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Bait Aversion and Oral Toxicity of Insecticides in a Field Strain of German Cockroach


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BAIT AVERSION AND ORAL TOXICITY OF INSECTICIDES IN A FIELD STRAIN OF GERMAN COCKROACH. By LINDA ANNE NCHERNE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2005 by Linda Anne Ncherne

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iii ACKNOWLEDGMENTS I would like to thank my husband for the love and support he gave me during the degree process. He never once complained when I was using him as a soundboard to work thorough technical problems, although he now knows more about cockroaches than he ever desired. I would al so like to thank my mother ; although deceased, I feel she helped guide me to this path and is proud of my accomplishments. Many thanks go my committee members: Dr Simon Yu for his editorial support and help with toxicology and Dr. Richard Pa tterson for his editorial support, wisdom, and guidance. Lastly thanks are due to my comm ittee chair, Dr. Philip Koehler. However, I feel thanks are not enough for all he has done. In addition to his vast knowledge of every aspect of urban entomology, he is a great ment or, pushing all of his st udents to aspire to greatness. Additionally, his ent husiasm for the subject, even after so many years, is truly inspiring. I sincerely feel honored to have been his graduate student. Additionally, thanks go the Urban entomo logy crew, especially Gilman Marshall for explaining chemistry related math to me so that I actually understood it something no high school or college professor had mana ged to do and to Joseph Smith for helping acclimate someone who had been out of academia for 8 years and for making me laugh. Lastly, but certainly not least, special tha nks go to Cynthia Tucker for bad day lunch commiserations, opening up her home to me when I was in need, and offering unconditional friendship a rarity in today’s world.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Biology........................................................................................................................ .3 Habitat........................................................................................................................ ...4 Pest Status.................................................................................................................... .5 Chemical Control of the German Cockroach...............................................................6 3 EVALUATION OF FEEDING DETTERRENCE IN SIX INSECTICIDAL GEL BAITS AND MORTALITY IN A FIELD STRAIN OF GERMAN COCKROACH, Blattella germanica (L)...................................................................11 Introduction.................................................................................................................11 Materials and Methods...............................................................................................12 Results........................................................................................................................ .15 Discussion...................................................................................................................18 4 ORAL TOXICITY OF INDOXA CARB AND SECONDARY MORTALITY FROM NECROPHAGY IN A SUSCEPTIBLE STRAIN AND A FIELD STRAIN OF GERMAN COCKROACH, Blattella germanica (L)...........................26 Introduction.................................................................................................................26 Materials and Methods...............................................................................................27 Results........................................................................................................................ .30 Discussion...................................................................................................................34 5 CONCLUSION...........................................................................................................48

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v LIST OF REFERENCES...................................................................................................50 BIOGRAPHICAL SKETCH.............................................................................................55

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vi LIST OF TABLES Table page 3-1 Mortality (4 d) and consumption of gel bait, dog food, and AI for 50 Orlando susceptible strain German cockroaches 24 h after gel bait placement.....................24 3-2 Mortality (4 d) and consumption of gel bait, dog food, and AI for 50 Daytona field strain German cockroaches 24 h after gel bait placement...............................25 4-1 Susceptibility of Orlando susceptible and Daytona field strains of German cockroach to two inge sted insecticides....................................................................41 4-2 Daily consumption of insecticide tr eated nymphs by Orlando susceptible strain German cockroaches................................................................................................42 4-3 Daily consumption of insecticide treated nymphs by Daytona field strain German cockroaches................................................................................................43 4-4 Cumulative daily mortality from inge stion of insecticide treated nymphs in Orlando susceptible strain German cockroaches......................................................44 4-5 Cumulative daily mortality from inge stion of insecticide treated nymphs in Daytona field strain German cockroaches................................................................45

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vii LIST OF FIGURES Figure page 4-1 Proportion fipronil and indoxacarb tr eated breadcrumbs eaten by Orlando susceptible strain cockroaches after a 24 h starvation period..................................39 4-2 Proportion fipronil and indoxacarb treat ed breadcrumbs eaten by Daytona susceptible strain cockroaches after a 24 h starvation period..................................40 4-3 Percent mortality of Orlando susceptible strain cockroaches from ingestion of insecticide treated nymphs.......................................................................................46 4-4 Percent mortality of Daytona field strain cockroaches from ingestion of insecticide treated nymphs.......................................................................................47

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viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BAIT AVERSION AND ORAL TOXICITY OF INSECTICIDES IN A FIELD STRAIN OF GERMAN COCKROACH By Linda Anne NcHerne August 2006 Chair: P.G. Koehler Major Department: Entomology and Nematology Control of German cockroaches is agai n becoming a severe problem. The usual method of control is the use of gel baits. In order for a bait to be effective, it must be palatable, attractive, highly toxic, an d provide secondary kill through coprophagy or necrophagy. Currently, strains of German cockroach exist th at are not being controlled by gel baits. It is unknown if this can be attributed to physiological or behavioral aversion. Daytona field strain German cock roach, was collected from an area that had reported control failure using gel bait in Da ytona, FL. To determine if Daytona field strain was behaviorally averse to gel baits, Daytona field strain a nd Orlando susceptible strain were given choice test using six different commerci ally available gel baits. Daytona field strain exhibited feeding deterr ence to three of the most common gel baits used: Avert, Maxforce and Maxforce FC as well as DPX-MP062-411a; however, Orlando susceptible strain exhi bited no feeding deterrence. In both strains, there is positive correlation between consumption and mortality. Bait aversion caused decreased

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ix consumption of three commonly used gel baits; however there were two formulations that overcame aversion. Physiological resistance was evaluated th rough an oral toxicity assay where active ingredient was fed to Daytona field strain and Orlando susceptible strain cockroaches on breadcrumbs. Daytona field strain exhibite d low resistance ratios to a new chemical, indoxacarb (3.5 LD50 and 4.4 LD90) and moderate to high resist ance ratios to fipronil (9.4 LD50 and 36.9 LD90). When using a palatable gel ba it formula, Daytona field strain ingested 1.5x more fipronil than necessary to kill 90% of the population. Due to evolving physiological resistance, palatable bait formulations may not control German cockroaches in the future. The ability of fipronil and indoxacarb to cause secondary mortality was evaluated on Daytona field strain and Orlando susceptibl e strain cockroaches. Cockroaches were offered insecticide treated nymphs as a food source both with and without a food choice. Necrophagy occurred in both choice and no c hoice tests for both strains. Significant mortality from ingesti on of fipronil treated nymphs only o ccurred in the no choice test for both strains; whereas significant mortality from ingestion of indoxacarb treated nymphs occurred in both choice and no choice tests for both strain s; however it occurred more rapidly in the no choice test. Overall, there have been significant cont rol problems in the field when using the most common gel baits. Control failures ar e due to the combination of physiological resistance and behavioral resistance, but can be overcome with the use of other formulations of gel baits or new active ingred ients. Control will be more effective when sanitation is used in co njunction with gel baits.

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1 CHAPTER 1 INTRODUCTION The German cockroach, Blattella germanica (L.), has adapted to cohabiting with the human species. Throughout the years, va rious methods and chemicals have been used to try to control this pest. While resu lts were as varied as the method of control, most treatments were ineffective. The firs t chemical treatments that really reduced cockroach populations and eff ected control were spray form ulations used in the 1950s; more chemical formulations were developed in the next two decades Ultimately, all of these chemicals became obsolete because, w ithin about five year s of exposure, the German cockroach became physiologically resistant (Cornwell 1976). Introduction of baits in the late eigh ties greatly reduced concerns about physiological resistance. Thes e baits contained new classes of chemicals that were so toxic that a lethal dose of ac tive ingredient was delivered in one meal (Wang et al. 2004), apparently preventing the development of physiol ogical resistance. It seemed to work as these new baits were effective at controlling the German cockroach. In a couple of years, bait formulas and applications changed from dry baits contained in a plastic case placed in a couple of random locations to gel bait fo rmulations that were placed drop-wise in many small spaces near harborages and foraging areas; which was even more effective at cockroach control. In the past ten years, control failures ha ve been reported from areas where gel baits had been used extensivel. It was determined that these failures were the result of cockroaches refusing to consume the bait. This behavioral aversion was initially determined to be due to glucose within the gel bait matrix. Although the problem of

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2 glucose aversion was overcome, failures were st ill reported. Currentl y, the exact cause of behavioral aversion is unknown. For my studies, a field strain of German cockroach was isolated from an area that had reported control failures. Control failure s are due to decreased mortality, which has many possible sources. Chapter 3 evaluates th e field strain collected to determine to what extent gel baits are feed ing deterrents and to determine if decreased consumption of gel bait affects mortality. Because the effectiveness of new classes of chemicals must be determined, two of the six gel baits tested in Chapter 3 c ontain a new chemical, indoxacarb Chapter 4 evaluated oral toxicity of indoxacarb, in both a susceptible strain and a field strain of German cockroach. One of the benefits of highly toxic chemicals contained in gel baits is that they can cause mortality in cockroaches that did not directly ingest the gel bait. Additionally, in chapter 4, sec ondary mortality due to necrop hagy was evaluated in both a susceptible strain and a fiel d strain of German cockroach. Behavioral changes in German cockroaches that cause them to eschew gel baits that once were effective is as important a surviv al strategy as physiologi cal resistance. The role of consumption of toxic gel baits ca nnot be understated. Consumption is what allows the active ingredient to have an effect, not only on primary mortality, but on secondary mortality as well. Documenting and understanding behavioral changes in cockroaches is the first step towards effecting new control measures.

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3 CHAPTER 2 LITERATURE REVIEW Biology German cockroaches, Blattella germanica (L.) (Blattaria: Blatellidae) are hemimetabolous, having only three life stag es: egg, nymph, and adult. When the young emerge as first stage nymphs, they are no l onger dependent on their mother and must find a way to acquire nutrition and water. Larger cockroaches ac complish this by foraging. Because first stage nymphs are very small a nd therefore easy prey for other insects, including other cockroaches, survival dictates they find another way. Their survival strategy stems from their gregarious nature. Instead of foraging out side the harborage, they remain sequestered inside the harbor age (Silverman, et al, 1991) consuming the excrement of other cockroaches; a proce ss known as coprophagy (Durier and Rivault 2000b, Gahlhoff et al. 1999; Kopanic et al. 2001). As they mature, coprophagy decreases as foraging and scavenging methods increa se. As with other insects, German cockroaches grow by the process of molti ng. The time between molts is known as a stadium. German cockroaches undergo 6-7 st adia and take an average of 103 days to mature into an adult. This time frame is dependent many factors such as temperature, nutritional status, and strain (Cooper and Schal 1992). Nymphs are dark brown in color with a large tan spot on their pronotum. They range in size from approximately 1.5 mm newly emerged to 1.6 cm as adults. Adults are light brown in color and have two dark stri pes on their pronotum extending longitudinally down the body under the wings. The male has a tapered abdomen while the abdomen of

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4 the female is rounded (Ebeling 1975). Although both sexes possess wings, they are incapable of flight. When a female German cockroach emerge s as an adult, it takes many days to become sexually active (Schal et al. 1997). When they do become sexually active, they often mate multiple times, although one mating is usually sufficient to fertilize all their eggs (Schal et al. 1997). This mated female produces, on average, 30-40 eggs per oothecal case (Willis et al. 1958). Adult females survive approximately 6 months and produce 4-6 broods in their lif etime (Schal et al. 1997). They are oviparous; however, they carry their ootheca until just prior to hatching (Schal et al 1997). This gravid cycle lasts approximately 21-28 days (Schal et al.1997) during which time the females eat sparingly or not at all (S chal et al.1997, Ross 1993). Habitat German cockroaches have a world-wide di stribution and, in addi tion to domiciles, can be found in restaurants (Rust and Reiers on 1991), hospitals (El gderi et al. 2006, Kitae et al. 1995), and even aboard naval vessels (Flynn and Schoof 1971). They are usually found in kitchens, bathrooms, or other areas wh ere water is readily av ailable. They are nocturnal scavengers capable of living off human waste foods tuffs (i.e. crumbs, residues on dishware, etc). During the day they usua lly remain sequestered in harborages. A harborage is any enclosed area allowing them protection to breed and survive. In homes this is often cabinetry, appliances, wall vo ids, or any clutter (boxes, stored goods, garbage, etc). This ability to sequester in very small spaces is often the mechanism for new infestation as introduction of cartons, boxes, or other materials harboring German cockroaches is transported to un-infested homes and businesses. Additionally, German cockroaches can infest apartments or offices connected by a common wall by moving

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5 from the infested apartment through wall voids or across conjoining plumbing systems to the new space (Owens and Bennett 1982). Pest Status German cockroaches commonly carry poten tially pathogenic bacteria such as Klebsiella, Enterobacter, Serralia, and Streptococcus (Elgderi et al. 2005; Kitae et al. 1995). No direct transmission of disease to humans has been demonstrated; however, cockroaches have the potential of transmitti ng these pathogens via contamination of food preparation surfaces and utensils (Kitae et al 1995). New data has found that a great number of pathogens isolated from wild strain cockroaches are multiple antibiotic resistant (Elgderi et al. 2005). Carrying pathogenic bacteria is not th e German cockroach’s only method of contaminating domiciles and causing harm to humans. Cockroaches have been linked to human allergies since the mid 1940s (Kang 1990). In infested households, German cockroach debris is second only to dust mite s in the composition of “house dust” (Silva 1990). The greatest sensitivity to cockroach alle rgens occurs in children seven to twelve years old (Garcia et al. 1993). As is the case with many allergens over time, cockroach sensitivities can develop in those that previously had none and existing sensitivity can increase (Steinberg et al. 1987, Kang 1990). Occasionally, the sensitivity becomes so bad that shellfish can no longe r be eaten, contact dermatitis occurs in the presence of infestation, (Silva 1990) or asthmatic respons es occur when cockroach allergens are inhaled (Garcia et al. 1993). When human hea lth risk is added to the psychological stress and stigma caused by German cockroach infestation, it is no wonder German cockroaches are considered a major pest worldwide.

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6 Chemical Control of the German Cockroach The oldest group of chemicals used in cockroach control is inorganic compounds such as boric acid and sodium fluoride. Th ese were slow acting powders that worked both via contact and orally when ingested during grooming (Ebeling et al. 1974). While designed for a variety of insects, these dus ts proved more effective on the larger cockroaches (Periplaneta ) and offered no real control of the German cockroach (Reid et al. 1990). The next group of insecticides used for German cockroach control was the chlorinated hydrocarbons such as DDT and chlordane. These compounds were very effective in both spray and dust form with long lasting residual effects. This class of chemical acted on the sodium channel causing re petitive firing. Physiological resistance to chlordane was first reported in 1951 and rapidly spread throughout the United States (Grayson 1964). Although chlorinated hydroc arbons have the differing modes of action, they all affected the nervous system. Worldwide resistan ce was documented to not just chlordane but many chlorinated hydrocarbons w ithin 10 years of the first report (Grayson 1954, Matsumura 1975). Due to environmental issues, the Environmental Protection Agency (EPA) cancelled the use of chlorinate d hydrocarbons in the late 1970s (Reid, et al, 1990). In the early 1960s, first organophosphates and, shortly thereafter, carbamates began replacing chlorinated hydrocarbons (Siegfri ed et al. 1990, Cochran 1982). Examples of organophosphates are diaznon, chlorpyrifos, and malathion; while examples of carbamates are bendiocarb and propoxur. Both of these classes of chemical were used primarily in spray formulations, inhibited acet ylcholinesterase, and were fairly toxic to vertebrates (Matsumura 1975). Just as with chlorinated hydrocarbons, German

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7 cockroaches developed resistance to both thes e classes of chemical; however, the time it took for them to develop resistance was less th an five years (Siegfried et al. 1990). For health issues, EPA no longer allows mo st of these chemicals to be used. The pyrethroids became popular for cockroach control in the 1970s. Prior to this time, pyrethrina natural pesticide derived from chrysanthemums in the Family Compositaehad been used to augment inor ganic pesticides. In the late 1950’s, pyrethroidsthe synthetic analogue of pyr ethrinsbecame commercially available; however these “Type I” pyrethr oids were not photo stabile and many cockroaches were able to metabolize the chemical and fully rec over. It was the “Type II” pyrethroids that became popular in the 1970s. These were phot o stabile, used in spray formulation, and had excellent residual effects against cockroaches. All pyrethroids acted on the sodium channe l to interfere with the transmission of nerve impulses. As with all the other classes of chemical, German cockroaches developed resistance pyrethroids (Cochran 1994). It was fou nd that cockroaches resistant to DDT also had inherent resistance, or cr oss-resistance, to many pyrethroids Scott and Matsumura 1982). Pyrethroids ar e still used today; however, they are used as flushing agents to drive cockroaches out of their harborag e (Fuchs 1988). Throughout the late 19802 and 1990s, many new classes of chemical have come on the market for cockroach control. The sa fest are Insect growth regulators, more specifically, juvenile hormone analogs such as hydroprene and pyriproxifen. These chemicals mimic insect juvenile hormone, a ffecting the endocrine balance and causing developmental disturbances such as molting inhibition, morphogenetic abnormalities, longer developmental time, and reproducti on suppression (King and Bennett 1989, Reid

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8 1994). While juvenile hormone analogs have application in the field, they have their limitations. They have no ability to suppr ess a population quickly (Zemen, et al 1991, Koehler and Patterson 1991) and unless more than 80% of the population is strongly affected by the juvenile hormone analog, vi able young can still be produced (Reid et al. 1994). Today, insecticidal gel baits are the main method of German cockroach control in the United States (Wang et al. 2004). Gel bait s contain many different chemical classes: Neonicotinoids such as imidicloprid and thiamethoxam, the avermectins such as abamectin, the aminidinohydrazones like hydromethylnon and sulfonomides such as sulfluramid, the phenyl pyrozoles such as fipronil, and the oxadiazines such as indoxacarb. With the exception of oxadiazine s, which have only appeared commercially in the past year, the chemical classes b ecame commercially available in the 1990s. One thing all gel baits have in common is that th ese newer active ingredients are highly toxic and take anywhere from hours to days to cause mortality (Scott 1991, Scott and Wen 1997, Koehler et al. 1991, Appel and Benson 1992) The benefit of slow acting active ingredients is that significant mortality occurs in cockroaches that di d not directly ingest the gel bait. This secondary mortality occurs through three different pathways. The first is from trampling: When contaminated co ckroaches return to the harborage, they defecate and orally secrete toxic metabolites Secondary mortality occurs when other cockroaches travel through these contaminat ed secretions and unwittingly ingest them during grooming (Durier and Rivault 2000b, Kopanic and Schal 1997). The second pathway is mortality from coprophagy: Since first stage nymphs survive almost exclusively on the feces of older nymphs a nd adults, when feces are contaminated by

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9 toxic metabolites, secondary mortality en sues (Kopanic and Schal 1999). The third pathway is from cannibalism: Even with pl enty of food, German cockroaches typically consume dead or dying cockroach conspecifi cs. During cannibalism of contaminated cockroaches, toxic metabolites are ingested a nd secondary mortality occurs (Gahlhoff et al. 1999, Durier and Rivault 2000b). Another benefit of the high toxicity of these new active ingredients regards physiologically resistance. When cockroaches consume gel bait, the ac tive ingredient is toxic enough to deliver a lethal dose in one meal. Since physiological resistance develops when sub-lethal doses of an activ e ingredient are metabolized, the heightened toxicity of these new active ingredients s hould prevent resistance from occurring (Wang et al. 2004). Indeed, in the past ten years, gel baits ha ve been the most common and effective control measure (Wang et al. 2004) Recently, there have been reports of German cockroach resistance to the active ingr edient fipronil; however, this resistance is apparently due to a cross resistance from cy clodienes and not severe enough to date to compromise the efficacy of gel baits c ontaining fipronil (Scott and Wen 1997, Holbrook et al. 2003). Even with the effectiveness and high toxicity of gel ba its and lack of significant physiological resistance, control failures have been reported. The first report occurred in the early 1990’s (Silverman and Bieman 1993) This isolated strain of German cockroach, T-164 strain, was found to be gl ucose averse. The active ingredients contained within the gel baits we re still toxic to this strain; however, they refused to eat the gel bait because it contained glucose (Silverman and Bieman 1993, Silverman and Ross 1994). This feeding deterrence in T-164 strain was so pronounced, they refused to

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10 ingest glucose even after a nine day st arvation period (Silverman and Selbach 1998). Recently, greater numbers of control failures have been reported. In a few cases, the wild cockroaches were harvested and subsequently reared in laboratory settings. Preliminary studies show some similarity to T-164 strain; specifically, they are still susceptible to the active ingredients contained within gel baits but they refuse to consume the bait. Unlike the T-164 strain, these strains are not glucos e averse. While the mechanism causing this bait aversion is currently unknown, it is obvious ly behavioral in na ture. Behavioral aversion is defined as evolved behaviors that allow an insect to survive in an otherwise lethal environment and often involves s timulus-dependent mechanisms such as repellency, irritation (Sparks et al. 1989), or in this case, feed ing deterrence. In the past, behavioral resistance was glossed over a nd considered far less important than physiological resistance (Sparks et al. 1989, Ro ss 1997). Today, more research is being done on behaviorally averse strains of German cockroach to try to find a way to overcome this resistance and again effectivel y manage German cockroach populations in the field.

