorescent bulb Fabric Blue 436 397586 35.5 GE daylight fluorescent bulb Fabric White 518 395725 248.6 GE daylight fluorescent bulb Fabric Y ellow 571 504585 153.9 GE daylight fluorescent bulb Fabric Green 544 450571 28.0 GE daylight fluorescent bulb Fabric Red 612 583752 28.0 GE daylight fluorescent bulb Fabric Black ** ** 19.4 GE daylight fluorescent bulb Plastic Blue 482 394583 59.2 GE daylight fluorescent bulb Plastic White 522 380812 232.5 GE daylight fluorescent bulb Plastic Yellow 575 502593 106.6 GE daylight fluorescent bulb Plastic Green 544 457570 28.0 GE daylight fluorescent bulb Plastic Red 610 578738 19.4 GE daylight fluorescent bulb Plastic Black ** ** 12.1 ** No measurable peak or wavelength.

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46 Table 3 2. Measured depolarization of female house fly equatorial region of right compound eye and median ocellus from white LED light source reflected off of plastic visual target Visual Target Compound Eye Ocelli Color Response (mV) Response (mV) White 8.3 + 0.97a 4.9 + 0.22a Blue 7.4 + 0.97ab 3.6 + 0.17b Yellow 6.8 + 0.94b 3.0 + 0.19c Red 5.2 + 0.64c 2.7 + 0.21c Green 4.3 + 0.49cd 1.8 + 0.12d Black 3.3 + 0.39d 1.7 + 0.05d Means within columns followed by different letters were significantly different at P=0.05. Table 3 3. Comparison of house fly response to f abric visual targets and plastic visual targets used in twosided light tunnel with GE daylight fluorescent bulb. Visual t arget Fabric target Plastic target color fly response fly r esponse White 67.0 + 3.86 a 67.3 + 4.12 a Blue 64.4 + 3.17 a 66.4 + 3.44 a Red 57.3 + 2.13 ab 55. 3 + 2.20 b Green 48.2 + 3.12 b 45.2 + 3.61 b Yellow 30.8 + 3.81 c 32.9 + 2.86 c Means within columns followed by different letters were significantly different at P=0.05. Figure 31. Typical electroretinogram elicited from female house fly equatorial region of compound eye A) and median ocellus B) after 0.5 s stimulus of white reflected light. A B

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47 Figure 3 2 Mean behavioral response of house flies to fabric visual targets compared to black after 2 h in two sided light tunnels. Figure 33 Mean behavioral response of house flies to fabric visual targets in two sided light tunnel; white vs. yell ow, blue vs. yellow and blue vs. white. 10 20 30 40 50 60 70 White Blue Red Green YellowMean response of house flies to color 0 10 20 30 40 50 60 70 White Yellow Blue Yellow Blue White Mean response of house flies to color a a ab b c

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48 Figure 3 4 Mea n behavioral response of house flies to different colored lines on blue backgrounds in twosided light tunnels compared to solid black. Figure 35. Mean physiological response of house flies from electroretinogram experiments with house fly behavioral response in two sided light tunnels. Third column of color group represents mean behavior response of house flies to plastic visual targets compared to black in TSLT. 10 20 30 40 50 60 70 80 Black Blue Red White Yellow Mean response of house fliesLine color on blue background a b b b c

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49 F igure 36. Spect ral reflect ance of sunlight from colored plastic visual targets Each visual target color is represented by its color of line; white is represented as a dotted line.

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50 CHAPTER 4 FLY KILL CAUSED BY T OXICANTS IN WOOL YAR N APPLIED TO A BLUE COLORED FLY CONTROL DEVICE Introduction Movement of house flies is primarily influenced by attraction to specific conditions, such as resting sites, food sources, and breeding media (Keiding 1965). House flies have been observed to prefer resting areas near the ceiling on electrical cords or the darkness of rafters in barns (Mathis et al 1967, Burg and Axtell 1984). These behaviors are often taken into account in order to optimally place traps. For inst ance, foraging flies are usually found close to the ground (Driggers 1971, Geden et al 2009), whereas resting flies are caught on sticky traps hung high in structures. Consequently, there has been mixed information on the best height placement of fly cont rol devices. In 1947, the observation was made that house flies preferred to rest on edges of various structures and objects (Baker et al 1947). Taking advantage of this type of fly behavior the use of insecticide treated cords was introduced. Cords wer e initially treated with DDT, but once commercially prepared, the insecticide treated cords were cotton and dipped in solutions such as parathion and/or diazinon (Fehn 1958). Insecticides that were comm only used on cords for fly control, such as organophosphate and organochlorine insecticides, are no longer registered by the Environmental Protection Agency (Rabari and Patel 1976) Research reviving the vintage insecticide treated cor d demonstrated that modern insecticid es were effective in killing flies w hen applied to wool cord (Hertz 2007). Baits and insecticide sprays are typically used as chemical methods f or house fly control but neither method provides long lasting residual effects. Insecticide treated cords has been viewed as an alternative to tra ditional insecticide sprays and have been

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51 success ful in controlling flies for long periods (Keiding 1976). In heavy infestations of flies the installation of 91 m/ 93 m2 of insecticide treated cords, was able to reduce the house fly population by 70% in one week and able to maintain control for at least a month (Fehn 1958). Insecticide resistance to a variety of active ingredients has been well documented in house flies (Georghiou and Lagunes Tejeda 1991, Liu and Yue 2000, Scott et al. 2000). In order to avoid resistance developing in house fly population, chemical groups of insecticides should be alternated in set time intervals routinely However, the number of registered insecticides for house fly control has drastically decreased making insecticide rotations a challeng e (Kaufman et al. 2001). Combining known house fly visual attractive colors, a blue back ground with black lines (Chapter 3), with an e ffective insecticide delivery method of wool cords (Hertz 2007) a fly attractant system wi th toxicant treated cords (FAST TC) can be constructed (Figure 4 1). To make this device available for use by military and civilian personnel it needs to be cost effective and versatil e. This studys objectives were to: 1) compare efficiency of active in gredients (AI) on FAST TC, 2) evaluate a less expensive alternative to wool cord, and 3) determine suitable height for use of FAST TC. Materials and Methods Insects Musca domestica (Linnaeus) used for these experiments were from the University of Florida Horse Teaching Unit and reared at the University of Florida, Gainesville, FL. Room conditions for all developmental stages were maintained at 26C and 55% RH with a 12:12 (L:D) photoperiod. Adult house flies were reared by placing eggs in a basin contai ning freshly mixed larval medium (1.5 liters of tap water, 250 ml Calf Manna pellets, Manna Pro Corp., St. Louis, MO, 15 ml methyl paraben, and 3

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52 liters of wheat bran). Larvae were allowed one week to pupate. Pupae were collected by floating the basin s content in tap water. Pupae were allowed to dry and placed in a screened cage along with provisioned granulated sugar, powdered milk, and water ad libitum Adult house flies, 3 5 days old, were collected from screened cages by aspiration using a hand vacuum with a modified crevice tool. After collection, house flies were anesthetized by being placed on ice for 30 min. AI comparison: No choice laboratory bioassay This experiment was conducted in laboratory cages (31 x 25 x 21 cm) constructed with PV C pipe (1.27 cm). A rubber band was used to establish individual treatment positions in center top of cage, one position for each cage. All pieces of yarn used were 10 cm in length and were attached to the treatment positions vertically using paper clips hanging in the top center of the laboratory cage. Eight treatments were evaluated: 1) cyfluthrin (CY Kick CS, Whitmire Micro Gen Research Laboratories, Inc., St. Louis, MO), 2) deltamethrin (Suspend SC, Bayer Environmental Science, Research Triangle Par k, NC), 3) fipronil (Termidor SC, BASF, Research Triangle Park, NC), 4) imidacloprid (Premise 75, Bayer Environmental Science, Research Triangle Park, NC), 5) indoxacarb (Indoxacarb 150 SC, Du Pont, Wilmington, DE), 6) thiamethoxam (Optigard Flex, Syngent a, Greensboro, NC), 7) chlorantraniliprole (E2Y45 200 SC, Du Pont, Wilmington, DE), 8) cyfluthrin and imidacloprid combination (C&I). All products diluted to 1% active ingredient when mixed with bait solution, except cyfluthrin and imidacloprid combination that contained 1% of each AI. Bait solution contained 10% sucrose and 10% powdered milk in tap water Controls consisted of wool yarn pieces dipped in tap water and dried overnight.

