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P-Element-Mediated Transformation of Drosophila melanogaster with the Yeast Metallothionein Gene CUP1 to Assess the Potential for Genetic Improvement of Beneficial Insects in Florida Citrus

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Title:
P-Element-Mediated Transformation of Drosophila melanogaster with the Yeast Metallothionein Gene CUP1 to Assess the Potential for Genetic Improvement of Beneficial Insects in Florida Citrus
Creator:
MEYER, JENNIFER L.
Copyright Date:
2008

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Drosophila ( jstor )
Eggs ( jstor )
Female animals ( jstor )
Insects ( jstor )
Microinjections ( jstor )
Pests ( jstor )
Plasmids ( jstor )
Transposons ( jstor )
Wildlife management ( jstor )
Yeasts ( jstor )
City of Orlando ( local )

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University of Florida
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University of Florida
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Copyright Jennifer L. Meyer. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
11/30/2005
Resource Identifier:
436098756 ( OCLC )

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P -ELEMENT-MEDIATED TRANSFORMATION OF Drosophila melanogaster WITH THE YEAST METALLOTHIONEIN GENE CUP1 TO ASSESS THE POTENTIAL FOR GENETIC IMPROVEMENT OF BENEFI CIAL INSECTS IN FLORIDA CITRUS By JENNIFER L. MEYER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 By Jennifer L. Meyer

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This document is dedicated to my wonderful husband, Jason Michael Meyer, for filling my life with love and laughter, and for always believing in me.

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ACKNOWLEDGMENTS I would like to thank my committee chair and advisor, Dr. Marjorie A. Hoy, for mentoring, guiding and supporting me during every step of this process; and my committee members, Dr. John Capinera and Dr. Marta Wayne, for providing fundamental information throughout my research. Dr. Ayyamperumal Jeyaprakash gave me valuable advice, support and much assistance with my experiments, especially with the construction of the P-element plasmid pAJ171 and the Southern blot analysis, for which I am grateful. I am very appreciative of Dr. Alfred Handler (USDA, Gainesville, FL) and his lab technicians, Robert Harrell and Pat Whitmer, for their time and assistance with the egg microinjections, and for the use of their lab and equipment. I would like to thank Dr. Heather McAuslane for help with the statistical analysis and Dr. Lawrence Winner (Department of Statistics, UF) for providing me with statistical consulting. I would like to thank Karla Addesso for helping to collect data during a key time. I am blessed for the love, support and guidance of my wonderful husband, Jason Meyer, and for my wonderful family and friends, especially my twin sister, Jessica Hettche, who continually encourage me and help me strive to be my best. The research was supported, in part, by the Davies, Fischer, and Eckes Endowment in Biological Control. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Introduction...................................................................................................................1 Copper in Florida Citrus...............................................................................................1 Metallothionein.............................................................................................................3 P-element-mediated Transformation............................................................................8 Genetic Transformation of Natural Enemies..............................................................13 Research Objectives....................................................................................................16 2 P-ELEMENT-MEDIATED TRANSFORMATION OF Drosophila melanogaster WITH THE YEAST METALLOTHIONEIN GENE CUP1 TO ASSESS THE POTENTIAL FOR GENETIC IMPROVEMENT OF BENEFICIAL INSECTS IN FLORIDA CITRUS...............................................................................................18 Introduction.................................................................................................................18 Materials and Methods...............................................................................................21 Drosophila melanogaster....................................................................................21 P-element Transformation Vectors.....................................................................21 Maternal Microinjection......................................................................................22 Egg Microinjection..............................................................................................24 Establishment of Putatively Transformed Lines.................................................25 Southern Blot Analysis........................................................................................25 Analysis of Copper Tolerance.............................................................................27 Inducement of the CUP1 Construct....................................................................29 Results and Discussion...............................................................................................30 Maternal Microinjection......................................................................................30 Egg Microinjection..............................................................................................31 High-fidelity PCR................................................................................................32 v

