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LE’BRARY Michigan State University ‘ PLACE ll RETURN BOX to romovo this checkout from your rooord. To AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE I—_II L—:_I ——I[____ [:27 MSU IoAnNflrmdlvo Action/Equal Opportunlty Immnlon mma THE BACHL US IHURHVGIENSLS' RESISTANT STRAIN OF COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE): A SEARCH FOR SYNERGISM EFFECTS USING NOVEL INSECTICIDES By Y. Andi Trisyono A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1994 ABSTRACT THE BACHL US IHURHVGENSLS' RESISTANT STRAIN OF COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE): A SEARCH FOR SYNERGISM EFFECTS USING NOVEL INSECTICIDES By Y. Andi Trisyono Colorado potato beetle has developed resistance to all synthetic and microbial insecticides registered for its control. After 34 generations of laboratory selection with the cryIIIA 5- endotoxin of Bacillus thuringiensis, a field derived strain was 714 times more resistant than the unselected (susceptible) strain. The resistance development has pleiotropic effects that reduce the size of egg masses and fecundity, reduce larval weight gain, and result in longer time to complete a generation. To determine correlation with B. thuringiensis resistance in Colorado potato beetle, the toxicity of four insect growth regulators (diflubenzuron, chlorfluazuron, cyromazine and azadirachtin) and a nerve poison, imidacloprid, were tested against both strains. Bioassays were conducted on second instars using leaf-dip or droplet assays. All five insecticides were efl'ective in controlling the susceptible as well as the resistant strains. The resistance strain was seven times more susceptible to diflubenzuron than the unselected strain. Yet the resistant strain demonstrated some degree of tolerance to chlorfluazuron, cyromazine, and azadirachtin in the early Observation period. Combinations of diflubenzuron or cyromazine with B. thufingiensis are not recommended since most of the tested combinations result in antagonism. This thesis is dedicated to my parents who introduced me to the difliculties of controlling insect pests, my wife and my children who always give me spirit, and my brothers and sister for their prayer. iv SO I say to you: Askanditwillbegiventoyou; seekandyouwillfind; knock and the door will be opened to you (Luke 11:9). Make every cfl‘ort to enter through the narrow door, because, many, I tell you, will try to enter and will not be able to (Luke 13: 24) ACKNOWLEDGMENTS I would like to thank to my major advisor, Dr. Mark E. Whalon, for his advice on a personal level and his guidance and support on an academic level throughout my master's program. I thank him for making me aware about the seriousness of the resistance problems and the importance of resistance management program I sincerely appreciate the suggestions and comments from the members of my graduate committee, Dr. Edward J. Grafius and Dr. Leah S. Bauer of the Department of Entomology, and Dr. James H. Asher of the Department of Zoology, Michigan State University. I would also like to thank to Mycogen, Uniroyal, Ciba-Geigy, W. R Grace, and Miles Corporations who provided technical grade and formulated insecticides to our laboratory. Finally, I thank to Utami Rahardja, Debbie Norris, Dr. Michael R Bush, and Pat Bill. Without their help, this research would not have been possible. TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................... x LIST OF FIGURES ....................................................................................................... xii CHAPTER I. GENERAL LITERATURE REVIEW ........................................................ 1 A INTRODUCTION ........................................................................................... 2 1. Insecticide Resistance in Colorado Potato Beetlle .................................. 2 2. Pest Management for Colorado Potato Beetle ....................................... 4 a Mechanical control .................................................................... 4 b. Cultural/mechanical control ....................................................... 5 c. Biological control ...................................................................... 5 d. Host plant resistance .................................................................. S c. Insecticidal control ..................................................................... 6 B. OVERALL GOAL AND OBJECTIVES ........................................................ 10 1. Overall Goal ........................................................................................ 10 2. Objectives ........................................................................................... 10 C. HYPOTHESIS .............................................................................................. 11 LITERATURE CITED ....................................................................................... 12 CHAPTER II. THE BACILLUS IHURINGIENSIS RESISTANT STRAIN OF COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE) ....... 17 A INTRODUCTION ................. ' ........................................................................ 1 s B. LITERATURE REVIEW .............................................................................. 20 l. Bacillus thuringiensis .......................................................................... 20 a. Classification ........................................................................... 20 b. Mode of action ........................................................................ 26 c. Host range of cryIIlA ............................................................... 29 2. Insect resistance to B. tlmrr‘ng‘iensis ..................................................... 29 C. MATERIALS AND METHODS ................................................................... 32 1. Cultures and Rearing ........................................................................... 32 a. Potato plants ............................................................................ 32 b. Colorado potato beetle ............................................................ 32 2. Bioassay .............................................................................................. 33 a. Selections ................................................................................ 33 c. Resistance progression ............................................................. 34 3. Life cycle ............................................................................................ 34 4. Data Analysis ...................................................................................... 35 D. RESULTS AND DISCUSSION .................................................................... 36 1. Selections ............................................................................................ 36 2. Resistance Progression ....................................................................... 37 3. Life Cycle ........................................................................................... 44 LITERATURE CITED ....................................................................................... 49 CHAPTER III. TOXICITY OF FOUR INSECT GROWTH REGULATORS AND IMIDACLOPRID AGAINST COLORADO POTATO BEETLE (COLEOPTERA' CHRYSOMELIDAE) ............................................................ 62 A INTRODUCTION ......................................................................................... 63 B. LITERATURE REVIEW .............................................................................. 67 1. Diflubenzuron ..................................................................................... 67 2. Chlorfluazuron .................................................................................... 69 3. Cyromazine ......................................................................................... 71 4. Imidacloprid ........................................................................................ 73 S. Azadirachtin ........................................................................................ 74 C. MATERIALS AND METHODS ................................................................... 77 1. Materials ............................................................................................. 77 a Insecticides .............................................................................. 77 b. The Colorado potato beetle ..................................................... 77 2. Methods .............................................................................................. 79 a Leaf-dip assay .......................................................................... 79 b. Droplet assay ........................................................................... 79 3. Data Analysis ................................................... , ................................... 80 D. RESULTS AND DISCUSSION .................................................................... 81 1. Results ................................................................................................ 81 a. Leaf-dip assay .......................................................................... 81 b. Droplet assay ......................................................................... 108 2. General Discussion ............................................................................ 112 LITERATURE CITED ..................................................................................... 115 CHAPTER IV. COMBINATIONS OF DIFLUBENZURON OR CYROMAZINE WITH BACILLUS THURINGIEN SIS AGAINST COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE) .......................................................... 125 A INTRODUCTION ....................................................................................... 126 B. LITERATURE REVIEW ............................................................................. 129 1. Risks and Benefits of Insecticide Mixtures ......................................... 129 2. Insecticide Interactions ...................................................................... 131 3. Combinations of Microbial and Chemical Insecticides ........................ 133 C. MATERIALS AND METHODS ................................................................. 136 1. Materials ........................................................................................... 136 a. Insects ................................................................................... 136 b. Insecticides ........................................................................... 137 2. Methods ............................................................................................ 137 a. Combination of diflubenzuron and Bacillus tlua'ingr'ensis. ...... 137 b. Combination of cyromazine and Bacillus tlna'ingiensis. ......... 138 3. Data Analysis .................................................................................... 140 D. RESULTS AND DISCUSSION .................................................................. 141 1. Results .............................................................................................. 141 a. Combination of difiubenzuron and Bacillus thufingiensis ....... 141 b. Combination Of cyromazine and Bacillus thuringiensis .......... 149 2. General Discussion ............................................................................ 157 LITERATURE CITED ..................................................................................... 161 CHAPTER V. GENERAL CONCLUSSIONS AND FUTURE RESEARCH POSSIBILITIES .............................................................................................. 165 A GENERAL CONCLUSSIONS .................................................................... 166 B. FUTURE RESEARCH POSSIBILTTIES ..................................................... 168 LITERATURE CITED ..................................................................................... 170 APPENDIX. COLORADO POTATO BEETLE VOUCHER SPECIMENS PLACED IN THE MICHIGAN STATE UNIVERSITY ENTOMOLOGY MU SEUM.... 171 LIST OF TABLES Page 2.1. Classification of insecticidal crystal proteins ............................................................. 22 2.2. Intoxication, ultrastrutural changes, and time course of poisoning between toxins active against lepidopteran and coleopteran insects ............................................... 28 2.3. Response of Colorado potato beetle to selection with cryIIIA 5-endotoxin of Bacillus thuringiensis at concentration of 741 mg a. i............/l. ...................................... 37 2.4. Progression of Colorado potato beetle resistance to Bacillus thuringiensis cryIIIA 8-endotoxin ...................................................................................................... 38 2.6. Summary statistics for the length of each stage, fecundity, and the % egg hatching for Bacillus thuringiensis resistant and susceptible strains of Colorado potato beetle .................................................................................................................. 45 3.1. Toxicity of diflubenzuron to second instars of Bacillus thufingiensis resistant and susceptible strains of Colorado potato beetle which were exposed for 96 h on treated leaves ................................................................................................................. 82 3.2. Toxicity of chlorfluazuron to second instars of Bacillus thra'ingiensis resistant and susceptible strains of Colorado potato beetle which were exposed for 96 h on treated leaves ................................................................................................................. 89 3.3. Toxicity of cyromazine to second instars of Bacillus thuringiensis resistant and susceptible strains of Colorado potato beetle which were exposed for 96 h on treated leaves ............................................................................................................................. 96 3.4. Toxicity of imidacloprid to second instars of Bacillus ihuringiensis resistant and susceptible strains of Colorado potato beetle which were exposedfor 96 h on treated leaves ............................................................................................................... 103 3.5. Toxicity of irnidacloprid using droplet assay to second instars of Bacillus thwingiensis resistant and susceptible strains of Colorado potato beetle ....................... 109 3.6. Toxicity of azadirachtin using droplet assay to second instars of Bacillus tlum’ngiensis resistant and susceptible strains of Colorado potato beetle ....................... lll 4.1. The mixtures ofdiflubenzuron and Bacillus tlmringiensis which were tested on second instar B. ihuringienmls resistant and susceptible Colorado potato beetles ........... 138 4.2. The mixtures of cyromazine and Bacillus thuringiensis which were tested on second instars B. tlmringiemis redstant and susceptible Colorado potato beetles .......... 139 4.3. Activity of diflubenzuron and Bacillus tluaingiensis separately, in a mixture, or when diflubenzuron was applied 24 h before treatment with B. thwr‘ngiensis (timed delay). All treatments were made to second instars of B. tluu'ingiensis resistant or susceptible of Colorado potato beetles ........................................................ 142 4.4. Activity of cyromazine and Bacillus thuringiensis separately, in a mixture, ‘ or when cyromazine was applied 24 h before treatment with B. tlmringiensis (timed delay). All treatments were made to second instars of B. tlun'ingiensis resistant or susceptible Colorado potato beetles ............................................................ 150 LIST OF FIGURES Page 2.1. Larval development of the Bacillus thuringiensis resistant strain of Colorado potato beetle treated with difi‘erent concentrations of cryIIIA 8-endotoxin Total heightofbarsindicatespercentmrvivalincludingallinstars ........................................... 40 2.2. Larval development of the Bacillus tlna'ingiensis susceptible strain of Colorado potato beetle treated with different concentrations of cryIIIA 8-endotoxin. Total height of bars indicates percent survival including all instar ............................................. 41 2.3. The relationship between concentration of cryIIIA 5—endotoxin of Bacillus tlum‘ngiensis and larval weight gain of Colorado potato beetle. S96 and $168: the susceptible strain at 96 and 168 h after treatment respectively. R96 and R168: the resistant strain at 96 and 168 h afier treatment respectively ....................................... 43 2.4. Larval development of Colorado potato beetle. S: the Bacillus thufingiensis susceptible strain. R: the Bacillus tlmringiensis resistant strain ....................................... 46 3.1. The relationship between concentration of diflubenzuron and larval weight gain of Colorado potato beetle. S96 and $168: the susceptible strain at 96 and 168 h after treatment respectively. R96 and R168: the resistant strain at 96 and 168 h alter treatment respectively ..................................................................................................... 84 3.2. Larval development of the Bacillus thuringiensis resistant strain of Colorado potato beetle treated with difi‘erent concentrations of diflubenzuron. Total height of bars indicates percent survival including all instars .......................................................... 86 3.3. Larval development of the Bacillus thufingiensis susceptible strain of Colorado potato beetle treated with difi‘erent concentrations of diflubenzuron. Total height of bars indicates percent survival including all instars .......................................................... 87 3.4. The relationship between concentration of chlorfluazuron and larval weight gain of Colorado potato beetle. S96 and 8168: the susceptible strain at 96 and 168 h afier treatment respectively. R96 and R168: the resistant strain at 96 and 168 h after treatment respectively ..................................................................................................... 92 3.5. larval development of the Bacillus tlruringiensis resistant strain of Colorado potato beetle treated with difi‘erent concentrations of chlorfiuazuron. Total height of bars indicates percent survival including all instars .......................................................... 93 3.6. Larval development of the Bacillus thuringiensis susceptible strain of Colorado potato beetle treated with difi‘erent concentrations of chlorfiuazuron. Total height of barsindicatespercentsurvivalincludingallinstars .......................................................... 94 3.7. The relationship between concentration of cyromazine and larval weight gain Of Colorado potato beetle. S96 and 8168: the susceptible strain at 96 and 168 h afiertreatmentrespectively. R96andR168: theresistantstrainat96and 168 hatter treatment respectively ..................................................................................................... 99 3.8. Larval development of the Bacillus tlua'ingiemis resistant strain of Colorado potato beetle treated with different concentrations of cyromazine. Total height of bars indicates percent survival including all instars ............................................................... 100 3.9. Larval development of the Bacillus tlmringiensis susceptible strain of Colorado potato beetle treated with different concentrations of cyromazine. Total height of bars indicates percent survival including all instars ............................................................... 101 3.10. The relationship between concentration of irnidacloprid and larval weight gain of Colorado potato beetle. $96: the susceptible strain at 96 h alter treatment. $168: the susceptible strain at 168 h alter treatment. R96: the resistant strain at 96 h after treatment. R168: the resistant strain at 168 h after treatment ......................... 105 3.11. Larval development of the Bacillus thuringiensis resistant strain of Colorado potato beetle treated with difl‘erent concentrations of irnidacloprid. Total height of bars indicates percent survival including all instars ............................................................... 106 3.12. Larval development of the Bacillus tlua'ingicnsis susceptible strain of Colorado potato beetle treated with difi‘erent concentrations of imidacloprid. Total height of bars indicates percent survival including all rnstars ........................... 107 4.1. Average weight of Bacillus tlmringiensis resistant Colorado potato beetle larvae alter treatment with different insecticides. Means followed by the same letter within a time period are not significantly difl‘erent using LSD test at or = 0.05. Upper-case letters correspond to companison of means at 96 h post treatment, and lower-case letters correspond to coman of means at 168 h post treatment ........................................ 144 xiv 4.2. Average weight of Bacillus tlua'ingiensis susceptible Colorado potato beetle larvae alter treatment with different insecticides. Means followed by the same letter within a time period are not significantly difi‘erent using LSD test at or = 0.05. Upper-case letters conespond to comparrison of means at 96 h post treatment, and lower-case letters correspond to comparrison ofmeans at 168 h post treatment ........................................ 145 4.3. Larval development of the Bacillus tlnm’ngiensis resistant Colorado potato beetle larvae 168 h after treatment with difi‘erent insecticides. Total height of bars indicates pereentsurvivalincludingallinstars .............................................................................. 147 4.4. Larval development of the Bacillus thuringiensis susceptible Colorado potato beetle larvae 168 h after treatment with difl‘erent insecticides. Total height of bars indicates percent survival including all instars ............................................................... 148 4.5. Average weight of Bacillus rlmringiensis resistant Colorado potato beetle larvae afier treatment with difl‘erent insecticides. Means followed by the same letter within a time period are not significantly difi‘erent using LSD test at or = 0.05. Upper-case letters correspond to comparrison of means at 96 h post treatment, and lower-case letters correspond to comparrison of means at 168 h post treatment ........................................ 152 4.6. Average weight of Bacillus tlmringiensis susceptible Colorado potato beetle larvae after treatment with different insecticides. Means followed by the same letter within a time period are not significantly different using LSD test at or = 0.05. Upper-case letters correspond to comparrison of means at 96 h post treatment, and lower-case letters correspond to comparrison ofmeansat 168 hposttreatrnent ........................................ 153 4.7. Larval development of the Bacillus tlmringiensis resistant Colorado potato beetle larvae 168 h after treatment with different insecticides. Total height of bars indicates percent survival including all instars ............................................................................. 155 4.8. Larval development of the Bacillus tluaingiensis susceptible Colorado potato beetle larvae 168 h after treatment with difi‘erent insecticides. Total height of bars indicates percent survival including all instars ............................................................... 156 CHAPTER I GENERAL LITERATURE REVIEW A. INTRODUCTION 1. Insecticide Resistance in Colorado Potato Beetle The Colorado potato beetle, Leptinorarsa decemlineata (Say) (Coleoptera: Chrysomelidae) was first documented as a pest ofpotatoes in Iowa in 1861 (Walsh 1865). Itspreadmotbastuesupotatofiddsmcreased,mdnhasbecomethemostimponam defoliating pest ofpotatoes in the Northeastern and Central United States for several decades (Grafius 1986, Casagrande 1987, Ferro and Geriernter 1989, Ferro 1993). Inthefirstseveralyearsasanewpotatopest, severalcontrolmeasuresotherthan insecticides (hand-picking, crop rotation, resistant varieties) had been used (Walsh 1865, Riley 1869, Bethune 1872). However, since Paris green was known to have an insecticidal efi‘cct on Colorado potato beetle (Riley 1871, Bethune 1872), it became the most preferable control method. Then, arsenical insecticides were substituted for Paris green and theywere used by Growersuntil the late 1940's (Gauthieret al. 1981). The arrival of DDT resulted in the displacement of arsenical insecticides. The use of insecticides increased continuously as other classes of insecticides such as organophosphates and carbamates were found. In the case of Colorado potato beetle, intensive spraying of insecticides has been a regular practice for many decades. Insecticide resistance problems in Colorado potato beetle have occurred repeatedly. Colorado potato beetle resistance to calcium arsenate was first noted in the mid 1940's on Long Island (Gauthier et al. 1981). This problem could be overcome with DDT. However, Colorado potato beetles became resistant to DDT, and this phenomenon was recognized first in 1952 (Gauthier et al. 1981). Eventually this pest developed resistance to almost all synthetic insecticides including arsenicals, organochlorines, carbamates, organophosphates, and pyrethroids (Gauthier et al. 1981, Forgash 1985). In Michigan, Ioannidis et al. (1992) demonstrated that most Colorado potato beetle populationswereresistantto atleastoneofthemajorgroupsofinsecticides (an'npbosmethyl, carbofuran, or permethrin) by 1991. Ithasbeengeneraflyacceptedthatthaearefmnmechmfismsofhuectredstance to insecficides: behavioral, reduced penetration, altered target site, and metabolic mechanisms. Behavioralmechanismsaretheabilityofinsectsto avoidtheinsecticide. For example, Anopheles mosquitoes avoid DDT treatments on walls whae they otherwise would rest (Hadaway 1950). Efl‘ectiveness ofmany insecticides is determined by their abilitytopenetratetheinsectcuticle. Increaseinthecuticlethicknessorachangeinthe Meal components of the cuticle could reduce insecticide penetration. As a result, the amount of insecticide that enters the insect body is not suficient to cause mortality. This mechanism ofien works together with increased insecticide detoxification processes. The acethylcholinesterase (AChE) enzyme in the neural synapses is the target site for organophosphate and carbamatc insecticides (Matsumura 1985). Reduced sensitivity of AChE to inhibition by organophosphate and carbamatc insecticides was considered to be due to structural alteration at the active site (Hams 1983). In addition, Van Rie et al. (1990) reported that resistance in Plodia interprmctella Hb. to Bacillus tlua'ingiensis was also due to alteration in the amnity of a midgut membrane receptor. Metabolic medunismsarethemostimportantresistancemechanismGlappandWang 1983) particularly in lepidopterous species. An increase in activity of detoxifying enzyme will increase the rate of insecticide detoxification. Several detoxifying enzymes have been investigated: aliesterases or carboxylesterases, mixed-function oxidases, GSH transferases, and DDT dehydrocholinesterase. Resistance in Colorado potato beetle to conventional insecticides has involved insensitivity of AChE to inhibition by insecticides (Argentine et al. 1989, Ioannidis et al. 1992, Wierenga and Hollingworth 1993) and detoxification by mixed-function oxidation (Argentine et al. 1989, Ioannidis 1992). Therateofrcsistancedevelopment to insecticidesisusuallydifl‘erentfiom species to species and from population to population within species. Selection for resistance to insecticide in fields' population is influenced by five factors: behavior, genetics, biology, ecology, and operatioml factors (Georghiou and Taylor 1976, Tabashnik and Croft 1982, RosenheimandTabashnik 1990, DenholmandRowland 1992). Thefactorsaredificultto madprdflehnmodanredstmcennmganentprogramsmaybeabletoaddresswmeof them (Denholm and Rowland 1992, McGaughey and Whalon 1992). In most cases, operational factorscanbemosteasilymanipulatcd sincethepredominantpestcontrol actions are made by people. Insight into resistance management strategies needs to be understood and implemented to prevent or, at least, to slowdown insects fiom becoming resistance to insecticides. 