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11 CHAPTER 3 EVALUATION OF FEEDING DETTERRENCE IN SIX INSECTICIDAL GEL BAITS AND MORTALITY IN A FIELD STRAIN OF GERMAN COCKROACH, Blattella germanica (L) Introduction The German cockroach, Blattella germanica L., is and has been a very important urban pest worldwide (Cornwell 1968, Abd-Elgha far et al. 1990). As such, it has a long and varied history of control. Early met hods of control utilized insecticidal spray formulations that began with the chlorinated hydrocarbons, progressed to organophosphates, carbamates, and then pyrethr oids. All suffered th e same fate: after about 5 years of intensive use, German cock roaches developed physiological resistance to the chemicals (Cornwell 1976). In the late 1970’s through th e 1980’s, strategies involving the use of insect growth regulators and biological contro l agents in the form of f ungal pathogens were employed. Nothing provided adequate measures of cont rol until toxic baits containing new classes of chemical compounds came on the market. While these classes all had differing modes of action, one thing they all had in common was th at they were toxic enough, when ingested orally, to deliver a lethal dos e of active ingredient in one meal (Wang et al. 2004). For ingestion to occur, the ba it had to be palatable enough to compete with other food sources. It was believed this combination of palatability and toxicity would make development of physiological resist ance less likely (Wang et al. 2004). In the early 1990’s, there were reports of control failures in the field. By 1993, Silverman and Bieman determined that th ese failures were not due to physiological

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12 resistance but due to behavioral resistance. They isolated the first bait averse strain of German cockroach. In this case, the aversi on was to glucose contai ned within the bait matrix. Since then, other strains exhibi ting glucose aversion have been isolated (Silverman and Ross 1994). In addition, many st rains have been isolated that are not simply glucose averse, but averse to one or more inert ingredients in the bait matrix (Silverman and Ross 1994, Ross 1997a, Wang et al. 2004). Silverman and Ross (1994) discovered a surprising level of genetic variabil ity in the strains they studied and Wang et al. found significantly different levels of c onsumption and mortality in the two strains they studied. Though there is variability among averse strains of cockroach, one thing remains the same: In all strains, decreased consumption of gel bait causes a decrease in mortality, possibly leading to control failure. Daytona field strain was collected from Daytona, FL after reports of control failure using traditional baits. The purpose of this study was to determine if gel baits caused feeding deterrence in Daytona field stra in, to determine the effect of consumption on mortality, and to determine if Daytona field strain is a bait averse strain of cockroach. Materials and Methods Insects. Orlando susceptible strain and Dayt ona field strain German cockroaches were obtained from laboratory colonies main tained at the urban entomology laboratory, University of Florida, Gainesville, FL. R earing containers and ha rborages for Orlando susceptible strain cockroaches were as described by Koehler et al. (1994) with the exception that the harborages contained with in the acrylic rack were looped cardboard sections (14 by 13 cm). Rat food (Purina Laboratory Rodent Chow, no. 5001, Ralston Purina, St. Louis, MO) and water was supplied ad libitum

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13 Rearing containers for Daytona field strain cockroaches were glass jars (7.57 liter, 22 cm diameter) greased on the inner rim w ith a petroleum jelly/ mineral oil (2:3) mixture to prevent cockroach escape. Jars were covered with co tton cloth and secured with rubber bands. Dog food (Purina One Puppy Growth and Development, Nestl Purina PetCare Company, St. Loui s, MO) and water was supplied ad libitum. Environmental conditions for both rearing rooms were 26oC and 55% relative humidity RH, with a photoperiod of 12:12 (L:D) h. Insectide baits. The following gel bait products were tested: Maxforce (2.15% Hydramethylnon, Bayer Environmental Science, Montvale, NJ), Avert (0.05% Avermectin, Whitmire Microgen Research La boratories, Inc, St. Louis, MO), DPXMP062-411a (0.6% Indoxacarb, Dupont Cr op Protection, Newark, DE), Advion Cockroach (0.6% Indoxacarb, Dupont Crop Protection, Newark, DE), Maxforce FC (0.01% Fipronil, Bayer Environmental Science, Montvale, NJ), Maxforce FC Select (0.01% Fipronil, Bayer Environmental Science, Montvale, NJ). Ge l bait (140 to 150 mg) was deposited from a syringe onto a piece of low nitrogen weighing paper (57 by 57 mm; Fisherbrand, Fisher Scientific Company, USA). Preference and consumption assay. Cockroaches were anesthetized with CO2 for 2 min and sorted through stacked 2.36 mm (No. 8, Fisher Scientific Company, Pittsburgh, PA) and 2.00 mm (No. 10, Fisher Scientific Company, Pittsburgh, PA) standard sieves. Cockroaches retained by the 2.00 mm sieve we re placed in a greased glass holding jar (3.79 liter, 17.5 cm diameter) containing har borage, dog food and water. Ninety percent of retained cockroaches were 2nd stage nymphs weighing 190.24 4.53 (Orlando susceptible strain cockroach) and 216.44 3.06 (Daytona field strain cockroach). To

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14 recover from the effects of CO2, cockroaches were held for 48 to 72 h before placement into foraging arenas. After the holding period, 50 cockroaches were aspirated and placed into a greased foraging arena containing wate r and harborage. Foraging arenas were clear plastic sweater boxes (26.5 by 9.5 by 19 cm, Pioneer Pl astics, Dixon, KY). Water was provided by a plastic vial (33 by 16 mm diameter) with a cotton stoppe r. Due to the ability of nymphs to enter inside the corrugations of cardboard, an index card (76.5 by 28.5 mm) folded in half lengthwise and secured with a staple was used for harborage. This process was repeated until there were seven arenas per strain. Cockroaches were starved for 24 h. At the end of the starvation period, two preweighed deposits of gel bait and two pre-weig hed pieces of dog food were added to each arena on a piece of weighing paper. One of the gel bait placements and one of the dog food placements were used as moisture loss stan dards. Both moisture loss standards were placed inside individual souffl cups (29.57 ml; Solo cup company, Urbana, IL.) and cups were covered with organdy and secure d with a rubber band to prevent cockroach entry. After 24 h, both food sources and both mo isture loss standards were reweighed and food sources were returned to the arena. Mortality was recorded 4 d after gel bait placement. Experimental set up was a ra ndomized complete block design of six treatments and one control. Experiment was a randomized complete block design with six gel baits and an untreated dog food control replicated ei ght times per strain using a total of 5,600 cockroaches. Data analysis. Gel bait consumption was calcu lated using the following formula: Consumption = A [A x (B D / B) C]

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15 where; A = Pre-consumption weight of exposed bait or food, B = Pre-consumption weight of comparable moisture loss standa rd, C = Post-consumption weight of exposed bait or food, and D = Post-consumption weight of comparable moisture loss standard. Consumption of gel bait product for each stra in and each treatment was analyzed via Student’s t -test (P, 0.005; [SAS Institute, 2001]). Amount of active ingredient consumed was calculated by multiplying the percent active ingredient in gel baits by the amount of gel bait consumed. Mortality data was corrected using Abbott’ s correction (Abbott, 1925) and percentages were arcs ine-root transformed before analysis. Both active ingredient per cockroach body weight and mortality were analyzed by Analysis of Variance and means separated by SNK (P, 0.005; [SAS Institute, 2001]). Results Within a few minutes of introducing f ood sources to the arenas, nymphs fed on either dog food or gel bait. Orlando normal strain had no significant preference for dog food control or dog food choice, indicating no location bias with in the arena (Table 3-1). There was no active ingredient in the control; therefore, no mortality occurred. All control cockroaches survived until the end of the experiment. Consumption of Maxforce gel bait was not significan tly different than dog food c onsumption. Mean amount of active ingredient consumed was 1009.5 ng per mg cockroach body weight and resulting mortality was 58%. Consumption of Avert ge l bait was not significantly different from dog food consumption. Mean amount of activ e ingredient consumed was 22.3 ng per mg cockroach body weight and resulting mort ality was 66%. Consumption of DPX-MP062411a gel bait was not significantly different from dog food consumption. Mean amount of active ingredient consumed was 271.9 ng per mg cockroach body weight and resulting mortality was 87%. Consumption of Advion ge l bait was not significan tly different from

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16 dog food consumption. Mean amount of ac tive ingredient consumed was 314.2 ng per mg cockroach body weight and resulting mortality was 88%. Consumption of Maxforce FC gel bait was not significantly different from dog food consumption. Mean amount of active ingredient consumed was 4.2 ng per mg cockroach body weight and resulting mortality was 81%. Consumption of Maxfor ce FC Select gel bait was not significantly different from dog food consumption. Mean amount of active ingred ient consumed was 4.6 ng per mg cockroach body weight a nd resulting mortality was 92%. Amount of active ingredient consumed per cockroach body wei ght significantly differed among products. Orlando normal stra in cockroaches consumed significantly more active ingredient from Maxforce gel ba it than all other products. The order of preference based on consumption of activ e ingredient was Maxforce >Advion = DPXMP062-411a = Avert = Maxforce FC = Maxf orce FC Select. Resulting mortality significantly differed among products. Highe st mortalities occu rred with Advion Cockroach, Dupont Formula 411a, Maxforce FC and Maxforce FC Select. Order of mortality was Advion Cockroach = Dupont Formula 411a = Maxforce FC = Maxforce FC Select >Avert = Maxforce > Control. Daytona field strain had no significant preference fo r dog food control or dog food choice, indicating no location bi as (Table 3-2). There was no active ingredient in the control; therefore, resu lting mortality did not occur. A ll control cockroaches survived until the end of the experiment. Consumpti on of Maxforce gel bait was significantly less than dog food consumption yielding a rati o of 1:5.2 (Maxforce:dog food), indicating feeding deterrence to Maxforce gel bait. M ean amount of active ingredient consumed was 326.2 ng per mg cockroach body weight and resulting mortality was 9%.

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17 Consumption of Avert gel bait was significan tly less than dog food consumption yielding a ratio of 1:9.4 (Avert:dog food), indicating fe eding deterrence to Avert gel bait. Mean amount of active ingredient consumed wa s 3.1 ng per mg cockroach body weight and resulting mortality was 20%. Consum ption of PX-MP062 411a gel bait was not significantly different than dog food consump tion. Mean amount of active ingredient consumed was 212.3 ng per mg cockroach body weight and resulting mortality was 49%. Consumption of Advion Cockroach gel bait was significantly greater than dog food consumption yielding a ratio of 1.8:1 (Advi on: dog food), indicating preference for Advion gel bait. Mean amount of active ingredient consumed was 450.3 ng per mg cockroach body weight and resulting mortalit y was 79%. Consumption of Maxforce FC gel bait was significantly le ss than dog food consumpti on yielding a ratio of 1:4.6 (Maxforce FC:dog food), indicating feeding de terrence to Maxforce FC gel bait. Mean amount of active ingredient consumed wa s 2.5 ng per mg cockroach body weight and resulting mortality was 15%. Consumption of Maxforce FC Select gel bait was significantly greater than dog food consumption 5.3:1 (M axforce FC Select:dog food), indicating preference for Maxfor ce FC Select gel bait. Mean amount of active ingredient consumed was 5.9 ng per mg cockroach body we ight and resulting mortality was 82%. Amount of active ingredient consumed per cockroach body wei ght significantly differed among products. Daytona field strain cockroaches consumed significantly more active ingredient from Advion gel bait than all other product s. The order of preference based on consumption was Advion > DPX-MP 062-411a = Maxforce > Avert = Maxforce FC = Maxforce FC Select. Resultant mortal ity significantly differed among products. Highest mortalies occurred with Advion gel bait and Maxforce FC Select gel bait while

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18 lowest mortality occurred with Maxforce FC gel bait. Order of mortality was Advion = Maxforce FC Select > DPX-MP062-411a > Maxforce > Avert = Maxforce FC > Control). Discussion Orlando susceptible strain co ckroaches have never shown feeding deterrence to any commercial gel bait formula tion. When Orlando susceptibl e strain cockroaches were simultaneously given a choice of six gel ba its, all baits were consumed equally (Silverman and Liang 1999). A significant consumption difference between gel baits would indicate feeding preference or feed ing deterrence. In my study, preference or feeding deterrence was determined by compar ison of consumptions between gel bait and dog food. Similar to Silverman and Lia ng’s study, Orlando susceptible strain cockroaches also exhibited no feeding deterren ce to six gel bait fo rmulations, including two formulations that have not been commonly used. A factor to take into account when perf orming a choice study is bait placement because location can affect consumption. German cockroaches locate food by random searching, so the more available and easily accessib le the bait, the more likely it is to be ingested (Rust and Reierson 1981). In my study, when Orlando susceptible strain was given a choice between two pieces of dog food th at had been placed equidistant from the harborage, both food sources were consumed equally, indicating no location bias in the experimental arena. Gel baits containing hydramethylnon have been a common treatment for susceptible strain cockroaches, do not cause feeding deterrence, and cause mortality through time. Adult male insecticide suscepti ble German cockroaches exposed for 3 d to Maxforce gel bait (2.15% hydramethylnon), read ily consumed the gel bait and exhibited

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19 no feeding deterrence; LT50 value for the uncontaminated gel bait was 4.1 d (Appel 2004). Scott (1991) determined LT50 was 76 h for adult male CSMA susceptible strain cockroaches fed bait containing 1.56% hydram ethylnon; Koehler and Patterson (1991a) determined LT50 was 4.5 d for adult male Orlando susceptible cockroaches fed bait containing 1.0% hydramethylnon. My study also showed no feeding deterrence to Maxforce (2.15 % hydramethylnon) in Orlando su sceptible strain cockroaches and the resultant 4 d mortality, was 58%; which was sim ilar to the mortalities reported above. When dealing with an averse strain of co ckroach, it is not the active ingredient that causes feeding deterrence but the gel bait matr ix. Adult male T-164 glucose averse strain cockroaches, given a choice between dog f ood and 2.0% hydramethylnon gel bait with glucose and without glucose, e xhibited feeding dete rrence to the gel ba it with glucose and not to the gel bait without gl ucose (Silverman and Liang 1999). Gel bait without glucose caused 14 d mortality of approximately 90%. Similarly, adult male Dorie and Cincy bait averse strains of cockroach, given a choi ce between rat chow a nd Maxforce gel bait (2.15% hydramethylnon), exhibited feeding dete rrence to the gel bait, resulting in only 10.0% and 33.3% 4 d mortality rates respectiv ely (Wang et. al. 2004). My study showed Daytona field strain exhibited feeding dete rrence to Maxforce gel bait, consuming 5 times less bait than food. Resulting 4 d mortal ity was 16%, which was similar to that of Dorie strain bait-averse cockroach. Gel baits containing abamectin have also been a common treatment for susceptible strain cockroaches. Koehler et al. (1991b) determined that abamectin caused feeding deterrence at concentrations above 0.500% The concentration of abamectin in commonly used gel bait is much lower than this; therefore, the gel baits containing

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20 abamectin do not cause feeding deterrence a nd cause cockroach mortality through time. Adult male insecticide susceptible German cockroaches exposed for 3 d to uncontaminated Avert gel bait (0.05% abamect in) readily consumed the gel bait and exhibited no feeding deterrence; LT50 for the Avert gel bait was 1.05 d (Appel 2004). Mixed sex and stage Orlando susceptible co ckroaches fed gel bait containing 0.05% abamectin readily consumed the gel ba it exhibited no feeding deterrence; LT50 values were 1.6 d for nymphs and 1.7 d for adult male s (Koehler et al. 1991b). Mixed sex and stage Navy 3 susceptible cockroaches in a ch oice test between gel bait containing 0.01% abamectin and dog food readily consumed the gel bait and exhibited no feeding deterrence; 14 d mortality rates were appr oximately 90% for adult males and 95% for small (<9 d old) nymphs (Ross1993). Because Av ert gel bait was consumed as readily as dog food by Orlando susceptible strain cockroach, my study also showed no feeding deterrence to Avert (0.05 % abamectin); however, my approximate LT50 for 2.15% hydramethylnon would be about 1 d more th an the value reported by Koehler and Patterson. Again, averse strains of cockroach exhibit feeding deterrence to the gel bait, not the active ingredient. Adult male Dorie and Cinc y bait averse strains of cockroach, given a choice between rat chow and Avert gel bait did not readily consume the gel bait and exhibited feeding deterrence, resulting in 4 d mortality rates of 69.3% and 0.0% respectively (Wang et al. 2004). In my study, Daytona field strain consumed more than 10 times less Avert gel bait than food, exhi biting feeding deterrence. Resulting 4 d mortality was 20%, which was similar to th e mortality of Cincy bait-averse strain cockroach.

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21 Fipronil is a fast acting chemical that is used in gel baits for treatment of susceptible strain cockroaches. These gel baits do not cause feeding deterrence and cause cockroach mortality through time. Adult male insecticide susceptible German cockroaches exposed for 3 d to uncontamina ted Maxforce FC gel bait (0.01% fipronil) readily consumed the gel bait and exhibited no feeding deterrence; LT50 for the uncontaminated gel bait was 2.1 d (Appel 2004). Adult male Orlando susceptible strain cockroaches, given a choice be tween dog food, 0.03% fipronil ge l bait with glucose and 0.03% fipronil gel bait without glucose, readily consumed bo th gel baits and resultant 14 d mortality was approximately 60% with an LT50 of 4.5 d (Silverman and Liang 1999). Adult male JWAX susceptible strain cockro aches, given a choice be tween rat chow and Maxforce FC (0.01% fipronil), readily consumed gel bait and had resultant 4 d mortality of 100% (Wang et al. 2004). In my st udy, Orlando susceptible strain consumed Maxforce FC and Maxforce FC Select (0.01% fipronil) gel baits as readily as dog food. Resultant mortality was 85% for Maxforce FC and 92% for Maxforce FC Select, which was similar to the susceptible strain used in Appel’s study and JWAX strain used in Wang et al.’s study. For averse strains of cockroach, it is th e gel bait that causes feeding deterrence. Adult male T-164 glucose averse strain co ckroaches, given a choice between dog food, 0.03% fipronil gel bait with gl ucose and 0.03% fipronil gel ba it without glucose, readily consumed the gel bait without glucose and e xhibited feeding deterrence to the gel bait with glucose; Resulting 14 d mortality for the bait with glucose was approximately 55% and the 14 d mortality for the bait without glucose was approximately 90% (Silverman and Liang 1999). When adult male Dorie a nd Cincy bait averse strains of cockroach

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22 were given a choice between Maxforce FC (0.01% fipronil) and rat chow, Dorie bait averse strain cockroach readily consumed the gel bait whereas Cincy strain exhibited feeding deterrence to the gel bait; resulting 4 d mort alities were 100.0% and 16.7% respectively (Wang, et. al. 2004). In my st udy, Daytona field strain consumed 21.5 times less Maxforce FC (0.01% fipronil) than dog food and mortality rates were 15%, which was similar to Cincy bait averse strain cockroach; However, Daytona field strain consumed 19 times more Maxforce FC Select gel bait (0.01% fipronil) than dog food. Resulting 4 d mortality was 82%, which was si milar to the 92% 4 d mortality of Orlando susceptible cockroach. Indoxacarb is a new chemical, class oxadi azine, which is biologically activated within the insect midgut. It has not been shown to cause f eeding deterrence and has been shown to cause cockroach mortality through time (Appel 2003). Adult male American Cyanamid susceptible strain German cockroaches had an LT50 value of .068 d to .025% indoxacarb gel bait (Appel, 2003). In my st udy, Orlando susceptible strain cockroaches readily consumed both DPX-MP062-411a ( 0.6% indoxacarb) and Advion gel bait. Resulting mortality for both gel baits was 87%. Because of the newness of indoxacarb, th ere are no published studies with bait averse cockroaches. In my study, Daytona field strain readily consumed DPX-MP062411a and consumed two times more Advion than dog food. Resulting mortalities were 49% for DPX-MP062-411a, which was less than Orlando susceptible strain cockroaches and 79% for Advion, which is similar to Orlando susceptible strain cockroaches. Orlando susceptible strain exhibited no feeding deterr ence or preference to any food source. One would expect gel bait to be preferred in order for it to be effective,

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23 especially in the field. It must be noted that this study had no location bias, whereas bait placement in the field has location bias. A dditionally, Since Orlando susceptible strain cockroaches have been reared in the laboratory since 1947 (Koehler, et. al. 1994), it is possible they have lost the ab ility to discriminate between food sources. Even with the lack of preference, Orlando susceptible st rain consumed enough gel bait to cause significant mortality for all products tested. Whereas on ly the gel baits specially formulated for bait averse cockroaches (A dvion Cockroach Gel and Maxforce FC Select) had mortality rates above 50% at 4 d for Dayt ona field strain cock roach, indicating that Daytona field strain is, in fact, a bait-averse strain of cockroach. Discovery of a bait-averse strain of cockro ach in Florida, so far from the strains found around Ohio, indicates that bait aversion is a more widespread phenomenon than once believed. This study determined consum ption of gel bait was positively correlated with mortality in German cockroaches. The ba it-averse strains in both this study and that of Wang et al. (2004) demonstrat ed that three bait-averse st rains find reformulated baits palatable; thus, feeding deterrence can be overc ome by reformulating bait matrices. It is unknown if bait averse strains of German cockroach also have physiological resistance; therefore, simply reformulating gel bait ma trices may not be enough for control in the future.

