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This is to certify that the thesis entitled IN VITRO AND IN VIVO EVALUATION OF CARBOXYESTERASE— BASED INSEC'I‘ICIDE RESISTANCE IN GREEN PEACH APHID (MYZUS PERSICAE (SUEZERH. presented by Dorothy Q' Hara has been accepted towards fulfillment of the requirements for Mastean—degree in mm Major professor Date 20 Novgtgber 1992 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State 1 University L___* PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE . 7 - ‘ H } v L i ‘ g :’ 1 I I {I "'r 'u ,4‘ I ‘1 I __1_{;_ MSU Is An Affirmative Action/Equal Opportunity inditution amount EV VITRO AND W VIVO EVALUATION OF CARBOXYESTERASE-BASED IN SECTICIDE RESISTANCE IN THE GREEN PEACH APHID, MYZ US PERSICAE (SULZER) (HOMOPTERA: APHIDIDAE). By Dorothy Shawn O'Hara A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE DEPARTMENT OF ENTOMOLOGY 1992 ABSTRACT BV VITRO AND EV VIVO EVALUATION OF CARBOXYESTERASE-BASED IN SECTICIDE RESISTANCE IN THE GREEN PEACH APIIID, MYZUS PERSICAE (SULZER) (HOMOPTERA: APHIDIDAE). By Dorothy Shawn O'Hara Green peach aphids (GPA) are efl‘ectively controlled by insecticides except in instances of resistance. The only known metabolic mechanism of resistance in GPA is increased carboxyesterase activity (Devonshire and Moores 1982). A rapid, accurate, and simple field test for resistance frequency quantification in GPA is needed. Resistance was diagnosed in this study by two difl‘erent approaches: an in vitro test quantifying esterase levels based on either a colorimetric method or polyacrylarnide gel electrophoresis of the enzymes and an in viva dosage mortality Bioassay was used to determine actual insecticide resistance levels. Strong positive correlations exist between the two sets of experiments, resulting in the conclusion that both of these sets of assays provide an efl‘ective measure of GPA carboxyesterase-based insecticide resistance. This information was then used to develop and evaluate a colorimetric carboxyesterase-based resistance diagnostic tool for pest management. iii This thesis is dedicated to the person who awakened my interest in science and believed I could accomplish a great deal. Thank you, Mom, I will never forget the many sacrifices you made for my firture. iv 0 Lord, our Lord, how majestic is your name in all the earth! (The Bible, NIV, Psalm 8:1). God has made everything beautifiil in His time. He has also set eternity in the hearts of men; yet they cannot fathom what God has done from beginning to end. I know that everything God does will endure forever; nothing can be added to it and nothing taken from it. God does it so men will revere him. (The Bible, NIV, Ecclesiastes 3:11, 14). AKNOWLEDGEMENTS I would like to gratefully acknowledge the assistance of my major professor, Dr. Mark E. Whalon, whose many hours of counsel and advice on both a personal and professional level have made the completion of this project possible. I thank him for seeing a potential in me that I could not see two years ago, and for the opportunity he has given me to learn about the basics of scientific research. I sincerely appreciate the suggestions, comments, and ideas submitted by the members of my graduate committee, Dr. James Asher of the Department of Zoology, and Dr. Edward Grafius and Dr. Edward Walker of the Department of Entomology, Michigan State University. Additional thanks are extended to all of the cooperators who aided my research by sending insects, to Dr. Alan Devonshire for his assistance in characterizing some of my strains, and to both the Mobay and FMC Corporations, who donated technical grade insecticide materials to our lab. I would also like to thank my fiiend Dr. Joel Wierenga for all of his toxicological insights and scientific advice regarding my project. The assistance of Utarni Rahardja, Debbie Miller, and Dr. Carlos Garcia-Salazar is sincerely appreciated. Special thanks are extended to the URC Women's Group for all of their love and support, and to Kaja Brix and Barb Strong for stress release and prayer, respectively. TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................ x LIST OF FIGURES ...................................................................................................... xii LIST OF ABBREVIATIONS ........................................................................................ xv CHAPTER 1. LITERATURE REVIEW AND INTRODUCTION .................................. 1 I. Introduction ..................................................................................................... 2 11. Integrated Pest Management ........................................................................... 3 A Definition and Approach ..................................................................... 3 B. Resistance Management ...................................................................... 3 C. The Use of Natural Enemies as IPM Control Strategies ........................ 6 D. Additional 1PM Techniques ................................................................. 7 II. The Green Peach Aphid .................................................................................. 7 A Geographic Distribution and Host Plants .............................................. 7 B. Biology ............................................................................................... 8 C. Genetic Variation: Recombinatin vs. Endomeiosis ............................... 11 D. Feeding Mechanisms ......................................................................... 13 E. Green Peach Aphid Pest Status .......................................................... 14 IV. The Virus Problem ................................................................................................... 15 A Virus Definition ............................................................................................. 15 B. Mode of Transmission .................................................................................... 15 C. Host Resistance .............................................................................................. 17 D. Viral Infections .............................................................................................. 17 E. Potato Viruses and Problems Associated With Them. ..................................... 19 V. Resistance ............................................................................... . ...... . ........................... 21 A Mechanisms .................................................................................................. 21 B. Green Peach Aphid Resistance Mechanisms ................................................... 24 C. The Problem .................................................................................................. 25 VI. Goals and Objectives ................................................................................................ 27 A Overall Goal .................................................................................................. 27 B. Objectives ..................................................................................................... 27 C. Hypotheses .................................................................................................. 27 D. Thesis Outline ............................................................................................... 28 CHAPTER 2. In Vitro Evaluation of Carboxyesterase-Based Insecticide Resistance ....... 30 I. Introduction .................................................................................................... 31 A Green Peach Aphid Identification ...................................................... 31 B. Resistance Evaluations ........................................................................ 31 C. Microplate Assay Introduction ............................................................ 37 D. PAGE Introduction ........................................................................... 40 E. Portable Introduction ......................................................................... 43 11. Materials and Methods ................................................................................... 44 A Strains ................................................................................................ 44 B. Microplate Survey .............................................................................. 50 C. Polyacrylamide Gel Electrophoresis .................................................... 53 D. Portable Evaluation of Carboxyesterase Levels ................................... 56 III. Results and Discussion .................................................................................. 58 A Strain and Microplate Survey Results ................................................. 58 B. Gel Results ......................................................................................... 81 C. Portable Data Results ......................................................................... 92 D. Conclusions ........................................................................................ 97 Chapter 3. IN VIVO EVALUATION OF CARBOXYESTERASE—BASED INSECTICIDE RESISTANCE ...................................................................................... 99 I. Introduction ................................................................................................ 100 II. Materials and Methods ............................................................................... 102 A Strain Selection ............................................................................. 102 B. Chemicals Selection Criterion ......................................................... 102 C. In vivo Assay Methodology ............................................................ 103 D. Data Analysis ................................................................................... 104 III. Results and Discussion ............................................................................... 104 A Strains Chosen. ................................................................................ 104 B. Chemicals Chosen ............................................................................. 106 C. In viva Bioassay ............................................................................... 110 D. General Discussion of in viva Resistance Analysis ............................. 124 CHAPTER 4. CORRELATION OF IN VIIRO AND IN VIVO EVALUATIONS OF CARBOXYESTERASE-BASED INSECTICIDE RESISTANCE ................................ 126 I. Introduction ................................................................................................. 127 II. Materials and Methods ................................................................................. 128 A Economic Evaluation ........................................................................ 128 B. Labor Evaluation .............................................................................. 128 C. Accuracy Evaluation ......................................................................... 129 D. Precision ......................................................................................... 129 E. Sensitivity ......................................................................................... 129 F. Overall Efl'ectiveness ......................................................................... 130 111me and Discussion ................................................................................. 130 A Economic Evaluations ...................................................................... 130 B. Labor Evaluations ............................................................................. 130 C. Accuracy Evaluations ....................................................................... 134 D. Precision Evaluations ........................................................................ 136 E. Sensitivity ......................................................................................... 136 F. Overall Efi‘ectiveness of Field Diagnostics ......................................... 136 IV. Conclusions ............................................................................................................ 137 V. Future Research Possibilities .................................................................................... 137 LITERATURE CITED ................................................................................................ 139 APPENDICES ............................................................................................................ 147 APPENDIX A Record of Depostion of Voucher Specimens ........................... 147 APPENDIX B. Equations of regression lines for standard curves found in the text .............................................................................................................................. 149 APPENDIX C. Portable standard curve of protein concentration versus abwmmcewovimserumalbunun)ug/100tdplonedagdnstabsorbance(609nm= protein). Thiscurveisqnvihnearandshowsthatndoesnotgivereadingswithafinear- typerelationshipuntiltheprotein concentration isator above 10 LIE/100M ................ 150 APPENDIX D. Toxicities of six difl‘ererrt insecticides (Ware 1983, Matsumura 1985) ........................................................................................................................... 151 LIST OF TABLES Page 1.1 Comparison ofaphid species which can act as vectors for four economically detrimental potato diseases while colonizing them (de Bokx 1987) .............................. 20 2.1 Code designation translations for green peach aphid strains ................................... 46 2.2 Strains, cooperators, host plants, and locations of populations on file in the Entomology Museum used for identification of strains as green peach aphid ................. 49 2.3 Listing of strains, cooperators, host plants, and locations of populations collected for the national carboxyesterase survey .......................................................................... 62 2.4 Results ofnational mrvey ofcarboxyesterase levels in green peach aphid using microplate assays: SEM (Standard Error ofthe Mean) and Tukey's test for significance ................................................................................................................ 76 2.5 Coeficients for correlation analysis of in vitro assays with microplate data across the eight main strains ........................................................................................................ 80 2.6 Comparison of azinphosmethyl resistance ratios (RR), densitometry unit values, portable values, and microplate values ......................................................................... 84 2.7 Summary of mean percent inhibition of green peach aphid strains using PAGE gels inhibited by carbaryl, oxydemetonmethyl, and permethrin (three replicates each) .......... 87 2.8 Coeficients for correlation analysis of in vitro assays with PAGE densitometry data across the eight main strains ..................................................................................... 91 2.9 Portable in vitro carboxyesterase assay results for eight main green peach aphid strains including Standard Error of the mean, and Tukey's Test for Pairwise Mean Comparisons with letters signifying statistically significant difi‘erences ......................... 96 2.10 Coefi'rcients for correlation analysis of in vitro assays with the portable carboxyesterase data across the eight main strains ...................................................... 98 3.1 Strains, cooperators, host plants, locations, and resistance levels for populations of green peach aphid used for in viva bioassay ............................................................. 105 3.2 Listing of insecticides used for the in viva bioassay of insecticide resistance in green peach aphid .......................................................................................................... 107 3.3 Listing of insecticide, compound class, and concentrations used for in viva bioassays on green peach aphid .............................................................................................. 111 3.4LC50valuesandslopesforeachinsecticideusedintheinviwbioassay ............... 118 3.5 Summary of resistance ratios for both LCso and LCgo's of six insecticides for eight strains of green peach aphid ........................................................................................ 122 4.1 Itemizedlistofcostsforinvitraassayprocedures ................................................. 131 4.2 Economics of the in viva bioassay for one chemical ............................................... 132 4.3 Labor evaluation of the four techniques for resistance diagnosis ............................. 133 4.4 Correlation coefiicients for regression analysis of in viva Bioassays with in vitro Assays ......................................................................................................................... 135 APPENDIX B Equations of regression lines for standard curves found in the text ....... 149 APPENDIX D Toxicities of six difl‘erent insecticides (Ware 1983, Matsumura 1985).. 151 LIST OF FIGURES Page 1.1 Schematicdiagramofthesisresearchproject ........................................................... 29 1.2 Basic external anatomy of the abdomen of an aphid (Aphididae) useful for identifying difi‘erent Families (Blackman and Eastop 1984) .............................................................. 32 2.2 Dorsalviewsofdifi‘erenttypesofaphid antennalmberclesusefirlforcomparison between Families: (a) undeveloped tubercles, (b) diverging tubercles, (c) well-developed diverging tubercles. (d) parallel. (e) conversing (We persicae), (t) well-developed median fi’ontal projection. Taken fiom Blackman and Eastop (1984) ............................. 32 2.3 Difl‘erent shaped of cauda found in Aphididae: (a) broadly rounded, (b) helmet shaped, (c) tongue-shaped, and (d) knobbed with a bilateral anal plate. Taken from Blackman and Eastop (1984) .................................................................................. 33 2.4 Aphid siphunculi of various shapes: (a) pore-like, (b) mammariform, (c) truncate, (d) tapering, (e) swollen proximally, (t) clavate, (g) with subapical zone of polygonal reticulation, and (h) with sharp spiky hairs. Taken from Blackman and Eastop (l984)...34 2.5 Structures and sequence for the reactions of carboxyesterases with naphthylacetate to yield naphthol. Naphthol reacts with O-dianisidine (tetrazotiaed) to yield a colored solution ....................................................................................................................... 39 2. 6 Structures of Acrylamide, N N’ methylene bisacrylamide, and Polyacrylamide gel2 (Hames and Rickwood 1984) ...................................................................................... 2.7 Map of locations for green peach aphid strains collected across the United States as part of the Microplate Carboxyesterase Survey ........................................................... 59 2.8 Map of green peach aphid strain collection sites in Michigan for the Microplate Carboxyesterase Survey ........................................................................................... 60 2.9 A paintbrush was used to transfer green peach aphids to clean potato plants for rearing in the greenhouse at Michigan State University ............................................... 61 2.10 Photograph of a slide mounted green peach aphid with the most visible taxonomic characters labeled for easy discrimination: (a) converging tubercles, (b) long antennae (as long as or longer than the body), (c) a tongue shaped cauda, ((1) long appendages, and (e) long, tapering or tubular siphunculi ............................................................................ 66 2.11 Rearing environment forgreen peach aphids in culture in agreenhouse at Michigan State University ....................................................................................................... 67 2.12 Microplate standard curves for a— (A) and B- (B) naphthol standard concentrations (rig)! 200 pl plotted against absorbance values (600 nm = a, 550 nm = B). Slopes of the lines are found in Appendix B ................................................................................ 68 2.13 Microplate standard curve for total protein (bovine serum albumin) standard concentrations (pg)! 10 ul plotted against absorbance values (600 nm = protein). The equationofthelineisfoundinAppendixB ............................................................ 70 2.14 Histograms of the cr- Naphthylacctate carboxyesterase assay showing total a- carboxyesterase levels, Tukey's Test for significance letters (a = .05, df= 110) ..... 71 2.15 Histograms of the B—Naphthylacetate carboxyesterase assay showing total B— carboxyesterase levels, Tuckey's test for significance letters (a = .05, df= 110)...74 2.16 Photograph of two native polyacrylamide gels strained for a-carboxyesterase activity. Strainsarelabeled aseitherRor S dependingontheirvaluesintheMicroplateAssay. The R strains of Gel A are: (1)MONTC1-MI, (2) MOXEEl-WA, (3) SALINAS-CA, (4) PRESQUE-ME, (5) STRATHAM-NH, (6) WOOSTER-OH, (7) CENTERZ-PA, (8) MADISON-WI, (9) ROCKINGHAMCO-NH. On Gel B the R strains are the same (1) - (6), with S strains of (7) TOPPENISH3-WA, (8) WILDERI-ID, and (9) PULLMAN- WA ...................................................................................................................... 