2. Pest Management for Colorado Potato Beetle IMegmtedPestManaganenth)isthemostappmpfiateapproachmmanageall pests including the Colorado potato beetle. It utilizes all compatible methods such as mechanical control, cultural control, biological control, host plant resistance, and chemical control to maintain the population below the economic injury level (Smith and Reynolds 1966), while limiting environmental and socioeconomic impact. The following are some of the potential control strategies and tactics for Colorado potato beetle. a. Mechanical control Hand-picking to control Colorado potato beetle was only used at the beginning of this insect's potato pest history (Walsh 1865). This technique was labor consuming and is not reliable for modern large potato fields. b. Cultural/mechanical control Croprotafionsandtheuseofeaflymamringvarieficshavebeenrccommendedto reduce the population of Colorado potato beetle. Nevertheless, these cultural controls havenotbeenattractiveforgrowers(Casagrande 1987). Otherculturaltacticsinchrding manipulation of planting dates, migration-barrier trenches, propane flamers, toxic barrier plantings, covercropsthatinterceptandarrestbcetleflights, etc., arecurrentlybeing evaluated. I c. Biological control Twenty-two natural enemies of Colorado potato beetle have been reported (Bethune 1972). Some of them have significant potential: 1)Edowarrputtleri Grissel as an egg parasitoid, 2) two pentatonrid predators, Perillus biocularus (F .) and Padisus nraculiwntris (Say), and 3) the parasitic mite, Chrysonrelobia labidornetae. Heavy applications of insecticide, however, have been one of the reasons why these natural enemies could not contribute much in regulating populations of Colorado potato beetle. In addition, annual agroecosystem arIe less stable than that in perennial crops (Southwood and Way 1970, van Emden and Williams 1974). As a result the presence and population development of natural enemies often lag far behind Colorado potato beetle population development in real field populations. Natural enemies could be a powerfirl tool if insecticides and other population suppressing strategies could be avoided and whenever an appropriate agroecosystern is maintained. (1. Host plant resistance Plantinginsect-resistantcropsareeconomical, environmentallysafe, andan efi‘cctive method to reduce the population of pest (Pedigo 1989). It has been noted since 1870's that some varieties of potato showed lower preference by Colorado potato beetle (Walsh 1866, Riley 1869). Dimock and Tingey (1985) stated that two defense mechanisms for Colorado potato beetle resistance were recognized. Toxic glycoalkaloids and glandular trichomes were the major population suppressing mechanism for Colorado potato beetle resistance in potatoes. However, complete Colorado potato beetle control was not possiblewithhostplantresistanceprovidedbythesepotatoes (Casagrande 1987). Inthe lastseveralyears, scientistshavebeenworkingontransgenicpotatoplantsbyputtingB. thrrringiensis toxin in the potato (Adang et al. 1993, Perlak et al. 1993). This may help to reduce the use of synthetic insecticides (Gould 1988). Appropriate deployment strategies areneededto conservetbeusefirlnessofuansgenicpmbecausemismanagementin using these plants could result in adaptation (Gould 1988, McGaughey and Whalon 1992). This technology would be more powerful if we could develop stirmrlo—deterrent (Dindonis and Miller 1980) transgenic potato plants. These plants could attract Colorado potato beetletoeatmdhyeggaMwhenevatheluvaeconmmetheleavestheydiebecwse they ingest the toxin. e. Insecticidal control History demonstrates that insecticides have made a large contribution to foOd production worldwide (Ware 1939). On the other hand, unjudicious application of insecticides has created problems of pest resistance, secondary pest outbreak pest resurgence, non target impacts, and environmental contamination . Application of insecticides as a single mortality factor has never yielded the best solution to controlling pestpopulation. Asimilarsituationmltwhenhostplantresistancewasdeployedasa single mortality factor. Several pests have shown that they have the ability to respond to any selection pressure directed at than. Therefore, efi‘orts to increase the mortality ofa particular pest like Colorado potato beetle must be as a result ofseveral mortality factors, and selection pressure fiom an individual control agent could be ameliorated. For the foreseeable future, insecticides will likely be a part of pest management programs. Theymayormaynotbeappliedataparticulartimedependingonthe population density, economic injury level, crop condition, and phenology. Pesticide of the mummstbeselecfivenon-tordctohumansandothernon-urgetorganism, andexhibit noadverseefi‘ectsontheenviromnent. Pesticidesthatmeettheserequirementsmaybe called 'biorational' (Ware 1989), and they were classified into two nnjor groups: microbial and chemical pesticides such as insect growth regulators (IGRs). e.l. Microbial insecticide Introduction of B. tlmringiensis to control agricultural pests including the Colorado potato beetle have enriched the existing alternative strategies to manage resistant and susceptible populations of insect pests. B. tlmrr‘ngiensr's var. tenebriom’s has been demonstrated to be efi‘ective in controlling Colorado potato beetle populations that have developed resistance to synthetic insecticides (F erro and Gelernter 1989, Zehnder and Gelernter 1989, Ferro 1993, Whalon et al. 1993). However, microbial insecticides also induce resistance. Eight species have been documented to be resistant after several selections either in the field or in the laboratory (MCGaughey and Whalon 1992). Diamondback moth, Plutella aylostella (L), is one example of an insect that has become resistant after repeated use of B. tlna'ingiemisinthe field (Tabashnik et al. 1990). In laboratory, Colorado potato beetle became 59 times more resistant to B. tlmringiensis than the unselected colony after 12 generations of selections (Whalon et al. 1993). Compared to synthetic insecticides, B. tlna'ingiensr's has several advantages: 1) it is more selective, 2) it has little or no hazard to human, 3) it is compatible with existing management programs, and 4) it is degradable (Deacon 1983, Flexner et al. 1986, Ferro 1993). These advantages will no longer beneficial to humans unless judicious applications of B. thm‘ngiemis can be achieved since mismanagement of microbial insecticides has caused pest resistance. Strategies specific to each pest need to be developed to prevent pests from becoming resistance to control measures (Ferro 1993) e2. Insect growth regulators Insect growth regulators are another example of biorational pesticides. They disruptinsectgrowthanddevelopmentinthreedifi‘erentways: asjuvenilehormones, as precosenes, andaschitinsyuthesisinhibitorsMare 1989). Theuseofinsect growth regulatorshasbecomemoreintenserecently(Adcocketal. 1993,Berryetal. 1993, Bull and Meola 1993, Haynes and Smith 1993, Sirota and Garfius 1994). They are very selective, andtheyafi‘ectthetargetdmingembryonic, larval, ornymplnldevelopment (Ware 1989). Some insect growth regulators were efi‘ective in preventing eclosion to the adult stage; for example, the third instar of cabbage maggot, Delia radian», treated with methoprene were not able to become adults (Gordon et al. 1989). Insect growth regulators are compatible with biological control agents such as predators and parasitoids (Oomen and Wagers 1984, Crofi 1990, Neimczyk et al. 1990) because they are quite specific. Some researchers have reported that some insect growth regulators were efi‘ective in controlling some pests that were resistant to conventional insecticides. Teflubenzuron and chlorfluazuron were reported as efi‘ective insecticides against diamondback moth resistant to conventional insecticides (Perng et al. 1987). House fly populations that were variously resistant to conventional insecticides were sensitive to cyromazine (Iseki et al. 1986). In addition, cyromazine was also effective against the insecticide-resistant Long Island strain of Colorado potato beetle at high concentration, but at low concentration this strain could live longer than the susceptible strain (Sirota and Grafius 1994). Like other insecticides, however, insect growth regulators may also select for resistance in some insects. After 20 generations of selection in the laboratory, larvae of the diamondback moth were 8 - 12 fold resistant to tefluberlzuron (Perng et al. 1988). They also reported that cross resistance with another insect growth regulator, chlorfluazuron, and a number of conventional insecticides like permethrin, fenvarelate, prothiophos, and carbofilran was not present. The house fly, Musca domestica, became 70 times more resistant to cyromazine than the unselected colony after 15 generations of selection (Bloomcamp et al. 1987). Nevertheless, the resistance level decreased after the selection discontinued. Microbial insecticides and Insect growth regulators have several advantages compared to conventional insecticides. However, continuous application has caused pest to develop resistance. Mixing or alteration insecticides that have different modes of action couldbeanefi‘ectivestrategyindelaying insectresistance. Mixturesarelikelytobemore durable than alterations (Roush 1989). B. OVERALL GOAL AND OBJECTIVES 1. Dual Goal The overall goal of this study was to find alternative control strategies for B. thwingiensr‘s susceptible and resistant strains of Colorado potato beetle using insecticides with novel modes of actions. 2. Objectives a To maintain a B. tharingr’ensr‘s resistant strain of Colorado potato beetle and to characterize some biological difi‘erences between the resistant and susceptible strains. b. To evaluate toxicity of four insect growth regulators and one neuroinsecticide against the susceptible and resistant strains, and to determine potential cross- resistance between B. tlmn’ngr’ensis and the five tested insecticides c. To evaluate the potency of combinations of B. (Ina-ingiensis with insect growth regulators. 10 C. HYPOTHESIS H1: The resistance level to B. thuringr‘ensis in Colorado potato beetle will increase if selection is maintained. H2: Biological characteristics (growth and development) of the resistant and susceptible strains will be different as a compensation of the resistant strain to develop its resistance mechanism. H3: The susceptible and resistant strains will respond similar to each insecticide, and the activity ofthe insect growth regulators will depend mainly on the period oftime post treatment. 114: The mortality efi‘ects of B. thurr’ngiensr‘s and insect growth regulator combinations will be influenced by application timing, and the relative potency of combinations will have similar mortality efl‘ects on susceptible and resistant strains. 11 LITERATURE CITED Adang, M. J., M S. Brody, G. Cardineau, N. Eagan, R T. Roush 1993. The reconstruction and expression of Bacillus tlnm'ngr’ensis cry 111A gene in protoplast and potato plants. Plant. Mol: Int. J. Mol. Biol. Biochem. Genet. Eng. 21(6): 1131-1148. Adcock, G. J., P. Batterham, L. E. Kelly, and J. A McKenzie. 1993. Cyromazine resistance in Drosophila melatogaster (Diptera: Drosophilidae) generated by ethyl methanesulfonate mutagenesis. J. Econ. Entomol. 86(4): 1001-1008. Argentine, 1A, J. M. Clark, and D. N. Ferro. 1989. Genetics and synergism ofresistance to azinphosmethyl and permethrin in the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82:698 Berry, R E., A F. Moldenke, J. C. Miller, and J. G. Wernz. 1993. Toxicity of diflubenzuron in larvae of Gypsy Moth (Lepidoptera: Lymantriidae): Effects of Host Plant. J. Econ. Entomol. 86(3): 809-814. Bethune, C. J. S. 1872. Report of the Entomological Society of Ontario for the year 1871. Hunter Ross, Toronto. Bloomcamp, C. L., R S. Patterson, and P. G. Koehler. 1987. Cyromazine resistance in the house fly (Diptera: Muscidae). J. Econ. Entomol. 80: 352-357. Bull, D. L. and R W. Meola. 1993. Effect and fate of the insect growth regulator pyriproxyfen after application to the horn fly (Diptera: Muscidae). J. Econ. Entomol. 86(6): 1754-1760. Casagrande, R A 1987. The Colorado potato beetle: 125 years of mismanagement. ESA Bulletin. Fall. Crofl, B. A 1990. Arthropod biological control agents and pesticides. John Wiley and Sons. New York pp. 305-333. Deacon, J. W. 1993. Microbial control for plants pests and disease. Van Nostrad Reinhold Woking. UK. 12 13 Denholm,IandM W. Rowland. 1992. Tacticsformanagingpesticideresistancein arthropods: theory and practice. Annu. Rev. Entomol. 37: 91-112. Dindonis,L. L. and]. R. Miller. 1981. Onionflyandlittlehouseflyhostfinding selectively mediated by decomposing onion and microbial volatiles. Journal of Chemical Ecology 7(2): 419-427. Dimoclg M. B. and W. M. Tingey. 1985. Resistance in Solaman spp. to the Colorado potato beetle: mechanisms, genetic resources and potential. Mass. Agric. Exp. Stn. Bull. 704: 79-106. Fer-re, J.,M. D. ReaLJ. VanRie, S. Jansens, andM Peferoen1991.Resistancetothe Bacillus thuringiensi.: bioinsecticide in a field population of Plutella aylastella is due to a change in the midgut membrane receptor. Proc. Nat. Acad. Sci. 88: 5119- 5123. Ferro, D. N. 1993. Potential for resistance to Bacillus thuringiensi: Colorado potato beetle (Coleoptera: Chrysomelidae)-A model system. American Entomologist. Spring. Ferro, D. N. and W. D. Gelertner. 1989. Toxicity of a new strain of Bacillus thuringiensis to Coorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82(3): 750-755. Flexner, J. L. B. Lighthart, and B. A Croft. 1986. The effects of microbial pesticides on non-target beneficial arthropods. Agric. Ecosyst. Environ. 16: 203-254. Forgash, A J. 1985. Insecticide resistance in the Colorado potato beetle. Mass. Agric. Exp. Stn. Res. Bull. 704: 33-52. ; Gauthier, N. L., R N. Hofinaster, and M. Semel. 1981. History of Coloradopotato beetle control, pp. 13-33. In J. H. Lashomb'and R A Casagrande [eds], Advances in potato pest management. Hutchinson Ross, Stroudsburg, Pa. Georghiou, G. P. and C. E. Taylor. 1976. Pesticide resistance as an evolutionary phenomenon, pp. 759-785. In Proc. 15th Int. Cong. Entomol., Washington, D. C. College Park, Md: Entomological Society of America. Gordon, R, T. L. Uoung, and M. Cornect. 1989. Effects of two insect growth regulators on the larval and pupal stages of the cabbage maggot (Diptera: Anthomyiidae). J. Econ. Entomol. 82(4): 1040-1045. Gould, F. 1988. Evolutionary biology and genetically engineered crops. BioScience 38: 26-33. 14 Grafius, E. 1986. Efl‘ects of temperature on pyrethroid toxicity to Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 79: 588-591. Hadaway, A B. 1950. Observations on mosquito behavior in native huts. Bulletin of Entomological Research 41: 63-78. Hama, H. 1983. Resistance to insecticide due to reduced sensitivity of acethylcholineesterase, pp. 299-331. In G. P. Georghiou and T. Saito [eds], Pest resistance to pesticides. Plenum Press, New York. Haynes, J. W. and J. W. Smith. 1993. Test ofa new growth regulator for boll weevils (Coleoptera: Curculionidae) by dipping and feeding. J. Econ Entomol. 86(2): 310- 3 13. Ioannidis, P. M., E. J. Grafius, J. M. Wierenga, M. E. Whalon, and R M. Hollingworth. 1992. Selection, inheretance and characterization of carbofuran resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae). Pestic. Sci. 35: 215-222. Ioannidis, P. M., E. J. Grafius, and M. E. Whalon. 1991. Patterns of insecticide resistance to azynphosmethyl, carbofilran, and permethrin in the Colorado potato beetle (Coleoptera; Chrysomelidae). J. Econ. Entomol. 84(5): 1417-1423. Iseki, A and G. P. Georghiuo. 1986. Toxicity of cyromazine to strains of the housefly (Diptera: Muscidae) variously resistant to insecticides. J. Econ. Entomol. 79: 1 192-1 195. ~ Matsumura, F. 1985. Toxicology of inseticides. Plenum Press, New York. 598 pp. McGaughey, WM. and M. E. Whalon. 1992.‘Managing insect resistance to Bacillus tlnm'ngiensis toxins. Science, vol. 258: 1451-1455. Neimczyk, E., M. Koslinnska, A Maciesiak, Z. Nowakowski, R Olszak., and A Szufa. 1990. Some growth regulators: their efl‘ectiveness against orchard pests and selectivity to predatory and parasitic arthropods. Acta-Hortic. Wageningen: International horticultural science. pp. 157-164. Oemen, P. A and G. L. Wagers. 1984. Selective efi‘ects of chitin synthesis inhibiting insecticide (CME-134) on the parasite Encam‘afonnosa and its whitefly host T naleurodes vaporarioum. Meded. Fac. Landbouwwet. Rijksuniv. Gent. 49: 745- 750. Pedigo, LP. 1989. Entomology and Pest Management. Macmillan Publishing Company, New York. pp.: 413-440. 15 Perlak, F. J., T. B. Stone, Y. M. Muskopf, L. I. Petersen, G. B. Parker, S. A McPherson, J. Wyman, 8. Love, G. Reed, and D. Biever. 1993. Genetically improved potatoes: protection fiom damage by Colorado potato beetles. Plant. Mol. Biol. 22(2): 313- 321. Perng, F. S. and C. N. Sun. 1987. Susceptlbility ofdiamondback moths (Lepidoptera: Phrtellidae) resistant to conventional inscticides to chitin synthesis inhibitors. J. Econ. Entomol. 80: 29-31. Perng, F. S., M. C. Yao, C. F. Hung, and C. N. Sun. 1988. Teflubenzuron resistance in diamondback moth (Lepidoptera: Phrtellidae). J. Econ. Entomol. 81 (5): 1227- 1282. Plapp, F. W. and T. C. Wang. 1983. Genetic origins of insectcide resistance, pp. 47-70. In G. P. GeorghiouandT. Saito [eds.],Pestresistancetopesticides PlenumPress, New York ’ Riley, C. V. 1869. First annual report on the noxious, beneficial, and other insects of the state of Missouri. Ellwood Kirby, J efl‘erson City, Mo. Riley, C. V. 1871. Third annual report on the noxious, benefial, and other insects ofthe state of Missouri. Horace Wilcox, Jefl‘orson City, Mo. Rosenheim, J. A and B. E. Tabashnik. 1990. Evolution of pesticide resistance: interactions between generation time and genetic, ecological, and operational factors. J. Econ. Entomol. 83(4): 1184-1193. Roush, R T. 1989. Designing resistance management programs: How can you choose?. Pestic. Sci. 26: 423-441. Sirota, J. M. and E. J. Grafius. 1994. Efl‘ects of cyromazine on larval survival, pupation, and adult emergence of Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87(3): 577-582. Smith, R F. and H. T. Reynolds. 1966. Principles, definitions and scope of integrated pest control. Proceeding of FAO (United Nations Food and Agriculture Organization) Symposium on thegrated Pest Control 1, 11-7. Southwood, T. R E. and M. J. Way. 1970. Ecological background to pest management, pp. 6-29. In R L. Rabb and F. E. Guthire [eds], Concepts of pest management. Raleigh: N. Carolina State Univ. Tabashnik, B. E. and B. A Crofi. 1982. Managing pesticide resistance in crop-arthropod complexes: interactions between biological and operational factors. Environ. Entomol. 11: 1137-1144. l6 Tabashnik, B. E., N. L. Cushing, N. Finson, and M. W. Johnson. 1990. Field development of resistance in Bacillus (Inningiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83: 1671-1676. vanEmden, H. F. andG. F. Williams. 1974. Insectstabilityanddiversityinagro- ecosystem. Annu. Rev. Entomol. 19: 455-475. Walsh, B. D. 1865. The new potato bug and its natural history. Pract. Entomol. 1: 1-4. Walsh. 1866. The new potato bug. Prac. Entomol. 11: 13-16. Ware, G. W. (1989. The pesticide book. Thomson Publications, California pp.: 237-253. Whalon, M. E., D. L. Miller, R M. Hollingworth, E. J. Grafius, and J. R Miller. 1993. Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain resistant to Bacillus thuringiensis. J. Econ. Entomol. 86(2): 226-233. Wierenga, J. M. and R M. Hollingworth. 1993. Inhibition of altered acethylcholinesterase from insecticide-resistant Colorado potato beetles (Coleoptera: Chrysomelidae). J. Econ. Entomol. 86(3): 673-679. Zehnder, G. W. and W. D. Gelernter. 1989. Activity of the M-ONE formulation of a new strain of Bacillus thufingiensis against the Colorado potato beetle (Coleoptera: Chrysomelidae): relationship between susceptibility and insect life stage. J. Econ. Entomol. 82(3): 756-761. CHAPTER II THE BA CILL US THURINGIENSIS RESISTANT STRAIN OF COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE) A. INTRODUCTION The incidence of insect resistance to insecticide has continuously increased. Even though the rate of resistance development varies fiom species to species, insects historically have developed resistant to all groups of insecticides. Third generation insecticides (insect growth regulators) are not exempt from the problem of resistance (Bloomcamp et al. 1987, Perng et al. 1988, Ishaaya and Klein 1990, Ismail and Wright 1991). On the other hand, the discovery of a new insecticide from discovery through initial marketing usually takes 8 - 10 years and it costs fi'om S 40 - S 60 million (Dill 1989). Therefore, insecticide resistance management is necessary to address the increasing rate of resistance development in key insect and mite pests. Four strategies of resistance management have been proposed: 1) reduced selection pressure, 2) diversity of mortality sources, 3) susceptibility managemmt, and 4) monitoring resistance development so that we can make good decisions (Denholm and Rowland 1992, McGaughey and Whalon 1992). In reality, adequate information to choose the precise tactic is sometimes not available. Prior research into the genetics, biochemistry, toxicology, and physiology of resistance can also be crucial (Leeper et al. 1986) to provide basic information for resistance management. The US Enviromnental Protection Agency (EPA) requires a lot of data for registering a new insecticide, however, some insecticides (e.g. insect growth regulators) have been marketed before their mode of actions has been clearly understood. It would be ideal if the mode of action and likely resistance mechanisms of a new insecticide could be initially research before the new insecticide is marketed. Ifthis information was available, more appropriate management tactics could be suggested before the new insecticide was generally marketed. l8 19 It is technically easier to develop and maintain resistance strains in the laboratory than in the field. However, unsuccessful attempts at selecting for resistance have been reported (Sneh and Schuster 1983, Goldman et al. 1986), and considerable controversies exist on the utility of laboratory selected populations for field resistance management programs (Roush and McKenzie 1987, Bloomcamp et al. 1987). The actual fi'equency of resistanceafldesinfieldpoprdafionsisdifiarhtomeamrebmitisanimponam parameter (Shufian and Whalon 1994, Rahardja and Whalon 1994). Successfirl laboratory selection for resistance to B. tlmringiensrs in several difl‘erent insects has been reported (McGaughey 1985, McGaughey and Beeman 1988, Tabaslmik et al. 1991, Whalon et al. 1993). Resistance in the Indianmeal moth, Pladia interpuncrella Hb., increased approximately 30-fold in only two generations of selection (McGaughey 1985). A moderately resistant strain of diamondback moth, Plutella xylostella L., selected for five generations in the laboratory became 5 to 7 times more resistant than the initial population (Tabashnik et al. 1991). In the Colorado potato beetle, Leprinorasa decemlineata (Say), 12 generations of selection resulted in a 59 fold increase in the resistance level (Whalon et al. 1993). These increases in resistance level indicate the presence of suflicient genetic variation in many species for resistance to B. thraingiensis (Tabashnik et al. 1991, Whalon et al. 1993). The intensity of selection, environmental, biological, ecological, and operational factors determines the rate of resistance development (Georghiou and Taylor 1976, McGaughey and Beeman 1988, Tabashnik et al. 1991). The objectives of this study were: 1) to monitor the resistance level of Colorado potato beetle under selection of cryIIIA b-endotoxin of B. tlnm‘ngr’ensis, and 2) to characterize the biological difi‘erences between resistant and susceptible strains. B. LITERATURE REVIEW 1. Baciuus thuringiensis a. Classification Five species of Bacillus have been identified as arthropod control agents: B. larvae, B. lentinrorbus, B. maericus, B. popilliae, and B. thuringienn’s. However, only the last three species are effective in controlling insect pests (Deacon 1983, Priest 1989, Tanada and Kaya 1993). B. tlmringiensis has captured the attention of many scientists fiom difi‘erent disciplines. Research is being conducted either to optimize the usefulness of existingsubspecies ortofindnewstrainsforcontrollinginsectsandotherpests. Many difi‘erent strains have been reported as efl'ective control agents for many difl’erent insects. It is well known that B. thuringiensis produces an insecticidal crystalline protein known as the 5—endotoxin (Deacon 1983, Priest 1989, Aronson and Geiser 1992). Each type of 8-endotoxin is toxic to a limited species of insect. On the bases of structural similarities and insecticidal activities, the 8—endotoxins have been categorized into six different 0188808 (WI. 00'”. CW1”. WW. WV. and WW) and 8 Cytob’tic crystal Pmtein (cyi) (Holte and Whiteley 1989, Adang 1991, Lereclus et al. 1993). These proteins are active against lepidopteran (cry!) (Hofle and Whiteley 1989, Hofle et al. 1990, MacIntosh et al. 1990, Moar et al. 1990, Van Rie et al. 1990, Visser et al. 1990, van Frankenhuyzen et al. 1991), dipteran and lepidopteran (cryll) (Donovan et al. 1988, Nicholls et al. 1989, Widner and Whiteley 1989, Wu et al. 1991), coleopteran (cryIII) (Kreig et al. 1983, Kreig et al. 1987, Hermstadt et al. 1986), dipteran (cryIV) (Thorne et al. 1986, Bourgouin et al. 1988, Delecluse et al. 1988), lepidopteran and coleopteran (ctyV) (Bleak et al. 1989, Edward et al. 1990, Tailor et al. 1990), and nematodes (004’?) (European Patent Ofice Application, Bone et al. 1987, Wharton and Bone 1989) pests, but they are non toxic to 20 21 birds and mammals. This classification including some examples of target insects is presented in Table 2.1. ThediscoveryofB. thra'ingiennsstrainswithactivityagainst noninsect havebeen reported many times. B. thuringiensis var. israelensis changed the permeability of the egg of Ikichosn'ongylas colabnformis in uptaking iodine (Bone et al. 1987) as well as disrupted the lipid layer of the egg-shell of a nematode (Wharton and Bone 1989). Casida (1989) reported that B. tlnrringr’ensis was active against protozoans. In addition, B. tlan'ingiensis and B. sphaericas were toxic to house dust mite, Demraraphagoides pterornrss'inas (Trouessart) (Saleh et al. 1991). Walters and English (1994) found that B. tlna'ingiensis (cryIIA, cryIIIA, and cryIVD) were all toxic to the adults of the potato aphid, Macrosipham eaphorbiae. 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The size ranged from 0.7 to 2.4 by 0.7 um, and the thickness varied from 0.15 to 0.25 um The nucleotide sequence of the gene encoding for B. tluaingiensis var. tenebriom’s 8—endotoxin was later found to be similar to that of B. tlwringr'ensis var. san diego and B. tintringiem var. EG 2158 (Hermstadt et al. 1986, Krieg et al. 1987, Donovan et al. 1988, Keller and Langenbruch 1993). However, the san diego designation has been dropped from the literature. The three dimensiorml structure of 601112! from B. thuringiensi.: var. tenebrionis has been determined (Li et al. 1991). It has three functional domains: a seven-helix bundle, a three sheet domain, and a B sandwich. The hydrophobic and amphipathic helices of domain I are responsible for pore formation. Domain II may contain the region that binds to receptors on the insect midgut, and domain III can create a defect in the membrane. The mode of action of B. (Inningiensr’s has been the subject of studies for decades, and it has been conducted mostly in lepidopteran insects. B. tinm'ngr‘ensis 5—endotoxins are stomach poison (Knowles and Dow 1993), therefore ingestion of toxins is required for intoxication in the susceptible insects. After toxin ingestion, intoxication can be separated into four steps: solubilization, conversion (activation), receptor binding, and pore formation (Home and Visser 1993, Knowles and Dow 1993, Visser et al. 1993). The midgut environment plays an important role in determining the emciency of solubilization process. Many lepidoptera have an alkaline midgut; in this condition the crystals dissolve, producing 130-140 kDa proteins (cry!) or 71 kDa proteins (ayII) (Knowles and Dow 1993, Visser et al. 1993). Unlike in lepidoptera, the pH of coleopteran midguts ( e. 3. Colorado potato beetle and Southern corn rootworm, Diabotn'ca 27 rmdeciwrmctata hawardi Barber) were 6.0 and 7.0 respectively (Slaney et al. 1991). Thesemthorsinsistcdthatinsteadopr, midgutdetergentsdcterminethecficiencyof ayIIIA sohtbilization. Using a universal bufi‘er, however, Koller et al. (1992) found that native crystals of B. tlmfingiensis var. san diego dissolve below pH 4 and above pH 10. In addition, these authors reported that cottonwood leaf beetle, Chrysomela scnpta F., had an acidic (pH between 4.5 and 6.0) midgut. This indicated that intoxication occurred under acidic environments (Koller et al. 1992). In lepidoptera, the solubilized crystal proteins, which are also called protoxins, are proteolytically cleaved or activated to yield lower molecular weight proteins, 68-55 kDa (Hofinann et al. 1988, Hofic and Whiteley 1989, Knowles and Dow 1993, Visser et al. 1993). However, in the Colorado potato beetle or southern corn rootworm, cryIIIA was not proteolytically converted into smaller polypeptides since this protein was already in an active form (Slaney et al. 1991). There is variation in the size ofprotoxins and active toxins, and these may be correlated with target specificity (Hoftc and Whiteley 1989). The activated toxin binds to specific receptors on the membranes of midgut ephitelium cells (Hofte and Whiteley 1989, Van Ric ct al. 1990, Fare ct al. 1991, Knowles and Dow 1993, Visser at al. 1993). These receptors may be glycoproteins (Knowles and Ellar 1986). Binding occurs at the apical microvilli of sensitive insects (Bravo et al 1992), and binding was essential for toxicity. Yet higher binding aflinity does not always result in higher mortality (Van Ric ct al. 1990, Bravo ct al. 1992, Visser ct al. 1993). Slaney et al. (1991) demonstrated that southern corn rootworm had three times the number of binding sites for cryIIIA than Colorado potato beetle. However, cryIIIA was less toxic to southern corn rootworm than Colorado potato beetle probably because pore formation was not as eflicient in southern corn rootworm as Colorado potato beetle. Aficrbindingthetordnsinscflirnothcplasmamembmncofthccellwhichis followed by pore formation in the co1umnar cell apical membrane (Knowles and Dow 1993, Honee and Visser 1993). This lead to an influx of K+ ions which was followed by 28 theinflwrofwaterintocolumnarcells(KnowlcsandDow1993).Thewaterinflwrcauses cohrmuceflsweflmgthatcvauuallyendsmcdllysismmwlesandDow1993,Honee andVrsser 1993).Celllysisopensthehemocoeloftheinsecttosecondaryscpficemia whichresultinparalysisanddeathoftheinsect. The mode of action of cryIIIA on coleopterans is difi‘erent from that of cry] on lepidopteransinseveral minorfeatures (Ebersoldetal. 