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24Table 3-1. Mortality (4 d) a nd consumption of gel bait, dog food, and AI for 50 Orlando susceptible strain German cockroaches 24 h after gel bait placement. Product Consumption (mg) Student’s t -test ng AI consumed per Gel Baita Dog food df t value P value mg body weight % Mortality Control 9.34 4.37 9.27 3.31 14 0.03 0.9742 N/A 0.00 0.00c Maxforce 10.16 4.92 9.81 2.07 14 0.19 0.8540 1009.52 172.83a 58.33 4.76b Avert 9.63 2.12 11.13 3.51 14 -1.03 0.3187 22.25 1.74c 66.22 4.24b DPX-MP062 411A 9.81 3.97 6.85 3.27 14 1.24 0.2368 271.89 38.93bc 87.24 2.58a Advion Cockroach 11.34 3.82 8.68 4.73 14 1.63 0.1259 314.23 37.44b 87.63 2.49a Maxforce FC 9.14 3.63 13.46 8.56 14 -1.31 0.2097 4.16 060c 81.16 7.12a Maxforce FC Select 9.92 3.75 5.56 5.10 14 1.95 0.0720 4.60 0.61c 91.81 1.17a aControl was a piece of dog food. Student’s ttest (P > 0.005, [SAS Institute, 2001]). Means in a column followed by the same letter are not significan tly different (P > 0.05; Student -Newman-Keuls sequential range test [SAS Institute, 2001]).

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25Table 3-2. Mortality (4 d) a nd consumption of gel bait, dog food, and AI for 50 Daytona field strain German cockroaches 24 h a fter gel bait placement. Product Consumption (mg) Student’s t -test ng AI consumed per Gel Baita Dog food df t value P value mg body weight* % Mortality Control 9.04 5.31 9.60 5.66 14 -0.20 0.8426 N/A 0.00 0.00e Maxforce 3.28 2.09 17.06 3.70 14 -9.17 <0.0001 326.21 73.42b 9.11 2.69d Avert 1.34 1.70 12.56 4.89 8.66 -6.14 0.0002 3.08 1.39c 20.34 3.46c DPX-MP062 411A 7.66 2.85 10.60 4.88 14 -1.47 0.1628 212.33 27.95b 48.97 4.77b Advion Cockroach 6.24 6.34 3.51 2.65 9.38 5.24 0.0005 450.27 62.12a 78.91 1.93a Maxforce FC 2.91 1.68 13.51 4.95 8.6 -5.74 0.0003 2.45 1.08c 15.04 2.09cd Maxforce FC Select 12.86 8.59 2.44 1.53 7.44 3.38 0.0107 5.89 1.41c 82.37 3.70a aControl was a piece of dog food. Student’s ttest (P > 0.005, [SAS Institute, 2001]). Means in a column followed by the same letter are not significan tly different (P > 0.05; Student -Newman-Keuls sequential range test [SAS Institute, 2001]).

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26 CHAPTER 4 ORAL TOXICITY OF INDOXACARB AND SECONDARY MORTALITY FROM NECROPHAGY IN A SUSCEPTIBLE STRAIN AND A FIELD STRAIN OF GERMAN COCKROACH, Blattella germanica (L) Introduction The German cockroach, Blattella germanica L., has been an important pest of all urban dwellings worldwide (Cornwell 1968, AbdElghafar et al. 1990). Over the years, attempts at control have largely shifted from applying residuals and sprays to placement of toxic baits (Reierson 1995). This was due in part to he ightened consumer awareness regarding pesticides and a ge neral trend to reduce pestic ide application (Kopanic and Schal 1997). Unlike sprays, baits are either contained in a protective unit or are placed in areas close to the harborages and foraging areas. This highly specialized application process has the advantage of targeting the pest spec ies while simultaneously allowing for use of less chemical (Kopanic and Schal 1997). Th ere are many active ingredients currently employed in baits used for German cockroach co ntrol, but bait is only effective if it is palatable and the active ingred ient is not a feeding deterr ent (Appel 1990). Current active ingredients are toxic enough to deliver a leth al dose in one meal (Wang et al. 2004). However, they act slowly enough to allow time for the cockroach to return to the harborage after feeding. Since German cockroaches are gregar ious and not social, it was once thought this was of no consequence. However, secondary transmission in the German cockroach has been well documented an d proved to be an important factor in control. Kopanic and Schal (1999) showed si gnificant mortality in first and second stage

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27 nymphs due to the ingestion of contaminat ed feces. Gahlhoff et al. (1999) showed significant mortality of a dult and nymphal cockroaches due to consumption of contaminated conspecifics. There is a new class of chemical availabl e in toxic baits, oxadiazines. Indoxacarb, the only current oxadiazine, is bioactivated (Wing 1999). The purpose of this study was to determine if indoxacarb was a feeding de terrent, determine the oral toxicity of indoxacarb, and evaluate seconda ry mortality from necrophagy in a susceptible strain and a field strain of German cockroach. Materials and Methods Insects. Orlando susceptible strain and Dayt ona field strain German cockroaches were obtained from laboratory colonies main tained at the urban entomology laboratory, University of Florida, Gainesville, FL. R earing containers and ha rborages for Orlando susceptible strain cockroaches were as described by Koehler et al. (1994) with the exception that the harborages contained within the acrylic rack we re looped cardboard sections (14 by 13 cm). Rat food (Purina Laboratory Rodent Chow, no. 5001, Ralston Purina, St. Louis, MO) and water was supplied ad libitum Rearing containers for Daytona field cock roaches were glass ja rs (7.57 liter, 22 cm diameter) greased on the inner rim with a pe troleum jelly/ mineral oil (2:3) mixture to prevent cockroach escape. Jars were covere d with cotton cloth and secured with rubber bands. Dog food (Purina One Puppy Growth a nd Development, Nestl Purina PetCare Company, St. Louis, MO) and water was supplied ad libitum. Environmental conditions for both rearing rooms were 26oC and 55% relative humidity RH, with a photoperiod of 12:12 (L:D) h.

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28 Oral toxicity assay. Adult male cockroaches were pulled without CO2 and placed into a greased plastic holding container ( 0.946 liter, 140 by 113 mm diameter) containing harborage and water. Cockro aches were starved for 24 h. During the starvation period, one breadcrumb was placed in the bottom of a cell culture cluster well (COSTAR Model number 3524, Corning Incorporated, Corning, NY). The breadcrumb was treated with 1 l of chemical solution. Solutions were derived from Termidor (9.1 % fipronil, Bayer Environm ental Science, Montvale, NJ), diluted in water and technical grade indoxacarb (56.2%, Dupont Crop Protection, Newark, DE), diluted in acetone. Each chemical had its own set of culture clusters The solution treated breadcrumb was allowed to dry for at least 4 hrs. The breadcrumb was then treated with 1 l of 10% sucrose soluti on and allowed to dry. At the end of the starvation period, indi vidual cockroaches were removed from the holding container and placed into souffl cups (59.147 ml, Polar size “G”, Polar Plastique, St-Laurent, QUE) containing treated breadcrumbs and moistened cotton balls (~5 mm diameter). Cup lids were immediatel y secured to prevent cockroach escape. Cockroach weights were recorded per treatment and concentration. Cups were set aside for 24 h to allow cockroaches time to feed. At the end of the feeding period, cockroaches that had consumed the entire breadcrumb, were released into a grea sed sweater box (26.5 by 9.5 by 19 cm, Pioneer Plastics, Dixon, KY). Each sweater box contai ned a plastic tube ( 33 by 16 mm diameter) of water with a cotton plug, and cardboard sections (14 by 13cm ) folded in half lengthwise secured with a staple for harborag e. There were separate arenas for each treatment concentration.

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29 Mortality was counted 4 d after end of feeding period. All moribund cockroaches (defined as an inability to walk) were considered dead. Necrophagy assay. Twenty adult male cockro aches were pulled without CO2 and placed into a foraging arena, which was a greased clear plastic sweater box (26.5 by 9.5 by 19 cm, Pioneer Plastics, Dixon, KY). Each arena contained a plas tic tube (33 by 16 mm diameter) of water with a cotton plug, and cardboard sections (14 by 13cm) folded in half lengthwise secured with a staple for harborage. This process was repeated until there were three arenas per strain. Cockroaches were starved for 24 hours. At the end of the starvation peri od, cockroaches were fed thawed 2nd-3rd stage nymphal German cockroaches that had previ ously ingested Formula 411a gel-bait (0.6% Indoxacarb, Dupont Crop Protecti on, Newark, DE) or Maxforce FC gel-bait (0.05% Fipronil, Bayer Environmental Science, Mont vale, NJ). Upon death, nymphs were frozen to preserve freshness. Control nymphs were gathered from the rearing containers and frozen. Initially, each arena received ten a ppropriately treated nym phs. Thereafter, each arena received seven nymphs daily for four days. Number of nymphs cannibalized was reco rded per day and uneaten nymphs were removed from arena. Moribund cockroaches un able to walk were considered dead. Evaluation was ended on day 5. Experiment was a randomized complete bloc k design with six replicates per strain per treatment for a total of 240 cockroaches. Statistical analyses. Percent consumption of breadcrumbs was analyzed using Analysis of variance and Student’s ttest (P<0.05; SAS Instit ute 2001). Lethal dose values were determined using probit analys is (SAS Institute 2001). Necrophagy and

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30 resultant mortality were analyzed via Analysis of variance and means separated with Student Newman Keuls (P<0.05; SAS Institute 2001). Results Oral toxicity assay. After 24 h in a souffl cup with only a treated breadcrumb as a food source, Orlando susceptible strain cockro aches ate 96% of all treated and untreated breadcrumbs whereas Daytona field strain ate 85% of untreated and fipronil treated breadcrumbs and 93% of indoxacarb treated breadcrumbs. Feeding deterrence in Orlando susceptible strain cock roaches was not caused by incr easing the concentration of fipronil or from increasing the concentrati on of indoxacarb (Fig 4-1). Feeding deterrence in Daytona field strain cockroaches was not caused by increasing the concentration of fipronil or from increasing the conc entration of indoxacarb (Fig. 4-2). Cockroaches that consumed the breadcrumb were included in an oral toxicity assay. Orlando susceptible stra in cockroaches had LD50 values of 0.072 ng per mg body weight and LD90 values of 0.108 ng per mg body weight for fipronil and LD50 values of 1.312 ng per mg body weight and LD90 values of 4.104 ng per mg body weight for indoxacarb (Table 3-2). Daytona fiel d strain cockroaches had LD50 values of 0.656 ng per mg body weight and LD90 values of 4.063 ng per mg body weight for fipronil and LD50 values of 4.653 ng per mg body weight and LD90 values of 18.045 ng per mg body weight for indoxacarb. Resistan ce ratios at the LD50 level/ LD90 level for Daytona field strain were 9.4/ 36.9 for fipronil and 3.5/ 4.4 for indoxacarb. Necrophagy assay. Upon placing treated nymphs into the arena, both strains of cockroach investigated the bodies, but no im mediate consumption was observed. When given a choice, Orlando susceptible strain co ckroaches consumed a constant amount of control nymphs (df = 4, F= 2.07, P =0.1152), ranging from 1.1 to 2.4 nymphs per day

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31 (Table 4-2), for a total 5 d consumption of 8.9 nymphs. Orlando susceptible strain cockroaches consumed 1.2 fipronil treated nymphs on day one of the choice experiment and consumption decreased significantly dur ing days two through five, ranging from 0.1 to 0.4 nymphs per day for a total 5 d consum ption of 2.1 nymphs. Orlando susceptible strain cockroaches consumed a constant amount of indoxacarb treated nymphs (df = 4, F= 1.97, P =0.1299) in the choice test, ranging from 0.2 to 1.2 nymphs per day, for a total 5 d consumption of 2.8 nymphs. When no choice was provided, Orlando susceptible strain cockroaches consumed a cons tant amount of control nymphs (df = 4, F= 0.82, P =0.5235), ranging from 4.8 to 6.3 nymphs per day, for a total 5 d consumption of 29.4 nymphs. Orlando susceptible strain cockroach es consumed a constant amount of fipronil treated nymphs (df = 4, F= 0.66, P =0.6225) in the no choice test ranging from 3.3 to 4.8 nymphs per day, for a total 5 d consumption of 19.1 nymphs. Orlando susceptible strain cockroaches consumed a constant am ount of indoxacarb treated nymphs (df = 4, F= 1.58, P =0.2095) in the no choice test, ranging from 2.8 to 5.0 nymphs per day, for a total 5 d consumption of 19.3 nymphs. When given a choice, Daytona field strain consumed a constant amount of control nymphs (df = 4, F= 0.77, P =0.5530), ranging from 0.4 to 1.2 nymphs per day (Table 4-3), for a total 5 d consumption of 4.1 nymphs. Daytona field strain consumed a constant amount of fipronil treated nymphs (df = 4, F= 0.31, P =8653) in the choice test, ranging from 0.3 to 0.6 nymphs per day, for a total 5 d consumption of 1.8 nymphs. Daytona field strain also consumed a constant amount of indoxacarb treated nymphs (df = 4, F= 1.22, P =0.3273) in the choice test, ranging from 0.3 to 0.8 nymphs per day, for a total 5 d consumption of 2.2 nymphs. When no c hoice was provided, Daytona field strain

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32 consumed a constant amount of control nymphs (df = 4, F= 2.11, P =0.1096), ranging from 1.5 to 4.3 nymphs per day for a total 5 d cons umption of 13.9 nymphs. Daytona field strain consumed a constant amount of fipronil treated nymphs (df = 4, F= 0.25, P =0.9044) in the no choice test, ranging from 1.3 to 1.9 nymphs per day, for a total 5 d consumption of 8.5 nymphs. Daytona field strain also consumed a constant amount of indoxacarb treated nymphs (df = 4, F= 2.10, P =0.1107) in the no choice test ranging from 1.0 to 2.7 nymphs per day, for a total 5 d consumption of 8.0 nymphs. Low mortality of 1.3% occurred in Orlando su sceptible strain cockroach at 5 d with choice control and there was no significant increase in mortality from 1 d (df = 4, F= 1.19, P =0.3393) (Table 4-4). Mortal ity of 6.0% occurred in Orlando susceptible strain cockroach at 5 d with choice fi pronil and there was no signifi cant increase in mortality from 1 d (df = 4, F= 2.41, P =0.0760). Mortality of 8.7% occu rred in Orlando susceptible strain cockroach at 5 d with choice indoxaca rb and there was signi ficant increase in mortality from day one. Orlando susceptible strain cockroach had mortality of 6.0% by day five with no choice cont rol and no significant increas e in mortality from 1 d ( F= 1.82, D F= 4, P<0.05). Mortality of 47.3% occurred in Orlando susceptible st rain cockroach at 5 d with no choice fipronil and there was signi ficant increase in mortality from 1d. Mortality of 36.7% occurred in Orlando sus ceptible strain cockroach at 5 d with no choice indoxacarb and there was significan t increase in mortality from day one. For Orlando susceptible strain cockroach, mortality increased linearly for control, fipronil, and indoxacarb in both choice and no choice experiments (Fig 4-3). Highest slopes, indicating highest mortality rates, were no choice fipronil and no choice indoxacarb. Lowest slope, indi cating least amount of mortalit y, was choice control. All

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33 slopes were negative, indicati ng a delay in mortality, however the largest delay, occurred with no choice indoxacarb. Intercepts indicate a delay of approximately 1 d after start of the experiment for all treatments with the exception of no choice i ndoxacarb, which had a delay of almost 1.5 d. Low mortality of 2.0% occurred in Daytona field strain cockroach at 5 d with choice control and there was no significant increase in mortality from 1 d (df = 4, F= 0.49, P =0.7413) (Table 4-5). Mortalit y of 8.0% occurred in Daytona field strain cockroach at 5 d with choice fipronil and there was no signi ficant increase in mortality from 1 d (df = 4, F= 1.76, P =0.1684). Mortality of 9.3% occurred in Daytona field strain cockroach at 5 d with choice indoxacarb and there was signifi cant increase in mo rtality from 1 d. Mortality of 20.8% occurred in Daytona field strain cockroach at 5 d with no choice control and there was significant increase in mortality from day one. Mortality of 31.6% occurred in Daytona field strain cockroach at 5 d with no choice fipronil and there was significant increase in mortality from 1 d. Mo rtality of 26.0% occurred in Daytona field strain cockroach at 5 d with no choice indoxa carb and there was si gnificant increase in mortality from 1 d. For Daytona field strain cockroach, mort ality increased linearly for control, indoxacarb, and fipronil in both choice and no choice experiments (Fig 4-4). Highest slope was no choice fipronil, followed by no choice indoxacarb and no choice control. Lowest slope was choice control. All slopes were negative; however, the largest delay, occurred with no choice indoxacarb and no choice control. Intercepts indicate a delay of approximately 1 d after start of the experiment for choice control, choice fipronil, and no choice fipronil. A delay of 1.25 to 1.5 d occurred with both no choice and choice

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34 indoxacarb. Greatest delay, approximately 2 d after start of experiment, was no choice control. Discussion Oral toxicity assay Insecticidal concentration can reduce palatability of a toxic bait base. When Orlando susceptible strain German cockroaches were included in a lethal time test with concentrations of abamectin ranging from 0.0025% to 0.1000%, LT50 values failed to significantly decrease a bove concentrations of 0.0500% (Koehler et al. 1991). It was determined this was due to feeding deterrence caused by abamectin in concentrations above 0.0500%. In my oral toxicity study, bait consumption did not significantly decrease as fipr onil concentrations increased 2 fold for Orlando susceptible strain cockroach and 13 fold for Daytona fi eld strain cockroaches. Additionally, bait consumption did not significantly decrease as indoxacarb concentrations increased 10 fold for Orlando susceptible strain cockro ach and 6 fold for Daytona field strain cockroaches. This indicates neither fipr onil nor indoxacarb are feeding deterrents. LD50 values are usually obtained by topical application of insecticides. Topical LD50 value for fipronil was 0.096 ng per mg body weight for Orlando susceptible strain cockroach (Valles et al. 1997). The same study also injected fipronil directly and determined the injected LD50 value was 0.081 ng per mg bo dy weight. My study, which required ingestion of fipronil, determined LD50 value for fipronil was 0.07 ng per mg body weight for Orlando susceptible strain co ckroach. Overlapping confidence intervals between Valles’ study and my study indicate LD50 value for ingested fipronil is equal to the LD50 value of injected fipronil. When Dorie and Cincy bait-averse strain cockroaches were compared to JWAX susceptible strain cockroach for topically ap plied fipronil, resist ance ratios at the LD50 /

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35 LD90 level were 8.7x / 44.9x for Cincy bait-ave rse strain cockroach and 9.3x / 52.7x for Dorie bait averse stain co ckroach (Wang 2004). Although my study determined Daytona field strain cockroach exhibited a resistance ratio at the LD50 level very similar to Dorie bait-averse strain cockroach, it must be noted that Orlando susceptible strain cockroach was used for comparison. There is inherent va riability in strains of German cockroach. When Orlando susceptible strain cockroach a nd JWAX susceptible strain cockroach were compared to each other in a lethal time test using five carbamates, susceptible strain cockroach exhibited 1.2 to 2 fold tolerance to four of the carbamates, indicating that overall, Orlando susceptible strain cockroach is slightly more tolerant of insecticides (Koehler and Patterson 1986). This was true in my study; upon comparison of LD50 and LD90 levels of Orlando susceptible strain co ckroach obtained in my study and values obtained for JWAX susceptible strain co ckroach in Wang et al’s study, Orlando susceptible strain cockroach exhibited 2.3 fold and 2.6 fold tolerance to fipronil respectively. Therefore, even though resistance ratios for Da ytona field strain cockroach were similar to Dorie bait-averse strain cockroach, when comparison strains are taken into account and direct comparison is made, Daytona field strain cockroach is 2.4x more resistant to fipronil than Dorie bait-averse strain cockroach. Add itionally, Daytona field strain cockroach exhibited a low level of resistance (RR50 = 3.5) to indoxacarb when compared to Orlando susceptib le strain cockroaches. When comparing resistance ratios, it is im portant to note differences of comparison susceptible strains used. It is also important to note method of application for lethal dose assays since it appears ingested lethal doses are significant ly different from topically applied. Even with the lower ingested LD50 value for fipronil and compared to a tolerant

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36 susceptible strain, Daytona field strain cockro ach had resistance ratios, especially at the LD90 level that could possibly interf ere with control in the field. Necrophagy assay. German cockroaches are known to consume conspecifics, a process known as cannibalism. More specif ically, when the cannibalized cockroaches are dead, it is known as necrophagy. When tw enty adult male Orlando susceptible strain cockroaches were given a no choice test wh erein the only food source was freshly dead nymphs, necrophagy was relatively low on the fi rst day and steadily increased (Gahlhoff et al. 1999). Conversely, n ecrophagy in my study remained constant over the 5 d period for both Orlando susceptible strain cockroach and Daytona field strain cockroach, with the exception of choice fipr onil in Orlando susceptible strain cockroach, which decreased. Although my experimental design wa s different from Gahl hoff et al’s (1999) in that I fed fewer nymphs over the course of only 5 d, used two different strains, and offered a choice in addition to no choice, our results were similar because in both cases, necrophagy occurred. German cockroaches can go several days be fore starving to deat h. In Gahlhoff et al.’s (1999) study, control mortality was approxi mately 2% on day five and increased to 10% on day seven, indicating st arvation. In my no choice study, control mortality was not significant at 5 d in Or lando susceptible strain cock roach; however, significant control mortality occurred at 4 d in Daytona field strain cockroach. Because adult male field cockroaches increase fo raging distance, time spent foraging, and movement velocity under the influence of starvation (Barcay and Bennett 1991), it is likely Daytona field strain cockroaches starved themselves wh ile foraging for more nutritious food while Orlando susceptible strain did not.