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Southern Blot Analysis........................................................................................32 Analysis of Copper Tolerance.............................................................................33 Inducement of the CUP1 Construct....................................................................36 Conclusions.................................................................................................................45 3 REFLECTIONS..........................................................................................................47 LIST OF REFERENCES...................................................................................................49 BIOGRAPHICAL SKETCH.............................................................................................58 vi

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LIST OF TABLES Table page 1 LC50 and LC95 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at 24C, ~75% RH and 14:10 L:D...........................................................................................................................35 2 The mean percentage survival and the mean number of days to emerge as adults for each D. melanogaster line in the water controls at 24C, ~75% RH and 14:10 L:D...........................................................................................................................36 3 LC50 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D in the non-induced and induced treatments......................................................39 4 LC95 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D in the non-induced and induced treatments......................................................40 5 The mean percentage survival and the mean number of days to emerge as adults in the water controls of each D. melanogaster line averaged over the non-induced and induced treatments when held at ~22-25C, ~50-75% RH, and 14:10 L:D.................................................................................................................43 6 The mean percentage survival and the mean number of days to emerge as adults of D. melanogaster pretreated with copper and then treated with water compared to flies treated only with water when held at ~22-24C, ~50-75% RH, and 14:10 L:D...........................................................................................................................43 7 The mean percentage survival for each D. melanogaster line averaged over the non-induced and induced treatment in the water controls at ~22-24C, ~50-75% RH, and 14:10 L:D...................................................................................................44 vii

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LIST OF FIGURES Figures page 1 Southern blots of the transgenic lines of D. melanogaster showing genomic integration of the CUP1 construct............................................................................33 2 Baseline concentration-mortality curve for the ww control line of D. melanogaster tested with CuOH at 24C, ~75% RH and 14:10 L:D.......................34 3 Concentration-mortality curves for the ww control line and transgenic lines 8 and 14 of D. melanogaster when treated with copper at 24C, ~75% RH and 14:10 L:D.................................................................................................................35 4 Concentration-mortality curves of the non-induced and induced treatments for the ww control line, and lines 8 and 14 of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D..................................................38 5 Concentration-mortality curves for the ww control line, and lines 8 and 14 of D. melanogaster in the non-induced and induced treatments when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D..................................................42 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science P-ELEMENT-MEDIATED TRANSFORMATION OF Drosophila melanogaster WITH THE YEAST METALLOTHIONEIN GENE CUP1 TO ASSESS THE POTENTIAL FOR GENETIC IMPROVEMENT OF BENEFICIAL INSECTS IN FLORIDA CITRUS By Jennifer L. Meyer May 2005 Chair: Marjorie A. Hoy Major Department: Entomology and Nematology The use of copper-containing fungicides, such as Kocide, may affect non-target beneficial insects in Florida citrus and limit Integrated Pest Management (IPM) programs. The insertion of the yeast metallothionein gene, CUP1, into the genome of beneficial insects could produce populations resistant to copper (Cu). To determine if CUP1 confers increased tolerance to Cu in insects, the model insect Drosophila melanogaster was transformed using a P-element construct carrying the Drosophila metallothionein promoter Mtn fused to the CUP1 open reading frame. Maternal microinjection was attempted but failed to produce transformants, so egg microinjection was used following traditional methods. The High-fidelity Polymerase Chain Reaction (PCR) and Southern blot analysis confirmed the transformation of two lines. The CuOH concentration-response curve for the ww control line was obtained and used as a baseline to assess the tolerance to Cu of the transgenic lines 8 and 14. Concentration-response curves of these transgenic lines showed approximately 1.5to 2-fold increases in ix