1977, PercyandFast 1983, Lancet al. 1989, Slatinetal 1990, Slaneyetal. 1991,BauerandPankratz 1992). These difl‘erences are summarized in Table 2.2. Table 2.2. Intoxication, ultrastuctural changes, and time course of poisoning between toxins active against lepidopteran and coleopteran insects Lepidoptera Colcoptera Process Observation Time‘ Observation Time‘ Ingestion arrested feeding 2 h arrested feeding 15 m paralysis 6 h paralysis 8-16 h Solubilization pH (alkaline) plays detergent plays important role important role Activation required not always Cell swelling disrupted microvilli 15 m no change microvilli 2 h Cell lysis vacuolization 30 m vacuolization 2-4 h l'h:hnurs,m:mimrtes Some strains also produce another toxic metabolite called the B—cxotoxin or thuringiensin which has a much wider spectrum of activity. B—exotoxin competes with ATP on the binding site of the enzyme, RNA polymerase (Deacon 1983). It is not only active in some insects, but also against vertebrates, therefore, it must be removed from commercial formulations (Deacon 1983, Mucler and Harper 1987, Priest 1989). Against 29 young larvae (3 to 6 h old) of Spodoptera fiugiperda (J . E. Smith), fi-exotoxin was 27 we toxic than B. timringr‘ensts (Dipel®), and combinations between those two toxins demonstratedsynergism(MuellerandHarper 1987). B—exotoxinwasalsotoxictolarvae of S. fivgiperda, Heliorhis sea (Boddie), Mchaplusia ni (Hubner) (Hornby and Gardner 1987), and Lygus Writs Knight (Tanigoshi et al. 1990). Even though the four species showeddifi'erentsusceptibility, thefirstinstarswercthcmost sensitiveineach species. c. Host range of GyIHA CryIIIA is efi‘cctive in controlling some coleopteran. Keller and Langenbruch (1993) documented 26 susceptible species; 21 species belong to chrysomclidac, three species from curmlionidac, one from coccincllidae, and one from tenebrionidae. In the chrysomclid family, toxicity of the toxin varies from species to species. Colorado potato beetle (Hermstadt et al. 1986, Ferro and Gelernter 1989, Keller and Langenbruch 1993, Whalon ct al. 1993) and cottonwood leaf beetle, C. scripta (Bauer 1990) are examples of susceptible species. However, the toxin is much less effective against larvae of southern corn rootworn (Slaney et al. 1991). This may be due to the lower afinity of cryIIIA binding to receptors in the midgut and reduced ability to enhance membrane permeability (Slaney et al. 1991). 2. Insect Resistance to Baciflus thuringiensi.: Since B. thrm‘ngienris occurs frequently in nature, insects lmve probably been naturally exposed and some selection may have resulted. Therefore, in some populations there may already be some level of resistance or at least extensive variability in susceptibility (Boman 1981). Therefore, if selection pressure (exposure) increases on these pro-selected populations, the population may shifi and a majority become resistant. Swaflresearchemhaveprovenmmmisproccssaauaflyocwmmnatmeflabmet 30 al. 1990, Tabashnik et al. 1992, Shelton ct al. 1993). McGaughey and Whalon (1992) listed six insect pests (diamondback moth, Colorado potato beetle, Indianmeal moth, Tropical warehouse moth Cadm mtella (Wlk.). Tobacco budworm Heliothis w‘rescens, Sunflower moth Homoeosoma electclhmt and two mosquito species Aedes aegipa and Culcx quinqucfmciattw) which have been selected for resistance either in the field or in the laboratory. Resistance mechanisms to CryIA(b) in Indianmeal moth and diamondbaclt moth havebeenresearched (VanRiectal.1990, VanRieetal.1992).'I’hesetworesistances were due to an alteration in toxin-membrane binding affinity or quantity. The resistant strain had a 50-fold reduction in the amnity with CryIA(b). 0n the other hand, both speciesbecamemorc sensitivcto CryIC. Thiswascausedbyanincreaseintbenumbcrof Crle receptors after several generations of selection. Unlike Indianmeal moth, the resistant strain of rice moth, Corcyra cephalom’ca (Stainton), produced a sticky layer that protected the insect from the toxin (Chiang ct al. 1986). ’In' addition, Oppel't et al. (1994) found that midgut enzymes (proteinases) in Indianmeal moth resistant to B. thufingiensis exhibited a five-fold lower activity than in the susceptible strain. These three studies have demonstrated that difl‘erent species may have similar or difi‘erent mechanisms of resistance. Therefore, strategies for preventing or delaying the development of resistance must be specific to the species and an understanding of the underlying mechanism for resistance would be useful in avoiding or delaying resistance. Until 1994, there was no published data mentioning Colorado potato beetle resistance to B. thufingiensis in the field. However, after 12 generations of laboratory selection, a field derived strain was 60 times more resistant than the unselected strain (Whalon et al. 1993). Whalon et al. reported that in the first four generations, the resistance ratios were very low (RR = 1) followed by a dramatic increase in the fifth generation (RR = 22). The resistance ratios fluctuated during the fifth to eleventh generations with little difi‘erence in resistance. In the twelfth generation, again the 31 resistanceratioincreased significantly(RR=60). Thus, further selectionenhancedthe resistance level dramatically. The presence of B. tintringiensis on the potato plants significantly reduced the mmber of eggs laid by a Colorado potato beetle females (Whalon et al. 1993), and the resistantstrainovipositedatarateofOJ £0.2eggmassesperday,buttheB. rlntringiensisstrscepnblesuainwasoostafic. Bothstrainsfailedto showanydifi‘erencesin feedingbchaviorexccptthattheybothincreased stemandpetiolcfeedinginthcprcsence of B. (Inningiensis. Further information about 001111! resistance mechanisrn(s) in Colorado potato beetle are not available, although considerable research is now underway. Understanding the mode of action should help to explain the resistance mechanism (Slaney ct al. 1991) by comparison of all the steps of intoxication in the susceptible and resistant strains. There are six hypothesized mechanisms (Boman 1981, Ferro 1993): 1) insensitivity of the receptor for the corresponding toxin, 2) detoxification of the toxin, 3) failure to move the toxin from the ingestion site to the target site, 4) the ability to avoid ingestion of the protoxin, 5) alteration of pH to prevent solubilization and activation, and 6) increased recovery mechanism. C. MATERIALS AND METHODS l. Cultures and Rearing a. Potato plants In this study, I used ”Superior” potato plants, and the plants were reared in the Pesticide Research Center (PRC) greenhouse. Seed potatoes were planted in 16-cm clay potsandwatcreddaily. Threeweeksaficrplanting,thcplantswcrcfertilizedandthc plants (6 to 8-wecks-old) were used for rearing, selection, and bioassays. Quality of plants were continuously maintained and unhealthy plants were discarded. Seed potatoes were stored in a cold room at 4°C. However, sprouting decreased as the seed aged. As a results, the vigor of potato plants oficn varied fi'om time to time (Whalon et al. 1993). b. Colorado potato beetle B. tlna'ingiensis susceptible and resistant strains of Colorado potato beetle adults were obtained from laboratory stocks at Pesticide Research Center, Michigan State University. Both strains were in their 29th generation (F29). Adults were originally collected during the Summer of 1987 and 1988 from potato farms located in Barry, Allegan, Clinton, Ingharn, Macomb, Montcalm, and Washtenaw counties in Michigan (Whalon ct al. 1993). They had been reared in the laboratory for 29 generations at 25¢2°Candaphotopcriodof 16 : 8(L : D). Theresistantstrainwassubsequcntly selected every generation (see below). Adults were placed in cages (0.4 by 0.4 by 0.45 m) with six pots per cage. Egg masses were collected daily then put in a 15-cm plastic petri dish until eclosion. Egg massesthathatched onthcsamedaywcreplaccdinpetridishes(5cggmassespcrpctri 32 33 dish)andmehrvaewaefedndthpotnofofiagemfilmethirdmstar.latemmm waemsfaredomopotatopmunfilaflhwaeunaedthesoflnhenfliepotatophms manandthepotswerccovaedadthapetfidishAdultsthnemergedfiomthesofl wereplacedincagesandtheovipositionpopulationwasmaintainedatZO-25 adultsper age.Whenevatoomanyaduhsanageddtuingashonpaiodoffimetheexcessaduhs wereputinacooleratS°Candheldtorestocktheovipositioncages.Theresistantand susceptible strains were reared in different buildings to avoid contamination. 2. Bioassay All bioassays were conducted with formulated cryIIIA 5—endotoxin of B. tlnuinglensis (SPUDCAP®, Mycogen Corporation, San Diego, California). The formulation consisted of 12.35 mg of active ingredient (AI) per liter. a. Selections The concentration of B. tlna'ingiensis solution used for selections was 60 ml of formulated product per liter (741 mg active ingredient per liter). The potato leaf-dipping methods was employed for selection (Whalon et al. 1993). Briefly, potato foliage was trimmed to the terminal five leaflets. The stem was wrapped with a sponge to avoid water spillageandinsertedinto a2-mlvialcontainingdistilledwater. Eachleaflctwasdippedin B. tlnm‘ngiensis solution 5 x for 30 seconds each and allowed to air dry before putting it intoa 15-cmpetridish. Eachpetridishhad20-25 secondinstars(3 -4dold)dcpending onthesizeofleaf,anditwashddat25¢2°Candaphotoperiodofl6 : 8(L:D). Iarvaewereexposedfor96hours(h)tothetreatedlcavcs. Thccxperimcntwaschecked dailyandwaterwasaddedtovialswhennecessary. Afler96h,thesurvivinglarvacwcre couMedfiomeachpefiidishmdUmsfaredMounfieatcdfofiagewhacwrvivomgrew untillatethirdinstars. Larvaewerethenplaced onuntrcatcdpotato plantsincagesuntil pupation. Adults emerging fi'om pupation were collected daily. 34 b.Resistanceprogression To measure resistance progression from generation to generation, bioassays were wnduaedusingwcondmstus(4dold).Theredstamemfiowucompmedbywmpadng theszooftheresistantstraindividedbythatofthcwscepfiblestrain Theleaf-dipping mahodwasusedupmnouslydesaibed.0nthebasisoftheprdiminuyuuy,fiveto sevensaiddilufiomwaesdeaedforthewmplaeauayiachwncennafionhadulean threereplicationswithtenlarvaeeach. Ifthe mortalityinthecontrol exceeded 20%,the atpaimentwasrepeated.Monafitywasassessedu96hafierexpomandconfimed every24huntil l68haficrtrcatment.Larvacwereweighedthreetimesduringthc experiment: 1) beforcbeingtransferredontotreated leaves, 2)96hafier exposure,and3) 168 h after exposure. Initially, all ten individual larvae per each replication, were weighed togetha.0nlyfivelarvaefiomeachpenidishwaeweighedwbsequenfly.hwal developmentwasrecordedbyinstarfi'om 48 to 168 h. 3. Life Cycle Tth35generationofresistantand susceptiblestrainswasutilizedinthclifccycle studies. The experiments were initiated from the egg stage by randomly selecting three egg masses per day over three successive days fiom both the resistant and susceptible colonies. Themmbermdviabifityofeggspaeggmasswerenoted.Ncwlyanergedhwaewcrc reared on fiesh potato leaves. Ten 1 d old larvae from each strain were selected for the larval development study. All selected larvae were weighed individually, and each larva was randomly placed in difi‘erent petri dishes and maintained on fresh potato leaves. Larval development was recorded until the larva entered into the soil for pupation. Each larva was weighed every otherdayto determineitswcightgain. Afterreachingthefourthinstar, thclarvawas transferred to a plastic pot filled with soil for pupation. All remaining leaves were removed 35 fromtheplasticpotsafierthelarvacntcredintothesoil.Thedateofadulternergcnce from pupation was noted and the adults were weighed individually. To study fecundity, seven pairs each from the susceptible and resistant strains were selected randomly from 40 adults. Each pair was placed in a plastic cage on one potato plam.Phntsweremaimainedbywataingasneccssmymdreplaccdthcmufithfiesh mataifluneededtomaintainfoodquafitylhemmbaofcggmassesandthemmbaof eggspercggmasswerecountcddaily. 4. Data Analysis LC50 s were determined by regressing log dose on probit mortality (Ming and Finney 1984). Data were corrected for control mortality using Abbott's formula (Abbott 1925) before they were submitted to probit analysis. LC50 s were computed on the bases ofthedatacollectedat168 hposttrcatrncnt. Asignificamdifi‘ercncebetweentwomso s was based on non-overlapping 95 % Confidence Interval values. Homogeneity of slopes between two regression lines were tested with SYSTAT (Wilkonson 1992). Difl‘erences betweentwomcanswereestablishedusingttestata=0.05 (GomezandGomcz 1984, MSTAT; Freed et al. 1991). D. RESULTS AND DISCUSSION 1. Selections B. tlmfingiensis selection on second instar Colorado potato beetle varied fi'om 5.3 to 17.3% survival across six generations (Table 2.3). At generation F3], 318 adults emerged from pupation and they represented approximately 15% of the number of initially selected larvae. Over the next two generations (F32 and F33), the survival was maintained at 5.9 and 5.3% respectively. Yet, survival in these two generations was still higher than that of generation F1 to F12 as reported by Whalon et al. (1993). The survival in generation F34 reached 17%, and it remained stable through the F3 5 generation. Survival declined in generation F36 to 7.5%. The lower survival rate were consistent with the results reported by Whalon ct al. (1993) since the percent survival in these selections (generation F1 to F12) also fluctuated fi'om generation to generation. A similar pattern of variable survival was reported in other laboratory selections with B. thuringiensis on diamondback moth (Tabashnik et al. 1991). Survival data for generation F13 to F30 were not available; therefore I was unable to make comparisons to these generation. Second instars showed difi‘erent susceptibility to B. thufingiensis (741 mg [All/l) within each generation. Survivorship as measured fi'om eggs through the termination of oviposition varied fi'om 45 - 65 % with the highest survival usually occurring in eggs laid during the middle of the oviposition period. Differences in vigor or viability through the oviposition period may have contribution to differences in adult survival between generations. Variation in the plants vigor may have caused this variation. 36 37 Table 2.3. Response of Colorado potato beetle to selection with cryIIIA 6- wdotoxin of Bacillus flnaingienn’s at concentration of 741 mg (AI)/l. No. No. % Generations Selection period larvae adults Survival exposed F3] 4 July 1993 - 26 July 1993 2150 318 14.8 F32 7 August 1993 - 9 September 1993 1170 69 5.9 F33 22 September - 21 Octobcr1993 1046 55 5.3 F34 22 November - 11 January 1994 1010 172 17.0 F35 23 February 1994 - 18 March 1994 595 103 17.3 F35 2 April 1994 - 1 May 1994 1035 78 7.5 2. Resistance Progression LC50 s of Fz-Flz generations were significantly lower than those of F17-F34 generations (Table 2.5). LC50 of generation F17 was significantly difi‘crent (P S 0.05) from that ofF2 - F12 and F31- F34 generations. Even though thercwas an increase in LC50 fiom F31 to F34 generations, they were not significantly difi‘crent (P > 0.05). This was due to high variability in the F31 generation as indicated by wider 95% CI. Anderson et al. (1989) also noted high variability in mortality within replicates when Colorado potato beetle was treated with Beauveria basriana (Balsarno). This phenomena was probably associated with the mode of action of these microbial insecticides. ClyIIIA 5- endotoxin of B. thuringiensi.: is very slow in initiating intoxication of the target insect (Bauer and Pankratz 1992) and this may contribute to high variability in dosage mortality experiment. As selection progressed, the resistance level increased dramatically (from 1 to 714- fold). These results indicated that resistance alleles were present in the population (Roush and McKenzie 1987, Whalon et al. 1993). Nevertheless, increasing resistance levels were not always followed by corresponding increase in the slope of regression line. The slopes Table 2.4. Progression of Colorado potato beetle resistance to Bacillus Mngiensis cryIIIA 5-endotoxin Generations“ Strain" n Lcsoc 95 % CI Slope RR" (mslAIl/l) (“ISIAH/l) F2 R 375 0.7: 0.3- 4.5 1.1 1 F7 R 420 24 b 20 - 30 1.9 14 F12 R 270 100 c 95 - 111 1.3 60 117 R 430 330 d 263 - 440 1.2 223 s 240 1.5 1.1- 2.1 1.5 F21 R 540 369 d 286 - 535 1.0 222 s 240 1.7 1.3- 2.3 1.9 F31 R 130 998 c 642 -2,283 1.2 525 s 150 1.9 1.1- 3.0 1.2 F34 R 280 1,071 e 334 -1,393 1.6 714 s 210 1.5 1.2- 1.8 2.7 “F2412 werecitedfi'omWhalonetal. (1993), F17-F21werecitedfiomRahsrdjaandWhalon(l994, inpess),F31: larvaewuecxposedonthetreatedleavesfor96h,F34:larvaewereexposedontreatedleavesfor96h,butthe leaveswuesubstimtedwithothertreatedleavesatflhaflaexponne bR:resistantsfiain;S:susceptiblcstrain. °LC50sfollowedbythemekflawithinachflrahuenasignifianflydifl'mbasedonnm-omlapping”% Confidence Intervals. dRR(resistanceratio)istheLC50forflseresistmnsnaindividedbyflieLCSOfcrfltemscqrtiblestainateach 39 fluctuated fiom 1.0-1.6 over 34 generations. Shallow slopes indicated that the resistant popuhfionhadmtstabflizedmdhadthepotenfidtomcreasemrednance(Seawfight 1972). Asimilarpattanoccunedinhouscflypoprflafiomsdectedwfithdiflubennnonand cyromazine (Keiding et al. 1991). . Homogeneity analysis of slopes (Wilkonson 1989) showed no significant difl‘erence (P> 0.05) betweentheF31 and F34 generationsintheresistant strain. IntheF31 generation, theregression slopesforthesusceptibleandtheresistant strainsweresimilar (P > 0.05); however, they were different in the F34 generation (P S 0.05). In the susceptible strain, increasing slope may have indicated that the F34 generation had less susceptible variability than the F31. Until the 29th generation, the laboratory had three subpupolations ofthe susceptible strain when they were mixed into one population. To avoid cross contamination the resistant and susceptible strains were subsequently reared in different building. Greater susceptible variability in generation F31 may have been due to contamination with the resistant strain in one of the subpopulations. Since there was no selection pressure and the generation progressed, the fiequcncy of resistant individuals decreased and the slope of regression line increased significantly. In both the resistant and the susceptible strains, increased B. tlum‘ngiensis concentrations delayed larval development (Figure 2.1 and 2.2). Concentrations used in the experiment for the resistant strain ranged fiom 92.6 to 1482.0 mg (AD/l. In the control treatment, all surviving larvae had become fourth instars at 168 b. However, at concentrations of 92.6, 185.3, 370.5, and 741 mg (AI)/l some surviving larvae were still in the third or fourth instars (Figure 2.1). In contrast, at the highest concentration (1482 mg (AI)/l) all surviving larvae were still in the third instar. For the susceptible strain, concentrations used in the bioassays ranged fiom 0.5 to 7.4 mg (AD/l. Like the resistant suainallsusccptiblelarvaeinthccontrolhadbecomefourthinstarsat 168 h. At concentration of 0.5 mg/l and above, the proportion of fourth instars decreased with ammonia c. use my (AM a. 370.5 mg (AM F. 1462.0 mg (AM ///////ln E. 741.0 mg (AM 188 120 144 Hours after treatment Colorado potato beetle treated with difi‘erent concentrations of cryIIIA 5—endotoxin. Figure 2.1. Larval development of the Bacillus thufingiensis resistant strain of Total height of bars indicates percent survival including all instars 41 B.0.Smg(AI)II n. 3.7 mg (AM mmmwwmo A$ve£5£§u§m£ w w a. a. mmm n m %m c. 1.0 mg (AM Hours after treatment E 1 4 mg (AM %///| a m m a a o m 0 38.58.3235 Hours altar treatment F igurc 2.2. Larval development of the Bacillus thuringiensis susceptible strain of Colorado potato beetle treated with different concentrations of cryIIIA 5—endotoxin. Total height of bars indicates percent survival including all instars. 42 increasing concentration (Figure 2.2). These results suggest that B. tlna'ingiemis not only killed the larvae, but also slowed the development of surviving larvae. B. ilnaingiensis delayed the larval growth of susceptible and resistant strains in a dose-dependent fashion. Increased concentration resulted in lower weight (Figure 2.3). Theaverageinitiallarvaweightrangedfrom 5.0-6.4mgfortheresistantstrainand5.0- 6.7 mg for the susceptible strain. Before regression analysis were done, the concentrations were log transformed to linearize the relationship between concentration andweightgain Thercgressionofwcightgainonconccntrationresultedinlinesforthe susceptible strain at 96 and 168 h ofy = 25.3 - 21.0 x with an r = - 0.92 andy=85.8 -79.1xwithanr=-0.87respectively,wherey=wcightgain,x=log concentration, and r = correlation cocficicnt. The regressions of the resistant larval weight gainwerc y=44.7-11.6xwithanr=-0.94at96handy=194.5-54.6xwithan r = - 0.99 at 168 h. Homogeneity of regression coeficicnts analysis (Wilkonson 1992) indicated that regression lines for 96 and 168 h within each strain were difl‘crent (P S 0.05). These difi‘erenccs probably resulted fi'om the level of stress delivered by different concentrations. Even though all surviving larvae were fed with untreated leaves afier 96 h of exposure, the efl‘cct of the difi‘erent concentrations was still observable. Thus surviving larvae fi'om low concentrations grew better than those from high concentrations. In other words, the weight of surviving larvae from low concentrations increased at a greater rate than those fiom high concentrations. As a result, the regression lines for 168 h in both strains were steeper than those for 96 h At 96 h post treatment, the slope ofthe susceptible strain was difi‘erent from that of the resistant strain (P S 0.05). However, at 168 h slopes ofthe resistant and the susceptible strains were not significantly difi‘crent (P > 0.05). These results confirmed that the susceptible strain grew faster than the resistant strain during this exposure period. However, afier the survivors were fed with untreated leaves both strains showed the same growth rate. This indicates that the relationship between concentration and weight gain in both strain is similar. 43 Log concentration (mg [Allll) Figure 2.3. The relationship between concentration of crylllA 5-cndotoxin of Bacillus thuringiensis and larval weight gain of Colorado potato beetle $96 and $168: the susceptible strain at 96 and 168 h after treatment respectively. R96 and R168: the resistant strain at 96 and 168 h after treatment respectively 3. Life Cycle The B. tlna'ingiensis resistant strain of Colorado potato beetle required a significantly longer time to complete a generation than the susceptible strain (Table 2.5). Comparedtothcsusceptiblcstrain, theresistantstrainrcquiredsignificantlylongertimeto completethelarval stadiaaswellasthelifccyclc, andhadlowcrfecundityand smallcregg masses. Thesefitnesscostsmaycontn‘butctotherapiddecreaseinresistancewhen selection is discontinued (Roush and McKenzie 1987, McGaughey and Whalon 1992, Tabashnik 1994). The resistant egg stadia averaged 5.6 days compared to the susceptible which was only 5.0 days, however, they were not significantly difl‘erent (P > 0.05). Percent egg hatch data were transformed using ‘1 x before analysis (Gomez and Gomez 1984). The resistant and susceptible percent egg hatch was very high and they were not significantly difi‘crcnt ( 97.8 and 98.4 % respectively). Seven out of nine egg masses completely batched in both strains. To complete the larval stadia (Table 2.5), the resistant strain required significantly (P 5 0,05) more time (13.1 :1: 0.9 days) than the susceptible strain (11.9 a: 0.7 days). The resistant instars l to 3 were longer than those the susceptible strain, and they were longer ineachlarvac stage except forthcthird instarwhichwas3.0:t0.5 daysand 21:1: 0.7 days for the resistant and susceptible strains respectively. Fourth instar in the resistant strain revealed a surprising trend toward a shorter development time when compared to the susceptible strain, yet they were not significantly difi‘crent (P > 0.05). In both the resistant and susceptible strains, thefourthinstarwaslongerthanthefirstthrecinstars. The resistant strain exhibited lower weight gain (P S 0.05 ) than the susceptible strain during larval development (F igurc 2.4). Newly emerged larva fiom the susceptible and resistant strains had similar weights, however, in the next stadia the susceptible larva were significantly heavier and required less development time than the resistant strain. These results indicated that there was a significant developmental cost in the resistant 45 Table 2.5. Summary statistics for the length ofeach stage and the % egg hatching for Bacillus thuringiensis resistant and susceptible strains of Colorado potato beetle. Stage Resistant Susceptible strain strain T test“ (x 3; SD.) (X 4: SD.) 1. Eg a. Length (days) 5.6 :l: 0.5 5.0 :l: 0.7 as b. Hatching (%) 97.8 :1: 4.5 98.4 :1: 3.3 ns 2. Larva (days) 13.1 d: 0.9 11.9 :1: 0.7 s a. First instar 3.4 i 0.5 3.1 i 0.3 as b. Second instar 2.6 :1: 0.5 2.4 :l: 0.5 as c. Third instar 3.0 :l: 0.5 2.1 :l: 0.7 s d. Fourth instar 4.1 :1: 1.2 4.3 t 0.5 ns 3. Prepupa to adult (days) 12.7 d: 0.8 12.7 i 0.5 ns 4. Female a Preoviposition (days) 4.6 :1: 1.3 3.9 :l: 0.7 as b. Number ofegg masses/female 28.6 t 13.0 41.6 :1: 16.5 as c. Number of eggs/female 444.4 1 148.7 1,208.3 :1: 850.1 s d. Number ofcggs/egg mass 16.3 :1: 2.4 27.8 :1: 11.3 s 5. Life cycle (egg to 688) 36.1 :1: 1.1 33.4 :1: 1.7 s 3rns:nosignificantdifl‘crencebetweenresistantandsusceptiblestrains,s:significantdifl‘erenceatts=0.05. 225 — T 200 T l / 175 150 125 100 Weight (mg/ larva) O 1 2 3 4 5 6 7 8 9 10 11 12 Age (days) Figure 2.4. Larval development of Colorado potato beetle. 