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37 Fipronil is a fast acting insecticide known to cause secondary mortality (Buczkowski et al. 2001, Durier and Rivau lt 2000b, Gahlhoff 1999). In Gahlhoff et al.’s (1999) study on cannibalization, mortality fr om fipronil was approximately 30% on day two and increased to 100% by day five. Si milarly, in my no choice study, mortality caused by ingestion of fipron il treated nymphs increased linearly over time. Linear increase probably occurred due to cons tant necrophagy. Additionally, significant secondary mortality from fipronil occurred in both Orlando susceptible strain cockroach and Daytona field strain cockroach at 3 d. No significant mortality occurred from fipronil treated nymphs in the choice test for either Orlando susceptible strain cockroach or Daytona field strain cockroach. Indoxacarb is a chemical that requires bioa ctivation to become effective (Wing et al. 1998). Using adult male American Cy anamid strain cockroaches, Appel (2003) determined the LT50 value of 0.25% indoxacarb was 0.68 days. In my study, only secondary mortality was recorded, and simila r to the findings of Appel (2003), mortality from ingestion of indoxacarb treated nym phs was delayed. Significant secondary mortality from indoxacarb treated nymphs o ccurred in the no choice study at 3 d for Orlando susceptible strain co ckroach and 4 d for Daytona field strain cockroach. Significant secondary mortality from indoxaca rb treated nymphs occurred in the choice study at 4 d for Orlando susceptible strain co ckroach and 5 d for Daytona field strain cockroach. Secondary mortality from ingestion of indoxacarb treated nymphs was delayed and significant in both choice and no choice expe riments for both Orlando susceptible strain cockroach and Daytona field strain cockro ach; however, in the no choice test, for

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38 Orlando susceptible strain, signi ficant secondary mortality occurred on the same day as significant secondary mortality from ingest ion of fipronil treated nymphs. This is perhaps due to the presence of already bio activated indoxacarb metabolite in the midgut and fat bodies of the cannibalized nymphs. Significant secondary mortality occurred in no choice tests for fipronil for both Orlando su sceptible strain co ckroach and Daytona field strain cockroach. Significant seconda ry mortality in choice experiments only occurred for indoxacarb in both Orlando suscep tible strain cockroach and Daytona field strain cockroach. Necrophagy causes significant secondary mortal ity in laboratory settings (Gahlhoff, et al. 1999). My study has shown that, while necrophagy occurs both in the presence or absence of food, availability of food is important to signi ficant secondary mortality. If another food source is readily available, German cockroaches engage less in necrophagy. In the absence of convenient food in a labor atory setting or a field setting, necrophagy could play a significant role in secondary mortality.

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39 y = 0.1135Ln(x) + 1.8674 R2 = 0.6354 0.81 0.83 0.85 0.87 0.89 0.91 0.93 0.95 0.97 0.99 1.01 0.00020.000250.00030.000350.00040.000450.00050.00055 Concentration (% AI)Proportion Eaten y = -0.0076Ln(x) + 0.9405 R2 = 0.1133 0.81 0.83 0.85 0.87 0.89 0.91 0.93 0.95 0.97 0.99 1.01 00.010.020.030.040.050.060.070.08Concentration (% AI)Proportion Eaten Figure 4-1. Proportion fipronil and indoxacar b treated breadcrumbs eaten by Orlando susceptible strain cockroaches after a 24 h starvation period. Orlando susceptible strain Indoxacarb Orlando susceptible strain Fipronil

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40 y = 0.0099Ln(x) + 0.907 R2 = 0.2237 0.81 0.83 0.85 0.87 0.89 0.91 0.93 0.95 0.97 0.99 1.01 00.0010.0020.0030.0040.0050.0060.0070.0080.009 Concentration (% AI)Proportion Eaten y = 0.0321Ln(x) + 1.0338 R2 = 0.1745 0.81 0.83 0.85 0.87 0.89 0.91 0.93 0.95 0.97 0.99 1.01 00.010.020.030.040.050.060.07 Concentration (% AI)Proportion Eaten Figure 4-2 Proportion fipronil and indoxacarb tr eated breadcrumbs eaten by Daytona susceptible strain cockroaches after a 24 h starvation period. Daytona field strain Indoxacarb Daytona field strain Fipronil

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41Table 4-1. Susceptibility of Orlando suscepti ble and Daytona field strains of German cockroach to two inge sted insecticides. Lethal dose (ng/mg)a Model fit Insecticide Strain n Slope SE LD50 (95% FL) LD90 (95% FL) RR50 b RR90 b x2 P Fipronil Orlando 300 7.220 0.954 0.072 (0.068-0.076) 0.108 (0.971-0.128) 1.0 1.0 0.7079 0.4001 Daytona 340 1.618 0.201 0.656 (0.535-0.822) 4.063 (2.634-8.071) 9.4 36.9 2.2410 0.5239 Indoxacarb Orlando 370 2.588 0.310 1.312 (1.128-1.488) 4.104 (3.329-5.634) 1.0 1.0 5.3696 0.2514 Daytona 354 2.177 0.301 4.653 (3.833-5.410) 18.045 (13.836-27.925) 3.5 4.4 0.9641 0.8099 a Dose (nanograms of insecticide per mg of insect) calculated based on body weights. Average body weights (mean SE) per strai n (n = 982) were Orlando, 47.29 0.28 and Daytona, 50.93 0.27 mg. b Resistance ratio based on LD50/ LD 90 va lues compared with Orlando strain.

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42Table 4-2. Daily consumption of insecticide treated nymphs by Orlando susceptible strain German cockroaches. Choice No Choice Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb 1 2.42 0.33 1.17 0.46a 0.83 0.17 6.17 0.79 4.75 0.94 5.00 1.28 2 1.08 0.27 0.42 0.24b 0.42 0.20 4.83 0.88 3.50 0.39 3.58 0.57 3 2.17 0.33 0.08 0.08b 0.17 0.11 6.00 0.56 3.92 0.69 4.50 0.65 4 1.58 0.44 0.17 0.17b 0.25 0.11 6.17 0.40 3.33 0.49 3.50 0.26 5 1.67 0.42 0.25 0.11b 1.17 0.60 6.25 0.51 3.58 0.80 2.67 0.44 Means in a column followed by the same letter are not significan tly different (P > 0.05; Student -Newman-Keuls sequential range test [SAS Institute, 2001]).

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43 Table 4-3. Daily consumption of insecticide treate d nymphs by Daytona field strain German cockroaches. Choice No Choice Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb 1 0.92 0.35 0.58 0.37 0.83 0.28 2.17 0.48 1. 83 0.40 1.58 0.27 2 1.00 0.41 0.33 0.17 0.42 0.20 1.50 0.52 1. 25 0.60 1.08 0.58 3 0.58 0.33 0.33 0.25 0.25 0.11 2.58 0.55 1. 92 0.61 1.67 0.56 4 0.42 0.20 0.17 0.17 0.25 0.17 3.42 1.02 1.75 0.59 2.67 0.49 5 1.17 0.42 0.42 0.33 0.42 0.27 4.25 0.95 1.75 0.31 1.00 0.29

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44 Table 4-4. Cumulative daily mortality from ingestion of insecticid e treated nymphs in Orlando sus ceptible strain German cockroa ches. Choice No Choice Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb 1 0.00 0.00 0.00 0.00 0.00 0.00a 0.00 0.00 3.33 1.23a 0.00 0.00a 2 0.00 0.00 2.67 0.84 3.33 1.91ab 1.33 0.84 11.33 3.17a 4.00 1.79a 3 0.37 0.67 4.67 1.61 4.00 1.79ab 1.33 0.84 22.00 3.39b 12.67 2.81b 4 1.33 0.84 5.33 1.98 6.00 1.71ab 4.00 2.53 33.33 3.37c 22.00 3.06c 5 1.33 0.84 6.00 2.25 8.67 2.40b 6.00 2.88 47.33 2.81d 36.67 3.78d Means in a column followed by the same letter are not significan tly different (P > 0.05; Student -Newman-Keuls sequential range test [SAS Institute, 2001]).

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45Table 4-5. Cumulative daily mortality from ingestion of insecticid e treated nymphs in Daytona field strain German cockroaches. Choice No Choice Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb 1 0.00 0.00 1.33 0.84 0.00 0.00a 0.67 0.67a 2.03 0.91a 1.33 0.84a 2 0.67 0.67 2.67 1.33 0.00 0.00a 0.67 0.67a 8.08 1.83ab 4.00 1.79a 3 1.33 1.33 6.00 3.06 3.33 1.23a 3.39 0.68a 13.50 3.63b 8.67 3.33ab 4 1.33 1.33 8.00 2.73 7.33 1.91b 12.84 1.22b 22.83 2.88c 18.00 5.54bc 5 2.00 1.37 8.00 2.73 9.33 1.33b 20.84 2.71c 31.61 2.99d 26.00 3.83c Means in a column followed by the same letter are not significan tly different (P > 0.05; Student -Newman-Keuls sequential range test [SAS Institute, 2001]).

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46 y = 11x 9.536 R2 = 0.9904 y = 9.134x 12.334 R2 = 0.959 y = 2.001x 1.603 R2 = 0.9658 y = 1.466x 0.664 R2 = 0.9094 y = 0.399x 0.591 R2 = 0.8659 y = 1.467x 1.869 R2 = 0.91650 5 10 15 20 25 30 35 40 45 50 12345 Days% Mortality Choice Control Choice Fipronil Choice Indoxacarb No Choice Control No Choice Fipronil No Choice Indoxacarb Figure. 4-3. Percent mortality of Orlando susceptible strain cockroaches from i ngestion of insecticide treated nymphs.

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47 y = 7.391x 6.563 R2 = 0.9866 y = 6.334x 7.402 R2 = 0.9549 y = 2.599x 3.799 R2 = 0.9389 y = 1.867x 0.401 R2 = 0.9246 y = 0.466x 0.332 R2 = 0.9421 y = 8.725x 22.543 R2 = 0.99770 5 10 15 20 25 30 35 12345 Days% Mortality Choice Control Choice Fipronil Choice Indoxacarb No Choice Control No Choice Fipronil No Choice Indoxacarb Figure. 4-4. Percent mortality of Dayt ona field strain cockroaches from in gestion of insecticide treated nymphs.

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48 CHAPTER 5 CONCLUSION German cockroaches are major pests of households and structures. The common method of control is the use of gel bait. Recently, reports of control failure were reported. In two cases, the cause of control fa ilure was attributed to behavioral aversion, a fairly new phenomenon in German cock roaches. It is unknown if the feeding deterrence exhibited by bait av erse strains of German cockroach can be overcome by reformulating the gel bait matrix. It is al so unknown if bait averse strains of German cockroach also have physiological resistance. For these studies, a field strain of cockroach was collected from Daytona, FL in an area that reported control failure using gel bait. This strain, Daytona field st rain, was determined to be bait averse. Chapter 3 evaluated feeding deterrence to six different commercially available gel bait formulations and concluded that cons umption was positively correlated with mortality. Daytona field strain cockroach exhibited feeding de terrence to four of the six gel baits. Two gel baits did not cause feed ing deterrence and therefore, had the highest levels of mortality; indicating feeding deterrence can be overcome by reformulating bait matrices Chapter 4 evaluated the oral toxicity of indoxacarb and fipronil to both Orlando susceptible strain cockroach and Daytona field strain cockroach. At the LD90 level, Daytona field strain cockroach exhibited lo w levels of physiological resistance to indoxacarb and high levels of resistance to fipr onil. Chapter 4 also evaluated the ability of fipronil and indoxacarb to cause seconda ry mortality from necrophagy. Necrophagy

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49 occurred in both strains in the presence and absence of food choice. Significant secondary mortality from ingestion of fipr onil treated nymphs only occurred in the no choice test for both strains. Significant sec ondary mortality from i ngestion of indoxacarb treated nymphs occurred in both choice and no choice tests for both strains; however, mortality occurred significantly faster in the no choice test fo r both strains. Overall, control failures in the field ca n be attributed to the combination of behavioral and physiological re sistance. Changing gel bait formulations and ingredients can overcome these failures. Because the availability of food significantly affects secondary mortality, control measures will be more effective if sanitation is practiced along with application of gel baits. Our bait averse field strain was collected in Florida whereas the only other isolated strains of bait averse cockroach were from Oh io. This indicates bait aversion is more widespread than once believed. While gel ba its can be formulated so that bait averse cockroaches consume them, physiological resistance and insignificant secondary mortality from necrophagy can still affect c ontrol in the long term. This study has revealed that there is more to behavior ally averse cockroaches than decreased consumption of gel bait.

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50 LIST OF REFERENCES Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265267. Abd-Elghafar, S.F., A.G. Appel, and T.P. M ack. 1990. Toxicity of several insecticide formulations against adult German cockro aches (Dictyoptera: Blattellidae). J. Econ. Entomol. 83: 2290-2294. Appel, A.G. 2004. Contamination affects the performance of insecticidal baits against German cockroaches (Dichtyoptera: Bla ttellidae). J Econ. Entomol. 97: 20352042. Appel, A.G. 2003. Laborator y and field performance of an indoxacarb bait against German cockroaches. J Econ. Entomol. 96: 863-870. Appel, A.G. 1990. Laborator y and field performance of consumer bait products for German cockroach (Blattodea: Blattellidae) control. J. Econom. Entomol. 83: 153159. Appel, A.G., and Benson, E.P. 1995. Perf ormance of abamectin bait formulations against German cockroaches (Dichtyoptera: Blattellidae). J. Econom. Entomol. 83: 153-159. Barcay, S.J., and G.W. Bennett. 1991. In fluence of starvation and lighting on the movement behavior of the German cockro ach (Blattodea: Blatte llidae). J. Econ. Entomol. 84: 1520-1524. Cochran, D.G. 1982. Cockroachesbiol ogy and control. WHO/VBC 82: 856-909. Cochran, D.G. 1994. Effects of synergists on pyrethroid resistance in the German cockroach. Resist. Pe st. Manage. 6: 16-17. Cooper, R.A., and C. Schal. 1992. Differential development and reproductionof the German cockroach (Dictyoptera: Blattellid ae) on three laboratory diets. J. Econ. Entomol. 85: 838-844. Cornwell, P.B. 1968. The cockroach, vol I. Hutchinson and Co., London. Cornwell, P.B. 1976. The cockroach, vol II. Hutchinson and Co., London.

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51 Durier, V., and C. Rivault. 2000. Secondary transmission of toxic baits in German cockroach (Dictyoptera: Blattellidae). J. Econom. Entomol. 93: 434-440. Ebeling, W., D.A. Reierson, R.J. Pence, and M.S. Viray. 1974. Silica aerogel and boric acid against cockroaches: exte rnal and internal action. Pesticide Biohem. Physiol. 5: 81-89. Elgderi, R.M., K.S. Ghengesh, and N. Berbash. 2006. Carriage by the German cockroach ( Blattella germanica ) of myultiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hosp itals and households in Tripoli, Libya. Annals of Tropical Medicine and Parasitology. 100: 55-62. Flynn, A. D., and H. F. Schoof 1971. Control og German cockroaches aboard US naval surface ships on the east coast. J. Econom. Entomol. 64:1176-1179. Fuchs, M.E.A. 1988. Flushing effects of pyr ethrum and pyrethroid insecticides against the German cockroach ( Blattella germanica L.) Pyrethrum Post 17: 3-6. Garcia, D.P., M.L. Corbett, J.L. Sublett. 1994. Cockroach allergy in Kentucky: a comparison of inner city, suburban, and ru ral small town populations. Ann allergy 72: 203-208. Gahlhoff, J.E. Jr., D.M. Miller, and P.G. Ko ehler. 1999. Secondary kill of adult male German cockroaches (Dictyoptera: Blatte llidae) via cannibalism of nymphs fed toxic baits. J. Econom. Entomol. 92: 1133-1137. Grayson, J.M. 1964. Resistance to insecticides in cockroaches in Proc. XII International Congress of Entomology. Holbrook, G.L., J. Roebuck. C.B Moore, M. G. Waldvogel, and C. Schal. 2003. Origin and extent of resistance to fipronil in the German cockroach, Blattella germanica (L.) (Dichtyoptera: Blattellidae) J. Econom. Entomol. 96: 1548-1558. Kaakeh, W., B.L. Reid, and G.W. Bennett. 1997. Toxicity of fipronil to German and American cockroaches. Entomol. Exp. Appl. 84: 229-237. Kang, B.C. 1990. Impact of cockroach alle rgens on humans. Proc. Natl. Conf. Urban. Entomol. 67-76. King, J.E., and G.W. Bennett. 1989. Comp arative activey of fenoxycarb and hydroprene in sterilizing the German cockroach (Dic tyoptera: Blattellidae). J. Econom. Entomol. 82: 833-838. Kitae, K., J.H. Jeon, and D. Lee. 1995. Various pathogenic bacteria on German cockroaches (Blattellidae, Blattaria) collect ed from general hospitals. Korean J. Entomol. 25: 85-88.

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52 Koehler, P.G.,and R.S. Patterson. 1986. A co mparison of insecticid e comparability in seven nonresistant strains of the German cockroach Blattella germanica (Dictyoptera: Blattellidae). Med. Entomol. 23: 298-299. Koehler, P.G., and R.S. Patterson. 1991a. Toxicity of hydramethylnon to laboratory and field strains of German cockroach (Orthopter a: Blattellidae). Fla. Entomol. 74: 345-349. Koehler, P.G., and R.S. Patterson. 1991b. In corporation of pyriproxifenin a German cockroach (Dictyoptera: Bla ttellidae) management program. J. Econom. Entomol. 84: 917-921. Koehler, P.G., T.H. Atkinson, and R.S. Pa tterson. 1991. Toxicity of abamectin to cockroaches (Dictyoptera: Bl attellidae, Blattidae). J. Econom. Entomol. 84: 17581762. Koehler, P.G., C.A. Strong, and R.S. Patterson. 1994. Rearing improvements for the German cockroach (Dictyoptera: Blatte llidae). J. Econom. Entomol. 81: 704-710. Kopanic, R.J. Jr, and C. Schal. 1997. Rela tive significance of direct ingestion and adultmediated translocation of bait to German cockroach (Dictyoptera: Blattellidae) nymphs. J. Econom. Entomol. 90: 1073-1079. Kopanic, R.J. Jr, and C. Schal. 1999. C oprophagy facilitates horizon tal transmission of bait among cockroaches (Dictyoptera: Blat tellidae). Enviro n. Entomol. 28: 431438. Kopanic, R.J. Jr., G.L. Holbrook, V. Sevala, and C. Schal. 2001. An adaptive benefit of facultative coprophagy in the German cockroach Blattella germanica. Ecol. Entomol. 26: 154-162. Matsumura, F. 1975. Toxicology of insecticides. Plenum Press, NY. Metzger, R. 1995. Behavior. pp 49-76. In M.K. Rust, J.M. Owens, and D.A. Reierson [eds.]. Understanding and controlling the German cockro ach. Oxford University Press, New York. Owens, J.M., and G.W. Bennett. 1982. Ge rman cockroach movement within and between urban apartments. J. Econ. Entomol. 75: 570-573. Reid, B.L., G.W. Bennett, and S.J. Barca y. 1990. Topical and oral toxicity of sulfluramid, a delayed-action insectic ide, against the German cockroach (Dichtyoptera: Blattellidae). J. Econom. Entomol. 83: 148-152. Reid, B.L., V. L. Brock, and G.W. Bennett. 1994. Developmental, morphogenetic, and reproductive effects of four polycyclic non-isoprenoid juvenoid in the German cockroach (Dichtyoptera: Blattellidae) J. Econom. Entomol. 29: 31-42.