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tolerance when held at 24C, ~75%RH and 14:10 L:D. In the water controls, lines 8 and 14 exhibited significantly lower survivorship and longer time to emerge as adults than the ww control line, which are indirect indications of fitness costs. Metallothionein genes (MTs) are induced by Cu at the transcriptional level so, to assess whether pretreatment would induce increased resistance, the ww control and transgenic lines were pretreated with 2.5 mM CuOH and held at ~22-24C, ~50-75% RH and 14:10 L:D. Each line (ww, 8 and 14) was more tolerant to Cu when pretreated, with line 14 exhibiting the greatest increase. The increased Cu tolerance of the transgenic lines could be a result of induced transcription of the CUP1 and native MTs, whereas in the ww control, the increase could be due to induced transcription of native MTs. As expected, lines 8 and 14 were more tolerant to Cu than the ww control line in both the non-induced (control) and induced treatments. Fitness assessment of the fly lines in each treatment of the water controls indicated that line 14 has greater fitness than the ww control line and line 8 when pretreated with Cu. Percentage survival of line 14 was significantly higher than the other two lines. However, lines 8 and 14 take significantly longer to emerge as adults than the ww control regardless of treatment. As expected, in the non-induced treatment of the water controls the ww control exhibited significantly higher survivorship and shorter time to emerge as adults than lines 8 and 14, indicating that the ww control has greater fitness in the absence of Cu. This decreased fitness of the transgenic lines reduces the potential risk of their persistence in the environment in the absence of Cu. The data suggest that the CUP1 gene may be useful for the genetic improvement of natural enemies where Cu is applied and may be a useful selectable marker when producing transgenic insects. x

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CHAPTER 1 LITERATURE REVIEW Introduction The success of Integrated Pest Management (IPM) programs is strengthened by the compatibility of chemical and biological control methods used in the system (Hoy 1990a, 1992a). This is challenging for complex agricultural systems where it is not possible to control key pests by methods other than chemical control (Hoy 1985, 1995a, 2000), which can result in incompatibility of biological and chemical control strategies causing target-pest resurgence, secondary pest outbreaks, and pesticide-resistant pest arthropods (Metcalf 1986, Hoy 1992a). The use of genetically transformed insects may provide new strategies for insect pest management (Beckendorf and Hoy 1985, Handler and O’Brochta 1991, Hoy 2000, Handler 2001). This review will describe the use of copper in Florida citrus, potential negative effects of copper to beneficial insects, and the causal agent of secondary pest resurgences. The potential use of genetic improvement to develop beneficial insect populations that are no longer negatively affected by the necessary use of copper-containing fungicides will be discussed. The methodology of producing genetically improved natural enemies will be described, as well as how they may be used to improve the compatibility of disease control and biological control in citrus. Copper in Florida Citrus Many potentially damaging fungal diseases threaten citrus production in Florida by attacking young leaves, fruit, twigs, and roots (McCoy et al. 2004). Fungicides must be 1

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2 applied to groves to protect trees from these pathogens. Common fungal diseases include citrus scab caused by Elsinoe fawcetti Bitancourt and Jenkins, melanose caused by Diaporthe citri Wolf, greasy spot caused by Mycosphaerella citri Whiteside, brown spot caused by Alternaria alternate (Fr.) Keissl., and brown rot caused by Phytophthora nicotianae van Breda de Haan and P. palmivora Butler (Aerts and Nesheim 2000). Greasy spot can be an especially damaging foliar and fruit disease, significantly reducing yield by inducing premature defoliation and leaving small, blemished fruit (Timmer and Zitko 1997). In citrus groves where the fruit is intended for either processing or fresh market, growers must consider applying a copper-containing fungicide in May-June to prevent infection (Timmer et al. 2004). Copper-containing fungicides, such as copper hydroxide, are the most widely used fungicides on Florida citrus (Childers 1994, Timmer et al. 2004, McCoy et al. 2004) because they are relatively inexpensive, highly toxic to fungal pathogens, and have a low toxicity to mammals (Cha and Cooksey 1991). As a result, the annual amount of copper applied in citrus groves may be as high as 16.7 to 20.3 pounds per acre (Timmer and Zitko 1997, Aerts and Nesheim 2000, Stover et al. 2002). In Florida, more acreage was treated with copper compounds than with any other fungicide between 1993 and 1999 (Florida Agricultural Statistics Service 2000). One drawback of the widespread use of these fungicides is their adverse effects on integrated pest management programs (Childers et al. 2001). Non-target effects in citrus due to the use of copper have been reported and include secondary pest outbreaks and mortality of predators and parasitoids. For example, the use of copper was attributed to secondary pest outbreaks of the citrus rust mite,