8: the Bacillus thuringiensis susceptible strain. R: the B. thuringiensis resistant strain fi“ 47 strain Thisphenomenavariesfrom speciesto species. Asimilarpatternwasreportedin B. thuringienrls var. inaclensis resistant strain of Culex quinquefascianrs (Georghiou 1989). On the other hand, H. virescens resistant and susceptible strains had no difl‘erences in larval weight (Gould and Anderson 1991). Both the resistant and susceptible strains took 12.7 days for prepupa to adult stage (Table 2.6). Since the Colorado potato beetle pupates in the soiL it was dificult to observcthelengthofprepupalandpupal stagesseparately,thuswhcnthelarvaentered thesoilwasinitiated forthepurposesofthescmcasurcrnent. Females fi'om the resistant strain had significantly lower fecundity and fewer eggs per egg masses than the female susceptible strain (P S 0.05). The resistant strain produced 289 to 607 eggs per female with an average 444 eggs per female; whereas the females susceptible strain laid between 402 - 2,718 eggs with an average 1208 eggs per female. In addition, females from the resistant strain only laid 16 eggs per egg mass, yet the susceptible strain was able to lay 28 eggs per egg mass on average (Table 2.6). Research on the efi‘ccts of B. thuringiensis on fecundity have been reported by several researchers. Treatment with B. thufingiensis significantly reduced the number of egg masses per day produced by the resistant and susceptible strains of Colorado potato beetle (Whalon ct al. 1993). C. quinquefasciatus (Georghiou 1989) and P. xylostella (Groetcrs et al. 1994) resistant to B. thuringiensis produced fewer eggs than the susceptible strain. On the other band, B. tlmringiens'is resistant and susceptible strains of sunflower moth did not differ in their fecundity (Brewer 1991). However, the sunflower moth exhibited very low (< 2 fold) resistance and this case is an anomaly (McGaughey and Whalon 1992). Thus the efi'cct of B. thla'ingiensis selection on fecundity usually caused reduced fecundity. To develop metabolic resistance mechanisms to a particular insecticide, insects may have to compensate by reducing or mitigating other defense mechanisms (Bower 1992). 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Diamondback moth resistance to Bacillus thuringiensis in Hawaii, pp. 175-183. In N. S. Talekar [ed], Diamondback moth and other crucifer pests. Proceedings of the second international workshop. Taipei, Taiwan: Asian Vegetable Research and Development Center. Tabashnik, B. E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39: 47-79. Tabashnik B. E., N. L. Cushing, N. Finson, M. W. Johnson. 1990. Field development of resistance to Bacillus rhrm‘ngiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83: 1671-1676. Tailor, R J. Tippett, G. Gibb, S. Pells, L. Jordan, and S. Ely. 1992. Identification and characterisation of a novel Bacillus tlna'ingiensis} 5—endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol. Microbiol. 6(9): 1211-1217. Tailor, R, J. Trippet, G. Gibb, S. Pells, D. Pike, L. Jordan, and S. Elly. 1992. Identification and characterisation of a novel Bacillus thuringiensis 5—endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol. Microbiol. 6(9): 1211- 1217. ~ Tanada, Y and H. K. Kaya. 1993. Insect pathology. Academic. San Diego. Tanigoshi, L. K., D. F. Mayer, J. M. Babcock, and J. D. Lunden. 1990. Eficacy of the B- exotoxin of Bacillus thuringiensis to Lygus havens (Heteroptera: Miridae): Laboratory and field responses. J. Econ. Entomol. 83(6): 2200-2206. Thorne, L., F. Garduno, T. Thompson, D. Decker, M. Zounes, M. Wild, A M. Walfield, and T. Pollock. 1986. Structural similarity nucleotide sequence the Lepidoptera- and Diptera-specific insecticidal delta-endotoxin genes of Bacillus tlna'ingiensis subsp. Imrsralci and israelensis. J. Bacteriol. 166: 801-811. Tungpradubkul, S., C. Settasateirl, and S. Panyim. 1988. The comlete nucleotide sequence of a 130 kDa mosqutio-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. iseaelensis. 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Entwistle, J. S. Cory, M. J. Bailey, S. Higgs, Bacillus thuringiensis, an environmental biopesticide: theory and practice. John Wiley & Sons, New York Visser, B., E. Munsterman, A Stoker, and W. G. Dirkse. 1990. A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crytal protein. J. Bacteriol. 172: 6783-6788. Waalwijck, C., A M. Dullemans, M. E. S. van Workum, and B. Visser. 1985. Molecular cloning and the nucleotide sequence of the Mr 28 000 crystal protein gene of Bacillus thuringiensis subsp. israelensis. Nucleic Acids Res. 13: 8206-8217. Wabiko, H., K. C. Raymond, and L. A Bulla, Jr. 1986. Bacillus rlmringiensis entomocidal protoxin gene sequence and gene product analysis. DNA 5: 305-314. Walters, F. S. and L. H. English. ? . Toxicity of Bacillus tlnrringiensrs 5—endotoxin toward the potato aphid in an artificial bioassay. Entomologia experimentalis et applicata. In press. Walters, F. S. and L. H. English. Toxicity of Bacillus thuringiensis 8-endotoxin tpward the potato aphid in an artificial diet bioassay. Entomologia experimentalis et applicata, in press. Ward, S. and D. Ellar. 1987 . Nucleotide sequence of a Bacillus thuringiensis var. israelensis gene encoding a 130 kDa deltaendotoxin. Nucleic Acids Res. 15: 7195. 61 Whalon, M E., D. L. Miller, R M Hollingworth, E. J. Grafius, and J. R Miller. 1993. Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain resistant to Bacillus tlnm'ngiensis. J. Econ Entomol. 86(2): 226-233. Wharton, D. A and L. W. Bone. 1989. Bacillus thuringiensis israelensis toxin afi‘ects egg-shell ultrastructure of T richaslrongylus colubrifonnis (N ematoda). Invertebr. Reprod. Dev. 15(2): 155-158. Widner, W. R and H. R Whiteley. 1989. Two highly related insecticidal crystal proteins of Bacills tlnrringiensis subsp. kurstaki posses different host range specificities. J. Bacteriol. 171: 965-974. Wilkonson, L. 1992. SYSTAT: the system for statistics. SYSTAT, Evanston, IL. Wu, D. X. L. Cao, Y. Y. Bay, and A I. Aronson. 1991. Sequnece of an operon containing a novel 8-endotoxin gene from Bacillus thuringiensis. FEMS Microbiol. Lett. 81: 31-36. Yamamoto, T., I. A Watkinson, L. Kim, M. V. Sage, R Stratton, N. Akanda, Y. Li, D. P. Ma, and B. A Rec. 1988. Nucleotide sequence of the gene coding for a 130 kDa mosquitodal protein of Bacillus tlnrringiensis israelensis. Gene 66: 107-129. CHAPTER III TOXICITY OF FOUR INSECT GROWTH REGULATORS AND IMIDACLOPRID AGAINST COLORADO POTATO BEETLE (COLEOPT ERA: CHRYSOMELIDAE) (It A. INTRODUCTION Thenumberofarthropods(insectsandmites)resistanceto pesticideshasincreased dramaticallyforthelastfortyyears, andmorethanSOO specieshavebecomeresistantto one or more pesticides (Georghiou 1990). The causes of this rapid increase appears to include genetic, biological, ecologicaL behavior, and operational factors (Georghiou and Taylor 1976, McGaughey and Beeman 1986, Roush and Tingey 1991, Tabashnik et al. 1991). The synthetic organic insecticides developed since 1945 are mostly neurotoxins with limited variation in their mode of action (Matsumura 1985, Hammock and Soderlund 1986, Ballantyne and Marrs 1992, Chambers 1992). For example, organophosphates and cmbamatesmtwodifl‘erentdassesofinsecficideabrntheyactutheumesiteu inhibitors of the acethylcholinesterase enzyme (Matsumura 1985, Ballantyne and Marrs 1992, Chambers 1992). As a result, rotations with these two classes do not usually reduce selection pressure. A similar situation also exists for most chlorinated hydrocarbons and pyrethroid insecticides (Chang and Plapp 1983, Hammock and Soderlund 1986, Eldefiawi and Eldefiawi 1990). Resistance to DDT, the prototype chlorinated hydrocarbon, protects the insect from pyrethroid insecticides as well. The limited rotation of compounds with difi‘erent modes ofaction may have contributed significantly to the rapid increase in the number of insects resistant to insecticides. Therefore, development of insecticides with novel modes of action has become increasingly important (Hammock and Soderlund 1986, Spuks 1990, Mayer et al. 1990, Mitsui et al. 1991, Kupatt et al. 1993, Miyamoto et al. 1993). Insecticides that target other sites ofaction beyond the nervous system have been the object of development over the last decade (Ware 1989, Mayer et al. 1990, Mitsui et 63 al. 1991, Elek and Longstafl'1993, Miyamoto et al. 1993). Insect growth regulators are a groupofinsecticidesthatdisruptgrowthanddevelopmentininsects. Onthebasisoftheir acfionimectgr'owthregrrlatorsareclassifiedintotwomajorgroups: asjuvenilehormoue analogsandaschitinsynthesisinhibitorsMare 1989,ElekandLongstafl‘1993, Miyamoto et al. 1993). Juvaulehomoneanalogsmptimmanrrcdevdopmemwdaduhemergencein arthropods. Juvenilehormones(JHO, I,Il,and4-methleH)oftheseclassesaretherefore unique to insects (Schooley and Baker 1985). Tlars, these insecticides including methoprene and kinoprene are nontoxic to mammals (Sparks 1990). Chifinsymhesisinhibitorspreventtheformafionofchitindmingmolting, an essential component of the arthropod cuticle. Chitin synthesis inhibitors consist of benzoylphmyl areas. polyxins and nikkomycins. “PM and Mphwlide fimsicidu. triazine, kitazin, tunicarnycins, avermectins, and plumbagin (Hajjar 1985). Difiubenzuron (Dimilin®) was the first commercial formulation fi'om benzoylpherlyl ureas (wtre 1989). Triazine, avermectin and plumbagin have been used as insecticides (Mayer et al 1990), whereaspolyoxinsandkitazinareusedasfirngicidesG-Iajjar 1985,Majeretal. 1990). The mseagrowthreglflmomhawbemmmunnglyusedforwnfioflingmydifl'aaum (Adcock et al. 1993, Bery et al. 1993, Bull and Meola 1993, Haynes and Smith 1993). In potato, Colorado potato beetle is the most important defoliating pest in many countries (Ferro et al. 1989, Heim et al. 1990, Malinowski and Pawinska 1992), and control has been dependent on insecticides since 1800's. Problems with resistance in this pest have occurred repeatedly. Colorado potato beetle was first recognized as resistance to DDT in 1952 (Gauthier et al. 1981), and has become resistance to all synthetic insecticides including pyrethroids on Long Island, New Jersey (Gauthier et al. 1981, Forgash 1985, Georghiou 1986). In Michigan, most Colorado potato beetle populations were resistant to at least one of the major groups of insecticides (chlorinated 65 hydrocarbones,organophosphates,carbamates,andlor synthetic pyrethroids)by 1987 (Ioannidisetal.1991). Asaremhoftheseredstmceproblenuwithwnvenfiomlnuuotordcinsecficides, reseachontheeficscyofinseagrowthmguluoahumceivedmonutenfionmcenfly. MalinowskiandPawinska(1992) reportedthatbenzoylphenylureas(chlorfluazuron, heaaflrmnnonteflubenwonniflunnnonandnovahnon)werevayefl‘ecfivein confioflthdondopomobeetleluvaemdredmingtheabifityofeggsmhatcth addifioncyromazineacompoundfiomthefiyazinefamily,wasdsoefl‘ectivein controllinglarvaeof Colorado potato beetle (Sirotaand Grafius 1994). Ithasalsobeenreponedthatmseagrowthngmnomwereefi’ecfivemconfiofling some insects that have been resistant to conventional insecticides. Teflubenzuron and chlorfluazuron were reported as efl‘ective against resistant diamondback moth (Perng and Sun 1987).Ahouseflystrainthatmsvafiouslyredthocomenfionalinsecfiddeswu smsifivemcyromazineyetmothasuainobtainedfiommueawhaecyromazinehad beenusedtoconfiolmotherinsectwumoda'atdyresistamasekiandGeorghiou 1986). Navanoetal.(1992)reponedthatmethopreneanddiflubenmroncmudbeusedto control a malathion resistant strain of Culcx quinquefasciatras. In these studies, diflubenzuron was more toxic than methoprene. Ontheothahmdpmblansofresistancetoinsectgrowthregulatomhasbeal naed.ResistancetodiflubennuonmthecodlingmothwasreponedbyMofinetal. (1988). In laboratory selection, diamondback moth larvae selected with teflubenzuron for 20generationsbecame8-12tirnesmoreresistantthananunselectedstrain(Perngetal. 1988). They also reported that the selected strain developed cross-resistance to the ovicidal efl‘ects of teflubenzuron. Another laboratory selection with cyromazine against house fly showed a similar situation. After 15 generations of selection, the house fly became 68.9 fold more resistant to cyromazinethan anunselected strain (Bloomcamp et al. 1987). Incidence of cross-resistance between one insect growth regulators with otha insect growth regulators or conventional insecticides depends on the resistance mechanism, previous insecticide exposure history, and species of insect. Cross resistance was not found between teflubenzuron and another insect growth regulator, chlorfluazuron, or several conventional insecticides (fenvalerate, mevinphos, permethrin, carbofuran, and prothiophos) in diamondback moth (Perng et al. 1988). In house fly, a cyromazine- resistant strainwassusceptibletoanotherinsectgrowthregulatormethoprene (Bloomcamp et al. 1987). However, in other research there was cross resistance between cymmazineanddiflubennuoninflnhouseflyashuya1992).1heuremhsmggeuthat insecticideresistancemanagernentisessential to managetheefiicacyofinsectgrowth regulators and species specific cross resistance information is needed. It has been reported that B. thuringiensis was efl‘ective in controlling Colorado potato beetle populations that have developed resistance to chemical insecticides (Ferro and Gelernter 1989, Zehnder and Gelernter 1989, Ferro 1993). Cross resistance of B. tlruringiensis to chemical insecticides including insect growth regulators have been explored (Whalon et al. 1993) but not found. Different modes of action and mechanisms of resistance may have resulted in lack of cross resistance between B. thuringiensis and chemical insecticides (Stone et al. 1991, Marrone and Maclntosh 1991). In order to explore new potential insecticides for Colorado potato beetle, I tested four difl‘erent insect growth regulators (diflubenzuron, chlorfluazuron, cyronmzine, and azadirachtin) and one conventional insecticide, irnidacloprid. All insecticides were evaluated against both of the B. thuringiensis susceptible and resistant strains of Colorado potato beetle larvae. The objectives of these experiments were: 1) to determine the LC50 and/or LD50 of these five insecticides to both strains, 2) to measure growth and development of surviving larvae, and 3) to explore potential cross resistance between B. thuringiensis and the five novel insecticides. B. LITERATURE REVIEW 1. Diflabenzuron. Diflubenzuroninhibitschifinsynthesiswhichisanessentialcompoundfor Weldon formation in insects (Ware 1989, Miyamoto et al. 1993). The target site of diflubenzuron is still unclear (Hajjar 1985, Miyamoto et al. 1993). Hajjar (1985) proposed seven mechanisms to explain the inhibition ofchitin synthesis: 1) interference with hormone metabolism which disrupts chitin synthesis, 2) inhibition of DNA synthesis, 3) blockingchitinsynthetasethatcatalyzesthefinal stepofpolymeriaationinchitin formation, 4) disruption of the synthesis of N-acetylglucosamine lipid-linked oligosaccharide, 5) inhibition of the activation of chitin synthesis zymogen, 6) interference with regulatory mechanism in the final step of polymerization in chitin formation, and/or 7) inhibition of UDP-N-acetylglucosamine transport. On the other hand, Miyamoto et al. (1993) hypothesized that there were only three possible target sites ofchitin synthesis inhibitors: 1) inhibition of chitin synthetase, 2) inhibition of the activation of chitin synthetase zymogen, and/or 3) inhibition of UDP-N-acetylglucosamine transport through themembrane. Inhibitionofchitin synthesisbydiflubenzuronwasstrongerintheinsect Wtbanintheperithrophicmembmnealthoughhvafiedfiomspeciesto species (Mulder and Gijswijt 1973, Clarke et al. 1977, Hajiar 1985), suggesting that the primary action involves inhibition in chitin synthesis within integument. Diflubenzuronisnotonlylarvicidal, butitisalsoasovicideinsomecases(Ware 1989). ItactseitherdirectlyontheeggorbyactionthroughthefemaleCThomson 1989). Itcanbeusedto controlmanyinsectsintheordersLepidoptera, Coleoptera, andDiptera (Miyamoto et al. 1993). Ware (1989) reported that female boll weevils exposed to diflubenzuron produce infertile eggs. Diflubenzuronalso had reduced egg hatchinDelia 67 _l— 68 radicurn(L)eggs(Youngetal.1987).Grosscrut(1978)statedthattheefl‘ectof diflubennuononeggswasdsoasmdatedwithdiuupfimofchifinformafiondruing embrional development. Diflubenmron(Dimilin®)wuthefirstcommacidformlhfionofabenmylphalyl meaaiajju1985)mdmsregistaedmtheUnhedStatesinl982fmcomroflinggypsy moth.bollweevil,andmostforestcsterpillars(Ware 1989).Atpresent,diflubenzuronis bdngusedmconnolsomeimpouammmforeuecosyuanmypsymoth),coum(bofl weevilsandarmyworms),soybeam(wybeancatapiflucomplexandMeldcanbean beetle), cabbage (cabbage caterpillars) and non-crop areas (flies and mosquitoes) (Thomson 1989). However, diflubenzuron is not efl‘ective in controlling sucking insects sinceitdoesnotworkasasystemicinsecticideflhomson1989).Thisinsecticidehasalso beentestedforotherpestsincitrus. Tamakietal. (1984)reportedthatdiflubenzuronwas efl‘ective in controlling Colorado potato beetle larvae. Survival of third instars fed with plantssprayedwithdiflubenzuronat50mg/lwasreducedby50%comparedtothe control. Increased concentration ofdiflubenzuron resulted in increased mortality. Even tbwghdiflubenmronshowedlessefl‘ecfivmessonfmnhmstusmantothirdmya application on fourth instars still suppressed their survival significantly. In most cases, application of diflubenzuron was compatible with biological control agentsCI'amakietal. 1984,Webbetal. 1989,Niemczyketal. 1990). Whenparasitized Coloradopotflobeedefouflhhflamwaefedwithdiflubenmronmeemagenceofthe pmsitoidDoophorqnlngadaWhorae (Riley), wasashighasthatof the control. However, diflubenzuronatconcentrationofabove 100mg/lreducedthesurvivalofthe parasitoid when it was applied to parasitised third instars of Colorado potato beetle (Tamakietal. 1984). Niemczyketal. (1990)foundthatcompatibility ofdiflubenzuronand naturdenemieshomhudecosyfiandepmdedonthespedesofmmrdmandthdr development stage. Application ofdiflubenzuron to control gypsy moth did not decrease the population of the parasitoid, Cotesia melanoscela (Ratzeburg) (Webb et al. 1989). 69 Aaneefl‘ectsonadrdtpuasitoidsandpredatomwaemMynmafl'eaeddhecdybrn side fiesta like decreased parasitoid emergence often resulted fi'om ingestion of residues fi'om the host (Croft 1990, Miyamoto et al. 1993). Likeotherinsecticides, contirnrousapplicatiomofdiflubenzuronselectsfor resistanceininsect. Younglarvaeof.$'padnptcmexigua(Hubner’)(1aeckeandDegheele 1991)and codlingmoth (Mofitt et al.1988)becameresistantto diflubenzuron In laboratoryselection, housefliesbecame 1 -5timesmoreresistantthananunselected colony (Keiding et al. 1991). Laecke and Degheele (1991) found that addition of profenofos and diethyl malcate increase the toxicity of diflubenzuron against diflubenzuron resistantstrainofS. exigua.'1‘hisindicatedthathydrolysiswasthemajorrouteof detoxificationaaeckeandDegheele 1991, LaeckeandDegheele1993). Several reports have indicated cross-resistance between diflubenzuron with other chitin synthesis inhibitors or conventional insecticides. Cross-resistance in house fly between diflubenzuron and organophosphates has been reported (Primprikar and Georghiou 1979). Shen and Plapp (1990) found cross-resistance between diflubenarron and cyromazine in house fly. 2. Chlorlluazuron Chlorfluazuron is another chitin synthesis inhibitor fiom the benzoylphenyl urea group (Hajiar 1985, Thomson 1989, Miyamoto et al. 1993). This insecticide resulted fi'om diflubmzuronmodification Themodificationwasexpectedto produceamoreactive compound than diflubenzuron (Miyamoto et al. 1993). The mode ofaction is similarto that of diflubenzuron with both contact and stomach-poison activity, and ovicidal efl‘ects. Like diflubenzorun, chlorfluazuron also has no systemic activity (Thomson 1989). Chlorfluazuron is efl‘ective against Lepidoptera, and it is considerably more toxic than diflubenzuron in various insects (Ishaaya 1992). Higher toxicity of chlorfluamron was due to higher retention in the insect (Miyamoto et al. 1993). Against Spodoptera 70 linnu, chlorfluazuron was 12 times more toxic than diflubenzuron, but less toxic than another benzoylphenyl urea, teflubenzuron (Miyamoto et al. 1993). In addition, chlorfluazuronwasalsoefi‘ectiveonColorado potatobeetlelarvaeMalinowski and Pawinska 1992). Toxicity of chlorfluazuron was as high as teflubenzuron and hexaflumuron, andtheacfivityofthoseinsecticidesincreasedasthefimeofobservafion increased. Resistancetochlorfluazuron, diflubenzuron, andteflubenzuronhasoccurredin diamomck moth in Taiwan and Malaysia (Perng et al.1988, Ismail and Wright 1991). After 29 generations of selection with teflubenmron in the Taiwan strain, the resistance levels increased 12 times (Perng et al. 1988). On the other hand, with 16 generations of selection in the Malaysian strain over 106 fold resistance to teflubenzuron was observed (Ismail and Wright 1991). The rate ofresistance development was influenced by the presenceofredumceafldesmddwrdafivefimessoftheredswugenotypesmoush and McKenzie 1937). In fact, teflubenzuron has not been used to control diamondback mothinthefield inTaiwan Therefore, initial selection forresistancetothat insecticidehas not occurred (Perng et al. 1988) except, perhaps, through cross-resistance. This might be thereasonwhyresistancedevelopmentintheTaiwan Strainwasslowerthaninthe Malaysian strain. Cross-resistance betweal chlorfluazuron and teflubenzuron was not consistent. InTaiwan, thediamondbackmothwasselectedwithteflubenzuron,butdid not show cross-resistance to chlorfluazuron (Perng et al. 1988). However, cross resistance between teflubenzuron and chlorfluazuron was apparent in the Malaysia strain (Ismail and Wright 1991). Studies with synergists, piperonyl butoxide and DEF, indicated that microsomal oxidases and esterases were involved in teflubenzuron detoxification (Perng et al. 1988, Ishaaya and Klein 1990, Isrmil and Wright 1991). 71 3. Cyromadne Cyromazineisminseagrowthregulatorfiomthefiiazinechamcdfamflyalajiu 1985, Thomson 1989). Likethebenzoylphwylmuiazinealsoinhibitschitinsynthesis with an undetermined mode of action (Hajjar 1985). However, several researchers reportedthatthetriazinefamilyhadlittleorhadnoefl‘ectonchitinformation(Cohenand Casida 1980, Hughesetal. 1989). Atriazinechitinsynthedsinhrbitor CGA 19255 wasa weakinhibitor ofchitinsynthetascin Ilibolirancasrareran(CohenandCasida1980). CyromazinefedmrdwassxtaflMidmtmdwechifinproducfionaIughesetd. 1989, Kotze and Reynolds 1991). Symptoms which resulted fi'om exposure to cyromazine includedbodyelongation, swellinginintersegmentalareas, ruptureofthebodywall, and death (Awad and Mulls 1984, Hughes et al. 1989, Sirota and Grafius 1994). Cyromazine is currently registered for controlling dipterous leafininers, celery, lettuce and manure fly pests (Thomson 1989, Anon. 1992). In addition, research showed that cyromazine was efl'ective in controlling Colorado potato beetle (Linduska et al. 1994, SirotaandGrafius1994, StoltzandMatteson1994).Cyromazinehadnoactivityasa feeding deterrent (Martinez and Moreno 1991, Kotze 1992), and its ovicidal activity dependedontheinsectspecies. AdultfemalesoftheMexicanfi'uitfly,Anasu-eplraludens (Loew),fedwithadiet containingcyromazineproducedsignificantlyfewereggsthan controlfernales,butthisinsecficidedidnotinterferewitheggferfilityoraduhmalessexual development(MartinezandMoreno 1991). Cyromazinelessenedthefecundityandfertility of the fruit fly Bactrocera cucurbirae (Coquillett) females (Stark et al. 1992). In contrast, Kotze (1992) stated tint cyromazine did not reduce the fecundity and fertility ofLucilia cuprina (Wiedemann), however, it sigrificantly inhibited subsequent larval development. InColorado potatobeetle, cyromazinewasveryefl‘ectiveonsecondinstars, anditreduced thesurvivalfiomfourthinstartopupation(SirotaandGrafius l994).1nveterinary applications, higheflicacycorrelatedwithcyromazineretentioninthebodyoffieated 72 animals. BrakeandAxtell(l990) reportedthatcyromazinewasstoredinthetissuesand organs of birds, and was released gradually. Sevadreponsindicatedefi‘ecfiveneuofcyromazineagainninseasredstmcew other Meeticides (Iseki and Georghiou 1986, Sirota and Grafius 1994). Some strains of houseflyresistmcemorganophosphneswaeuwscepnbleuthemscepnblesuainm cyromazine (Iseki and Georghioul986).1nsecticide-resistant and susceptible Long Island strains ofColorado potato beetle responded similarly to cyromazine even though the resistantstraincould survivelongerafierexposurethanthesusceptiblestrain. Allthe larvae were dead at 10 d (Sirota and Grafius 1994). Researchdataontheefl‘ect ofcyromazineonnaturalenerniesisstilllimited. Pupation of endoparasitoids, Psyrmlia incisi (Silvestri), P. fletcheri (Silvestri), Diaclrasrninrorpha longicaudaia (Ashmead), and D. ryroni (Cameron), was not afi'ected by exposure of their hosts (fruit flies) to cyromazine (Stark et al. 1992). However, data on the efl‘ects of direct exposure was not available. With limited data, this research demonstrated the compatrbility of cyromazine with biological control agents. Resistanceto cyromazinehasbcenreportedinhouseflyasekiandGeorghiou 1986, Bloomcamp et al. 1987). House flies collected fiom the field where cyromazine had beenused showedafivefoldincreaseinresistanceto cyromazinecomparedtothe laboratory susceptible strain (Iseki and Georghiou 1986). Wild and laboratory conventional insecticide-resistant strains were moderately resistant (2.9 - 4.5 fold) to cyromazine (Bloomcamp et al. 1987). They also reported that 15 generations of selection withcyromazineresultedinanincreasedresistancelevelof69.8. Incontrast, Keidinget al. (1991)reportedthatselectionwithcyromazineonhouseflyinthelaboratorydidnot increasetheresistancelevel. Theyalsofoundthattwoyearsoflarvicidecyromazine applications in the field had no effect on the development of resistance. However, Keiding hadimplementedaresistancemanagement programonhouseflybeforecyromaa'newas used. Thesedifl‘erenceswerepresumably duetodifl‘erencesincyroman'neapplication 73 meafiequencyandthepresenceofredstamflldes.Appficafimofcyromzimthmgha feed-thwghhsecfiddefomdafimmixedflopoulfiyfeedasddandGeorghhmlm) gaveasfiongasdecfionpresmtohouseflythandirectappflcafimwmamrmmdding ud.1991).Labomorysdecfionmpponedthepresenceofresistamdldesinthe populafionRapiddwdopMofredstmceundahbomorysdecfimmloommpetd. l987)n'mplyindicatedanincreaseinthefi’equencyofresistantalleles. Studywith synergists, piperonyl butoxide (PB)anddiethylmaleate(DEM), suggestedthatresistance medufiunstocyromazimwaenmduemmetabohcdegradafionasekiandGeorghiou 1986). 4. Imidacloprid Imidacloprid is a neuroinsecticide acting as an inhibitor of the acethylcholine receptor in the post synaptic binding process (Liu and Casida 1993). Recently, very intensive research has been carried out to determine the efi‘ectiveness of irnidacloprid to difl'erent target insects; thirty-two experiments have been conducted and reported in 'Arthropod Management Tests: 1994, Vohrme 19". Imidacloprid has a broad spectrum of targets inchlding insects fi'om the orders Homoptera, Hemiptera, Lepidoptera, and Coleoptera It exhibits contact and systemic activity that allows control of a range of feedingstrategiesinpestsincludingsuckinginsectlees, Technicalandsafety information sheets, 1992). Imidacloprid is being developed to control various pests on vegetables, turf, field crops, ornamentalsandfiuit. Itcanbeappliedthroughsoil application, seedtreatrnent, chernigation, or foliar application (Miles 1992). However, registrations have mostly pursued soil and foliar treatments. No cross-resistance between inridacloprid and other conventional insecticides on vafimuinsedshubemrepofledfldflfin1993),yathewopeofmsimsuaintesfing has been fairly limited. Some insects resistant to organophosphates or carbamates were 74 macepfibkmhmmclopddmbatad.1990).Thismaybeduetodifl'aemmrgetdtes between those insecticides, acethylcholincesterase is the target site of organophosphate andcubmnuemsecfiddesahtwmun1985),whaushmmdopddactsbybbcldngthe acethylcholinereceptor(Mullin 1993).Laboratoryselectionwithdifl‘erentconcurtrations ofinfidaclopfidonMyarspersicaefordmoumnetygenaafionsdidnotsdectfor resistance(Mullin1993).However,thediversityofclonallinesatthestartofselection wuundear.T1nlghdidnotindicatethatmsectswouldnotdevdopredstancew imidadopridbecausethaemayhavebeenmabsenceofredstammdesintheimw population. Since the mode of action of irnidacloprid is similar to that of nicotine (Mullin 1993), itisplausiblethatcross-resistancebetweenthosetwo insecticides could occur. Thus application of imidacloprid to insects that have exhibited resistance to nicotine may lead to cross-resistance to imidacloprid. 