83 2.17 Photograph of two native polyacrylamide gels stained for a-carboxyesterase activity. Gel A is the control gel, which was not incubated in any inhibitor (insecticide). Strains are labeled as R or S depending upon their classification by Microplate Assay. Strains are also numbered: (1)MONTC1-MI, (2)MOXEE1-WA, (3) WOOSTER-OH, (4) PRESQUE- ME, (5) STRATHAM—NH, (6) SALINAS-CA, (7) CENTERZ-PA, (8) MADISON-WI, (9) PULLMAN-WA, and (10)WILDER1-ID ....................................................... 86 2.18 Portable in vitro assay standard curves for cr- (A) and B—naphthol (B) standard concentrations (mg)/ 100 ml plotted against absorbance values (609 nm = a, 555 nm = B). TheequationsofeachlinearefoundinAppendixB ................................................... 93 2.19 Histograms of portable assay results across eight main strains. Letters signify Tuckey's Means Separation Test for significant difi‘erences between strains ................ 94 3.1 Regression lines depicting the relationship between probit values and lethal concentrations for azinphosmcthyl (A) and oxydemetonmethyl (B) ............................ 112 3.2 Regression lines depicting the relationship between probit values and lethal concentrations for parathion (A) and permethrin (B) ................................................. 114 3.3 Regression lines depicting the relationship between probit values and lethal concentrations for methomyl (A) and carbaryl (B) .................................................... 116 xiv APPENDIX C. Portable stande curve of protein concentration versus absorbance (bovine serum albumin) M 100 pl plotted against absorbance (609 nm = protein). Tldsanveiscrnvflinearandshowsthatndoesnotgivereadingswithafinear— typerelationshipuntiltheprotein concentrationisator above 10 ug/ IOOuI ............... 150 GPA PAGE Bisacrylamide sns IPM OP GSH XV LIST OF ABBREVIATIONS green peach aphid polyacrylamide gel electrophoresis N,N,N‘,N‘-tetramcthylethylenediamine N,N-methylene bisacrylamide sodium dodecyl sulfate Integrated Pest Management Acetylcholinesterase Biological Control microliter organophosphate insecticide Mixed function oxidase system Glutathion S-Transferase CHAPTER I INTRODUCTION CHAPTER 1. LITERATURE REVIEW AND INTRODUCTION I. Introduction The green peach aphid (GPA), Myzus persicae (Sulzer), is a principal pest on many important food crops both in the United States and throughout the world (Way 1971, Cancelado and Radclifi‘ 1979, Devonshire 1989). Millions of dollars are spent annually in an attempt to control this insect. On potato, Solarium tuberoswn (L), GPA is a major pest because of its vector potential and plant feeding damage (Kennedy et a]. 1962), and there is a direct correlation between GPA population density (including migration intensity) and the problem of virus transmission (Galecka and Kajak 1971). Until recently, growers were able to control this insect with soil systemic insecticides, such as aldicarb (Temik®). However, the voluntary removal of this compound (and others like it) fi'om agriculture due to suspected ground water contamination has resulted in the use of other systenrics followed by less efi'ective foliar aphicides (Preston et a]. 1990, Reed et al. 1990). With the fiequency of foliar applications, insects (GPA) can develop high levels of resistance (Tabashnik and Craft 1982). Resistance is the ability of sections of a population to tolerate or avoid potentially lethal or reproductively detrimental factors that would negatively influence a normal population (Pedigo 1989). Resistance results from the strong directional selection caused by repeated insecticide applications or use. The use pattern of foliar aphicides causes high levels of mortality in biological control organism populations (Reed et a1. 1990). At this time, potato growers are reporting increasing resistance problems. Prior to the period before efi‘ective control by aldicarb (1974), control of GPA was difficult to maintain. Currently registered insecticides did not control virus transmission (Adams 1950, Hille Ris Lambers 1953, Powell 1973, Bacon 1976), and the primary source of such viruses as PLRV is infected seed (Flanders et al. 1991). Although reduction is possible, complete prevention of viral transmission to potatoes by insects is not possible via insecticides alone, including aldicarb (V rllacarlos 1963, Powell and Mondor 1973, Woodford et al. 1983). For this reason, a resistance monitoring system by which growers can detect the incidence of resistance in vectors (including GPA) in their fields could aid agriculture greatly by substantiating whether or not spraying of a specific chemical is the most efi‘ective pest elinrination technique, or if other management methods need to be employed because the pests are resistant and are likely to survive the chemical application II. Integrated Pest Management A. Definition and Approach Integrated Pest Management (IPM) is a comprehensive approach to pest management which allows growers to make informed judgments with regards to crop production that are specifically tailored to their own fields. Geier and Clark outlined many of the pest management principles in 1961 (Geier and Clark 1961, Geier 1966), of which a key aim is the conservation of enviromnental quality while maintaining economically productive crop yields and efi‘ective pest control. IPM utilizes a holistic approach for decision making that incorporates multiple tactics with an extensive understanding of the entire agro-ecosystem of which each crop is a part. Included in this need for data is an evaluation of an individual growers crop situation, economic needs, yield capacity, pest level and pest resistance potential, and many of the other biotic and abiotic factors involved in crop production. In pest management decisions, the more information that is obtained regarding a crop ecosystem, the more effective decisions the grower is able to make. B. Resistance Management Inherent in IPM is the concept of pesticide resistance management. This is a method of controlling the development of resistance in pests. As stated previously, insecticidal compounds exhibit strong directional selection pressures on insect populations: they remove susceptible genotypes from a population. Resistance management involves many factors including selection of the appropriate insecticides for use on a specific pests and timing of chemical applications. Use and timing of insecticides, especially foliar sprays, are important because not only do these compounds vary in overall toxicity and mode of action, they also difi‘er in their efi‘ect on non-target organisms (Croft and Brown 1975). Ofien when fields are sprayed, populations of biological control organisms die in greater numbers than the targeted resistant pests as a result of both insecticide poisoning and loss of food supply (Croft and Brown 197 5). As a result, growers may begin to observe other species exhibiting characteristics detrimental to the crop that were heretofore unnoticed because the population size (and thus amormt of damage) was controlled efl‘ectively by natural enemies. Once these natural controls are eliminated, the species is able to flourish and cause significant damage to the crop (Peterson 1963, Radclifi‘ 1972, 1973, Mackauer and Way 1979). In addition to the elimination of biological control organisms, a strange phenomenon of resurgence is associated with certain chemicals: they have the ability to cause accelerated growth and increased fecundity (greater numbers of ova produced) in some pest species that are resistant to the chemical (Peterson 1963). In this case, the pest population is not killed off, but increases significantly as a result of the spray. Chemicals targeted at other pests can also significantly accelerate the development of resistance in non-target pests. For this reason, care must be taken to choose the correct insecticide and spray at optimum times for highest target-pest mortality. This may help to eliminate many of the problems associated with sprays and can enable more efl‘ective pest control. One factor in resistance management is the practice of attempting to reduce unwarranted sprays. This is important for maintaining the lowest frequency of resistance possible in pest species while sustaining an econonrically feasible level of pest population suppression (Stern er a1. 1959). In order to determine what is economically feasible pest damage, the concept of a pre-determined economic threshold for spraying crops was established. When pest populations are below a set "threshold", damage caused by pest species results in less financial loss than spraying, and therefore no spray is necessary. However, if pest population numbers are at or above the economic threshold, sprays are necessary to reduce the pest species population because above this threshold level the pests are in significant enough numbers to cause extensive damage to the crop. An example of this is that in Minnesota, there is a 10 GPA/ 100 leaves threshold in seed potato production (Cancelado and Radclifl‘ 1979). The use of economic thresholds helps to eliminate urmecessary sprays, but it is not as simple to implement as a calendar spray system. This type of pest management involves pest monitoring (sampling and resistance testing) by trained scouts, a detailed knowledge of pest and natural enemy populations present in fields, resistance frequencies of pest species, and similar types ofdecision criteria. The use of resistance management techniques such as IPM and economic thresholds results in retarded resistance development because pest populations are controlled at the optimum times resulting from monitoring and reduction of urmecessary sprays causing accelerated resistance development (Cancelado and Radcliff1979). Another key concept in the elucidation of IPM strategy and resistance management is the extension of pesticide use-life. Pesticide use-life relates closely to avoidance of unnecessary sprays and the use of econonric thresholds. Multiple sprays quickly shorten the use life of pesticides and can necessitate the use of synergists or nrixtures of several insecticides to increase the toxic action of the compound on the targeted pest insects. Pesticide use-life is the length of time an insecticide (or pesticide) can be used to control the targeted pest species effectively. In order to lengthen pesticide use-life beyond the initial span of time, some growers resort to insecticide nrixtures to promote higher mortality levels in insects, hoping for a multiplicative toxic effect on the insects. Others utilize synergists, such as piperonyl butoxide to promote higher morbidity in pest species. Although synergists and mixtures do increase the toxicity of pesticides, there is much doubt as to the advisability of these methods, especially regarding rrrixtures of several insecticides. There is some evidence that mixtures of insecticides applied to a crop tend to increase the development of cross resistance in insects exposed to them, and can even accelerate the development of resistance more strongly than a single insecticide alone. C. The Use of Natural Enemies As IPM Control Strategies Another IPM strategy involves the promotion of natural enemies, or biological control organisms. This can be challenging in sprayed ecosystems since most commonly biological control organisms are more susceptible to insecticides than pest species. Although some claim that natural enemies are not effective for controlling insect vectors at an economically productively level once they are present on plants (Broadbent 1953) others disagree, claiming that these natural enemies can be relatively efl‘ective for control of GPA (Tarnaki and Weeks 1972, Tarnaki 1973, Powell et a]. 1974). Among the most important predators of GPA are coccinellids, clnysopids (green lacewings), and syrphid flies (Croft 1989). Some claim that predation has a strong effect on GPA mortality while others disagree. In a population dynamics study conducted in Sicily in 1972, Barbagallo et 01. observed that few aphids were killed by fungi and parasitoids, while predators were the causative agent in 21% of the total aphid loss. Additionally, 26.4% of the aphid population loss was caused by emigration, and Barbagallo et a1. claim that this shows that predation is not a reliable means of controlling the pest levels below the economic threshold (Barbagallo er al., 1972). By contrast, Mack and Smilowitz (1978, 1981) claim that biological control organisms can be effective at controlling GPA populations. They found eighteen species of GPA predators in a field, with coccinellid adults and lacewing adults as the two most important types (Mack and Smilowitz 1978, 1981). Syrphid fly adults, however, were rare. It seems that although natural enemies can be an effective means for GPA control, alone they are not enough to control the spread of viruses to potato by GPA D. Additional IPM Techniques In addition to the pest resistance management methods previously mentioned, there are multitudes of other management tactics. Some of these strategies involve use of the difl’erent cultrual practices used on crops. Cultural applications can include practices to control pest plants (weeds) and insects, planting (such as monoculture or polycultures and rotation), fertilizing, irrigation, and sanitation practices, selection of tolerant or resistant (to the pest) crop varieties, and any other factors which could influence the crop's quality and resistance or promotion to pest activity. In order to know when to implement these, pest monitoring is a necessary part of both the culture practices and the IPM system as a whole. III. The Green Peach Aphid A. Geographic Distribution and Host Plants Green peach aphids have a very broad distribution and host plant range. They are found throughout most of the world, although most connnonly in the northern parts of the temperate zone. This range includes most of Europe, the United States, East Asia, and much of Central America (Blackman 1974, 1984, and Connnonwealth Institute of Entomology 1954). GPA are considered polyphagous leaf aphid species with an extremely large host plant range (Dixon 1985). They are heteroecious and holocyclicl between peach, the primary host (and other Pnams species), and secondary hosts such as potato (I-Ielle et al. 1987). GPA is (able to feed on a variety of hosts) on secondary species where peach is absent, and in temperate climes (Blackman 1974). The considerable flexibility (climate and host-wise) of this insect is believed by some to contribute to its ability to evade control measures successfully and to rapid adaptation to new habitats. GPA are found on most cultivated plants. Some potential hosts are: plum, cherry, prune, citrus, cabbage, dandelion, endive, mustard greens, parsley, turnip, tobacco, rose, spinach, peppers, beets, celery, lettuce, chard, and potato (Blackman 1984). The sermal generation of GPA oviposit overwintering eggs on peach, plum, and cherry in colder climates (Blackman 1984). In warmer areas of some states, such as Arizona, California, Oregon, and Washington they overwinter as adults, and some believe they do so in colder climes under the snow (Takada 1974). B. Biology Greenpeachaphidsareactiveinthespringandsunnner, andthroughthefall. Theyare found clustered together in an unequal distribution on the plant's rapidly growing or senescent leaves (Bradley 1952, Taylor 1953, 1962, Mack and Smilowitz 1981, Jansson and Smilowitz 1985, Nderitu and Mueke 1989). The reason that they prefer senescent tissue may be that phloem sap concentrations change as the leaves senesce (Thomas and Stoddart 1980) and they contain higher nitrogen concentrations (Kennedy 1958, Jansson and Smilowitz 1985). This in turn induces more rapid growth and reproduction in GPA (Jansson and Smilowitz 1985). Additionally, GPA are found in a clustered distribution lHeterocious (alternating host plants) and holocyclic (having a sexual generation) because they reproduce parthenogenetically throughout much of the summer, and once an aphid finds a suitable site for feeding, they remain sessile. They also maintain close proximity with other GPA for protection purposes because they do not move very rapidly and their defense mechanisms are linrited. The life cycle of GPA is a sequence of different morphs (fornrs) triggered by environmental factors (Lees 1966, Hille Ris Larnbers 1966). GPA have two principle forms in their life cycle: alate (winged) and apterous (wirrgless), and they exhibit considerable variation in color. Takada observed the occurrence of both green and red aphids in a sympatric population of GPA with red color controlled by a dominant allele (Takada 1981). The apterous adults are light to medium green with a yellow tint in summer (Blackman 1984). In the fall the cormnon color morph is red. Alate GPA are brown to black with a yellow abdomen. GPA is also known to some as the tobacco aphid (has been defined as a separate solely parthenogenetic species) or the spinach aphid. The cyclical lifecycle of GPA with several parthenogenetic generations in spring and summer with an armual sexual generation in the fall is called "cyclical parthenogenesis" (Blacknran 1974). However, some adults are thought to overwinter under the snows in colder climates (Takada 1974), and other alates could migrate north on wind currents in the spring. Ideally, after several asexual generations, the sexual adults lay eggs in the fall. Those eggs laid the previous spring overwinter and emerge, and the cycle begins anew with parthenogenetic generations the following spring. This alternation of sexual and asexual forms is called ”holocycly" by Blackman, while anholocyclic parthenogenetic populations, such as greenhouse populations, are derived fiom the holocylic populations (Blackman 1974). Any population can exhibit holocycly, anholocycly, or a combination of the two, according to Blackman (1974). 10 The reproductive category (ovipara or viviparaz) is usually deternrined by environmental stimuli, usually photoperiod (Helle 1987). Generally, the first generation, which is found during springtime with high humidity, are asexual apterous females that are larger than the other stages, and called fundatrices or stem mothers (Helle 1987). Not only is the fimdatrixofGPAlargerinsizeandplumperthanfirtureprogeny, withsmallereyesand head, shorterantermae, legs, cauda, andsiplnmculi,itisalsomorefecundduetoitshaving more ovarioles (Blackman 1978, Helle 1987). These characters are called the fundatrix facies (Les 1961). Later generations are asexual or sexual depending on temperature, photoperiod, plant quality, and possible humidity. After the fundatrix generation, GPA commonly reproduce parthenogenetically. Some believe that GPA parthenogenetic reproduction is a form of paedogenesis, or reproduction by sexrrally irmnature or larval forms of the insect (Urchanco 1924). Others, such as Takahaslri disagree, explaining that GPA parthenogenesis is not paedogenesis (1924) but a form of asexual reproduction that is not paedogenesis. Additionally there has been much debate as to whether GPA are aponrictic (parthenogenesis which results in progeny genetically identical to the mother) or autonrictic (allowing for recombination of a sort and thus genetic variation within parthenogenetic lines) (Helle 1987). In line with this, Cognetti coined the term endomeiosis for the form of autonricty found in aphids (1961). Endomeiosis involves alleles crossing over, or the exchange of alleles between homologous chromosomes during prophase of meiosis within the nucleus of the oocyte (Cognetti 1961, Beranek and Berry 1974, Helle 1987). This allows for a segregation of the alleles at the loci for which the mother is heterozygous (Helle 1987). Blackman 2Ovipara (Producing eggs which hatch to produce young) and Vivipara (producing live young). ll claimed that there was no evidence for endomeoisis in GPA (1978). On a more basic line, Suomalainen et a1. argued that this was not actual recombination because the alleles are combined within the same nucleus (1980). However, others disagree, claiming that Suomalainen et al. (1980) overlook the fact that recombined chromosomes would be lost to the polar body at the maturation division (Helle 1987). Baker claims that endomeiosis could occur, however it must be either rare or require special circumstances. It cannot, according to Baker, account for the large changes of resistance frequencies found in the electrophoresis of biotypes over short time intervals in the field (Baker 1978). C. Genetic Variation: Recombination vs. Endomeiosis Because of their ability to reproduce parthenogenetically, aphid lines have the potential to develop a virtually unlimited nmnber of alterations to the ancestral karyotype. Over time these segregated lines can become an all but separate line of progeny (Helle 1987). Aphids are expected to show more variation within species than many other organisms because of the holocentric nature of their chromosomes (no localized centromeres). This allows fiagrnerrts of their clnomosomes to perpetuate themselves through many generations because the entire chromosome has centromeric activity, permitting them to orient correctly at the equatorial plate at mitosis and tlnrs replicate nommlly and pass into the two daughter cells (Helle 1987). One example of such variation is the genus Ampharaphora, which shows a range of clnomosome numbers form n=2 to n=36. Population difl‘erences are due entirely to dissociations and fusions of the autosomes, as the X-chromosomes remain unaffected throughout (Blackman 1980). In the field there may be a mixture of (GPA) clones (from difl'erent mothers) during the summer generations that are genetically isolated and can recombine only in the sexual (fall) generation (Reinink et al., 1989). One example of this type of segregative 12 adaptation is that although as a species GPA are highly polyphagous’, clones from one mother can become restricted to certain host plants because of adaptations (Weber 1985). Takada agreed with these findings in a study he conducted in Japan (Takada 1979) This same adaptability seems to apply to insecticide resistance and other similar genetic adaptations, not just to feeding preferences. Thus, a population of GPA may be extremely resistant to one chemical or class of chemicals (such as organophosphates), but susceptible toanotherchemicalfi’omadifl’ererrt(oreventhesame) classofcompounds. In the sexual generation progeny have genes contributed by both parents, thus yielding classical genetic recombination This is significant in that the most fit parthenogenetic linesarecontinued, otherswillbeselectedagainstandelinrinatedduringthesexual generations. The most successfirl lines will continue to contribute to the population season alter season (Blackman 1974). According to Blackman, populations that are continuously parthenogenetic (such as greenhouse populations) do not have this fitness selection mechanism available by genetic recombination (Blackman 1974). He states that parthenogenetic populations appear to have a reduced ability to respond to selection pressures, since adaptive mutations that arise in individuals are not shared with other individuals (do not enter the gene pool) because they cannot recombine by sexual reproduction (Blackman 1974). Blackman claims that although endomeiotic recombination would help to achieve homozygousity, and therefore show the adaptive significance of recessive alleles, more often than not heterozygous individuals are superior (Blackman 1974). Heterozygousity can be maintained in autonrictic populations as long as selection favors those individuals (Asher 1970), but maintenance of heterozygousity 3Polyphaguusrrreanstlurttheseinsectsarecapableoffeedingonawidevarietyofhostplants. 