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3 Phyllocoptruta oleivora Ashmead, in two of the three field experiments in which copper-only treatments were included (Childers 1994). Citrus rust mite densities were significantly higher in copper-treated fields as compared to the untreated field, leading to a conclusion that copper either prolonged high citrus rust mite populations or caused an increase. These results may be due to the negative effect of copper compounds on the entomopathogenic fungus Hirsutella thompsonii Fisher (Eger et al. 1985, McCoy and Lye 1995). However, copper has been shown to have a negative impact on Agistemus industani Gonzalez, a predator of the citrus rust mite and other phytophagous mite pests (Childers et al. 2001). In field studies, applications of copper hydroxide combined with petroleum oil caused 50% or greater reduction in egg production by A. industani and larval mortalities were more than 50% higher than in the untreated field controls. In two laboratory studies, copper was attributed to increased mortality of a predator and a parasitoid. Michaud (2001) found that a mixture of copper and petroleum oil fungicide caused significant mortality to Cycloneda sanguinea Linnaeus larvae, a coccinellid predator of the brown citrus aphid, Toxoptera citricida Kirkaldy, and the Asian citrus psyllid, Diaphorini citri Kuwayama, suggesting that the widespread use of copper may result in a significant decrease of this beneficial insect in citrus. Copper hydroxide (Kocide101), when used at the lowest recommended field rate in clip-cage bioassays, reduced survival of Ageniaspis citricola Logvinovskaya, parasitoid of the citrus leafminer, Phyllocnistis citrella Stainton, by 60.8% (Villanueva-Jimnez and Hoy 1998). Metallothionein All organisms require metals such as zinc and copper to perform a variety of cellular functions (Cousins 1994). These metals can be obtained from the environment

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4 and are essential to sustain life. Both zinc and copper are vital for oxidation, electron transfer, and the activity of various enzymes such as polymerases, transcription factors, and metalloenzymes (Hanas et al. 1983, Hamer et al. 1985, Evans and Hollenberg 1988). Over 300 different proteins depend on zinc for proper folding and function (Klaassen et al. 1999). These essential metals can be toxic if obtained from the environment in excess amounts. For example, excess zinc can cause prostate cancer in humans (Hasumi et al. 2003) and excess copper inhibits cell growth and development in Drosophila melanogaster (Meigen) (Maroni and Watson 1985). Other metals present in the environment, including cadmium, mercury, silver and lead, are harmful non-essential metals that can be toxic in any amount (Tohoyama et al. 1995) by altering enzyme specificity, disrupting cellular functions, damaging the structure of DNA (Hamer et al. 1985) or inducing mutations (Jeyaprakash et al. 1991). The ability to tolerate reactive heavy metal ions and to utilize some of them for metabolic processes is an evolutionary success made possible by metal-binding macromolecules such as metallothioneins (Kagi and Schaffer 1988). Metallothioneins (MTs) belong to a family of metal-binding proteins first discovered in the equine kidney cortex in 1957 (Margoshes and Vallee 1957) and found in all eukaryotes and many prokaryotes (Palmiter 1998). These small cysteine-rich proteins form metal/sulfur clusters through thiolate bonds (Cherian and Goyer 1978, Tohoyama et al. 1995) and are believed to function in metal detoxification (Beach and Palmiter 1981, Silar and Wegnez 1990). MT synthesis is a very useful system for studying eukaryotic gene regulation and metal homeostasis because the protein is inducible at the transcriptional level by both essential and non-essential heavy metals