5. Azadirachtin Azadirachtin was originally found fiom the extracts ofneern, Azadirachta indica A Juss, andthistreeiscommonlyfoundinAsiaandAfiicaaiepburn 1989, National ResearchCouncil1992).Eventhoughallpartsoftheneemtreehaveazadirachtin, the seed kernel has the most concentrated azadirachtin (National Research Council 1992). Azadhachfinwuthemostacfivepesfiddecomparedtootherwmpomdsfoundinnean (Hepburn 1989, National Research Council 1992, BIRC 1993). A study comparing the activity of neem seed kernel extracts and synthetic azadirachtin on Plutella xylosrella L. showed similar toxicity. This indicated that azadirachtin was the major insect toxic component in neem (Verkerk and Wright 1992). Seven other limonoids (salanin, 3- deacetylsalannin, salannol, meliantriol, nimbin, nirnbidin, and deacetylazadirachtinol) had been isolated. Salannin, 3-deacetylsalannin, salannol, and meliantriol inhibited feeding on locusts, b11ttheydonotinfluenceinsectmolts.lntobarx:obudworm, descetylaasdirachtinol demonstrated similar activity to azadirachtin. Nimbin and nimbidin 75 hadantiviralactivity. Theyafi‘ectedpotatovirusx, miniavirus, andfowl poxvirus (National Research Council 1992). Severalresearchershavereportedthatneemextractscontrolledatleast 125 difl‘erentspeciesfi'omseveralinsectordersincludingColeoptera, Diptera, Orthoptera, Hemiptera, Homoptera, Isoptera, and Lepidoptera (Hepburn 1989, National Research Council 1992, BIRC 1993). Four formulated neem products (Margosan®, Antin®, BioNeern®, and Align®) have been marketed in the United States for controlling many difi‘a'artinsectsmosenbaum, personalconnmrnication). Thefirstthreeproductsarebeing used for non-food crops, but Align has just received an exemption from residue tolerances on food crops. Align® was reported to be efl‘ective for controlling citrus leafrniner (Stansly and Knapp 1994). The mode of action of azadirachtin has not been clearly described, but it has been reported as a growth regulator, a feeding deterrent, a repellent, a sterilant, and as a mating and communication disruptant (Rembold et al. 1984, Hepburn 1989, National Research Council 1992, BIRC 1993). Azadirachtin inhibited larval development of six noctuids (Actebiafennica [Tausch], Manrestra copyigurata Walker, Perish-om saucia Hubner, Melanchra picta [Hart], Spodoptera litura [Fab], and T richoplusia ni Hubner), and it had antifeedant efi‘ects on these species as well (Isman 1993). Only P. saucia and S. litura were able to distinguish between a diet containing azadirachtin and a control diet. Against mole cricket, Scapreriscus vicinus Scudder, azadirachtin slowed the nymphs] growth in a dose-dependent fashion (Braman 1993). Azadirachtin also affected the growth and feeding rate of Pieris brassicae L. First instars fed with leaves that had been dipped in 10 ppm of azadirachtin solutioncould notreachsecondinstarswithin72h(ArpaiaandvanLoon 1992). Azadirachtin was reported safe to several non target species (National Research Council 1992, BIRC 1993). Three endoparasitoids of fiuit flies were not afl‘ected by exposure of fruit flies to azadirachtin. Psytallia incisi (Silvestri) and Diaclrasmirnorpha 76 longicaudata (Aslunead) could develop and eclose fi'om Dacus dorsalis Hendel larvae thathadbeenerposedtosandneatedwithaudimchfin.DiacluwnimorpluW (Cameron) also successfirlly eclosed from Ceraritis capitara (Wiedemann) treated with azadirachtin (Stark et al. 1992). Application or azadirachtin (Margosan-O®) slightly reducedthetotal populationofnon-targetinvertebratesinaturfgrassecosystem, but it was much less detrimental compared to the conventional insecticide, clorpylifos (Stark 1992). Difl‘erent taxons showed difl‘erent susceptibility to azadirachtin, and oribatid mites were more susceptible to azadirachtin than clorpyrifos. C. MATERIALS AND METHODS 1. Materials a. Insecticides Five formulated insecticides were tested against the B. thuringiensis susceptible and resistant strains of Colorado potato beetle in morbidity assays. Four of the tested insecticides were insect growth regulators including; diflubenzuron (Dimilin® 25 Wetable Powder [WP]; Uniroyal, Naugatuck, Connecticut), chlorfluazuron (CGA 112913 WP; Ciba-Geigy, Greensboro, NC), cyromazine (Trigard® 75 wp, Ciba-Geigy, Greensboro, NC), and azadirachtin (Margossn-o®; w. R Grace, Columbia, MD). Imidacloprid (BAY NTN 33893; Miles, Kansas City, MO), a nerve poison, was the fifth insecticide tested. All tested insecticides have oral LD50 in rat > 4,500 mg (AD/kg; therefore, they fall into category 111 (slightly toxic). b. The Colorado potato beetle B. thuringiensis susceptible and resistant strains of Colorado potato beetle were obtained fi'om the laboratory stocks at Pesticide Research Center, Michigan State University. Adults were originally collected during the Summer of 1987 and 1988 from potato fields located in Bary, Allegan, Clinton, Ingharn, Macomb, Montcalm, and Wastenaw counties in Michigan (Whalon et al. 1993). They had been reared in the laboratory for 30 generations at 25 :l: 2°C and a photoperiod of 16 : 8 (L : D). The resistant strain was subsequently selected every generation with B. thufingiensis solution at concentration of 60 ml of formulation product per liter (SPUDCAP, Mycogen Corporation, 5451 Oberlin Drive San Diego, California 92121). 77 78 "Superior'potatoseedswereplarnedin16-cmclaypots,andpotatoplants,6to8- weeksoldwueusedforrearingandbioassays. Adultswereplacedinacage(0.4by0.4 by0.45 m)withsixpots.Eggmasseswerecollecteddailythenputina lS-cmplasticpetri dishunfiledosionThemwepfibkluvuwaefedwithunneuedpmatofofiageunfilthe thrrdrnstar Potatofoliagewassubstitutedeverydaytomaintainthequalityoffood. Late pMswueanandcoveredwithapenidishAdlfltsthatemagedfiomthesoflwae placedinthecagesandthepopulafionwasmaimainedatZO-ZSaduhspacage. Approximately 1,000resistantlarvaeineach generationwae selected forthenext _generation,andtherestswereusedforthisstudy.Earlysecondinstarswereselectedwith B. rlna-ingicnsis solution at the concentration of 60 ml of formulated product (741 mg [AU/l). The potato leaf-dipping was employed for selection (Whalon et al. 1993). Potato foliagewastrimmedtotheterminalfiveleaflets.Thestemwasinsertedintoa2-mlvial containingdistilledwatcr.Eaehleatletwasdippedinthesolutionavetimesrorsoseconds andallowedairdry. Twentytotwentyfivelarvaewereexposedintothetreatedfoliage for96h1hewrvivinghrvaewaefimsfmedhounfiefledfofiagemthepenidish.The latethirdinstarswerereleasedontopotatoplantsinacageuntilalllarvaeenteredthesoil. Potatoplantswerecutandcoveredwithapetridish.Adultsthatemergedfi'omthesoil werereleasedinacageandthepopulationwasmaintainedatZO-ZS adultspercage. ThisstudywasconductedusingF31 andF32 generations. Secondinstarof Colorado potato beetle (4 d old) were chosen through a two steps process. First, larvae of similar size were collected from the B. tlnrringiensis resistant strain and susceptible populations. Second, larvaewererandomly allocated to bioassaysandtrcatrnentswithin assays. Conwnfiafiomofinsecficidewaeetpnssedinmgorugofacfiveingredientper liter. 79 LMethoda a.Leal'-dipassay Thepotatoleaf-dipmethodwasemployedfordeterminingtheLCsoof diflubenzuron, chlorfluazuron, cyromazine, andirnidaclopridagainstsusceptibleand resistantsfiainsofColoradopotatobeetlthalonetal. 1993).Potatofoliagewas uhnmedmthetanunalfiveleaflets.Thenanwuwnppedndthaspongetoavoidwata spiflageandinsatedhuan-mlvidcomdmngdisfifledmfiachleafluwasdipped huoaueafinausohlfion5xf0r30secondsandaflowedmairdrybefmephcinghhnoa lS-cmpetridish.Alltreatmentswereat25:l:2°Candaphotoperiodof16:8(L:D). Larvaewereexposedfor96hours(h)onthetreatedleavesandthesurvivinglarvae transferredontountreated potato leaves. Themortalitywasassessedat96, 120, 144, and 168hposttreatment.Waterwasaddedtovialsasnecessary. Onthebasisofapreflnfinarybioassay,fivetonineserialconcentrafionswere selected. Each concentrationwasreplicatedthreeto fourtimeswith tenlarvaeperdosage perreplicate.Ifthemonalityinthecontrolexceeded20%,theexperimentwasredone. Initially, all ten larvae were collectively weighed in each replication. Only live larvae fi'om eschtreatrnent/replicate were weighedat96and 168 hafter exposure. Larval developmentwasrecordedbyinstarfi'om48to 168h. b. Droplet assay A droplet assay method was used to determine the LD50 ofirnidacloprid and azadirachtin. Allassayswerecaniedoutonthesecondinstar(4dold). Initially, allten larvaepertreatment/replicatewerecollectivelyweighed, andattheendoftheexperiment (168hafiertreatrnent), thesurvivinglarvaewereweighed. Mortalitywasobservedat48h after application and continued every 24 h until 168 h afier treatment. Plastic microtitre plates with 24 wells held individual larvae. Whatrnan filter papers (1.5 cm diameter) excised with scissors were placed in each well to provide a moisture 80 source.EachfilterpapewasmoisturedwichOulofdisfifledwatetopreveuwiltingof thepotatoleafdisks.Apotatoleafdisk(diameter3mm)wasexcisedusingasmallcork boredandputinto eachwell. Irnidaclopridandazadirachtinsohrtionsweremadein 10% sucrose which acted as a phagostimulant and ensured complete consumption of the leaf diskLarvaewerestarvedforzhpriortotreatrnenttoensuretheconsumptionofthetotal potato leafdisks. Using a micro applicator (Hamilton), 0.2 ul of treatment formulation wuspphedMeachbsfdiskandtennndomlysdectedhrvaweerdeasedMoflnwefls. Afieafloftheleafdisksweeconamed(abmn3h),thehrvaefiomeachconcenuafion treatment/replicate were put into a 15 cm petri dish and ofl‘ered untreated potato leaves. 3. Data analysis LC50 s and LD50 s were determined by regressing log dose on probit mortality (Ming and Finney 1984). Data was corrected for control mortality using Abbott's formula (Abbott 1925) before they were submitted to probit analysis. LCso s (leafdip assay) were assessed at 96, 120, 144, and 168 h, whereas LDso a (droplet assay) wee estimated at 48, 72, 96, 120, 144, and 168 h after treatment. A significant difl‘erence between two LC50 s or two LD50 s was based on non-overlapping 95 % Confidence Interval values (P S 0.05). Homogeneity of slopes between two regression lines were tested with SYSTAT (Wilkonson 1992). Iarvalwdghtwasregreasedonlogcomenfiafionofinsecfiddeonthebasesofthe datacollectedat96and 168hposttreatment. Regression, correlationanalysis, and homogeneity of slopes wee tested using SYSTAT (Wilkonson 1992). D. RESULTS AND DISCUSSION 1. Results a. Leaf-dip assay Difluheazaron. TheLCso ofdiflubenzuronat96hfortheresistantstrainwas 15.4mg/l anditdcclinedt02.6mg/lat168 haltertreatmthl‘able 3.1). TheLCso sat 144 and 168 h were significantly different from LC50 at 96 h, but they were not different at 12011 Thisindicatedthattheinifialefl‘eeofdiflubenmmnwassfiflpresentalthough the surviving larvae were ofi‘ered untreated leaflets. This observation supported previous research (Malinowski and Pawinska 1992) in which the mortality of diflubenzuron on Colorado potato beetle was dependert on time. For the susceptible strain, the LC 50 of diflubenzuronat96hwas32.4mylanditdecreasedto18.2mg/lat120handremained constantthrough168 hafiertreatmert. TheLCsovaluesacrossthefourdifl‘erent obsevation times were not significantly difl‘erent in the susceptible strain At the earlier observations (96 and 120 h), the LC50 s ofdiflubenzuron against the resistant and susceptible strains were not significantly different, but they diverged at 144 and 168 h alter treatment (Table 3.1). Compared to the susceptible strain, the LC 50 in the resistant strainwastwotimeslowerat96handprogressedto seventimeslowerat 168 h. This result suggests a negatively cross-resistance correlation between B. tlnrringiensis and diflubenzuron. In other words, the B. tlnrringiensis resistant strain was more susceptible to diflubenzuron than the B. thuringiensis susceptible strain. Negatively correlated cross- resistance between insect growth regulators and organophosphate insecticides has been reported previously. Carte' (1975) also found that diflubennrron was more efi‘ective in controlling resistant stored grain beetles. Auda and Degheele (1985) reported that Spodoprcra littoralis (Boisduval) resistant to monochrotophos were 0.6 fold more 81 82 Table 3.1. Toxicity of diflubenzuron to second instar of Bacillus thuringiensis resistant and susceptible strains of Colorado potato beetle which were exposed for 96 h on treated leaves Strain it Time LCso“ 95 % CI Slopcb 12 c RR" 01) ms (AD/l m3 (AD/l Resistant 210 96 15.4aA 9.3-22.o 1.4aA 4.4 0.5 120 6.0abA 1.3-10.7 1.1aA 3.7 0.3 144 3.3bB 0.5- 6.9 1.1m 2.3 0.2 168 2.6bB 0.3- 5.7 1.2aA 1.1 0.1 Susceptible 150 96 32.4aA 13.3-37.4 0.9aA 4.1 120 18.2aA 7.9 - 41.9 0.9aA 9.1 144 17.6aA 7.2 - 37.6 1.1aA 9.1 163 18.5aA 6.3 - 40.4 1.2aA 13.3 “Signifimmeofdifiembemnwsosmsbaaedmmnmeflapping95%Cmfidenmmvfl values. LCsos foflowedbyfliesameloweeaseletewerenmdifl’eremfithinamainwhflewsos foflowedbymesameuppereaselenewemnmdifl’eentbeweensuaimatflnsamefime. bsmafoflmedbymemlowemmleneweenmdifieemwiminaanhfledopmfoflomdby flesannuppecaselenewemnmdifiemntbeweensuaimmthesamefinnahmogenetyof regression coefiicientstest; Wilkonson1992). ‘Theresistantstrain:thetabularxzvaluewithtldf.andat5%levelofsignificanceis9.49.The Sinceptiblcstrain:thetabularxzvaluewichdfandat5%leveldsignificanceiSS.99. dRR(mdeamemfio)msmledmdbydifidingtheLC50dflwmdmmMnbytheLCwofme susceptiilestrain 83 wscwtible to chlorfluazuron than the susceptible strain. Because of this negative correlation, diflubenzuron could be used as an alternative insecticide where B. WW3 resistance is suspected in Colorado potato beetle. Homogeneity analysis of slopes (Wilkonson 1992) indicated that probit regression lines within a strain were not significantly difl‘erent (P > 0.05). In the earlier observation (96 and 120 h), slopes ofthe resistant strain were steeperthan those ofthe susceptible strain; however, they were not significantly difi‘erent (P > 0.05) based on homogeneity analysis of slopes (Table 3.1). These may indicate that there was considerable genetic variability in both strains. Increased time of observation did not increase the slope of probit line for the resistant strain, but it did slightly effect the susceptible line. Besides killing the larvae, ingestion of diflubenzuron reduced the weight of surviving larvae (Figure 3.1) and weight reduction was negatively correlated with concentration. The regression of larval weight on treatment concentration resulted in lines for the susceptible strain at 96 h (S96) and 168 h (8168) ofy = 39.0 - 6.3 x with an =-0.86andy= l32.2-35.7xwith anr=-O.78 respectively, wherey=weightgain, x = log concentration, and r = correlation coeficient. The regressions of the resistant larval weightgainwerey= 57.3 -19.9xwithanr=-O.68 at96h(R96)andy=128.2- 50.8x with an r = - 0.95 at 168 h (Rl68). The r values were relatively high (> 0.65); however, they were not significant (P > 0.05) except for R168 based on simple linear correlation analysis (Gomez and Gomez 1984, Wilkonson 1992). In biological research, a r greater than 0.6 is ofien considered representing a linear relationship (Gill 1987). Homogeneity analysis of slopes (Wilkonson 1992) showed that regression lines for 96 and 168 h observation periods were difl‘erent in the resistant strain (P < 0.05) yet were not difl‘erent for any observation periods in the susceptible strain (P > 0.05) . The regression lines for R96 and S96 were not significantly difi‘erent (P > 0.05). A similar result occurred when the regression coeficients of R168 and 8168 were compared (P > 0.05), Differences in the final weight of resistant and susceptible strains in any concentration were originally Log concentration (mg [Aim Figure 3.1. The relationship between concentration of diflubenzuron and larval weight gain of Colorado potato beetle 896 and $168: the susceptible strain at 96 and 168 h after treatment respectively. R96 and R168: the resistant strain at 96 and 168 h after treatment respectively. 85 duetodifi‘erenceingmwthbetweenresistantand susceptiblestrains(seeFigure2.4 CW2). Diflubenzuron delayed the larval development of the resistant and susceptible straim(lt“1gure3.2and3.3). Intheeontrol, allresistantlarvaemoltedintofourthinstarsby 168 h after application (Figure 3.2). At concentrations of6.3 and 12.5 mg (AD/L approximately5% ofthelarvaewerestillthirdinstars. Incontrast,inmuchhigher concentratiortsof25.0t0200.0mg(Al)/l, noresistantlarvaewereabletodeveloptothe fourthinstar. Aslighflydifi‘erentremhocwnedmthemscepfiblestrainliketheresistam strain, aflsuscepfiblelarvaeinthecontroltreatmentmoltedmfomthinstarby 168 h after application (Figure 3.3). In addition, all surviving larvae from the concentrations of 0.2 and 0.8 mg (AD/l also molted into fourth instar by 168 h. At a concentration of 12.5 mg(AI)/l, 55% ofthelarvaesmviveduntil 168 hobservationwhereS‘Ve ofthemwere third instars. Unlike the resistant strain, 3% ofthe susceptible larvae were to become fourthinstarsatconcentrationsofSOandZOOmg(AI)/l. Theseresultsindicatethatthere was difi‘erent susceptibility to diflubenzuron among individuals in the susceptible strain. BJJMMM AW m x/ z/ , , M/ , /// . x // m; , 120 144 188 120 144 188 n wwwwmo 11 GS €0.58; .335 D. 50.0w (AM c. 12.5 mg (AM 120 144 168 Hours attartreatment m m m a m 6 3535352. F. zoonmuAM E. 100.0 mg (AM 120 144 188 7296 Hours after treatment mmwwmo Al as c8353»... 120 144 16. / //-: '"J’, ',:’/,.x 72 N Figure 3 .2. Larval development of the Bacillus theingiensis resistant strain of Colorado potato beetle treated with difi‘erent concentrations of diflubenzuron. Total height of bars indicates percent survival including all instars. 87 m mat M. m ///////fm m m M J m s. , 9am mwwwwmo mmwwwmo 1 1 1 al as 3533.392. as 5.35.. .32. mm % / rn/h 144 168 120 Hours after treatment E 50 0 mg (AM 120 144 168 w /////////,/// u a //////////////////// n m c.0.8mg(Al)ll Hours after treatment Hours after treatment Colorado potato beetle treated with different concentrations ofdiflubenzuron. Total Figure 3.3. Larval development of the Bacillus rhufingiensis susceptible strain of height of bars indicates percent survival including all instars. Chlerllnaauren. The LC50 ofclorfluazuron at 96 h for was 101.8 mg/l and it declined significantlyto 32.2mg/lat 120 h. TheLCsoat 144hwas 16.6mylandit remained constant until 168 h (Table 3.2). [£50 3 at 120, 144, and 168 h were significantlylowerthanthe96hobservation. Eventhoughtherewasatrendofdecreasing LCsofi’om96to l68hinthemsceptiblestmimLCsoswerenot significantlydifl‘erent. CompuedtothemwepnblesnainLCsosoftheresistamurainwerehigher: however, Lcsoswerenotsignificantlydifi’erentexceptat96h. At96h,theresistanceratiowas 3.7, whereas at 120, 144, and 168 h resistance ratios were 1.7, 1.1, and 1.2 respectively. Bventhoughtheremsmdifi‘ermcebaweenmeredstmmmscepfiblemainumehst observation period, the resistant strain showed some degree of resistance to chlorfluazuron as indicated by a prolonged time for the insecticide to kill the resistant larvae. This difi‘erences may involve a cross-resistance mechanism to B. tlnm’ngiensis or vigor tolerance. ShaflowslopesintheresisMandsuscepfiblesfiainsdemonstratethatthe populations were fairly heterogeneous in the response to chlorfluazuron (Seawright 1972). Inboth strains, thevalues ofslopesincreasedlwith increasingtimes ofobservation However, homogeneity analysis of probit regression coefficients (Wilkonson 1992) indicatednosignificamdifi‘erences(P>0.05)amongslopesintheresistamstrain In contrast, significant differences were present in the susceptible strain (P S 0.05). At 168 h, theslopewassignificantlysteeper(P $0.05)thanthatof96or120h,butitwasnot difi‘erent(P>0.05)fi'omthatof144 h. Comparedtotheresistant strain,theslopesofthe susceptible strain were significantly steeper (P S 0.05) except at 120 h after treatment. These results indicate that the susceptible strain was more sensitive to chlorfluazuron than Table 3.2. Toxicity of chlorfiuazuron to second instar of Bacillus thin-inglensis resistant and susceptible strains of Colorado potato beetle which were exposed for 96 h on treatedleaves Strain 11 Time LCso" 95 % c1 Slope” ch and (h) ms(AI)/| ms(AI)/| Resistant 270 96 101.8aA 63.3-1771 0.91041 6.0 3.7 120 33.23.41 21.3- 49.9 1.1m 7.3 1.7 144 16.6bA 10.3- 24.3 1.3aA 6.9 1.1 168 16.5bA 10.4- 24.6 1.4m 4.7 1.2 Susceptible 130 96 27.31113 13.5- 41.3 1.4a 1.1 120 19.4aA 13.0- 23.2 1.6aA 2.0 144 15.2aA 10.6- 21.3 1.9an 0.6 168 13.8aA 8.6- 20.3 2.5bB 0.9 “SignifimnuofdiflemcubamLCsosmhmdmmnmmpping95%ConfidemeInmrvfl vahres.LC503 fouowedbymesamlmeaselenermenmdifiaemfitMnasnmmwsos foflowedbymesameuppereaselenerwemnmdiflaentbaweenaraimatmesamefime. bsmrwmmmmrmmmmmMMMamwmmmmw thesameuppercaselenawemnmdiflenntbaweenmaimumeamefimeabmogunityof regress’on coeficients test; Wilkonson 1992). c‘l‘heresistantstrainzthetabular 2valuewith6df.andat5%levelofsignificanceislz.59.11re susceptrblestrainzthetabularx valuewith3d.f.andat5%levelot‘signifieanceis7.81. dRR(mdmmemfio)wumlqflamdbydividingtheLC5odtMredmmmflnbytheLC500fthe arscepfiblestrain. Likediflubenzuron, chlorfiuamronalsoreducedtheweightofsurvivinglarvaeof both strains(Figure 3.4). Theregression oflarval weightontrestment concentration resultedinlinesforthe896and S168 ofy=88.9-34.7xwithanr=-0.96and y- 176.3 -69.5 xwithanr=-0.88 respectively. Theregressions oftheresistantlarval weightgainwerey=38.0-9.0xwithanr=-O.88fortheR96andy= 131.1 -4l.2x withanr=-0.92fortheR168. Simplelineareorrelationanalyais(GomezandGomes 1984, Wilkonsosn 1992) indicated that the r values for the three regression lines (S96, R96, and R168) were highly significant (P < 0.01). No significance at $168 was due to smallnumberofsamples(n=3)sincenolarvaewerealiveatthetwohighest concentrations. Homogeneity analysis of slopes (Wilkonson 1992) indicated a difi‘erence (P S 0.05) between S96 and S168, R96 and R168, S96 and R96, and 8168 with R168. Even though aflsurvivinglarvaewerefedvfithuntreatedleavesafier96hofexposure,theefl‘ectsof thedifl‘erentconcentrationsadministered resultedindifi‘erentweightgainuntilthelast observation period. Higher concentrations yielded reduced larval weight gain. As a result, the slope ofthe168 h observation periodwas steeperthanthat of96 hfor each strain. Shallow slopes in the resistant strain indicated that they were less sensitive than the susceptible strain and perhaps more geneticaly variable for chlorfiuazuron selection. In other words, the same concentration resulted in smaller weight reduction in the resistant strainthanthatinthesusceptible strain. Atthehighest concentrations, nolarvawasalive at 168 h in either strain. Chlorfiuazuron at sub-lethal concentrations delayed larval development in both strains, and at high concentrations treated larvae died before reaching the fourth instar. In bothstrains, survivalinthecontrolwasgreaterthan85%andall survivinglarvaewere fourth instars at 168 h (Figure 3.5 and 3.6). The proportion ofthird instars to total surviving larvae increased with increasing concentration of chlorfluazuron, thus chlorfluazuron consistently delayed development (Figure 3.5). At the higher 91 concentrations, 100.0and 250.0 mg(AI)/l, onelarvasuccessfirllymolted into afourth instar, however, itdied 24hlater. Atthehighest concentration (625 mg(AI)/I)alllarvae died at 120 hafierapplication Susceptible larvaetreated withchlorfluazuron concentration of 2.6 and 6.4 mg(AI)/l developed similarlywiththecontrol (Figure 3.6). Atconcentrationof40.0mg(AI)/l, oneoutoffoursurvivinglarvaewasathirdinstaruntil the 168hobservationtime. AlllarvaetreatedwithconcentrationoflOOandZSOmg(AI)/l diedat 144and168hafiertreatment. 92 2m 180' ISOGS‘IGBROGFH” 150* A140" 0 0 gm, )- -100’ E 30- 3 W ”C 20_ '0 Log concentration (mg [Allin Figure 3.4. The relationship between concentration of chlorfluazuron and larval weight gain of Colorado potato beetle. S96 and $168: the susceptible strain at 96 and 168 h after treatment respectiver R96 and R168: the resistant strain at 96 and 168 h after treatment respectively. 93 e.1.omg(A1yI D. 40.0” (AM 39 53.3% .335 c. 6.4 mg (AM Hours after treatment ms after treatment m F. 625.0 mg (AM E. 100.0 mg (AM . . . s p m w n w w m o 1! as 39 £858.53“. .385 10. as 120 144 Hours attertreatment Figure 3.5. Larval development of the Bacillus thuringiensis resistant strain of Colorado potato beetle treated with difi‘erent concentrations of chlorfluazuron. Total height of bars indicates percent survival including all instars. B. 2.6 mg (AM o. 40.0 mg (AM o. 6.4 mg (AM 120 144 188 0487208 mwmmwmo al al as cage .92. th instar rd instar nd rnstar g 120 144 188 .96. Hours alter treatment W///////////////////////%e mmmwwmo Gav egg—u afic— Hours after treatment F. 250.0 mg (AM E. 100.0 mg (AM 4”“ “/2”; WV; 4572 0 48 72 98 120 144 188 mmlwwwmo 38 3.59.5“. .32.. Hours alter treatment Hours alter treatment Figure 3.6. Larval development of the Bacillus thufingiensis susceptible strain of Colorado potato beetle treated with different concentrations of chlorfluazuron. Total height of bars indicates percent survival including all instars. 95 Cyromazine. Theresponseoftheresistantstraintocyromazineand chlorfiuazuronwas similar. TheLC50 ofcyromazine at 96 hwas 136.5 mg]! and it decreased abruptlywith increasingtime. However, theLCso at 144 was not significantly difi'erentfromthatat 168 h. Thesusceptiblestrainshowedasimilartrendtotheresistant strainhnflredecreaseinLCsoswasnotasdramaficasintheresistamstrain. TheLCso at96hwasnot determinedsincethecomputedLCsoexceededthehigbestconcentrafion usedinthebioassay.TheLC50at120hwas3.6mg/landitdeaeased significantlyto 2.2 mylat 168 h. ComparedtotberesistantstraintheLCSOat 120hinthesusceptiblestrain was significantly lower", however, they were not difi‘erent at 144 and 168 h. This may indicate that the resistance strain has considerably greater protection fi'om cyromazine than the susceptible strain. A similar situation was reported by Sirota and Grafius (1994) with the Long Island strain that was resistant to many conventional insecticides. The long Island strain treated with low concentrations could live longer than the susceptible strain, butalllarvaeinbothstrainsdiedafier 10d. Theystatedthathighervigorinthelong Islandstraincouldbethemajorcauseofdifl‘erencesinsurvival. Incontrast,theB. tlrun'ngiena’s resistant strain was less vigorous than the susceptible strain (see Chapter 11). Therefore, tolerance to cyromazine in the B. thwingiensis resistant strain may be due to the existing resistance mechanism and not vigor. Homogeneity analysis of slopes (Wilkonson 1992) indicated no difi'erence (P > 0.05) among slopes within a strain. However, the slopes of the susceptible strain were significantly steeper (P S 0.05) than those of the resistant strain. These results may have demonstrated population heterogeneity in the resistant strain. Cyromazine reduced the weight of surviving larvae of both strains, and reduction in weight was concentration dependent (Figure 3.7). The regression lines for the S96 and the 168hwerey=48.5 -43.9xwithanr=-0.91 andy= 131.1 -66.6xwithan = - 0.90 respectively. The regressions of the resistant larval weight gain were Table 3.3. Toxicity of cyromazine to second instar of Bacillus tlnrringiensis redstantandsusceptiblestrainsofColorado potatobeetlewhichwereexposedfor96hon treated leaves Strain n Time LC50" 95 v.01 Slope” x2” and (h) ms(AI)/| ms(AI)/l Resistant 270 96 136.5a 83.3 - 290.9‘ 1.0a 5.4 120 37.6bA 21.8 - 72.3 0.7aA 5.0 10.4 144 4.20A 0.7 - 9.7 0.5aA 12.5 1.5 168 1.4cA 0.2 - 3.6 0.6aA 11.7 0.6 Susceptible 150 96 > 7.5 120 3.6aB 2.9 - 4.6 3.0aB 0.9 144 2.8abA 2.1 - 3.4 3.7aB 4.1 168 2.2bA 1.8 - .2.6 5.2aB 0.2 “SignifimddiflenncsbawwnLCsosmbasedmmnMapping95%Confidmmvd values.LC503 fouowedbythesamebwa’caselenerwaenmdifl’emmfiminamainwhilewsos fdlowedbythesannuppereaselenerwemnmdifiaembaweensuaimatflnsamefime. bprufoflowedbythemhmmleflerwuenddiflMwiMaanhfleshpafdhwedby thesannrmpermselaterwemnadifluembawemsuaimflthemmefimefihmogeneityd regression coefficients test; Wilkonson 1992). chreSistamSuainzmemhflmxzvalmwith6d£andat5%kvdofsignificanceis12.59.11): suscepnblestrainzthetallrlarxzvaluewichdf.andat5%levelofsignificancei35.99. dRR(rednamemfio)msmlwaydivfiingtheLC500fdnredaameytheLC50dthe susceptiblestrain. 97 l y=19.5-7.2xwithanr=-0.89at96handy=59.l -28.8xwithanr=—0.80at168h (R168). Analysis of linear correlation (Gomez and Gomez 1984, Wilkonson 1992) signified no difference (P > 0.05) in the susceptible regression lines (S96 and $168). No difi'erencemaybeduetothesmallnumberofsurvivors(4and3 respectively)sincether values were relatively high (0.91 and 0.90). Compared to the susceptible strain, the r values ofthe resistant strain were lower, however, they were highly significant (P S 0.01) sincethetabtdurvaluesdecreasedwithincreasingmtmbaofsamples(n=8). Rangeof theselected concentrationsintbesusceptiblestrainwasnarrow, therefore, thetested concentrations were small. Homogeneity analysis of slopes (W rlkonson 1992) indicated a difi‘erence (P S 0.05) between S96 and S168, R96 and R168, S96 and R96, and 8168 with R168. Higher concentrations resulted in more restricted larval growth. As a result, the slopeatthe 168 hobservationwas steeperthanthatatthe96hobservationperiod inboth strains. Shallow slopes in the resistant strain may have indicated that they were less sensitive than the susceptible strain. In other words, the same increasing concentration resulted in smaller weight reduction in the resistant strain than in the susceptible strain. The major final difi‘erence in weight between the susceptible and the resistant strains was probably due to different growth rates (see Figure 2. 4, Chapter II). It was clear that some concentrations of cyromazine inhibited larval development (Figure 3. 