MU"! _.u 4..4x.A _ -o'V s ‘3 "a. w. ‘ a w. .- ‘1‘ u. “121 .4», wna 5w “"5... ,-... vi {3 ‘53 ‘93! t t >—. -o«-.; a - “any”... lfigmu 5 ‘ ‘Ivn'l' .253 36': 5:3) p X! ;' 1 l zéhfiéfi $555 *’ ‘ up?! .. .. .m.v-‘-. ’7- p. vr .— . . '4'- y : ,4;- 5. 2.0 .i‘ ~,..4....¢ . «agony... “.4c... M. o » é-n «iv .,,-.. a n. a... :A' . .5. ' "1H1: ‘ Y.P3,_, Q‘Efl,’ ‘ :.~ 3'4"; 'J‘w" ' ‘ .m- n4 "5-”. u, now......o..~‘ mud. .. . v4 unmynm y ."‘\I A-\ :v‘ - ’51.", «num- .. .. “mm... - »— “mama-n. 3‘- . 1 ‘nzg'rJ . 4‘ l ,1 r v, a“ , ‘ . 93K? , ’ up. - “5‘ ' :, :4 ‘ . q . '1' 5! L.Y‘a‘.i§x§¢‘7‘-§§a ’ ,- , “as? i‘~ :r’llfl .1: ... .n x-- t‘: A. .3 I firs ! v -. :1 13."... ,3“ "- so“; u. var-v .— J ~ :‘ .t i u , :9 “"TQL' ’ a ‘19:: ‘9 ‘ :3" l! .w: \l .1 t. 1". 3’ , ”y . ', 0,. 1,: v 35314:? ,4; x , ~ t..';x. ~ : a fix.“ a y "s 53'; :a;.~.' I . f 3:). ‘ 5:3? r "53:2: r2 3‘94 q .I ~ . $532.;zgmu ' ’ ‘ 2:5 uvgh‘fi‘ ‘3'}. W~O§1n ‘I’IQ "A m IHHWI {HIIHHU11111111111sl‘HlIlHHlHll 1293 01388 This is to certify that the dissertation entitled GENETIC CHARACTERIZATION OF A CDLORADO POTATO BEETLE STRAIN RESISTANT TO THE COLEOPTERAN SPECIFIC -ENDOTOXIN OF Bacillus thuringiensis subspecies tenebrionis presented by Utami Rahardja has been accepted towards fulfillment of the requirements for _D9§:1:Qral_ degree in M Datew MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE ll RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or betore dete due. DATE DUE DATE DUE DATE DUE MSU le An Affirmative ActlorVEquel Oppommlty Inetltutlon Wane-n: GENETIC CHARACTERIZATION OF A COLORADO POTATO BEETLE STRAIN RESISTANT TO THE COLEOPTERAN SPECIFIC 8-ENDOTOXIN OF Bacillus thun'ngiensis subspecies tenebrionis by Utami Rahardja A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1995 ABSTRACT GENETIC CHARACTERIZATION OF A COLORADO POTATO BEETLE STRAIN RESISTANT TO THE COLEOPTERAN SPECIFIC 5-ENDOTOXIN OF Bacillus thuringiensis subspecies tenebrionis By Utami Rahardja The inheritance of Colorado potato beetles, Leptinotarsa decemlineata (Say), resistance to Bacillus thuringiensis CryIIIA B-endotoxin was investigated. Analysis of probit lines from the F1 reciprocal crosses indicated that inheritance of B. thuringiensis resistance was not influenced by the sex of the parents. The degree of dominance (D) of 0.77 and 0.76 for the (R X S) and (S X R) F1 generations, respectively, indicates that B. thuringiensis CryIIIA 8-endotoxin resistance is conferred by a partially dominant gene(s). 12 analysis of mortality responses of backcrossed offspring suggested that resistance might be caused by more than one genes. When the selection pressure was removed from colonies being selected for resistance, the resistance level of the selected colony decreased after five generations. Ifselective pressures were for another 12 generations, resistance levels did not revert to the resistance levels observed in susceptible colonies. The realized heritability of resistance to B. thuringiensis CryIIIA S-endotoxin Colorado potato beetles was estimated for over 29 generations. The estimates reached the highest value at generation four, and then decreased and reached values with very little fluctuation afier the tenth generation. The heritability estimates in the first 12 generations showed significant correlation with the increment of the resistance ratio. There was no significant correlation between standard deviation of LC 50 and the number of generations over 29 generations in both selected and unselected strains. The mean estimated heritability value after 29 generations is relatively low (h2 = 0.10). DNA markers for genes conferring resistance to B. thuringiensis CryIIIA endotoxin in Colorado potato beetles were developed by means of Polymerase Chain Reaction (PCR) technique. Primers R-14 (5’-ACAGGTGCTG-3’) and R-17 (S’- CCGTACGTAG-3’) gave 650 and 1800 basepairs fragments, respectively, which are specific markers for the resistant in our laboratory colony. The linkage between the genetic markers for the dominant gene(s) and the resistant phenotype was determined. The 650 basepairs marker was not linked to the gene conferring resistance to CryIIIA endotoxin. The 1,800 basepairs fragment identifies a genetic region that significantly linked to the gene. The recombination fractions of the 650 and 1,800 fragments were 0.46 and 0.20, respectively. Population of Colorado potato beetle from nine different locations at Michigan were used to validate the usefulness of the primers (R-14 and R—17) for field detection. The susceptibility of the field samples were determined and a total of 1000 field sampled beetles were tested. Nine field populations in Michigan tested with R—l4 showed the diagnostic marker representing 0.02% of the total populations tested. Two of the nine field populations tested with R-17 showed the diagnostic markers. The R-17 marker appears to be reliable in detecting resistant population. Ten different 10 to 15 oligomer primers used in twenty five different combinations of primers were used to detect polymorphism through Reverse-Transcript-Polymerase Chain Reaction (RT-PCR) analysis. Two sets of the primers exhibited polymorphism that were related to the resistance to B. thuringiensis CryIIIA endotoxin in Colorado potato beetle. "And I will restore to you the years that the locust hath eaten, the cankerwonn, and the caterpillar, and the palmerworrn, My great army which I sent among you. And ye shall eat in plenty, and be satisfied, and praise the name of the LORD your God, that hath dealt wondrously with you: and My people shall never be ashamed. And ye shall know that I am in the midst of Israel, and I am the LORD your God, and none else: and My people shall never be ashamed. And it shall come to pass afterward, that I will pour out my Spirit upon all flesh ...... " Joel 2: 25-28 To my mother and father, Sumijati and Rahardjamulja who introduced me to the beautiful and interesting creature, insects vi ACKNOWLEDGMENTS I am gratefiil to the member of my guidance committee: Dr. James Asher, Dr. Leah Bauer, Dr. Edward Grafius and Dr. Robert Hollingworth, for their advice, suggestions, interest and criticism. I would like to thank my major advisor Dr. Mark Whalon, for his patience, guidance and for trusting me in conducting my research. His prayers and support throughout my doctoral program helped me to persevere when I was discouraged. I also benefited from the Department of Entomology Research Assistantship, and USDA grants 1994 and 1995. Special thanks go to Debbie Norris for her assistance in maintaining the Colorado potato beetle strains and Pat Bills for his assistance in solving technical problems with the computer. I am thankfirl to Dr. Joel Wierenga, Dr. Kevin Shufran and Dr. Mike Bush for their critical suggestions and encouragement. I especially thank my parents Sumijati and Rahardjamulja for without their understanding and support I would not been able to stand the distance from my family. I am very grateful to my husband to-be, Ralph DiCosty, for the understanding and prayers. His constant love and encouragement helped me to keep going when I was in a mental block. My deepest honor goes to God for His faithfulness and provisions. He has given me the opportunity to study insects, the creature that I was interested in and has helped me to firlfill my goal. "Trust in the Lord with all your heart and lean not on your own understanding." Proverbs 3:5 vii TABLE OF CONTENTS LIST OF FIGURES . . . . . . . . . . . . . . . . . xi LIST OF TABLES . . . . . . . . . . . . . . . . . xii LIST OF APPENDICES . . . . . . . . . . . . . . . . xiii GENERAL 1N TRODUCTION . . . . . . . . . . . . . . 1 Colorado Potato Beetle: The Need for Better Pest Management. . 2 Bacillus thuringiensis : Promising Bioinsecticide?. . . . . . 3 Bacillus thuringiensis : Are We Losing The Promise?. . . . . 10 Thesis Objectives. . . . . . . . . . . . . . . . 15 References Cited . . . . . . . . . . . . . . . . 19 CHAPTER I : Inheritance of Resistance to Bacillus thuringiensis subspecies tenebrionis CryIIIA 8-endotoxin in Colorado Potato Beetle (Coleoptera: Chrysomelidae) 27 Introduction. . . . . . . . . . . . . . . . . . 28 Materials and Methods . . . . . . . . . . . . . . . 29 CryIIIA S-Endotoxin Sources . . . . . . . . . . . 29 Beetle Strains, Rearing and Selection . . . . . . . . . 3O Inheritance and Stability Studies . . . . . . . . . . . 30 Bioassays . . . . . . . . . . . . . . . . . 31 Data Analysis . . . . . . . . . . . . . . . . 32 Results . . . . . . . . . . . . . . . . . . . 33 Resistance Selection . . . . . . . . . . . . . . 33 Resistance Inheritance. . . . . . . . . . . . . . 33 Resistance Stability . . . . . . . . . . . . . . . 36 Discussion . . . . . . . . . . . . . . . . . 39 ReferencesCited................42 viii CHAPTER H : Heritability Estimation of Resistance to Bacillus thuringiensis subspecies tenebrionis CryIflA 8-endotoxin in Colorado Potato Beetle (ColeOptera: Chrysomelidae) Introduction Materials and Methods Insect Rearing and Selection . Bioassays . Data Analysis. Results . Discussion . References Cited . CHAPTER III : Random Amplified Polymorphic DNA Markers For Genes Conveying Resistance To Bacillus thuringiensis subspecies tenebrionis CryIIIA 5-endotoxin In Colorado Potato Beetle (Coleoptera: Chrysomelidae) Introduction . Materials and Methods . A. Detection of Colorado Potato Beetle Resistance to Bacillus thuringiensis subspecies tenebrionis: Linkage Analysis of RAPD Markers DNA Isolation . DNA Amplification Linkage Analysis. B. Detection of Colorado Potato Beetle Resistance to Bacillus thuringiensis subspecies tenebrionis: Validation and Field detection of RAPD Markers Field evaluation . DNA Extraction from Field Samples. Results . Development of DNA Markers . ix 46 48 43 48 49 51 61 69 71 74 74 75 75 77 78 78 78 Linkage Analysis Validation and Field detection of RAPD Markers . Discussion . References Cited . CHAPTER IV : Isolation And Identification Of mRNA Conferring Resistance To Bacillus thuringiensis subspecies tenebrionis CryIIIA 5-endotoxin In Colorado Potato Beetle (Coleoptera: Chrysomelidae) Introduction . Objectives Materials and Methods Insect Rearing and Selection . RNA Extraction . mRNA Differential Display System . Results . Discussion . References Cited . SUMMARY AND GENERAL CONCLUSION . 81 84 86 90 113 115 116 116 116 117 117 120 122 126 LIST OF FIGURES Figure 1.1. Probit values were plotted against concentration of a toxic agent (log concentration). Figure 11.1. The rate of change in Colorado potato beetle to selection by Bacillus thuringiensis S-endotoxins CryIIIA. Each dot represents the gain (deviation of response of the resistant colony from the unselected colony) from the selection in each generation. Figure 11.2. Standard deviation (l/slope) of LC 50 values of the Bacillus thuringiensis 5—endotoxin selected and unselected strains of Colorado potato beetle Figure II. 3. Coefficient of Inbreeding (F) values of Colorado Potato Beetle strains after 29 generations of selection . . . . Figure [1.4. Effects of heritability and percentage mortality on the average response per generation of Colorado potato beetle to selection with Bacillus thuringiensis 8-endotoxin . FigureIII.1. The genotype of the progeny of the backcross between F1 (from reciprocal cross) and susceptible strain . . . . . . . . Figure 111.2. PCR products of Colorado Potato Beetles amplified by R14. Each lane represents one individual. Lane 1 is lambda HindIII marker, lane 2-10 are resistant individuals, lane 11-19 are susceptible individuals Figure 111.3. PCR products of Colorado Potato Beetles amplified by R17. Each lane represents one individual. Lane 1-10 are resistant individuals, lane 11- 20 are susceptible individuals . Figure 111.4. Diagram of the expected and observed phenotype of F1 progenies from reciprocal cross between susceptible and resistant parents 2, 020 is the RAPD-PCR fragment diagnostic for susceptible phenotype and 1 ,800 rs the RAPD-PCR . . . . . Figure 111.5. Locations of nine populations of Colorado potato beetle tested with the RAPD primers for detecting Bacillus thuringiensis endotoxin resistance 39 54 55 59 64 76 79 80 83 85 Figure IV. 1. Differential display of total RNA isolated from Bacillus thuringiensis 8-endotoxin susceptible and resistant Colorado potato beetles. 3 and 5: resistant samples, 4 and 6 : susceptible samples. a, b, c, and d are unique fragments.....................119 xii LIST OF TABLES Table 1.1. Progression of resistance in Colorado potato beetle to the CryIIIA 8- endotoxin of Bacillus thuringiensis . . . . . . . . . Table 1.2. Response of susceptible and resistant Colorado potato beetle and the genetic crosses to the CryIIIA 8-endotoxin . . Table 1.3. Chi-square analysis of mortality statistics from F 1 X susceptible backcrosses of Colorado potato beetle (test monogenic model). Table 1.4. Resistance stability to CryIIIA 5-endotoxin in Colorado potato beetle. Table [1.1. Estimation of realized heritability (h2) of resistance to Bacillus thuringiensis 8—endotoxin in Colorado potato beetle. Table 11.2. Correlation regression analysis between standard deviation of LC 503 and generation number. . Table 11.3. Coefficient of Inbreeding (F ) values. Table 11.4. Approximate values of the heritability of various characters in various animal species. The estimates are rounded to the nearest 5 per cent; their standard errors range from about 2 per cent to about 10 per cent . Table 11.5. Estimates of realized heritability (hz) of insecticide resistance. Table 111.1. History of Colorado potato beetle field populations tested with the RAPD primer R-17 for to Bacillus thuringiensis CryIIIA S-endotoxin resistance . . . . Table 111.2. Chi-square analysis of RAPD-PCR products of back cross progeny. Tabel IV .1. 