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53 Reierson, D.A. 1995. Baits and baiting. pp 231-266. In M.K. Rust, J.M. Owens, and D.A. Reierson [eds.]. Understanding and controlling the German cockroach. Oxford University Press, New York. Ross, M.H. 1993. Laboratory studies on the response of German cockroaches (Dichtyoptera: Blattellidae) to an abamectin gel bait. J. Econ. Entomol. 86:767771. Ross, M.H. 1997. Evolution of behavior al resistance in German cockroaches (Dictyoptera: Blattellidae) se lected with a toxic bait. J. Econom. Entomol. 90(6): 1482-1485. Ross, M.H. 1998. Responses of behavi orally resistant German cockroaches (Dichtyoptera: Blattellidae) to the active ingredient in a commercial bait. J. Econom. Entomol. 91: 150-152. Rust, M.K., and D.A. Reierson. 1981. Attrac tion and performance of insecticidal baits for German cockroach control. Int. Pest Control. 23: 106-109. Schal, C, G.L. Holbrook, J.A.S. Bachmann, and V.L. Selva. 1997. Reproductive biology of the German cockroach Blattells germanica: Juvenile hormone as a pleiotropic master regulator. Arch. Ins ect Biochem. Physiol. 35: 405426. Scott, J.G. 1991. Toxicity of abamectin and hydramethylnon to insecticide susceptible and resistant strains of German cockroach (Dictyoptera: Blattellidae). J. Agric. Entomol. 8: 77-82. Scott, J.G., and F. Matsumura. 1982. Evidence for two types of toxic actions of pyrethroids on susceptible and DDT-resi stance German cockroaches. Pest. Biochem. Physiol. 19: 141-150. Scott, J.G., and Z. Wen. 1997. Toxicity of fipronil to suscep tible and resistant strains of German cockroaches (Dichtyoptera: Bl attellidae) and hous e flies (Diptera: Muscidae). J. Econom. Entomol. 90: 1152-1156. Siefried, B.D., J.G. Scott, R.T. Toush, a nd B.C. Zeichner. 1990. Biochemistry and genetics of chlorpyrifos resi stance in the German cockroach, Blattella germanica(L). Pest. Biochem. Physiol. 38:110-121. Silva, J.M. 1990. Achoo! Must be the roaches! Pest Manage. 9: 21-22, 24-25. Silverman, J., and D.N. Bieman. 1993. Gl ucose aversion in the German cockroach Blattella germanica J. Insect Physiol. 39: 925-993. Silverman, J., and M.H. Ross. 1994. Behavior al resistance of field-collected German cockroaches (Blattodea: Blattellidae) to baits containing glucose. Environ. Entomol. 23(2): 425-430.

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54 Silverman, J., and H. Selbach. 1998. Feeding behavior and survival of glucose-averse Blattella germanica (Orthoptera: Blattoidea: Blatte llidae) provided glucose as a sole food source. J. Inse ct Behavior 11(1): 93-102. Silverman, J., and D. Liang. 1999. Effect of fipronil on bait formulation-based aversion in the German cockroach (Dichtyoptera: Blattellidae). J. Econom. Entomol. 92(4): 886-889. Sparks, T.C., J.A. Lockwood, R.L. Byford, and B.R. Leonard. 1989. The role of behavior in insecticide resist ance. Pestic. Sci. 26: 383-399. Steinberg, D.R., D.I. Bernstein, J.S. Galla gher, L. Arlian, and I.L. Bernstein. 1987. Cockroach sensitization in laboratory workers. J. Allergy Clin. Immunol. 80: 586590. Valles, S.M., P.G. Koehler, and R.J. Brenne r. 1997. Antagonism of fipronil toxicity by piperonyl butoxide and S,S,S,-tribu tyl phosophorotrithioate in the German cockroach (Dichtyoptera: Blattellidae) J. Econom. Entomol. 90: 1254-1258. Willis, E.R., G.R. Riser, and L.M. Roth. 1958. Observations on reproduction and development in cockroaches. Ann. Entomol. Soc. Am. 51: 5369. Wang, C., M.E. Scharf, and G.W. Bennett. 2004. Behavioral and physiological resistance of the German cockroach to ge l baits (Blattodea: Blattellidae). J. Econom. Entomol. 97(6): 2067-2072. Wing, K.D., M.E. Schnee, M. Sacher, and M.Connair. 1998. A novel oxadiazine insecticide is bioactivated in lepidopteran larvae. Arch. Insect Biochem. Physiol. 37: 91-103.

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55 BIOGRAPHICAL SKETCH Linda Anne NcHerne, daughter of Harry and Jessie Riley, was born in 1969. She graduated from Bayshore High School in Braden ton, FL, in 1987. She enlisted in the US Army in 1988 and performed her duties as a laboratory technician in Landstuhl, Germany. She graduated from the University of South Florida in 1996 with a bachelor’s in environmental science/ zoology. Sh e joined the US Navy in 1998 and was commissioned in December of that year. During her time, she ran an office for the training of future military pilots in Pensacola, FL, and was a deck officer and a weapons officer onboard the USS Germantown stationed in Sasebo, Japan. Desiring to become a Medical Entomologist with the US Navy, she en tered the University of Florida’s graduate program in August of 2003.


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BAIT AVERSION AND ORAL TOXICITY OF INSECTICIDES IN A FIELD STRAIN
OF GERMAN COCKROACH.















By

LINDA ANNE NCHERNE


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

UNIVERSITY OF FLORIDA


2006



























Copyright 2005

by

Linda Anne Ncherne















ACKNOWLEDGMENTS

I would like to thank my husband for the love and support he gave me during the

degree process. He never once complained when I was using him as a soundboard to

work thorough technical problems, although he now knows more about cockroaches than

he ever desired. I would also like to thank my mother; although deceased, I feel she

helped guide me to this path and is proud of my accomplishments.

Many thanks go my committee members: Dr. Simon Yu for his editorial support

and help with toxicology and Dr. Richard Patterson for his editorial support, wisdom, and

guidance. Lastly thanks are due to my committee chair, Dr. Philip Koehler. However, I

feel thanks are not enough for all he has done. In addition to his vast knowledge of every

aspect of urban entomology, he is a great mentor, pushing all of his students to aspire to

greatness. Additionally, his enthusiasm for the subject, even after so many years, is truly

inspiring. I sincerely feel honored to have been his graduate student.

Additionally, thanks go the Urban entomology crew, especially Gilman Marshall

for explaining chemistry related math to me so that I actually understood it- something

no high school or college professor had managed to do and to Joseph Smith for helping

acclimate someone who had been out of academia for 8 years and for making me laugh.

Lastly, but certainly not least, special thanks go to Cynthia Tucker for bad day lunch

commiserations, opening up her home to me when I was in need, and offering

unconditional friendship- a rarity in today's world.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iii

L IST O F TA B LE S .............. ............................ ............ ... ......... .......... vi

L IST O F FIG U R E S .... ....... ................................................ .... ..... .. ............. vii

A B S T R A C T ......... .................................. ................................................... v iii

CHAPTER

1 IN TR O D U C TIO N ......................................................................... .... .. ........

2 LITER A TU R E R EV IEW ............................................................... ...................... 3

B biology ............................................................. . 3
H a b ita t ................................................................................. 4
P est S tatu s .................................................................. .............................. 5
Chem ical Control of the Germ an Cockroach ........................................ ............... 6

3 EVALUATION OF FEEDING DETTERRENCE IN SIX INSECTICIDAL GEL
BAITS AND MORTALITY IN A FIELD STRAIN OF GERMAN
COCKROACH, Blattella germanica (L) ................................. ..............................11

In tro d u ctio n ............................................................................................. 1 1
M materials an d M eth o d s .......................................................................................... 12
R e su lts ...........................................................................................1 5
D isc u ssio n .............................................................................................................. 1 8

4 ORAL TOXICITY OF INDOXACARB AND SECONDARY MORTALITY
FROM NECROPHAGY IN A SUSCEPTIBLE STRAIN AND A FIELD
STRAIN OF GERMAN COCKROACH, Blattella germanica (L) ........................26

In tro d u ctio n .......................................................................................2 6
M materials an d M eth od s .......................................................................................... 2 7
R e su lts .................................................................................................................... 3 0
D isc u ssio n .............................................................................................................. 3 4

5 CONCLUSION..................... ..................48



iv










L IST O F R E F E R E N C E S ........................................................................ .....................50

B IO G R A PH IC A L SK E T C H ...................................................................... ..................55


























































v















LIST OF TABLES


Table page

3-1 Mortality (4 d) and consumption of gel bait, dog food, and AI for 50 Orlando
susceptible strain German cockroaches 24 h after gel bait placement ...................24

3-2 Mortality (4 d) and consumption of gel bait, dog food, and AI for 50 Daytona
field strain German cockroaches 24 h after gel bait placement. ...........................25

4-1 Susceptibility of Orlando susceptible and Daytona field strains of German
cockroach to two ingested insecticides. ...................................... ............... 41

4-2 Daily consumption of insecticide treated nymphs by Orlando susceptible strain
G erm an cockroaches. .......................... ...... .................... ........ ...... .... 42

4-3 Daily consumption of insecticide treated nymphs by Daytona field strain
G erm an cockroaches. .......................... ...... .................... ........ ...... .... 43

4-4 Cumulative daily mortality from ingestion of insecticide treated nymphs in
Orlando susceptible strain German cockroaches.......................................... 44

4-5 Cumulative daily mortality from ingestion of insecticide treated nymphs in
Daytona field strain German cockroaches..................................... ............... 45















LIST OF FIGURES


Figure page

4-1 Proportion fipronil and indoxacarb treated breadcrumbs eaten by Orlando
susceptible strain cockroaches after a 24 h starvation period. ................................39

4-2 Proportion fipronil and indoxacarb treated breadcrumbs eaten by Daytona
susceptible strain cockroaches after a 24 h starvation period. ................................40

4-3 Percent mortality of Orlando susceptible strain cockroaches from ingestion of
insecticide treated nym phs. ........................................................... .....................46

4-4 Percent mortality of Daytona field strain cockroaches from ingestion of
insecticide treated nym phs. ........................................................... .....................47















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

BAIT AVERSION AND ORAL TOXICITY OF INSECTICIDES IN A FIELD STRAIN
OF GERMAN COCKROACH

By

Linda Anne NcHerne

August 2006

Chair: P.G. Koehler
Major Department: Entomology and Nematology

Control of German cockroaches is again becoming a severe problem. The usual

method of control is the use of gel baits. In order for a bait to be effective, it must be

palatable, attractive, highly toxic, and provide secondary kill through coprophagy or

necrophagy. Currently, strains of German cockroach exist that are not being controlled

by gel baits. It is unknown if this can be attributed to physiological or behavioral

aversion. Daytona field strain German cockroach, was collected from an area that had

reported control failure using gel bait in Daytona, FL. To determine if Daytona field

strain was behaviorally averse to gel baits, Daytona field strain and Orlando susceptible

strain were given choice test using six different commercially available gel baits.

Daytona field strain exhibited feeding deterrence to three of the most common gel baits

used: Avert, Maxforce and Maxforce FC, as well as DPX-MP062-41 la; however,

Orlando susceptible strain exhibited no feeding deterrence. In both strains, there is

positive correlation between consumption and mortality. Bait aversion caused decreased









consumption of three commonly used gel baits; however there were two formulations that

overcame aversion.

Physiological resistance was evaluated through an oral toxicity assay where active

ingredient was fed to Daytona field strain and Orlando susceptible strain cockroaches on

breadcrumbs. Daytona field strain exhibited low resistance ratios to a new chemical,

indoxacarb (3.5 LD50 and 4.4 LD90) and moderate to high resistance ratios to fipronil (9.4

LD50 and 36.9 LD90). When using a palatable gel bait formula, Daytona field strain

ingested 1.5x more fipronil than necessary to kill 90% of the population. Due to evolving

physiological resistance, palatable bait formulations may not control German

cockroaches in the future.

The ability of fipronil and indoxacarb to cause secondary mortality was evaluated

on Daytona field strain and Orlando susceptible strain cockroaches. Cockroaches were

offered insecticide treated nymphs as a food source both with and without a food choice.

Necrophagy occurred in both choice and no choice tests for both strains. Significant

mortality from ingestion of fipronil treated nymphs only occurred in the no choice test for

both strains; whereas significant mortality from ingestion of indoxacarb treated nymphs

occurred in both choice and no choice tests for both strains; however it occurred more

rapidly in the no choice test.

Overall, there have been significant control problems in the field when using the

most common gel baits. Control failures are due to the combination of physiological

resistance and behavioral resistance, but can be overcome with the use of other

formulations of gel baits or new active ingredients. Control will be more effective when

sanitation is used in conjunction with gel baits.














CHAPTER 1
INTRODUCTION

The German cockroach, Blattella germanica (L.), has adapted to cohabiting with

the human species. Throughout the years, various methods and chemicals have been

used to try to control this pest. While results were as varied as the method of control,

most treatments were ineffective. The first chemical treatments that really reduced

cockroach populations and effected control were spray formulations used in the 1950s;

more chemical formulations were developed in the next two decades. Ultimately, all of

these chemicals became obsolete because, within about five years of exposure, the

German cockroach became physiologically resistant (Comwell 1976).

Introduction of baits in the late eighties greatly reduced concerns about

physiological resistance. These baits contained new classes of chemicals that were so

toxic that a lethal dose of active ingredient was delivered in one meal (Wang et al. 2004),

apparently preventing the development of physiological resistance. It seemed to work as

these new baits were effective at controlling the German cockroach. In a couple of years,

bait formulas and applications changed from dry baits contained in a plastic case placed

in a couple of random locations to gel bait formulations that were placed drop-wise in

many small spaces near harborages and foraging areas; which was even more effective at

cockroach control. In the past ten years, control failures have been reported from areas

where gel baits had been used extensively. It was determined that these failures were the

result of cockroaches refusing to consume the bait. This behavioral aversion was initially

determined to be due to glucose within the gel bait matrix. Although the problem of









glucose aversion was overcome, failures were still reported. Currently, the exact cause of

behavioral aversion is unknown.

For my studies, a field strain of German cockroach was isolated from an area that

had reported control failures. Control failures are due to decreased mortality, which has

many possible sources. Chapter 3 evaluates the field strain collected to determine to

what extent gel baits are feeding deterrents and to determine if decreased consumption of

gel bait affects mortality.

Because the effectiveness of new classes of chemicals must be determined, two of

the six gel baits tested in Chapter 3 contain a new chemical, indoxacarb Chapter 4

evaluated oral toxicity of indoxacarb, in both a susceptible strain and a field strain of

German cockroach. One of the benefits of highly toxic chemicals contained in gel baits

is that they can cause mortality in cockroaches that did not directly ingest the gel bait.

Additionally, in chapter 4, secondary mortality due to necrophagy was evaluated in both

a susceptible strain and a field strain of German cockroach.

Behavioral changes in German cockroaches that cause them to eschew gel baits that

once were effective is as important a survival strategy as physiological resistance. The

role of consumption of toxic gel baits cannot be understated. Consumption is what

allows the active ingredient to have an effect, not only on primary mortality, but on

secondary mortality as well. Documenting and understanding behavioral changes in

cockroaches is the first step towards effecting new control measures.














CHAPTER 2
LITERATURE REVIEW

Biology

German cockroaches, Blattella germanica (L.) (Blattaria: Blatellidae) are

hemimetabolous, having only three life stages: egg, nymph, and adult. When the young

emerge as first stage nymphs, they are no longer dependent on their mother and must find

a way to acquire nutrition and water. Larger cockroaches accomplish this by foraging.

Because first stage nymphs are very small and therefore easy prey for other insects,

including other cockroaches, survival dictates they find another way. Their survival

strategy stems from their gregarious nature. Instead of foraging outside the harborage,

they remain sequestered inside the harborage (Silverman, et al, 1991) consuming the

excrement of other cockroaches; a process known as coprophagy (Durier and Rivault

2000b, Gahlhoff et al. 1999; Kopanic et al. 2001). As they mature, coprophagy decreases

as foraging and scavenging methods increase. As with other insects, German

cockroaches grow by the process of molting. The time between molts is known as a

stadium. German cockroaches undergo 6-7 stadia and take an average of 103 days to

mature into an adult. This time frame is dependent many factors such as temperature,

nutritional status, and strain (Cooper and Schal 1992).

Nymphs are dark brown in color with a large tan spot on their pronotum. They

range in size from approximately 1.5 mm newly emerged to 1.6 cm as adults. Adults are

light brown in color and have two dark stripes on their pronotum extending longitudinally

down the body under the wings. The male has a tapered abdomen while the abdomen of









the female is rounded (Ebeling 1975). Although both sexes possess wings, they are

incapable of flight.

When a female German cockroach emerges as an adult, it takes many days to

become sexually active (Schal et al. 1997). When they do become sexually active, they

often mate multiple times, although one mating is usually sufficient to fertilize all their

eggs (Schal et al. 1997). This mated female produces, on average, 30-40 eggs per

oothecal case (Willis et al. 1958). Adult females survive approximately 6 months and

produce 4-6 broods in their lifetime (Schal et al. 1997). They are oviparous; however,

they carry their ootheca until just prior to hatching (Schal et al 1997). This gravid cycle

lasts approximately 21-28 days (Schal et al. 1997) during which time the females eat

sparingly or not at all (Schal et al. 1997, Ross 1993).

Habitat

German cockroaches have a world-wide distribution and, in addition to domiciles,

can be found in restaurants (Rust and Reierson 1991), hospitals (Elgderi et al. 2006, Kitae

et al. 1995), and even aboard naval vessels (Flynn and Schoof 1971). They are usually

found in kitchens, bathrooms, or other areas where water is readily available. They are

nocturnal scavengers capable of living off human waste foodstuffs (i.e. crumbs, residues

on dishware, etc). During the day they usually remain sequestered in harborages. A

harborage is any enclosed area allowing them protection to breed and survive. In homes

this is often cabinetry, appliances, wall voids, or any clutter (boxes, stored goods,

garbage, etc). This ability to sequester in very small spaces is often the mechanism for

new infestation as introduction of cartons, boxes, or other materials harboring German

cockroaches is transported to un-infested homes and businesses. Additionally, German

cockroaches can infest apartments or offices connected by a common wall by moving









from the infested apartment through wall voids or across conjoining plumbing systems to

the new space (Owens and Bennett 1982).

Pest Status

German cockroaches commonly carry potentially pathogenic bacteria such as

Klebsiella, Enterobacter, Serralia, and Streptococcus (Elgderi et al. 2005; Kitae et al.

1995). No direct transmission of disease to humans has been demonstrated; however,

cockroaches have the potential of transmitting these pathogens via contamination of food

preparation surfaces and utensils (Kitae et al. 1995). New data has found that a great

number of pathogens isolated from wild strain cockroaches are multiple antibiotic

resistant (Elgderi et al. 2005).

Carrying pathogenic bacteria is not the German cockroach's only method of

contaminating domiciles and causing harm to humans. Cockroaches have been linked to

human allergies since the mid 1940s (Kang 1990). In infested households, German

cockroach debris is second only to dust mites in the composition of "house dust" (Silva

1990). The greatest sensitivity to cockroach allergens occurs in children seven to twelve

years old (Garcia et al. 1993). As is the case with many allergens over time, cockroach

sensitivities can develop in those that previously had none and existing sensitivity can

increase (Steinberg et al. 1987, Kang 1990). Occasionally, the sensitivity becomes so

bad that shellfish can no longer be eaten, contact dermatitis occurs in the presence of

infestation, (Silva 1990) or asthmatic responses occur when cockroach allergens are

inhaled (Garcia et al. 1993). When human health risk is added to the psychological stress

and stigma caused by German cockroach infestation, it is no wonder German

cockroaches are considered a major pest worldwide.









Chemical Control of the German Cockroach

The oldest group of chemicals used in cockroach control is inorganic compounds

such as boric acid and sodium fluoride. These were slow acting powders that worked

both via contact and orally when ingested during grooming (Ebeling et al. 1974). While

designed for a variety of insects, these dusts proved more effective on the larger

cockroaches (Periplaneta) and offered no real control of the German cockroach (Reid et

al. 1990).

The next group of insecticides used for German cockroach control was the

chlorinated hydrocarbons such as DDT and chlordane. These compounds were very

effective in both spray and dust form with long lasting residual effects. This class of

chemical acted on the sodium channel causing repetitive firing. Physiological resistance

to chlordane was first reported in 1951 and rapidly spread throughout the United States

(Grayson 1964). Although chlorinated hydrocarbons have the differing modes of action,

they all affected the nervous system. Worldwide resistance was documented to not just

chlordane but many chlorinated hydrocarbons within 10 years of the first report (Grayson

1954, Matsumura 1975). Due to environmental issues, the Environmental Protection

Agency (EPA) cancelled the use of chlorinated hydrocarbons in the late 1970s (Reid, et

al, 1990).