8 and 3.9). Survivors of the resistant larvae treated with concentration of 1.9 mg (AI)/l were able to molt into fourth instars by 168 h alter application. However, survivors fiom concentrations of greater than 3.8 mg (AI)/l prevented molting into fourth instars (Figure 3.8). In the susceptible strain, larvae treated with 0.9 mg (AI)/l exhibited similar development to the untreated larvae (Figure 3.9). Some survivors fi'om higher concentrations (1.9 and 3.8 mg [An/l) reached the fourth instar. At the highest concentration (7.5 mg [AI]/l), survivors (10 percent) reached the fourth instar at 144 h afier application, but all died 24 h later. These results indicate that cyromazine was slower than the other compounds (diflubenzuron and chlorfluazuron) in afi‘ecting growth 98 puanntasfl'hislutaremhmmpponedbypreviousobsavedefi‘easonthehwsefiy (Brakeetal.1991). 180 , 150 r I soasrssassnrss -I— '9‘ -o— + r40L E120 ~ 0 ‘9 100L V 80_ ‘e’. . + 3 60- 4o— 20- o 1 -1 0 1 2 3 Logconcentratlon(mg[Alyl) Figure 3.7. The relationship between concentration of cyromazine and larval weight gain of Colorado potato beetle. S96 and $168: the susceptible strain at 96 and 168 h after treatment respectiver R96 and R168: the resistant strain at 96 and 168 h after treatment respectively. e.1smg(A1v1 100 D.30.0mg(Alyl 72 06 120 144 188 Hoursaitertreatment F. 200.0mg(Al)ll mmwwwmo m , m- mm m //////////mm w a m. /////////////////ma m I. .. mmmmwmo mmmmmmo Colorado potato beetle treated with different concentrations of cyromazine. Total Figure 3.8. Larval development of the Bacillus thuringiensis resistant strain of height of bars indicates percent survival including all instars. 101 e.osmg(ary| AW ”3 Cow/fl . ,7/// .v %//%////V%///////////M/ a D. 3.6 mg(Al)l| mmwwwmo 9’3"” W m m m at M m.. W mw Mr %mm m am an //////////////ar .” um n ////////////////nm e an aa/za/é/fm on I. Colorado potato beetle heated with different concentrations of cyromazine. Total Figure 3.9. Larval development of the Bacillus thuringiensis susceptible strain of height of bars indicates percent survival including all instars. 102 ImidadopfidEventhoughtherewasadecreaseinLCsowithincreasingtime, LCso 3 within each strain were not significantly difi‘erent (Table 3.4). The LC50 of imidaclopridatthe96hobservationintheresistantstrainwas4.6mg/landdeclinedto3.5 mg/latl681LWiththesuscepfiblestrain,theLC50atthe96hobservafionwas3.1mg/l anddecreasedto2.9mgllat168h.LittlechangeinLC50withincreasingtimewas observed. Asanervepoison, imidacloprid causedmortalityinlesstimeaachell1992) than the three insect growth regulators (diflubenzuron, chlorfluamron, and cyromazine). Therefore, iftheluvaemrvivedthe96hofexpoa1repaiod,theysurvivedthroughthe complete trial. Compared to the susceptible strain, the LC50 s in the resistant strain were higher, however, the LC50 s between strains were not significantly difi‘erent except at the 96 h observation period. The resistance ratio declined from 1.5 x at the 96 h obervation to 1.2xatthe168hobservafionpedod.Thisarggestsd1attheresistaMandthesuscepfible strains have the same level ofsusceptibility and the existing resistance mechanisms do not provide protection to imidacloprid. Inbothstrains, probitregressioncoeficientswererelativelyhigh(>4.0). Homogeneity analysis of slopes (Wilkonson 1992) indicated no difi‘erence (P < 0.05) withinastrainaswellasbetweenstrains. Theseresultsindicatethattheresistant poprflafionisashomogeneousasthewscepfiblepoprflafionmthdrresponsesto imidacloprid intoxication. The efi‘ectiveness of cyromazine on both strains makes it a valuable addition in controlling Colorado potato beetle where resistant to B. thrm'ngiensis is suspected. The efi‘ect of imidacloprid on weight gain of surviving larvae demonstrated a concentration dependent (Figure 3.10) response. The regression lines for the S96 and the 168hwerey=22.2-26.8xwithanr=-0.99andy=97.6-106.2xwithanr=-0.97 respectively. The regressions ofthe resistant larval weight gain were y = 14.9 - 16.0 x with anr=-0.97atthe96hobservationandy=59.4-60.2xwithanr=-0.99atthe168h observation period. Analysis of linear correlations (Gomez and Gomez 1984, Wilkonson 103 Table 3.4. Tordcity of imidacloprid to second instar of Bacillus thrm'ngiensis resistantandsusceptiblestrainsofColorado potatobeetlewhichwereexposedfor96hon treated leaves Strain 11 Time 1.0500 95 °/. (:1 Slopeb 12 c and (11) ms (AD/I ms (AD/I Resistant 130 96 4.6aA 3.9-5.3 4.0aA 3.3 1.5 120 3.7aA 3.2-4.3 4.8aA 0.7 1.2 144 3.5aA 3.1-4.1 4.5m 2.5 1.2 168 3.5». 3.1-4.0 4.1m 3.3 1.2 Susceptible 150 96 3.1aB 2.7-3.5 5.3aA 0.4 120 3.1aA 2.7 - 3.5 5.3aA 0.4 144 2.9m 2.5 - 3.4 4.3aA 2.1 168 2.9aA 2.5 - 3.4 4.3aA 2.1 “SignifimmeofdiflembdwemLCsosmhsedmmnevuhpping95%Confidmcelm«vd values.LC503 fouowdbyflnsamelowucaselenerwerenmdiflaemwithinasuainwhihwsos foflowedbyflwsameuppercaselaterwennmdifl'emmbaweensuaimatmesamefime. bsmmumwthemrmmrenamnmmnathMaminwmmmumw mesannuppereaselaterwemnmdifienmbaweensuaimflmemmefimabmogendtyof regression coeficients test; Wilkonson1992). cSlopesfoflowedbythesamelowercaselenerwerenmdifi'aemwithinasuain,whileslopesfollowedby umwmummmamwmwmmm Wilkonson 1992). dRR(mdflanmrafio)mmhflMbyfifidingtheLC50d‘theredmmdnbytheLC5oofthe suswptiblestrain. 104 ' 1992)mdicaedmuthenmplehnearconaanoneoeaieimuwaehighryngnineamas 0.01) for the S96, R96, and R168 and significant (P S 0.05) for the 8168. This suggests that increased concentrations resulted in lower weight gain Homogeneity analysis of slopes(Wilkonson1992)indicatedadifi‘erence(PS0.05)withinastrain(betweenthe S96andtheSl68 orbetweentheR96andtheR168)andbetweenstrains(the S96andthe R960rbetweenthe Sl68andtheR168). Theslopeforthesusceptiblestrainatthe96h observationwas-26.8 anditdecreasedto-106.2atthe168hobservationperiod showing almostan4tfold change. Asunththemscepfiblestraintheslopeoftheresistantstrain decreased fourtimesfrom -16.0 atthe96hobservation to -60.2 at 168 h. This maymean that the relationship is similar, but the relative value is very difi‘erent. Survivors fi'om treated larvae developed slower than control larvae and the susceptible and resistant strains showed similar responses (Figure 3.11 and 3.12). In low concentrations(1.5 and 2.0 mg/l [AI]), most ofthe surviving larvae reached fourth instar by the 168 h observation period in both strains. However, at higher concentration (3.0 mg [AI]/l), most ofthesurvivinglarvaeremainedinthethirdinstar. Atthehighest concentration (5.0 mg [AI]/l), imidacloprid prevented larvae from reaching the fourth instar. These results suggested that application of imidacloprid at sub-lethal doses slowed larval deve10pment in both strains in a similar fashion. 105 120 100— 3 Wale!“ (Indium) 8 8 o a 1 a l 1 l s l a 0 0.2 0.4 0.8 0.8 1 Log concentration (mg [1014) Figure 3.10. The relationship between concentration of imidacloprid and larval weight gain of Colorado potato beetle S96 and $168: the susceptible strain at 96 and 168 h after treatment respectiver R96 and R168: the resistant strain at 96 and 168 h after treatment respectively 106 B. 1.5muAM y//////////%w D. 3.0 mg (AM c. 2.0 mg (AM Hours alter treatment F. 6.0 mg (AM mwmwwmo E 4.0mg(AI)l| i ’1 08 /////I n Hours after treatment Hours after treatment Figure 3.11. Larval development of the Bacillus thuringiensis resistant strain of Colorado potato beetle treated with different concentrations of imidacloprid. Total height of bars indicates percent survival including all instars. 107 B.1.5mg(Al)ll D. 3.0 mg (AM c. 2.0 mg (AM treatment Hours alter E. 5.0 mg (AM 0 4s 72 06 120144166 Hoursattertreatment Figure 3.12. Larval development of the Bacillus thuringiensis susceptible strain of Colorado potato beetle treated with different concentrations of imidacloprid. Total height of bars indicates percent survival including all instars. 108 b. Droplet assay Imidacloprid. Using the droplet assay method, the LD50 s of the resistant strain were 1.0-1.3 times higher than those ofthe susceptible strain Based on non-overlapping 95% ConfidencelntervalstheLDsoswerenot significantlydifi‘erentwithinthe susceptible strain. A significant difi‘erence (P s 0.05), however, was only present between Lbsosatthe24hand1680bservationperiodsintheresistantstrain TheLDsosofthe resistanstrainwerenotsignifantlydifl‘erentfromtboseofthe susceptiblestraina‘able 3.5). Bothstrainswereverysusceptibleto imidaclopridandtherewasasimilartrendin thattheLDsosdecreasedwithincreasingtimeofobservation. Theseresultswere consistent with the results from leaf-dip assays and suggested that B. tluningr‘ensis selection did not result in cross—resistance to imidacloprid. Homogeneity analysis of slopes (Wilkonson 1992) indicated no difi‘erence (P > 0.05) within a strain and a difference (P < 0.05) between strains. In general, the slopes of the susceptiblestrainswere steeperthanthose oftheresistantstrainand significant difl‘erences occurred at the 72, 144, and 168 h observation periods. Compared to the results fiom the leaf-dip assay, the slopes fiom the droplet assay were significantly lower (P5005). Thismaybeduetothedifi‘aencesintheamMofimidaclopridingestedby larvae. Intheleaf-dip assay,thelarvaewereexposedontreatedleafietsfor96hduring which time they could have ingested imidacloprid. On the other hand, in the droplet assays the larvae ingested imidacloprid which was deposited on the small leaf disk. In other words, repeated ingestion did not occur in the droplet assay. Thus, the larvae treated with leaf-dip assay may have consumed a higher dose than the larvae treated with droplet assay. As a result, the slopes in the leaf-dip assay were steeper than those in droplet assay. 109 Table 3.5. Toxicity of imidacloprid using droplet assay method to second instars of Bacillus tluningiensis resistant and susceptible strains of Colorado potato beetle Strain n Time 1.13509 95 v. CI Slopeb 12 c RRd (h) 113(AD/lam u3(AI)/larva Resistant 323 24 0.0026aA 00019-00041 1.5aA 2.9 1.3 43 0.0017abA 00013-00026 1.4m 3.7 1.0 72 0.0016abA 00011-00027 1.1aA 3.0 1.1 96 0.0014abA 00010-00022 1.1aA 5.5 1.0 120 0.0016abA 00011-00026 1.1». 6.2 1.1 144 0.0014abA 00009-00023 1.1aA 3.7 1.2 168 0.001le 00007-00013 1.0aA 3.6 1.0 Susceptible 230 24 0.0020aA 0.0015 -0.0029 1.7aA 4.2 43 0.0017aA 0.0013 - 0.0023 1.8aA 4.3 72 0.0015aA 0.0011 - 0.0021 1.6aB 1.5 96 0.0014aA 0.0011 - 0.0021 1.5m 2.3 120 0.0014aA 0.0011 - 0.0021 1.5aA 2.3 144 0.0012aA 0.0009 - 0.0017 1.5aB 1.6 168 0.0011aA ‘ 00003-00016 1.5aB 1.3 “Sigrifimofdiflemncesbawemwwsmsbamdmmnmhpping95%Confidenmmwfl values. 1.0503 foflowedbythesamelowercaselaterwennmdifi‘uemwithinastrainwhflewsos bfouowedbymesameuppercaselenerwemnmdiflenmbaweensnaimmflwsamfime. bSlopesfouowedbythesamelowercmelenerwerenotdifi‘eremfithinasuain,whileslopesfollowedby desameuppemseleuerwennmdiflemmbaweenmaimmthemfimealomogendtyof regression coeficients test; Wilkonson1992). . cheresistantStrain: thetabular 2valuewith3d£andat5%levelofsignificanceis7.81. The dsusceptiblestrain: thetabularx valuewith2d£andat5%levelofsignificanceis$.99. dRR(redfianmmfio)mmlaumdbydifidingmeLC5odthemdmmmainbytheLCsoofthe susceptiblestrain 110 Azadirachtin. Based on the non-overlapping 95% Confidence Intervals, the IDsosofazadirachtinintheresistantstrainwere significantlydifi‘erent across observation times which no difi'erence was found in the susceptible strain (Table 3.6). In theresistantstrain,theLD50atthe48hobservationwaso.21 ug/larvaanddeclined 10 foldto0.02ug/larvaatthe168 h Inthesusceptiblestrain, adeclineinLDsowith inausingobservafionfimeswasobsaved,hnitwasmtudramaficuintheredstam strain TheLDsosintheresistantstrainweresignificantlyhigherthanthoseinthe susceptible strainintheearlierobservationperiods(48, 72, and96h), however, theywere notdifl‘erentattheendoftheobservationperiod. Theseresultsindicatedthattheresistant strain could live longer than the susceptible strain under azadirachtin treatment. In other words, the resistant strain may have considerably greater protection from azadirachtin than the susceptible strain. Overall slopeswithinastrainwerenot significantlydifi‘erent(P>0.05)basedon the homogeneity analysis of slopes (Wilkonson 1992). However, slopes of the susceptible strainwere steeperthanthose oftheresistant strainand significant difl‘erenceswere observed at the 72 and 120 h observation periods (Table 3.6). Shallow probit line slopes for the resistant strain may have indicated heterogeneity in this population to azadirachtin. In other words, some individuals in the resistance strain may have had lower morbidity responsethaninthesusceptible strain. Inaddition, theresistant strainLDsoatthe48h observationwasseventimeshigherthanthatofsusceptiblestrain, andtheratio decreased to two times higher at the 168 h alter treatment. This data showed that some appreciable crosspresistance exists between B. tinningr’ensr’s and azadirachtin. 111 Table 3.6. Toxicity of azadirachtin using droplet assay method to second instar of Bacillus tlmringiensis resistant and susceptible strains of Colorado potato beetle Strain n Time Losoa 95 % CI Slope” 120 and (h) u3(AI)/larvn sum/lam Resistant 210 43 0.21». 0.12-0.55 1.0m 4.3 7.0 72 0.03am 005-014 1.2». 2.0 4.0 96 0.06bA 0.04-0.09 12». 1.6 3.0 120 0.05bcA 003-007 1.3m 1.5 2.5 144 0.04bcA 0.03 -006 1.4m 0.9 2.0 163 0.02cA 001-003 1.2». 2.5 2.0 Susceptible 140 43 003.13 0.02-0.06 1.6aA 6.3 72 0.02m 0.01 - 0.03 2.6aB 5.3 96 0.02aB 0.01 - 0.03 2.0m 3.6 120 002». 0.01 - 0.03 2.4a 3.9 144 002m 0.01 - 0.03 2.2m 2.4 163 0.01 - 0.02 1.9m 4.6 0.01aA “Signifimd'difiaembdmwsosmbuedmmvvahpping95%®nfidmlmvfl values.I.D5os foflowedbythesamelowucaseleuawuenmdifl'aemwithinasuainwhilewsos fouowedbymesameuppacaseleuermnmdifiuentbaweenmaimmthesameflme. bSlopesfonowedbythesamelowercaseleuerwerenctdifl'aemWithinastrain.whileslopesfiollowedby thesameuppercaseleuerwemnmdifiemntbdwemmaimatheamefimeahmogendtyof regression coeficients test; Wilkonson 1992). ‘ cTheresistantstrainzthetabular 2valuewith4d.£andat5%levelofsignificanceis9.49.1‘he arsceptiblestrainzthetabularx valuewithSdtZandatflfilevelol'significanceisllD‘l. dRR(redaanmrafio)wumlmlmdbydifidingtheLC50dthemdsmmmfinbytheLC50dme susceptiblestrain. 112 b. General Discussion Alltestedinsecticidefldiflubenmron, chlorfiuamron,cyroman’ne,azadirachtin, andirnidacloprid)werenotequallyefl‘ectiveincontrollingtheB. Wandsresistant strainofColoradopotatobeetle. TheLCso sorLDsosintheresistant strainwerehigher thmthoseinthemscepfibkstrahuhowevafiwywaenmdgmficanflydifl‘aemmmost cases. Theresistantstrainshowedsomedegreeoftolerancetochlorfluanu'on, cyroman'ne, andazadirachtinasindicatedbyhigherLCsoorLDsodm'ingtheearly observationperiods. Inothu'wordatheseinsecticidestookalongertimetokillthe resistantlarvaethanthesusceptiblelarvae. Thisphenomenonmayinvolvetheexistenceof across-resistancemechanismintheB. rlumrgr'emisstrain. Vigortolerance isnotlikely since the resistant strain exhibited lower fecundity and longer develoment (see Chapter II). A similar situation was reported by previous researchers with different insecticides. The Long Island strain of Colorado potato beetle resistant to conventional insecticides lived longer tlmn the susceptible strain after treatment. with low dosages of cyromazine (Sirota and Grafius 1994). Incontrast, difiubenmronwassignificantlymoretoxictotheresistantstrainthan the susceptible strain. Negatively cross-resistance between B. thunngiensis and chemical insecticides have been reported several times. Van Rie et al. (1990) reported that P. interprmctella resistant to the cryIA (b) of B. tlurrr’ngiensis were more susceptible to the cryICtoxin. AsimilarsituationwasalsoreportedinthehouseflymallandArakawa 1959) and in the permethrin- and methomyl resistant strains of H. virescens (Ignofo and Roush 1986). This may be a significant finding if B. tlnrringr’enas field resistance developed in Colorado potato beetle. The resistant and susceptible strains responded similarly to imidacloprid. This may beduetothefactthatbothstrainshadnotbeenexposedtonervepoisonsformorethan 30 generations. Thus B. thrm’ngr‘ensis selection apparently does not impact significant advantage to Colorado potato beetle exposed to imidacloprid. 113 Therewasasignificamwrrelationbetweentimeafieru'eaunmandmortality partiarlarlyforinsectgrowthregulatorssuchasdiflubenmron, chlorfiuazuron, cyroman’ne, andazadirachtin OnthebasesoftheLCsosattheéshobservationperiod, cymmazinewasthemosttordcimecficidetoColondopotatobeetlefoflowedby imidacloprid, chlorfiuamron, and diflubenzuron Using the droplet assay, however, azadirachtinwaslesstoxicthanimidacloprid. 'l'hesecomparisonsweremadeonthebases oftoxicitytothemscepfiblestrainsincetheresistantstrainshowed somedegreeof resistancetochlorfluanrron, cyromazine, orazadirachtin. Highertoxidtyofcyromaa’ne maycorrelatewithitspersistenceasreportedbyBrakeetal. (1991). Diflubenzuronwas less toxic than chlorfluazuron which supports similar efi‘ects observed in S. littarulis (Ishaaya 1992). In this case, lower toxicity was due to lower retention time in the insect (Ishaaya 1992). The efi‘ectiveness of the five tested insecticides against B. thra'ingr'ensr’s susceptible and resistant Colorado potato beetle may provide valuable additional information for managing the field populations of Colorado potato beetle. In addition, these results give some insight into the development of future program for Colorado potato beetle. Under an overall IPM program, McGaughey and Whalon (1992) proposed four difi'erent strategies for resistance management: 1) diversity mortality sources, 2) reduced selection pressure, 3) manage susceptibility, and 4) monitoring the resistance development sothatinformedcontroldecisionscouldbemade. Inpractice, tacticscouldbederived from these strategies. The following discussion will focuse on the possibilities of using the results fi'om this study to delay resistance development in Colorado potato beetle. In considering these compounds in Colorado potato beetle IPM programs, they may have several advantages over current synthetic organic insecticides. Even though research on the side-efi‘ects of these insecticides to natural enemies is limited, they seem to be compatible with biological control agents (Tamaki et al. 1984, Webb et al. 1989, Niemczyk et al. 1990, Stark 1992, Stark et al. 1992a, Stark et al. 1992b). Natural enemies 114 can reduce the frequency ofinsecticide applications. In other words, natural enemies can indirectly slow resistance development (Tabashnik 1986). At sub-lethal doses, tested insectgromhregulatorscoulddelaylarvaldevelopment. Thistacticcouldresultina reduction in the number ofgenerations per season (McGaughey and Whalon 1992). On theotherhand, reductionintherateofapplicationcouldreduceoreliminatethe side efi‘ectsto naturalenemiesandpmvideagreaterwindowofoppommityforbiological control to work Theefi‘ecfiveneuandreliabifityoflowdosagetacficsdependsonthemof target insects and the contribution of other mortality sources (McGaughey and Whalon 1992). In the case of Colorado potato beetle, relyance on the low dosage insecticide applications may not be an efl‘ective approach since most populations are naturally above the economic threshold. Additional mortality factors such as the use of transgenic potatoes with low expression (McGaughey and Whalon 1992), manipulation of planting date, early maturing varieties (Riley 1871) could also contribute. Other culmral tactics including migration-barrier trenches, toxic barrier plantings (transgenic plants), and cover crops to _ prevent emigration fiom previous potato field into new potato planting are currently being evaluated. 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Can. Ent. 116: 197-202. Tatchell, G. M. 1992. Influence of imidacloprid on the behaviour and mortality of aphids: vectors of Barley Yellow Dwarfvirus, pp. 409-422. In M. Esters, Papers on the mechanism and mode of action of imidacloprid. Bayer AG, Leverkusen. Thomson, W. T. 1989. Agricultural chemical book I: Insecticides, acaricides and ovicides. Thomson, California. 288 p. Van Laecke, K., D. Degheele, and M. Auda. 1991. Detoxification of diflubenzuron and teflubenzuron in the larvae of the beet armyworm (Spodoptera exigua) (Lepidoptera: Noctuidae). Pestic. Biochem. Physiol. 40: 181-190. Van Rie, J. W. H. McGaughey, D. E. Johnson, B. D. Barnett, and H. Van Mellaert. 1990. Mechanism of insect resistance to the microbial insecticide Bacillus tlna'ingiensis. Science 247 : 72-74. Verkerk R H. J. and D. J. Wright. 1992. Biological activity ofneem seed kernel extracts and synthetic azadirachtin against larvae of Plutella xylostella L. Pestic. Sci. 37: 83-91. Ware, G. W. 1989. The pesticide book. Thomson, California. 340 p. Webb, R E., M. Shapiro, J. D. Podgwaite, R C. Reardon, K. M. Tatrnan, L. Venables, and D. M. Kolodny-Hirsch. 1989. Effect of aerial spraying with dimilin, dipel, or gypcheck on two natural enemies of the gypsy moth (Lepidoptera: Lymantriidae). J. Econ. Entomol. 82(6): 1695-1701. 124 Whalon, M. E., D. L. Moller, R M. Hollingworth, E. J. Grafius, and J. R Miller. 1993. Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain resistant to Bacillus thuringiensis. J. Econ Entomol. 86(2):226-233. Wilkonson, L. 1992. SYSTAT: the system for statistics. SYSTAT, Evanston, IL. Young, T. L., R Gordon, and M. Cornect. 1987. Efl‘ects ofseveral insect growth regulators on egg hatch and subsequent development in the cabbage maggot Delia radicunr (L.). Can Eat. 119: 481-489. Zehnder, G. W. and W. D. Gelernter. 1989. Activity of thr M—One formulation of a new strain of Bacillus thuringiensis against the Colorado potato beetle (Coleoptera: Chrysomelidae): relationship between susceptibility and insect life stage. J. Econ. Entomol. 82: 756-761. CHAPTER IV COMBINATIONS OF DIFLUBENZURON OR CYROMAZINE WITH BACILL US IHURINGIENSIS AGAINST COLORADO POTATO BEETLE (COLEOPTERA: CHRYSOMELIDAE) A. INTRODUCTION Resarchondevdopmgnrategiesformamgingpestresistancetomsecfiddeshas receivedmoreattentioninthelastdecade, andseveralconceptshavebeenformulated (Georghiou 1983, Roush 1989, Tabashnik 1989, Croft 1990, Denholm and Rowland 1992, McGaughey and Whalon 1992). There were similarities and difi'erences among those concepts. Georghiou (1983) proposed three general strategies: 1) ”management by moderation”, 2) ”management by saturation”, and 3) ”management by multiple tactics”. The tactics were developed fi'om those strategies centered on manipulation of insecticide use (Georghiou 1983). Croft (1990) suggested another strategy of promoting the development of natural enemy's resistance. Reduction of insecticide use has hear the major concern for resistance management, and strategies as well as tactics were developed within the philosophy of IPM (Denholm and Rowland 1992, McGaughey and Whalon 1992). These authors suggest four general strategies for combating resistance: 1) reduce selection pressure , 2) maintain the susceptible population, 3) diversify mortality sources, and 4) monitor resistance development. Even though there are some difl‘erences among the authors, they agree that combinations of two insecticides could be an efl‘ective tactic for delaying some types of resistance development (Georghiou 1983, Roush 1989, Tabashnik 1989, Croft 1990, Denholm and Rowland 1992, McGaughey and Whalon 1992). In practice, two insecticides can be alternated, applied sequentially, applied together as a mixture, or applied with spatial variation (”mosaics") (Tabashnik 1989). Alteration or rotation is intended to retard resistance development by limiting the length of exposure to each insecticide. This tactic assumes that the fi'equency of individuals resistant to one insecticide decreases when application of the insecticide is discontinued (Georghiou 126 127 1983, Tabashnik 1989, Denholm and Rowland 1992, McGaughey and Whalon 1992). The dedineinfiequencyofreduaMgenesmightbeduemimnngrafionofwwepuble homozygotes orreducedfiMsofredstMindividualsoracombinafionostetactics (Tabashnik 1989, Denholm and Rowland 1992, McGaughey and Whalon 1992). Mosaicmahodshavenotbeenusedwiddysmcethuemethodspmvidenobenefit overalterationsintheabsenceofrdeasesofsusceptiblemales(€mtis 1985, Comins1986, Roush1989, Tabashnik 1989, Denholm and Rowland 1992). When insecticide A and B mWfieuseofAforthefirstgenaufionwillaflowcontrolthesecond generation with B. With mosaic methods, however, each insecticide may not give more than 50% control in the second generation if resistance is dominant and both insecticides have been used in the first generation (Roush 1989). He developed a simulation model for comparing the effectiveness of alteration and mosaic methods in delaying resistance development. With an assumption that mating is completely random, the simulation model demonstrates that alternation will never be worse than mosaic. In addition, when there is interbreeding within the areas in the mosaic, the mosaic and alteration methods give the same result. Theoretically, mixtures could be more efl'ective than rotations or sequences for insecticide resistance management (Tabashnik 1989, Maui 1990, Denholm and Rowland 1992, McGaughey and Whalon 1992). The assumptions underiying the use of mixtures are: l) resistance to insecticides is rare and independent (Curtis 1985), and 2) individuals that survive one insecticide in the mixture are killed by another insecticide (Georghiou 1983, Comins 1986). This latter concept is termed ”redundant killing". To achieve the optimum resistance retardation, both insecticides should have equal persistence and efl‘ectiveness to kill the susceptible individuals, there should be immigration of susceptible individuals from untreated areas, and resistance genes should be rare ( Curtis 1985, Mani 1985, Roush 1989). Ifone of the first two conditions can not be met, unexpected results 128 suchasacceleration ofresistancetooneorbothinsecticides (cross-resistance) could occur(Denholm andRowland 1992, McGaugheyandWhalon 1992). Chapterflldemonstratedthatdiflubenwonandcyromazinewereefl‘ecfiveto controlB. tlmringiensis ausceptibleandresistant strainsofColorado potatobeetle (see Chapter 3). In orderto search for synergism, combinations of diflubenzuron-B. tlnaingiemisandcyromazine-B. Wandswere studied. Efi‘ectsofcombinationswere evahmedonthebasisofhrvdmomfity,weightgainofauvivinghrvaemdluval development. B. LITERATURE REVIEW 1. Risks and Benefits ofinsecticide Mixtures Tabashnik (1989) identified three possible disadvantages of using insecticide mixturesincluding: 1)theymayconfercross-resistance, 2)theymaybemorelnrmfirlto non-targetinsectsinchidingnaturalenemies, and3)theymaypromoteresistancein secondary pests. Evidence ofcross-resistance caused by insecticide mixtures were reported in the German cockroach, Blattella gemrica (L.), to a combination of chlordane-malathion (Burden et al. 1960), and Indianmeal moth to a combination of B. tlnrringiensis isolates EDI and HD-l33 (McGaughey and Johnson 1992). To the best of myknowledge,thereisnopublishedpaperontheefi‘ectsofinsecticidembrmresonnon- target species or secondary pests. Using insecticidemixturesforcontrollinginsectshavetwoadvantages. First, mixtures can retard resistance development (McGaughey and Johnson 1992, McKenzie and Byford 1993). Second, ifsynergisticjoint actions can be achieved the application rate of each component can be reduced (Salama et al. 1984). Simulation models and laboratory research demonstrate that insecticide mixtures can delay the resistant development but are not always better than alterations or sequences (Mani 1985, Comin 1986, Denholm and Rowland 1992, McGaughey and Johnson 1992, McGaughey and Whalon 1992, McKenzie and Byford 1993, Yamamoto et al. 1993). In the situation where insecticides haveequal persistence, equal efi‘ectiveness, and someofthepopulationescapefiom exposure, a mixture would be a powerfiil tool for retarding resistance development (Mani 1985). Laboratory erqieriments have demonstrated that the application of a mixture of B. tlurringienals isolates ID] and HD-133 against Indianmeal moth yielded resistance to both isolates; however, the rate of resistance development in the mixture was slower than 129 130 that of each isolate alone (McGaughey and Johnson 1992). McKena'e and Byford (1993) danonmuedmuannxtumofpamahin-dhzimnrotafiomofpamahfin-diazinonma pamethin-ivamahrinmhmneremhedmredumceonemsevengenaafiomlatathan resistancedevelopedtoeachinsecticidewhenappliedalone. Synagisficacfionbetweentwoinsecficideshasbeenknownformmydecades. Yamamotoet al. (l993)reportedrecentlythat amixtureofN-methylcamabate- oxadiaaolonerearltedinsynergismtogreenleafliopper. However, theefi‘ectsofthe mixturesonresistancedevelopment havenotbeenresearched. Amixtureofpermethrin and chlorpyrifos on pyrethroid-resistant horn flies, Haematobia im'tans (L.), was 2.9-fold as toxic as permethrin alone and 5.9-fold as toxic as chlorpyrifos alone (Byford et al. 1987). The mixture resulted in synergism with the susceptible strain. 