13 does not imply adaptability, since most adaptations involve a loss of some heterozygousity (for example: resistance). On an opposite tack, Carson (1967) maintains that parthenogenesis maintains heterozygousity. Blackman claims that although endomeiosis could bring about one more adaptive step, its evolutionary significance is dubious and for this reason the sexual generation is very important for maintaining heterozygosity and therefore adaptability within a holocylic aphid population (Blackman 1974). Darlington (1937) and Suomalainen (1950) claimed that parthenogenesis is an evolutionary dead end. Blackman fails to note that resistance is associated with greenhouse populations of parthenogenetically reproducing aphids, and many resistance researchers feel that field resistance may have originated in greenhouse populations (Mark Whalon, personal cormnunication). He seems to ignore the fact that although heterozygousity increases an organism's ability to adapt, in insecticide resistance situations those individuals homozygous for resistance tend to be better fit to withstand chemical treatment, even without the help of genetic recombination found only in sexual reproduction. D. Feeding mechanisms GPA have piercing and sucking mouthparts for feeding. They ingest food by insertion of their mouthparts (styli) into the phloem tissue of the plant, and this enables them to extract fluids. The mechanism of their feeding is such that upon insertion of their styli (the maxillary and mandibular styli) there is a pharyngeal pump which forces the plant fluids into the foregut through the food canal (Ponsen 1987). The sequence is as follows: liquid food is forced through the maxillary and mandibular styli, the pharyngeal duct, and the pharyngeal pump, through the head and over the tentorium. Next it flows into the foregut (where there are spherical symbionts), then to the midgut and on to the hindgut. The lrindgut connects to the arms (or rectum), fi'om which honey dew is excreted. The aphid 14 filter chamber takes the place of the Malpighian tubules which are found in other insects (Ponsen 1987). Wastes, constituted of excess sugars ("honeydew") flows out of the anus. Honeydew is a sticky substance which can be used as a carbohydrate source by other insects (and sooty or other molds) because most of the sugars found in plant phloem are left behind by the aphids' digestive tract (aphids are generally nitrogen-limited rather than carbohydrate-limited). Ants in particular are known for cultivating the aphids that produce honeydew, even to the point of "farming" the aphids, providing shelter in the winter and protection from predators. These ants have developed a "milking" behavior which involves stroking the GPA until they ermde some of the honeydew. Thus they have evolved a symbiotic relationship that is mutually beneficial to both parties. E. Green Peach Aphid Pest Status GPA are considered secondary pests on many crops, because most of the damage is not direct but a result of viral infection. This is true of the crop of focus for this study, the potato (Salomon tuberasum L.). Approximately thirty million dollars is spent arnnrally in attempts to control this pest (Anon. 1991). Damage is caused by both viral infections and direct feeding, although the feeding damage is not economically significant. The primary damagecausedbyGPAisthat ofvirus transmissionto the host plant duringfeeding. Aphids are the largest group of vectors that transmit disease to plants, transmitting more than 300 known viruses (de Bokx 1987). Barbagallo er a]. consider GPA to be the most active vectors of potato viruses (Barbagallo er a]. 1972, Nderitu and Mueke 1989). The most economically important viruses that GPA vectors to potato are Leafi'oll Virus (PLRV) and Potato Virus Y (PVY) (Beemster 1987, Nderitu and Mueke 1988). 15 IV. The Virus Problem A. Virus Definition Anatomically, viruses are simple particles. Viruses are made up of virions, or virus particles, which consist of nucleic acids in the form of double or single stranded DNA or RNA Encasing the nucleic acids is a protein coat (envelope), or capsid, which helps protect the genetic material inside (van der Want 1987). These particles require the metabolic capabilities ofa cell inorderto reproduce. Theytherrlysethecell and spread to surrounding cells, steadily reproducing and lysing, thereby causing the outward symptoms of dead and necrotic tissue and other forms of disease found in plants (and animals). There has been much debate over whether or not viruses are living organisms or just organized conglomerations of nucleic acids and proteins. Much of this stems fiom the fact that although these particles can reproduce, they require the use of another cell's metabolic machinery to accomplish this. Other than the reproductive factor, and the fact that viruses contain mrcleic acids, they exhibit no characteristics necessary to define them as living. B. Mode of transmission Viruses are transmitted in various ways. The three types of transmission are (1) mechanicaL (2) circulative, and (3) propagative. Mechanical transmission of the virus depends on some sort of physical transfer, such as the aphid feeding for a short time on several plants (testing them) before settling on one (Sylvester 1949). The virus can be transmitted by physical contact, such as on the mouthparts (stylets). Some examples of mechanically transmitted viruses are Potato virus M (PVM), and Potato virus S (Rich 1983). Ifthe insect feeds on several plants the virus can be spread to each plant. In this case the virus is often (but not always, as is the case with PVM) stylet-borne, and non- persistent (Sylvester 1969). l6 Circulative transmission occurs by a persistent means when the plant is colonized. Potato leafi’ollvirus(PLRV)isgenerallytermed apersistentvirus, sinceit istransferredbyaphids which colonize the host plant. In this case the virus may be stylet-borne, but may circulate in the insect, but the virus does not infect the insect, it simply "circulates". The last type of viral transmission is called propagative transmission. This means that the virus multiplies within the aphid as well as within the host plant (Sylvester 1969). PLRV can also be a propagative virus, but only in GPA (Stegrnan and Ponsen 1958). In this case, not only the host plant, but the aphid are infected with the virus. For this type of transnrission, the virus has to have entered the aphid previous to transmission. It requires a period of time for the aphid to obtain a virus while Mug on an infected plant. This period is called the "acquisition period”. The virus then enters the aphid's digestive tract, moves tluough the intestinal wall and into the hemolymph, through the accessory gland and into the salivary canal. In the salivary canal the virus becomes mixed with the saliva and is excreted. Thus it enters the next plant's phloem during feeding (Black 1959). There is often a latent, or "incubation" period, between acquisition and the next viral transmission This occurs because the viral nucleic acids (genome) must be liberated and take control of the cell's metabolic protein synthesis mechanisms in order to replicate. The viral genome causes the cell's protein synthesis mechanisms to form complete replicates of the viral genome, down to the protein coat (van der Want 1987). All of this takes time; to travel through the insects body and to replicate for further transmission. The inoculation period is the time of feeding (by the vector) required for the virus to be transmitted fiom the aphid to a new host plant. 17 C. Host Resistance To difi‘erentiate the various abilities of hosts to withstand virus replication, terminology has been adapted to describe hosts that resist virus infection. A virus can infect one of two kinds of hosts: the susceptible host or the resistant host (Swenson 1969). The susceptible host allows a virus to infect it and multiply within its cells. The resistant host may allow a virus to infect it, however the virus cannot replicate within the host's cells (van der Want 1987). In order for a virus to infect a host (to replicate within a host's cells), it must be able to cross a series of barriers. [fit is unable to cross any one of these barriers, it cannot infect the organism. There are four main barriers in GPA: (1) the uptake of the virus by the mouthparts during feeding, (2) passage through the gut wall, (3) passage in the body of the aphid via the hemolymph, and (4) the passage into the salivary glands and out into the next plant. The most crucial barrier is the passage through the salivary glands (Rochow 1969). This may be because aphids have no Malpighian tubules, and so the salivary glands may act in part as excretory organs (Rochow 1969). And this is one way the virus can be transmitted (circulatively or propagatively. D. Viral Infections The virus can infect the plant either locally or systemically. Ifthe infection is local, the virus is confined to a specific area and it cannot spread throughout the plant. Local infection is to the plant's advantage, because if the part of the plant infected with the virus dies ofi‘, so will the virus. In the case of the systerrric infection, however, the virus has spread throughout the plant and the plant is infected for its lifetime. Thus, vegetatively reproducing plants (like potatoes) often transmit the virus to their progeny. Ifthe plant is infected as part of the initial inoculation into the field, it is known as a prirrrary infection. If the plant was infected as a result of spreading from the primary infection throughout the field, it is known as a secondary infection (van der Want 1987). 18 Plants have many means of dealing with viruses and the symptoms they cause. In some cases,theplantwillnot showanyobvious symptomsthatithasbeeninfectedwithavirus. This is called an asymptomatic infection, and could be beneficial because lack of symptoms shows that the host plant is tolerant of the virus (van der Want 1987). The other type of infection is called symptomatic, and this is what is generally recognized as a "viral infection”, ordiseasecausedbyavirus. Generally, leafrolling ornetnecrosis(tuber darkening in potatoes) are examples of symptomatic viral infection. Some species of plants may be more tolerant of certain viruses than others, and although this does not eliminate the virus, it limits the damage done by the virus (economic thresholds). Another response is hypersensitivity to the virus, which is comparable to an allergic reaction in humans. Hypersensitive individuals have an advantage because they are so sensitive that the material where the virus is located may die ofi‘ and thus localize the infection. Some plants wall ofl‘ infected cells, sacrificing those cells and allowing the virus tokillthemandthennmoutofcellstoconsumeintheareaandthereforedieofl‘. This localizes the infection, and eventually eliminates it. In this way the plant has a kind of field resistance to the virus (van der Want 1987). Other mechanisms used by plants include ”pitching out", which involves a release of resins fiom the plant, or secondary compounds for use both in combating the virus and in keeping the vector away. Some plants have specialized hairs or trichomes in order to keep the vectors away, a few types of these can even release sticky or toxic substances to aid in elinrinating vectors. Other plants have a thickened waxy layer (cuticle) to combat pests (and prevent water loss) which can assist the plant by making it more diflicult for the vector to transmit the virus. Since most viruses are obtained by plants through some type of injury or another, the waxy layer can assist in keeping such injuries from occurring (de Bokx 1987). 19 E. Potato Viruses and Problems Associated With Them Although seldom lethal in potato, viruses reduce both yield and quality (N deritu and Mueke 1989). One example of a virus transmitted to potato by GPA is potato leafroll virus (PLRV). In a symptomatic infection, PLRV has an outward manifestation of potato phloem necrosis and potato leafioll. The results of infection by PLRV are smaller tubers and net necrosis, with losses up to and even exceeding halfof the yield. Transmission of this virus is accomplished in a persistent manner solely by aphids in nature, with GPA as the most eficient vector (Peters 1987, Nderitu and Mueke 1989). There are many other viruses which GPA and other aphids transmit which are major problems. Some of these are shown in Table 1.1 (de Bokx 1987). One point of interest is that potato viruses, according to Avery Rich (1983) are not transmitted through the true seed, but are tuber-perpetuated through clonal propagation. This means that every new cultivar should be flee from viruses (in theory) until infected, which must happen during the testing and vegetative seed increase processes before they are sold to producers. However, the main source of viruses in the field is infected seed (Flanders et al. 1991), therefore infection of seed is occurring at some point. Potato crops are protected fiom virus infection by three factors: maintaining and planting only virus-flee certified seed potatoes, control of aphid virus vectors, and restricting the sources of overwintering viruses (reservoirs) to prevent firture re infection the following year (Bishop 1967). With the loss of aldicarb and other soil-systemic insecticides, control of viruses relies largely on maintenance of virus-free certified seed potatoes, cultural practices (sanitation and rotation) and timing of foliar insecticide applications to control aphids at the optimum times (Reed et al. 1990). Reed et al. (1990) suggest strong insecticide control measures early in the season to eliminate the virus vectors from plants 20 Table 1.1—Comparison of aphid species which can act as vectors for four economically detrimental potato diseases while colonizing them (de Bokx 1987). Aphid species PLRV‘ PVY" PVAc PVM‘I Aphis craccivara Koch . - A. goswiifrangulae complex - a: - a: A. nasturtii Kaltenbach + + + + Aulacorthum solam' (Kaltenbach) + a :h + Macrosiphum eupharbiae (Thomas) + + + + Myzus ascalanicus Doncaster + - Myzus persicae (Sulzer) + + + + If' + + Rhopalasrphoninus Ian’sipharr (Davidson) aPotato LeafRoll Virus, 5Potato Virus Y, cPotato Virus A, “potato Virus M. +aphidisavector,:l:aphidcanbeavector, -aphid is notavector, . notknown 21 newly emerging form the soil. Such procedures as these can help contain the spread of viruses. V. Resistance A. Mechanisms There are many mechanisms of resistance in insects, they can involve metabolic detoxification or avoidance 'of mortality factors. There are four main classifications recognized as mechanisms of insecticidal resistance: behavioral, reduced or slowed penetration, altered target site, and metabolic mechanisms. 1. Behavioral Behavioral resistance is defined by Lockwood er a1. (1984) as "those actions, evolved in response to the selection pressure exerted by a toxicant". These behaviors can involve the use of refirges during pesticide sprays, avoiding certain types of foods at certain times, and other avoidance types of behaviors. A classic example of behavioral resistance is the avoidance of DDT-treated walls and ceilings of huts in Afiica during the malaria eradication programs. Certain Anopheles mosquitoes would not light on the walls and ceilings of huts whereas other mosquitoes would land on the walls and ceilings. After treatment with DDT, those mosquitoes which would not land in the huts were selected for and survived in greater numbers than those which would land, illustrating the advantage of difi‘erent behaviors for survivability (and the development of resistance). 2. Slowed Penetration Penetration resistance involves the presence of barriers of some sort (generally physical) which prevent the uptake and concentration of toxicants to lethal levels. This can involve things like thickened cuticles or changes in the chemical structure of the cuticle on insects, 22 which prevent both absorption of toxins and injuries to the exoskeleton that promote absorption. 3. Altered Target Site Altered target site types of resistance involve some type of change to the target macromolecule so that it is less sensitive to the action of the toxicant (Vlfierenga 1992). Altered target site insensitivity has been documented in several species (Hama and Iwata 1978, Devonshire and Moores 1984, Oppenoorth 1985) including the Colorado potato beetle (\Vrerenga 1992). An example of altered target site resistance is altered acetylcholinesterase (AchE) in the Colorado potato beetle, as an insecticide resistance mechanism against carbofirran (Merenga 1992). 4. Metabolic Metabolic resistance involves the prevention of toxicants from reaching lethal levels by enzyme degradation and sequestration or secretion This type of resistance means that toxins are being attacked by some enzyme found in the body of an insect to produce an excretable metabolite that is not necessarily less toxic to the insect as the parent compound. In some cases production of an excretable metabolite first involves activation, which produces a more toxic substance that is then detoxified. An example of this is malathion detoxification: first malathion is activated to form malaoxon, then this is detoxified. Metabolic resistance is one of the most irnportant forms of insecticide resistance. There are four main types of metabolic resistance mechanisms. These mechanisms include the glutathion transferases, mixed function oxidases (MFO's), and esterases. 