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53 Experimental design was configured in a Latin square, conducted under continuous light at room temperature of 23oC. Fifty female house flies were released in each laboratory cage and m ortality of house flies was recorded 1, 2, 3, and 6 h. House fly mor tality data was analyzed by oneway analysis of variance. Mea ns were separated using Fisher s protected LS difference test (JMP 2007) AI comparison: Choice laboratory bioassay. This experiment was c onducted in laboratory cages used in AI no choice bioassay Three rubber bands were used to establish twelve posit ions for wool yarn pieces that were uniformly distributed along the top of the laboratory cage, four pieces of yarn per row. All pieces of yarn used were 10 cm in length and were attached vertically to the treatment positions using paper clips. One of the twelve pieces of yarn was treated with AI and randomly placed in center row. The AI treated piece of yarn rotated position with each replicate. Seven treatments were evaluated: 1) cyfluthrin, 2) deltamethrin, 3) fipronil, 4) imidacloprid, 5) indoxacar b, 6) thiamethoxam, 7) cyfluthrin and imidacloprid combination ( C&I ) All AI treatments and control pieces of yarn were prepared in same manner as in nochoice experiment. Experimental design was configured in a Latin square, but blocking for treatment position for AI impregnated yarn treatment. The experiment was conducted under continuous light at room temperature of 23oC. Fifty female house flies were released in each laboratory cage. Two 59 ml containers with cotton wicks containing 10% sugar water were provided ad libitum at each end of the cage. Mortality of house flies was recorded 1, 2, 3, 6, and 24 h. House fly mor tality data were analyzed by oneway analysis of variance; data was arcsine

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54 square root t ransformed. Means were separated using Fishers protected LS difference test (JMP 2007) AI comparison: Field bioassay This experiment was c onducted in field cages (1.8 x 3.7 x 1.8 m; Outdoor Cage, #1412A, 18 x 14 mesh, Bioquip, Rancho Dominguez, CA) with translucent plastic sheeting (6 mi l) used to cover the floor built on a grassy area shaded by pine trees. Food (1 L of 10% sucrose), water (1 L), and 60 ml container of spent oviposition media (covered with paper towel to prevent house fly access) were placed in the cages to provide nutrition and to compete with the treatments for fly activity. Treatments consisted of FAST TC (Figure 4 1) treated with different active ingredients. Each device was constructed with two blue corrugated plastic boxes (54 x 8 cm, Coroplast, blue, number: C505L8 000.9000; Dallas, TX) each folded and hot glued into an open box with a 13 x 13 cm piece of blue plastic glued in place to close one side. The two boxes were connected by one 30 cm piece of wool yarn (Wool Ease color# black 153; Lion Brand Yarn Company, New York, NY.) centered on each side and hot glued into place. The top box was mounted inverted with the open end facing down and bottom box was mounted with the opening facing up. Across the open end of the bottom box two 13 cm pieces of wool yarn were crisscrossed and hot glued in placed over 170 g of food source (Calf Manna pellets, Manna Pro Corp., St. Louis, MO). Six active ingredients treatments were evaluated: 1) cyfluthrin, 2) deltamethrin, 3) fipronil, 4) imidacloprid, 5) indoxacarb, 6) thiamethoxam. All AI and control wool yarn

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55 was prepared in same manner as in nochoice and choice bioassay s. In the morning the experiment was started, AI tre ated yarn was hot glued to its correspondi ng device. All treatments were placed in the field cages 1 h after 350 house flies of mixed sex were released in each cage. Treatments were hung 1 m in front of the cage entrance and with the top of the filth fly control device 1.2 m from the floor of field cage. At 6 and 24 h fly counts were conducted, dead flies were collected from the floor of each cage and placed in a Mason jar (118 ml) to allow knocked down flies to recover in the cage. At each sampling time, flies on the floor of the cage, those remaining in the jar, and those collected in the bottom of the FAST TC were counted. Percent m ortality data for each time after trap placement was analyzed by oneway analysis of variance after arcsine square root transformation. Means were separated using Fishers protected LS difference test (JMP 2007) Wool cord vs. wool yarn. This experiment was c onducted in laboratory cages (31 x 25 x 21 cm) constructed with PVC pipe (1.27 cm). A rubber band was used to establish individual treatment at the center of cages. Treatments consisted of 10 cm wool cord pieces ( Twisted, Natural Cord, Wooded Hamlet Designs, Greencastle, PA; 0.6 cm diameter) and 10 cm wool yarn pieces (Patons Classic Merino Wool Yarn, Ontario Canada, winter white, #N4W3H3). Nine pieces of wool yarn were braided to match the diameter of one wool c ord. Wool cords and wool yarn were prepared using 2.5% imidacloprid bait (Maxforce Fly Spot bait, Bayer CropScience, Kansas City, MO 64120) Cords and yarn pieces were dipped for 1 min in the insecticide solution and then dried overnight in fume hood. Treatments were secured in position vertically using paper clips, one treatment per cage. Controls consisted of 10 cm wool cord pieces that

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56 were dipped in tap water and dried in fume hood overnight. Laboratory cages were enclosed with a transparent plast ic bag (3716 cm2, 1 mil poly, Uline, Waukegan, IL). Thirty female house flies were released in each laboratory cage and allowed 1 hour for acclimation before first mortality count Mortality of house flies was recorded at 1, 3, 5, and 24 h after fly release in cages Four replications were conducted under continuous light at room temperature of 23oC. The experiment was c onducted in a randomized complete block design. Mortality data was analyzed by analysis of variance, and m eans were separated using Fis hers protected LS difference test (JMP 2007) Trap height test : indoor bioassays. Comparison of three trap heights simultaneously was conducted in laboratory room (4.8 x 20.1 x 2.7 m) with ambient temperature of 23oC. Treatments consisted of sampling cards (10 x 10 cm white corrugated plastic, Coroplast, number: C505L8000.9000; Dallas, TX) assembled with a piece of fly ribbon (Aeroxon fly catchers fly strips, #01844100005 3, The Tanglefoot Company, Grand Rapids, MI) pinned horizontally across each s ide. Sampling cards were arranged in three locations equally spaced from one another around the center of the room. Sampling cards were hung vertically from ceiling with fishing line at three heights of 0.91, 1.8, and 2.44 m simultaneously per location. The laboratory window was blocked out with aluminum foil and closed blinds to prevent external light from entering room. Entry door was sealed from inside with masking tape gasket to prevent house flies from escaping. Three hundred house flies of mixed s ex were released in the center of the room on top of a fixed, centrally located counter (0.79 x 2.9 x 0.91 m). After the house flies were