5'-primers and 3'-primers used for identification and isolation of mRN A conferring resistance to Bacillus thuringiensis S-endotoxin in Colorado potato beetle . xiii 34 35 37 38 53 56 58 63 67 81 84 121 LIST OF APPENDICES APPENDIX 111.1. Primers screened for developing DNA marker for gene/s responsible for resistance to Bacillus thuringiensis 8-endotoxin in Colorado potato beetle . 93 APPENDIX 111.2. PCR-RAPD products of individual sample of Colorado potato beetle amplified by primer R-14 . . . . . . . . . 102 APPENDIX 111.3. PCR-RAPD products of individual sample of Colorado 107 potato beetle amplified by primer R—17 . xiv GENERAL INTRODUCTION GENERAL INTRODUCTION Colorado Potato Beetle: The Need for Better Pest Management The Colorado potato beetle, Leptinotarsa decemlineata (Say), was first described by Thomas Say, a young entomologist at the Philadelphia Museum of Science, in 1823. The beetle was found feeding on a weed, Solanum rostratum, growing in arid areas of the Colorado River basin in the western United States (Berenbaum 1983). It is suggested that the Colorado potato beetle and its host plant originated in Mexico. The beetle invaded cultivated potatoes (Solanum tuberosum L) when settlers moved west and planted potatoes in the foothill of the Rocky Mountains (Casagrande 1985). Approximately 30 years after first being identified, the Colorado potato beetle was considered as a pest of potatoes in the western United States. It was particularly a problem in western part of Missouri. The Colorado potato beetle is now considered the most destructive pest of potato in many parts of the United States. A variety of approaches have been used to control the Colorado potato beetle. Chemical control was initiated when the first synthetic stomach poison, Paris Green, was invented in the 1860's. This copper acetoarsenate became the leading insecticide for many leaf-feeding pests, including the beetle, until the discovery of lead arsenate. DDT was introduced during World War 11, and was adopted as the universal insecticide. This insecticide was very effective for controlling potato beetles and was widely used by potato growers across the country (Gautheir et al. 1981). Following the failure of DDT to control this pest, Dieldrin showed a significant degree of insect control during the 1950's (Hofinaster 1965). During the 1960's, azinphosmetyl and carbaryl were widely used with excellent results for a number of years (Gautheir et al. 1981). Currently, the controls of the Colorado potato beetle rely heavily on multiple applications of insecticides, cultural control, bacterial insecticides and even flaming with propane devices. Insects, like any other organism, evolve through selection as a result of their changing environment. A population becomes adapted through natural selection to its environment. Resistance may be an unavoidable consequence of insecticide use. Insect pests have evolved mechanisms that enable them to circumvent toxic agents. The gene pool of most of the insect pests may contain genes that enable the insect pests to detoxify or resist the toxic agents. Heavy reliance on insecticides for control has resulted in insecticide resistance, environmental contamination, and suppression of natural enemies. Many insect pests have adapted to chemical controls by developing metabolic and behavioral defense mechanisms against insecticides. Insect resistance to insecticides was reported over 70 years ago, but the greatest increase and most important development in resistance has occurred during the last 40 years since the discovery of synthetic insecticides (reviewed by Georghiou 1986). The Colorado potato beetle was one of the first pests to exhibit resistance to DDT (Gautheir et al. 1981). Insecticides often failed to control Colorado potato beetles within one or two years of introduction because of cross-resistance (F orgash 1981, Grafius 1986). To date, in many parts of the United States, the Colorado potato beetle has developed resistance to all classes of conventional insecticides including organochlorines, organophosphates, carbamates, and pyrethroids (Gautheir et al. 1981, Georghiou 1986, Grafius 1986, Ioannidis et al. 1991, 1992). Bacillus thuringiensis 8-endotoxin: A Promising Bioinsecticide? Microbial insecticides, such as B. thuringiensis 8-endotoxins, have been considered the best alternative for controlling insect pest populations. The 8-endotoxins have limited specificity (Haider et al. 1986) and have no known detrimental effects on humans or wild or domestic animals and they tend to be short-lived in the environment (Stone et al. 1989, McGaughey & Beeman 1988). Therefore, these bioinsecticides are attractive alternatives to chemical insecticides which are perceived to be more environmentally disruptive and hazardous to applicators and consumers. In addition, the limited range of activity of the 8-endotoxins toward insects means that often they will kill the target pest species but have no effects on predatory species. This feature makes them highly desirable for use as components in integrated pest management (1PM) programs. A group of bacteria possesses insecticidal activities. This includes species with the ability to infect healthy insects and rapidly multiply in their hosts following the infection. The major species of bacteria with those abilities are spore forming bacilli. Among the spore forming bacilli, B. thuringiensis and B. sphaerius are two major groups that produce protoxin. Bacillus lhuringiensis is an aerobic spore forming Gram positive soil bacterium. The crystal 5-endotoxin is produced as large protein molecules during sporulation. These proteins are deposited as parasporal inclusion. The crystalline inclusion, also known as 8- endotoxin, is a gut poison for larva of more than 100 insect species belonging to the order of Lepidoptera, Diptera and Coleoptera (HOfte & Whiteley 1989). Many toxic proteins with varying similarity, or degrees of homology in amino acid sequence, have been isolated from worldwide collections of B. thuringiensis strains (Hofte & Whiteley 1989, Ishii & Ohba 1993, Kaelin 1994). These proteins are encoded by the curl, cryII, cryIII, cryIV and cyt genes. All of these cry 5—endotoxin genes are thought to have a common evolutionary origin because of the highly conserved amino acid composition. The C-temiinal part of CryI, CryIVA and CryIVB B-endotoxins that does not contain toxic segments shows the most highly conserved domain of the protein. The N-terminal half of the protein is essential for toxicity. In the amino acid sequence corresponding to the toxic domain, five highly conserved sequence fragments can be distinguished in all but CryIIA, CryIIB and CryIVD S—endotoxins. These fragments are separated by highly variable sequences of various lengths for different crystal proteins. With the exception, of CryII and CryIVB. 8-endotoxins, a stretch of hydrophobic amino acids at a comparable position within the 120 N-terminal amino acids of all 8-endotoxins is remarkably conserved. This feature indicates a significant function. It has been proposed that the conserved hydrophobic region plays a role in an interaction. between the 8- endotoxins and the membrane of midgut epithelial cells (Schnepfet al. 1990). A protoxin comprising 130 - 140 kDa polypeptides is the predominant parasporal component of most B. thuringicnsis subspecies. Digestion process yields smaller proteinase resistant 8-endotoxins which are derived from the N-terminus of the protoxin (Hofie et al. 1986, Choma & Kaplan 1990). ' CryI (lepidopteran 8-endotoxins) in general, CryII (lepidopteran and dipteran 8-endotoxins) and CryIV and cytA (dipteran 8- endotoxins) protoxins are solubilized and digested into smaller proteins ranging from 30- 80 kDa (Lilley et al. 1980, Huber & Luthy 1981, Hofie & Whiteley 1989). In contrast, CryII, CryIII and CryIVD 8-endotoxins do not undergo protease mediated C-terminal degradation. Three subspecies of B. thuringiensis, known to produce coleopteran-specific 8-cndotoxin, release crystaline protein that does not need to be further cleaved to activate the 5—endotoxin (Caroll et al. 1989, Herrnstadt et al. 1986). These proteins appear to be naturally truncated at the C-terrninus of the fi-endotoxins. Removal of a small C-terminal fragment of 11 amino acids causes a loss of the activity of CryIIA S-endotoxins (Widner & Whiteley 1989). The C-terminus of CryIIIA and CryIVD 5-endotoxins contains a conserved domain that is required for toxicity in several other CryI-activated &endotoxins (Caroll et al. 1989, Widner & Whiteley 1989). Degradation of these C-tenninal domains will likely result in the loss of insecticidal activity of CryIIIA and CryIVD 5-endotoxins. However, these naturally truncated proteins may undergo protease cleavage in the insect midgut. A smaller protein of 55 kDa fi'om ClyHIA 8-endotoxin was produced from a 67 kDa 5-endotoxin exposed to the digestive juice of T en'ebrionis molitor (Slaney et al. 1992). This 55 kDa CryIIIA was fully active. Removal of up to 159 amino acid residues fi'om the N-terrninus of CryIIIA S-endotoxins did not decrease its insecticidal activity (Caroll et al. 1989, McPherson et al. 1988). The role of protease in Coleoptera is still in question (Li et al. 1989, Carroll et al. 1989). B. thuringiensis subspecies tenebrionis produces the crystal that contains a major polypeptide of 67 kDa and minor polypetides of 73, 72, 55 and 46 kDa (Caroll et al. 1989). During sporulation, the minor polypeptide of 73 kDa decreases, while the concentration of 67 kDa crystal protein increases. The finding that a smaller trypsin treated S-endotoxin, 55 kDa, is as toxic as the native CryIIIA 8-endotoxin raised the question of the significance of the endogenous protease activity after the crystal release (Carroll et a1 1989). There is extensive variation in the size and structure of the inclusion proteins, the intermediate protoxins, and the active 8-endotoxins. These wide variations are presumed to relate to the insect specificity of the Svendotoxins. Generally, however, the activated 8- endotoxin is comprised of three structural regions: an N-terminal region, the toxic domain consisting of several conserved hydrophobic regions and a conserved C-terminal region. A variable region between the toxic domain and the conserved C-terrninal region contains most of the residue differences (Aronson et al. 1986, Hofie & Whiteley 1989, Choma & Kaplan 1990). The most recent publication of the molecular structure of a 5-endotoxin was on CryIIIA (Li et al. 1991). The 67 kDa CryIIIA S-endotoxin that aligns with the N- terrninal half of 130-140 kDa Cry 8-endotoxins is composed of three distinct domains. Domain 1 is a bundle of seven amphipathic helices. The as helix is the center of the bundle surrounded by the outer six helices. These outer six helices are comprised of hydrophobic residues on the side facing the 015.helix The amphipathic helix is a protein domain commonly found in transmembrane pores. Therefore, this domain may facilitate the membrane insertion and pore formation. Domain II is composed of three B-sheets laid side by side. The non-conserved region of the CryIII 8-endotoxin is mainly part of the domain 11. This domain probably deterrninis the specificity of the receptor binding domain. Residues fi'om segments within domain 11, responding to sheets which are responsible for the specificity of the 8-endotoxins, are found also in CryI and CryII 8- endotoxins. Domain 111 is responsible for strands forming two sheets of the B—sandwich. A high degree of conservation of the C-terminal region of most Cry 5—endotoxins is found in this domain. The buried strands of an inner anti parallel sheet in domain III seem to be the core of C-terminal that are resistant to proteolysis. Beyond structural stability and integrity of the 8-endotoxin, the function of domain III remains unclear. The conserved block 4 in the CryIa 8—endotoxin was observed to be involved in'ion conductance indicating a functional role of the domain III (Chen et al. 1993). All insecticidal proteins of B. Ihuringiensr’s 8-endotoxins are toxic only after ingestion by the susceptible insect. The sign of intoxication following ingestion of 8- endotoxin is cessation of feeding as a result of paralysis of the gut and mouth parts. In comparison to other microbial 5—endotoxins, this response is extremely rapid, especially when we consider that the crystals have to be dissolved and activated in the gut juice, and the active, moiety has to reach the