23 The Glutathion S-Transferases (GSH) are a family of compounds involved in detoxification. Substrates for GSH must be somewhat hydrophobic, rrrust contain an electrophillic carbon atom, and they must react non-enzymatically with glutathion at some rate that is measurable (Klaassen 1986). These compounds can react with insecticides to form excretable metabolites (generally detoxification reactions Williams and Weisberger 1986) and thus enable the insect to be resistant to that compound. The Cytochrome-P450—containing monooxygenases, mixed function oxidases (MFO's or cytochrome-P450) are part of a coupled enzyme system that contains both cytochrome- P450 and NADPH-cytochrome-P450-reductase (Sipes and Gandolfi 1986). This is one of the most important groups of enzymes involved in the biotrarrsforrnation of toxins (such as insecticides) (Sipes and Gandolfi 1986). The MFO system has many functions, including catalyzing the oxidation of various compounds. Some examples are desulfirration, oxidative dehalogenation, sulfoxidation, and deamination of toxins (Sipes and Gandolfi 1986). Often compounds are activated by this system, resulting in oxon compounds, which may be more toxic than their parent compound. A common mechanism of resistance in insects, MFO's have not been implicated in GPA insecticide resistance. Esterases are compounds that hydrolyze ester bonds to form a carboxyl group and an alcohol (Sipes and Gandolfi 1986). Esterases commonly hydrolyze bonds in such substances as organophosphate insecticides. There are several classifications for esterases: (l) arylesterases, which preferentially hydrolyze aromatic esters, (2) carboxylesterases (or carboxyesterases), which hydrolyze aliphatic esters, (3) acetylesterases, which preferentially hydrolyze acetyl esters, and (4) cholinesterases, which hydrolyze esters that have choline as the alcohol moiety (Sipes and Gandolfi 1986). These classifications do allow for some overlap, however, as carboxyesterases also catalyze the hydrolysis of aromatic esters. 24 Increased metabolism by nonspecific esterases is a common mechanism of resistance to organophosphate insecticides in insects (Oppenoorth 1985), especially in homopterans, such as aphids and whiteflies. The only known mechanism of metabolic resistance in GPA is carboxyesterase hydrolysis of the toxin (Devonshire and Moores 1982). This mechanism is due to amplification of the E4 gene which causes overproduction of esterase-4 (FE4) or E4 (due to an A1,3 translocation) (Devonshire 1977, Devonshire and Sawicki 1979, Devonshire and Moores 1982, Devonshire et al. 1986). Resistance due to the increased FE4 is stable and non-inducible (Blackman and Takada 1975, Devonshire er al. 1986). Resistance associated with the A1,3 translocated gene (E4 enzyme) is not stable (Blackman et al. 1978), due to the fact that the gene can be methylated and thus produce lower levels of carboxyesterase (show a more susceptible phenotype) (Field at al. 1989) after several generations in the absence of selection (Bauernfeind and Chapman 1985, Georghiou 1963, Dunn and Kempton 1966). This revertancy and recovery (with selection) is associated only with the A1,3 translocated gene (Ffrench-Constant et al. 1988), which is related to extremely high levels of resistance (Devonshire and Sawicki 1979, Sawicki et al. 1980, Devonshire et al. 1986). Kirknel and Reitzels (1973) and Dunn and Kempton (1966) did not note any revertant tendencies in their populations, however the time of revertancy seems to vary with population, perhaps it is due to genotypes or to external factors other than chemical selection B. Green Peach Aphid Resistance Mechanisms Insecticide resistance was first reported in aphids in 1928 (Boyce 1928). At this time it was artificially induced in Aphis gossypii Glov. by selection with hydrocyanic acid. Resistance in the field was reported by Michelbacher et al. in 1954 with Chromaphis juglandicala (Kalt) to parathion. Insecticide resistance was first documented in GPA in 1954 by Anthon. Later, Shirk (1960), Georghiou (1963), Gould (1966), Wyatt (1967), 25 FAQ (1967, 1969), Baranyovitz and Ghoush (1969), and Hurkova (1970) confirmed the observation that GPA had begun to exhibit insecticide resistance. In GPA, resistance is correlated to elevated carboxylesterase level (Needham and Sawicki 1971, Beranek 1974, Devonshire 1975, Devonshire and Needham 1975). However, this is not true in all insect species. In the resistant mosquito, higher carboxylesterase activity was associated with low B—naphthyl benzoate hydrolysis and the opposite was true in the case of the two-spotted spider mite (Motoyama and Dauterman 1974). According to Needham and Devonshire, in all GPA populations tested, resistance was associated with increased carboxylesterase activity (Needham and Devonshire 1974). One interesting thing about this is that it certainly seems to refirte the concept of Macro evolution, while supporting Micro evolution. If Macro evolution were the driving force in insecticide resistance, one would expect all insects to have carboxyesterase resistance mechanisms, MFO's, and the like. Additionally, one would expect all insects with carboxyesterases to have similar mechanisms, so that one test would work on all resistant species. But this is not the case, therefore, it looks like evolution on the rnicroscale has more involvement in this case than Macro evolution C. The Problem Green peach aphids are a serious pest in both Michigan and the rest of the United States. In Michigan alone, GPA are estimated to have caused over $0.5 million worth of damage annually, chiefly to the seed potato industry. The conventional and once successful current measures of control were soil-applied granular systemic insecticides united with foliar applications. These chemicals are especially important in seed potato production because other biological control or natural control mechanisms are not suficient to prevent PLRV and PW transmission at a high enough level. 26 Several years ago, GPA were relatively well controlled with aldicarb (Temik®), and other insecticides. It appeared that the agricultural industry had achieved a realistic technique for controlling GPA above the economic threshold. Unfortunately, aldicarb and other soil-applied granular insecticides have been implicated in groundwater contamination. Some of these more than twenty insecticides have been removed by the EPA, and others have been removed voluntarily by their producers. Since aldicarb and other granular insecticides are no longer available most growers have resorted to foliar applied insecticides to control GPA These foliar insecticides involve a greater frequency of spraying and a relative lack of eficacy (when compared to aldicarb). The more fiequent sprayings can lead to a greater opportunity for resistance development thus compounding the problem. Experience demonstrates that the current measures for controlling GPA lead to resistance development. With the system as it stands now, insecticide-induced resistance is a major problem not only because of selection due to spraying, but because the natural enemies of such pests are lost due to higher susceptibility to sprays. A difl'erent strategy must be employed to control the development of resistance in agricultural systems. IPM is one efl‘ective system that integrates population ecology and pest management (Smith 1970). The goal is to use pesticides effectively and yet maintain the lowest possible level of resistance in populations, while preserving the environment. (Waterhouse 1969). According to Smith, this can be done by avoiding all but necessary applications and utilizing as many alternative control procedures as possible so that agriculture is not entirely dependent upon insecticides (Smith 1970). Additionally, monitoring is one of the most effective tools at our disposal. Knowledge of when sprays would be effective and when they would not is a necessary component of and integrated management system, and as much knowledge about a pest population as can be obtained would be most beneficial. 27 VI. Goals and Objectives A. Overall Goal The overall goal of this study is to develop and test a field-monitoring tool usefirl as a resistance diagnostic technique by implementing currently available technology into a simple protocol, by which resistance can be diagnosed quickly and reliably by field workers. B. Objectives: 1) to develop an in vitro carboxyesterase-based insecticide resistance monitoring system for GPA using a modification of the 0010rimetric system developed by Gomori in 1953. 2) to bioassay (in viva) using standard slide dip techniques and probit analysis to determine the actual resistance level of eight GPA strains using insecticides fiom each of the three major classes (organophosphates, carbamates, and synthetic pyrethroids). 3) to correlate the carboxyesterase levels in Objective 1 with resistance levels from the probit mortality assays in Objective 2 using polyacrylarnide gel electrophoresis and a nricroplate assay. C. Hypotheses Four relevant hypotheses were developed for testing this work: H1: Strains of GPA with different LC 50 values will not have the same carboxyesterase enzyme activity (amount). H2: Resistant and susceptible strains of GPA will show quantitative and qualitative differences in polyacrylamide gel electrophoresis of enzymes. 28 H3: Resistant and susceptible insects show quantifiable differences in both microplate carboxyesterase assays and portable carboxyesterase assays. H4: There is a relationship between carboxyesterase level and resistance in populations of GPA in the United States. D. Thesis Outline Figure 1.1 outlines the overall format of this study. First, a literature review is necessary to gain an understanding of the problem. Next, in order to completely evaluate the in vitro field resistance monitoring tool, three steps must be evaluated. The first of these steps is to conduct a national resistance survey for an overall concept of resistance frequencies as well as to find suitable strains for further investigations. Second, the portable carboxyesterase tool was developed using the microplate assay as a design guide. Third, the portable tool was evaluated on the basis of the microplate assay, polyacrylarnide gel electrophoresis, and an in viva bioassay for resistance. This data next enabled economic, labor, accuracy, and simplicity assessments of each test and then a ranking of each tool as a field diagnostic device for carboxyesterase-based insecticide resistance. 29 Flow Diagram of Thesis Resistance Monitoring Tool M * orrelat ‘t In \IIVO Test System 1 Field Evaluation Figure 1.1-Schematic diagram of thesis research project. CHAPTER 2 IN VITRO EVALUATION OF CARBOXYESTERASE—BASED INSECTICIDE RESISTANCE 30 31 CHAPTER 2. UV VITRO EVALUATION OF CARBOXYESTERASE-BASED INSECI'ICIDE RESISTANCE. I. Introduction A. Green Peach Aphid Identification Green peach aphids (GPA) have several salient external anatomical characteristics which aid in distinguishing them from other members of the Homopteran Family Aphididae. GPA have a non-pigmented dorsal abdomen (that usually looks yellowish) and long antennaewithsixormoresegments. Theterminal antennal segmentislongerthanthe base of the last segment (Blackman and Eastop 1984). Figure 2.1 shows some of the basic external anatomy of aphids, including two inrportarrt structures helpful for identification purposes: the cauda and the siphunculi. Well-developed, converging tubercles are useful characteristics for differentiation between GPA and other aphids, and some comparisons are depicted in figure 2.2. Figure 2.2 (e) is a pictorial example of the converging tubercles found on GPA. The shape of the cauda is another character of good taxonomic value, and Figure 2.3 is an illustration of a tongue-shaped cauda as Opposed to a rounded, helmet, or knobbed shaped cauda GPA siphunculi are longer than the cauda andpale(ordarkonlyondistalhalf), usuallytaperingortubular, arrdaboutfourtimesas long as the basal diameter (Blackman and Eastop 1984). An example of such siphunculi is found in Figure 2.4 (d) (Blackman and Eastop 1984). B. Resistance evaluations Over the past few decades, the amount of crop lost annually to insects has risen to thirteen percent of the total (May and Dobson 1986). Even with increased pesticide usage and better chemistry, insects still manage to take their toll on crOps. This is due to the phenomenon of insecticide resistance which is found in over 447 species of insects (Georghiou 1986). In the past thirty years the judicious use of pesticides has been 32 Figure 2.1-Basic external anatomy of the abdomen of an aphid (Aphididae) useful for identifying difl'erent F arnilies. Taken fiom Blackman and EaStOp (1984). Figure 2.2-Dorsal views of difl'erent types of aphid antenrral tubercles useful for comparison between Families: (a) undevelOped tubercles, (b) diverging tubercles, (c) well- developed diverging, (d) parallel, (e) converging (Myzu: persicae), (t) well-developed median fiontal projection Taken fiom Blackman and Eastop (1984). 33 (cl (bl (a) Figure 2.3-_—Difl‘erent shapes of cauda found in Aphididae: (a) broadly rounded, (b) helmet shaped, (c) tongue shaped, and (d) knobbed with a bilateral anal plate . Taken fiom Blaclcman and Eastop (1984). 34 (cl I I; I l / '0 ,.’. N ”I;' z I, a» ‘ W . ~ .I‘ ./ air-13¢" ' '3‘ r . <27”! "- ‘1: I Ja I _ i . ‘0 s t . '1 \3. t‘ “3. (hi Figure 2.4~Aphid siphunculi of various shapes: (a) pore-like, (b) mammariform, (c) truncate, (d) tapering, (e) swollen proximally, (t) elevate, (g) with a subapical zone of polygonal reticulation, and (h) with sharp spiky hairs. Taken fiom Blackman and Eastop (1984). 35 formulated into a mechanism for controlling resistance development called Integrated Pest Management(IPM). OneofthekeyoonceptsofIPMistheuseofpesticidesonlywhen dmgecwsedbypestspedespassesaprevioudydefinedlhfitcafledflreecomnfic thresholdOIannnockandSoderlund,1986). Routineresistancemonitoringnmstbe donetoqumfifypestmmbasordunagefitisvaluabletohavemesthnateofthe fiequencyofredsmmmdividudspremnmapoprdafionmannnockmdSodaiund 1986). Levin(l986)assatsflNconfimousmomtofingofresistmcefiequendesshouldbem integralpartofallprogramstomanageresistance. Atoolforassessingtheinsecticide resistance levels of samples is necessary to enable such monitoring. Most insecticide resistance evaluations are determined by either field failure ofinsecticides on crops or by bioassays ofthe insects in a laboratory (Sawicki eta]. 1977, Brown and Brogden 1987). Bothofthesemethodsareextremelytimeconsmningandinvolvealarge number of insects (Devonshire and Needham 1975). According to Sawicki et al. (1977), routine biochemical methods ofresistance detection are rare and seldom feasible. For this reason, manyresearchers soughtalesslabor-intensiveandcostlymethodbywhichto diagnose insecticide resistance both in the field and in the laboratory (Gomori 1953, Brown and Brogden 1987, Brogden 1988, Sawicki er al. 1977, van Asperen 1962, Brogden and Dickinson 1983, Pasteur and Georghiou 1981). A rapid biochemical method of resistance detection would be most valuable for such determinations (Sawicki et al. 1977, Hammock and Soderlund 1986). Increased detoxification by nonspecific esterases is a connnon mechanism of resistance to organophosphate insecticides in insects (Oppenoorth 1985), particularly Homoptera In GPA, the only known metabolic mechanism of resistance is enzymatic hydrolysis and sequestration of insecticidal esters by carboxyesterases (Needham and Sawicki 1971, Beranek 1974, Sawicki et al. 1978, Takada 1979, Devonshire and Moores 1982) also 36 called carboxylester hydrolases by Brown and Brogden (1987). Mixed function oxidase (MFG) activity may have some impact on resistance, however MFO's have not been implicated in insecticide resistance in strains of GPA (Beranek and Oppenoorth 1977). Devonshire (1973) suggests that this lack ofMFO detectability may be due to inhibitors andpredictsthattheseenzymesmaystillbepresent. AlthoughMFO'smaynotbe attributedasresistancemechanismsinGPA, theystillplayalargeroleinchemieal detoxification (oxidation) in other insects and are required in some cases to enable certain carboxyesterases to react more effectively with compounds. Carboxyesterases hydrolyze carboxyl esters and amides, such as some organophosphate insecticides. This usually results in the formation of a non—toxic (or less toxic) acid, althoughinsomecasesenzymehydrolysiscanproduceamoretoxic secondarycompound (Motoyama and Dauterman 1974, Brown and Brogden 1987). The properties of carboxyesterases vary considerably with the species of insect studied (Motoyama and Dauterman 1974). For instance, housefiies show a negative correlation between carboxyesterases and organophosphate insecticide resistance (Van Asperen and Oppenoorth 1959). In GPA, resistance shows a positive correlation with carboxyesterase levels (Needham and Sawicki 1971, Al Khatib 1985, Pasteur and Georghiou 1989). This resistance to organophosphate and carbamate compounds is associated with a quantitative increaseintheamountofcarboxyesterase, notinanincreasedafinityoftheenzymefora substrate (Devonshire 1978, Devonshire and Sawicki 1979, Devonshire and Moores 1982). This quantitative increase is due to the amplification on the E-4 gene (Devonshire and Sawicki 1979, Devonshire and Moores 1982), and is associated with the incrmd ability of aphid homogenates to hydrolyze naphthyl acetate (Needham and Sawicki 1971, Devonshire 1989). 37 C. Microplate Assay Introduction Theabifityofcarboxyeflmsestohydmlyzeeflawmpmrflsmaddscanbeexplohedm develop asensitivetest forresistancedueto elevatedesterase levels. Several ofthese camoxyeuaasesmeablemdegradeMthylestasasweuasinsecfidddeuasmdtlfis pmpatycanbemedtoidanifyresismminseasquanfimfivdybyawlofimetficenzyme assay type of test (Gomori 1953, Needham and Sawicki 1971, Devonshire and Needham 1975, Pasteur and Georghiou 1981, Brogden and Dickinson 1983, Raymond et al. 1985, Hemmingway et al. 1986, Brogden and Barber 1987, Brown and Brogden 1987, Brogden 1988, Moores er a1. 1988, Pasteur and Georghiou 1989, Dary et at 1991). There is a posifivecondafionbetweenmsecfiddemsiswneinGPAmdmm'easedhydmlysisby carboxyesterases, andthispropertyiswhatcanbeusedtomonitorresistancemeedham and Sawicki 1971, Al Khatib 1985). In 1953, Gomori publishedastudyofamethodforthequantitativeanalysisofesterases basedonacolorimetric changeresultingwhennaphtholproducedbythereactionof esterases hydrolyzing naphthyl acetate coupled with an azo-dye (Gomori 1953). Gomori's assay has been modified since by Brown and Dickinson (1983) and Dary et al. (1990) to detect general esterase activity and by Devonshire eta]. (1986), on a more specific level. Pasteur and Georghiou descrrbe a filter paper test (1981) and an improved filter paper test for detection and quantification of increased esterase activity (1989). This test is accurate, but dificult to use in field tests and for determination of intermediate levels of resistance (Pasteur and Georghiou 1989). The method involves two reactions: the first is the reaction of the insect homogenate (including carboxyesterases) with rat-naphthyl acetate to form or-naphthol and acetic acid. In a second reaction the a-naphthol couples with Fast Garnet GBC (or other azo-dye) to give a colored precipitate (or solution) visible to the naked eye. This color change is then read by a densitometer or microplate reader (Pasteur and Georghiou 1989, Brown and Brogden I987). 38 Other researchers have used similar procedures. Van Asperen studied housefly esterases usingavery similar methodology (vanAsperen 1962, OppenoorthandvanAsperen 1961). To stain the naphthol produced by the esterasenaphthyl acetate reaction, Van Asperen used a diazoblue-sodium laurylsulphate solution The sodium lauryl sulfate solution denamresthemzymemdcwsesthewlm-changereacfiontopmceedmomquicklymd theproducttobemorestable. Thisissimilartotheefi'ectsofsodirnndodecylsulfate (SDS) and other detergents. The reaction sequence ofcarboxyesterases with naphthyl acetate and then the coupling with O-dianisidine for a color change is shown in Figure 2.5. This reaction has been used by many researchers to test for carboxyesterasebased insecticide resistance levels in insects (Gomori 1953, Pasteur and Georghiou 1981, 1983, Brogden et al. 1983, 1984). Pasteur and Georghiou (1981) also exploited this reaction to test for resistance in mosquitoes using a squash test. Brogden er al. (1983) used a rnicrotitre plate assay for meawreeflemseacfifitymdpmtemlevdsmverysmaflsmnplessevaaldifi‘eremfimes (Brogden 1984). The level of carboxyesterase activity estimated densitometrically appears to be related to the level of insecticide resistance (at LCgo) in some insects (Sudderuddin 1973, Beranek 1974, Devonshire and Needham 1975, Devonshire 1975, Pasteur and Georghiou 1989) and is probably responsible for organophosphate degradation (Beranek and Oppenoorth 1977). Although Oppenoorth and Voerman (1975) observed no correlation between esterase activity and insecticide resistance, Pasteur and Georghiou (1989) did observe a significant linear increase in staining intensity as esterase concentration increased, up to optical densities of 0.45. The test appears to be compatible for GPA because these insects have the same organophosphate-detoxifiing esterases that hydrolyze naphthyl acetate (Needham and Sawicki 1971, Brookes and Loxdale 1987). 39 Z=Z O-DIANISIDINE or B O OCH 3 Figure 2.5—Structures and sequence for the reactions of carboxyesterases with naphthylacetate to yield naphthol. Naphthol reacts with O-dianisidine (tetrazotized) to yield a colored solution. 