0n the other hand, synergism was not detected with this mixture on chinch bugs, Bliasus leucopterous Ieocopterous (Say) (Wilde et al. 1984). Liu et al. (1984) reported that fenvalerate, permethrin, deltamethrin, and cypermethrin were synergized by butacide on diamondback moth. Thesestudiesindicatethattherewhsofambaurearealsodeterminedbythe species of insect. Synergism between B. tlum’ngiensis and conventional insecticides have been demonstrated on some insects. Combinations of B. thuringiensi.: with some organophosphate or pyrethroid insecticides showed synergistic interaction on Spodoptera littoralis (Boisduval) (Salama et al. 1984). Dabi et al. (1988) also found synergism against Heliothis amrigera Hubner. when B. tlmfingiensis was mixed with malathion, endosulfan, monochrotofos, and carbaryl. Permethrin at the median concentrations enhanced the efi‘ect of B. tlmfingienfis on the cabbage looper, Tfichoplusia m’ (I-Iubner) (J aques et al. 1989). Many IPM workers recognize that insecticide resistance management is a tactic with an IPM program (Philips et al. 1986, Tabashnik 1989, Denholm and Rowland 1992, McGaughey and Whalon 1992). Therefore, tactics for combating insects' resistance to insecticides must be compatible with other components of IPM; for example biological 131 connolaganasimepredatonandpmsitoidscmddheefi‘ccfivemolsforredstmce management (Croft 1989) by diversifying mortality sources. Inmdatodinnnatedisadvamageaofusingmsecfiddesnfinureaacompnhensive smdyshmddbewnmraedonthewmplexecosyuanwhaetheuuisintended.Anflyfis ofbenefit-costrafiosshmddhearpandeanonlyontheurgetspcdeshndwonmn- mgaspedesmdudingmmrdenamestaflthereswchshouldhewnduaedhefom aparticularinsecticidemixtureisrecommendedtogrowers. 2. Insecticide interactions Results of insecticide mixtures are determined by the relative importance of the second insecticide to the biological limitations (absorption, distribution, and excretion) of the first insecticide whui it is applied alone (Wilkonson 1976). The species of insect and itsage, sex, andthetimeintervalhetweenapplicationsofthetwo insecticides shouldbe considered because they may mask or enhance synergism or antagonism. Wilkonson (1976) identified four sites where the one insecticide could afi'ect the other: 1) during the absorption processes, 2) while being distributed in the tissues, 3) during hiotransformation, or 4) at the target site. Onthebasisofthe mode ofaction,joint actionhetweentwo insecticidescanhe classified into two types: independent and similar joint actions (Bliss 1939, Finney 1971). Independentactionmcansthatthetwo insecticideswork independentlyhecausethcyhave difl‘erent modes of actions. Similar joint action occurs when two insecticides result in similarefl‘ects. Inotherwords, thetwo insecticideshaveasimilarmodeofaction Therefore, one insecticide can be substituted at a constant proportion for the other insecticides. Forindependentjointaction, theexpectedmortalityofann'xturecanhecalculated as P=P1+P2(l -P1)(1-r)(Blissl939);wherePismortalityofmixture,P1is mortalitycausedbythefirstinsecficideatthedosegiveninthemixturePzismortality 132 causedhythesecondinsecticideatthedosegiveninthemhrtureifPl isgreaterthaan, and r is the degree ofcorrelation between susceptibilities to the two insecticides. Ifr =1, thentheformulasimplycanbewrittenas P=P1.'I'hismeansthattheexpectedmortality ofthemhrturewillheequaltothatofthemoreactiveinsecticide. Thenbycomparingthe obmedmoflahtyandflwarpeaedmonafitymdfingfiomthemhameaconclusioncan hednwn(Tmnmes1964).Iftheobsavedmonafltyisgreaterthantheexpected mortality, themhrtureresultsinsynergisrn. Incontrast,iftheobservedmortalityislessthanthe expectedmortality,themixtureresultsinantagonisrn. Theresultwillhecalled independent if the observed mortality is equal to the expected mortality. In similar action, when each insecticide is tested alone, they will show parallel log dose probit mortality regression lines (Bliss 1939, Finney 1971). The presence of a second insecticidewilladdtothemortalityandtheadditionalmoflalityremltingfiomthesecond insecticide depends on the relative toxicity of the second insecticide to the first insecticide (Finney 1971, Tabashnik 1992). For example, the relative toxicity of the second insecticidetothefirstinsecticideisp, andamixturecontainingamount "a” ofthefirst insecticide and ”b" of the second insecticide. Conversion of the mixture into an equivalent dose of the first insecticide can be calculated as ("a" + p ”b”). Using the regression probit line ofthe first insecticide, the expected mortality ofa mixture can be predicted. The action of a mixture will be called synergistic if the observed mortality is greater than the expected mortality. Incontrast, iftheobservedmortalityis smallerthantheexpected mortality, then the action is called antagonistic. A mixture is said to show similar action (additive action) if the observed mortality is equal to the expected mortality (Tammes 1964, Finney 1971). Ifthe insecticides have similar efi‘ects, synergism studies can also he conducted using a series of dosage-mortality curves, and each curve represents a mixture in which the insecticides occur in a fixed proportion (Finney 1971, Tabashnik 1992). Methods for 133 evaluafionofsynagiunmdiswssedinthefoflowingpmgmphsasdeacfihedhy Tabashnik(1992). First, computethepotencyofasecondinsecticide(B)relativetothatofthefirst insccticidc(A).Itcanhcestimatedas PB=ID50(A)/LD50(B)wherePB istherclative potencyofinsecticideBtoA Second, predictflrepotencyofthemhrhrreWrelafivetothatofinsecficideA 'I‘hePMiscornputedusingaforrmrlaof PM=rA+(erPB)wherePM‘-'=thepotency ofthemixture,rA=proportionofinsecticideAinthemhrtureandrB=proportionof insecticideBinthemixture. Third, computetheexpected LD50 of the mixture (LD50(M) with aformula: LD50(M) =LD50(A)/P1w 'I’heobservedLDsoofthemixturecanhecalwlatedfi’omtheexperimentandby comparingtheobser'vedandtlieexpectedLDSOS,theactioncanbelmown. Ifthe observedLDsoissmallerthantheexpectedLDso,itmeansthattheinsecticidesinteract synergistically. Ontheotherhandiftheobservedmsoisgreaterthantheexpected LD50, theinteraction is called antagonism. Whenthe expected mm is equal to the observed LD50, it demonstrates that the insecticides have additive action. 3. Combinations of Microbial and Chemical Insecticides Researchers have examined compatibility and synergism of combinations between microbial and chemical insecticides. Anderson et al. (1984) noted that the tests with ahamecfinfiifiummonthufingiensinandcubuylshowedmsigrfificamfiectin inhibition of colony growth of the fiingal biological control agent, Beauveria basn'ana. These combinations showed additive interactions on Colorado potato beetle. Results from combinations of permethrin and Autographa califomica (Speyer) nuclear polyhedrosis virus (ACNPV) to T ficaplusr’a ni (Huhner) varied depending on the concentration of each 134 component (Jaques et al. 1989). Synergistic actions occurred when high concentration of permethrin was combined with low or moderate concentration of ACNPV. TheuseofB. Wensrlsforconn'oflinginsectpestshashecomeintensivesince thismicrobialinsecticideisquitespecific. SeveralattemptstoenhancetheactivityofB. Wantshawheenwnduaedbywmmismsecfiddewithmhamsecfiddes. Jaquesetal. (l989)reportedthatacombinationof B. tllurtngienrisandpermethrin resulted in syncrgismagainst II niand antagonism againstArtogsia (=Pieris)rqpae (L.). Thenggeaedmuthcdifi‘aenceswaedinmthedifl'auumcepfibihtyofthosemo species to B. tlnm'ngl'ensrs. Both pyrethroids (fenvalerate, cypermethlin, and permethrin) and organophosphates (phoxim, dimethoate, profenofos, and chlorfenvinphos) enhanced the activity of B. tlnrfingiensis var. galleriae on Spodoptera littoralis (Boisduval). The same results were achieved when pyrethroids were combined with B. tlmringiensis var. entomocidus, but dimethoate and profenofos only showed additive eaeots with this strain (Salama et al. 1984). In field experiments, combinations of pyrethroids (cypermethl'in, dcltametrin, fenvalerate, and permethrin) with B. tlnm'ngl'ensis var. hastaki showed high eficacy to winter moth, Operophtera bn'anata (L.) (Hardman and Gaul 1990). Salalna et al. 1984 also reported that when] (a carbamate insecticide) and diflubenzuron (an insect growth regulator) had an additive efi‘ect when combined with B. tlnm’ngienns var. entomocidus. However, combination of carbar'yl with B. tlnm’ngl'ensis var. kurstaki yielded synergistic efi‘ect on Heliothis armigera Hubner (Dabi et al. 1988). Dabi et al. 1988 found that the three other combinations, endosulfan, malathion, or monocrotophos with B. tlnrringiensis var. hrrstala' resulted in synergism. Efl‘ects fi'om a combination of phenthoate with B. thrm'ngl'ensis, however, varied depending on time. Synergistic efi‘ect only occurred before 48 h post treatment. By 72 h this combination resulted in antagonism. Most of the combinations of neem-seed-kernel- extract with B. thlaingiensis against S. fl-ugiperda showed an additive efi‘ect, but some 135 combinations resulted in synergism (Hellpap and Zebitz 1986). Actions resulted by combination of insect growth regulators (AIM 120 EC, Alsystin 48 SC, Cascade 5 EC, Dimilin 25 WP, Nomolt 15 SC, and Sonet 5 EC) andB. tlnm'ngiensis againstLymantria dispar L. were variable (Novotny et al. 1990). Antagonistic reactions occurred when insectgromhregulatorswerecombinedatthehighestdosage(1000mgofa.i. perha) withB. tlrurtngiaasisatarateoflOngerha. Some synergistic reactionswere achieved whenB. tlwfingiensiswaemixedwithAMNomoldorCascadeatarateofIOOmgof a. i. pcrha. Thisresearchshowedthatresultsfi'omthcsamecombinationscouldbe difi‘eremdependingonspeciesofinsectsmdconcennafionofeachcomponent. In addition, these studies indicated that combinations between microbial and chemical insecticides could give synergistic efi'ects. C. MATERIALS AND METHODS 1. Materials a. Insects B. tlnatngtenris susceptible and resistant strains of Colorado potato beetle adults wereobtainedfi'omlaboratorystocksatthePesticideResearchCenter, Michigan State University. The resistant strain had been selected for at least 32 generations with B. thatngtensis and was more than SOC-fold resistant. Adults were originally collected during the summer of 1987 and 1988 fi'om potato farms in Barry, Allegan, Clinton, Ingham, Macomb, Montcalm, and Washtenaw counties in Michigan (Whalon et al. 1993). The Colorado potato beetles were reared in the laboratory at 25 i 2°C and a photopeliod of 16 : 8 (L : D). Adults were placed in cages (0.4 by 0.4 by 0.45 m) with six potspercage.Eggmasseswerecollecteddailythenputina15-cmpetridishuntil eclosion and the larvae were fed with potato foliage. The susceptible larvae were reared in the petri dishes until the third instar. Late third instars were transferred onto potato plants in a cage until all larvae entered the soil to pupate. Potato plants were cut and covered with a petri dish. Adults that emerged from thesoilwereplacedincagesandthepopulationwasmaintainedat20-25 adultspercage. Early second instars ofthe resistant strain were selected withB. tlna'ingl’ensr's solution at the concentration of 60 ml of formulation product per liter (741 mg AI per liter).'I'he potato leaf-dipping method was employed for selection (Whalon et al. 1993). Potato foliage was trimmed to the terminal five leaflets. The stem was wrapped with a sponge and inserted into a 2-ml vial containing distilled water. Each leaflet was dipped in the B. thufingiensis solution five times for 30 seconds and allowed air dry before putting itintoa lS-cmpetridish. EachpetridishhadZO-ZS secondinstarsandthelalvaewere 136 137 orposedfor96honthefieatedleavesflhemrvivinghrvaewu’emnsfaredomo untreatedfoliageinpetlidishes.Iatethirdinstarswerereleasedontopotatoplantsina cageunfilaflluvaeauaedthesofltopupue.AdNuanagedfiomthesoflwerephced inacage. b. Insecticides All bioassays were done with fornnllated insecticides: B. tlmringr‘ensis var. tcmbrl'om's (SPUDCAPG; Mycogen Corporation, San Diego, California), diflubenzuron (Dimilin® 25 wp [Wettable Powder]; UniroyaL Naugatuck, Connecticut), and cyromazine ('I‘rigard® 75 WP; Ciba, Greensboro, NC). 2. Methods a. Combination of dil'lubenzuron and Baciflus thufingiensis Sub-lethal concentrations were chosen for each component obtained fi'om the previous probit lines (see Chapter III). Twodifl‘ermt concentrations of diflubenzuron were combined with a concentration of B. tlnrrl'ngl'enris for each strain, and the combinations were presented in the Table 4.1. Thecxperimentwasdesignedusingfivetreannentsincludinganuntreated, diflubenzuron or B. tlnm'ngiensis alone, a mixture of diflubenzuron and B. tlnm'ngiensis, and diflubenzuron plus B. tlmringiensis where the B. thuringiensis was administered 24 h after diflubenzuron (Timed delay). The experiments were conducted at 25 :l: 2°C and a photopeliod of 16 : 8 (L : D). 138 Table 4.1. ThemixtmesofdiflubenzuronandBacilIustImringienuswhichwere tested onsecondinstarB. tlnrringiensisresistantandsusceptibleColoradopotatoheetles. Insecticides Strain Diflubenzuron (mg [AH/l) B. tluatngtensis (mg [AH/l) Resistant 5.3 620.7 15.4 620.7 Susceptible 5.9 1.4 32.4 1.4 Studies were carried out using leaf dip method with second instars of Colorado potato beetle (Whalon et al. 1993). Each leaflet was dipped in a solution five times for 30 seconds each, and allowed to air dry before putting it into a 15-cm petri dish. Ten second instarsofthesameage(4dold)weretransferredontotrcatedleavesineachpetridishand each treatment was replicated at least three times. Alter 24 h, the leaflets for the timed delay treatment were dipped in a B. tlnm’ngiensr‘s solution five times for 30 seconds and allowedairdrybefore puttingthemhackinthepetridishes. , At96haftertreatment, survivinglarvaefromeachunitweretransferred onto untreated foliage and left to grow until 168 h after treatment. Mortality was assessed at 96 and 168 h after treatment. A larva was considered dead ifit did not move when repeatedly touched with forceps. At the same times, the live larvae from each experimwtal unit were weighed together. Larval development was recorded by instar from 48 to 168 h after treatment. b. Combination of cyromazine and Baciflus thuringiensis Sub-lethal concentrations were selected for each insecticide. Two different mixtures of cyromazine and B. tlna'ingiensis were applied against resistant and susceptible strains (Table 4.2). 139 Table 4.2. The mixtures of cyromazine and Bacillus tlnm'ngiensis which were tested on second instar B. tlnm’ngiensis resistant and susceptible Colorado potato beetles. Insecticides Strain Cyromazine (mg [AI]/l) B. tlna'ingl‘ensis (mg [All/l) Resistant 30.1 620.7 136.5 620.7 Susceptible 5.6 1.4 12.1 1.4 The experiment was designed using five treatments including an untreated, cyromazine or B. tharingr‘ensis alone, a mixture of cyromazine and B. thafingiensis, and cyromazine plus B. thuringiensi.: where the B. tlntringl'ensis was administered 24 h after diflubenzuron (Timed delay). Each leaflet was dipped in a solution five times for 30 seconds each and allowed to air dry before putting it into a lS-cm petri dish. Ten second instars were released onto treatedleavesineachpetridishandeachtreatmernwasreplicated4-12times. Afier24h, the leaflets for the timed delay treatment were dipped in a B. thunngiensis solution five times for 30 seconds and allowed air dry. Afier96hofexposme,thesurvivinglarvaefiomeachunitwereflansferred onto untreated foliage and left to grow until 168 h alter treatment. Mortality was assessed at 96 and 168hafiertreatrnent. Atthesametimes,thelivelarvaefiomeachexperimentalunit were weighed together. Larval development was recorded by instar fi'om 48 to 168 h alter treatment. 140 3. Data Analysis Since diflubenzuron, cyromazine, and B. tlna'r'ngiensis have difl‘erent modes of action, joint actions between diflubenzuron and B. tlna'ingiemis or cyromazine and B. tlnrringicnsiswereanalyzedusingBhss‘sfomadawiththeassumpfionr=1;whereristhe degreeofcorrelationbetweensusceptibilitiestothetwo insecticides. Thesameformula wasusedbyJaquesetal. (l989)toevahlatethceficacyofmicrobial insecticidesand permethrinmixturesagaimt 71 niandA. mJI'heexpectedmonalityfi‘omthemhrture, thereforewiflbesimilartothcmortalityfi‘omthemoreactivecomponem. Abbott's formula (Abbott 1925) was used to account for control mortality during the experiment, and the mortality data were subjected to Are Sine ‘1 percentage transformation before the data were submitted to ANOVA (Gomez and Gomez 1984, MSSTATMSU,1990).Thedataweretransformedhecauscthevariancewas heterogeneous and the percentage data ranged from 0 to 100%. Comparisons between means were carried out using MSTAT (MSU 1990) with LSD test at a = 0.05. ANOVA andLSDwerealsoutilizedtocomparetheweightgainofsurvivinglarvaefi'omeachof thediflflmtumts. Datafiomlarvaldevclopmentwaspooledforeachtreatmentand numbers of each instar were presented in percentage. The efl‘ects of insecticides on the larvaldevelopmemweresfiflobservableunfiltheendoftheobservafionpaiod (168h posttreatmm). Thuscomparisonsbetweentreatmentswerebasedonthedatafromthc 168 h observation period. D. RESULTS AND DISCUSSION 1. Results a. Combinations of diflnbenznron and Racial: thafingiensis Mortality. Low (5.3 mg [AH/l) or high (15.4 mg [All/l) concentrations of diflubenzuron with B. tlnuingiensis (620.7 mg [AI]/l) for the resistant strain of Colorado potato beetle did not result in synergism (Table 4.3). Even though mortality resulted by mixtures (low and high concentrations of diflubenzuron) tended to be lower than the mortality yielded by each component alone, they were not significantly different. These resultsindicatethat ”redundant killing” wasnot presentinthesemixtures. Application of low and high concentrations of diflubenzuron 24 h before B. tharingiensis (the timed delay treatment) resulted in antagonism (Table 4.3). Mortality fromthesetreatrnentswas significantly lowerthanmortality fromthemixtures or each component alone, demonstrating that diflubenzuron interferes with the B. thrm‘ngiensis intoxication. Increased concentration of diflubenzuron resulted in an approximately 4 fold increase in mortality compared with mortality at the lower concentration (Table 4.3). In mixture and timed delay, the increases were approximately two and three times respectively. With the susceptible strain, the mixture ofdiflubenzuron at a low concentration (5.9 mg [AI]/l) and B. tlna'ingiensis (1.4 mg [AU/l) resulted in synergism (Table 4.3). However, synergism was not present when concentration of diflubenzuron was increased to 32.4 mg (AI)/l. 141 142 gagged-noo—saoogogo 6833292... .ooo«cassomogfineeagofiasecasfiaésgogsaéaggfisoasfissfiao iguana-ofiasnasasrisifisné.mtefiosahgocficaiaanssaoageztgze . 3 « hoo o hon u hm... on o h: u. on" o ho a ho on 3% Bap . he « o2. e o.o_ « o.oo on s ofl « o.oo on ha « hon on 2:32 . o no a he o no u h: on . on u on. on ha u. «:2 on acoustic .m c ha. u go o o.o_ n. o.oo on n he s woo e no" « o9. on 838.35 :3 we I u ensue: .m =9: we homo u ensues: .m an :3 us to... t 538359 8a :3 ma v.2 u eeafioosn o 4.2 a has o o: u oé oo o 3 e 2 o ho a ho oo 38 8.5 c mom « 3o . In a «.3. oo c on u hon c ho a. ho_ oo 9:32 n can u hon on o.: a 3" on c nos « hon c he on hon oo fineness .m o o.o~ u h: on non n. 33 oo o. h: n" o.o~ o h: u 2: oo =23..on <93 me E u assume: .m 2258 homo u hegemonic .m Ba :3 we 3 u 53832 33 <9: as ho u Sassoon—on o oo_ o oo o: o oo_ o oo on snow spoooosm seem assess $895859 ado « xv noose: x 3:02. 838 2.8200 mo 032—033 .8 .558.— oenufizg .m meg—ocean. Seven—803%: Aha—ovooauvgummeg .mfimagbflemonavm 3:938? 838.3% so; so «has? n a eases assesses 3:38 o5 83.33% oo esoo... .3 2.3 143 Comparedtodiflubenwonalonetherewasatendencytoward decreased mondhyoramagomnnwiththefimedddayedUeatmemmthewscepnblesminCrahle 4.3). At 96 h, themoltalityresultingfi'omlow concentration of diflubenzuronwas28.8% anditwasapproximatelytwotimeshigherthanthatfi‘omthetimeddelay.Withthehigh concennafionofdiflubenmronthemonafityhecameWAmdhwassigmficmflyhigha thanthatinthetimeddelay. Yet,themortalityresultingfi'omthesetwotreatmentswas notsignificantlydifl‘erentatthe 168hobservationpeliod. Theseresultsindicatethatthe efi'ect of diflubenzuron to B. tluatngr’ensis intoxicationwasonly observedintheearly observationperiod. In both strains, progressive mortality was noted in all treatments. Mortality resulted by mixture and timed delay treatments developed difl‘erently over time. As in the resistammairhmonahtyfiomthembdmewusigfificanflyhigherthanthatfiomthe timed delay. This might be due to difl‘erences in leaf consumption. Larva weight. Resistant larvae that survived fi'om 5.3 mg (AI)/l diflubenzuron gained weight at the same rate as the control larvae (Figure 4.1. [A]). However, the high concentration of diflubenzuron (15.4 mg [AI]/l) resulted in significantly more growth retardation than with the lower concentration (Figure 4.1. [B]). Surviving larvae from B. thrm’ngl’ensis, mixtures, and timed delayed treatments had significantly lower weight than the control larvae (Figure 4.1. [A] and [B]). These results demonstrated that at low concentration of diflubenzuron, inhibition of larval growth caused by the mixture and the timed delay treatments were mostly as the result of B. thuringiensis. At high concentration, however, diflubenzuron was as strong as B. tlnm‘ngiensis in inhibiting the larval growth. 144 A. Diflubenzuron (5.3 mg [A|]l|) and Bacillus thuringiensis (620.7 mg [All/l) 120 . a lash-lash A 1m § .. 3 . é eo -------------- gt ,0 ............... 2°_ """ / o /// 4% /% /://// (0 WM “M M “MM 8. Diflubenzuron (15.4 mg [All/l) and Bacillus thuringiensis (620.7 mg [All/l) 120 ‘ a IOBhI‘IBOh A 1“) T § .. 5, . é so :05: . -— 40 0 g . 20 0'00““ /// //////: //////. W A A Figure 4.1. Average weight of Bacillus thafingiensis resistant Colorado potato beetle larvae after treatment with difi‘erent insecticides. Means followed by the same letter within a time period are not significantly difi'erent using LSD test at a = 0.05. Upper-case letters correspond to comparison of means at 96 h post treatment, and lower-case letters correspond to comparison of means at 168 h post treatment. 145 A. Difluhenzuron (5.9 mg [A|]/|) and Bacillus thuringiensis (1.4 mg [A|]/l) 140 a 120 T I I96hl168h I ’8 a 100 E B: so E, E w .............. 2’ g 40 20 / 7%sz . H ”/9? ' H onwflflsouw‘” it” «MW B. Diflubenzuron (32.4 mg [Al]/l) and Bacillus thuringiensis (1.4 mg [Al]/|) .96h-168h 120 b Weight (mg/larva) Figure 4.2. Average weight of Bacillus thufingiensis susceptible Colorado potato beetle larvae after treatment with different insecticides. Means followed by the same letter within a time period are not significantly different using LSD test at or = 0.05. Upper-case letters correspond to comparison of means at 96 h post treatment, and lower-case letters correspond to comparison of means at 168 h post treatment. 146 Asinflieresistammainthesdectedconcenfinfionsofdiflubenwonddayedthe growth of susceptible larvae. The high concentration resulted in stronger tendency to delay growth than the low concentration (Figure 4.2 [A] and [B]). B. tlnrringiensr's demonstrated stronger tendency in growth retardation than low concentration of diflubenzuron. In contrast, the high concentration of diflubenzuron inhibited the larval growth stronger than B. tlnrrr’ngl’emis. Surviving larvae fiorn B. tlnrn'ngr'enns demonstrated difl‘erent response. In the first experiment (Figure 4.2. [A]), the larvae gained 30 mg on average and in the second experiment (Figure 4.2 [B]) the average weight was 70 mg per larva. This may indicate thatthelarvaefromthesecondexperimenthavehigherrateofrecoverymechanismthan those fiom the first experiment. Larval development. Resistant larvae treated with diflubenzuron at a low concentration were mostly fourth instars alter 168 h (Figure 4.3. [A]), but the high concentration delayed development and most were in the third instar after 168 h (Figure 4.3. [B]). All larvae that survived from B. tlnrringr’ensrlr were third instars, whereas only some ofthesurvivinglarvaefromthemixtureandtimed delaytreatrnentsbecamefourth instars (Figure 4.3. [A] and [B]). These data suggest that at the treatment concentrations, B. thrm'ngr’ensr’s had stronger efl‘ects than diflubenzuron in slowing down the growth and development of surviving larvae. With the susceptible strain, surviving larvae treated with diflubenzuron at a low concentration were mostly fourth instars (Figure 4.4. [A]). As in the resistant strain, the high concentration delayed development (Figure 4.4. [B]). In the first experiment (Figure 4.4. [A]), approximately 50% of the surviving larvae from B. thafingiensis treatment were thirdinstars,whereasinthesecondexperiment(Figure4.4. [B])allsurvivinglarvacwere able to become fourth instars. This indicates that there remains considerable genetic 147 A. Diflubenzuron (5.3 mg [All/I) and Bacillus thuringiensis (620.7 mg [All/l) d 8 8 8 E ! lnstar distribution (%) 8 o B B. Diflubenzuron (15.4 mg [All/l) and Bacillus thuringiensis (620.7 mg [A|]/|) Instar distribution (%) s 8 8 8 o 8 wwntflwlsmws “W“ «MM Figure 4.3. Larval development of the Bacillus thuringiensis resistant Colorado potato beetle larvae 168 h after treatment with different insecticides. Total height of bars indicates percent survival including all instars. 148 A. Diflubenzuron (5.9 mg [All/l) and Bacillus thuringiensis (1.4 mg [All/I) lnstar distribution (%) B. Diflubenzuron (32.4 mg [Al]/|) and Bacillus thuringiensis (1.4 mg [All/l) A 1‘” I3ldinatar-4thinstar ES w _ ............................... T" ’ ........................................................ E m ....................................................................................... “0‘ sis to to“ solutes» «t on" Figure 4.4. Larval development of the Bacillus thraingiensis susceptible Colorado potato beetle larvae 168 h after treatment with different insecticides. Total height of bars indicates percent survival including all instars. 149 variationinthesusceptiblestrainasexpected. Survivinglarvaefi'omthemixtureandtimed delayweremostlyinthirdinstars(Figure 4.4. [A] and [B]). b. Combinations ofcyromazine and Baciuas thuringiensis Mortality. Mixtures of cyromazine at either low (30.1 mg [All/l) or high (136.5 mg [AU/I) concentration with B. tlnningiensis at 620.7 mg [AI]/l did not resultinsynergisrnwiththeresistantstninCl‘able4A). Mortalityresultingfi’om mixtureortimeddelaytreaunentswasashighasmortalityrenlltingfi'om cyromazine alone. This indicates that combination of cyromazine and B. thrm'ngiensis acted independently or cyromazine masked the activity of B. tlurn'ngiemis. Therefore, ”redundant killing" was not observed. Application of cyromazine 24 h before B. thuringiensis treatment yielded monahtyashighasinthemhnureneatmentaable4.4).1hismaydemonsuate that the presence ofcyromazine in the insect midgut had little interference on B. thla'ingiensis intoxication or the larvae had ingested suficient amount of cyromazine before they stopped eating as a result of B. tlrraingr’enris ingestion A visual observation indicated that cyromazine did not cause larvae stop eating. However, the treated larvae consumed less foliage than the untreated larvae. Increasing concentration of cyromazine enhanced mortality in cyromazine, themixture, andthetimeddelaytreatrnentsandtheywerenot statistically difl'erent. Progressive mortality was noted in all treatments, indicating that the effects of the insecticides were still observable until the 168 h observation period. On the bases ofthe 168 h observation period, combinations ofcyromazine at a low (5.6 mg [AH/l) or high (12.1 mg [AI]/l) concentration and B. thuringiensis (1.4 mg [AI]/l) resulted in antagonism with the susceptible strain (Table 4.4). A similar trend occurred in both combinations. Cyromazine alone yielded the highest mortality and then followed by the mixture and the timed delay. 