site of action. Subsequent to the paralysis of the gut and mouth, ingested 5-endotoxins are solubilized and, in most cases, proteolyticaly digested to the active form. The activation takes place in the gut. The combined action of alkalinity of gut pH and protease activity are responsible for the dissolution of protoxin. Histological studies have demonstrated that Cry 5-endotoxins disrupt the midgut epithelium of susceptible larvae (Luthy & Ebersold 1981, Endo & Nishiitsutsuji-Uwo 1980, Percy & Fast 1983, Bauer & Pankratz 1992). Typical histophatological alteration results in swelling of the cytoplasma, the mitochondria, the Golgi complexes and the endoplasmic reticulum with subsequent loss of their characteristic structure. The content of mitochondria is dissolved, leaving only a membranous fragment. The nuclei also swells. Vacuolization occurres between the outer and the inner membrane of the nuclei (Luthy & Ebersold 1981). Subsequently, connections between cell membranes are disrupted resulting in the separation of cells from each other. The cells burst and release their cytoplasmic content into the lumen (Luthy &'Ebersold 1981, Endo & Nishiitsutsuji-Uwo 1980, Bauer & Pankratz 1992). The 8-endotoxin apparently binds to specific receptors localized on the brush border midgut epithelium and induces pore formation. Binding seems to.be anessential step in'toxicity. Studies demonstrated a close correlation between binding afiinity and toxicity (Endo & Nishiitsutsuji-Uwo 1980, Hofinann & Lilthy 1986, Hofinann et al. 1988a, 1988b, Van Ric et al. 1989, 1990a, 1990b, Ferré et al. 1991, Hofie and Whiteley 1989, Li et al. 1990, Knight et al. 1994). However, more recent studies have demonstrated a degree of nonspecific binding in certain insect species (Garczynski et al. 1991). It appears, in some species, that high affinity binding may occur without killing the insect. Thus binding is necessary, but not sufficient for toxicity. Binding of CryI 8-endotoxins to the midgut epithelium of European corn borers, Ostrinia nubilalis, larva was characterized. Two independent binding receptors were observed to be found in the brush border gut epithelium (Denolf et al. 1993). CryIA(b) and CryIA(c) 8-endotoxins were recognized by the same receptor. A competitive binding study showed that CryIB b-endotoxin replaced neither CryIA(b) nor CryIA(c) 8- endotoxins. The study indicated a different receptor for the crystal protein exists in the midgut of European corn borers. Different levels of toxicity of the three proteins were observed in bioassay tests. The different toxicities between CryIA(b) and CryIA(c) 5- endotoxins appeared because of affinity differences of those proteins to the same receptor. As demonstrated here, the existence of receptors with different specificities could be a very useful feature for delaying the development of resistance to S-endotoxins. The binding study of CryIIIA b-endotoxin with 125I was done with Colorado potato beetles and Southern corn rootworrns, Diabrotica undecimpunctata lrowardi Barber (Slaney et al. 1992). CryIIIA 5-endotoxin showed higher affinity on’the midgut brush border of Colorado potato beetles than on Southern corn rootworrns. It appears that the susceptibility to 5-endotoxin is associated with the affinity of solubilized protein to the receptors residing in the midgut brush border. ' The binding of the 8-endotoxin to the receptors causes a conformational change in domain I (which is an amphipathic helix) so that the hydrophobic residues are in close proximity with the membrane and initiate pore formation. The small pores in the membrane cause an increase in potassium ion permeability of the midgut epithelium. Increased permeability to K+ would have an effect of decreasing membrane potential and increasing the intercellular pH. The leak of K+ subsequently is followed'by the leakage of water into midgut cells causing the cells to swell and lyse. This mode of activity has been demonstrated in Lepidoptera (Sacchi et al. 1986, Knowles and Ellar 1987, Hofte and Whiteley 1939, English et al. 1991). The disruption of midgut structure and function leads to ion and pH imbalances in the hemolymph, total body paralysis and eventually death. Investigation on .CryI 8-endotoxin showed that the 8-.endotoxins alter the permeability of lepidopteran midgut apical membranes for monovalent cations (Sacchi et al. 1986, Crawford & Harvey 1988, Wolfersberger 1989, Carroll & Ellar 1993). Changes in the membrane permeability of Manduca secta midgut brush-border membrane vesicles after addition of Cr-yI 5-endotoxin was also studied. The permeability of the membrane was significantly affected by CryIA B-endotoxin. The change was relatively non-selective among solutes. Cations, anions and neutral solutes all traverse the membrane to an increased extent in the presence of CryIA(c) 8-endotoxin. No effect on brush-border membrane vesicles was observed when CryIB S-endotoxin was used in a similar experiment (Carroll & Ellar 1993). 10 The biological action, of CryIIIA 5-endotoxin has also been studied (Slatin et al. 1990, Li et al. 1991). The ability of CryIIIA Seendotoxin to induce ion leakage in planar lipidbilayers has been demonstrated (Slatin et al. 1990). The leakage of id and H20 is probably caused by induced 8-endotoxin pore formation in the plasma membranes. Domain 1 of the CryIIIA 8-endotoxin has been proposed as the portion of the 8-endotoxin molecule that penetrates the membrane. The membrane might internalize the hydrophobic surfaces of the protein bringing the protein in closer to the membrane. The close contact of the hydrophobic surfaces of the protein with the cell membrane may cause conformational changes in the 8-endotoxin (Li et al. 1991). The confOrrnational changes might be responsible for the formation of the pores or channels in planar lipid bilayers. Consequently, ion flow occurs with eventual vesicle or cell lysis. The pore formation process is thought to be initiated by the interaction of the CryiiiA‘o-endotoxin with putative receptors, phospholipids on the surface of the membrane (English et al. 1990, Li et al. 1991, Gazit & Shai 1993). Bacillus thuringicnsis 5-endotoxin: Are We Losing The Promise? The field applications of B. lhuringiensis 8-endotoxin in controlling agriculturally important insect pests is just beginning to. lead to the selection of resistance insects (McGaughey 1985, Tabashnik et al. 1990, McGaughey & Whalon 1992, Whalon et a1. 1993). Yet development of field resistance toward B. thuringiensis B-endotoxin has been slow. Several factors have probably contributed to this delayed evolution of resistance. These may include a limited selection pressure on pest populations due to marginal field eficacy. Intensity of selection pressurein the field has not been very strong because of: 1) limited use of the microbial insecticide, 2) very short residual activity, 3) new plant growth '11 that does not have residues of B. rhuringiensis 8—endotoxin, and 4) the reservoir of susceptible genotypes dispersing into sparse fields that would 'flood out' resistant genotypes (Stone et al. 1989, McGaughey & Beeman 1988). Insects do have, however,“ the capacity to develop resistance to B. thuringiensis 8- endotoxin. A few important insect pests fi'om orders Diptera, Lepidoptera and Coleoptera were reported to. develop resistance. Harvey and Howell (1965) reported a selection with B. tlntringiensis 8-endotoxin on a laboratory colony of house flies, Musca domestica L. After continuous laboratory selection for 50 generations, the colony became resistant. The resistance was not stable, however, and it appeared to decline during 20 subsequent generations without selection. Resistance to B. thuringiensis 8«endotoxin in Drosophila melanogaster was obtained in the laboratory (Carlberg & Lindstorm 1987). Laboratory colonies of fruit flies were used to start the selection. At least fivefold resistance in 70 generations to the 5- endotoxin of the B. rhuringiensis serotype H-l was observed in the fi'uit fly colonies. Another dipteran, Aedes aegypti, was reported to develop resistance to 8-endotoxin produced by B. thun'ngiensis subspecies israelensis. The mosquito strains were raised in the laboratory for five generations before being used to start the selection. After fourteen generations of selection, a small but statistically significant increase in resistance was observed (Goldman et al. 1986). An almond moth (Cadra vautella (Walker)) colony was started from an infested bin insouthem Texas and reared in the laboratory for at least 130 generations before being used for selection (McGaughey & Beeman 1988).. Selection was done by exposing the neonates with B. thuringiensis subspecies kurstaki 5-endotoxin incorporated in their diet. Resistance increased seven-fold after- 21 generations of intensive selection. A sunflower mOth (Homoseosoma electellum (I-Iulst)) colony, maintained on a wheat germ-based diet for at least three years, was used for selection with B. thuringiensis subspecies kurstaki 8- endotoxin (Brewer 1991). A newly hatched larva was placed on a diet topically treated 12 with suspension of the 8-endotoxin. A significantly higher tolerance to the 5-endotoxin was first observed in generation eight. A major lepidopterous pest on cotton, Spodoptera littoralis (Boisduval), was subjected to a laboratory selection (Salama & Matter 1991). The cotton leafworrn was raised in the laboratory for several years before being selected with 8-endotoxin from B. thw'ingiensis subspecies kurstaki I-lD-l (Dipel). After eight generations of selection, the colony was significantly more resistant than the unselected one. Another Lepidoptera, Charistoneurafumiferana (L.), was observed to develop resistance (< ten fold resistance) after laboratory selection for eight generations (see Tabashnik 1994). Laboratory selection of a stored grain pest resulted in resistance to B. thw'ingiensis subspecies kurstaki 8-endotoxin(McGaughey 1985). Larvae or pupae of Plodia interpunctella were collected from bins of B. thuringiensis 5-endotoxin-treated grain and had been maintained for 16-26 generations before the selection studies began. The colonies were subcultured for several successive generations on a B. thuringiensis 8- endotoxin-treated diet. The diet contained the 8-endotoxin at concentrations that caused 70-90% larval mortality. Within three generations, the LC 50 of one of the colony was 29 times higher than the unselected colony. The level of resistance increased to more than 100 fold in sixteen generations. When selection pressure was continued for another 20 generations, the level of resistance increased to greater than 250 fold relative to the unselected parent colony (McGaughey & Beeman 1988). Laboratory colonies of tobacco budworrns, Helicoverpa virescens (F .), in which wild males were introduced annually, were used, for selection. A genetically engineered Pseudomonasfluorescens expressing CryI fi-endotoxin was used to select the neonates. The colony was reared for three generations prior to subculturing the neonates on a 5- endotoxin-treated diet. Significant tolerance was first observed at generation three. By generation seven, the level of resistance increased to 24 fold (Stone et al. 1989). Different populations of tobacco budworrn were selected against B. thuringiensis 8-endotoxin 13 (Gould et al. 1992). A fourth generation colony was used to start the selection with a CryIA(c) treated diet. After ten generations, the level of resistance was about ten fold. The resistance ratio between selected and unselected colonies increased to 50 fold after seventeen generations of selection. Genetic analysis of this strain indicated that the resistance was inherited as a partially recessive trait. Further study showed that this particular colony exhibited cross-resistance to B. thuringiensis 8-endotoxins (CryIA(b), CryIIA). . There is very little information on B. thuringiensis 8-endotoxin resistance in field populations. The only documented species showing resistance to B. thuringiensis 6- endotoxin in a field population was the diamondback moth, Plutella xylostella (L.) (T abashnik et al. 1990, 1991 1992). Tabashnik et al. (1990) reported results from a survey of responses of diamondback moths collected fi'om commercial fields of watercress, cabbage, and broccoli in Hawaii. In a laboratory bioassay, diamondback moths collected from watercress fields intensively treated with B. thuringieusis subspecies hirstaki firendotoxin were observed to be 25 times more resistant than the susceptible laboratory colonies. This field population was further selected in the laboratory. After nine generations of selection, the resistance level was significantly greater (up to 36 fold resistance) than the susceptible laboratory colonies (Tabashnik et al. 1991). Rapid development of resistance may be a result of heavy treatment with B. thuringiensis subspecies kurstaki 5-endotoxin on field populations of diamondback moths. Various crosses between selected and unselected colonies were conducted to determine the mode of inheritance of the resistance (Tabashnik et al. 1992). Responses of