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5 (Hamer et al. 1985, Seguin and Hamer 1987, Tohoyama et al. 1995). The conserved protein structure and the metal sequestering capabilities support the theory that MTs play a role in cellular metabolism by regulating essential metals and facilitating the passage of all metals through cells thus providing protection against metal toxicity (Kagi and Schaffer 1988, Cousins 1994). In D. melanogaster, the metallothionein gene Mtn is expressed in the malpighian tubules of larvae, pupae and adults, and copper and cadmium ions strongly induce Mtn mRNA accumulation in the midgut, indicating that the MTN protein may be involved in absorption and excretion of metallic ions (Bonneton et. al 1995). Metallothioneins are divided into three classes based on structural relationships. Class I includes MTs from mammals, crustaceans and mollusks, class II is found in most invertebrates, fungi and cyanobacteria (Valls et al. 2000), and class III consists of phytochelatins (Steffens et al. 1986, reviewed by Cobbet and Goldsbrough 2002). Research on class I MTs from mammals, and class II MTs from both Drosophila and baker’s yeast, Saccharomyces cerevisiae Hansen, have provided insight into the structure and function of this family of proteins. MT proteins from these organisms have a highly conserved region of Lys-Lys-Ser-Cys-Cys-Ser, and the cysteine residues are characteristically organized in a cys-X-cys motif where X represents any other amino acid residue (Kagi and Schaffer 1988, Tohyama et al. 1995, Valls et al. 2000). Despite the conserved secondary structure, MTs vary in the number of amino acids (AA), the number of cysteine residues, the metals that can bind to the protein, and the metals that induce transcription. Mammalian MT has 61 AA, 20 of which are cysteine, is induced by Zn, Cu and Cd, and can bind 7 Zn or Cd, or 12 Cu ions (Tohoyama et al. 1995). Yeast

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6 metallothionein protein, CUP1, has 53 AA, 12 of which are Cys, is induced by Cd (Jeyaprakash et. al. 1991) and Cu, and can bind to 4 Zn or Cd , and 8 Cu ions (Winge et al. 1985). Drosophila metallothionein protein, MTN, has 40 AA, 10 of which are Cys, is induced by Cu and Cd, and can bind to 4 Zn or Cd or 8 Cu ions (Lastowski-Perry et al. 1985, Valls et al. 2000). The tertiary structure of mammalian MT differs from CUP1 and MTN in that mammalian MT forms a two-domain structure () while CUP1 and MTN form a one-domain structure resembling the domain of MT. The domains tightly enclose the metals and shield them from the environment (Fischer and Davie 1998). Saccharomyces cerevisiae and D. melanogaster populations that vary in tolerance to metal have been isolated and provide insight on how MT plays a role in metal detoxification. Natural populations of copper-resistant yeast were discovered in 1955 and resistance was found to be due to a single gene, CUP1, which encodes the yeast metallothionein protein (Brenes-Pomales et al. 1955). Since this discovery, the purification of CUP1 from a highly copper-resistant yeast strain led to the isolation and amino acid sequence of the protein (Winge et al. 1985). These naturally occurring copperand cadmium-resistant strains of S. cerevisiae produce a large amount of CUP1 protein when induced by metals (Tohoyama et al. 1995, 1996), which made its isolation possible. Research on metal-resistant strains from natural and transgenic populations of yeast indicates that resistance to heavy metal poisoning is a result of enhanced production of the CUP1 gene product due to both gene amplification and increased transcription (Fogel and Welch 1982, Tohoyama 1996). In copper-resistant strains, the addition of 50-100 M copper ions to liquid yeast cultures causes a 10to 20-fold increase in CUP1