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57 released, the room was sealed and left undisturbed with continuous illumination by ceiling mounted fluorescent light After 24 h, all sampling cards were collected and the captured house flies were counted for each height /location combination. S ix replications were conducted. Percentage of flies caught at each height was calculated by dividing the total number of fli es caught at each height by the total number of flies released. Statistica l analysis of percentage of house flies caught at the three heights was done with oneway analysis of variance after being arcsine square root transformed and m eans were separated using Fishers protected LS difference test (JMP 2007) Comparison of trap heights was also conducted at each height individually This experiment was conducted in rearing rooms (2.6 x 3.5 x 2.7 m ) with temperatures maintained at 23 26oC Each room received a modified FAST TC, co nsisting of only the bottom box which was hung at the appropriate height The trap was c onstructed with blue corrugated plastic (54 x 8 cm, Coroplast, blue, number: C505L8000.9000; Dallas, TX) folded and hot glued into an open box with a 13 x 13 cm piece of blue plastic glued in place as the boxs floor. Across the open end of the blue box were two 30 cm pi eces of black 2.5% imidaclopridtreated yarn that crisscrossed and was hot glued in place. Treated bl ack yarn was prepared the night before by soaking in 2.5% imidacloprid (Maxforce Fly Spot bait, Bayer CropScience, Kansas City, MO 64120) for 1 m in Untreated wool yarn was dipped in tap water. All wool yarn pieces were dried overnight in a fume hood be fore attachment to blue box. Treated black yarn extended the opening of the box forming a plus sign as well as extending over the center of box sides down to bottom edge of box. Blue boxes were hung open side up by fishing line

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58 from ceiling and held 50 g of food attractant (Calf Manna pellets, Manna Pro Corp., St. Louis, MO). Rooms had no windows and the entry doors were sealed from inside with masking tape gasket to prevent house flies from escaping. Three treatments were evaluated: 0.91, 1.8, and 2.44 m. Each treatment trap was hung from center of the ceiling in each room. The experiment was conducted using a 4x4 Latin square configuration. Three hundred mixed sex house flies were released in the center of room at floor level floor level and left undisturbed. Fly counts were taken after 24 h by counting dead flies recovered on the floor and from within the modified bottom box of the filth fly control device. House fly mortality percentage data was arcsine square root transformed and oneway analys is of variance was performed. Means were separated using Fishers protected LS difference test (JMP 2007) Trap s uitable height tests : Semi o utdoor experiments Comparison of individual heights in semi outdoor conditions were conducted in a four room screened shade house (3.1 x 3.7 x 2.7 m per room) constructed with a metal frame and screen mesh walls and ceiling. Atmospheric conditions were sunny and clear with average temperature during the day of 29oC during the four days this experiment was conducted. Treatments consisted of a modified bottom box of the FAST TC as described previously. Three treatment heights were evaluated: 0.91, 1.8, and 2.44 m, and each modified trap was hung from center of ceiling in each screened room. The experiment was co nducted using a 4x4 Latin square configuration. Three hundred mixed sex house flies were released in center of room at the floor level and left undisturbed. Fly counts were taken after 24 h counting dead flies recovered on floor and from within the

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59 modif ied bottom box of the filth fly control device. House fly mortality percentage data was arcsine square root transformed and oneway analysis of variance was performed. Means were separated using Fishers protected LS difference test (JMP 2007) Results A I comparison. House fly mean mortalities were not significantly different between indoxacarb (100%), fipronil (96%), cyfluthrin and imidacloprid combination (92% ), and imidacloprid (92% ) when house flies were give no choice of yarn pieces (Figure 4 2 A). In all four treatments, the population was quickly reduced with in 6 h. The mortality of cyfluthrin (88%) without the addition of the imidacloprid killed just about the same number of flies when combined with imidacloprid Deltamethrin (56%) and thiamethoxam (70%) did not kill as many house flies in six hours but did eliminate over 50% of the population. However, chlorantraniliprole was significantly less effective than t he other treatments with a mean mortality only matching the control. When additional untreated pieces of yarn were added to the lab cages, the mean mortality was similar at 6 h between cyfluthrin and imidacloprid combination (84%), fipronil (86%), and cyfluthrin (78% ) but these treatments caused significantly higher m ortalities than the other treatm ents (Table 41). Thiamethoxam (48%), indoxacarb (42%), imidacloprid (38%), and deltamethrin (22% ) killed about the same number for flies. Deltamethrin eliminated the lowest proportion of the house fly population. The high est mean mortalities in the choice test at 24 h were caused by indoxacarb (94%), fipronil (100%), cyfluthrin and imidacloprid combination (98%), imidacloprid (90%) and deltamethrin (88% ) (Figure 4 2 B) with no significant differences. The lowest mean mortality was caused by cyfluthrin (82% ) which was not significantly different from deltamethrin, thia methoxam (84%) imidacloprid, or indoxacarb. The cyfluthrin and

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60 imidacloprid combination were not significantly more effective tha n imidacloprid alone in 1 h, only knocking down 8% of the fly population. Cyfluthrin alone knocked down ~20% of the house fly population in 1 h but by 24 h had lower mortality than both imidacloprid and the combination of imidac loprid and cyfluthrin. In the field cages with AI applied to FAST TC, treatments reduced the house fly population by greater than 88% at 24 h compared to the 3% decrease of the control (Figure 4 2 C). No significant difference was found between number of dead flies by deltamethrin (94%), thiamethoxam (97%), imi dacloprid (94%), and indoxacarb (93% ). There was no significant difference between fipronil (91%) and deltamethri n, imidacloprid, and indoxacarb. Cyfluthrin (89% ) was not significant ly different from fipronil and indoxacarb. Wool cord vs. wool yarn. No difference was found between the uses of the wool cord as an insecticide delivery method verses the wool yarn (Figure 4 3 ). T here was no significant difference at each mortality count Both treatments eliminated ~70% of the fly population within 24 h co mpared to close to 0% mortality in the control Suitable height I n the laboratory test with the three heights simultaneously in the room significantly more house flies (~50%) were caught at 0.91 m (Figure 4 4 A) tha n the other heights. T here was no sig nificant difference in the number of flies caught at 1.8 (~20%) and 2.44 m (~30%). There were no differences among the room locations of treatments H eight test conducted with individual heights per room (Figure 4 4 B) showed significantly more flies kille d (95%) when traps were placed at 2.44 m c ompared to 1.8 m (~70%), but neither height was significant ly different from traps at 0.91 m that killed

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61 80% of the flies Individual heights evaluated in the semi outdoor conditions of the screened shade house ( Figure 4 4 C) demonstrated there was no significant difference between the three heights having an aver age mortality rate of ~90% at 24 h. Discussion Varieties of house fly strains have shown resistance to tetrachlorvinphos, permethrin, cyfluthrin, and methomyl (Kaufman et al. 2001). Permethrin elimination for 30 generations of house flies under laboratory conditions stopped the natural selection for permethrin resistant house flies and a reversal in insecticide resistance levels was observed (Meyer et al. 1987). A control technique to avoid flies developing insecticide resistance is to change the class of AI after set periods of time such as one month rotations In order for the FAST TC to be a useful tool in fly control, a variety of active ingredients should be used to produce adequate fly mortality. This would allow AI rotation to avoid or counteract potential insecticide resistance in fly populations. In the no choice comparison s, seven out of ei ght AIs decreased the fly population in laboratory cages by > 50% in 6 h. Chlorant r aniliprole, which is anthranilimide insecticide, was the only AI that did not cause significant fly mortality This insecticide was dropped from subsequent tests. Fipronil a phenylpyrazole insecticide that causes insect death by ingestion and absorption through the cuticle is not registered for house flies It caused no mortality within the first hour of the choice assay but then close to 50% mortality at 3 h. Indoxacar b, an oxdiazine insecticide als o not registered for house fly control, c auses mortality of insects by ingestion and cuticle absorption. It caused mortality similar to that of fipronil. However, house fly reduction with indoxacarb did not reach 45% until 6 h.