the progeny fi'om reciprocal crosses were not different. The results indicate an autosomal inheritance. The LCsos of the F1 offsprings were not significantly greater than LC 50 of the unselected colony. This result showed that the resistance in diamondback moths is inherited as a recessive trait. Further field survey of resistance in diamondback moths was done in six 14 states of the United States and in Indonesia (Shelton et al. 1993). This survey concluded that intensive use of B. thuringr‘ensis 8—endotoxin increases the resistance problems. Several theories on possible physiological mechanisms of B. thuringiensis 8- endotoxins resistance are suggested. Most of the studies have been done on lepidopteran species (Endo & Nishiitsutsuji-Uwo 1980, Sacchi et al. 1986, Knowles & Ellar 1987, Hofie & Whiteley 1989). Altered binding affinity is one of the hypothesized mechanisms of insect resistance to B. thuringiensis 8-endotoxin. A study on P. interpunctella showed evidence that resistance to B. thuringiensis CryI 8-endotoxin is due to a change in binding afini'ty of receptors or alteration of the binding sites (Van Rie et al. 1990). Resistance to CryIA(b) B-endotoxin is associated with the reduction of the affinity of the receptors for 8-endotoxin. Furthermore, resistant P. intetpunctella gained sensitivity to CryIC 6- endotoxin. Changes in afiinity of the receptors to 8-endotoxins have also been observed in the resistance of H. virescens (Gould et al. 1992). However, a different population of H. vifescensWacIntosh et al. 1991) showed the contrary result. No significant difference in CryIA(b)- 8-endotoxin binding affinity between selected and unselected strains was found. Furthermore, cross-resistance with CryIA(c) 3—endotoxin was observed in these strains suggesting that a different mechanism of resistance may be involved. A study was conducted to determine how changes in protease inhibitors would affect the proteolytic processing of the 5—endotoxin. Very low effects of the inhibitors on response of P. xylostella (both resistant and susceptible strains) to B. thuringiensis 8- endotoxin suggested that altered proteolytic processing was not a major mechanism of resistance (T abashnik et al. 1992). Possible behavioral avoidance of formulated and/or purified B. thuringiensis 8-endotoxin has been reported in P. xylostella (Gould & Anderson 1991, Scwartz et al. 1991). The laboratory choice tests suggested that there is no evidence for behavioral resistance. 15 The complex mode of action of CryIIIA 8-endotoxin suggests the possible mechanisms of resistance in Colorado potato beetles, including a decrease in 8-endotoxin solubilization (assuming that protease is critical for CryIII 8-endotoxin solubilization), conformation changes of solubilized protein in the insect gut, membrane turnover, and recovery. Thesis Objectives The developments of insect-resistant plants would provide more effective, less costly,.and more environmentally attractive pest control. This process has been successfully accomplished through the Agroliacterium tumefaciens binary vector system. The field expressions of the S-endotoxin genes in transgenic plants have also been demonstrated to be effective in suppressing insect pest populations (Adang et al. 1987, Fischhoff et al. 1987, Vaeck et al. 1987, Delannay et al. 1989). This progress raised growing concern about the durability of B. rhuringiensis 8-endotoxin (McGaughey & Whalon 1992). The ability of several major insect pests to tolerate B. thuringiensis 8- endotoxin has been demonstrated in laboratories. Therefore, insect resistance will be a critically important consideration as B. thuringiensis 6—endotoxin applications (transgenic plant releases or conventional 8-endotoxin sprays) increase. Without a cautious and wise resistance management program, the loss of the effectiveness of B. thuringiensis 8- endotoxin will occur in only a few years. Resistance management is any attempt to prevent or delay adaptation of insect pests to toxic agents. The strategy in managing pest resistance is to maintain resistant alleles at very low frequencies. This could be achieved by preserving a sufficient population of susceptible individuals or alleles, and by reducing the rate and probability of resistance development. Tactics to implement these strategies might include: 1) variation 16 in dose or rate of insecticide applications, 2) reducing the frequency of applications, 3) sinmltaneous use of two or more chemical with different modes of action or target site, 4) rotation, alteration or sequential applications of insecticides with different modes of action or target sites. Eventually, resistance in Colorado potato beetle populations will undoubtedly develop in the field, when conventional B. thuringiensis fi—endotoxin and transgenic B. tharingiensis 8-endotoxin plants are used widely on potato, tomato, egg plants and other hosts. Therefore, understanding of B. thuringiensis 8-endotoxin resistance is critically important to developing strategies for managing resistance. The goal of this action should be toreduce the selection pressure of this bioinsecticide and to prolong the utility of this environmentally safe bioinsecticide (Croft 1990). The ideal strategy is to develop resistance management as early as possible, or even before the resistance has evolved in field population of the targeted insect. .Altemative controls are urgently needed to extend the efi‘ectiveness of bioinsecticides. Applications of these insecticides to selectively manage resistance development is an obvious necessity. ' Resistance monitoring. is one of the key features of any resistance management programs. An appropriate monitoring system should provide for early detection of resistance as it begins in the field. This initial warning will allow resistance managers the time to take necessary steps to abate resistance evolution. In 1987, Whalon et al. (1993) initiated field selection of Colorado potato beetle with CryIIIA fi-endotoxin of Bacillus thuringiensis, and subsequently selected them in the laboratory resulting in over 200 fold resistance. Understanding the mode of inheritance of this resistance phenotype in a Colorado potato beetle population is critical to in-depth knowledge of the resistance. The management of this resistance will depend on whether the resistance is inherited in a discrete manner or as a continuously quantitative trait. When the resistance is a mono- or oligogenic. trait, the number of alleles at the resistance 17 determining loci and the dominance relationships among these alleles should be determined (Curtis et al. 1978).. The identification of interactions between genes conferring resistance as well as the modifying loci are an impOrtant step in understanding the evolution of insecticide resistance (Uyenoyama 1986). It is particularly critical to know the mean levels of resistance, the phenotypic variances and the additive or non-additive genetic variance (Via 1986). This study was done to characterize the B. thuringr'ensis 8- endotoxin resistance in Colorado potato beetles, and to apply the knowledge gained to developing a strategy of early prevention of resistance evolution in field populations. Biotechnology has provided techniques for qualitative and quantitative detection of insecticide resistance. These techniques allow researchers to determine polymorphism in insecticide resistance within a species, and also among different species in different pOpulations. Nearly five years ago, a new genetic assay was developed by two different laboratories (William et al. 1990, Welsh & McClelland 1990). This technique, Random Amplified Polymorphic DNA (RAPD), detects nucleotide sequence polymorphism in the genome of a wide variety of different species. A small amount of genetic variation is revealed by amplification of certain regions of the genomic DNA. The genomic DNA, as a template, is subjected to amplification using the polymerase chain reaction (PCR) system and RAPD oligonucleotide primers. This technique makes use of a single oligonucleotide primer (usually ten base pairs in length) that hybridizes and amplifies'arbitrary regions of a genome. . With these advanced molecular techniques, DNA-based genetic fingerprinting of numerous species has already been reported (Black IV et al. 1992, Landry et al. 1993). Two years after the original invention, a new adoption of the RAPD technique was developed (Liang & Pardee 1992) and was called Reverse Transcription-PCR (RT-PCR). The procedure uses an additional primer that will anchor the poly(A) tail at the 3' end of total mRNA The cDNA resulting from this amplification is then used as a template for subsequent RAPD assays. Developing genetic markers for gene(s) conferring resistance to B. thuringiensis 6-endotoxin in Colorado potato beetles will lead to further 18 understanding of the inheritance of resistance. Furthermore, the markers developed will be very useful tools for early detection or monitoring of the development of resistance in field populations. The objectives of this research were: 1) to examine the continued selection of Colorado potato beetle with B. thuringiensis 8-endotoxin CryIIIA, 2) to'identify the mode of inheritance ’of resistance to B. rhuringiensis 8-endotoxin CryIIIAin Colorado potato beetle, 3) to determine the heritability of resistance in Colorado potato beetle selected with the B. .thuringiensis 5—endotoxin CryIIIA, and 4) to identify markers that can serve as diagnostic probes for B. thuringiensis. 8- endotoxin resistance in Colorado potato beetle field populations. 19 REFERENCES CITED Adang, M. J., J. Firoozabady, J. Klein, D. DeBoer’, V. Sekar, J. D. Kemp, E. Murray, T. A Rocheleau, K. Rashka, G. Staffeld, C. Stock, D. Sutton, & D. J. Merlo. 1987. Expression of a Bacillus thuringiensis insecticidal crystal protein gene in tobacco plants, pp. 345-353. In UCLA Symposium on molecular and cellular biology, vol. 48, Molecular strategies for crop protection. Alan R. Liss, Inc., New York. Aronson, A 1., W. Beckman, & P. Dunn. 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev. 50: 1-24. Bauer, L. S. & HS. Pankratz. 1992. Ultrasructural effects of Bacillus thuringiensis var. san diego on midgut cells of the cottonwood leaf beetle. 1. of Invertebr. Pathol. 60:15-25. Berenbaum, M. 1983. Pests: The Colorado potato beetle. Horticulture. Vol. 7. Number 7:48-50. Black W, W. C., N. M. DuTeau, G. J. Puterka, J. R. Nechols, & J. M. Pettorini. 1992. Use of random amplified polymorphic DNA polymerase chain reaction (RAPD- PCR) to detect DNA polymorphism in aphids (Homoptera: Aphididae). Bull. Entomol. Res. 82: 151-159. Brewer, G.J. 1991. Resistance to Bacillus thuringiensis subsp. kurstaki in the sunflower moth (Lepidoptera: Pyralidae). Environ. Entomol. 20: 316-322. Carlberg, G. & R. Lindstrom. 1987. Testing fly resistance to thuringiensin produced by Bacillus thuringiensis, serotype H-l. J. Invertebr. Pathol. 49: 194-197. Carroll, 1., J. Li, & D. J. Ellar. 1989. Proteolytic processing of a Coleopteran-specific 5- endotoxin produced by Bacillus thuringiensis var. tenebrionis. Biochem. J. 261: 99-105. Carroll, 1., & D. J. Ellar. 1993. An analysis of Bacillus thuringiensis 5—endotoxin action on insect-midgut-membrane permeability using a light-scattering assay. Eur. J. Biochem. 214: 771-778. Casagrande, RA. 1985. The Iowa Potato beetle, its discovery and spread on potatoes. Bulletin of the ESA. Summer 1985:27-29. Crawford, D. N., & W. D. Harvey. 1988. Barium and calcium block Bacillus thuringiensis subspecies kurstaki 8—endotoxin inhibition of potassium current across isolated midgut of larval Manduca sexta. J. Exp. Biol. 137:277-286. 20 Chen, X.J. M.K.Lee, & D.H. Dean. 1993. Site-directed mutation in a highly conserved region of Bacillus thuringiensis 5—endotoxin affect across Bombix mori midguts. Proc. Natl. Acad. Sci. USA. 90: 9041-9045. Choma, C. T., & H. Kaplan. 1990. Folding and unfolding of the protoxin fi'om Bacillus thuringiensis: evidencethat the toxic moiety is present in an active conformation. Biochem. 29: 10971-10977. Croft, BA. 1990. Developing a philosophy and program of pesticide resistance management, pp. 277-296. In: R.T. Roush & BE. Tabashnik [eds], Pesticide resistance in arthropods. Chapman and Hall, NY. Curtis, C.F., L.M. Cook, & R]. Wood. 1978. Selection for and against insecticide resistance and possible method of inhibiting the evolution of resistance in mosquitoes. Ecol. Entomol. 32273-287. Delannay, X., B. La Vallee, R. Proksch, R. Fuchs, S. Sims, J. Greenplate, P. Marrone, R. Dodson, J. Augustine, J. Layton, & D. Fischhoff 1989. Field performance of transgenic tomato plants expressing the Bacillus thuringiensis var. kurstaki insect control protein. Science 7: 1265-1269. Denolf, P., S. Jansens, M. Peferoen, D. Degheele, & J. Van Rie. 1993. Two different Bacillus thuringiensis delta-endotoxin receptors in the midgut brush border membrane of the European corn borer, Ostrinia nubilalis (I-Iubner) (Lepidoptera: Pyralidae). Appl. and Environ. Microbiol. 59:1828-1837. Endo, Y., & J. Nishiitsutsuji-Uwo. 1980. Mode of action of Bacillus thuringiensis 5— endotoxin: histopathological changes in the silkworm midgut. J. Invertebr. Pathol. 