In the early 1960s, first organophosphates and, shortly thereafter, carbamates began

replacing chlorinated hydrocarbons (Siegfried et al. 1990, Cochran 1982). Examples of

organophosphates are diaznon, chlorpyrifos, and malathion; while examples of

carbamates are bendiocarb and propoxur. Both of these classes of chemical were used

primarily in spray formulations, inhibited acetylcholinesterase, and were fairly toxic to

vertebrates (Matsumura 1975). Just as with chlorinated hydrocarbons, German









cockroaches developed resistance to both these classes of chemical; however, the time it

took for them to develop resistance was less than five years (Siegfried et al. 1990). For

health issues, EPA no longer allows most of these chemicals to be used.

The pyrethroids became popular for cockroach control in the 1970s. Prior to this

time, pyrethrin- a natural pesticide derived from chrysanthemums in the Family

Compositae- had been used to augment inorganic pesticides. In the late 1950's,

pyrethroids- the synthetic analogue of pyrethrins- became commercially available;

however these "Type I" pyrethroids were not photo stabile and many cockroaches were

able to metabolize the chemical and fully recover. It was the "Type II" pyrethroids that

became popular in the 1970s. These were photo stabile, used in spray formulation, and

had excellent residual effects against cockroaches.

All pyrethroids acted on the sodium channel to interfere with the transmission of

nerve impulses. As with all the other classes of chemical, German cockroaches

developed resistance pyrethroids (Cochran 1994). It was found that cockroaches resistant

to DDT also had inherent resistance, or cross-resistance, to many pyrethroids Scott and

Matsumura 1982). Pyrethroids are still used today; however, they are used as flushing

agents to drive cockroaches out of their harborage (Fuchs 1988).

Throughout the late 19802 and 1990s, many new classes of chemical have come on

the market for cockroach control. The safest are Insect growth regulators, more

specifically, juvenile hormone analogs such as hydroprene and pyriproxifen. These

chemicals mimic insect juvenile hormone, affecting the endocrine balance and causing

developmental disturbances such as molting inhibition, morphogenetic abnormalities,

longer developmental time, and reproduction suppression (King and Bennett 1989, Reid









1994). While juvenile hormone analogs have application in the field, they have their

limitations. They have no ability to suppress a population quickly (Zemen, et al 1991,

Koehler and Patterson 1991) and unless more than 80% of the population is strongly

affected by the juvenile hormone analog, viable young can still be produced (Reid et al.

1994).

Today, insecticidal gel baits are the main method of German cockroach control in

the United States (Wang et al. 2004). Gel baits contain many different chemical classes:

Neonicotinoids such as imidicloprid and thiamethoxam, the avermectins such as

abamectin, the aminidinohydrazones like hydromethylnon and sulfonomides such as

sulfluramid, the phenyl pyrozoles such as fipronil, and the oxadiazines such as

indoxacarb. With the exception of oxadiazines, which have only appeared commercially

in the past year, the chemical classes became commercially available in the 1990s. One

thing all gel baits have in common is that these newer active ingredients are highly toxic

and take anywhere from hours to days to cause mortality (Scott 1991, Scott and Wen

1997, Koehler et al. 1991, Appel and Benson 1992). The benefit of slow acting active

ingredients is that significant mortality occurs in cockroaches that did not directly ingest

the gel bait. This secondary mortality occurs through three different pathways. The first

is from trampling: When contaminated cockroaches return to the harborage, they

defecate and orally secrete toxic metabolites. Secondary mortality occurs when other

cockroaches travel through these contaminated secretions and unwittingly ingest them

during grooming (Durier and Rivault 2000b, Kopanic and Schal 1997). The second

pathway is mortality from coprophagy: Since first stage nymphs survive almost

exclusively on the feces of older nymphs and adults, when feces are contaminated by









toxic metabolites, secondary mortality ensues (Kopanic and Schal 1999). The third

pathway is from cannibalism: Even with plenty of food, German cockroaches typically

consume dead or dying cockroach conspecifics. During cannibalism of contaminated

cockroaches, toxic metabolites are ingested and secondary mortality occurs (Gahlhoff et

al. 1999, Durier and Rivault 2000b).

Another benefit of the high toxicity of these new active ingredients regards

physiologically resistance. When cockroaches consume gel bait, the active ingredient is

toxic enough to deliver a lethal dose in one meal. Since physiological resistance

develops when sub-lethal doses of an active ingredient are metabolized, the heightened

toxicity of these new active ingredients should prevent resistance from occurring (Wang

et al. 2004). Indeed, in the past ten years, gel baits have been the most common and

effective control measure (Wang et al. 2004). Recently, there have been reports of

German cockroach resistance to the active ingredient fipronil; however, this resistance is

apparently due to a cross resistance from cyclodienes and not severe enough to date to

compromise the efficacy of gel baits containing fipronil (Scott and Wen 1997, Holbrook

et al. 2003).

Even with the effectiveness and high toxicity of gel baits and lack of significant

physiological resistance, control failures have been reported. The first report occurred in

the early 1990's (Silverman and Bieman 1993). This isolated strain of German

cockroach, T-164 strain, was found to be glucose averse. The active ingredients

contained within the gel baits were still toxic to this strain; however, they refused to eat

the gel bait because it contained glucose (Silverman and Bieman 1993, Silverman and

Ross 1994). This feeding deterrence in T-164 strain was so pronounced, they refused to









ingest glucose even after a nine day starvation period (Silverman and Selbach 1998).

Recently, greater numbers of control failures have been reported. In a few cases, the wild

cockroaches were harvested and subsequently reared in laboratory settings. Preliminary

studies show some similarity to T-164 strain; specifically, they are still susceptible to the

active ingredients contained within gel baits but they refuse to consume the bait. Unlike

the T-164 strain, these strains are not glucose averse. While the mechanism causing this

bait aversion is currently unknown, it is obviously behavioral in nature. Behavioral

aversion is defined as evolved behaviors that allow an insect to survive in an otherwise

lethal environment and often involves stimulus-dependent mechanisms such as

repellency, irritation (Sparks et al. 1989), or in this case, feeding deterrence. In the past,

behavioral resistance was glossed over and considered far less important than

physiological resistance (Sparks et al. 1989, Ross 1997). Today, more research is being

done on behaviorally averse strains of German cockroach to try to find a way to

overcome this resistance and again effectively manage German cockroach populations in

the field.














CHAPTER 3
EVALUATION OF FEEDING DETTERRENCE IN SIX INSECTICIDAL GEL BAITS
AND MORTALITY IN A FIELD STRAIN OF GERMAN COCKROACH, Blattella
germanica (L)

Introduction

The German cockroach, Blattella germanica L., is and has been a very important

urban pest worldwide (Cornwell 1968, Abd-Elghafar et al. 1990). As such, it has a long

and varied history of control. Early methods of control utilized insecticidal spray

formulations that began with the chlorinated hydrocarbons, progressed to

organophosphates, carbamates, and then pyrethroids. All suffered the same fate: after

about 5 years of intensive use, German cockroaches developed physiological resistance to

the chemicals (Cornwell 1976).

In the late 1970's through the 1980's, strategies involving the use of insect growth

regulators and biological control agents in the form of fungal pathogens were employed.

Nothing provided adequate measures of control until toxic baits containing new classes of

chemical compounds came on the market. While these classes all had differing modes of

action, one thing they all had in common was that they were toxic enough, when ingested

orally, to deliver a lethal dose of active ingredient in one meal (Wang et al. 2004). For

ingestion to occur, the bait had to be palatable enough to compete with other food

sources. It was believed this combination of palatability and toxicity would make

development of physiological resistance less likely (Wang et al. 2004).

In the early 1990's, there were reports of control failures in the field. By 1993,

Silverman and Bieman determined that these failures were not due to physiological









resistance but due to behavioral resistance. They isolated the first bait averse strain of

German cockroach. In this case, the aversion was to glucose contained within the bait

matrix. Since then, other strains exhibiting glucose aversion have been isolated

(Silverman and Ross 1994). In addition, many strains have been isolated that are not

simply glucose averse, but averse to one or more inert ingredients in the bait matrix

(Silverman and Ross 1994, Ross 1997a, Wang et al. 2004). Silverman and Ross (1994)

discovered a surprising level of genetic variability in the strains they studied and Wang et

al. found significantly different levels of consumption and mortality in the two strains

they studied. Though there is variability among averse strains of cockroach, one thing

remains the same: In all strains, decreased consumption of gel bait causes a decrease in

mortality, possibly leading to control failure.

Daytona field strain was collected from Daytona, FL after reports of control

failure using traditional baits. The purpose of this study was to determine if gel baits

caused feeding deterrence in Daytona field strain, to determine the effect of consumption

on mortality, and to determine if Daytona field strain is a bait averse strain of cockroach.

Materials and Methods

Insects. Orlando susceptible strain and Daytona field strain German cockroaches

were obtained from laboratory colonies maintained at the urban entomology laboratory,

University of Florida, Gainesville, FL. Rearing containers and harborages for Orlando

susceptible strain cockroaches were as described by Koehler et al. (1994) with the

exception that the harborages contained within the acrylic rack were looped cardboard

sections (14 by 13 cm). Rat food (Purina Laboratory Rodent Chow, no. 5001, Ralston

Purina, St. Louis, MO) and water was supplied ad libitum.









Rearing containers for Daytona field strain cockroaches were glass jars (7.57 liter,

22 cm diameter) greased on the inner rim with a petroleum jelly/ mineral oil (2:3)

mixture to prevent cockroach escape. Jars were covered with cotton cloth and secured

with rubber bands. Dog food (Purina One Puppy Growth and Development, Nestle

Purina PetCare Company, St. Louis, MO) and water was supplied ad libitum.

Environmental conditions for both rearing rooms were 26C and 55% relative humidity

RH, with a photoperiod of 12:12 (L:D) h.

Insectide baits. The following gel bait products were tested: Maxforce (2.15%

Hydramethylnon, Bayer Environmental Science, Montvale, NJ), Avert (0.05%

Avermectin, Whitmire Microgen Research Laboratories, Inc, St. Louis, MO), DPX-

MP062-41 la (0.6% Indoxacarb, Dupont Crop Protection, Newark, DE), Advion

Cockroach (0.6% Indoxacarb, Dupont Crop Protection, Newark, DE), Maxforce FC

(0.01% Fipronil, Bayer Environmental Science, Montvale, NJ), Maxforce FC Select

(0.01% Fipronil, Bayer Environmental Science, Montvale, NJ). Gel bait (140 to 150 mg)

was deposited from a syringe onto a piece of low nitrogen weighing paper (57 by 57 mm;

Fisherbrand, Fisher Scientific Company, USA).

Preference and consumption assay. Cockroaches were anesthetized with CO2 for 2

min and sorted through stacked 2.36 mm (No. 8, Fisher Scientific Company, Pittsburgh,

PA) and 2.00 mm (No. 10, Fisher Scientific Company, Pittsburgh, PA) standard sieves.

Cockroaches retained by the 2.00 mm sieve were placed in a greased glass holding jar

(3.79 liter, 17.5 cm diameter) containing harborage, dog food and water. Ninety percent

of retained cockroaches were 2nd stage nymphs weighing 190.24 4.53 (Orlando

susceptible strain cockroach) and 216.44 3.06 (Daytona field strain cockroach). To









recover from the effects of CO2, cockroaches were held for 48 to 72 h before placement

into foraging arenas.

After the holding period, 50 cockroaches were aspirated and placed into a greased

foraging arena containing water and harborage. Foraging arenas were clear plastic

sweater boxes (26.5 by 9.5 by 19 cm, Pioneer Plastics, Dixon, KY). Water was provided

by a plastic vial (33 by 16 mm diameter) with a cotton stopper. Due to the ability of

nymphs to enter inside the corrugations of cardboard, an index card (76.5 by 28.5 mm)

folded in half lengthwise and secured with a staple was used for harborage. This process

was repeated until there were seven arenas per strain.

Cockroaches were starved for 24 h. At the end of the starvation period, two pre-

weighed deposits of gel bait and two pre-weighed pieces of dog food were added to each

arena on a piece of weighing paper. One of the gel bait placements and one of the dog

food placements were used as moisture loss standards. Both moisture loss standards were

placed inside individual souffle cups (29.57 ml; Solo cup company, Urbana, IL.) and

cups were covered with organdy and secured with a rubber band to prevent cockroach

entry. After 24 h, both food sources and both moisture loss standards were reweighed and

food sources were returned to the arena. Mortality was recorded 4 d after gel bait

placement. Experimental set up was a randomized complete block design of six

treatments and one control. Experiment was a randomized complete block design with

six gel baits and an untreated dog food control replicated eight times per strain using a

total of 5,600 cockroaches.

Data analysis. Gel bait consumption was calculated using the following formula:

Consumption = A [A x (B D / B) C]









where; A = Pre-consumption weight of exposed bait or food, B = Pre-consumption

weight of comparable moisture loss standard, C = Post-consumption weight of exposed

bait or food, and D = Post-consumption weight of comparable moisture loss standard.

Consumption of gel bait product for each strain and each treatment was analyzed via

Student's t-test (P, 0.005; [SAS Institute, 2001]). Amount of active ingredient consumed

was calculated by multiplying the percent active ingredient in gel baits by the amount of

gel bait consumed. Mortality data was corrected using Abbott's correction (Abbott,

1925) and percentages were arcsine-root transformed before analysis. Both active

ingredient per cockroach body weight and mortality were analyzed by Analysis of

Variance and means separated by SNK (P, 0.005; [SAS Institute, 2001]).

Results

Within a few minutes of introducing food sources to the arenas, nymphs fed on

either dog food or gel bait. Orlando normal strain had no significant preference for dog

food control or dog food choice, indicating no location bias within the arena (Table 3-1).

There was no active ingredient in the control; therefore, no mortality occurred. All

control cockroaches survived until the end of the experiment. Consumption of Maxforce

gel bait was not significantly different than dog food consumption. Mean amount of

active ingredient consumed was 1009.5 ng per mg cockroach body weight and resulting

mortality was 58%. Consumption of Avert gel bait was not significantly different from

dog food consumption. Mean amount of active ingredient consumed was 22.3 ng per mg

cockroach body weight and resulting mortality was 66%. Consumption of DPX-MP062-

41 la gel bait was not significantly different from dog food consumption. Mean amount

of active ingredient consumed was 271.9 ng per mg cockroach body weight and resulting

mortality was 87%. Consumption of Advion gel bait was not significantly different from









dog food consumption. Mean amount of active ingredient consumed was 314.2 ng per

mg cockroach body weight and resulting mortality was 88%. Consumption of Maxforce

FC gel bait was not significantly different from dog food consumption. Mean amount of

active ingredient consumed was 4.2 ng per mg cockroach body weight and resulting

mortality was 81%. Consumption of Maxforce FC Select gel bait was not significantly

different from dog food consumption. Mean amount of active ingredient consumed was

4.6 ng per mg cockroach body weight and resulting mortality was 92%.

Amount of active ingredient consumed per cockroach body weight significantly

differed among products. Orlando normal strain cockroaches consumed significantly

more active ingredient from Maxforce gel bait than all other products. The order of

preference based on consumption of active ingredient was Maxforce >Advion = DPX-

MP062-41 la = Avert = Maxforce FC = Maxforce FC Select. Resulting mortality

significantly differed among products. Highest mortalities occurred with Advion

Cockroach, Dupont Formula 41 la, Maxforce FC, and Maxforce FC Select. Order of

mortality was Advion Cockroach = Dupont Formula 411 a = Maxforce FC = Maxforce

FC Select >Avert = Maxforce > Control.

Daytona field strain had no significant preference for dog food control or dog food

choice, indicating no location bias (Table 3-2). There was no active ingredient in the

control; therefore, resulting mortality did not occur. All control cockroaches survived

until the end of the experiment. Consumption of Maxforce gel bait was significantly less

than dog food consumption yielding a ratio of 1:5.2 (Maxforce:dog food), indicating

feeding deterrence to Maxforce gel bait. Mean amount of active ingredient consumed

was 326.2 ng per mg cockroach body weight and resulting mortality was 9%.









Consumption of Avert gel bait was significantly less than dog food consumption yielding

a ratio of 1:9.4 (Avert:dog food), indicating feeding deterrence to Avert gel bait. Mean

amount of active ingredient consumed was 3.1 ng per mg cockroach body weight and

resulting mortality was 20%. Consumption of PX-MP062 41 la gel bait was not

significantly different than dog food consumption. Mean amount of active ingredient

consumed was 212.3 ng per mg cockroach body weight and resulting mortality was 49%.

Consumption of Advion Cockroach gel bait was significantly greater than dog food

consumption yielding a ratio of 1.8:1 (Advion: dog food), indicating preference for

Advion gel bait. Mean amount of active ingredient consumed was 450.3 ng per mg

cockroach body weight and resulting mortality was 79%. Consumption of Maxforce FC

gel bait was significantly less than dog food consumption yielding a ratio of 1:4.6

(Maxforce FC:dog food), indicating feeding deterrence to Maxforce FC gel bait. Mean

amount of active ingredient consumed was 2.5 ng per mg cockroach body weight and

resulting mortality was 15%. Consumption ofMaxforce FC Select gel bait was

significantly greater than dog food consumption 5.3:1 (Maxforce FC Select:dog food),

indicating preference for Maxforce FC Select gel bait. Mean amount of active ingredient

consumed was 5.9 ng per mg cockroach body weight and resulting mortality was 82%.

Amount of active ingredient consumed per cockroach body weight significantly

differed among products. Daytona field strain cockroaches consumed significantly more

active ingredient from Advion gel bait than all other products. The order of preference

based on consumption was Advion > DPX-MP062-41 la = Maxforce > Avert = Maxforce

FC = Maxforce FC Select. Resultant mortality significantly differed among products.

Highest mortalies occurred with Advion gel bait and Maxforce FC Select gel bait while









lowest mortality occurred with Maxforce FC gel bait. Order of mortality was Advion =

Maxforce FC Select > DPX-MP062-41 la > Maxforce > Avert = Maxforce FC >

Control).

Discussion

Orlando susceptible strain cockroaches have never shown feeding deterrence to any

commercial gel bait formulation. When Orlando susceptible strain cockroaches were

simultaneously given a choice of six gel baits, all baits were consumed equally

(Silverman and Liang 1999). A significant consumption difference between gel baits

would indicate feeding preference or feeding deterrence. In my study, preference or

feeding deterrence was determined by comparison of consumption between gel bait and

dog food. Similar to Silverman and Liang's study, Orlando susceptible strain

cockroaches also exhibited no feeding deterrence to six gel bait formulations, including

two formulations that have not been commonly used.

A factor to take into account when performing a choice study is bait placement

because location can affect consumption. German cockroaches locate food by random

searching, so the more available and easily accessible the bait, the more likely it is to be

ingested (Rust and Reierson 1981). In my study, when Orlando susceptible strain was

given a choice between two pieces of dog food that had been placed equidistant from the

harborage, both food sources were consumed equally, indicating no location bias in the

experimental arena.

Gel baits containing hydramethylnon have been a common treatment for

susceptible strain cockroaches, do not cause feeding deterrence, and cause mortality

through time. Adult male insecticide susceptible German cockroaches exposed for 3 d to

Maxforce gel bait (2.15% hydramethylnon), readily consumed the gel bait and exhibited









no feeding deterrence; LT50 value for the uncontaminated gel bait was 4.1 d (Appel

2004). Scott (1991) determined LT50 was 76 h for adult male CSMA susceptible strain

cockroaches fed bait containing 1.56% hydramethylnon; Koehler and Patterson (1991a)

determined LT50 was 4.5 d for adult male Orlando susceptible cockroaches fed bait

containing 1.0% hydramethylnon. My study also showed no feeding deterrence to

Maxforce (2.15 % hydramethylnon) in Orlando susceptible strain cockroaches and the

resultant 4 d mortality, was 58%; which was similar to the mortalities reported above.

When dealing with an averse strain of cockroach, it is not the active ingredient that

causes feeding deterrence but the gel bait matrix. Adult male T-164 glucose averse strain

cockroaches, given a choice between dog food and 2.0% hydramethylnon gel bait with

glucose and without glucose, exhibited feeding deterrence to the gel bait with glucose and

not to the gel bait without glucose (Silverman and Liang 1999). Gel bait without glucose

caused 14 d mortality of approximately 90%. Similarly, adult male Dorie and Cincy bait

averse strains of cockroach, given a choice between rat chow and Maxforce gel bait

(2.15% hydramethylnon), exhibited feeding deterrence to the gel bait, resulting in only

10.0% and 33.3% 4 d mortality rates respectively (Wang et. al. 2004). My study showed

Daytona field strain exhibited feeding deterrence to Maxforce gel bait, consuming 5

times less bait than food. Resulting 4 d mortality was 16%, which was similar to that of

Dorie strain bait-averse cockroach.