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62 Imidacloprid fly spot bait which is registered for house fly control kills flies by ingestion and is fast acting and has not shown any signs of house fly insecticide resistance (Gao et al 2007). In comparisons with spinosad and methomyl, ho use flies were affected as quick ly as 30 min by the imidacloprid. However, house flies exposed to imidacloprid had a higher recovery rate tha n flies exposed to other insecticides (White et al 2007). Additionally house flies force fed imidacloprid to house flies in laboratory test have shown insecticide resistance (Gerry and Zhang 2009). An attempt to enhance imidacloprid by combining it with cyfluthrin in laboratory conditions showed no synergistic or additive effect. The FAST TC eliminated > 88% of the field ca ge house fly population within 24 h with all six AIs evaluated. Field cage findings suggest that the FAST TC can be used with a wide range of AIs available on the market This may prove useful for control of insecticide resistant fly populations, or in p revention of insecticide resistance. In an evaluation of natural and synthetic materials, wool proved effective in reducing house fly populations by 50% in 18 h when used as an insecticide treated cord (Hertz 2007). However, the handmade wool cord previously used was cost ly at ~$5.10/m, whereas store bought 100% wool knitting yarn cost ~$0.03/m The comparison of the handmade wool cord and store bought wool knitting yarn of the same diameter showed that both materials eliminated ~90% of the hous e f ly po pulation. Along with improvements to its design and material selection, the height the FAST TC during its operation was evaluated. It has been demonstrated in field cage studies with light traps that twice as many house flies were caught at the height of 0.6 m than at higher heights (Driggers 1971). House fly monitoring devices in field conditions have

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63 shown to be more effective at heights of 1 m or less (Geden 2005). Also, commonly used sticky fly trap s have been shown to work best for other Muscid ae at 0.3 m (Broce 1988). Typically, house flies feed and emerge from organic matter locat ed on the ground (West 1951), w hich makes it logical that more house flies would be caught near the ground if all of the breeding and foraging sources are located th ere. When house flies were indoors and simultaneously given a choice of sugar based attractants at three heights The sticky cards hung at 0.91 m were near est the floor where the house flies were released and the sticky cards hung at 2.4 m were near the visually attractive fluorescent lights mounted in the ceiling This experiment suggests that house flies can be attracted to a colored target hung at any height I ndividual height test s conducted in rearing rooms demonstrated that t here was no difference between the fly mortality at 0.91 m and the other two heights. This leads to the conc lusion that the height is not important when hanging a fly control device inside a building. I n semi outdoor conditions in a screened shade house there was no differenc e in house fly mortality at any of the three heights. In compariso n of the three experiments, it was concluded that the FAST TC at all three heights was visually attractive enough to lure foraging and resting house flies to it. Using the combination of attractive colors, the FAST TC can be constructed for house fly control. The FAST TC can be effective with a variety of insecticides and can be constructed at low cost. In addition, when in use the FAST TC is visually attractive to be used at a wide rang e of heights indoor or outdoor conditions.

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64 Table 41. H ouse fly mortality in choice laboratory cage study using t welve 10 cm pieces of black wool yarn distributed uniformly across ceiling of cage. O ne of the center four pieces impregnated with 1% AI and 10% sucrose and powdered milk. AI 1 h 3 h 6 h Deltamethrin 1.5 + 0.3 bc 4.5 + 3 .0 de 11.3 + 4.4 bc Thiamethoxam 3.8 + 1.1 b 15 .0 + 0.7 bc 24.3 + 4.9 b Cyfluthrin 10 .0 + 6.2 a 39.8 + 7.6 a 38.8 + 6.5 a Imidacloprid 4.3 + 1.3 b 13.3 + 2.7 cd 18.5 + 2.3 b C&I 3.5 + 0.3 bc 13.3 + 0.6 cd 42.8 + 2 .0 a Fipronil 0 .0 + 0 c 23.8 + 4 .0 b 42.8 + 2.1 a Indoxacarb 0 .0 + 0 c 4.5 + 3.2 de 20.8 + 8.9 b F = 7.19 P = .0001 F = 12.91 P < .0001 F = 10.87 P < .0001 Treatments within a column followed by the same letter are not significantly different.

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65 Figure 41. Schematic drawing of collapsible FAS T TC. a. Plastic cable for hanging. b&c. Blue cor rugated plastic. d. Insecticidetreated wool yarn. e. Insecticide treated wool yarn crisscross opening of box over food source.

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66 A Figure 42 Mortality of flies exposed to cords treated with different active ingredient. A. No choice laboratory cage study with solitary 10 cm black wool yarn impregnated with 1% AI, 10% sucrose, and 10% powdered milk. B. Choice laboratory cage study with eleven untreated 10 cm pieces of black wool yarn distributed uniformly across the cage, one center piece treated with 1% AI and 10% sucrose and 10% powdered milk. C. F ield cage study with FAST TC 0 5 10 15 20 25 30 35 40 45 50 Number of dead flies A AB AB B C D E E AB

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67 B C Figure 42 Continued 0 5 10 15 20 25 30 35 40 45 50 Number of dead flies 0 50 100 150 200 250 300 350 Number of dead flies ABC BC AB C A AB D ABC AB ABC C B ABC D A

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68 Figure 43 Mean number of dead house flies after 1, 3, 5, and 24 h exposure to w ool cord or wool yarn treated with 2.5% imidacloprid fly bait. 0 5 10 15 20 25 30 1 3 5 24 Hours Average dead house flies Control Wool Cord Wool Yarn

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69 A B Figure 44. Indoor and semi outdoor trap height comparisons over 24 h. A. Number of flies caught on s ampling cards hung at three heights simultaneously indoors. B. Number of dead house flies from modified FAST TC hung in doors C. Number of dead house flies from modified FAST TC hung semi outdoors. 0 100 200 300 0.91 1.80 2.44 Height of sticky sampling cards (m) Number of house flies caught on sticky card 0 100 200 300 0.91 1.80 2.44 Height of modified FAST-TC (m) Number of dead house flies A B B AB A B

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70 C Figure 44. Continued 0 100 200 300 0.91 1.80 2.44 Height of modified FAST-TC (m) Number of dead house flies A A A

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71 CHAPTER 5 FIELD EVALUATION OF A FLY ATTRACTANT SYSTEM WI TH TOXICANT TREATED CORDS Introduction The house fly is an important pest in markets, stores, and animal facilities due mostly to its annoyance (Hogsette 2003). Controlling house fly populations is important because they can serve as m echanical vectors for one hundred known pathog ens (Greenberg 1971). The scuttle flies are known to br eed in minimal amounts of various decaying organic matter and are often a pest of dirty floor drains and mausoleums (Christenson 1988; Disney 2008). Wher eas the blow fly is often associated with decaying animals and other high protein organic matter ( Hall and Gerhardt 2009). The scuttle and blow flies are w idely considered an annoyance pest, but can also cause facultative myiasis; an invasion of vertebrate tissue by the larval stages (Day et al 2004; Hall and Gerhardt 2009). A fter natural disasters and during wars, fly populations can become quite large, and control options may be limited (West 1951). Baited fly traps are considered monitoring t ools and can not be used as a control method alone (Geden 2005). However, these fly traps are still commonly used to monitor fly populations during concurrent use of various control measures to determine the effectiveness of a management strategy. Baited traps work with two basic components: visual attraction and chemical attraction (Pickens 1995). The visual component attracts flies from a distance and the chemical attractant lures the flies into the trap. The fly attractant system with toxicant treated cords (FAST TC) was developed from research conducted at the University of Florida (Chapter 4). This device was constructed using a visually attractive blue corrugated plastic background with black