4o Thepmnnseuponwhichthistestisbasedisthefactthatinsecfidde-resistmn GPAhave greater total esterase levels than susceptible insects (Needham and Sawicki 1971, Devonshire 1975). Total carboxylesterase activity is readily determined in individual GPA, anddifl‘erencesare distinct, providedthat activityiscorrected forbytheweight of individual aphids (Sawicki et al. 1977) or total protein concentration However, in other insects, somesdenfistshavehadpmblemsacmratdydifi‘erenfiafingimqmediueeaerase activitylevel strainsinfieldsurveyswasteurandGeorghiou I989). Theincreased carboxyesterase levels are associated with insecticide resistance (Devonshire 1975) and have been documented as the only known mechanism for GPA metabolic insecticide resistance (Devonshire and Moores 1982). C. PAGE Introduction Any charged group or ion will migrate in an electric field (Hames 1981). Since proteins carryanet chargeatanypHotherthantheirisoelectric point, theywillmigrateandthe rate ofmigration depends on the ratio ofcharge and mass ofthat protein (Hames 1981). The application of an electric field to a mixture of proteins in solution will result in the different proteins migrating towards one or the other of the electrodes at difi‘erent rates, depending upon their charge (thus depending on the pH of the bufi‘er, as well) (Barnes 1981). This property is the basis of polyacrylamide gel electrophoresis (PAGE). A supporting medium can be used rather than liquid solution (free electrophoresis) to minimize the disruptive efi‘ects of such things as convection (caused by heating) and difi‘usion (which would prevent efi‘ective separation) and to achieve stable, permanent separation of the proteins. Electric current is run through the medium, and proteins migrate along this stable matrix according to charge and density. The resulting banding patterns are stable and permanent and are easily evaluated quantitatively and qualitatively (Hames 198 l ). 4] Although various media can be used for electrophoresis, polyacrylamide is an excellent one because it not only prevents diflirsion and convection, it interacts physically with the proteins for a molecular sieving efl‘ect, separating proteins physically by size, as well as by charge and mass. This sieving effect is can be enhanced because it depends on the pore size of the gel chosen, which in turn depends on the gel concentration used and the type or percentage crosslinker used (I-Iames 1981). Polyacrylamide is a polymer (repetitive groups of multiple units of a monomer) made of acrylamide monomer (single acrylamide units) formed into long chains and cross linked with various substances, in this case MAP-methylene bisacrylamide (Bis). The structures of acrylamide and Bis, as well as the structure of polyacrylamide can be found in Figure 2.6. The polymerization reaction occurs as a result of the crosslinkers reacting with functional groups (double bonded areas) at the ends of chains (Hames 1981). The polymerization reaction is catalyzed by either ammonium sulfate or riboflavin. When riboflavin is used, light is necessary to initiate polymerization (by photodecomposition of riboflavin to form fi'ee radicals) and MMN',N’-Tetramethylethylenediamine (TEMED) is usually added to insure polymerization. PAGE when used as a native gel is a useful tool for the separation of proteins and active enzymes (Kasciinitz et al. 1968, Zacharius et al. 1969). Some researchers have used the PAGE-SDS system, which denatures proteins, to study carboxyesterases in GPA This ' system does not provide suflicient information about enzymes' activity levels and the effects of insecticides and other inhibitors on the various bands found in the gel. For this reason, native gel PAGE was conducted to determine the banding patterns of several strains of GPA and to elucidate the effect of insecticidal compounds on the enzymes ability to metabolize them. These techniques offer valuable information for strain resistance level characterization and evidence that the enzymes responsible for reaction with the or- 42 CH2=GH CH2=GH =0 $=0 hmz fiH an é=o I CH2=CH i g g... N,N' - methylene bisacrylamide -CH2-CH-(CH2-C|H-)nCH2-CH-(CH2-GH-)nCH2- ('30 (F0 $0 NHZ IfiH NHZ ('IHZ h'lH (f0 -CH2-CH-(CH2-('3H-)nCH2-CH-(CH2-(|IH-)n-CH2- .0 $30 ('30 yH Iflu iMfl CH2 11m éo -CH2-éH-(CH2-CH-)nCH2-CH-(CH2-CH-)n-CH2- 'CO (:20 | O imz NH NHZ Polyacrylamide gel Figure 2.6-Structures of Acrylarnide, N,N' - methylene bisacrylamide, and Polyacrylamide gel. (Hames and Rickwood 1984) 43 naphthyl acetate to form a-naphthol are the same enzymes responsible for breakdown of insecticides, and subsequently responsible for insecticide resistance. E. Portable Introduction Theabilityofcarboxyesterasesto hydrolyzeestercompoundsto acidshasbeenusedinthe past to develop colorimetric tests for resistance diagnosis (Gomori 1953, Needham and Sawicki 1971). Manyofthesetestsnmstbeconductedbyspeciallyu'ainedtechnicians, closeto laboratoryfacilities, andinvolvingagreatdeal oftimeandexpensivematerialsor equipment. Some researchers have made efl'orts to design a simple, field-oriented, memensiwassaymahodbywlnchmsistmcecmbediagnosedbymn-sdenfisthbom (Pasteur and Georghiou 1981, Pasteur et al. 1984). A monitoring tool of this type would be extremely helpful to growers and would make great strides in assisting IPM decision- makers in resistance monitoring and subsequent pest control on potato. Simplicity is a key element in developing a portable test. Unfortunately, many field- oriented tests are still too complicated. The development of a novel, extremely simple, highly effective resistance level evaluation tool was still needed. The technique designed here was developed as an extension of the carboxyesterase characterization study and involves the use of a portable colorimetric carboxyesterase assay for detection of field insecticide resistance levels. The microplate assay developed in the first section of this chapterwasusedto alargeextent forthedesignofthetest. Theportable assayalso needs to be evaluated by several different testing methods for accuracy, precision, economics, labor time, and simplicity. ILMaterialsandMetlrods A.Strains Forthisstudyasnainisdefinedasapowlationofindividualskeptincultmethat originatedfiomaspecificareaoftheUnitedStates. Generallystrainswerebegunfiom thirtyormoreGPA However,somepopulationswererearedfromonlyoneorafew aphids. In addition, as with any laboratory-reared colony, there were bottlenecks in which some of the population died off due to an inability to survive under laboratory conditions, parasitism, or plant quality difl‘erences. Additionally, each strain has a specific insecticide redflancekvdwlnchwasmeaauedmthehbomtorymdusedasameanstochssifir them Overonelumdredandtwentytelephonecallsandletterswere sentto potential cooperators in targeted areas of potato production in the continental United States. Potato (and selected other crop) growing regions in the following states were solicited: Alabama, Califomia, Colorado, Delaware, Florida, Georgia, Idaho, Indiana, Iowa, Maine, Maryland,Massachusetts, Michigan, MimesotaNebraska, NewHampshire,NewYork, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, V'nginia, Washington, Wisconsin, and Wyoming Additionally, some strains were hand-collected by persons employed by or associated with our laboratory. Shipped with the appropriate USDA and Animal Health Inspection Service permits, GPA were sent by cooperators on plant material in plastic (50 ml) centrifuge tubes. Parafilmm covered the opening, containing the aphids yet allowing for gas exchange. The tubes were contained within brown cardboard shipping containers insulated with paper towels or tissue paper. Upon receipt of shipping containers with GPA from cooperators, the insects were removed fiom the shipping container, identified positively as GPA using the 45 diagnostic characters identified previously, and placed on clean potato plants contained in two-liter plastic cages. UponarrivaLallstrainsweregivenacode. Themostirnportarrtfactorsirrvolvedinthe codewerethecropandthelocationstrainswerecollectedfi'om, includingcityandstate. ThecodedesignationcanbefoundinTableZJ. Peach,theinsects'primaryhost,and potato,thesecondaryhost,werethetwocropsthestudyfocusedon. Samples of GPA fiom each of the eight major colonies listed in Table 2.2 were mounted on slides for identification. Initially, GPA were soaked in a 10% potassium hydroxide solution overnight and warmed for three hours to degrade soft tissues. Next, GPA were transferred to a series offour serial increasing concentrations ofethanol (70-100°/o) to remove water from the insects' tissues. The GPA were subsequently transferred to clove oil for the addition of color, which enabled easier microscope viewing. They were then mounted in Euparol® fixative (ASCO Laboratories, Manchester, England) between a microscope slide and cover, and allowed to dry for a few weeks (Blackman and Eastop 1984, Bob Kriegel personal communication). Finally, they were sent to the USDA/ARS Systematic Entomology Laboratory in Beltsville, Maryland for identification to species (special thanks to Manya Stoetzel and Mary Lacey-Theison for identification services). GPA were reared on insect and disease-free Superior potato plants (Belding, Michigan). Whalon and Smilowitz (1978) observed that the optimum temperature for rearing GPA with highest survival (87.2%) is 239°C. For this reason, GPA were cultured in a greenhouse with a temperature range of approximately 21 to 25°C with a light/dark ratio of 16:8 hours. Table 2.1—Code designation translations for green peach aphid strains. DESIGNATION FULL NAME tion: MOXEE-WA Moxee, WA PRESQUE-ME Presque Isle, ME SALINAS-CA Salinas, CA WOOSTER-OH Wooster, OH STRATHAM—NH Stratham, NH MONTC-MI Montcalm, MI PULLMAN-WA Pullman, WA NEWMAN-CA Newman, CA IMPCO-CA Imperial County, CA GAINESVL-FL Gainesville, FL WILDER-ID Wilder, 1D . PARMA—ID Parma, 1D BINGHAM-ID Bingham County, ID MOSCOW-ID Moscow, ID UNKNOWN-II) Unknown, ID GRDRAPIDS-MI Grand Rapids, MI CARLETON-MI Carleton, MI MONROE-MI Monroe, MI RKINGHM-NH Rockingharn County, NH BERRIEN-MI Berrien County, MI 47 Table 2.1—Continued. DESIGNATION FULL NAME ti n: GRATIOT-MI Gratiot County, MI IDAHO Idaho AN SON-MI Anson, MI MUN GER-MI Munger, MI STJOI-IN‘S-M] St. John's, MI AUGRES-MI Au Gres, MI TRAVERSE-MI Traverse City, MI BRDGPORT-NB Bridgeport, NB ALLIANCE-NB Alliance, NB I-IEMINGFD-NB Hemmingford, NB BEAUFORT-NC Beaufort, NC VAN ORA-OR Vanora, OR I-IERMI STON-OR Herrniston, OR CENTER-PA MARYHIL-WA QUINCY-WA YAKIMA-WA TOPPEN-WA RVRSIDE-CA WALLULA-WA MADISON-WI Center County, PA Maryhill, WA Quincy, WA Yakima, WA Toppenish, WA Riverside, CA Wallula Junction, WA Madison, WI 48 Table 2.1»Continued. DESIGNATION FULL NAME Crop: P1 Peach P2 Potato T Tobacco 0 All other crops 49 Table 2.2--Strains, cooperators, host plants, and locations of populations on file in the Entomology Museum used for identification of strains as green peach aphid. Strain Cooperator Host Location MOXEEl-WA—Pl L. Fox Peach Moxee, WA PRESQUE-ME-PZ G. Sewell Potato Presque Isle, ME SALINAS-CA-O L. Fox Other Salinas, CA WOOSTER-OH-P2 C. Hoy Potato Wooster, OH STRATHAM-NH-PZ M. Campbell Potato Stratham, NH MONTCl-MI-P2 M. Otto Potato Montcalm, MI PULLMAN-WA-O T. Mowry Other Pullman, WA WILDERl -ID-P1 L. Fox Peach Wilder, ID 50 B. Microplate Survey Standard curve data were determined by seven serial dilutions of a- and B-naphthol in potassium phosphate bufl‘er (KH2P04 + KZHPO4, 0.05 M, pH 7.0) ranging fiom l ng/lO ul to 20 ng/lO ul similar to a procedure used by Brogden eta]. (1983, Brogden1984). 10 ul ofeach naphthol serial dilution were pipetted into a micnotitre plate and 190 pl of potassiumphosphatebufl’erwereadded. No incubationperiod is necessary, andthe 50 ul of fieshly made O-Dianisidine (0.3% O-dianisidine dilute in H20), also called Fast Blue B (Aldrich Chemical Co., Milwaukee, Wisconsin) was added. Three replicates were used for each dilution, and the entire assay was conducted in firll six times. Microtitre plates were read on an automated Microplate minireaderm MR590 (Dynatech Laboratories Inc., Alexandria, V'u‘ginia) at 600 nm (A600) for a-naphthol and 550 (A550) for B-naphthol. Thesedatawerethenanalyzedbyregressionanalysis(CohenandCohen l983)using Systat (Systat Inc., Evanston, Illinois) and the Systat Manual (Wilkinson 1990). Proteinstandardcurvedatawereobtainedbytheserial dilutionof lOmgBovineAlbumin Serum (BSA) per ml potassium phosphate bufi'er. Controls consisted of 50 ul of potassium phosphate bufl‘er and 200 ul of dilute (1:4) BioRad Protein Reagent, which is a type of Coomassie Brilliant Blue stain (Bradford 1976). Eight serial dilutions ranged fiom 0.1 rig/10 pl —10 rig/10 pl. 10 ul ofeach protein dilution was pipetted into a well ofa microtitreplate, 40 wofpotassirimphosphatebufl‘erwasaddedto eachwell, and200 ul of dihrted BioRad Protein Reagent was added. Three replicates were taken of each BSA dilution and the microtitre plate was read after five minutes on a Dynatech automated minireaderm MR590 (or Dynatech microplate reader) at 600 nm (A600). The entire assay wasreplicatedinfull sixtimesandthedatawereanalyzedby Systatusingmultiple regression analysis (Cohen and Cohen 1983). 51 The methods ofGomori (1953) were used to examine each strain ofGPA (with modifications similar to Brogden eta]. 1983) as follows: controls were replicated six times andconsistedofSOulpotassiumphosphatebufi‘erandlSOuleithera-orB-naphthyl acetate(consistentwiththerestofthephte)addedtothefirstsixwellsintheArowofa 96-well microtitre plate. Alter a ten minute incubation (in concert with the rest of the plate) 50 pl O-dianisidine (0.3% O-dianisidine diluted inHZO) were added and the plate wasincubatedagainfortenmimrtes. ControlswerereadasblanksintheDynatech minireaderateither600nm(a)or550nm(B)priortotherestoftheplate. Individual GPA were homogenized in 150 pl ofpotassium phosphate bufl‘er (0.5M, pH 7.0) within each of 24 wells of a 96 well microtitre bioassay plate (Costar Corp, Cambridge, Massachusetts) with a plastic pestle (Kontes, Vineland, New Jersey). A 50 ul aliquot was pipetted fiom the original homogenate and diluted with 150 pl of potassium phosphate bufl’er. Three replicates from each well consisting of 25 ul of diluted homogenate were transferred to a new 96-well rnicrotitre plate, beginning at row B (controls were always pipetted into row A). Each aliquot was diluted further with 25 pl potassium phosphate bufi‘er. 150 pl of either a— or B-naphthyl acetate (diluted to 0.6% in acetoneandthen100-foldinbufl’er)wasaddedtoeachwellinafivesecondsequence. Theplatewasincubatedforatotal oftenmimrtes, startingwiththefirstwell, andthen 50 ul of color reagent (0.3% 0—dianisidine dilute in H20) was pipetted into the wells in the same timed 5 second sequence. This was followed by a second ten minute incubation prior to quantitation. Microtitre plates were read on a Dynatech microplate reader at an absorbance density of 600 nm (A600) for a- and 550 nm (A550) for B-naphthyl acetate. Protein determinations were conducted to correct for difl‘erences in protein quantities in GPA bodies. Total protein concentrations of each sample were later to be used as a control for standardizing a- and B-naplrthyl acetate sample concentrations. The procedure 52 of Bradford (1976) was followed, using a type of Coomassie Brilliant Blue stain designed by BioRad, which is stable over time and not influenced by protein molecular weights or PI values (Brogden and Dickinson 1983). Controls were replicated twelve times to dinnnueposdblevadafimmmeprmdnraganandwnsistedofsotdhrfl‘erandzootd diluted BioRad Protein Reagan", with a five minute incubation period observed along withtherestoftheplate. TheywerepipettedintotheentireArowofa96-wellsterile micr'otitre plateandwereread asblanksinorderto standardizetheDynatech microplate reader. For the actual protein assay, 25 ul ofthe original homogenate (first dilution) was pipetted inthreereplicatesto eachwellbeginningwithrowB ofa sterile96-wellmierotitreplate. Next 25 ul ofpotassiumphosphatebufl‘erwasaddedandthenZOO ulofdilutedBioRad Protein Reagent” (dihrted 1:4 with H20) were pipetted into the wells. After a five minute incubation period, the plate was read on the Dynatech microplate reader at an optical density of 600 nm. Protein values were first tested using ANOVA (Shefl‘e 1959) in Systat to obtain mean and standard error values for each individual insect (every three wells originated from one insect). This was done in order to help eliminate some of the variation in protein values due to mispipetting, which is a common error when working with mimite volumes. These data were then entered into Systat's Edit spreadsheet and replicated three times (one for each well of the plate), so that each insect would have one protein value and three difl’erent or- and B-naphthyl acetate values. These data were next corrected using the standard curve data that were analyzed by regression analysis. Results of the correction calculations were then transformed to correct for difi‘ering protein concentrations and then mathematically computed using 53 molecular weight equivalents to nmol or- or B-naphthol/ ug protein] 20 minutes. Next theywereanalyzedbyANOVAusingSystat forthemeananderrormeansquaredvalues (Carmer and Swanson 1968, Bernhardson 1975, Shefl‘e 1959). Then they were analyzed using Tukey's Means Separation test in MSTAT—C (or = .05, df= 166) (Michigan State University, Departments of Crop and Soil Sciences and Agricultural Economics, East Lansing, Michigan 1990) (Tukey 1977) and plotted in histogram form Foraccuracycomparisons, mier0platedatawerecomparedwithotherin vitmdatausing correlation analysis in MSTAT-C and Systat (Systat, Inc., Evanston, Illinois) (both Pearson's linear correlation and Spearman's rho rank correlation test (Cohen and Cohen 1983). Correlation coeficients were determined for each comparison, then tested by using Steel and Torrie's table (A.13) ofr values. A x3 test for global significance was also conducted (Steel and Torrie 1980). C. Polyacrylamide Gel Electrophoresis 1. PAGE methodology For this technique, a 10% solution of acrylamide was used, composed of acrylamide- bisaerylamide (acting as a crosslinker) (30:0.8) in solution with distilled water. This was then added to 12 ml of his-glycine bufl‘er ([Sx], pH 8.64), and 25 ml of double distilled H20. After degassing for five minutes under vacuum pressure of 15 psi to remove oxygen, which inhibits polymerization (Barnes 1981), 150 pl of 10% ammonium persulfate and 15 ul of TEMED (MMMM-Tetramethylethylenediamine) were added as a catalyst and accelerator, respectively. The 10% gel was then injected into electrophoresis molds by the use of a syringe, and care was taken to eliminate bubbles from the gel. Combs were then added to the liquid gel, and it was allowed to polymerize into a solid for sixty to ninety minutes. The rapidity of the 54 reaction depended largely on the concentrations of acrylamide used, the amount of ammomumpu'udfateorTEMEDaddedandflretemperamreandlmmidityofthe laboratory. Sample solution was pipetted into the gel's sample wells when it was fully polymerized, approximately two hours. The sample solution consisted of his-glycine bufi‘er ([Sx], pH 8.64), glyceroL Bromophenol Blue stain, and double distilled H20. Samples were preparedby maceratingGPAinISOMTris-glycinebufl‘er([5x])overiceusingaplastic pestle(Kontes, Vineland, Newlersey)inamicrocentrifirgenrbe. Thentheaphid/bufl‘er homogenflewascenfiifiigedat3000rpmforfivemhnnesat4°Cinannmcenuifirge (Eppendorf5415C ). After centrifirgation, 75 ul ofthe supernatant (equivalent to one-half ofaGPA)waspipettedintoasterilemierocentrifiigetubeandanequalvohnne(75 ul)of preparation sohrtionwas added. Next, 75 ul ofthe combined aphid homogenate/buffer andsamplesohrtionwereaddedtoeachwellofthegel(onefourthofaGPA), onestrain ofGPAperwell. Allofthesestepsareconductedovericewhenpossible(butitisnot necessary) to retard carboxyesterase enzyme degradation Electrophoresis was conducted at 150 volts (constant voltage) for 17 hours with constant mirdngofthebufl‘erusingamagnetie stirbar. Theternperaturewasmairrtainedat2°C, in a cold room, to retain a pH of8.4 in the temperature-dependent Tris-glycine bufl‘er. After electrophoresis, the gels were stained with or-carboxyesterase stain (0.4 M Tris-glycine bufi‘er solution, Fast Blue RR Salt, acetone, a-naphthyl acetate, and double distilled H20) for 45 minutes. Then they were washed briefly with double distilled H20 to remove excess stain solution and preserved by one of several methods (explained later). 55 2. Insecticide Inhibition in PAGE Fortln'sexperimentonechemicalwasusedfiomeachofthethreeclassesofinseeticides theinvr'mbioassayswereperformedwith Thechendcalswereasymheficpyrethroida carbamate, and a systemic (oxon) organophosphate. The compounds used for the inhibition study were permethrin, carbaryl, and oxydemetonmethyl. Using the Basic 10% PAGE protocol, several experiments were conducted. Duplicate gelswerepouredand sampleswereloadedasperthegeneral 10%geltecbniqueandrun for 17 hours at 150 volts (Constant Voltage) in a cold room (2°C). However, before staimngoneofthegelswasincubated for 10mimrtesin500mlofoneofthree 5.0mM concentrations of insecticides. Either permethrin, carbaryl, or oxydmetonmethyl was usedastheinhibitor. Priorto staining, theinhibited gelwasrinsedwithdouble distilled H20. The duplicate gel was used as a control and was not inhibited with anything. Both theinhibitedgelandthecontrolwereplacedina—carboxyesterasestahratthesamefimein separateglassdishes. Eachsetofexperiments(consistingofthetwogels)wasrepeated tlneetimeswiththesameinsecticidal compound. 