36: 90-103. English, L. H., T. L. Ready, & A. E. Bastian. 1991. Delta-endotoxin-induced leakage of 36Rb+-K+ and H20 from phospholipid vesicle is catalyzed by reconstituted midgut membrane. Insect Biochem. 21: 177-184. Ferré, J., M. D. Real, J. Van Rio, 8. Jansens, & M. Peferoen. 1991. Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor. Proc. Natl. Acad. Sci. USA 88: 5 1 19-5 123. Fischhoff, D. A, K. S. Bowdisch, F. J. Perlak, P. G. Marrone, S. H. McCormick, J. G. Niedermeyer, D. A. Dean, K. Kusano-Kretzmer, E. J. Mayer, D. E. Rochester, S. G. Rogers, & R. T. Fraley. 1987 . Insect tolerant transgenic tomato plants. Bio/Technology 5: 807-813. 21 Flint, M., & R. van den Bosch 1981. Introduction to integrated pest management. Plenum Press, New York. 240p. - Forgash, AJ. 1981. Insecticide resistance of the Colorado potato beetle, Leptinotarsa decenilineata (Say). pp. 34-46. In Advances in Potato Pest Management. J .H. Lashomb & R Casagrande [eds]. Stroudsburg, Pa. Hutchington Ross Publ. Company. Garczynski, S. F ., J. W. Crim, & M. J. Adang. 1991. Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis 5- endotoxin by protein blot analysis. Appl. Environ. Microbiol. 57: 2816-2820. Gazit, E.& Y. Shai. 1993. Structural and functional characterization of the 015 segment of Bacillus thuringiensis 6-endotoxin. Biochem. 32:3429-3436. Georghiou, G. 1986. The magnitude of resistance problem. pp. 14-43. In Pesticide resistance, strategies and tactics for management, U.S. committee on strategies for management of pesticide resistant pest population [eds]. National Ac. Press. Washington, DC. Goldman, I.F., J. Arnold, & B.C. Carlton. 1986 Selection for resistance to Bacillus thuringiensis subspecies israelensis in field and laboratory populations of the mosquito Aedes aegmti. J. Invertebr. Pathol. 47: 317-324. Gould, F: A. Martinez-Ramirez, A. Anderson, J. Ferre, F. J. Silva, & W. J. Moar. 1992. Broad-spectrum resistance to Bacillus thuringiensis 5-endotoxins in Heliothis virescens. Proc. Natl. Acad. Sci. USA 89: 7986-7988. Grafius, E. 1986. Effects of temperature on Pyrethroid toxicity to Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 79: 588-591 Gautheir, N.L., R. Hofrnaster, & M. Semel. 1981. History of Colorado potato beetle control. pp. 13-33. In Advances in Potato Pest Management. J.H. Lashomb & R. Casagrande [eds]. Stroudsburg, Pa. Hutchington Ross Publ. Company. Haider, M. 2., B. H. Knowles, & D. J. Ellar. 1986. Specificity of Bacillus thuringiensis var. colmeri insecticidal 5—endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. Eur. J. Biochem. 156: 531-540. Harvey, T. L., & D. E. Howell. 1965. Resistance of the House Fly to Bacillus thuringiensis Berliner. J. Invertebr. Path. 7: 92-100. Herrnstadt, C.,‘ G. G. Scares, E. R. Wilcox, & D. L. Edwards. 1986. A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/1' echnol. 4: 305-308. 22 Hofinann, C., P. Li’lthy, R Hi’itter, & V. Pliska. 1988a. Binding of the 8-endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae). Eur. J. Biochem. 173: 85-91. Hofinann, C., H. Vanderbruggen, H. Hofte, J. Van Ric, S. Jansens, & H. Van Mellaert. 1988b. Specificity of Bacillus thuringiensis 5-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc. Natl. Acad. Sci. USA 85: 7844-7848. Hofinann, C., & P. Liithy. 1986. Binding and activity of Bacillus thuringiensis 5- endotoxin to invertebrate cells. Arch. Microbiol. 146: 7-11. Hofinaster, R.N., & R.L. Waterfield. 1965. The Colorado potato beetle problem in Virginia and the present status of control measures. Trans. Peninsula Hortic. Soc. 1965:20-26. Hofie, H., H. Greve, J. Seurinck, S. Jansens, J. Mahillon, C. Ampe, J. Vanderkerckhove, H. Vanderbruggen, M. van Montagu, & M. Zabeau. 1986. Structural and filnctional analysis of a cloned delta endotoxin of Bacillus thuringiensis Berliner 1715. Eur. J. Biochem. 161: 273-280. Hofte, H., & H. R. Whiteley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242-255. Huber, H.E., & P. Luthy. 1981. Bacillus thuringiensis delta endotoxin: composition and activation. pp. 209-234. In Pathogenesis of invertebrate microbial diseases. E. W. Davidson [ed]. Allenheld, Osmun Publisher. Totowa. NJ. Ioannidis, P. 1., E. J. Grafius & M. E. Whalon. 1991. Patterns of insecticide resistance to azinphosmethyl, carbofuran, and pennethrin in the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 84: 1417-1423. Ioannidis, P. M., E. J. Grafius, J. M. Wierenga & R. Hollingworth. 1992. Selection, inheritance and characterization of carbofuran resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae). Pestic. Sci. 35: 215-222. Ishii, T., & M. Ohba. 1993. Diversity of Bacillus thuringiensis environmental isolates showing larvicidal activity specific for mosquitoes. J. of General Microb. 139: 2849-2854. Kaolin, P., P. Morel, & F. Gadani. 1994. Isolation of Bacillus thuringiensis from stored tobacco and Lasioderma‘serricome (F .). Appland Environ. Microbiol. 60: 19- 25. 23 Kirsch, K., & H. Schmutterer. 1988. Low efficacy of a Bacillus thuringiensis (Berl.) formulation in controlling the diamondback moth, Plutella xylostella (L.), in the Philippines. J. Appl. Entomol. 105: 249- 255. Knight, P.J., N. Crickmore, & D. J. Ellar. 1994. The receptor for Bacillus thuringiensis CryIA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is amino peptidase N. Mol. Microb. 11(3):? ? Knowles, B. H., '& D. J. Ellar. 1987. Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis 8—endotoxin with different insect specificity. Biochem. Biophys. Acta 924: 509-518. Landry, B. S., L. Dextraze, & G. Boivin. '1993. Random amplified polymorphic DNA markers for DNA fingerprinting and genetic variability assessment of minute parasitic wasp species (Hymenoptera: Mymaidae and Trichogrammatidae) used in biological control programs of phytopha'gous insects. Genome 36: 580-587. Liang, P. & A B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971. Li. 1., J. Carroll, & DJ. Ellar. 1991. Crystal structure of insecticidal 8-endotoxin fi'om Bacillus thuringiensis at 2.5 A resolution. Nature 353: 815-821. Lilley, M., RN. Ruffell, & H.J. Somerville. 1980. Purification of the insecticidal 8- endotoxin in crystal of Bacillus thuringiensis. J. Gen. Microbiol. 118: 1-11. Lilthy, P. & H. R. Ebersold. 1981. Bacillus thuringiensis delta-endotoxin: - histopathology and molecular mode of action. In Pathogenesis of invertebrate microbial diseases. Chapter 9. E. W. Davidson, [ed]. Allanheld, Osmun, Totowa, NJ. MacIntosh, S. C., T. B. Stone, R S. Jokerst, & R. L. Fuchs. 1991. Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens. Proc. Natl. Acad. Sci. USA 88: 8930-8933. McPherson, s. A, r. J. Perlak,-R. L. Fuchs, P. G. Marrone, P. B. Lavrik & D. A. Fischhoff. 1988. Characterization of the protein gene of Bacillus thuringiensis var. tenebrionis. Bio/Technology 6: 61-66 McGaughey, W. H. 1985. Insect resistance to the biological insecticide Bacillus thuringiensis. Science 229: 193-195. McGaughey, .W. H., & R W. Beeman. 1988. Resistance to Bacillus thuringiensis in colonies of Indian meal moth and almond moth (Lepidoptera: Pyralidae). J. Econ. Entomol. 81: 28-33. 24 McGaughey, W. H. & M. E. Whalon. 1992. Managing insect resistance to Bacillus rhufingiensis 8-endotoxins. Science 258: 1451-1455. Percy, J. & P. G. Fast. 1983. Bacillus thuringiensis crystal 8—endotoxin: ultrastructural studies of its effect on silkworm midgut cells. J. Invertebr. Pathol. 41: 86-98. Sacchi, V.F., P. Parenti, GM. Hanozet, B. Giordana, P. Lt‘lthy, & MG. Wolfersberger. 1986. Bacillus thuringiensis 8—endotoxins inhibits K+ gradient-dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells. FEBS Lett. 204: 213-218. Salama, H. S. & M. M. Matter. 1991. Tolerance level to Bacillus thuringiensis Berliner in the cotton leafwonn Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae). J. Appl. Entomol. 111: 225-230. schnepf, H. E., K. Tomczak, J. P. Ortega, & H. R. Whiteley. 1990. Specificity- detennining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis. J. Biol. Chem. 265:20923-20930. Shelton, A. M., J. L. Robertson, J. D. Tang, C. Perez, S. D. Eigenbrode, H. K. Preisler, W. T. Wilsey, & R. J. Cooley. 1993. Resistance of diamondback moth Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field. J. Econ. Entomol. 86: 697-705. Slaney, AC, H. L. Robbins, & L. English. 1992. Mode of action of Bacillus thuringiensis 8-endotoxin CryIIIA: an analysis of toxicity in Leptinotarsa decemlineata (Say) and Diabrotica undecimpunctata hawardi Barber. Insect. Biochem. Molec. Biol. 22: 9-18. Slatin, S. L., C. K. Abrams, & L. English. 1990. Delta-endotoxins from cation selective channels in planar lipid bilayers. Biochem. Biophys. Res. Comm. 169:765-772. Steinhaus, E. A. 1949. Principles of insect pathology. McGraw-Hill, New York. pp. 757. Stone, T. Bi, 8. R. Sims, & P. G. Marrone. 1989. Selection of tobacco budworrn for resistance to a genetically engineered Pseudomonasfluorescens containing the 8- endotoxin of Bacillus thuringiensis subsp. kurstaki. J. Invertebr. Pathol. 53: 228- 234. Tabashnik, B. E., N. L. Cushing, N. Finson, & M. W. Johnson. 1990. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83: 1671-1676. 25 Tabashnik, B. E., N. Finson, & M. W. Johnson. 1991. Managing resistance to Bacillus thufingiensis: Lessons from the diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 84: 49-55. Tabashnik, B. E., J. M. Schwartz, N. Finson, & M. W. Johnson. 1992. Inheritance of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 85: 1046-1055. Tabashnik, B. E., N. Finson, & M. W. Johnson. 1992. Two protease inhibitors fail to synergize Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 85: 2082-2087. Tabashnik, B. E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39: 47-79. Uyenoyarha, M. K. 1986. Pleiotropy and the evolution of genetic systems conferring resistance to pesticides. pp. 207-221. In. Pesticide resistance: strategies and tactics for management. National Research Council, National Academy of Sciences, Washington, DC. Van Ric, J., S. Jansens, H. Hofte, D. Degheele, & H. Van Mellaert. 1989. Specificity of Bacillus thuringiensis 8—endotoxins: Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur. J. Biochem. 186: 239- 247. Van Ric, 1., S. Jansens, H. Hofte, D. Degheele, & H. Van Mellaert. 1990a. Receptors on the bI'USh border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis 5-endotoxins. Appl. Environ. Microbiol. 56: 1378-1385. Van Ric, J., W. H. McGaughey, D. E: Johnson, B. D. Barnett, & H. Van Mellaert. 1990b. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science 247: 72-74. Vaeck, M., A. Reynaerts, H. Hofie, S. Jansens, M. De Beukeleer, C. Dean, M. Zabeau, M. Van Montagu, & J. Leemans. 1987 . Transgenic plants protected from insect attack. Nature 328: 33-37. Via, S. 1986. Quantitative genetic models and the evolution of pesticide resistance. pp.222-235. In Pesticide resistance, strategies and tactics for management, US. committee on strategies for management of pesticide resistant pest population, [eds]. National Ac. Press. Washington, DC. Whalon, ME, D.L. Miller, RM. Hollingworth. E.J. Grafius, & JR. Miller. 1993. Selection of resistant Colorado potato beetle (Coleoptera: Chrysomelidae) to Bacillus thuringiensis. J. Econ. Entomol. 86: 226-233. 26 Widner, W. R. & H. R. Whiteley. 1989'. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol. 171: 965-974. William, J.G.K., AR. Kubelik, K.J. Livak, J.A. Rafalski, & S.V. Tingey. 1990. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18: 6531-6535. ' CHAPTERI Inheritance of Resistance to Bacillus thuringiensis subsp. tenebrionis CryIIIA 5—endotoxin in Colorado Potato Beetle (Coleoptera: Chrysomelidae) 27 28 INTRODUCTION Coleopteran specific 8-endotoxin, CryIIIA (Hofte & Whiteley 1989), a product of Bacillus thuringiensis subsp. tenebrionis (Krieg et al. 1983, 1984; Sekar et al. 1987; McPherson et al. 1988), has been formulated into several commercial insecticides (F erro & Gelemter 1989, MacIntosh et al. 1990) that are active on the Colorado potato beetle, Leprinotarsa decemlineata (Say), and have been genetically engineered into potatoes (Gasser & Fraley 1989, Brunke & Meeusen .1991). This 5—endotoxin of B. thuringiensis appears to be an environmentally sound alternative to broad spectrum synthetic organic insecticides for controlling Colorado potato beetle, the most destructive pest on potatoes. Recent development of Colorado potato beetle resistance to synthetic organic insecticides in the eastern and midwestern United States (Ionnidis et al. 1991) have contributed to an increase in the use of B. thuringiensis CryIIIA 8-endotoxin. For > 20 yr, various B. thuringiensis isolates have been used commercially to control insect pests. However, one species has developed resistance in the field (T abashnik et al. 1990). Resistance in the tobacco budworrn, Heliotlu's virescens (F .), and Colorado potato beetle (Whalon et al. 1993) has been reported only fi'om laboratory selection experiments, but the capacity for resistance in these species is of concern because of the great economic significance of these two pest species. The recent discovery that several important species of pest insects can develop resistance to B. thuringiensis 5- endotoxins now raises concerns regarding the long-term use of this biological insecticide in pest control. Questions of longevity are especially critical in the rapidly advancing area of plant genetic transformation, which has emphasized the use of B. thuringiensis 5- endotoxin genes to impart pest resistance in several major crop species (Gasser & F raley 1989, Boulter et al. 1990, Brunke & Meeusen 1991). Regardless of how the B. 29” tlna'ingiensis 8-endotoxins is delivered, insect pests might develop tolerance. The intensive application of B. thuringiensis 8-endotoxin through conventional sprays or transgenic plants deployment could seriously diminish the economic value of this technological development and force continued reliance on other chemical insecticides (Gould 1988a, b; Stone et al. 1989; Tabashnik et al. 1992). . The genetic basis of laboratory-selected resistance to B. thuringiensis 8-endotoxin was examined in diamondback moth, Plutella xylostella (1..) (Tabashnik et al. 1992) and Indianmealo moth, P. interpunctella (Hubner) (McGaughey 1985, McGaughey & Beeman 1988). This genetic knowledge may be usefiil in devising strategies to slow resistance evolution. However, inheritance of laboratory-selected resistance may differ from resistance selected in the field (Roush & McKenzie 1987, Tabashnik et al. 1992). . Beginning in 1987, we selected a field and laboratory colony of Colorado potato beetle with the CryIIIA 8-endotoxin of B. thuringiensis (Whalon et al. 1993). The objective of the study reported here was to examine and report the continued selection and the inheritance of resistance. MATERIALS AND METHODS CryIIIA fi-Endotoxin Sources M-One insecticide (AI: B. thuringiensis subsp. tenebrionis 8—endotoxin, Mycogen, San Diego, CA) containing 40,900 Coloradopotato beetle International Units (CPB 1U)/mg of formulation and 8,800 i 10% ug (mean :t SEM) CryIIIA 8-endotoxin per milliliter of formulation was initially used for selection and bioassays. Spud-Cap Bioinsecticide (AI: B. thuringiensis subsp. renebrionis 8-endotoxin encapsulated in killed Pseudomonasflorescens cells, Mycogen, San Diego, CA) was used beginning in June 1989. Spud-Cap contained 83,400 CPB IU/mg formulation and 12,300 + 10% 11g (mean :1: SEM) CryIIIA 8-endotoxin per milliliter of formulation. 30 Beetle Strains, Rearing, and Selection Strains of Colorado potato beetle used for the experiment were maintained in our laboratory at 25 i 2°C and a photoperiod of 16:8 (LzD) h. The origin and maintenance of both susceptible and resistance strains were described earlier by Whalon et al. (1993). Susceptible and resistant strains were fi'om the same stock collected in 1987 and 1988 fiom seven counties in Michigan. Second instars (3 d old) were selected in every generation at a level of CryIIIA 8—endotoxin concentration that prevented >98% fiom reaching the adult stage. Beetles were exposed for 2 - 3 d on 'Superior’ potato foliage treated with a CryIIIA 5—endotoxin concentration of 1,500 - 741,000 ug/liter of water (Whalon et al. 1993). To select beetles, potato petioles (5 leaflets) were inserted into 2-ml vials filled with water, dipped five times into the CryIIIA S-endotoxin solution, and allowed to air dry before being transferred to individual petri-dishes (15 cm diameter). Twenty larvae were placed on each leaf and held at 25 :1: 2°C and a photoperiod of 16:8 (LzD) h in a grth chamber. Foliage was checked daily and water was replenished as needed. Larvae were placed on the foliage within 30 min after the CryIIIA 5-endotoxin application had air dried; they were allowed to move and feed freely. After exposure to CryIIIA 8—endotoxin, surviving larvae were transferred to untreated foliage of potted potato plants within rearing cages for 5 - 10 (1 until pupation. Pair-by-pair cultures were used to increase homogeneity for genetic analysis of the strains. The sex of newly emerged adults from selections was determined; they were paired before being placed on potted potato plants covered with a cage made of a 2-liter soda bottle. Progeny from each pair of resistant strains was selected and maintained separately for five generations before genetic analysis. 31 Inheritance and Stability Studies Resistance dominance, sex linkage, and chromosomal inheritance were determined by performing reciprocal crosses as pair by pair matings between susceptible and resistant individuals. Investigations were done with G17 - 634 beetles. Newly emerged virgin females and males of G29 of each strain were separated until use. One male and one female were paired on caged potato plants. Progeny (F1) from each pair of each cross (S X R and R X S) was tested. A portion of the F] progeny was reared to maturity for further experimentation. The experiment was done with nine pairs for reciprocal crosses. Four pairs of backcrosses between adult F1 and susceptible parent were done to determine whether resistance was inherited as a monogenic or polygenic trait. We tested the hypothesis that the resistance was inherited by a single dominant gene. To test this hypothesis, we calculated the expected mortality of the F2 at concentration (c) as (percentage of expected mortality of F1 at c + percentage of expected mortality of suceptible at c)/2. Mortality from each backcross was observed; chi-square analysis was used to compare observed mortality with mortality expected assuming simple Mendelian inheritance. Larvae from the G17 generation were separated from the selected colony and maintained for seventeen generations as described above on untreated potato plants in 2-liter cages. Second instars in each generation were tested to determine the stability of the resistance. Bioassays Activity of CryIIIA 5-endotoxin on Colorado potato beetle was assessed with 3 d old larvae. Five or six serial dilutions of CryIIIA 5-endotoxin were made to give concentrations ranging from 0.125 to 16 times the expected LC 50 (Whalon et al. 1993). Potato foliage was trimmed to the terminal five leaflets and the petiole was inserted into a 2-ml vial containing water. Each petiole was dipped five times into one of the CryIIIA 8- 32 endotoxin solutions and allowed to air dry before being placed in a 15-cm petri-dish. At least five petioles were prepared per concentration; we used no more than 10 larvae on each leaf at each concentration and at least 30 larvae per concentration. Larvae were held at 25 :t 2°C, 50 - 60% RH, and a photoperiod of 16:8 (LzD) h in a growth chamber. Mortality was assessed 96 h after treatment. Data Analysis Data were analyzed by probit regression (F inney 1971); Abbott's (1925) formula was used to correct for control mortality. LC 505 were compared between generations to monitor the progress of resistance. Failure of 95% CL to overlap was used as the criterion for significant differences at LC50 (P < 0.05). If confidence limits did not overlap, the LC 50 values were considered to be significantly different. The resistance ratio was calculated by dividing the LC 50 of the selected with the LC 50 of the unselected strain within each generation. Concentration - mortality relationships obtained for F1 crosses were used to determine the autosomal or sex-linked nature of resistance inheritance. The dominance level (D) of CryIIIA 8—endotoxin resistance in F1 progeny was estimated with the index given by Stone (1968). X0- XC D: (where Xa =log10 (LC50) of the resistant colony, X b =log10 (LC50) of the heterozygous colony, and Xc =log10 (LC 50) of the susceptible colony). This formula will result in a value of -1 if resistance is completely recessive, a value of 0 if there is no dominance, and a value of +1 if resistance is completely dominant. The standard error was estimated by taking the square root of variance of D (Preisler et al. 1990): 33 4 (Xb-Xc)2 (’Yb")(a)2 0 = -——- 0 + -——— o + —— o D (XC'Xa)2{ Xb (Xa'Xc)2 X0 (Xa' Xc)2 X0} The D is significantly different from £1 when the approximate 95% CL value (D i 2 SEM) includes :1. Chi-square values from observed and expected mortality of the F1 backcrossed progeny were calculated to determined the monofactorial inheritance of resistance. Expected mortality was estimated assuming simple Mendelian inheritance, where backcross mortality of any given dose was the sum of half of the mortality of the parental strains. The chi-square test for goodness-of-fit for expected mortalities was calculated with formulas obtained from Finney (1971). RESULTS Resistance Selection Table 1.1 shows the progression of resistance from the F13 through F29 generations under selection with CryIIIA 8-endotoxin. Significantly higher LC 50s than the susceptible colony were observed in generations F17 and F 21. A 3-fold increase in resistance ratio was observed from the F13 to the F29; this increase represented a >200- fold difference between the susceptible and resistant strains. The LC95 of F13, F15 and F29 were significantly different from LC95 of the F15, F 17, and F21, respectively. The unselected strain, which was also tested throughout as a control, had LC 505 that did not vary significantly from F13 to F34 (1.69 at 0.20 mg CryIIIA 6-endotoxin per liter of water). Resistance Inheritance The probit regression statistics of the parental strains and their F1 progeny (Table 1.2) indicated no significant differences in lethal concentrations were observed between the two reciprocal F1 generation crosses. 34 Table 1.1. Progression of resistance in Colorado potato beetle to the CryIIIA 8- eudotoxin of Bacillus thuringiensis. Generation“ n Slopel: SEM LC501’(95% CL) RR" x2 df G13 780 1.27:1:0.07 133a (111 - 160) 82 2.32 3 UN13 240 1.92:t0.29 1.62 (1.22 - 2.19) 2.77 5 G15 480 1.07:1:007 162a (125 - 215) 98 1.33 3 UN15 240 1.19i0.11 1.66 (1.18 - 2.52) 2.72 5 G16 480 18610.16 241ab (204 - 291) 146 2.33 3 UN16 240 1.69:1:0.11 1.65 (1.25 - 2.20) 0.57 5 G17 480 1.16i0.09 330bc (263 - 440) 223 2.48 3 UN17 240 1.46:t0.09 1.49 (1.11 - 2.08) 2.00 5 G21 540 095320.07 369bc (286 - 535) 222 0.31 3 UN21 240 1.90:t0.29 1.66 (1.25 - 2.27) 2.32 5 G29 168 2.30i0.12 4840 (379 - 625) 293 1.16 3 UN29 192 1.29i0.08 1.65 (1.11 - 2.42) 1.03 5 Values followed by the same letter are not significantly different if their 95% CL between generations of resistance colonies overlap. a F ilial generation. Gnresistant strain generation 11; UN“ unselected strain generation n b mg CryIIIA 5-endotoxin per liter of water. 0 Resistance ratio = LC50 of Gn-I- LC50 of UNn 35 Table 1.2. Response of susceptible and resistant Colorado potato beetle and the genetic crosses to the CryIIIA 5—endotoxin Strain and Crossa n SlopetSEM chob (95% CL) RR" 2:2 df Resistant parent 168 2301012 484a (379 — 625) 293 1.16 3 Flmfx Sm) 288 2.101090 255b (208 - 318) 154 7.89 9 F1(sfom) 300 1.871016 2456 (191 - 314) 148 8.58 lo Values followed by the same letter are not significantly different if their 95% CL overlap. a S, susceptible; R, resistant; f, female; and n1, male. 5 mg CryIIIA 5-endotoxin per liter of water. c Resistance ratio = LC50 of resistant parent or F1 + LC 50 susceptible strain. 36 In Colorado potato beetle, we thus concluded that resistance to CryIIIA 5-endotoxin is autosomaly inherited and that there is no maternal influence (assuming that the mode of sex determination is XY, XX). Approximately the same degree of dominance values (D :1: SEM) were observed in these F 1 crosses (R X S and S X R), which were 0.77 :1: 0.06 and 0.76 :1: 0.059, respectively. These values were significantly different from -1 , indicating that the resistance trait was not completely recessive. The D values ranged fiom 0.66 to 0.89 and 0.64 to 0.87 respectively indicating that resistance might be inherited through incompletely dominant gene(s). Mortality of backcross progeny with susceptible parents deviated from expected mortalities. The observed mortality were higher at all concentrations (Table 1.3). The test of monogenic model showed no significant deviation between observed and expected mortality at three of six concentrations. Unlike other studies (Halliday & Georghiou 1985, Roush et al. 1986), significant deviation (P < 0.01, df = 1) from expected values Were produced at low concentrations ($29.64 mg/liter). Deviations from expected mortality in the backcross experiment suggested that more than one locus may be responsible for resistance. Resistance Stability After selection pressure with CryIIIA S-endotoxin was relaxed, we noticed a downward trend in the resistance ratio. The resistance ratio decreased from 200 to 48- fold in >10 generations. The LC 50 decreased significantly five generations after the selection pressure was removed (Table 1.4). When the colony was continously raised without selection with 5—endotoxin for another 12 generations (G22 - G34), the resistance ratio declined within a range of 48 - 81 fold. The LC50 of G22 - G34 were significantly lower than G17. Except for F29, the 95% CL of the LCsos of those generations overlapped. The last generation tested (G34) showed significantly higher tolerance to the 5-endotoxin than the unselected strain. 