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72 lines made of the insecticide treated wool cord (Hertz 2007). This objective of this study was to evaluate the field effectiveness of the FAST TC in areas with known fly populations of the : 1) house fly Musca domestica, 2) scuttle fly Megaselia scalaris and 3) blow fly Lucilia cuprina. Materials and Meth ods Fly attractant system w ith toxicant treated cords (FAST TC ) Treated yarn Treated yarn was prepared by soaking yar n (6 mm diameter, Wool Ease color# 153 black ; Lion Brand Yarn Company, New York, NY.) in 2.5% imidacloprid solution (Maxforce Fly Spot bait, Bayer CropScience, Kansas City, MO) for 1 min and allowing the cords to drain and dry in a fume hood overnight Fly attractant system assembly The fly attractant system wa s constructed with two cylinders connected by black insecticidetreated wool yarn. The cylinders were made with a blue 38 x 11 cm strip of plastic sign board (Coroplast, blue, number: C505L8000.9000; Dallas, TX) and hot glued to form a 12.5 cm diameter cylinder A 237 ml clear plastic deli cup (8 oz clear plastic container #APCTR08; American Plastic Industries Ltd. Chattanooga, TN) was hot glued inside each cylinder with the opening pointed towards the center of the system. A single 13 cm piece of black treated yarn was extended across the center of the open end of the to p cylinder and hot glued in place. Across the open end of the bottom portion, two 13 cm pieces of black treated yarn were cris scrossed and hot glued in place over 170 g of additional food source (Calf Manna pellets, Manna Pro Corp., St. Louis, MO). The two cylinders were vertically connected by four evenly spaced parallel 30 cm long treated pieces of wool yarn extending from the top to bottom cylinders of the device, separated by a 11 cm space.

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73 Field evaluation procedure A FA ST TC was suspended 0.3 m from the ground over a plastic collection tray (46 x 76 x 10.5 cm) for five consecutive days in various field locations The collection tray was placed under the device to catch dead flies falling from the FA ST TC to assess mortality estimates. Choice of location was determined by live population sampling prior to placement of FA ST TC following the device placement suggestion of Lysyk and Axtell ( 1986). Live population sampling consisted of observing and counting live flies resting and landing in predetermined sampling areas for 5 m in For smaller fly species the use of sticky cards (Sticky Aphid trap, Seabright Laboratories Emeryville, CA) was employed. Fly counts and dead fly collections were conducted every approximately 24 h at the same time every day, beginning one day prior to FAST TC placement extending through five days of its deployment, and two days after its removal. Flies were considered dead if found ataxic in the collection pan, in the bottom cyl inder containing calf manna or on the FAST TC device itself Dead flies were collected and placed into labeled zipper locked bags to be counted in the laboratory. Dead flies were collected each day after live fly counts were recorded. Treatment r eplica tes consisted of a nine day period during which fly populations (live and dead) were monitored. Each day received a number represent ing the presence (+) or absence ( ) of th e FAST TC, 1, 0, + 1, + 2, + 3, + 4, + 5, 2, 3 The zero represent ed the day of FAS T TC placement. Days 1 and 0 were considered pretreatment counts and days + 1, + 2, + 3, + 4, + 5, 2 and 3 were considered post treatment counts.

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74 Field sites by species. The house fl y There were three sites used to test FAST TC for control of house flies. A herpetology facility i n Alachua County, Florida that produces and distributes ~10,000 snakes per month had a large house fly population breeding around the facilitys garbage dumpster. This dumpster was only emptied once every two weeks and cont ained organic debris including animal waste and snake corpses, providing optimal feeding and breeding sites for house flies. The garbage dumpster lid ( lid area 2.6 x 1.5 m ) served as t he live fly sampling area at this facility. A 1 m Sheppards hook was used to suspend the FA ST TC at this site o ver a collection pan 2 m on the north side of the dumpster. The herpetology facility also operated a rat rearing building, also with waste receptacles located on its east side of its rat rearing building. These receptacles contained rat feces and used cage bedding, and were also emptied once every two weeks However, they were occasionally over looked by sanitation workers resulting in larger periods of accumulated waste. The live fly sampling areas near the rat rearing facility were marked on the top of two receptacles (0.8 x 0.8 m each) one on each side of the treatment location. A 1 m Sheppards hook was used to suspend the FA ST TC over a collection pan 2 m to the east of the two receptacles used for live fl y sampling I n Chesapeake, VA a compost pile located in the backyard of a residential house maintained a continuous population of house flies. Two live fly sampling areas (0.5 x 0.5 m each) were marked on the ground with wooden stakes and string on each side of the compost pile. The FA ST TC was suspended from a 1 m Sheppards ho ok over a collection pan 2 m on west center side of compost pile.

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75 D uring military field exercises conducted at U.S. Army Fort McCoy, WI, militar y preventive medicine personnel eva luated the FA ST TC around the i r military berthing and dining tents. For evaluation at this site, the wool yarn on the FAST TC was treated at the location by the Army medical entomologist. This was done using a spray bottle to appl y a 2.5% imidacloprid spot bait suspension instead of dipping the cords prior to FAST TC assembly. The FA ST TC were hung from the tent lines on the side of the dining tent and between the two berthing tents and were monitored following the field protocol. The s cuttle fl y. The a forementioned Alachua County, F L herpetology facility experimental site also had a severe infestation of scuttle flies which persi sted for many years prior. T he flies were present in the snake egg incubators and ovipositioned on dead snake eggs and other organic materials The incubators provided optimal conditions that allow ed M. scalaris to breed in high numbers (Trumbl e and Pienkowski 1979). T housands of adult flies were released in the room whenever the incubators were opened, dispersing flies which bred on the snake fecal waste and in other incubation chambers. The FA ST TC devices were evaluated in the two main snake rearing rooms (213 m2 each) referred to as the Annex and Colubrid rooms A wall separated the Annex and Colu brid room s with a single door connecting the m which was kept closed when not in use. Destructive sampling was used to monitor in house live adult scuttle fly populations. N ine sticky cards were evenly distributed throughout each room with three cards loc ated on each of two edge and one center aisle. Sticky cards were spaced evenly from one another, one card being in the center and one on each side of the aisle.

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76 Sticky cards were placed in a vertical orientation on snake cage shelves, 1.5 m from floor Sticky cards were removed and replaced every 24 h during sampling periods. Cards were returned to the laboratory where flies were counted under a dissecting microscope. Due to staff traffic and normal oper ations of the facility, the FAST TC in each room w as hung from a plant hook 0.4 m from the wall over a collection pan. In the Annex, the FA ST TC was placed next to the west wall 0.2 m from the staff restroom and a door leading to a smaller rearing room. This location was near the southwest corner of the Annex and was 1 m from the closest set of shelves containing snake cages. In the Colubrid room the FA ST TC was p laced on the center of the south wall between two windows directly 1 m from the south edge aisle of the shelves containing snake cages. The blow fly A carport located i n Gainesville, FL, at a residential lot w h ere the resident s set out paper plates daily containing moist cat food experienc ed waves of blow fly invasions w hen cat food was present. T wo of the plates used for the cat food were designated as live fly sampling areas. The FA ST TC was suspended from the railing of a boat parked in the carport, over a collection pan 0.5 m from the plates of cat food. Data analysis. Live f ly live population data was used to calculate the percent control for each treatment. Percent control was calculated as the average number of live flies sampled two days prior to FA ST TC placement (days 1 and 0) divided by the average live population o ver the seven days post FAST TC deployment (days 1, 2, 3, 4, 5, 2, and 3). A Paired t test was performed on live fly numbers sampled during preand post treatment. Average number of dead flies was calculated collectively by