3. Preservation of Gels and Data Analysis Data were analyzed by densitometry using the AMBIS Radioanalytical Imaging System (San Diego, California) (with background readings subtracted) and then tested using correlation analysis in MSTAT-C and Systat (both Pearson's correlation analysis and Spearman‘t rho rank correlation test) for correlations with other in vitro data using the r values (Steel and Torrie 1980) significance tests (Cohen and Cohen 1983, Fischer and Yates 1949, Pearson and Hartley 1954). A global x2 test was also conducted. Gels were preserved using BioGel Wrap” drying apparatus (BioDesign Inc., Carmel, New York), which encases the gel in clear plastic, or a simple sealed Seal-A—Meal bag (Dazey 56 Corp., Industrial Airport, Kansas) . Pictures were taken of gels as a permanent record of data. Gelswereanalyzedinoneofthreeways: byvisualmethods(pictmes),by densitometry analysis using AMBIS, and by statistical analysis of the densitometry data using ANOVA procedures in Systat. D. Portable Evaluation of Carboxyesterase Levels Standard curve data were determined by seven serial dilutions of a— or B—naphthol in potassium phosphate bufi‘er (KHzPo4 + KZHPO4, .05 M, pH 7.0) with a range fi'om 1 ng I10 ul to 20 ng/10 pl. Controls consisted of2.5 ml ofpotassium phosphate bufl'er and wereusedtosetpercenttransmissionwasat 100%. Thiswasreplieatedsixtimesfor consistency. For the standard curve data, 1500 ul a- or B-naphthol solution were pipetted into sterile 16 x 100 mm test tubes (VWR Scientific, San Francisco, California) and 500 pl potassium phosphate bufi‘er was added. Next 500 pl of O-Dianisidine/SDS (0.3% tetrazotized 0- dianisidine dilute in H20 + 1% sodium dodecyl sulfate) (Aldrich Chemical Co., Milwaukee, Wisconsin), was pipetted into the test tubes. Each dilution was replicated three times and then read on the CHEMetrics USA Portable Photometer (Calverton, Virginia) (for percent transmittance with the correct filter (a=609 nm and B=555 nm). The results were subtracted fiom 1.00 to obtain percent absorbance values, then the data were analyzed by regression analysis using Systat (Cohen and Cohen 1983). Bovine Albumin Serum (BSA) was the standard used in serial dilution to obtain values for the protein standard curve. The BSA stock solution consisted of 10 mg BSA/1 ml (10,000 ppm) potassium phosphate buffer. Eight serial dilutions were used with a range in concentrations of0. 156 ng/10 ul to 100 ng/10 ul. Controls consisted of2.5 ml bufl'er alone, which was used to set the photometer at 100% transmittance. This was replicated 57 six times to maintain consistency. For the actual data, 500 ml of the BSA dilutions were pipetted into sterile test tubes in three replicates. Next, 500 ml of potassium phosphate bufl‘erwasaddedandthenZOOOmlofdihnedBioRadProteinReagem(dihned1:4with H20). Results were read afier five minutes on the CHEMetrics USA portable photometer witha609nmfilter. 'Ihesedatawerethensubtractedfrom 1.00toobtainpercent absorbance and then analyzed by regression analysis (Cohen and Cohen 1983). To obtain standard curve regression lines, absorbance (Y) was plotted versus protein concentration (rig/100 ul) (X) in Sygraph and Systm. For this procedure the protocol for Microplate Assay was used with several modifications. Both a- and B—naphthyl acetate assays were conducted but protein assays were not. Sodium dodecyl sulfate (SDS) was introduced into this assay for stability purposes, as well as shortening the total incubation time fiom twenty mimrtes prior to reading the absorbance (or transmittance) to ten minutes. The protocol is as follows: one GPA was homogenized in 500 pl potassium phosphate bufl‘er (pH 7.0, 0.05 M) in a sterile test tube with a glass rod. 0.6% a— or B—naphthyl- acetate was diluted lOO-fold and then 1500 141 of it was added to the test tube with homogenate. This was subsequently incubated for ten mimrtes after which time 500 pl 0.3% tetrazotized O-dianisidine (Fast Blue B) with 1% SDS was added to the test tube and a color change was observed. A CHEMetrics USA portable spectrophotometer was calibrated with a clear buffer solution to 100% transmittance and then percent transmission was read (555 nm for or- and 609 nm for B—naphthyl acetate). Data were subtracted from 1.00 to adjust percent transmittance to percent absorbance. Next the results were corrected using the standard curve regression values and then calculated with correct conversion factors (inverse of standard curve correlation 58 coeficients) to obtain nmol naphthol per insect per ten minutes (incubation time) in Systat. Finally Tukey's test for difl‘erences between means was conducted on the data in MSTAT-C. (a=.05, df= 16) (T ukey 1977) and the results tabulated in both histogram and table form. Theportabledatawere comparedwiththeotherinvia'odatausingcorrelationanalysisin MSTAT-C and Systat (Pearson's correlation analysis and Spearman's rho rank correlation analysis) (Cohen and Cohen 1983). , and the r values were tested for significance using a table (A 13) fiom Steel and Torrie ( 1980). The 1} values were also tested as a global test of significance (Fisher and Yates 1949, Pearson and Hartley 1954). III. Results and Discussion A. Strain and Microplate Survey Results Strains were received from thirteen states: California, Florida, Idaho, Maine, Michigan, Nebraska, New Hampshire, North Carolina, Ohio, Oregon, Pennsylvania, Washington, and Wisconsin (Figure 2.7). The high concentration of strains acquired from potato growing regions of the United States, such as Idaho, Washington, Oregon, and parts of Michigan was intentional, as the potato is the major crop of focus for this study. Often multiple samples were taken fi'om these areas of the country. Figure 2.8 shows a detailed map of strain locations in Michigan. Multiple strains were collected from the central Michigan (Montcalm County) area, as this is a strong potato producing area of Michigan, as well as the St. John's and Munger, Michigan areas. Fifty-five strains were received from cooperators and Figure 2.9 shows the procedure used upon receipt of samples fi'om a cooperator. Insects were received in containers, removed and placed on potato plants using the method shown here. Table 2.3 is a listing of the 59 xotsm owflofiobthu owe—e063 2: me can 8 mogm e225 05 $28 38:8 23?. Bees :82. :35 com 28:82 go ens—Ind 2&5 8% cocoa—.8 5.5m O 3:. 03.3%» co eBozm © \ . tr .e- ' u \ J . nu .aotnm goumoxxopao owe—e822 05 com 3522 E 8% eaten—.8 5.5.». 25% :83 :0on mo EEImN 822m a saw Seoo=oo sea a 61 Figure 2.9—A paintbrush was used to transfer green peach aphids to clean potato plants for rearing in the greenhouse at Michigan State University. 62 Table 2.3-Listing of strains, cooperators, host plants, and locations of populations collected for the national carboxyesterase survey. Strain Cooperator Host Location MOXEEl-WA-Pl L. Fox Peach Moxee, WA PRESQUE-ME-P2 G. Sewell Potato Presque Isle, ME SALmAS-CA-O L. Fox Other Salinas, CA WOOSTER-OH-P2 C. Hoy Potato Wooster, OH STRATHAM-NH-P2 M Campbell Potato Stratham, NH MONTCl-MI-P2 M. Otto Potato Montcalm County, MI PULLMAN-WA-O T. Mowry Other Pullman, WA WILDERl-ID—Pl L. Fox Peach Wilder, ID NEWMAN-CA-O L. Fox Other Newman, CA IMPCO-CA-O C. Farrar Other Imperial County, CA GAINESVL-FL-T F. Johnson Tobacco Gainesville, FL WILDERZ-ID-Pl L. Fox Peach Wilder, ID PARMA-ID-O T. Mowry Other Parma, ID BIN GHAM-ID-O T. Mowry Other Bingham County, ID MOSCOW-ID-O T. Mowry Other Moscow, II) UNKNOWN-ID-PZ T. Mowry Potato Unknown, ID GRDRAPIDS-MI-Pl M. Resch Peach Kent County, MI CARLETON-MI-O P. Marks Other Carleton, MI MONROE-MI-P2 P. Marks Potato Monroe County, MI BERRIEN-MI—PZ C. Garcia Potato Berrien County, MI Table 2.3-Continued. 63 Strain Cooperator Host Location GRATIOT-MI-P2 D. O'Hara Potato Gratiot County, MI MONTC2—MI-P2 D. Miller Potato Montcalm County, MI MONTC3-MI-P2 M. Otto Potato Montcalm County, MI MONTC4-MI-P2 M. Otto Potato Montcalm County, MI MONTCS-MI—P2 M Otto Potato Montcalm County, MI AUGRES-MI-P2 K. Kernstock Potato Au Gres, MI TRAVERSE-MLPZ M. Harmon Potato Grand Traverse Co., MI BRDGPORT-NB-P2 M. Whalon Potato Bridgeport, NB ALLIANCE-NB-P2 M. Whalon Potato Alliance, NB HEMINGFD-NB-PZ M. Whalon Potato Hemmingford, NB BEAUFORT-NC-O K. Sorenson Other Beaufort, NC VAN ORA-OR-O L. F ox Other Vanora, OR HERMISTON—OR-P2 G. Reed Potato Hermiston. OR CENTERl-PA-P2 Z. Smilowitz Potato Center County, PA MARYHILLl-WA-O L. Fox Other Maryhill, WA QUINCY-WA-Pl L. Fox Peach Quincy, WA YAKIMA-WA-Pl L. Fox Peach Yakima, WA TOPPENl-WA-O L. Fox Other Toppenish, WA TOPPENZ-WA-O L. Fox Other Toppenish, WA WALLULA-WA-O L. Fox Other Wallula Junction, WA MADISON-WI-PZ J. Wyman Potato Madison, WI Table 2.3-Continued. Strain Cooperator Host Location IDAHO-P2 T. Mowry Potato Idaho AN SON-MI-P2 C. Garcia Potato Anson, MI MONTC6-MI-P2 M. Otto Potato Montcalm County, MI MONTC7-WI-P2 D. Ragatz Potato Montcalm County, MI MUNGER-MI-P2 U. Rahardja Potato Munger, MI STJOHN‘S-MI-PZ D. O'Hara Potato St. John's, MI RVRSIDE-CA-O T. Unrue Other Riverside. CA MARYHlLLZ-WA-O L. Fox Other Maryhill, WA MOXEEZ-WA-Pl L. Fox Peach Moxee, WA MONTC8-MI-P2 M Otto Potato Montcalm County, MI TOPPEN 3 -WA-O L. Fox Other Toppenish, WA CENTER2-PA-P2 Z. Smilowitz Potato Center County, PA TOPPEN4-WA-P2 L. Fox Other Toppenish, WA RCKINGHAM—NH-P2 J. Bowman Potato Rockingham County, NH 65 code, crop, location, and cooperator for each strain of GPA The codes were defined in the Materials and Methods section of this chapter. There were seven strains collected fiom peach (Pl) and thirty fi'om potato (P2), which are the primary and secondary host plant species of GPA, respectively. One strain was collected on tobacco, and eighteen were collected on various other crops. Slide mounted aphids from each oftheeight strainsused fortheInvr'vo studyare preserved as slide mounted specimens in the Michigan State University Department of Entomology Museum Collection, Voucher #1992—05, found in Appendix A. These same eight strains were identified as Myzus persicae (Sulzer) (GPA) by the USDA/ARS Systematic Entomology Laboratory in Beltsville, Maryland (September 28, 1992). Figure 2.10 is a photograph of a slide mounted GPA with identifying characters labeled for easy viewing. Strains were maintained for two years (some cultures were lost due to poor plant condition and parasitism) in the greenhouse at Michigan State University. Figure 2.11 illustrates the manner in which the strains of GPA were reared and kept flee from contamination. Each strain was kept in a separate two-liter plastic cage which was screened with fine gage aphid-proof netting and maintained in water approximately three- quarters of an inch deep. Plants were changed (with clean plants) as needed. Figure 2.12 shows the standard curves for both the or- and B-naphthol standards. There appears to be a linear progression of increasing optical density with increasing naphthol concentration. Although both absorbance values increase with increasing naphthol concentration at similar same SIOpes, the B—naphthol curve definitely has a steeper slope than the a-naphthol curve, as is evidenced by the two difl‘erent Absorbance scales. For example, at 100 ng B-naphthol/ 200 pl, the absorbance value is approximately 0.25, Figure 2.10-Photograph of a slide mounted green peach aphid with the most visible taxonomic characters labeled for easy discrimination: (a) converging tubercles, (b) long arrtemrae (as long as or longer than the body), (c) a tongue shaped cauda, (d) long appendages, and (e) long, tapering or tubular siphunculi. 67 Figure 2.11-Rearing environment for green peach aphids in culture in a greenhouse at Michigan State University. 0.4 ABSORBANCE 0.6 0.5 0.4 0.3 ABSORBANCE 0.2 0.1 0.0 50 100 150 T I B > a p a a , .. > , a L l 50 100 150 CONCENTRATION N APHTHOL Figure 2.12—Microplate standard curves for or- (A) and B- naphthol (B) standard concentrations (ng)/ 200 pl plotted against absorbance values (600 nm= a, 550 nm= B). Slopes of the lines are found in Appendix B. 69 whereas for 100 ng cit-naphthol] 200 ml, the absorbance value is approximately 0.16. The B-naphthol absorbance readings increase at a greater rate than the a—naphthol absorbance values do for the same amounts of naphthol. Thus, there is a greater colorimetric change with the B—naphthol than there is for the same amount of or-naphthol. This shows that more a-naphthol is required than B-naphthol is to make the same change in absorbance. RegressioncoefidenQobminedmtheanalysisofmestmdaMWdatawemusedto correct the absorbance values of the microplate assays. The cr-regression coeflicient was 0.002 and the B was 0.003. The inverse of these, 500 and 333.33, respectively, was multiplied with the data obtained from the microplate assay to standardize the assays. Figure 2.13 shows the standard curve for the protein standards. The standard solution usedwasbovineserumalbuminmSA). Theproteinstandardcurvedoesnotbeginat zero, but above it, at approximately 0.80. This agrees with Bradford (1976) and the BioRad "microtecbnique" results, which show a curve that begins slightly above zero on the Y-axis. The protein curve has a large s10pe value, which can be observed in the figure by the slope of the line. There is a linear relationship between increasing protein concentration and absorbance values in the microplate assay, and this is clearly observable in the Figure. The protein standard curve values were analyzed by regression analysis to obtain the regression coefficient of 0.175. The inverse of this, 5.714, was multiplied with the protein absorbance values to obtain corrected values. Figure 2.14 shows the results of the ct-carboxyesterase microplate assay in histogram form, along with Tukey's Test letters. The susceptible strains are PULLMAN-WA-O and GAINESVILLE-FL-T. The extremely resistant strains are MOXEEZ-WA-Pl and HERMISTON-OR-PZ. Both of the susceptible and most of the low rat—carboxyesterase 70 0.6 r r ABSORBANCB PROTEIN CONCENTRATION Figure 2.13-Microplate standard curve for total protein (bovine serum albumin) standard concentrations (ug)/ 10 ul plotted against absorbance values (600 nm = protein). The equation of the line is found in Appendix B. 71 Figure 2.14--Histograms of the a-Naphthylacetate carboxyesterase assay showing total a—carboxyesterase levels, Tukey's test for signifance letters (or = .05, df = 1 10). ...,....r.r.......r..........O. 7' VOOOOOOOOOOOQu B '00.DOD.DODODODO’ODODODOOODODC. 9nOWOMOWOMOWOWOMOWOWOMOMOMA 72 3.9- 261— 1.3- it ”WW WW T 5 6 3:5 9. Z— 8 3 E c 6 2 Q2 . _ ennnnm ESE. union—Eagle .532 r .o»rototototototototototfi. .«ewewesetercretg vacueuetewowowewou vu’t 104949404.” .nonononononona addicted mowewowoweu. vOOOOOOOOOD. cvewewewewes. P’W’..M.~.L. a» VO'ODODO. mosaics”. newswows 3000090., . v0.6.0.1. .666». Renews... 6.5 52 - 3.9 P 2.6 - 13 - 0.0 STRAINS 73 levels originated on host plants other than peach or potato. Most moderate and high a- carboxyesterase levels originated on peach or potato, however, and both of the extremely high a-carboxyesterase levels originated on either the primary host (peach) or the secondary (potato). Figure 2.15 shows the results of the B—carboxyesterase microplate assay, also in histogram form. The B-assay was used to gain a perspective of the total carboxyesterase level found in each strain. Although some strains have a relatively low a—carboxyesterase level, they may have a rather high B-carboxyesterase level, or general carboxyesterase level. An example of this is the WILDER-Z-ID-Pl strain, which was moderate in the a- carboxyesterase assay and extremely high in the B-carboxyesterase assay. Two strains which maintained extremely high levels in both aspects of the assay were the MOXEEZ- WA—Pl and HERMISTON-OR—PZ strains. Table 2.4 elucidates the mean carboxyesterase levels and confidence limits (i standard error of the mean or SEM), resistance levels, and Tukey's test values for each strain (or = .05, df= 110). Strains have been divided into five categories based on the Tukey's tests: susceptible, low, moderate, high, and extreme levels of or-carboxyesterases. The category boundaries, however, are flexible and were designed solely for ease of discussion and understanding, not as absolute measures. Sawicki et a]. conducted a similar survey in 1976 of GPA in Great Britain, where 258 populations were collected and assayed for insecticide resistance. Of all the populations collected, only three did not contain demethoate-resistant insects. In 197 of the samples, more than 76% of the GPA were resistant (Sawicki eta]. 1978). Sawicki et a]. believe that although bioassays are effective for resistance detection in insects with a large 74 Figure 2.15--Histograms of the B-Naphthylacetate carboxyesterase assay showing total B- carboxyesterase levels, Tuckey's test for signifance letters (or = .05, df = 110). 86 O W” were” MINUTES 3 '3 8 8 (D NMOL B—NAPHTHOLIug PROTEIN/20 o 3 '3 8 3 8 with” 75 5.. a. .‘Q‘ 1 are Ir ‘. «0“» MW“ I I r I I I I I I I I I I r .- h - ’a‘ o‘ s‘. 53 ft '0 '0‘ 0‘. t! 34 570' a. '0'3' r r r r r - h - E? ti 33 EE ii W 0" "v. 9 ' 5.3.. 9... ”A I l .. .. - 91*: his: ”WW M STRAINS w c 94?. ’M M 76 Table 2.4-Results of national survey of carboxyesterase levels in green peach aphid using microplate assay 3: SEM (Standard Error of the Mean) and Tuckey's test for significance. Strain Mean a-carboxyesterase Mean B-carboxyesterase value i SEM value A: SEM MOXEEl-WA-Pl 1.53 i 0.16 gh 2.74 :h 0.01 uvw PRESQUE-ME-P2 1.29 :t 0.12 hi 2.99 d: 0.15 rst SALINAS-CA-O 0.53 i 0.08 qrs 5.30 at 1.24 f WOOSTER-OH-P2 0.40 d: 0.04 stuv 3.80 :i: 0.30 hijk STRATHAM-NH-PZ 1.75 i 0.34 fg 7.42 :t 0.80 d MONTC 1-MI-P2 0.74 d: 0.32 mnopq 2.94 i 0.66 stu PULLMAN-WA—O 0.03 :h 0.01 x 1.97 :l: 0.10 @% WILDERl-ID-Pl 0.23 :t 0.03 uvwx 2.85 :t 0.13 tuv NEWMAN-CA-O 0.82 i 0.12 mno 3.56 :1; 0.52 lmn IMPCO-CA—O 3.16 3: 1.36 c 3.37 A: 0.34 nop GAINESVL—FL-T 0.03 st 0.004 x 2.85 :l: 0.13 tuv WILDERZ-ID-Pl 0.80 i 0.12 mno 28.34 i: 9.67 a PARMA-ID-O 2.84 :1: 0.54 d 4.64 a: 0.48 g BINGHAM-ID—O 0.79 i 0.13 mno 4.03 i 0.90 h MOSCOW-ID—O 1.20 :t 0.10 ij 3.03 i 0.24 rst UNKNOWN-ID—PZ 0.73 i 0.22 mnopq 2.68 :1: 0.30 vwx GRDRAPIDS-Ml-Pl 0.60 d: 0.05 opqrs 1.19 i 0.09 # CARLETON-MI-O 0.37 d; 0.05 stuvw 2.82 i 0.11 tuv MONROE-Ml-PZ BERRIEN-Ml-PZ 0.73 i 0.06 mnopq 0.41 :l: 0.08 stu 3.13 i 0.15 qrs 2.47 a: 0.26 xyz* 77 Table 2.4-Continued. TRAVERSE-MI-PZ BRDGPORT-NB-P2 ALLIANCE-NB-P2 HEMINGFD-NB-PZ BEAUFORT-N00 VAN ORA-OR-O HERMISTON-OR-PZ CENTER] -PA-P2 MARYHILL l -WA-O QUINCY-WA-P 1 YAKIMA-WA-P 1 TOPPENI -WA-O TOPPENZ-WA-O WALLULA-WA-O 1.67 i 0.61 gh 0.65 i 0.07 nopqr 1.06 :t 0.09 jk 0.80 :1: 0.05 mno 0.17 i: 0.02 vwx 0.70 :1: 0.08 mnopqr 5.51 :r: 0.70 a 1.51 d: 0.20 jkl 0.54 :L- 0.17 qrs 0.60 :1: 0.10 opqrs 0.55 i: 0.05 pqrs 0.92 i 0.12 klm 3.16 d: 0.65 c 1.49 :1: 0.49 hi Strain Mean or-carboxyestcrase Mean B—carboxyesterase value :1: SEM value 3: SEM GRATIOT—MI-PZ 0.85 i 0.30 lmn 1.90 d: 0.37 % MONTCZ-MI-P2 1.17i0.11j 3101:021qu MONTC3-MI-P2 2.20 :t 0.23 e 3.92 d: 0.25 hi MONTC4-MI-P2 3.15 i 0.38 c 5.09 :l: 0.47 f MONTCS-MI-P3 1.49 :1: 0.19 hi 4.01 :1: 0.55 h AUGRES-MI-O 0.73 a 0.61 mnopq 3.80 a 0.24 hijk 4.62 :1: 1.20 g 2.86 i 0.38 tuv 2.64 i 0.20 vwxy 2.35 :t 0.15 2*! 1.31 :l: 0.14 # 2.18 i 0.12 !@ 18.21 a: 0.53 c 2.44 :1: 0.32 yz“ 3.88 a: 0.37 hij 2.51 i 0.25 wxyz 2.25 i 0.09 *l 1.95 :1: 0.16 @% 3.76 :t 1.39 ijkl 4.49 :1: 1.45 g 78 Table 2.4--Continued. Strain Mean or-carboxyesterase Mean B-carboxyesterase value :1: SEM value 2t SEM MADISON-WI-P2 0.53 i 0.06 qrs 3.48 i 0.14 mno IDAHO-P2 0.69 :1: 0.04 mnopqr 3.99 i 0.23 hi ANSON-MI-P2 0.79 i 0.17 mno 3.67 :1: 0.23 klmn MONTC6-MI-P2 0.78 d: 0.17 mnop 3.30 :t 0.25 opq MONTC7-MI-P2 1.91 a: 0.28 f 3.58 a 0.29 klmn MUNGER-MI-PZ 0.29 :h 0.09 tuvw 3.22 d: 0.54 pqr STJOHN'S-MI—PZ 0.47 :1: 0.06 rst 2.35 :1: 0.11 2*! RVRSIDE-CA-O 1.52 :1: 0.16 ghi 2.15 i 0.10 !