Gel baits containing abamectin have also been a common treatment for susceptible

strain cockroaches. Koehler et al. (1991b) determined that abamectin caused feeding

deterrence at concentrations above 0.500%. The concentration of abamectin in

commonly used gel bait is much lower than this; therefore, the gel baits containing









abamectin do not cause feeding deterrence and cause cockroach mortality through time.

Adult male insecticide susceptible German cockroaches exposed for 3 d to

uncontaminated Avert gel bait (0.05% abamectin) readily consumed the gel bait and

exhibited no feeding deterrence; LT50 for the Avert gel bait was 1.05 d (Appel 2004).

Mixed sex and stage Orlando susceptible cockroaches fed gel bait containing 0.05%

abamectin readily consumed the gel bait exhibited no feeding deterrence; LT50 values

were 1.6 d for nymphs and 1.7 d for adult males (Koehler et al. 1991b). Mixed sex and

stage Navy 3 susceptible cockroaches in a choice test between gel bait containing 0.01%

abamectin and dog food readily consumed the gel bait and exhibited no feeding

deterrence; 14 d mortality rates were approximately 90% for adult males and 95% for

small (<9 d old) nymphs (Rossl993). Because Avert gel bait was consumed as readily as

dog food by Orlando susceptible strain cockroach, my study also showed no feeding

deterrence to Avert (0.05 % abamectin); however, my approximate LT50 for 2.15%

hydramethylnon would be about 1 d more than the value reported by Koehler and

Patterson.

Again, averse strains of cockroach exhibit feeding deterrence to the gel bait, not the

active ingredient. Adult male Dorie and Cincy bait averse strains of cockroach, given a

choice between rat chow and Avert gel bait did not readily consume the gel bait and

exhibited feeding deterrence, resulting in 4 d mortality rates of 69.3% and 0.0%

respectively (Wang et al. 2004). In my study, Daytona field strain consumed more than

10 times less Avert gel bait than food, exhibiting feeding deterrence. Resulting 4 d

mortality was 20%, which was similar to the mortality of Cincy bait-averse strain

cockroach.









Fipronil is a fast acting chemical that is used in gel baits for treatment of

susceptible strain cockroaches. These gel baits do not cause feeding deterrence and cause

cockroach mortality through time. Adult male insecticide susceptible German

cockroaches exposed for 3 d to uncontaminated Maxforce FC gel bait (0.01% fipronil)

readily consumed the gel bait and exhibited no feeding deterrence; LT50 for the

uncontaminated gel bait was 2.1 d (Appel 2004). Adult male Orlando susceptible strain

cockroaches, given a choice between dog food, 0.03% fipronil gel bait with glucose and

0.03% fipronil gel bait without glucose, readily consumed both gel baits and resultant 14

d mortality was approximately 60% with an LT50 of 4.5 d (Silverman and Liang 1999).

Adult male JWAX susceptible strain cockroaches, given a choice between rat chow and

Maxforce FC (0.01% fipronil), readily consumed gel bait and had resultant 4 d mortality

of 100% (Wang et al. 2004). In my study, Orlando susceptible strain consumed

Maxforce FC and Maxforce FC Select (0.01% fipronil) gel baits as readily as dog food.

Resultant mortality was 85% for Maxforce FC and 92% for Maxforce FC Select, which

was similar to the susceptible strain used in Appel's study and JWAX strain used in

Wang et al.'s study.

For averse strains of cockroach, it is the gel bait that causes feeding deterrence.

Adult male T-164 glucose averse strain cockroaches, given a choice between dog food,

0.03% fipronil gel bait with glucose and 0.03% fipronil gel bait without glucose, readily

consumed the gel bait without glucose and exhibited feeding deterrence to the gel bait

with glucose; Resulting 14 d mortality for the bait with glucose was approximately 55%

and the 14 d mortality for the bait without glucose was approximately 90% (Silverman

and Liang 1999). When adult male Dorie and Cincy bait averse strains of cockroach









were given a choice between Maxforce FC (0.01% fipronil) and rat chow, Dorie bait

averse strain cockroach readily consumed the gel bait whereas Cincy strain exhibited

feeding deterrence to the gel bait; resulting 4 d mortalities were 100.0% and 16.7%

respectively (Wang, et. al. 2004). In my study, Daytona field strain consumed 21.5 times

less Maxforce FC (0.01% fipronil) than dog food and mortality rates were 15%, which

was similar to Cincy bait averse strain cockroach; However, Daytona field strain

consumed 19 times more Maxforce FC Select gel bait (0.01% fipronil) than dog food.

Resulting 4 d mortality was 82%, which was similar to the 92% 4 d mortality of Orlando

susceptible cockroach.

Indoxacarb is a new chemical, class oxadiazine, which is biologically activated

within the insect midgut. It has not been shown to cause feeding deterrence and has been

shown to cause cockroach mortality through time (Appel 2003). Adult male American

Cyanamid susceptible strain German cockroaches had an LT50 value of .068 d to .025%

indoxacarb gel bait (Appel, 2003). In my study, Orlando susceptible strain cockroaches

readily consumed both DPX-MP062-41 la (0.6% indoxacarb) and Advion gel bait.

Resulting mortality for both gel baits was 87%.

Because of the newness of indoxacarb, there are no published studies with bait

averse cockroaches. In my study, Daytona field strain readily consumed DPX-MP062-

41 la and consumed two times more Advion than dog food. Resulting mortalities were

49% for DPX-MP062-41 la, which was less than Orlando susceptible strain cockroaches

and 79% for Advion, which is similar to Orlando susceptible strain cockroaches.

Orlando susceptible strain exhibited no feeding deterrence or preference to any

food source. One would expect gel bait to be preferred in order for it to be effective,









especially in the field. It must be noted that this study had no location bias, whereas bait

placement in the field has location bias. Additionally, Since Orlando susceptible strain

cockroaches have been reared in the laboratory since 1947 (Koehler, et. al. 1994), it is

possible they have lost the ability to discriminate between food sources. Even with the

lack of preference, Orlando susceptible strain consumed enough gel bait to cause

significant mortality for all products tested. Whereas only the gel baits specially

formulated for bait averse cockroaches (Advion Cockroach Gel and Maxforce FC Select)

had mortality rates above 50% at 4 d for Daytona field strain cockroach, indicating that

Daytona field strain is, in fact, a bait-averse strain of cockroach.

Discovery of a bait-averse strain of cockroach in Florida, so far from the strains

found around Ohio, indicates that bait aversion is a more widespread phenomenon than

once believed. This study determined consumption of gel bait was positively correlated

with mortality in German cockroaches. The bait-averse strains in both this study and that

of Wang et al. (2004) demonstrated that three bait-averse strains find reformulated baits

palatable; thus, feeding deterrence can be overcome by reformulating bait matrices. It is

unknown if bait averse strains of German cockroach also have physiological resistance;

therefore, simply reformulating gel bait matrices may not be enough for control in the

future.













Table 3-1. Mortality (4 d) and consumption of gel bait,
after gel bait placement.


dog food, and AI for 50 Orlando susceptible strain German cockroaches 24 h


Product Consumption (mg) Student's t-test ng AI consumed per

Gel Baita Dog food df t value P value mg body weight % Mortality



Control 9.34 4.37 9.27 3.31 14 0.03 0.9742 N/A 0.00 + 0.OOc

Maxforce 10.16 + 4.92 9.81 + 2.07 14 0.19 0.8540 1009.52 172.83a 58.33 + 4.76b

Avert 9.63 2.12 11.13 3.51 14 -1.03 0.3187 22.25+ 1.74c 66.22 4.24b

DPX-MP062 411A 9.81 + 3.97 6.85 + 3.27 14 1.24 0.2368 271.89 38.93bc 87.24 2.58a

Advion Cockroach 11.34 3.82 8.68 4.73 14 1.63 0.1259 314.23 37.44b 87.63 2.49a

Maxforce FC 9.14 3.63 13.46 8.56 14 -1.31 0.2097 4.16 060c 81.16 7.12a

Maxforce FC Select 9.92 + 3.75 5.56 + 5.10 14 1.95 0.0720 4.60 + 0.61c 91.81 1.17a


"Control was a piece of dog food.
Student's t- test (P > 0.005, [SAS Institute, 2001]).
Means in a column followed by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls sequential range test
[SAS Institute, 2001]).














Table 3-2. Mortality (4 d) and consumption of gel bait,
gel bait placement.


Product


Control


Maxforce


Avert


DPX-MP062 411A


Advion Cockroach


Maxforce FC


Maxforce FC Select


Consumption (mg)
Gel Baita Dog food

9.04 5.31 9.60 5.66


3.28 + 2.09 17.06 + 3.70


1.34 + 1.70 12.56 + 4.89


7.66 + 2.85 10.60 + 4.88


6.24 6.34 3.51 2.65


2.91 + 1.68 13.51 4.95


12.86 + 8.59 2.44 + 1.53


dog food, and AI for 50 Daytona field strain German cockroaches 24 h after


Student's t-test ng Al consumed per


Student's t-test
df t value P value

14 -0.20 0.8426


14 -9.17 <0.0001


8.66 -6.14 0.0002


14 -1.47 0.1628


9.38 5.24 0.0005


8.6 -5.74 0.0003


7.44 3.38 0.0107


ng AI consumed per
mg body weight*

N/A


326.21 + 73.42b


3.08 + 1.39c


212.33 + 27.95b


450.27 62.12a


2.45 + 1.08c


5.89 + 1.41c


% Mortality

0.00 + 0.00e


9.11 + 2.69d


20.34 + 3.46c


48.97 4.77b


78.91 + 1.93a


15.04 + 2.09cd


82.37 + 3.70a


"Control was a piece of dog food.
Student's t- test (P > 0.005, [SAS Institute, 2001]).
Means in a column followed by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls sequential range test
[SAS Institute, 2001]).














CHAPTER 4
ORAL TOXICITY OF INDOXACARB AND SECONDARY MORTALITY FROM
NECROPHAGY IN A SUSCEPTIBLE STRAIN AND A FIELD STRAIN OF
GERMAN COCKROACH, Blattella germanica (L)

Introduction

The German cockroach, Blattella germanica L., has been an important pest of all

urban dwellings worldwide (Comwell 1968, Abd-Elghafar et al. 1990). Over the years,

attempts at control have largely shifted from applying residuals and sprays to placement

of toxic baits (Reierson 1995). This was due in part to heightened consumer awareness

regarding pesticides and a general trend to reduce pesticide application (Kopanic and

Schal 1997).

Unlike sprays, baits are either contained in a protective unit or are placed in areas

close to the harborages and foraging areas. This highly specialized application process

has the advantage of targeting the pest species while simultaneously allowing for use of

less chemical (Kopanic and Schal 1997). There are many active ingredients currently

employed in baits used for German cockroach control, but bait is only effective if it is

palatable and the active ingredient is not a feeding deterrent (Appel 1990). Current active

ingredients are toxic enough to deliver a lethal dose in one meal (Wang et al. 2004).

However, they act slowly enough to allow time for the cockroach to return to the

harborage after feeding. Since German cockroaches are gregarious and not social, it was

once thought this was of no consequence. However, secondary transmission in the

German cockroach has been well documented and proved to be an important factor in

control. Kopanic and Schal (1999) showed significant mortality in first and second stage









nymphs due to the ingestion of contaminated feces. Gahlhoff et al. (1999) showed

significant mortality of adult and nymphal cockroaches due to consumption of

contaminated conspecifics.

There is a new class of chemical available in toxic baits, oxadiazines. Indoxacarb,

the only current oxadiazine, is bioactivated (Wing 1999). The purpose of this study was

to determine if indoxacarb was a feeding deterrent, determine the oral toxicity of

indoxacarb, and evaluate secondary mortality from necrophagy in a susceptible strain and

a field strain of German cockroach.

Materials and Methods

Insects. Orlando susceptible strain and Daytona field strain German cockroaches

were obtained from laboratory colonies maintained at the urban entomology laboratory,

University of Florida, Gainesville, FL. Rearing containers and harborages for Orlando

susceptible strain cockroaches were as described by Koehler et al. (1994) with the

exception that the harborages contained within the acrylic rack were looped cardboard

sections (14 by 13 cm). Rat food (Purina Laboratory Rodent Chow, no. 5001, Ralston

Purina, St. Louis, MO) and water was supplied ad libitum.

Rearing containers for Daytona field cockroaches were glass jars (7.57 liter, 22 cm

diameter) greased on the inner rim with a petroleum jelly/ mineral oil (2:3) mixture to

prevent cockroach escape. Jars were covered with cotton cloth and secured with rubber

bands. Dog food (Purina One Puppy Growth and Development, Nestle Purina PetCare

Company, St. Louis, MO) and water was supplied ad libitum. Environmental conditions

for both rearing rooms were 26C and 55% relative humidity RH, with a photoperiod of

12:12 (L:D) h.









Oral toxicity assay. Adult male cockroaches were pulled without CO2 and placed

into a greased plastic holding container (0.946 liter, 140 by 113 mm diameter) containing

harborage and water. Cockroaches were starved for 24 h.

During the starvation period, one breadcrumb was placed in the bottom of a cell

culture cluster well (COSTAR Model number 3524, Corning Incorporated, Corning,

NY). The breadcrumb was treated with 1 [tl of chemical solution. Solutions were derived

from Termidor (9.1 % fipronil, Bayer Environmental Science, Montvale, NJ), diluted in

water and technical grade indoxacarb (56.2%, Dupont Crop Protection, Newark, DE),

diluted in acetone. Each chemical had its own set of culture clusters. The solution treated

breadcrumb was allowed to dry for at least 4 hrs. The breadcrumb was then treated with

1 [tl of 10% sucrose solution and allowed to dry.

At the end of the starvation period, individual cockroaches were removed from the

holding container and placed into souffle cups (59.147 ml, Polar size "G", Polar

Plastique, St-Laurent, QUE) containing treated breadcrumbs and moistened cotton balls

(-5 mm diameter). Cup lids were immediately secured to prevent cockroach escape.

Cockroach weights were recorded per treatment and concentration. Cups were set aside

for 24 h to allow cockroaches time to feed.

At the end of the feeding period, cockroaches that had consumed the entire

breadcrumb, were released into a greased sweater box (26.5 by 9.5 by 19 cm, Pioneer

Plastics, Dixon, KY). Each sweater box contained a plastic tube (33 by 16 mm diameter)

of water with a cotton plug, and cardboard sections (14 by 13cm) folded in half

lengthwise secured with a staple for harborage. There were separate arenas for each

treatment concentration.









Mortality was counted 4 d after end of feeding period. All moribund cockroaches

(defined as an inability to walk) were considered dead.

Necrophagy assay. Twenty adult male cockroaches were pulled without CO2 and

placed into a foraging arena, which was a greased clear plastic sweater box (26.5 by 9.5

by 19 cm, Pioneer Plastics, Dixon, KY). Each arena contained a plastic tube (33 by 16

mm diameter) of water with a cotton plug, and cardboard sections (14 by 13cm) folded in

half lengthwise secured with a staple for harborage. This process was repeated until there

were three arenas per strain. Cockroaches were starved for 24 hours.

At the end of the starvation period, cockroaches were fed thawed 2nd-3rd stage

nymphal German cockroaches that had previously ingested Formula 411 a gel-bait (0.6%

Indoxacarb, Dupont Crop Protection, Newark, DE) or Maxforce FC gel-bait (0.05%

Fipronil, Bayer Environmental Science, Montvale, NJ). Upon death, nymphs were frozen

to preserve freshness. Control nymphs were gathered from the rearing containers and

frozen. Initially, each arena received ten appropriately treated nymphs. Thereafter, each

arena received seven nymphs daily for four days.

Number of nymphs cannibalized was recorded per day and uneaten nymphs were

removed from arena. Moribund cockroaches unable to walk were considered dead.

Evaluation was ended on day 5.

Experiment was a randomized complete block design with six replicates per strain

per treatment for a total of 240 cockroaches.

Statistical analyses. Percent consumption of breadcrumbs was analyzed using

Analysis of variance and Student's t-test (P<0.05; SAS Institute 2001). Lethal dose

values were determined using probit analysis (SAS Institute 2001). Necrophagy and









resultant mortality were analyzed via Analysis of variance and means separated with

Student Newman Keuls (P<0.05; SAS Institute 2001).

Results

Oral toxicity assay. After 24 h in a souffle cup with only a treated breadcrumb as a

food source, Orlando susceptible strain cockroaches ate 96% of all treated and untreated

breadcrumbs whereas Daytona field strain ate 85% of untreated and fipronil treated

breadcrumbs and 93% of indoxacarb treated breadcrumbs. Feeding deterrence in

Orlando susceptible strain cockroaches was not caused by increasing the concentration of

fipronil or from increasing the concentration of indoxacarb (Fig 4-1). Feeding deterrence

in Daytona field strain cockroaches was not caused by increasing the concentration of

fipronil or from increasing the concentration of indoxacarb (Fig. 4-2).

Cockroaches that consumed the breadcrumb were included in an oral toxicity assay.

Orlando susceptible strain cockroaches had LD50 values of 0.072 ng per mg body weight

and LD90 values of 0.108 ng per mg body weight for fipronil and LD50 values of 1.312 ng

per mg body weight and LD90 values of 4.104 ng per mg body weight for indoxacarb

(Table 3-2). Daytona field strain cockroaches had LD50 values of 0.656 ng per mg body

weight and LD90 values of 4.063 ng per mg body weight for fipronil and LD50 values of

4.653 ng per mg body weight and LD90 values of 18.045 ng per mg body weight for

indoxacarb. Resistance ratios at the LD50 level/ LD90 level for Daytona field strain were

9.4/ 36.9 for fipronil and 3.5/ 4.4 for indoxacarb.

Necrophagy assay. Upon placing treated nymphs into the arena, both strains of

cockroach investigated the bodies, but no immediate consumption was observed. When

given a choice, Orlando susceptible strain cockroaches consumed a constant amount of

control nymphs (df=4, F=2.07, P=0.1152), ranging from 1.1 to 2.4 nymphs per day









(Table 4-2), for a total 5 d consumption of 8.9 nymphs. Orlando susceptible strain

cockroaches consumed 1.2 fipronil treated nymphs on day one of the choice experiment

and consumption decreased significantly during days two through five, ranging from 0.1

to 0.4 nymphs per day for a total 5 d consumption of 2.1 nymphs. Orlando susceptible

strain cockroaches consumed a constant amount ofindoxacarb treated nymphs (df=4,

F= 1.97, P=0.1299) in the choice test, ranging from 0.2 to 1.2 nymphs per day, for a total

5 d consumption of 2.8 nymphs. When no choice was provided, Orlando susceptible

strain cockroaches consumed a constant amount of control nymphs (df=4, F=0.82,

P=0.5235), ranging from 4.8 to 6.3 nymphs per day, for a total 5 d consumption of 29.4

nymphs. Orlando susceptible strain cockroaches consumed a constant amount of fipronil

treated nymphs (df=4, F=0.66, P=0.6225) in the no choice test, ranging from 3.3 to 4.8

nymphs per day, for a total 5 d consumption of 19.1 nymphs. Orlando susceptible strain

cockroaches consumed a constant amount ofindoxacarb treated nymphs (df=4, F=1.58,

P=0.2095) in the no choice test, ranging from 2.8 to 5.0 nymphs per day, for a total 5 d

consumption of 19.3 nymphs.

When given a choice, Daytona field strain consumed a constant amount of control

nymphs (df 4, F 0.77, P=0.5530), ranging from 0.4 to 1.2 nymphs per day (Table 4-3),

for a total 5 d consumption of 4.1 nymphs. Daytona field strain consumed a constant

amount of fipronil treated nymphs (df=4, F 0.31, P=8653) in the choice test, ranging

from 0.3 to 0.6 nymphs per day, for a total 5 d consumption of 1.8 nymphs. Daytona

field strain also consumed a constant amount of indoxacarb treated nymphs (df=4,

F=1.22, P=0.3273) in the choice test, ranging from 0.3 to 0.8 nymphs per day, for a total

5 d consumption of 2.2 nymphs. When no choice was provided, Daytona field strain









consumed a constant amount of control nymphs (df=4, F=2.11, P=0.1096), ranging from

1.5 to 4.3 nymphs per day for a total 5 d consumption of 13.9 nymphs. Daytona field

strain consumed a constant amount of fipronil treated nymphs (df=4, F=0.25, P=0.9044)

in the no choice test, ranging from 1.3 to 1.9 nymphs per day, for a total 5 d consumption

of 8.5 nymphs. Daytona field strain also consumed a constant amount of indoxacarb

treated nymphs (df=4, F=2.10, P=0.1107) in the no choice test, ranging from 1.0 to 2.7

nymphs per day, for a total 5 d consumption of 8.0 nymphs.