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77 species per sampling day. The total number of dead flies collected on a particular samp ling day was divided by the total number of dead flies collected. Numbers of dead flies were not analyzed statistically Results and Discussion The house fl y The FA ST TC was very effective in controlling house flies with an overall estimated control of >70 % for all sites (Table 51) Approximately 50% of all flies were killed during the first 24 h of the FAST TC deployment at each location (Figure 51). The herpetology facility had the largest number of house flies observed during this study. The house fly populations at the dumpster and behind t he rat rearing faci lity declined rapidly after FAST TC deployment. At both sites, the average number of dead flies collected everyday decreased while FAST TC was deployed, suggesting a decline in the house fly population, as demonstrated by the estimated percent control. At the VA residential compost pile FA ST TC also reduced the house fly population with in the first 24 h. However, the average number of dead flies collected on the fourth day of FAST TC de ployment increased suggesting a new house fly emergence that was also eliminated within 24 h. The FA ST TCs used at Fort McCoy during a U .S. Army field exercise were hung throughout the camp. Unlike the other field evaluations with the FAST TC the insectic ide was mixed in a spray bottle and sprayed on the wool yarn until it was saturated. The application of insecticide resulted in equal killing efficiency as when cords were dipped into pesticide solution. This demonstrates that the FA ST TC can be recharged if needed with insecticide spray applications. Two days after treatment was removed, a small percentage of house fly mortality was observed, likely due to the residual imidacloprid spot bait that was left behind on the tent cord.

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78 The s cuttle fl y. The s cuttle fly is the most medically important phorid fly as they are associated with sporadic cases of facultative human myiasis (Hall and Gerhardt 2009). Before FAST TC deployment the average number of scuttle flies captured i n a 24 h period on an individual sticky cards was >500. After five days of the FA ST TC deployment, the average number of scuttle flies captured on an individual sticky card was < 20. T he average overall estimated control fo r both snake rearing rooms was 79% (Table 51) The FAST TC e fficiently reduced the scuttle fly population, killing >40% of the total number of scuttle flies in the first 24 h (Figure 52). Scuttle flies are opportunistic and can breed in small amounts of organic matter (Disney 2008). Prior to the FA ST TC testing the herpetology use of insecticide or other control measures by facility staff was limited Unsuccessful attempts to control the unmanageable fly population were made with aphid sticky cards. The sticky cards offered no noticeable control but did prove to be an excellent populationmonitoring device while the FAST TCs were used. The blow fl y. Blow flies are normally associated with dead animals and were highly attracted to during moist cat food as it likely mimicked carrion (Scholl et al 2009). The average estimated percent control for blow flies did not show reduction, although the onsite observation s indicated that there were fewer flies in the area when the FAST TC was deployed. The number of dead flies collected near the moist cat food while the F AST TC was deployed also demonstrated that the blow fly population fluctuated (Figure 53). This may be due to blow fly emigration from off site areas. Lucilia spp usually appear s within minutes on remains of animals (Anderson 2010). In

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79 addition, the p resence of the cat food was dependent on time of placement, and on the speed of consumption by the cats. In summary, to reduce a filth fly population by 5090%, traps must remove 2458% over 24 h (Weidhaas and Haile 1978). Using the visually attractive blue background in combination with the insecticidetreated black yarn proved effective in controlling two species of filth flies. The FA ST TC demonstrated that it could be used in conditions where insecticide use is limited as well as having the ability to be recharged by spray applications when necessary. This novel approach can be refined as a useful tool for filth fly control.

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80 Table 51. Estimated percent fly control calculated per FAST TC location separated by fly species. Replicate Pre treatment li ve sampling Post treatment live sampling Percent fly control House fly Garbage dumpster 1 17.0 23.0 35% 2 69.0 35.9 48% 3 23.5 15.1 36% 4 30.0 19.1 36% 5 26.0 18.4 29% 6 117.0 19.3 84% 7 169.0 18.4 89% 8 123.5 20.1 84% 9 130.0 20.3 84% 10 233.0 22.9 90% 11 206.5 20.4 90% Rat rearing facility 1 107.5 12.1 89% 2 92.5 10.9 88% 3 137.0 19.7 86% 4 61.5 34.7 44% 5 59.5 13.1 78% Residential compost pile 1 34.0 4.0 88% 2 16.5 2.4 85% 3 5.5 4.0 27% Ft McKoy military camp 1 10.0 3.7 63% 2 21.3 7.7 64% 3 1.7 1.5 10% 4 8.0 1.2 85% 5 11.0 0.8 92% 6 23.7 0.8 96% 7 11.0 1.5 86% 8 14.0 2.3 84% Total mean pre treatment count = 65.1 Total mean post treatment count = 19.0 Total estimated house fly control = 71% *Pre treatment live sampling consist of sampling days 1 and 0. **Post treatment live sampling consist of sampling days 1,2,3,4,5, 2, and 3. Bold numbers represent increase in fly population.

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81 Table 51 continued. Replicate Average Pre treatment count Average Post treatment count ** Percent fly control Scuttle Flies Annex snake rearing room 1 1284.5 297.6 77% 2 756.5 127.6 83% 3 334.0 108.6 67% CLB snake rearing room 1 267.0 99.9 63% 2 193.5 309.9 60% 3 968.0 122.6 87% 4 1772.5 360.3 80% 5 1660.0 579.1 65% 6 1163.0 686.4 41% 7 1484.0 730.1 51% 8 908.0 763.9 16% 9 1751.0 796.3 55% 10 745.0 790.0 6% Total mean pre treatment count = 1022.0 Total mean post treatment count = 213.5 Total estimated scuttle fly control = 79% Blow flies Near moist cat food 1 16.0 28.0 75% 2 48.5 10.1 79% 3 8.5 23.4 176% 4 81.5 34.1 58% 5 7.0 45.3 547% 6 11.0 46.4 322% 7 12.5 43.6 249% 8 8.0 38.6 382% 9 15.5 5.6 64% 10 15.5 5.7 63% 11 5.5 4.1 25% 12 7.0 5.6 20% 13 6.0 5.7 5% 14 2.0 5.7 186% 15 7.5 5.7 24% Total mean pre treatment count = 16.8 Total mean post treatment count = 11.1 Total estimated blow fly control = 34%

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82 Figure 51. Daily percent of dead house flies collected under FAST TC in relation to total dead flies collection (based on 44,598 total flies) Figure 52. D aily percent of dead scuttle flies collected under FAST TC in relation to total dead flies collection (based on 61,705 total flies). 0% 10% 20% 30% 40% 50% 60% 1 0 1 2 3 4 5 2 3Percent of dead house fliesSampling days 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 1 0 1 2 3 4 5 2 3 Percent of dead scuttle fliesSampling days

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83 Figure 53. Daily percent of dead blow flies collected under FAST TC in relation to total dead flies collection (based on 3,136 total flies). 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 1 0 1 2 3 4 5 2 3Percent of dead blow fliesSampling days