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7 transcription (Jeyaprakash et al. 1991). Southern blot analysis of sensitive and resistant strains of yeast reveal that they differ in the number of repeating CUP1 genomic DNA segments and consequently exhibit enhanced resistance levels directly proportional to the number of repeats (Fogel and Welch 1982). Copper-resistant strains typically contain 11 to 15 copies of the 2 kb-repeats in tandem array, while copper-sensitive strains contain one to 10 copies. The metal resistance of these strains is attributed to the metal sequestering ability of CUP1 (Mehra and Winge 1991, Wlostowski 1993). Yeast strains constructed to have a disrupted CUP1 gene (cup1) are sensitive to 0.3 M CuSO4 and can be subsequently transformed with the CUP1 gene or MT genes from other organisms to compare metal resistance (Ecker et al. 1986). This technique has been used to show that the CUP1 protein is able to detoxify copper and cadmium (Ecker et al. 1986), and that gene transcription is inducible by both copper and cadmium (Jeyaprakash et. al. 1991). To determine whether structurally dissimilar MTs are functionally analogous, mammalian and D. melanogaster MTs have been inserted into yeast (Thiele et al. 1986, Silar and Wegnez 1990). Transformation of cup1 yeast strains with a plasmid containing a CUP1 promoter and monkey MT-I and MT-II cDNAs showed a 40and 100-fold increase in tolerance to copper, respectively, as compared to yeast strains with a single CUP1 copy (Thiele et al. 1986). Monkey MT in yeast performs both of the known functions of endogenous yeast MT, copper detoxification and autoregulation of transcription (Thiele et al. 1986). Transformation of cup1 yeast strains with the Drosophila MT gene, Mtn, when under the control of the CUP1 promoter also confers higher resistance to copper (resistance up to 1000 M CuSO4) due to the increased production of the metallothionein protein (Silar and Wegnez 1990). MT

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8 proteins from humans, Drosophila and yeast perform closely related functions when inserted into yeast. Natural populations of D. melanogaster containing multiple copies of Mtn show increased tolerance to metals (Otto et al. 1986, Theodore et al. 1991), whereas wild-type populations with a single copy of the Mtn gene are metal sensitive, showing a significant reduction in viability and growth rate (Maroni and Watson 1985, Maroni et al. 1986) when exposed to metals. The first amplification of the Drosophila Mtn gene was detected in a laboratory strain selected for cadmium resistance, which resulted in a single Mtn duplication and a two-fold increase in Mtn RNA (Otto et al. 1986). Since then, Mtn duplications have been found in natural populations from four different continents; these populations produce 1.7-2.1 times the amount of Mtn RNA and showed a 1.5-fold increase in tolerance to copper and cadmium as compared to the wild-type strain carrying only one copy of Mtn (Maroni et al. 1987). Transgenic D. melanogaster containing an extra copy of Mtn, introduced by P-element-mediated transformation also produced more Mtn RNA than strains with one copy of Mtn and lived significantly longer on 0.1 mM, 0.5 mM and 1 mM of cadmium chloride (Maroni et al. 1995). P-element-mediated Transformation The ability to genetically transform arthropods is a valuable tool for studying genetics, biochemistry, development, and behavior (O’Brochta and Atkinson 1997) and has the potential for improving integrated pest management strategies (Beckendorf and Hoy 1985, Handler and O’Brochta 1991, Hoy 2000, Handler 2001). The first successful genetically transformed insect was achieved by P-element-mediated transformation in 1982 when foreign DNA was microinjected into embryos of Drosophila melanogaster (Rubin and Spradling 1982, Spradling and Rubin 1982). The Drosophila gene for wild

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9 type eye color, rosy+, was stably introduced into the genome of a rosy mutant strain by a P transposable element vector and the resulting red eye color was stably inherited in subsequent generations. Transposable elements (TEs) are DNA sequences, found in every organism studied, that can move to new chromosomal locations, invert, and either be deleted or amplified (Kidwell and Lisch 2001). They were first discovered in maize in the 1940s as elements capable of regulating gene expression (McClintock 1984) and are believed to be a fundamental part of the genome of most species (Sentry and Kaiser 1992, Kidwell and Lisch 2001). Many different families of TEs have been discovered and characterized in all species sufficiently examined (Kidwell and Lisch 2001). The opposing opinions of TEs as merely selfish (junk) DNA or as mobile DNA with evolutionary benefits are still debated, but it is clear that these elements have a complex relationship with their hosts. TEs are classified into two classes according to their repeating structures and transposition mechanisms. Class I elements transpose by an RNA intemediate, encode a reverse transcriptase and consist of both short and long interspersed nuclear elements. Class II elements transpose by a DNA intermediate and have either short or long terminal inverted repeats. The position and numbers of these elements vary among individuals of the same species (Engels 1992). An individual D. melanogaster may carry TEs from 50 different transposon families with 100 copies per family, representing as much as 20% of the genome (Engels 1992). The P transposable element has been developed as an important a molecular tool in Drosophila genetics. P is a highly mobile class II TE with short inverted terminal repeats that are 31 bp in length. P excision and chromosomal integration is by a cut-and-paste mechanism that