37 Table 1.3. Chi-square analysis of mortality statistics from F1 X susceptible backcrosses of Colorado potato beetle (test monogenic model) Concentration % Mortality mg/liter Expected Observed x2 P“ 463.125 97 98 1.29 0.28 185.25 86 95 3.14 0.08 74.10 71 79 2.98 0.09 29.64 46 60 7.74 <0.05* 11.85 22 44 23.73 <0.0l"' 4.74 9 24 32.35 <0.01* a Values followed by "' are significantly different at P $0.05 38 Table 1.4. Resistance stability to CryIIIA 8-endotoxin in Colorado potato beetle Generation“ 71 Slope-tSEM LC50(95% CL)b RR9 x2 or G17 240 1.16d:0.09 330a (263'- 440) 223 2.48 3 UN17 240 1.461009 l.49(1.11 - 2.08) 2.00 5 019 240 0.83:1:006 230ab (155 - 410) 140 0.82 4 UN19 240 1.27s0.09 1.62(1.16 - 2.33) 0.31 5 (332 , 210 0.754007 100bc (65 - 186) 60 1.83 3 0sz 240 2,130.43 1.67(l.29 - 2.23) 5.30 5 623 210 1.361014 l34bc (98 - 202) 81 0.97 4 UN23 210 1.60:1:016 1.65(1.18 - 2.22) 0.37 4 629 240 2.17s0.47 79c (64 - 102) 48 1.11 3 UN29 192 l.29i0.08 1.65(1.11 - 2.42) 1.03 5 G34 168 3.25:l:O.64 121bc (98 - 158) 55 2.77 4 UN34 . 320 2.92a0.49 2.21 (1.86 - 2.65) 4.15 5 Values followed by the same letter are not significantly different if their 95% CL overlap. ' a G“: generation 11; UN“: unselected generation 11. The selection was relaxed in the G17 generation for seventeen generations (G34). b mg CryIIIA 5-endotoxin per liter of water. c Resistance ratio = LC50 of Fx + LC 50 unselected strain. 39 DISCUSSION A graph of the percentage response (mortality) against a stimulus (toxin) will give a steadily rising curve. However the rate of increase in mortality per unit increase in toxin concentrations is frequently very low in the region of zero or 100% mortality, but higher in the intermediate region so that the curve is actually sigmoidal. When the toxin concentration is measured in metametrix units (log concentration), the curve takes a characteristic normal sigmoid curve. The transformation of percent mortality into probit values also helps to linearize the normal sigmoid curve. In some situations, however, the correlation between the probit values and log concentration is more complicated than previously mentioned. 0- e U i. 1 3- e 21 e V I ‘l 1 0.01 0.1 1 10 100 Concentration Figure I. l. Probit values were plotted against concentration of a toxic agent (log concentration). Some of the transformed data gave plots as shown in the figure above. The more concentrated the toxin, the less mortality was observed. One explanation could be the dimerization or polymerization of the crystal protein. As a consequence, less toxin would have been active to cause disruption; thus less mortality would have resulted. 40 Previous genetic and biochemical studies of B. thuringiensis 8-endotoxin indicate that resistance in insects is commonly inherited as a partially or fiilly recessive trait (Gould et al. 1992). Study of CryIIIA S-endotoxin resistance on Colorado potato beetle indicates a difl‘erent conclusion. Our results suggest that an incomplete dominant gene(s) is involved in CryIIIA 5—endotoxin resistance in Colorado potato beetle. Bioassays of reciprocal crosses produced lines that were close to those of resistant parents. When incomplete or partial dominance exists, at least two different gene interactions can occur. The presence of other genes may hide the effect of the heterozygote resistance gene(s). In addition, epistasis can occur as a consequence of increased or decreased enzyme activities, or changes in pH that effect a particular phenotype (Strickberger 1985). Epistasis could increase the ability of the insect to tolerate the toxic agent by, for example, changing receptor-ligand kinetics, or changing the gut acidity or physiology. Crow (1957) suggested that epistatic interactions can evolve under close inbreeding. This explanation pertain to our selection process with Colorado potato beetle, 1.8. a simple relationship between genes in which each makes a contribution to the resistance character. Thus, the introduction of genes from the susceptible to the resistant colony would dilute the effects of these genes. Production of heterozygotes also may result in reduced fitness because of interference between detoxification pathways, binding sites, or normal metabolic processes (Uyenoyama 1986). The results presented here may indicate that resistance to 8-endotoxin segregates in non monofactorial fashion in backcrosses to susceptible parents. The bioassay of backcross progeny did not produce evidence of monofactorial segregation. When the backcross progeny were exposed to the discriminating concentration (:60 mg CryIIIA 5-endotoxin per liter of water, which was expected to kill 100% of the susceptible larvae), >50% mortality was observed. These results also suggested that a genetic network controls the resistance mechanism. In the backcross experiments to the susceptible strain, we observed a significantly higher mortality than expected over the three lower 41 concentrations ($29.64 mg/liter). With the lower concentrations, the larvae may have continued feeding on the treated leaves longer, resulting in more mortality. We have observed this phenomena is subsequent bioassays of feeding behavior. Loci for genes conferring CryIIIA B—endotoxin resistance might also be separately segregated. Our resistance stability experiment demonstrated that the resistance level significamly declined over a five generation period when the selection pressure was removed. However, continued breeding for another 12 generations without selection did not produce further reduction in resistance level. Because further breeding did not produce firrther reduction in level of resistance, homozygosity apparently had been reached for the majority of the genes determining resistance. The effect of continued inbreeding, as occurs in many laboratory colonies, might cause the reduction of heterozygosity up to certain level that further reduction does not occur. Reintroduction of the susceptible genes may result in further reduction of resistance level. Our results suggest that resistance management of Colorado potato beetle (Whalon et al. 1993) may be possible in potato production if resistance management begins before resistance factors are fixed in targeted p0pulations. 42 REFERENCES CITED Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265 - 267. Boulter, D., J. A. Gatehouse, A. M. R. Gatehouse & V. A. Hilder. 1990. Genetic engineering of plants for insect resistance. Endeavour, New Series 14: 185 - 190. Brunke, K. J. & R. L. Meeusen. 1991. Insect control with genetically engineered crops. Tibtech 9: 197 - 200. Crow, J. F. 1957. Genetics of insect resistance to chemicals. Annu. Rev. Entomol. 2: 227 - 246. ‘ Ferro, D. N. & W. D. Gelernter. 1989. Toxicity of a new strain of Bacillus thuringiensis to the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82: 750 - 755. Finney, D. J. 1971. Probit analysis, 3rd ed. Cambridge University Press, Cambridge. Gasser, C. S. & R. T. Fraley. 1989. Genetically engineering plants for crop improvement. Science (Washington, DC) 244: 1293 - 1299. Gould, F. 1988a. Evolutionary biology and genetically engineered crops: consideration of evolutionary theory can aid in crop design. BioScience 38: 26 - 33. 1988b. Genetic engineering, integrated pest management and the evolution of pests. pp. 15 - 18. In Planned release of genetically engineered organisms. Elsevier, Cambridge. Gould, F., A. Martina-Ramirez, A. Anderson, J. Ferre, F. J. Silva & W. J. Moar. _ 1992. Broad-spectrum resistance to Bacillus thuringiensis 5-endotoxins in Heliothis virescens. Proc. Natl. Acad. Sci. USA. 89: 7986 - 7990. Halliday, W. R. & G. P. Georghiou. 1985. Inheritance of resistance to perrnethrin and DDT in the southern house mosquito (Diptera: Culicidae). J. Econ. Entomol. 78: 762 - 767. Hofte, H. & H. R.Whiteley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242 - 255. Ioannidis, P. I., E. J. Grafius & M. E. Whalon. 1991. Patterns of insecticide resistance to azinphosmethyl, carbofuran, and perrnethrin in the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 84: 1417 - 1423. 43 Krieg, V. A., A. M. Huger, G. A. Langenbrucb & W. Schnetter. 1983. Bacillus tlmringiensis var. tenebrionis: ein neuer, gegenuber Larven von Coleoperen wirksamer Pathotyp. Z. Angew. Entomol. 96: 500 - 508. Krieg, V. A., A. M. Huger, G. A. Langenbruch & W. Schnetter. 1984. Neue ergebnisse fiber Bacillus thuringiensis var. tenebrionis unter basonderer Ben'icksichtigung seiner wirkung auf den Kartofi‘elkafer. Anz. Schaedlingsk. 57: 145 - 150. . McGaughey, W. H. 1985. Insect resistance to the biological insecticide Bacillus thuringiensis. Science (Washington, DC) 229: 193 - 195. McGaughey, W. H. & R. W. Beeman. 1988. Resistance to Bacillus thuringiensis in colonies of Indianmeal moth and almond moth (Lepidoptera: Pyralidae). J. Econ. Entomol. 81: 28 - 33. McGaughey, W. H. & M. E. Whalon. 1992. Managing insect resistance to Bacillus thuringiensis 8-endotoxins. Science (Washington, DC) 258: 1451 - 1455. MacIntosh, S.C., T. E. Stone, S. R. Sims, P. L. Hunts, J. T. Greenplate, P. G. Marrone, F. J. Perlak, D. A. Fischhoff’ & R. L. Fuchs. 1990. Specificity and efficacy of purified Bacillus thuringiemis proteins against agronomically important insects. J. Invertebr. Pathol. 56: 258 - 266. McPherson, S. A. F. J. Perlak, R. L. Fuchs, P. G. Marrone, P. B. Lavrik & D. A. Fischhol'f. 1988. Characterization of the coleopteran-specific protein gene of Bacillus thuringiensis var tenebrionis. Biotechnology 6: 61 - 66. Preisler, H. K., M. A. Hoy & J. L. Robertson. 1990. Statistical analysis of modes of inheritance for pesticide resistance. J. Econ. Entomol. 83: 1649 - 1655. Roush, R. T. & J. A. McKenzie. 1987. Ecological genetics of insecticide resistance. Annu. Rev. Entomol. 32: 361 - 380. Roush, R. T., R. L. Combs, T. C. Randolph, J. MacDonald & J. A. Hawkins. 1986. Inheritance and effective dominance of pyrethroid resistance in the horn fly (Diptera: Muscidae). J. Econ. Entomol. 79: 1178 - 1182. Sekar, V., D. V. Thompson, M. J. Maroney, R. G. Bookland & M. J. Adang. 1987. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc. Natl. Acad. Sci. U. S. A 84: 7036 - 7040. Stone, B. F. 1968. A formula for determining degree of dominance in cases of monofactorial inheritance of resistance to chemical. Bull. WHO. 38: 325 - 326. 44 Stone, T. B., S. R. Sims, & P. G. Marrone. 1989. Selection of tobaco budworrn for resistance to a genetically engineered Pseudomonasfluorescens containing the 5— endotoxin of Bacillus thuringiensis subsp. kurstaki. J. Invertebr. Pathol. 53: 228 - 234 Strickberger, M. W. 1985. Genetics, 3rd ed. MacMillan, New York. Tabashnik, B. E., N. L. Cushing, N. Finson & M. W. Johnson. 1990. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83: 1671 - 1676. Tabashnik, B. E., J. M. Schwartz, N. Finson, & M. W. Johnson. 1992. Inheritance of Resistance to Bacillus “thuringiensis in Diamondback Moth (Lepidoptera: ‘ Plutellidae). J. Econ. Entomol. 85: 1046 - 1055. Uyenoyama, M. K. 1986. Pleotropy and the evolution of genetic systems conferring resistance to pesticides. pp. 207 - 221. In Pesticide resistance: strategies and tactics for management. National Research Council, National Academy of Sciences, Washington, DC. Whalon, M. E. , D.L. Miller, R. M. Hollingworth, E. J. Grafius & J. R. Miller. 1993. Selection of resistant Colorado potato beetle (Coleoptera: Chrysomelidae) to Bacillus thuringiensis. J. Econ. Entomol. 