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84 CHAPTER 6 CONCLUSION House fly control is best achieved using integrated management strategies combining cultural, mechanical, biological, and chemical control methods. T ypically, in an effort to break the life cycle chain of house flies the rem oval of fly breeding and resting areas is achieved with sanitation. When sanitation fails insecticides are utilized to spray or bait areas, where flies feed or rest. The development of the fly attractant system with toxicant treated cords (FAST TC) gives the ability to target flies that are seeking foraging or harborage sites while utilizing minimal amounts of insecticides In addition, upon removal of the F AST TC no residual chemicals will be left to be harmful to the environment. House flies perceive reflected sunlight from objects as visual input for survival. This gives flies the ability to navigate through their environment to avoid obstacles while flying, find food, find harborage, and be able to recognize resting areas. Neurological responses o f house flys compound eye and ocelli were measured from reflected light from colored visual targets using a electroretinogram. Light of the same intensity reflected from multiple plastic visual targets showed that house fly compound eye and ocellus were capable of color vision. The greatest neurological responses for the compound eyes and ocelli were to white, blue, and yellow (Chapter 3) Behavioral responses to colored plastic and fabric visual targets compared to black were determined using a twosi ded light tunnel. The house fly attraction to colored visual targets was White = Blue > Red > Green > Yellow. In a paired test the blue visual target was more attractive than white. Yellow visual targets proved to be repellent to house flies. An observ ation of more house flies on the black visual target

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85 side of the two sided light tunnel when compared to yellow further demonstrated the yellow visual target repellency. The addition of yellow lines on to an attractive surface also repelled flies. However, the addition of black lines, which simulated a harborage area, enhanced the attractiveness of the blue background. The visual attraction to the black lined blue background s uggest s that house flies are able to form images to guide their behavior (Chapt er 3) Combining visually attractive color patterns with previously studied wool insecticide treated cords led to the construction of the FAST TC (Chapter 4) In a laboratory cage study with house flies, knitting wool yarn performed as well as previously used wool cord as a insecticide delivery method. The comparison of active ingredients, (deltamethrin, thiamethoxam, imidacloprid, indoxacarb, fipronil, and cyfluthrin) applied to FAST TC in field cages showed a reduction of house fly population > 88% in 24 h compared to the 3% decrease of the untreated control Bioassay conducted with sticky cards hung simultaneously at three heights caught 50% of the house flies released in a laborat ory room at the lowest level, demonstrating that house flies ten d to fly low. Modified version of the FAST TC was able to attract house flies that were foraging at the low heights and flies that were seeking resting areas at the higher heights. The FAST TC was field tested and with very low amounts of insectici de and was able to control two species of flies in a variety of conditions (Chapter 5) Due to the efficiency that the FAST TC demonstrated in the field in controlling house, scuttle, and blow flies the University of Florida submitted a patent application for this device.

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86 The research conducted here to develop the FAST TC will provide a novel approach in the control of filth flies and will provide a useful tool in the arsenal of fly control. Hopefully this research will stimulate further investigation of fly v ision to help in the improvements and development of other control measures.

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87 APPENDIX A EFFECTIVENESS OF FLY TRAPS AND TREATED CO RDS IN CATCHING AND KILLING HOUSE FLIES (DIPTERA: MUSCIDAE) Introduction There is some debate whether baited traps fit into a fly management program as monitoring or control devices. To reduce a fly population by 5090%, traps must remove 2458% of the house flies daily (Weidhaas and Haile 1978); however, traps alone have not been shown to reduce fly populations to acceptable levels (Smallegange 2004). Insecticide treated cord can also be used to control house flies. Cords impregnated with organophosphate and chlorinated hydrocarbon insecticides were introduced in 1947 and were found to provide good house fly control (Smith 1958). However, use of treated cords fell in popularity due to the ease of applying residual insecticide sprays to structures. Low concentrations of new insecticides, such as 0.1% fipronil and 1.2% indoxacarb impregnated in cords (Hertz 2007), have been s hown to effectively kill flies in field cages. The objective of this study was to evaluate the enhancement of a fly trap using insecticide treated cord to control house flies in field cages. Materials and Methods Insects For the laboratory experiments, house flies were collected from the University of Florida Horse Teaching Unit about 1 month prior to experiment, reared in the laboratory, and used at the F3 through F5 generation. Flies were reared following a method modified from Hogsette et al. (2002) w ith no powdered egg yolk in the adult diet, and Calf Manna (MannaPro Products, Chesterfield, MO) substituting for alfalfa meal and corn meal in the larval diet. Larvae and adults were held in separate

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88 containers at 26.2 0.5C and 51.2 3.5% RH with a 12:12 (L: D) photoperiod. When collectin g flies from the colony, 3 to 5 d old house flies were aspirated and placed in a freezer (30C) until inactive (~1 5 min). Fly trap enhancement with treated cord. Enhancement of fly traps with treated cords was evaluated on populations of flies established in field cages. Field cages (1.8 x 3.7 x 1.8 m; Outdoor Cage, #1412A, 18 x 14 mesh, Bioquip, Rancho Dominguez, CA) with translucent plastic sheeting (6 mil) used to cover the floor were built on a grassy area shaded by pine trees. Food (1 L of 10% sucrose), water (1 L), and 60 ml container of spent oviposition media (covered with paper towel to prevent fly access) were placed in the cages to provide nutrition and to compete with the treatments for fly activity. M usca Doom Disposable Fly Traps (Farnam Companies, Inc. Product code 057117; Phoenix, AZ 85067) (identical to Trap n Toss fly traps) were used in this experiment. The label sticker was removed from each trap to allow maximum visual fly response to the trap. Six treatments were evaluated: 1) fly trap, no attractant, untreated wool cord, 2) fly trap, attractant, no cord, 3) fly trap, attractant, untreated cord, 4) fly trap, attractant, treated cord, 5) fly trap, no attractant, treated cord, and 6) no fly tr ap, no attractant, treated cord hung in the shape of a halo. Treatments with attractant were prepared the morning of the experiment by filling fly traps with 5 ml of MuscaDoom attractant solution mixed, as directed by the manufacturer, in tap water. Tre ated cords were prepared using imidacloprid bait (Maxforce Fly Spot bait, Bayer CropScience, Kansas City, MO 64120) with 25 g of product dissolved in 100 ml of tap water. Wool cord pieces (Twisted, Natural Cord, Wooded Hamlet Designs, Greencastle, PA; 0. 6 cm diameter) were cut to the length (46 cm) to match the

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89 circumference of the outside center of the fly trap and treated by dipping for 1 min in the insecticide solution. Untreated cords were dipped in tap water. All cords were dried overnight in a fum e hood. Cords were then wrapped around the fly traps or formed into a halo for the treatment without traps. All treatments were placed in the field cages 1 h after ~ 300 flies (35 mL volumetrically) were released in each cage, corresponding to ~ 45 flies per m2 of cage area. Fly traps were hung 1 m in front of the cage entrance and 14 cm from the cage ceiling; the cord halo was hung 28 cm from the cage ceiling. At 1, 24, and 48 h after trap placement, flies were collected from the floor of each cage and placed in a Mason jar (118 ml) to allow knocked down flies to recover in the cage. At each sampling time, flies on the floor of the cage, those remaining in the jar, and those collected in traps were counted. After mortality data were collected, a fly ann oyance index was estimated by a person walking around the inside of the field cage for one complete revolution while tapping the cage mesh. During the following 1 m, annoyance was rated using the following scale: 1 = no flies (0 per m2), 2 = few flies seen in cage (~10 per m2), 3 = several flies around face and arms (~20 per m2), 4 = many flies around face and arms (~30 per m2), 5 = numerous flies around the face, arms (~40 per m2), and many seen in cage. Mortality data for each time after trap placement were analyzed by analysis of variance, and means were separated using Student NewmanKeuls test (P = 0.05; SAS 2001). Linear regression was used to correlate the annoyance index with the number of flies observed in cage. Results Flies released into field cages initially oriented to the top sides and corners of the cage. Eventually, flies were attracted to the traps or cords, food, water, and oviposition