@ MARYHILL2-WA—O 0.29 d: 0.02 tuvw 2.48 d: 0.17 xyz" MONTC8-MI-P1 0.80 :h 0.10 mno 5.64 st 1.07 e TOPPEN3-WA—O 0.16 :1: 0.04 wx 1.38 d: 0.07 # CENTER2-PA-P2 1.12 d: 0.08 j 3.29 :1: 0.25 opq TOPPEN4-WA—P2 0.59 :1: 0.04 opqrs 2.75 :l: 0.09 uv RCKINGHAM—NH-P2 2.25 at 0.77 e 2.66 d: 0.44 vwxy MOXEEZ-WA-Pl 4.88 :1: 0.45 b 19.46 3: 1.19 b 79 difl‘erence between the resistant (R) and susceptible (S) individuals, when phenotypes overlap or have intermediate levels, bioassays are not as efi‘ective (1977). The diazoblue or tetrazotized O-dianisidine (Fast Blue B) that van Asperen used is the samethatwasusedinmyesteraseprotocol. However,Idid notusesodium laurylsulphate, which van Asperen used to enhance the color of his naphthol solutions as well as to stop the esterase-naphthyl acetate reaction (van Asperen 1962). Also in contrast, van Asperen used B-naphthyl acetate as substrate only occasionally, whereas I usediteachtimetogetanideaofthetotal carboxyesteraseamomrtsineachstrainof insect. Wedidusethesamebufi‘ersolutionsande-I vanAsperendid not mention proteinassays, whichareanintegralpartofmy study, astheyaidincomparisonsof amount of naphthol per insect using total protein variations. Ruud et a]. (1988) note that in their studies of est-m, an esterase found in chrysomelid beetles, that there may be a problem with the BioRad and other total protein staining determinations. This was observed while being unable to stain est-m with Coomassie Blue stain during Polyacrylamide Gel ElectrOphoresis (PAGE). I also observed this in my experiments with GPA carboxyesterases during PAGE, but not in the microplate assays. I would like to point out that carboxyesterase resistance is associated with increased activity of the enzyme. This is overall activity, not specific activity. It is believed that carboxyesterase resistance in GPA is associated with increased levels of carboxyesterases. This is difl‘erent than specific activity, which in this case would be associated with a mutant form ofthe enzyme that has an increased affinity for the substrate or a faster catalytic activity level. Table 2.5 illustrates the correlation coeflicients and their significance between carboxyesterase levels and the other in vitro carboxyesterase experiments. These other 80 Table 2.5-Coeficients for correlation analysis of in vitro assays with microplate data across the eight main strains. ct-Portable Assay B-Portable Assay PAGE a—Microplate Assay .898" - .762" B—Microplate Assay - .405 .568 Significance was tested at a = .05 (‘) and at = .01 C“) levels. Global 13 test = 21.091 ° (significant) 8l techniques involved Basic 10% PAGE banding patterns and a portable carboxyesterase assay tool for field work. Microplate carboxyesterase assays were compared with the 10% PAGE gels to seek possible correlations between them. The global x2 test was significant (21.091). Table 2.5 shows the correlation coeficients for the comparisons. The total densitometry units show a strong correlation coeficient (0.762) with a—carboxyesterase levels (significant at a = .05). The B-carboxyesterase microplate data does not show a significant correlation (0.548) with the PAGE densitometry data The correlation analysis of the microplate a-carboxyesterase level with portable or- carboxyesterase level was 0.898, significant at or = 0.01. The portable B—carboxyesterase level (with the B-microplate assay) showed a positive correlation coeficient of 0.405, which is not significant. However, there is an overall positive correlation which shows that as carboxyesterase level increases with the microplate assay, it also increases with the portable assay. This shows that the portable a-carboxyesterase assay is a valid measure of carboxyesterase level as defined by the microtitre esterase assay, as the correlations are significant ata= .01. B. Gel Results 1. Basic 10% Gel Results Adult GPA were electrophoresed using PAGE in order to characterize each strain and determine difl‘erence in banding patterns or intensity. Mack and Smilowitz found no difference in the protein banding patterns for GPA in PAGE gels (1980). Sudderuddin discusses the use of staining with substrates, such as 1-naphthyl acetate as well as the use of inhibitors like dichlorvos (Sudderuddin 1972). Other inhibitors, such as insecticides could be used as well. 82 Figure 2.16 is a picture of the two gels used to elucidate the basic banding patterns of each strain Difl‘erencesbetweenstrainscanbeobservedwiththenakedeyeaswellas documented using densitometry techniques. In the figure, susceptible strains (as defined by microplate carboxyesterase data) appear to lack (or it is very faint) one or both of the two bands found in the strains with greater resistance levels. The significant band is the bottom (second band), which is missing in all susceptible strains and present in all resistant ones. Although some resistant strains lack the top band, they still retain the bottom band. Additionally, the top band stains more darkly with B-naphthyl acetate than it does with a, and the bottom more darkly with a—naphthyl acetate. This fiirther illustrates the concept that the second band is the band asmciated with carboxyesterase resistance due to elevated esterase levels in GPA The banding difl‘erences are quantified by AMBIS (AMBIS Inc., San Diego, CA), a powerful new image acquisition and analysis system. This data is shown in Table 2.6, which elucidates the various levels of AMBIS analyzed densitometry peaks for each carboxyesterase band and compares them with a—Portable values, a—Microplate values, and resistance ratios (azinphosrnethyl LCgo). In this way, comparisons can be observed between densitometry units and the other measures. For instance, the two lowest values for the complete densitometry units, PULLMAN-WA and WILDERl-ID are also the two lowest values for the or-Microplate Assay and the ct-Portable Assay, and are the two lowest resistance ratio values. The highest densitometry value, SALINAS-CA, is not the highest in any other category, suggesting some error in the gel. However, the highest resistance ratio value, STRATHAM-NH (6.6), also has the highest (tied) or-Portable value and the second highest or-Microplate Value. This strain is also quite close to the densitometry value for SALINAS—CA. 83 .<3-z<2.::m 5 as .94me43» As 43-83588 5% as... w e? .61: use 2.. a. see. a 2.. m .8 no .mzoozéezgooe 6 $2853 5 5.2328 6 flog—$83 § .mzééeéem 5 gembommé 5 .5 -9238 5 £345on 5 .5650: S on < co 8 ease m 2:. sea 22885 2: 5 32.; as: S 588% m ._o M gonzo ms eo_o€_ 0.3 835m 53:8 SahuaoEoanid new 353» new neg—bonanza 023: 95 mo :eafiBoiiB .N 233m 84 Table 2.6-Comparison of azinphosrnethyl resistance ratios (RR), densitometry unit values, portable values, and microplate values. STRAIN RR DUV1 PV2 MV3 PULLMAN-WA 1 .0 0 0.000 0.000 SALINAS-CA 1.3 34600 0.046 0.496 MONTC l-MI 1.5 39900 0.017 0.709 WOOSTER-OH 2.7 11000 0.035 0.370 MOXEEl-WA 3.4 52300 0.052 1.503 PRESQUE-ME 4.5 39500 0.048 1.258 STRATHAM-NH 6.6 58600 0.052 1 .720 WILDERI -ID - 0 0.000 0.204 1DU Value = dcnsitomctry units (DU) of each strain - DU of susceptible strain (PULLMAN-WA). 29 Value = a—portablc results (nmol a—naphthol/ insect! 10 minutes) ofeach strain - nmol (it-naphthol for susceptible strain PULLMAN-WA. 3M Value = o- microplate results (nmol a-naphthol/ ng protein/ 20 mimrtcs) ofeach strain — nmol or.— naphthol for susceptible strain PULLMAN-WA. 85 2. Insecticide-Inhibited Gel Results Figure 2.17 is a picture of two 10% PAGE gels, one inhibited with an insecticide, one (control) not inhibited. This gel is an example, each insecticide inhibition and control was replicated three times in order to obtain data that was valid for statistical analysis. In the insecticide-inhibited gel there is an absence of bands and in the control gel there is a strong presence of handing, both of which can be observed with the naked eye, as well as in densitometric analysis in the AMBIS Radioanalytic Imaging System Table 2.7 shows the percent inhibition for the mean densitometry values (total value minus background - inhibited densitometry units/ uninhibited densitometry units "' 100) found for all three replicates of each insecticide. According to the table, the highest percent inhibition was 100%, and the lowest was 66.5% (CENTERZ-PA). A visual observance of the gels shows that there were no hands or else very faint ones on the insecticide inhibited gel. The densitometry data confirms this, showing lower values for the inhibited gels than for the controls. This means that the substrate a-naphthol is actually the correct substrate for studying the enzymes that hydrolyze 3. Discussion Physically, gels can be in many difl'erent forms. Vertical slab gels were chosen for these experiments for the ease of preparation and reproducibility. Heat is more easily dissipated in slab gels than in other types (such as rod) and their shape allows procedures such as densitometry to be conducted for quantification purposes. Lastly, many samples can be run under identical purposes on the same gel, which is not true of rod gel type gels (Hames 1981). Electrophoresis of insect esterases on polyacrylamide gel has been less widely used than starch and agarose gels (Ogita 1963, Cook and Forgash 1965, Salkeld 1965, Benton 1967, 86 Figure 2.17--Photograph of two native polyacrylamide gels stained for a—carboxyesterase activity. Gel A is the control gel, which was not incubated in any inhibitor (insecticide). Strains are labeled as R or S depending on their classification by Microplate assay. Strains are also numbered: (1) MONTCl-MI, (2) MOXEEl-WA, (3) WOOSTER—OH (4) PRESQUE—ME, (5) STRATHAM-NH, (6) SALINAS-CA, (7) CENTERZ-PA, (8) MADISON-WI, (9) PULLMAN-WA, and (10) WILDERl-ID. Gel B is the inhibited gel, which was incubated in one of three insecticides before staining. 87 Table 2.7-Summary of mean percent inhibition of green peach aphid strains using PAGE gels inhibited by carbaryl, oxydemetonmethyl, and permethrin (three replicates each). STRAINS‘ azinphosmethyl % INHIBITIONC RR” CHEM] CI-IEMZ CHEMsd MONTCl-MI 1.5 82.6 80.1 90.4 MOXEEl-WA 3.4 73.7 98.4 100 SALINAS-CA 1.3 82.7 100 100 PRESQUE-ME 4.5 100 100 100 STRATHAM-NH 6.6 95.5 100 100 WOOSTER-OH 2.7 100 100 100 WILDERl-ID — 94.2 95.4 100 PULLMAN-WA 1.0 100 97.4 100 MADISON-WI -. 80.6 77.9 96.8 CENTERl-PA .- 66.5 71.2 82.1 ‘STRAINS correspond to Table 2.3, which outlines the alphabetic and numerical codes for each strain of green peach aphid. I’le is the methomyl resistance ratio (LC50 strain] LC50 susceptible strain PULLMAN-WA)obtained by in viva assays. ‘% INHIBITION is calculated by uninhibited total densitometry units (UDU) for each strain - inhibited densitometry units (IDU) for the same strain, divided by UDU and multiplied by 100 ‘CHEMl=earbaryl, CHEM2=oxydemetonmethyL and CI-IEM3=‘permethrin. 88 Clements 1967, Matsumura and Sakai 1968, Cook et al. 1969, Katzenellenbogen and Kafatos 1971, Sudderuddin I973). Blackman and Devonshire conducted horizontal starch gel electrophoresis (along with a stain system similar to mine in 1978 (Blackman and Devonshire 1978). However, Sims (1965) studied the esterases of Drosophila on PAGE, as did others (Price and Bosman I966, Arurkar and Knowles 1967, 1968, Ahmad 1968, Ozaki 1969). Sudderuddin (1972, 1973) observed a higher quantity of one band ("E3 ") in resistant strains of GPA The major change was only in activity of est-4 (E4), the enzyme involved in resistance, not in mutation, just amount (Devonshire 1977, Sawicki et al. 1980). Devonshire observed in 1977 that the E4 enzyme, responsible for resistance, stains more heavily in PAGE in resistant individuals than susceptibles, even when the insects are only slightly resistant (Devonshire 1977). He showed that the carboxyesterase degrades organophosphate insecticides and ct-naphthyl acetate more rapidly in resistant than susceptible insects (Devonshire 1977). Pasteur and Georghiou (1989) used polyacrylamide gel electrophoresis (PAGE) to determine increased or unincreased levels of esterases. They claim that a gap of several optical density units separates susceptible individuals from those with increased levels of esterases (1989). Sawicki er. a1 did note that gel results were semi-quantitative and the levels of resistance could not always be determined by the intensities of the E4 band, and for this reason, they conducted other tests for resistance determinations (1977), as I did. These results were then correlated with the data from dosage mortality assays and carboxyesterase assays. Sawicki et al. (1977) explained that although PAGE is suitable for rapidly estimating the proportion of resistant individuals in a population, the biochemical esterase determination is more reliable for determining resistant types of each individual. I noted excellent correlations with the resistance indicators (ct—naphthyl acetate) and rather inconclusive 89 results with the general esterases, which is to be expected as staining was selective only for a-naphthyl acetate. The results show that PAGE is a valid measure of carboxyesterase difi‘enences, although not as quantitatively accurate as is preferable. Sudderuddin observed that there were (quantitative) differences between resistant and susceptible strains of GPA by their electrophoretic patterns (Sudderuddin 1973). Mack and Smilowitz notedno difi’erencebetweenthe numberand location ofbands, onlythe banding intensity (Mack and Smilowitz 1980). Total carboxyesterase activity assessed associated with est-4 (Blackman et al. 1977). Sudderuddin notes "interesting" results when gels were first inhibited with dichlorvos then stained for esterases. He notes some slight activity and suggests that this may relate to the fact that they may be arylesterases (Sudderuddin 1972). He also notes that the banding patterns of GPA reveal a relatively constant pattern though quantitative fluctuations can occur over time in laboratory populations (1972). I also noted relatively consistent banding patterns in the PAGE gels tested, and some very slight activity on the insecticide inhibited gels. Mack and Smilowitz noted no difi‘erences in banding patterns on two GPA biotypes although there were differences in banding intensity (Mack and Smilowitz 80). I note quantitative difi'erences between strains on my gels, but no qualitative. For a molecular weight standard, Mack and Smilowitz used Bovine serum albumin (BSA) as a dye marker (mw 68,000) (1980). They do not report dificulties in staining the carboxyesterase as I did and as Ruud et a1. did (1988). They used SDS gels, however, which could suggest that there is a structural anomaly to the carboxyesterase which is not present when the enzyme is active. However, Field et al. report that the molecular weight of esterase E-4 is 65,000 with a polypeptide portion of 57,000. Devonshire and Sawicki report E-4 as 65,000 kDa, and FE-4 as 66,000 kDa (Devonshire er al. 1986, Field et al. 90 1989). In the mutant strains of GPA with esterase FE-4, the molecular weight of the polypeptide portion is 58,000 (the whole molecule is a glycoprotein) and the catalytic center of activity is slightly difi‘erent (Field er al. 1988). In an attempted experiment, I had dificulty with the Coomassie Brilliant Blue dye not stain my carboxyesterases. Ruud er a1. attributes this fact to the carbohydrate form of some carboxyesterases of other structural peculiarities somehow preventing the Coomassie blue (and in his case silver stain as well) form reacting with enzyme moieties essential to stain uptake. This could also reflect low levels of esterase on the gel (Ruud et al. 1988). Ruud eta]. also suggest thatthis may also meanthattheinabilityto stainthe esterase in the gels may also reflect on the eficacy of staining with BioRad Protein Reagent" for total protein (Ruud er al. 1988). 3. Basic Gel Correlations with other in vitro tests Table 2.8 illustrates the correlation results between the basic gel densitometry data and the carboxyesterase microplate and portable assays. There were positive correlations with the carboxyesterase assay data sets, especially the (Jr-carboxyesterase values. The portable or- values were high 0.831 (Pearson correlation coeficients), and the portable B- values were also high at 0.914 (Pearson correlation), both of which are significant at or = .01. The ct- carboxyesterase microplate correlation values were significant (0.762) at or = .05 (Without WILDERl-ID, the correlation coeflicient was 0.919, which is highly significant. This shows that WILDERI -ID is skewing the data to some degree). For the B-microplate assay, the correlation coeflicient was not significant (0.548). This is not surprising as the stain system is selective for a-carboxyesterases. There is an overall positive correlation between the a-carboxyesterase assays and PAGE, as the data shows. 91 Figure 2.8—Coeficients for correlation analysis of in vitro assays with PAGE densitometry data across the eight main strains. PAGE a—Microplate Assay 0.919‘ B-Microplate Assay 0.548 ct-Portable Assay 0.831" BcPortable Assay 0.914" Significance was tested at a = .05 (*) and at = .01 C") levels. Global 13 test = 21.091 * (significant) 92 C. Portable Data Results In Figure 2.18, the regression lines for the standard curves of both the a— and B—naphthol standard curves are shown. Both data sets were replicated three times for statistically correct values for analysis. Standard curve data is useful as a transforming character for standardizing absorbance (or transmittance values of a photometer). Aprotein standard curvewasattemptedbut discardedastheyarenot integral toa functional field assay, but are for a more exact scientific interpretation All the field workers and pest management decision makers will need is to know the nmol naphthol per insect (GPA), not per ug protein. Additionally, some dificulties were encountered in calibration of the portable photometer for protein readings, as it quickly reaches the maximum value for accurate readings. According to the graph shown in the appendix, the protein standard curve does not appear to be a linear relationship, but more like a curvilinear one. Additionally, protein values will only be accurately read at protein concentrations above 10 ug/ 100 pl. This curve can be found in Appendix C. For a graphical representation of the portable assay results, the histograms in Figure 2.19 are one means of elucidation. The amount of naphthol per insect per ten minutes is plotted for each strain. The highest a-carboxyesterase level strain was MOXEEl-WA-Pl. The lowest values were WILDERl-ID-Pl and PULLMAN—WA-O, and this is true for the fi-carboxyesterase level as well. The highest B-carboxyesterase level strains are SALINAS-CA, PRESQUE-ME-PZ, and MOXEEl-WA-Pl. For Tukey's tests of differences between means, Table 2.9 was set up with mean nmol a- and B-naphthol per GPA per ten minutes :t standard error of the mean (SEM), and Tukey's test for significant difi‘erences between means. 93 15- 10- ABSORBANCB III 0 E, - m M o —l 8 < _ 0.0 J .. _0.1 L l l J 0 5 10 - 15 20 25 NAPHTHOL CONCENTRATION Figure 2.18—Portable in vitro assay standard curves for ct- (A) and B—naphthol (B) standard concentrations (ug)/ 100 1.11 plotted against absorbance values (609 nm = a, 555 nm = B). The equations of each line are found in Appendix B. 94 Figure 2.19-Histograms of portable assay results across eight main strains. Letters signify Tukey's Means Separation Test for significant differences between strains. ab “‘.‘...‘...‘..“.‘.“““‘. w6%bfléflbfibfibflbfléfléfififlbfléfléfiéfl do900600090.900809060090000! a5b555bbbbbbbbbbbbbbbbbbbbbbfi .............................. n333fihfihfifihfififihhhhfifihhhhhfifih adflflfififififlfififififififififififififififififififl ASflflfiflflflflfififififlflfiflfiflgyfiwV. oooooooooooooooos%s%ah%s €%€€fi§€§fi§fififififififivflvh€o%€ .5.9.0.9.90.0.9.0.9.990.99.90.93.990.99.. O “Njfifififififififihflfifihfifififihfl c.§????§38¢££¢&¢§8€¢€6 .ooooooooooooooooooooo be II"-(}1 + CH 3 It): \ / azinphosmethyl HS‘CH 2.2K @ \ 109 Although carbaryl is not a very efi‘ective aphicide, it is used on potato for controlling leaflioppers, Tarnished Plant Bug, cutworms, and Flea beetles (MSU Extension Bulletin #312, 1992). Oxydemetonmethyl is a systemic oxon-organophosphate. For this reason, the compound isveryefi’ectiveonceitenterstheinsect’sbcdy. Oxoncompoundsaregcnernllymore toxic than their non-activated parent compounds. Generally, insecticides are activated by the Mixed Function Oxidase (MFO) system, such as when parathion is activated to the more toxic paraoxon before enzymatic degradation This chemical is commonly applied to potato fields at the rate of 2 pt (SC)! acre for control of GPA, leafhoppers, and Flea beetles (Epitrix cucmnerix (Hanis)). It is particularly noted as effective for problem infestations of GPA (MSU Extension Bulletin #312, 1992). Perrnethrin is a synthetic pyrethroid containing two aromatic rings. This compound was patterned afier the botanical insecticides, such as pyrethrum, but it has the advantage of being more stable in stmlight. Although not a very toxic compound to GPA, it is still used for control of leafhoppers in potato fields (MSU Extension Bulletin #312, 1992). Methomyl is a carbamate compound. Unlike carbaryl, methomyl has no aromatic rings. Methomyl, or Lannate®, is commonly used for control of GPA, cutworms, flea beetles, and leaflioppers in potato fields at a field rate of 0.5 lb/acre (MSU Extension Bulletin #312, 1992). Azinphosmethyl is an organophosphate with two rings, one containing nitrogen. Azinphosmethyl is commonly known as Guthion® and although it is generally not used for GPA control on potato, it is used for control of other pests such as Colorado Potato Beetle, flea beetles, and leafltoppcrs (MSU Extension Bulletin #312, 1992). 