86: 226 - 233. CHAPTER II Heritability Estimation of Resistance to Bacillus thuringiensis subsp. tenebrionis CryIIIA 8-endotoxin in Colorado Potato Beetle (Coleoptera: Chrysomelidae) 45 46 INTRODUCTION The potential development of field resistance to pesticides depends on genetic and biological characteristics of the pests as well as the environmental characteristics of the particular population (Keiding 1986). When selection pressure is applied on a particular population, the frequency of the particular genes conferring the resistant trait will change. The change in the proportion of resistance, therefore, is due to genotypic change resulting fiom selection. For a given trait, selection gain can be predicted for several initial generations of selection. As the selection progresses, however, the gains are being generally below those anticipated. Eventually the selection brings no response, and 'plateau' is reached (Dobzhansky 1970). Reproductive advantage may affect the selection gain. Gain under selection is determined by the selection advantage of the desired genotype and the reproductive potential of the population. Any gain in laboratory selections does not represent the response of the population in nature under equal selection pressure. The following are several factors that may contribute to the different results between field and laboratory selections. The rare resistance genes and additional genes may be missing in a laboratory colony as the gene pool is smaller. Furthermore, effects of inbreeding also become more pronounced under intense laboratory selection, because the size of the breeding population is greatly reduced. The difl'erence in response often result in lower mortality in laboratory colony. In a laboratory colony, an artificial selection may result in exploitation of polygenetic variance. On the other hand, field selection tends to act on alleles of single resistance genes (Keiding 1986) 47 Laboratory selections have been conducted under many different environmental conditions to gain more understanding about resistance development. However, there are some limitations in predicting the progress of selection. In some cases, environmental factors may have significant influence on the response of the individuals in a population toward the selection pressure. In nature, selection may operate much more strongly in favor of individuals able to survive for themselves and overcome the unexpected environmental stress. A fixed and constant environmental condition of artificial selection may decrease the chance survive of individuals with disadvantage environmental condition. The study of inherited factors uncovered by manipulating laboratory colonies may provide information about the nature of the alleles conferring resistance and their anticipated fate in the population. Laboratory selection for resistance, despite its limitations, is usually included as one element in predicting the probability of pesticide resistance development. The selection gain can be predicted from the heritability of the selected phenotype fi'om each generation (Dobzhansky 1970). Heritability is defined as the ratio of additive genetic variance to phenotypic variance (Falconer 1989). The value of the heritability thus represents the overall genetic inheritance characteristics of a given population. Information collected from artificial selections can be used to estimate the heritability of the traits being selected. Heritability determined from generation to generation of artificial selection can be useful in predicting the potential of resistance development in other populations, especially if the inheritance of genes for resistance is known and markers to detect the presence of the gene(s) in the field p0pulation are available. Although no research has yet demonstrated a clear linkage between field population monitoring and laboratory heritability, this is a long-term goal of our laboratory. In 1987 we initiated field selection of Colorado potato beetle with CryIIIA 8- endotoxin of Bacillus thuringiensis (Whalon et al. 1993), and subsequently selected them in the laboratory which resulted in over 200 fold increase in resistance (Rahardja & 48 Whalon 1995). We report here the estimation of realized heritability from each generation of Colorado potato beetle, Leptinotarsa decemlineata (Say), artificially selected with B. thuringiensis CryIIIA 8—endotoxin. MATERIALS AND METHODS Insect Rearing and Selection Colorado potato beetle strains (susceptible and resistant) used for this study were reared and selected under conditions as described elsewhere (Whalon et al. 1993, Rahardja & Whalon 1995). Second-instar Colorado potato beetles were selected by exposure for 96 h on potato foliage dipped in a B. thuringiensis 5—endotoxin concentration of 1.5-741 mg/liter of water. Twenty larvae were placed on each treated potato leaf and held at 25 :L- 2 °C, 50—60% RH and a photoperiod of 16:8 (LzD) h in a growth chamber. After exposure to CryIIIA 8~endotoxin, surviving larvae were transferred to untreated fresh leaves in clean petridishes and maintained until third-instar. The larvae then were placed on potted untreated potato plants within rearing cages until pupation. The percentage of surviving adults was observed and documented. Bioassay Details on bioassay were previously reported elsewhere (Whalon et al. 1993, Rahardja & Whalon 1995). Groups of ten to fifteen medium (06-08 g) second instars were exposed to treated potato leaves dipped in B. thuringiensis 5-endotoxin at 5-6 serial concentrations. Each bioassay was replicated three times at 25 :t 2 °C, 50—60% RH, and a photoperiod of 16:8 (LzD) h. The mortality was observed and documented 96 h afier exposure, and the data subjected to probit analysis (F inney 1971). 49 Data Analysis The method described here follows Tabashnik (1992). The realized heritability values are calculated as h2= 3‘3- (1) where 122 is the realized heritability, R or the response to selection is the difference in mean phenotype between the ofi‘spring of the selected parents and the parental generation before selection, and S or the selection differential is the average difference in mean phenotype between the selected parents and the parental generation before selection. R was estimated as R = rogggso Eu 1!; log (Lemm- ) (2) where LC 50Fn is the LC50 of offspring after n generations of selection and LC 50Fi is the LC50 of the parental generation before n generations of selection. S was estimated as S = i op ( 3 ) where i is the intensity of selection estimated using Appendix A of Falconer (1989) based on the percentage surviving selection. The average percentage of adults emerged fi'om unselected colony was 30%. The proportion of mortality was adjusted using Abbot formula (Abbot 1952, Tabashnik 1992). The phenotypic standard deviation, op, was estimated as 6p: (slopeigslopeny1 (4)} where slopei is slope of the initial probit regression line (F inney 1971) before selection, and slopen is final slope afier n generations of selection. The responses to selection of each generation were also estimated by calculating the deviation of the log LC 50 of resistant from the unselected colonies (Falconer 1973). 50 The correlations between the estimated and the cumulative selection deferential were plotted to determine the direction and the rate of the evolution of resistance. The genotypic variance was observed following Tanaka & Noppun (1989). The standard deviations of the phenotype LC 50 (l/slope) over all generations were calculated to determine the possibility of elimination of genetic variances as a consequence of inbreeding and selection. The Coefficient of Inbreeding of both susceptible and resistance strains was estimated to determine the genetic variance of the strains. The inbreeding coefficient was calculated as a function of true population size (Wright 1942). F, = 1/(2N) + (1-1/(2N))F.,.. (5) The backcross studies indicated that incompletely dominant gene(s) were involved in the resistance. The Colorado potato beetle strain was selected against CryIIIA endotoxin with a concentration of the toxin that produced over 95% mortality. If we assume that the selection causes elimination of the recessive individuals (SS), the proportion of S can be estimated from the number of adults that survived. S = 1- (% adult survived/100) In equilibrium populations with inbreeding consisting of two components: inbred individuals and random individuals (Li 1971). The average fitness of the inbred (W1) and random (Wk) components is W, which is the sum of WI and WR where W,=1-sS,andWR=l-SSZ and W=FW1+(l-F)WR Sl =r=(1- 38) + (1 - F)(1- s32) Small 8 is the intensity of the selection for the recessive individuals and F is the coefficient of inbreeding. The new fiequency of the recessive (S') gene in the next generation (Wright 1942) is: S'=1/W{RS(1-F)+S2(1~F)(l-s)+SF(1-s)} (6) When s is 1, the equation (6) will be S'=1/W{RS(l-F)} (7) and the average fitness will be W=F(1- S)+(1-F)(1- 32) (8) RESULTS The percentage of adults surviving after selection is reported: in (Table 11.1). There was a dramatic decrease in adult emergence at generations F 10. However, the resistance ratio (RR = LC 50 resistant colony/LC50 susceptible colony) kept increasing every generation. The 29th generation of the resistant strain is 380 to 625 times more tolerant to B. thuringiensis CryIIIA 8-endotoxin than the susceptible strain (Rahardja & Whalon 1995). The average responses to selection (R) for every generation were calculated. The values ranged fiom 0.01 (Fl) to 0.49 (F4). The R values decreased after the fourth generation and reached values with very little fluctuation after the tenth generation. The deviations of the response (log LC 50) of the resistant colony from the unselected colony every generation were plotted against cumulative selection differentials, S (Figure II. 1). The graph shows that in the first five generations, the resistance gains fluctuated 52 and then the values are very closely approximated to a straight line (r=0.98, slope=0.083, F=210, and P<0.001). The estimate of realized heritability 012) of resistance to B. thuringiensis CryIIIA 8—endotoxin in Colorado potato beetle based at 29 generations was 0.17 (Table H.1). The highest value was 0.21 (F4) and the lowest was 0.005 (F 1). After the tenth generation, the h2 reached values which fluctuate very little (Figure 11.2). The values fluctuated in a very narrow range (5%) over the last 12 generations. The estimated h2 increased substantially at generation 29, in which the selection pressure was low (% adults emerged was 41). 53 Table IL]. Estimation of realized heritability (hz) of resistance to Bacillus thuringiensis E-endotoxin in Colorado potato beetlea ADULT LC50 EMERGED mg/Iiter SLOPE R S *2 (°/°) G] 0.90 1.63 1.80 0 1.50 0 62 0.40 0.68 1.10 -0.18 2.04 0 G4 0.40 147 0.90 0.49 2.19 0.21 Gs 1.07 37 1.53 0.27 1.58 0.16 G6 1.88 25 1.30 0.20 1.57 0.12 G7 2.08 24 1.86 0.17 1.31 0.12 G3 2.60 65 1.49 0.20 1.41 0.13 G9 2.30 51 1.47 0.16 1.45 0.11 G10 0.85 45 1.70 0.14 1.56 0.09 (312 1.00 101 1.25 0.15 1.75 0.08 613 2.54 133 1.27 0.15 1.52 0.09 615 2.49 162 1.07 0.13 1.63 0.08 G16 2.64 272 1.86 0.13 1.27 0.10 G17 6.36 330 1.16 0.13 1.32 0.11 621 2.50 369 0.95 0.11 1.70 0.06 G29 41 484 2.30 0.08 0.49 0.17 a . . . . . G: Generatron; Slope: the slope of the probrt lrne; R or response to selectron rs the difi‘erence in mean phenotype between the offspring of the selected parents and the parental generation before selection; S or selection differential is the average difference in mean phenotype between the selected parents and the parental generation before selection. 54 2.5 8 5 21 0 § 15 0 a: ' a 0 h- e 1 = E. 0.51 .2 > 0. 4'. r 5 F’ a -0.5 0 5 10 15 20 25 Cumulative Selection Differential Figure DJ. The rate of change of response in Colorado potato beetle to selection by Bacillus thufingiensis 8—endotoxins CryIIIA. Each dot represents the gain (deviation of response of the resistant colony from the unselected colony) from the selection in each generation. 55 There was no significant correlation between the heritability estimates and the increment of resistance (r=0.27, P>0.05). However, the heritability estimates in the first 12 generations showed a relatively significant correlation with the increment of the resistance ratio (r=0.80, P=0.01). Stande deviations of LC 505 over all generations (Figure 11.2) were not significantly different between selected and unselected colonies. (t=-1.84, df =25, P=0.08). This indicates that the standard deviations in both strains are of the same magnitude. ,__ 1-4 ‘ O Resistant 3% 1'2 ‘ O O I Susceptible .5 1 . 8 I 8 O D 0.8 ~ 0 I I) I I E 0.6 ‘ I O O a. I E 0 4 . 0' I 'l o 53 ° I I ' 0.2 i 0 t 4 4 ‘ T Generation Figure 11.2. Standard deviation (l/slope) of LC50 values of the Bacillus thuringiensis 8-endotoxin selected and unselected strains of Colorado potato beetle. Phenotypic standard deviations are not significantly correlated with the number of generations (Table 11.2). This indicates that there were no dramatic changes in phenotypic variances over many generations in both the selected and unselected populations. However, the variance does appear to decrease slowly through the experiment in both 56 strains. The negative slopes of the regression lines between the number of generations and standard deviations of both colonies indicate that the genetic variance decrease over generations. In general, genetic variance decreases as a result of directional selection (Falconer 1989). Table 11.2. Correlation regression analysis between standard deviation of LC50s and generation number STRAIN N r slope P Resistance 16 O. l 1 -0.003 0.69 Susceptible l 8 O. 17 -0.004 0.50 The principle effect of inbreeding in a population is to increase the frequency of homozygous genotypes. Thus, inbreeding measures the reduction of the frequency of the heterozygous individuals. Fixation is the consequence of inbreeding. Fixation occurs in a line when the line becomes homozygous for the same allele at a particular locus. F will be equal to one when the same allele is fixed in a population. The frequency of the homozygous individuals is 1, too. CryIIIA endotoxin resistance selection in Colorado potato beetle was started in six Michigan field sites representing different insecticide use histories. The density of Colorado potato beetle larvae was estimated to be between 2 - 6 million. After 2 years of 57 field selection, approximately 25,000 surviving beetles were collected and transported to the laboratory. The field selected beetles were maintained and randomly mated for 95:3 boas—one— ..om com: seaflocow can .383: omfiozw .8 women vows—330 m u HAEHUmDmh "<2 co woman was 882.8 as. 838.8 m 3.056%: "<3..— aseaa as. 88.836 a assesses n my... asses 6: .23 858:8 m 338F309»: n 2.5— “<2 no woman Baa—:28 m HA