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90 media. Within 1 hour, significant differences in fly mortality (df = 5, F = 5.12, P = 0.0016) were seen among treatments (Figure A 1 ); treatments with treated cords had higher mortality (21%) than those with traps alone (1%). After 24 h, all treatments containing treated cords had significantly higher mortality (68%) than fly traps alone (12%) (df = 5, F = 16.82, P < 0.0001). There were no significant increases in trap kill when attractant was added to either the trap alone (14% with attractant vs. 6% without attractant) or traps with treated cords (64% with attractant vs. 70% without attractant). After 48 h, there was a significant difference between the treatments with treated cords (97% fly mortality) and the ones without cords (17% fly mortality) (df = 5, F = 87.41, P < 0.0001). Traps with treated cords killed 97% of flies; whereas, traps without treate d cords killed only 17% The annoyance index (Figure A 2 ) decreased as flies per m2 decreased; traps with the treated cords decreased the annoyance index to 1 after 48 h. Discussion Whether baited house fly traps can be efficient as a control device or just a monitoring device depends on the house fly population reduction it can provide (Weidhaas and Haile 1978). This experiment showed that, total catches with commercial traps were within a range insufficient to provide fly population control. Traps used according to the manufacturers instructions only retained a very small percentage of the fly population after 48 h. Therefore, trap performances must be enhanced if they are to be used for control, rather than as monitoring devices only. The addition of an insecticide element, such as imidacloprid treated cords, increased fly trap efficacy. Imidacloprid has residual effects (Pospischil et al. 2005) that in combination with the wool cord reduced the fly population quickly. In this experiment, the treat ments with the imidaclopridtreated wool cord also reduced the fly population

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91 quickly as observed previously with imidacloprid use (White et al. 2007). The fly annoyance index was negatively correlated with fly mortality in cages; as flies per m2 decreased the annoyance decreased. Furthermore, the treated wool cord alone was shown to be just as effective as the traps with the treated cord addition, demonstrating that the trap itself was not a critical factor in reducing the fly population. This research has demonstrated that house fly trap design is important for attraction, trapping, and retaining flies, and that these factors can greatly influence whether a trap can be an efficient method for fly population reduction. Once a house fly has been attracted to and trapped in a fly trap, the traps capability to remove attracted insects from the infesting population can be improved by increasing the traps killing power. The addition of insecticidetreated cord is a simple method to improve traps efficacy in the field, and may provide sufficient improvement to make house fly traps into control devices rather than simple population monitor. However, insecticidetreated cords alone or in combination with other visual and odor attractants, but without a trapping mechanism, may cause sufficient fly mortality to provide efficient fly control.

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92 Figure A 1. Mortality of house flies at 1, 24, and 48 h after initial exposure to different fly trap treatments in field cages. Figure A 2. Correlation between annoyance index and number of surviving flies observed in field cages. 0 50 100 150 200 250 300 350 1h 24h 48h 1h 24h 48h 1h 24h 48h 1h 24h 48h 1h 24h 48h 1h 24h 48h Trap, Attractant, No Cord Trap, Attractant, Untrt Cord Trap, No Attractant, Untrt Cord Trap, Attractant, Trt Cord Trap, No Attractant, Trt cord No Trap, No Attractant, Trt Cord Total house fly mortality y = 0.0969x + 1 R = 0.9628 1 2 3 4 5 0 10 20 30 40 50Annoyance indexMean flies per m2 AB A A A B B a a a b b b

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93 APPENDIX B CONCISE DESCRIPTION OF THE INVENTION Fly Attractant System with Toxicant Treated Cords This fly attractant system incorporates a visual and chemical attraction to lure nuisance flies to the device. From a distance, flies are attracted visually to the color blue and the edges of the device. As t he fly approaches the device the black yarn against the blue background, give the appearance of a crack or crevice, which a fly will perceive as shelter and fly even closer. Once the fly is in close proximity to the device the chemical attractant lures th e fly in closer attracting the fly to land on the device with the assumption it is a food source. The fly will investigate the black yarn and will feed. Once it feeds on the yarn that is treated with imidacloprid insecticide, the fly will die within seconds to minutes. Treated yarn. Treated yarn was made by soaking yarn (Wool Ease color# black 153; Lion Brand Yarn Company, New York, NY.) in 2.5% imidacloprid bait (Maxforce Fly Spot bait, Bayer CropScience, Kansas City, MO 64120) for 1 min. Fly attra ctant system assembly. Cylindrical fly attractant system with toxicant treated cords is constructed with two cylinders connected by black insecticidetreated wool yarn. The top portion of the attractant system was made with blue plastic sign board (Coroplast, blue, n umber: C505L8000.9000 ; Dallas, TX) cut in 38 x 11 cm strip and rolled to form a cylinder with the diameter of 12.5 cm, with ends hot glued together. A 237 ml clear plastic deli cup (8 oz clear plastic container, #APCTR08; American Plastic Industries Ltd. Chattanooga, TN) was hot glued inverted to the bottom of the t op portion of the device. A 13 cm piece of black treated yarn was extended across the center of the open end of the deli cup and glued in place with hot glue. The bottom

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94 portion of the attractant system was prepared as described above for the top portion with a 38 x 7 cm blue plastic sign board. A deli cup was hot glued inside the bottom portion of the device. Across the open end of the bottom portion two 13 cm pieces of black treated yarn were crisscrossed and hot glued in placed. The two cylinders were connected by four evenly spaced parallel 30 cm long treated pieces of wool yarn 5 mm in diameter extending from the top to bottom portions of the device, which were separated by a 11 cm space.

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95 Figure B 1. Cylindrical fly a ttractant system with toxi cant treated c ords

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96 Figure B 2. Fly attractant system with toxicant treated c ords placed near fly infested garbage cans, which eliminated 3006 flies in 24 hours.

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97 Figur e B 3. Dead flies killed by the fly attractant system with toxicant treated cords near a house fly infested dumpster. Device placed near dumpster at snake farm 0 50 100 150 200 250 300 350 400 -1 0 1 2 3 4 5 -1 -2 -1 0 1 2 3 4 5 -1 -2 Number of dead flies

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104 BIOGRAPHICAL SKETCH Joseph W. Dicl aro II was born in the coal mining city of Beckley, West Virginia. He was the second youngest of four children. After leaving home at a young age, he moved to Cincinnati, Ohio and graduated from Mt. Healthy High School in 1989. In 1990, he enlisted in t he United States Navy and was proud to become a Hospital Corpsman. Over the last 20 years, he has served overseas at Naval hospitals, with Naval Mobile Construction Battalion, United States Marine Corps, as well as various shore duties. M ost recently, he was assigned to the medical staff at the Attending Physician Office at the United States Capitol. He received his Associate of Science degree in general s tudies from Northern Virginia Community College in 2000. He received his Bachelor of Science in int erdisciplinary studies in biology from Mountain State University, Beckley, West Virginia in 2004. He attended George Mason University and received his m aster s in b iodefense in 2007 In 2006, he was the second enlisted Sailor selected to study entomology under the Medical Service Corps Inservice Procurement Program (MSC IPP) and became the first to obtain a PhD in this program. Upon graduation HM1 (SCW ) Joseph W. Diclaro II will be commissioned to the rank of Lieutenant as a medical entomologist in the Medical Service Corps. He enjoys the time he can spend with his kids and looks forward to seeing them succeed. He is also a proud active member of Centennial Lodge #174 of Ancient Free and Accepted Masons located in Upper Marlboro, Maryland.