Low mortality of 1.3% occurred in Orlando susceptible strain cockroach at 5 d with

choice control and there was no significant increase in mortality from 1 d (df=4, F 1.19,

P=0.3393) (Table 4-4). Mortality of 6.0% occurred in Orlando susceptible strain

cockroach at 5 d with choice fipronil and there was no significant increase in mortality

from 1 d (df 4, F=2.41, P=0.0760). Mortality of 8.7% occurred in Orlando susceptible

strain cockroach at 5 d with choice indoxacarb and there was significant increase in

mortality from day one. Orlando susceptible strain cockroach had mortality of 6.0% by

day five with no choice control and no significant increase in mortality from 1 d (F 1.82,

DF=4, P<0.05). Mortality of 47.3% occurred in Orlando susceptible strain cockroach at

5 d with no choice fipronil and there was significant increase in mortality from Id.

Mortality of 36.7% occurred in Orlando susceptible strain cockroach at 5 d with no

choice indoxacarb and there was significant increase in mortality from day one.

For Orlando susceptible strain cockroach, mortality increased linearly for control,

fipronil, and indoxacarb in both choice and no choice experiments (Fig 4-3). Highest

slopes, indicating highest mortality rates, were no choice fipronil and no choice

indoxacarb. Lowest slope, indicating least amount of mortality, was choice control. All









slopes were negative, indicating a delay in mortality, however, the largest delay, occurred

with no choice indoxacarb. Intercepts indicate a delay of approximately 1 d after start of

the experiment for all treatments with the exception of no choice indoxacarb, which had a

delay of almost 1.5 d.

Low mortality of 2.0% occurred in Daytona field strain cockroach at 5 d with

choice control and there was no significant increase in mortality from 1 d (df=4, F=0.49,

P=0.7413) (Table 4-5). Mortality of 8.0% occurred in Daytona field strain cockroach at

5 d with choice fipronil and there was no significant increase in mortality from 1 d (df=4,

F=1.76, P=0.1684). Mortality of 9.3% occurred in Daytona field strain cockroach at 5 d

with choice indoxacarb and there was significant increase in mortality from 1 d.

Mortality of 20.8% occurred in Daytona field strain cockroach at 5 d with no choice

control and there was significant increase in mortality from day one. Mortality of 31.6%

occurred in Daytona field strain cockroach at 5 d with no choice fipronil and there was

significant increase in mortality from 1 d. Mortality of 26.0% occurred in Daytona field

strain cockroach at 5 d with no choice indoxacarb and there was significant increase in

mortality from 1 d.

For Daytona field strain cockroach, mortality increased linearly for control,

indoxacarb, and fipronil in both choice and no choice experiments (Fig 4-4). Highest

slope was no choice fipronil, followed by no choice indoxacarb and no choice control.

Lowest slope was choice control. All slopes were negative; however, the largest delay,

occurred with no choice indoxacarb and no choice control. Intercepts indicate a delay of

approximately 1 d after start of the experiment for choice control, choice fipronil, and no

choice fipronil. A delay of 1.25 to 1.5 d occurred with both no choice and choice









indoxacarb. Greatest delay, approximately 2 d after start of experiment, was no choice

control.

Discussion

Oral toxicity assay. Insecticidal concentration can reduce palatability of a toxic

bait base. When Orlando susceptible strain German cockroaches were included in a

lethal time test with concentrations of abamectin ranging from 0.0025% to 0.1000%,

LT50 values failed to significantly decrease above concentrations of 0.0500% (Koehler et

al. 1991). It was determined this was due to feeding deterrence caused by abamectin in

concentrations above 0.0500%. In my oral toxicity study, bait consumption did not

significantly decrease as fipronil concentrations increased 2 fold for Orlando susceptible

strain cockroach and 13 fold for Daytona field strain cockroaches. Additionally, bait

consumption did not significantly decrease as indoxacarb concentrations increased 10

fold for Orlando susceptible strain cockroach and 6 fold for Daytona field strain

cockroaches. This indicates neither fipronil nor indoxacarb are feeding deterrents.

LD50 values are usually obtained by topical application of insecticides. Topical

LD50 value for fipronil was 0.096 ng per mg body weight for Orlando susceptible strain

cockroach (Valles et al. 1997). The same study also injected fipronil directly and

determined the injected LD50 value was 0.081 ng per mg body weight. My study, which

required ingestion of fipronil, determined LD50 value for fipronil was 0.07 ng per mg

body weight for Orlando susceptible strain cockroach. Overlapping confidence intervals

between Valles' study and my study indicate LD50 value for ingested fipronil is equal to

the LD50 value of injected fipronil.

When Dorie and Cincy bait-averse strain cockroaches were compared to JWAX

susceptible strain cockroach for topically applied fipronil, resistance ratios at the LD50 /









LD90 level were 8.7x / 44.9x for Cincy bait-averse strain cockroach and 9.3x / 52.7x for

Dorie bait averse stain cockroach (Wang 2004). Although my study determined Daytona

field strain cockroach exhibited a resistance ratio at the LD50 level very similar to Dorie

bait-averse strain cockroach, it must be noted that Orlando susceptible strain cockroach

was used for comparison. There is inherent variability in strains of German cockroach.

When Orlando susceptible strain cockroach and JWAX susceptible strain cockroach were

compared to each other in a lethal time test using five carbamates, susceptible strain

cockroach exhibited 1.2 to 2 fold tolerance to four of the carbamates, indicating that

overall, Orlando susceptible strain cockroach is slightly more tolerant of insecticides

(Koehler and Patterson 1986). This was true in my study; upon comparison of LD5o and

LD90 levels of Orlando susceptible strain cockroach obtained in my study and values

obtained for JWAX susceptible strain cockroach in Wang et al's study, Orlando

susceptible strain cockroach exhibited 2.3 fold and 2.6 fold tolerance to fipronil

respectively. Therefore, even though resistance ratios for Daytona field strain cockroach

were similar to Dorie bait-averse strain cockroach, when comparison strains are taken

into account and direct comparison is made, Daytona field strain cockroach is 2.4x more

resistant to fipronil than Dorie bait-averse strain cockroach. Additionally, Daytona field

strain cockroach exhibited a low level of resistance (RR5o = 3.5) to indoxacarb when

compared to Orlando susceptible strain cockroaches.

When comparing resistance ratios, it is important to note differences of comparison

susceptible strains used. It is also important to note method of application for lethal dose

assays since it appears ingested lethal doses are significantly different from topically

applied. Even with the lower ingested LD50 value for fipronil and compared to a tolerant









susceptible strain, Daytona field strain cockroach had resistance ratios, especially at the

LD90 level that could possibly interfere with control in the field.

Necrophagy assay. German cockroaches are known to consume conspecifics, a

process known as cannibalism. More specifically, when the cannibalized cockroaches

are dead, it is known as necrophagy. When twenty adult male Orlando susceptible strain

cockroaches were given a no choice test wherein the only food source was freshly dead

nymphs, necrophagy was relatively low on the first day and steadily increased (Gahlhoff

et al. 1999). Conversely, necrophagy in my study remained constant over the 5 d period

for both Orlando susceptible strain cockroach and Daytona field strain cockroach, with

the exception of choice fipronil in Orlando susceptible strain cockroach, which

decreased. Although my experimental design was different from Gahlhoff et al's (1999)

in that I fed fewer nymphs over the course of only 5 d, used two different strains, and

offered a choice in addition to no choice, our results were similar because in both cases,

necrophagy occurred.

German cockroaches can go several days before starving to death. In Gahlhoff et

al.'s (1999) study, control mortality was approximately 2% on day five and increased to

10% on day seven, indicating starvation. In my no choice study, control mortality was

not significant at 5 d in Orlando susceptible strain cockroach; however, significant

control mortality occurred at 4 d in Daytona field strain cockroach. Because adult male

field cockroaches increase foraging distance, time spent foraging, and movement velocity

under the influence of starvation (Barcay and Bennett 1991), it is likely Daytona field

strain cockroaches starved themselves while foraging for more nutritious food while

Orlando susceptible strain did not.









Fipronil is a fast acting insecticide known to cause secondary mortality

(Buczkowski et al. 2001, Durier and Rivault 2000b, Gahlhoff 1999). In Gahlhoff et al.'s

(1999) study on cannibalization, mortality from fipronil was approximately 30% on day

two and increased to 100% by day five. Similarly, in my no choice study, mortality

caused by ingestion of fipronil treated nymphs increased linearly over time. Linear

increase probably occurred due to constant necrophagy. Additionally, significant

secondary mortality from fipronil occurred in both Orlando susceptible strain cockroach

and Daytona field strain cockroach at 3 d. No significant mortality occurred from

fipronil treated nymphs in the choice test for either Orlando susceptible strain cockroach

or Daytona field strain cockroach.

Indoxacarb is a chemical that requires bioactivation to become effective (Wing et

al. 1998). Using adult male American Cyanamid strain cockroaches, Appel (2003)

determined the LT50 value of 0.25% indoxacarb was 0.68 days. In my study, only

secondary mortality was recorded, and similar to the findings of Appel (2003), mortality

from ingestion of indoxacarb treated nymphs was delayed. Significant secondary

mortality from indoxacarb treated nymphs occurred in the no choice study at 3 d for

Orlando susceptible strain cockroach and 4 d for Daytona field strain cockroach.

Significant secondary mortality from indoxacarb treated nymphs occurred in the choice

study at 4 d for Orlando susceptible strain cockroach and 5 d for Daytona field strain

cockroach.

Secondary mortality from ingestion of indoxacarb treated nymphs was delayed and

significant in both choice and no choice experiments for both Orlando susceptible strain

cockroach and Daytona field strain cockroach; however, in the no choice test, for









Orlando susceptible strain, significant secondary mortality occurred on the same day as

significant secondary mortality from ingestion of fipronil treated nymphs. This is

perhaps due to the presence of already bioactivated indoxacarb metabolite in the midgut

and fat bodies of the cannibalized nymphs. Significant secondary mortality occurred in

no choice tests for fipronil for both Orlando susceptible strain cockroach and Daytona

field strain cockroach. Significant secondary mortality in choice experiments only

occurred for indoxacarb in both Orlando susceptible strain cockroach and Daytona field

strain cockroach.

Necrophagy causes significant secondary mortality in laboratory settings (Gahlhoff,

et al. 1999). My study has shown that, while necrophagy occurs both in the presence or

absence of food, availability of food is important to significant secondary mortality. If

another food source is readily available, German cockroaches engage less in necrophagy.

In the absence of convenient food in a laboratory setting or a field setting, necrophagy

could play a significant role in secondary mortality.
















y = 0.1135Ln(x) + 1.8674
R2 = 0.6354


1.01

0.99

0.97

- 0.95
I 0.93

o 0.91
.-
. 0.89
" 0.87

0.85

0.83

0.81


0.0002 0.00025 0.0003 0.00035


0.0004 0.00045 0.0005 0.00055


Concentration (%AI)


y = -0.0076Ln(x) + 0.9405
R2 = 0.1133
* *


Orlando susceptible strain
Indoxacarb


0 0.01 0.02 0.03 0.04 0.05
Concentration (% AI)


0.06 0.07 0.08


Figure 4-1. Proportion fipronil and indoxacarb treated breadcrumbs eaten by Orlando
susceptible strain cockroaches after a 24 h starvation period.


Orlando susceptible strain
Fipronil


1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81











Daytona field strain
Fipronil





y = 0.0099Ln(x) + 0.907
R2 = 0.2237

**


0 0.001 0.002 0.003 0.004 0.005 0.006
Concentration (% AI)


0.007 0.008 0.009


Daytona field strain
Indoxacarb


1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81


y = 0.0321 Ln(x) + 1.0338
R2 = 0.1745
_


0 0.01


0.02


0.03


0.04


0.05


0.06


0.07


Concentration (% Al)



Figure 4-2. Proportion fipronil and indoxacarb treated breadcrumbs eaten by Daytona
susceptible strain cockroaches after a 24 h starvation period.


1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81













Table 4-1. Susceptibility of Orlando susceptible and Daytona field strains of German cockroach to two ingested insecticides.

Lethal dose (ng/mg)a Model fit
Insecticide Strain n Slope + SE LD5o (95% FL) LD9o (95% FL) RR50b RR9b x2 P

Fipronil Orlando 300 7.220 + 0.954 0.072 (0.068-0.076) 0.108 (0.971-0.128) 1.0 1.0 0.7079 0.4001

Daytona 340 1.618 + 0.201 0.656 (0.535-0.822) 4.063 (2.634-8.071) 9.4 36.9 2.2410 0.5239



Indoxacarb Orlando 370 2.588 0.310 1.312 (1.128-1.488) 4.104 (3.329-5.634) 1.0 1.0 5.3696 0.2514

Daytona 354 2.177 0.301 4.653 (3.833-5.410) 18.045 (13.836-27.925) 3.5 4.4 0.9641 0.8099



a Dose nanogramss of insecticide per mg of insect) calculated based on body weights. Average body weights (mean SE) per strain (n = 982) were Orlando,
47.29 0.28 and Daytona, 50.93 0.27 mg.
b Resistance ratio based on LD50/ LD 90 values compared with Orlando strain.












Table 4-2. Daily consumption of insecticide treated nymphs by Orlando susceptible strain German cockroaches.

Choice No Choice

Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb

1 2.42 + 0.33 1.17 + 0.46a 0.83 + 0.17 6.17 + 0.79 4.75 0.94 5.00 + 1.28

2 1.08 + 0.27 0.42 + 0.24b 0.42 0.20 4.83 + 0.88 3.50 + 0.39 3.58 + 0.57

3 2.17 0.33 0.08 0.08b 0.17 0.11 6.00 0.56 3.92 0.69 4.50 0.65

4 1.58 + 0.44 0.17 + 0.17b 0.25 + 0.11 6.17 + 0.40 3.33 + 0.49 3.50 + 0.26

5 1.67 0.42 0.25 0.11b 1.17 0.60 6.25 0.51 3.58 0.80 2.67 0.44


Means in a column followed by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls sequential range test
[SAS Institute, 2001]).














Table 4-3. Daily consumption of insecticide treated nymphs by Daytona field strain German cockroaches.

Choice No Choice

Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb

1 0.92 + 0.35 0.58 + 0.37 0.83 + 0.28 2.17 + 0.48 1. 83 + 0.40 1.58 + 0.27

2 1.00 0.41 0.33 0.17 0.42 0.20 1.50 0.52 1.25 0.60 1.08 0.58

3 0.58 + 0.33 0.33 + 0.25 0.25 + 0.11 2.58 + 0.55 1. 92 + 0.61 1.67 + 0.56

4 0.42 + 0.20 0.17 + 0.17 0.25 + 0.17 3.42 + 1.02 1.75 + 0.59 2.67 0.49

5 1.17 + 0.42 0.42 + 0.33 0.42 0.27 4.25 0.95 1.75 0.31 1.00 + 0.29














Table 4-4. Cumulative daily mortality from ingestion of insecticide treated nymphs in Orlando susceptible strain German cockroaches.

Choice No Choice

Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb


1 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00a 0.00 0.00 3.33 + 1.23a 0.00 0.OOa

2 0.00 + 0.00 2.67 + 0.84 3.33 1.91ab 1.33 + 0.84 11.33 + 3.17a 4.00 + 1.79a

3 0.37 + 0.67 4.67 + 1.61 4.00 + 1.79ab 1.33 + 0.84 22.00 + 3.39b 12.67 + 2.81b

4 1.33 + 0.84 5.33 + 1.98 6.00 + 1.71ab 4.00 + 2.53 33.33 + 3.37c 22.00 3.06c

5 1.33 + 0.84 6.00 + 2.25 8.67 + 2.40b 6.00 + 2.88 47.33 + 2.81d 36.67 3.78d


Means in a column followed
[SAS Institute, 2001]).


by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls sequential range test












Table 4-5. Cumulative daily mortality from ingestion of insecticide treated nymphs in Daytona field strain German cockroaches.

Choice No Choice

Day Control Fipronil Indoxacarb Control Fipronil Indoxacarb


1 0.00 + 0.00 1.33 + 0.84 0.00 0.OOa 0.67 0.67a 2.03 + 0.91a 1.33 + 0.84a

2 0.67 0.67 2.67 1.33 0.00 0.00Oa 0.67 0.67a 8.08 1.83ab 4.00 1.79a

3 1.33 + 1.33 6.00 + 3.06 3.33 1.23a 3.39 + 0.68a 13.50 + 3.63b 8.67 + 3.33ab

4 1.33 + 1.33 8.00 + 2.73 7.33 1.91b 12.84 + 1.22b 22.83 + 2.88c 18.00 + 5.54bc

5 2.00 + 1.37 8.00 2.73 9.33 1.33b 20.84 2.71c 31.61 2.99d 26.00 3.83c


Means in a column followed
[SAS Institute, 2001]).


by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls sequential range test















S y = 11x- 9.536
0 R2 = 0.9904


* Choice Control
* Choice Fipronil
Choice Indoxacarb
No Choice Control
x No Choice Fipronil
* No Choice Indoxacarb


y =9.134x 12.334
R2 = 0.959


50

45

40

35

. 30

0
20

15

10

5

0


Figure. 4-3. Percent mortality of Orlando susceptible strain cockroaches from ingestion of insecticide treated nymphs.


y =2.001x 1.603
R2 = 0.9658
y =1.466x 0.664
R2 = 0.9094
y = 1.467x 1.869
R2= 0.9165
y = 0.399x 0.591


1 2 3 4 5

Days













35
Choice Control
Choice Fipronil y = 7.391x -6.563
30 Choice Indoxacarb R2 = 0.9866
No Choice Control
25 No Choice Fipronil y = 6.334x 7.402
No Choice Indoxacarb R2 = 0.9549
S 20 y = 8.725x 22.543
SR2 = 0.9977
0
15
y = 1.867x 0.401
10 R2 = 0.9246
y = 2.599x 3.799
R2 = 0.9389
5-
y = 0.466x 0.332
0 R2 = 0.9421

1 2 3 4 5

Days


Figure. 4-4. Percent mortality of Daytona field strain cockroaches from ingestion of insecticide treated nymphs.














CHAPTER 5
CONCLUSION

German cockroaches are major pests of households and structures. The common

method of control is the use of gel bait. Recently, reports of control failure were

reported. In two cases, the cause of control failure was attributed to behavioral aversion,

a fairly new phenomenon in German cockroaches. It is unknown if the feeding

deterrence exhibited by bait averse strains of German cockroach can be overcome by

reformulating the gel bait matrix. It is also unknown if bait averse strains of German

cockroach also have physiological resistance. For these studies, a field strain of

cockroach was collected from Daytona, FL in an area that reported control failure using

gel bait. This strain, Daytona field strain, was determined to be bait averse.

Chapter 3 evaluated feeding deterrence to six different commercially available gel

bait formulations and concluded that consumption was positively correlated with

mortality. Daytona field strain cockroach exhibited feeding deterrence to four of the six

gel baits. Two gel baits did not cause feeding deterrence and therefore, had the highest

levels of mortality; indicating feeding deterrence can be overcome by reformulating bait

matrices

Chapter 4 evaluated the oral toxicity ofindoxacarb and fipronil to both Orlando

susceptible strain cockroach and Daytona field strain cockroach. At the LD90 level,

Daytona field strain cockroach exhibited low levels of physiological resistance to

indoxacarb and high levels of resistance to fipronil. Chapter 4 also evaluated the ability

of fipronil and indoxacarb to cause secondary mortality from necrophagy. Necrophagy









occurred in both strains in the presence and absence of food choice. Significant

secondary mortality from ingestion of fipronil treated nymphs only occurred in the no

choice test for both strains. Significant secondary mortality from ingestion of indoxacarb

treated nymphs occurred in both choice and no choice tests for both strains; however,

mortality occurred significantly faster in the no choice test for both strains.

Overall, control failures in the field can be attributed to the combination of

behavioral and physiological resistance. Changing gel bait formulations and ingredients

can overcome these failures. Because the availability of food significantly affects

secondary mortality, control measures will be more effective if sanitation is practiced

along with application of gel baits.

Our bait averse field strain was collected in Florida whereas the only other isolated

strains of bait averse cockroach were from Ohio. This indicates bait aversion is more

widespread than once believed. While gel baits can be formulated so that bait averse

cockroaches consume them, physiological resistance and insignificant secondary

mortality from necrophagy can still affect control in the long term. This study has

revealed that there is more to behaviorally averse cockroaches than decreased

consumption of gel bait.
















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BIOGRAPHICAL SKETCH

Linda Anne NcHerne, daughter of Harry and Jessie Riley, was born in 1969. She

graduated from Bayshore High School in Bradenton, FL, in 1987. She enlisted in the US

Army in 1988 and performed her duties as a laboratory technician in Landstuhl,

Germany. She graduated from the University of South Florida in 1996 with a bachelor's

in environmental science/ zoology. She joined the US Navy in 1998 and was

commissioned in December of that year. During her time, she ran an office for the

training of future military pilots in Pensacola, FL, and was a deck officer and a weapons

officer onboard the USS Germantown stationed in Sasebo, Japan. Desiring to become a

Medical Entomologist with the US Navy, she entered the University of Florida's graduate

program in August of 2003.