150 §§§afio§ofioouoao§o “3.325520 .oo.ouaaasn3!!ensee%§§nau§assuabocsseogfianaaoaggnsoessfiao gggomEnssasroasasassnfiao.mtanooofipgcaocaacsanoaouaaeztge o Sm u o.oo o ems « o.mn on c mo u o.oo on he u o.om oe 3% 85. on 2: u o.mo o. m.m_ « o.mm oe c m.» u o.oo o. o.2 u o.mm oe chi: o m.m_ a. mom o m.m_ u 2m 3. . o mom on o.mm o 92 u on oe chafing .m c o... u o.oo o oo « o.oo oo o o.o u o.mm e m.» u o.oo oc oengoso :3 ma .1 u Eng .m :3 ma homo u censuses .m Ba <9: on _.m_ u Sansone nae <9: we o.oa n 83826 o on « o.mm o oo— « o.m_ on e o.: u o.oo o o.» u ho_ om_ Eco Baa. o no u in e e: u o.om on e 2m u o.oo c m.m_ u o.mm om_ 23x3 o ms a 2m on a: n o.om oo o o.o_ u o.mm n he u o.mm om_ easing .m e 3. « o.oo on e.» u o.mm on e mm on hmo on o.o_ u mom om_ oencaoso 53 ma 5 u sang .m E3 on. homo u chafing .m can Sgoaoouoengoao can :3? homuoencaoao u o oo_ o oo .e o m2 a oo on £86 03a§m £93m «Snead n «8830.5. a do u x o $.52 .x. .8303 280a eon—o.oo 03.3083 .5 gap. ogumwng .m mo Sea 888 o. coca so; neonate .2 or? ooaoo eugenics .h as, essence sous o cm soon. as Senses e33 6 .238 c a .533» seasons: sheen o8 8382s mo esooo. .2. one. 151 The mixture resulted significantly higher mortality than B. thw'ingiensis alone (Table 4.4). These data indicated that mortality in the mixture was due to cyromazine or a combination of cyromazine and B. tlmringiensis activities. Since B. flmfingiendscausedthelawaeceasedeafingmdcyromazinedidmgtheluvae treatedwiththembrnireingestedless cyromazinethanlarvaetreatedwith cyromazine alone. As a result, mortality resulting fiom cyromazine treatment was higher than that fiom the mixture. Mortalityinthetimeddelaywassignificantlylowerthanthatinthe mixture. This might suggest that the presence ofcyromazine in the midgut inhibited B. thuringiensis intoxication. This data might also support that mortality in the mixture was partially due to B. thuringiensis since the larvae treated with the mixture might ingested smaller amount of cyromazine than the larvae fiom the timed delay treatment. Larva weight. In weight gain comparisons with resistant larvae, control larvae weighed significantly more than larvae in other treatment (Figure 4.5. [A] and [B]). At the low concentration of cyromazine, larval weights fi'om cyromazine, B. tlnm’ngiensis, the mixture, and the timed delay were not significantly difi‘erent (Figure 4.5. [A]). Increasing concentration of cyromazine resulted in significant weight gain reduction (Figure 4.5. [B]). Susceptible larvae treated with cyromazine, B. (Intringiensis, the mixture, or the time delay gained significantly less weight than untreated larvae (Figure 4.6. [A] and [B]). The surviving larvae treated with the mixture of cyromazine and B. tinm'ngiensis had significantly less weight than those treated with cyromazine, B. thuringiends, or the timed delay (Figure 4.6. [A]). This indicates tint the mixture of these compounds yielded synergistic action in retarding larval growth. At the high concentration of cyromazine, however, synergism decreased (Figure 4.6. [B]). 152 A. Cyromazine (30.1 mg [All/I) and Bacillus thuringiensis (620.7 mg [All/l) a lash-teen Weight (mg/larva) B. Cyromazine (136.5 mg [A|]l|) and Bacillus thuringiensis (620.7 mg [AI]/l) i .Whllaflh Weight (mg/larva) Figure 4.5. Average weight of Bacillus thuringiensis resistant Colorado potato beetle larvae afier treatment with difl‘erent insecticides. Means followed by the same letter within a time period are not significantly difl‘erent using LSD at a = 0.05. Upper-case letters correspond to comparison of means at 96 h post treatment, and lower-case letters correspond to comparison of means at 168 h post treatment. 153 A. Cyromazine (5.6 mg [A|]I|) and Bacillus thuringiensis (1.4 mg [AI]/l) 160 a 140 ’ 1' l.a§.b.l.1.aa h g 120 g 100 E, so E . .9 5° m . g 40 20 o B. Cyromazine (12.1 mg [All/l) and Bacillus thuringiensis (1.4 mg [Al]ll) s a “o man-Jean g 120} ab g 100 ‘ E, 80’ bc 3 so A be T g 40— --------- T B ..... 20 _. _...._...‘3 ....... c: C C o Figure 4.6. Average weight of Bacillus thuringiensis susceptible Colorado potato beetle larvae after treatment with difl'erent insecticides. Means followed by the same letter within a time period are not significantly difi‘erent using LSD at a = 0.05. Upper-case letters correspond to comparison of means at 96 h post treatment, and lower-case letters correspond to comparison of means at 168 h post treatment. 154 This may have resulted fiom the greater mortality efi‘ects of cyromazine at the higher rate masking the developed into synergism. Larval development. Most of the resistant larvae in the control treatment had developed to the fourth instar by 168 h post treatment (Figure 4.7. [A] and [B]). Incontrast,aflmwivinglarvaetreatedwithcyronnzinealoneorinthe combination treatments were delayed in the third instars. Some larvae treated with B. tlurringr'ensr‘s alone were able to complete development into fourth instar. These datasuggest thatcyromazinewasmore activethanB. dum’ngr’emisatthese concentrations. With the susceptible larvae, all control larvae had become fourth instars at the 168 observation period. Surviving larvae from cyromazine were mostly in the third instar. On the other hand, most of surviving larvae fi'om B. thuringiensis treatment became fourth instars (Figure 4.8. [A] and [B]). Some larvae treated with the mixture were able to complete development into fourth instar. These data suggest that inhibition of larval development in the mixture treatment was a result of cyromazine and B. tinm'ngiensis activities. When the concentration of cyromazine was increased, most of the larvae treated with cyromazine alone, the mixture, or the timed delay treatment was delayed in the third instar (Figure 4.8. [B])- A. Cyromazine (30.1 mg [All/l) and Bacillus thuringiensis (620.7 mg [All/l) //% /% WflMW ///m. W// B. Cyromazine (136.5 mg [All/I) and Bacillus thuringiensis (620.7 mg [All/l) 'l/ll/l/Il VIII/II” .m. m m w m . c at co_sn_=m_u .32. m m w m m o 38 559.5% .39.. resistant Colorado potato beetle larvae 168 h after treatment with different insecticides. Total height of bars indicates Figure 4.7 . Larval development of the Bacillus thuringiensis percent survival including all instars. 156 A. Cyromazine (5.6 mg [A|]/|) and Bacillus thuringiensis (1.4 mg [All/l) lard instar-4th instar lnstar distribution (%) 8 B. Cyromazine (12.1 mg [All/l) and Bacillus thuringiensis (1.4 mg [Al]/|) I3rd instar-4 th instar lnstar distribution (%) Figure 4.8. Larval development of the Bacillus thuringiensis susceptible Colorado potato beetle larvae 168 h after treatment with different insecticides. Total height of bars indicates percent survival including all instars. 157 2. General Discussion MixturesofdiflubenzuronwithB. tlnaingiensisyieldeddifi‘erentrenrlts. Mostofthetreamtestedacted independently. Inotherwords, mortality readfingfiomthemixhrreswasashighasthemoflalityresrfltingfiomthemme activeinaecticide. Synergistic interactionwasachievedonlyinthemsceptible strainwhenalowconcentrationofdiflubermu'onwasmixedwithB. Wm Thismaybeusefirlinfield simationswhereB. Wandsresistancehasnot developed. Ontheotherhand, antagonisticefl‘ectsoccm'redwhendiflubenwon wasadministered 24hbeforetreatmentwithB. Mngr‘ensis. Thepresence of diflubenzuron might change the physiology, perithropic membrane or pH of the Colorado potato beetle midgut. For example, if the diflubenzuron changed the pH, B. tlnm‘ngr'ensis may no longer be soluble in the midgut environment, preventing binding and pore formation. Salama et al. (1984) reported that a mixture of difiubenzuron with B. thuringiensis var. gallerie or var. entauocr'dus demonstrated an additive effect on S. littoralis. On the other hand, Novotny et al. (1990) found that a mixture of difiubenzuron and B. thuringiensis var. harsrakr resulted in lower mortality to Lwrantn‘a dim than where B. tlrrm'ngiensis was applied alone. These results suggestthatinteractionbetweenthesetwoinsecticidesisdependent onthevariety of B. tlmrr‘nglensis used, the species of insect, the concentration of each component used in the mixture, and degree of susceptibilities or resistance to the insecticides The model used to compute the expected mortality may have contributed some variation in these results. Dabi et al. (1988) calculated that the expected mortalityresultingfi‘omamixnnebyaddingthemonalityyieldedbyeach component when applied alone. Salama et al. (1984) followed the model developedbyFinney(l964);P=P1+P2(1-P1)whereP=theexpected 158 mortality, P1 =mortalityfi'omthefirstinsecticidewhenitisapplied alone, ansz =mortalityfromthesecond insecticidewhenitisappliedalone. Jaquesetal. (1989) used the model proposed by Bliss (1939) to compute the expected mortalitsz=P1+P2(l -P1)(1-r)whereP,P1, anszwerethesameasabove and r = the degree of correlation between susceptibilities to the two insecticides. Wheneva'thereisnoconelafionmmscepfibifity(r=0),theforunflasdevdoped byFinneyandBlissaresimilar. Ifacorrelationispresentandthecorrelationis positive(r>0), theefi‘ectsofthesecondinsecticidereducebecausesome susceptible individuals have received a lethal dose of the first insecticide. Therefore, in this situation, Bliss's formula is more appropriate because there is a correction factor considering the susceptibility of the population to the two insecticides. Synergisrn between cyromazine and B. tlwringiensr’s on Colorado potato beetle did not occur. The resistant strain responded to both dosages of cyromazine similarly. Mortalityresultingfromthemixtureandthetimedelayedtreatments were primarily from cyromazine. This probably indicates that cyromazine was more active or faster activity than B. thuringiensis under these treatment conditions. With the susceptible strain, the mixture demonstrated considerable antagonism, and antagonistic efl'ects were more evident when cyromazine was administered before treatment with B. tlnm'ngr‘ensis. This may also indicate that the presence of cyromazine changed the midgut environment that leads to prevent B. thm‘ngiensis intoxication No previous studies using this mixture have been reported, therefore I was unable to make further comparison. Difiubenzuron, cyromazine, and B. tlnm'ngiensis delayed growth and development of surviving larvae. The mixtures of diflubenzuron and B. tlnrringr‘ensr's acted independently in retarding the larval growth. The surviving lawaefiomflrembauresgainedweiglnatsimflarmtewiththesurvivinglarvae 159 fromthemoreactiveinsecticide. Therewasatendencythatthesurvivinglarvae fiomthefimedelayedtresflnemwadhigherweightthanthosefi‘omthembnures. Withtheresistarfistrainthesurvivinglarvaefiomthemixmreandtimed treatments. At a low concentration ofcyromazine, however, the mixture ofthese compounds yielded synergistic action in retarding the larval development on the susceptiblestrain Whentheconcentrationofcyromazinewasincreased, synergism didnotoccur. Yetthesurvivinglarvaefi’omthemixturetreatmenthadlessweight than those from the more active insecticide. Thisstudyindicatesthatconcentrafionsofinsecficidesusedinthemixmre and times of application play an important :role in determining the results of the combinations. Larvae of Colorado potato beetle had stopped feeding by 5 h after exposure (Hough-Goldstein et al. 1991) so that we should be able to administer a suficient amount of the second insecticide in order to result in mortality. Insect populations may have high degree of correlationin susceptibilities to the insecticides particularly if they have not been exposed to the insecticides. Therefore, ingestion of both insecticides may not always result in mortality equal with the total mortality from each insecticide when they are applied alone. Dabi et al. (1988) reported that combination of the conventional insecticides (carbaryl, endosulfan, rnalathion, and monochrotofos) and B. thuringiensis on H. armigera resulted in synergism. In contrast, this study demonstrated that most of the tested combinations between diflubenzuron or cyromazine with B. thuringiensis resulted in independent or antagonistic efl‘ect. The difi‘erences may be due to the mode of action. The conventional insecticides have faster activity than insect growth regulators. Therefore, if the tested larvae ingest sufiicient amount of the conventional insecticides they would die before the efi‘ect of B. thuringiensis occurs. In addition, B. tlnm'ngr’ensis may kill the 160 individualsthat survivefiomtheconventionalinsecticides. Inthisstudy,boththe insect growth regulators and B. tlum‘ngiensis have slow efi'ects. Therefore, at the sametimebothinsecticidesworkinthesameinsects. Asaresult,thepresenceof thesecondmsecficidehumefi‘ectifthemseashavereceivedalethddoseofthe first insecticide unless both insecticides interact and produce a more toxic compound. Themixhrreoflowdifiubennn‘onorcyromazinewithB. tlwn’ngiensr’smay onlybeusedinthefieldsimationswhereB. Wendsresistancehasnot developed and each insecticide must be applied at low rate. Application of the insecticides at low rate may benefit natural enemies, particularly endoparasitoids. This gives more windows for biological control agents to work Diflubezuron and cyromazine demonstrated as efl'ective insecticides for B. thrm’ngiensis resistant and susceptible Colorado potato beetle (see Chapter III). Therefore, they can be valuable additional alternatives to resistance management programs for Colorado potato beetle. LITERATURE CITED Abbot, W. S. 1925. Amethodforcomputingtheefl'ectivenessofaninsecticide. J. Econ Entomol. 18:265-267. Anderson, T. E., D. R Laing, andH. E. L. Maw. 1989. Eficacy ofmixtures ofmicrobial insecticides and permethrin against cabbage looper (Lepidoptera: Noctuidae) and the imported cabbageworm (Lepidoptera: Pieridae). Can. Ent. 121: 1511-1515. Ball, W. L., J. W. Sinclair, M. Crevier, and K Kay. 1954. Modification of parathion's toxicity for rats by pretreatment with chlorinated hydrocarbons insecticides. Can. J. Biochem. Physiol. 32: 440. Bliss, C. I. 1939. The toxicity of poisons applied jointly. Ann. Appl. Biol. 26: 585-615. Brake, J. and R. C. Axtell. 1991. Retention oflarvicidal activity afier feeding cyromazine (Larvadex) for the initial 20 weeks of life of single comb white leghorn layers. Poul. Sci. 70: 1873-1875. Burden, G. S., C. S. Lofgren, C. N. Smith. 1960. Development of chlordane and malathion resistance in the German cockroach. J. Econ. Entomol. 53: 1138. Byford, R. L., J. A Lockwood, S. M Smith, T. C. Sparks, and D. G. Luther. 1987. Insecticide mixtures as an approach to the management of pyrethroid-resistant horn flies (Diptera: Muscidae). J. Econ. Entomol. 80: 111-116. Comins, H. N. 1986. Tactics for reistance management using multiple pesticides. Agric. Ecos. Environ. 16: 129-148. Crofi, B. A 1990. Developing a philosophy and program of pesticide resistance management, pp. 277-296. In R T. Roush and B. E. Tabashnik, Pesticide resistance in arthropods. Chapman and Hall, New York. Curtis, C. F. 1985. Theoretical models ofthe use ofmixtures for the management of resistance. Bull. Entomol. Res: 75: 259-265. 161 162 Dabi,RK.,M.KPuri,H. C. Gupta,andS.K Shanna 1988. Synergisticresponseof low rate of Bacillus rlarringiensis with sub-lethal dose of insecticides against Heliotlris armigera Hubner. Indian J. Ent. 50(1): 28-31. Denholm, I. and M. W. Rowland. 1992. Tactics for managing pesticide resistance in arthropods: theory and practice. Annu. Rev. Entomol. 37: 91-112. Dtmkle, R. L. and B. S. Shasha 1989. Response of starch-encapsulated Bacillus tluaingiensis containing ultraviolet screens to sunlight. Environ Entomol. 18: 1035-1041. Finney, D. J. 1971. Probit analysis: a statistical trestrnent ofthe sigmoid response curve, second edition Cambridge University, London 318 p. Frawley, J. P., H. N. Fuyat, E. C. Hagan, J. R Blake, and O. G. Fitzhugh. 1957. Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharrnacol. Exp. Ther. 121: 96. Georghiou, G. P. 1983. Management of resistance in arthropods, pp. 769-792. In G. P. Georghiou and T. Saito, Pest resistance to pesticides. Plenum, New York. ! Gomez, K. A and A A Gomez. 1984. Statistical procedures for agricultural research. An Internatioanl Rice Research Institute book. John Wiley & Sons, New York. 680 p. Hardman, J. M. and S. 0. Gaul. 1990. Mixtures ofBacillus tlna'ingiensis and pyrethroids control winter moth (Lepidoptera: Geometridae) in orchads without causing outbreaks of mites. J. Econ. Entomol. 83(3): 920-936. Hellpap, V. C. and C. P. W. Zebitz. 1986. Combined application of neem-seed-kernel- extract with Bacillus thruingiensis—products for the control of Spodoptera fi'ugiperda and Aedes (0301'. J. Appl. Ent. 101: 515-524. Jaques, R P., D. R Laing, and H. E. L. Maw. 1989. Eficacy of mixtures of microbial insecticides and permethrin against the cabbage looper (Lepidoptera: Noctuidae) and the imported cabbageworm (Lepidoptera: Pieridae). Can. Ent. 121: 809-820. Liu, M. Y., J. S. Chen, and C. N. Sun. 1984. Synergisrn of pyrethroids by several compunds in larvae of the diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 77: 851-856. Mani, G. S. 1985. Evolution of resistance in the presence of two insecticides. Genetics 109: 761-783. Mani, G. S. 1989. Evolution of resistance with sequential application of insecticides in time and space. Proc. R Soc. Lond. B 238: 245-276. 163 Marshall, D. B., D. J. Pres, and B. D. McGarvey. 1988. Efi'ects ofbenzoylphenylures insect growth regulators on eggs and larvae ofthe spotted tentiform leafininer Plryllonorycler blamdella (Fabr.) (Lepidoptera: Gracillariidae). Can Ent. 120: 49-62. McGaughey, W. H and D. E. Johnson 1992. Indianmeal moth (Lepidoptera: Pyralidae) resistance to difi‘erent strains and mixtures ofBacillus tlmringiensis. J. Econ Entomol. 85(5): 1594-1600. McGaughey, W. H andM E. Whalon. 1992. MamginginsectresistancetoBacillus Wm toxim. Science 258: 1451-1455. McKenzie, C. L. and R L. Byford. 1993. Contirarous, alternating, and mixed insecticides afi‘ect development ofresistance in the horn fiy (Diptera: Muscidae). J. Econ Entomol.: 86(4): 1040-1048. MSTAT Development Team MSU. 1991. MSTAT-C: a microcomputer program for the design, management, and analysis of agronomic research experiment. MSU. Murphy, S. D., R L. Anderson and K. P. DuBois. 1959. Potentiation of the toxicity of malathion by triorthotolyl phospate. Proc. Soc. Exp. Biol. Med. 100: 483. Novotny, Julius, M. Svestka, and P. Kanecka 1990. The eficacy of mixtures of Bacillus thuringiensis Berl. and insect growth regulators. Biol. Abstr. 90(7): AB-306. Ref. No. 74052. Philips, J. R, J. B. Graves, and R G. Luttrell. 1989. Insecticide resistance management: relationship to integrated pest management. Pestic. Sci.: 459-464. Roush, R T. 1989. Designing resistance management program: How can you choose?. Pestic. Sci. 26: 423-441. Salama, H. S., M. S. Foda, F. N. Zaki, and S. Moawad. 1984. Potency of combinations of Bacillus tlna'ingiensis and chemical insecticides on Spodoptera littoralis (Lepidoptera: Noctuidae). J. Econ. Entomol. 77: 885-890. Seume, F. W. and R D. O'Brien. 1960. Metabolism of malathion by rat tissues\ prepration and its modification by EPN. J. Agr. Food Chem. 8: 36. Stewart, J. G., J. E. Lund, and L. S. Thompson. 1991. Factors afi‘ecting the eficacy of Bacillus thuringiensis var. san diego against larvae of the Colorado potato beetle. Poc. Ent. Soc. Ont. 122: 21-25. 164 Tabashnik, B. E. 1989. Managing resistance with multiple pesticide tactics: theory, evidence, and recommendations. J. Econ Entomol. 82(5): 1263-1269. Tabashnik, B. E. 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Applied and Environmental Microbiology 58(10): 3343-3346. Tames, P. M 1964.1soboles, a graphic representation ofsynergism in pesticides. Neth. J. Plant. Path. 70: 73-80. Whalon, M E., D. L. Miller, R M Hollingworth, E. J. Grafius, and J. R Miller. 1993. Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain reistant to Bacillus tlna’ingiemis. J. Econ Entomol. 86(2): 226-233. Wilde, G., A Kadoum, and T. Mime. 1984. Absence of synergism with insecticide combinations used on chinch bugs (Heteroptera: Lygaeidae). J. Econ. Entomol. 77 : 1297-1298. Wilkonson, C. F. 1976. Insecticide interactions, pp. 605-647. In C. F. Wilkonson, Insecticide biochemistry and physiology. Plenum, New York. Yamamoto, I. N. Kyomura, and Y Takahashi. 1993. Negatively correlated cross resistance: combinations of N-methylcarbamate with N-propylcarbamate or oxadiazolone for green rice leafhopper. Archives of Insect Biochemistry and Physiology 22: 277-288. CHAPTER V GENERAL CONCLUSIONS AND FUTURE RESEARCH POSSIBILITIES A. GENERAL CONCLUSIONS This research demonstratedthat continued laboratory selectionswithBacillus tlmringiensisinColondopotatobeetleincreasedflreredstancelevel. Afier34generations ofselection, afield derivedstrainwas>700timesmoreredstantthantheunselected strain. Thisinfomafionisimpoflaminunderatandingifandatwhatrateredstancemay develop in field populations. Even though laboratory selection does not always represent what will happen in the field (Roush and McKenzie 1987, Bloomcamp et al. 1987), information achieved from laboratory studies can be used to develop programs for delaying or reversing resistance development in the field (McGaughey and Whalon 1992). Since laboratory selections can be carried out before a particular insecticide becomes available for commercial use, successful resistance selection in the laboratory can act as a catalyst for responsible deployment and development of resistance management programs. Colorado potato beetle resistance development to B. tlna'ingiensis has pleiotropic efi‘ects (fitness costs) that required longer time to complete a generation, reduced fecundityandeggmasssize, andareduced rateoflarvalweightgain. Theresistantlarval stages required significantly longer to develop than the susceptible larvae, but there was no difl'a‘ence in the other stages. These fitness costs may lead to selection for susceptibility when selection is discontinued. Thus when the resistance level was still low (60 fold), the resistance level decreased in the absence of exposure to B. tlnaingiensis (Whalon et al. 1994). The four insect growth regulators (diflubenzuron, chlorfluazuron, cyromazine, and azadirachtin) and the nerve poison, imidacloprid, were efi'ective insecticides in morbidity assays conducted on Colorado potato beetles which were resistant to B. rluaingiensis. At sub-lethal dosages, the five insecticides also inhibited the growth and the development of 166 167 both resistant and susceptible larvae. Thus these insecticides may provide effective control when used in an alteration strategy with B. tlmringiensis b-endotoxin crleIA. At the end ofthe morbidity assay observation period (168 h), the resistant strain was7timesmorearscepfibletodiflubenzuronthanthesuscepfiblesh'ain Incontrast, the resistant strain showed some degree of tolerance to chlorfluazuron, cyromazine, and azadirachtin in the early observations with the resistance ratios of3.7, 10.4, and 7.0 respectively. In otherwords, thethreeinsecticidestook longertirnetokilltheresistant strainthanthesusceptiblestrain Attheendoftheobservationperiod(168 h),thesethree insecticides demonstrated similar toxicity to both strains. In other words, the three insecticides took longer time to kill the resistant strain than the susceptible strain. Imidacloprid was very toxic insecticide and both strains responded similarly. The results fi'om assays employing combinations of difiubenzuron or cyromazine with B. thuringiensis cryIIIA were variable depending on the insecticide, concentration, and the strain of Colorado potato beetle tested. Combinations or mixtures of diflubenzuron or cyromazine with B. tlna'ingiensrls, therefore, are not recommended since most of the tested combinations did not result in synergism. However, synergism was observed when a low concentration mixture of difiubenzuron (5.9 mg/l) and B. thuringiensis cryIIIA (1.4 myl) was applied to the susceptible strain. Some of diflubenzuron and B. thuringiensis cryIIIA mixtures studies demonstrated independently. In other words, the mortality resulting fi'om mixtures was as high as the mortality fi'om difiubenzuron alone. Mixtures of cyromazine and B. thrm'ngiensis demonstratedanindependentefi'ectontheresistantstrainandanantagonisticefl‘ectonthe susceptible strain. Pretreatment with diflubenzuron 24 h before applications of B. thuringiensis cryIIIA remhedinanMagonismintheresistantstrainandanindependenceinthe susceptible strain. On the other hand, pretreatment with cyromazine resulted in an independence in the resistant strain and an antagonism in the susceptible strain. B. FUTURE RESEARCH POSSIBILITIES TheavailabilityofourB. tlaaingiendslaboratoryresistantstrainwillmakeother researchposableindudingflrestabflityofresiflanceresistancemechadsms fitnesscosts, cross-resistanceto othertoxinsorchemicalinsecticides, and synergists. Whalonet al. (1994) reportedthattheresistancetoB. tlmrirrgiendsinColoradopotatobeetlewas unstable when the resistance was still limited (60 fold). Now, the resistant strain is over 700 fold more resistant than the susceptible strain. At this level, the stability of resistance isnotknown. Anotherstudyaboutfimesscostsisnecessarysincetlusstudydidnotcover some other possible difl'erences such as mating behavior, oviposition behavior, and survival with large number of samples. The resistance mechanisms to B. thrm’ngiensis in diamondback moth Plutella xylostella (1..) , Indianmeal moth Plodia interprmctella (Hubner), and rice moth Corcyra cephalonica (Stainton) have been researched. These studies indicated that difi‘erent species may have similar or difi’erent mechanisms. Thus our resistant strain provides valuable material to study the resistance mechanisms in Colorado potato beetle. The previous studies have demonstrated cross-resistance among toxins. In diamondback moth and Indianmeal moth, the pattern of cross resistance correlated with the binding sites (Van Ric et al. 1990, Ferre et al. 1991, Tabashnik1994).lftwo toxins have difi‘erent binding sites, cross-resistance is unlikely to happen. Ifthe resistance mechanisms are known, the pattern of cross-resistance may be predictable. Therefore, understanding the resistance mechanisms will open an opportunity to find blocking strategies using synergists. The five tested insecticides were potential insecticides for Colorado potato beetle. However, previous studies have indicated that some of these insecticides select resistance 168 169 inotherinsectpests. Thuslaboratory selectionswiththeseinsecticidesonColoradopotato beetle may be essential to provide information related with resistance development. Further studies on combinations of insect growth regulators and B. rim-inglensis areneeded. Thepreviousstudiesaswellasthisshrdyindicatedthattheresultsof combinations were variable. Thus it may be interesting to conduct in vitro studies to explainthesedifi‘erencesanddesignthecombinations. LITERATURE CITED Bloomcamp, C. L., R S. Patterson, and P. G. Koehler. 1987. Cyrimazine resistance in the house fly (Diptera: Muscidae). J. Econ. Entomol. 80: 352-357. Ferre, J. M D. Real, J. VanRie, S. Jansens, andM Pefcroen 1991. Resistancetothe Bacillustlnuingiensisbioinsecticdeinafield population ofPlutellaxylostellaisdue to a change in a midgut membrane receptor. Proc. Natl. Acad. Sci. USA 88: 5119- 5123. McGaughey, W. M. and M. E. Whalon. 1992. Managing insect resistance to Bacillus thuringiensis toxins. Science 258: 1451-1455. Roush, R T. and J. A McKenzie. 1987. Ecological genetics of insecticide and acaricide resistance. Annu. Rev. Entomol. 32: 361-380. Tabaslmik, B. E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39: 47-79. Van Ric, J., W. H. McGaughey, D. E. Johnson, B. D. Barnett, H. Van Mellaert. 1990. Mechanim of insec resistance to the microbial insecticide Bacillus thuringiensis. Science 247: 72-74. Whalon, M. E., U. Rahardja, and P. Verakalasa 1994. Selection and management of Bacillus thuringiensis resistant Colorado potato beetle, pp. 309-321. In G. W. Zehnder, M. L. Powelson, R K. Jnason, and K. V. Raman [eds], Anvances in potato pest biology and management. APS, Minnesota 170 APPENDIX COLORADO POTATO BEETLE VOUCHER SPECIMENS PLACED IN THE MICHIGAN STATE UNIVERSITY ENTOMOLOGY MUSEUM APPENDIX 1 Record of Deposition of Voucher Specimens* The specimens listed on the following sheet(s) have been deposited in the named museum(s) as samples of those species or other taxa which were used in this research. Voucher recognition labels bearing the Voucher No. have been attached or included in fluid-preserved specimens. Voucher No. : 1994 ' 4 Title of thesis or dissertation (or other research projects): The Bacillus thuringiensis Resistant Strain of Colorado Potato Beetle (Coleoptera: Chrysomelidae): A Search for Synergism Effects Using Novel Insecticides Museum(s) where deposited and abbreviations for table on following sheets: Entomology Museum, Michigan State University (MSU) Other Museums: Investigator's Name (8) (typed) Andi Trisyono Date 1-10-1995 *Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in Nbrth America. Bull. Entomol. Soc. Amer. 24:141-42. Deposit as follows: Original: Include as Appendix 1 in ribbon copy of thesis or dissertation. Copies: Included as Appendix 1 in copies of thesis or dissertation. Museum(s) files. Research project files. This form is available from and the Voucher No. is assigned by the Curator, Michigan State University Entomology Museum. 172 APPENDIX 1.1 VOucher Specimen Data 1 of 1 Pages Page wumw HOumuso mmmHIOHIH mama muwmum>fics mumum amw«:0fiz Una :H moamv pom mamanmam vmumwa w>onm mcu vm>wmumm oco>mwua and v I vmmd .oz uo£o=o> Avmn%uv Amvmemz m.uoumwfiumm>cH “hummmmowa ma muwwsm Hmcoaufivvm mmpv mm: m CH sz tumm .wcoaoo .nmq A>mmv mummcfidsmuwc mmumuocflumwa moo? pwufiwommv cam com: no kuomHHoo coxwu uwsuo no mmwowam m e r r m m e .m % mcwanmam How mumv Honda erOdEIIapWS S e 0.8 .h u u D. m .5 u.n e t t .d .d u a .8 M w.d.1 .U .A .A p. N .L "n "mo amnesz 173