110 The six compounds were not only chosen fi'om several difi‘erent classes of compounds, but withcaretakenthattheyalsovariedintoxicitylevel. Forthisreason, theyhave difi‘erent reaction sequences in GPA and perhaps difi‘erent modes of action. The mammalian toxicities of each compound were also taken into account solely for a general range of toxicity. However, mammalian and insect toxicities are not necessarily related, this was justusedasabasis. Carewastakento selectcompoundsnot onlyonthebasisofagood diversityofcompound classes, butalsowithrespecttoavariablerangeoftoxicities. C. In vivo Bioassay Prelimary assays were conducted solely to gain an understanding of the range of efi‘ective concentrations to be used for each compound. The compound concentrations used are elucidated in Table 3.3 They vary extensively from compound to compound depending on toxicity and resistance levels to the compounds found in strains tested. Concentrations were chosen on the basis that they produced approximately 90% mortality or higher at the highest dose, with decreasing mortality percentages with decreasing concentration The data are presented in ppm in double distilled 1120. Figure 3.1 elucidates the probit data for azinphosmethyl and oxydcmetonmethyl (two organophosphates) in graphical form. Graph A shows the probit lines for azinphosmethyl (Guthion). Each strain in this graph shows a similar, relatively flattened slope of the line except for MONTC l-MI, which shows a more vertical line. Generally the flatter the slope of the line, more heterogenous the population is. Additionally, there is greater resistance potential forastrainthathasaflatslopethanamorevertieal sloped linethathasthe same LC 10 value. Therefore, of the seven strains tested with azinphosmethyl, MONTC I-MI has the most heterogenous population. The strain that has the lowest log concentration values is PULLMAN-WA, at the LC 10, LC 50, and LC90 values consistently. Closely parallel to PULLMAN-WA is SALINAS-CA, also susceptible to azinphosmethyl. The two strains 111 Table 3.3—Listing of insecticide, compound class, and concentrations used for in vivo bioassays on green peach aphid. Chemical Compound Class ConcentrationI #5 #4 #3 #2 #1 azinphosmethyl organophosphate 420 168 109 84 29 ethyl parathion organophosphate 88 57 31 11 7 carbaryl carbamate 442 178 115 62 22 permethrin synthetic pyrethroid 1892 1135 757 378 151 oxydcmetonmethyl organophosphate 1288 644 386 129 26 methomyl carbamate 1750 875 525 175 35 lConcentrations measured in ppm. 112 1 l T I l A s — - 5 8" 4 ----- woman-on .. — — mamas-run ‘ '- --------- saunas-ca y --—- mum-rm ‘ - - reasons-Isa — — MOXBBl-WA 3 I I I L —— isomer—m 1 to 100 tons roses 1m 1 I I I ‘ —l g s d —— woosraa—on ----- ernanr-rn ‘ - — — strum-an --------- saunas-ca —--- PULLMAN-WA - - raucous-us — — MOXBBl-WA 3 I I L —— MON'I'Cl-Ml 1 10 100 ms roses Figure 3.1—Regression lines depicting the relationship between probit values and lethal concentrations for azinphosmethyl (A) and oxydcmetonmethyl (B). 113 have similar slopes, although SALINAS-CA shows a slightly steeper slope than does PULLMAN-WA The most resistant strain at LC“) is MONTCl-MI. However, due to MONTC l -MI's extremely steep slope, by the LC90 concentration, MONTC 1-MI has teh third lowest LCgo value. The other four strains: PRESQUE-ME, MOXEE l-WA, WOOSTER-OH, and STRATHAM-NH are closely grouped alter the LC50 value and although there is some difi‘erence in slope, they remain close alter this point. Although there was is a wide spread prior to the LCso point, this rapidly bottlenecks and the strains become grouped hereafter. Figure 3.1B illustrates the probit line for oxydcmetonmethyl, a systemic organophosphate. Inthisgraphthereislittle crossingoverofstrainsfi'omtheLClototheLCmanchgo values. The strain with the highest resistance potential is MONTCl-MI, due not only to the extremely flat slope of the line but also to the fact that this strain yielded the highest LClo, LC 50, and LC90 values. When compared with the azinphosmethyl probit line, the opposite was true: MONTC 1 -MI was the strain with the lowest reistance potential of the seven tested. However, in both graphs, MONTCl-MI began as the most resistant strain at the LC 10 level, and it was not until the LC50 point that the slope-associated difi‘erence was elucidated. In Figure 3.1B (oxydcmetonmethyl) the strain with the most vertical slope of the line is WOOSTER-OH. This strain was among the closely grouped strains on the azinphosmethyl probit graph. The first three strains found on the oxydcmetonmethyl graph: MOXEEl-WA, PRESQUE-ME, and PULLMAN—WA, are very closely grouped, with similar lethal concentrations and slopes. Figure 3.2 is a set of two graphs illustrating the probit lines of parathion (A) and permethrin (B). Graph A elucidates the probit lines for ethyl parathion, a very commonly 114 7 I T I I A 5 b —. 5 o 5 ’- -I E ----- woos'ras-on . ,_ _ — — mam-rm , --------- saunas-ca ' —-—- PULLMAN-WA "' - MODE-KB _ — MEI-WA 3 I l l l ‘-—"' ”mi-HI 1 lo I“ 1m 1m 1m 7 I I I I 6 —I —"—" WMR-on ----- WILDBll-ID _ — — STEAM-NH ‘ -------- - saunas-ca ‘---"' PULLMAN"A "' "’ “QUE—IE — - MOXEE-WA 3 l 1 I 1 —— MONTCl-Ml lo I” 1000 1000!! 1m 1m MW" Figure 3.1-Regression lines depicting the relationship between probit values and lethal concentrations for parathion (A) and permethrin (B). 115 used organophosphate compound, and the most toxic insecticide used for the in viva Bioassay (Ware 1983). The strain in this graph with greatest vertical slope is PULLMAN- WA, itisalsothemost susceptiblestrainatthecholevel, showingthatithasthelowest resistance potential of all strains tested. STRATI-IAM-NH is the most susceptible strain at the LC“, level, but it has a more horizontal slope and therefore crosses over the PULLMAN-WA strain at approximately the LC 50 level and becomes the second most susceptrblestrain AtI£90,themostresistam8trainisPRESQIJE-ME,bmthisisduem part the the flat slope ofit's probit line. At the LC“) level, it is only a moderately resistant strain, partofagroup ofsixmoderatestrains. Fiveofthesestrainsarestillgroupedatthe LC” level, and four remain grouped at the LCgo level. Figure 3 .2B illustrates probit lines for the least toxic compound used, permethrin, a synthetic pyrethroid. The strain with the most vertical slope is PULLMAN-WA The most resistant strainatLCgo, STRATHAM-NH, isbyfarthemost resistant strainofall tested. Inaddition, thisstrainhasthegreatest resistaneepotential, duetotheveryflat slope of it's probit line. At the LC“, level this strain is the most susceptible, as well, showing that this strain is very heterogeneous for resistance to permethrin The second most resistant strain is MONTCl-MI, which has yielded the highest resistant results to both azinphosmethyl and oxydcmetonmethyl, but not to parathion. The other five strains remain closely grouped throughout the graph. Figure 3.3 is an illustration of the probit lines for methomyl (A) and carbaryl (B), both carbamate insecticides. Graph A shows a very closely grouped association of lines. The highes value at LC“, and LC50 appears to be SALINAS-CA, however, there do not appear to be any significant difi‘erenees between lines or slopes of lines in this graph. All eight strains have fairly similar slopes of their probit lines. One exception is 116 1 I T I A 6 r- q 5 s - .. 3 .0 —— woosrsa—on ----- ernanI-rn 4 _, — — smmn—nn I --------- saunas-ca ——-- rum-rm — - masons—Isa -— — mar-WA 3 L I I — Harmer-1111 1 10 100 1000 10000 1 I l r s - - E 5 II— u-t ----- woman-on ‘ _ _ — — mamas-rm --------- saunas-ca —-—- mum-rm - - masons—us — -' MOXBBI-WA 3 L I I MON'I’CI-MI 0.001 1.000 1000000 1000000000 .1ssssss+10 CONCENTRATION Figure 33 --Regression lines depicting the relationship between probit values and lethal concentrations for methomyl (A) and carbaryl (B). 117 PULLMAN-WA, the strain with the lowest LC 50 value, and the most susceptrble strain across all chemicals. All other strains are very closely grouped. Figure 3.313 is another carbamate, carbaryl (Sevin®). This graph has the greatest variation and the highest concentrations due to its relatively low toxicity. This chemical yielded broad ranges for concentrations at all levels and a high error level. Dificulties occurredinobtaininga90%mortalityrateacrossallstrains. Asaresult, somecurious probit lines are shown Strain STRATHAM-NH yielded the flattest slope, however it was paralleled closely by MOXEEl-WA, the strain with the greatest LC50 and LCgo values. Thestrainwiththesteepest slopeofthelinewas SALINAS-CAandthiswasgroupedat the LC 50 level with PULLMAN-WA (the susceptible for all other chemicals) and PRESQUE-ME. However, the slope of these two strains lines was more horizontal than that of SALINAS-CA, and therefore they did not yield as low LC values. The calculated LC 50 and LC90 values and 95% confidence limits for each strain and chemical are listed in Table 3.4. In addition, so are the slopes of the lines. Overlapping confidence limits of LC50's were compared, but a multiple range test was not run because it is not reliable unless weighted. Data for carbaryl is not discussed due to a high error level (very broad confidence limits). This error was due not only to a natural variability in GPA strains but to the low level of toxicity of the compound to GPA Letters signify difi‘erences between strains. Overall, the most susceptible strain was PULLMAN-WA, maintaining the lowest for LC 50 values. In the case of azinphosmethyl, PULLMAN-WA was significantly lowest of all strains (LC 50 = 65), with SALINAS-CA the second-most susceptible (LC50 = 99). These two strains also had the lowest LC50 level for parathion, but not for oxydcmetonmethyl (MOXEE 1 -WA, PRESQUE-ME, and PULLMAN-WA), methomyl, or permethrin. The strain most resistant to azinphosmethyl was PRESQUE- ME (LC50 = 334), as well as for parathion (LC50 = 381). For methomyl, the most 118 -- .. -- -- 9-23.55 6.»: a; 13.3 on c a: 23 xmecme «a: 2.2m 20-. :35 382.32 @3558qu 38.88:... m «3.... 121 resistant strain was SALINAS-CA (LC50 = 135), and for permethrin the highest LC50 was 1709 for STRATHAM-NH. However, this overlapped confidence limits with MONTCl- MI (1455), so that both strains are considered the most resistant at LG». MONTCl-MI was also the most resistant strain at LC50 for oxydcmetonmethyl, a systemic organophosphate. Overall, data showed excellent difl‘erences across strains and chemicals. Carbaryl was the only chemical which showed a high error level and therefore wide 95% confidence limits. Even carbaryl yielded some difl‘enences across strains. The most toxic (to mammals) compound tested, parathion, did not show the lowest LC50's. Oxydemetonmethyl, the systemic oxon-organophophate compound had the lowest LC50 values, ranging from 10 to 114, but an average of 31 ppm. Parathion's average LCso was 209 ppm. This is most likely due to two factors: the fact that oxydcmetonmethyl is a systemic oxon- organophosphate and therefore probably more toxic to aphids than a regular organophosphate (Ware 1983, Matsumura 1985). Resistance ratios were computed to aid in comparing between strains. These resistance ratios (RR) are listed in Table 3.5 and are solely indexing tools to observe trends. Some strains maintain approximately the same RR fiom one chemical to another (PULLMAN- WA, WIIDERl-ID, SALINAS-CA), whereas others vary highly (MONTCl-ML MOXEEI-WA, STRATHAM—NH). Differences also can exist between LC50 and LCgo RR's, such as STRATHAM-NH shows in carbaryl: a range of 8.4 to 32269. Other strain RR's remain the same for a certain chemical whether it is LC 50 or LCgo, such as MONTC l-Ml exemplifies for azinphosmethyl a range of 1.5 to 1.3. 122 .23.z<2.::& 03285 83 Eaa 88 E 838.8 a 83 5... .23-z<2§e 035985 33 Beam a3 3 8.23.8 a 83 mm. 3 I n. E -- -- 9-28.55 3 3 3 3 3 3 <3.z<_5.5s 3. 2 S 2 32 can moemhmooa an: 3. 2 n. 33% v.» :z-2<:._. PAGE > microplate assay > portable assay. With equipment, the ranking is Microplate Assay > PAGE > in viva bioassay > portable assay. B. Labor Evaluations Labor was evaluated on a per papulation sample basis. Table 4.3 shows an estimate of labor time necessary for experimental, data analysis, and sampling. The in viva bioassay takes approximately 25.5 hours for 190 insects to be assayed (including incubation time of l3 1 Table 4.1-Itemized list of costs for in vitro assay procedures. MICROPLATE PORTABLE PAGE Chemicals $ 15.00 $ 10.00 $ 20.00 Basic Supplies $ 9.50 $ 6.00 $ 2.00 Labor Solutions $ 30.00 $ 30.00 $ 60.00 Experiment $ 52.50 $ 37.50 $ 255.00 Data Analysis 3 75.00 $ 25.00 $ 37.50 Equipment EIA Reader $3000.00 3 450.00 Gel Apparatus $1095.00 Power Supply $1095.00 Drying Apparatus $ 150.00 Portable Reader $ 450.00 Portable Filters $ 60.00 Pipettes + Tips $ 603.00 $ 603.00 TOTAL without equipment $ 182.00 $ 108.50 $ 374.50 TOTAL with equipment $3785.00 $ 618.50 $3317.50 132 Figure 4.2-Economics of the in viva bioassay for one chemical. COMMODITY BIOAS SAY COST Chemicals $ 50.00 Basic Supplies $ 100.00 Labor Solutions 3 30.00 Experiment 5 250.00 Data Analysis $ 50.00 Equipmwt Microscope $ 800.00 Pipettes + Tips $ 603.00 TOTAL without equipment 3 480.00 TOTAL with equipment $1883.00 133 Table 4.3-—Labor evaluation of the four techniques for resistance diagnosis. Microplate Assay Portable Assay PAGE in viva Bioassay Sample 30 min 30 min 15 min 5 hours Solutions 2 hours 2 hours 4.5 hours 30 min Experiment 3 .5 hours 1 hour 5 hours 5 hours Data Analysis -- -- 9 hours 18 hours TOTAL 6 hours 3.5 hours 18.75 hours 28.5 hours 134 eighteen hours). The portable assay takes only 3.5 hours for 20 insects to be assayed, including sample time. The total hours are ranked as: in viva bioassay > PAGE > microplate assay > portable assay. C. Accuracy Evaluations Table 4.4 shows the correlation coeficients and an asterisk for significance of the F-test (and t-test), with " indicating significance at a=.05,” a.=.02, and "‘ a=.01 for R2 values withonedegreeoffieedominthenumeratorandsixinthedenominator. Thistableshows the relationships between a- and B-carboxyesterase level and LC50 for the in viva bioassays (microplate a— and B—). The permethrin LCgo correlates strongly (85%"”) with the microplate a values. Weaker correlations exist with the Basic PAGE densitometry units (“76%"). There is a strong correlation between methomyl and the a-microplate assay (94%*"), the a—portable assay (99%”*), and PAGE (95%*"). Parathion also showed strong correlations with all three (Microplate = 87%", Portable 100°/s"""", and PAGE 94%*"). Azinphosmethyl showed and extremely strong correlation with the (Jr-portable assay (99%*") and a fairly strong correlation with PAGE (86%“) but none with the ot- microplate assay. These data show that correlations exist between the in vitro carboxyesterase evaluations ofresistanceandtheinvivabioassays. Anaccuracyratingforthesetests, takinginto account chance for experimental error, would follow a format such as this (fi'om most accurate to least accurate): microplate assay > PAGE > portable assay > in viva bioassay. All tests are actually quite close in accuracy, however, the reason the portable assay is next to last is that the handheld spectrophotometer probably does not have as high quality lens as it should for laboratory work. The in viva bioassay is last because there is a 135 Table 4.4—Correlation coeficients for regression analysis of in viva Bioassays with in vitro Assays. a-Microplate Assay a—Portable Assay PAGE LC90 Perrnethrin .85 " .50 .758‘ LC90 Methomyl .94 ”* .99 "t _95 see LC90 Parathion .87 "W 1.00 a” .94 an L0» Oxydemetonmethyl .82 " .46 .73 " LCgo Azinphosmethyl .68 .99 am .86 n. Both a t-test (partial) and an F-test were conducted on the data to test for significance. "' signifies significance at a = .05, “ signifies significance at a = .025, and “" signifies significance at a = .01. 136 great deal of room for experimental error, such as inexact pipetting, dificulty in difi‘erentiation between dead and alive insects, and other such errors. D. Precision Evaluation ThemostpreciseassaytypewastheMicroplate, followedbythePortable Carboxyesterase Assay, followed by PAGE, and finally the in viva Bioassay. This is becausealthoughtheMicmplateandPortableAssayswouldbeeasyto makeanerror srrchasnrispipetfingtherepeatabilityofbothassaysisexcellem. Therepeatabilityof PAGEisquitehighaswell, however,thistechniqueissosensitiverepeatabilitycanbe compromised, and inconsistencies in polymerization of the gel can strongly afi‘ect results. TheBioassayislastduetothedificultyrepeatingthistypeofassay, andtheeasewith which errors could occur in multiple replicates. E. Sensitivity The technique that is the most precise is the Microplate Assay, then the PAGE evaluation, then the Portable Assay, finally the in viva Bioassay. The Microplate Assay needs apprordmately 1/6 of an aphid, and the PAGE system utilizes approximately 1/4 of an aphid. ThePortableassayusesabout l aphid,butis sensitivewithaslittleas 1/4 ofan aphid. The in viva Bioassay utilizes at least 540 aphids for each insecticide. Thus the ranking is Microplate Assay > PAGE > Portable > in viva Bioassay. F. Overall Effectiveness for Field Diagnostics Upon overall evaluations of each technique, there are many factors to consider. First economics, time for labor, and accuracy were examined. In this case, the Portable Assay is ranked first, although it is not as accurate as either the Microplate Assay or PAGE. However, when one takes into account that this is a field—diagnostic tool and it is not 137 expected to be as accurate as the laboratory, the Portable wins the rank of first overall. The rank is: Portable > Microplate > PAGE > in viva Bioassay. IV. Conclusions Themonitoringtooldesignedinthis studyhasmetallofthegoalsoutlinedinthe Introduction An in vitro carboxyesterase resistance monitoring tool was developed using cumdyavailabletechnologymddamfoundmthemfiondcarboxyenerasemey. An in viva dosage mortality bioassay was conducted on eight strains of GPA to determine actualresistancelevel ofeachstrain. Thisdatawastested forcorrelationswiththe diagnostic tool and the other in vitro tools used in designing the portable. The study has proven that the Portable Carboxyesterase Assay is a valid measure of carboxyesterase levels in GPA The strong correlations with insecticide resistance (in viva Bioassay data) prove that the Portable Carboxyesterase Assay is valid for determining resistance levels due to elevated carboxyesterases. For these reasons, the Portable Carboxyesterase tool has excellent potential as a field resistance diagnostic tool. This tool will aid in distinguishing resistance frequencies in the field and therefore 1PM decisions. V. Future Research Possibilities In the firture, more emphasis should be placed on IPM techniques rather than the quick fix of foliar insecticide sprays. Sprays should be a last ditch effort at control and other tactics such as more effective promotion of biological control organisms and the use of such chemicals as insect growth regulators (IGR) like Kinoprene. According to Bauernfiend and Chapman, Kinoprene can cause high levels of progeny disruption in GPA (1984). 138 Additionally, if spraying is continued, which I believe it will be, it is important to maintain susceptible individuals in a population, so the use of such things as susceptible population refuges is an excellent idea. Future research into the enzyme-insecticide system could involve the use of selected substrates for determining actual hydrolysis products of insecticides This would involved the addition of an insecticide to a homogenized aphid followed by a stain which would stainonlyforaspecificproduct. Thusifthesohrtionshowedacolorchangetheprimary metabolite would be the predicted product. A reaction such as this would firrther elucidate the mode of action of insecticides and show the primary site of action for the enzyme (such as esterases). This could aid in designing better insecticides with greater efi‘ectiveness. The Portable Carboxyesterase Assay could also use further research The development of a more accurate photometer would aid in error elimination The use of other strategies to make the system more portable, such as pie-substrate saturated filter paper (Pasteur and Georghiou 1981, 1989) would be of some use and would help to reduce costs. 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Analytical Biochemistry 30:148-152. APPENDICES APPENDIX A 147 APPENDIX 1 Record of Deposition of Voucher Specimens* The specimens listed on the following sheet(s) have been deposited in the named museum(s) as samples of those species or other taxa which were used in this research. Voucher recognition labels bearing the Voucher No. have been attached or included in fluid-preserved specimens. Voucher No.: 1992-05 Title of thesis or dissertation (or other research projects): IN VIVO AND IN VITRO EVALUATION OF CARBOXYESTERASE-BASED INSECTICIDE RE$$STANCE IN GREEN PEACH RPHID (Myzus persicae (Sulzer) (HOMOPTERA: APHIDIDAE). Museum(s) where deposited and abbreviations for table on following sheets: Entomology Museum, Michigan State University (MSU) Other Museums: Investigator's Name (3) (typed) Dorothy O '1;1_ara Date 20 November 1992 *Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in Narth America. Bull. Entomol. Soc. Amer. 24:141-42. Deposit as follows: Original: Include as Appendix 1 in ribbon copy of thesis or dissertation. Copies: Included as Appendix 1 in copies of thesis or dissertation. Museum(s) files. Research project files. This form is available from and the Voucher No. is assigned by the Curator, Michigan State University Entomology Museum. 148 APPENDIX 1.1 Voucher Specimen Data Pages of Page mum: Houmuzo Nam.” :02 ON mumm— .E=om=z honoEousm >uumuo>wcb oumum cowanofiz unu ca uHmonov you mcwanoam vmumfia o>onm ozu vo>uooom Mama .0 >588 moummmn .oz nono=o> Avoa>uv Amvusmz m.uoumwwumo>cH Axumwmwooc ma muwonm Hmcowuwvcm omsv % 1. m QHIHmeAHn . mm ¢3I2¢zqum :OImmamooz axlmawH moommmm =2nz¢ma¢mam “M > III 0 _ Z < m M O on _ a: 4 0.3 l ' 0 50 100 150 PROTEIN CONCENTRATION Appendix CuPortable standard curve of protein concentration versus absorbance (bovine serum albumin) pg] 100 pl plotted against absorbance (609 nm = protein). This curve. is. curvilinear and shows that it does not give readings with a linear-type relationship until the. protein concentration is at or above 10 ug/ 100 111. APPENDIX D 151 Appendix D—Toxicities of six difi'erent insecticides (Ware 1983, Matsumura 1985). Chemical Oral rat Lb” Dermal rabbit LDso azinphosmethyl 5 220 carbaryl 307 2000 ethyl parathion 3 6.8 methomyl 3.6 1000 oxydemetonmethyl 17 100