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10 creates a duplication of the insertion site (Engels et al. 1990, Kaufman and Rio 1992, Engels 1997). P excision is catalyzed by transposase, the P-element-encoded enzyme required for transposition, at the inverted repeats on the P element. This leaves behind a double-stranded gap that is usually repaired using the P element sequence on the sister chromatid as a template, thus replacing the missing P element (Gloor et al. 1991, Sentry and Kaiser 1992, Daniels and Chovnick 1993). The excised P element may insert randomly into a new position in the genome, which may cause mutations. P elements were discovered in the 1970s due to their role in hybrid dysgenesis, which occurred when males from a wild population mated with females from a laboratory population. The progeny had numerous genetic defects such as sterility, mutations, broken chromosomes, and developmental abnormalities (Bingham et al. 1982, Kidwell et al. 1997). P transposable elements, present in the wild strain (P cytotype), but absent in the laboratory strain (M cytotype), were found to be the causal agents (Anxolabhre et al. 1988). When P strain males mate with the M strain females, the P elements are released from regulation of the P cytotype as the sperm enters the egg. Transposase is expressed due to the lack of a repression system and the mobilized P elements cause mutations (Bingham et al. 1982, Robertson et al. 1988). P elements are hypothesized to have invaded D. melanogaster relatively recently on the evolutionary time scale. Anxolabhre et al. (1988) screened 100 D. melanogaster strains that had been collected from several continents at different times during 60 years and found the earliest detection of P occurred in colonies collected during the 1950’s. The youngest strain devoid of P was collected in 1974 from the USSR. Due to the absence of P in laboratory strains established prior to the 1950s, the P element is thought

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11 to have invaded D. melanogaster within the past 50 years by horizontal gene transfer from D. willistoni (Daniels et al. 1990). This hypothesis is supported by the high degree of conservation of P element DNA sequences in both species, the rapid spread of the element in strains lacking P, and the discovery that the two species may not have come in contact with each other until just prior to when P was speculated to have been introduced to D. melanogaster (Daniels et al. 1990, Engels 1992). It is possible that the invasion of P occurred by a virus that infected a transposon-bearing D. willistoni, and the transposable element, which entered the genome of the virus, was transmitted into the cells of D. melanogaster (Engels 1992). Another way that P may have been horizontally transferred is by the parasitic mite, Proctolaelaps regalis DeLeon. Houck et al. (1991) found evidence of P-element sequences in P. regalis grown in a P -strain culture of D. melanogaster, demonstrating the mites can acquire P sequences. P-element-mediated transformation has been a successful transformation system due to several factors. D. melanogaster is a good model insect because it has a short life cycle, it is cheap and easy to culture in large numbers, and it has been extensively researched. Another factor that makes P-element-mediated transformation successful is the ability to use cloned wild-type genes as markers to detect transformation success when the genes are inserted into a fly strain lacking the wild-type gene. The average transformation rate of this system is approximately 1.6% (Spradling 1986) making it necessary to screen thousands of flies before finding one transformant. Visible markers, such as the rosy+ and white+ gene, present on the P-element vector allow for efficient identification of transgenic red-eyed progeny when introduced into mutant fly strains with rosy or white eyes. Another crucial factor is the existence of laboratory strains

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12 devoid of P elements with the M cytotype. The repressive cellular environment of the P cytotype inhibits transposition of P elements making transformation of this cytotype almost impossible (Engels 1996). The development of a vector-helper system was a vital development that facilitates stable integration of the transgene (Karess and Rubin 1984). The P helper plasmid encodes transposase but does not have the ability to integrate. The co-injection of the P helper plasmid and the P-element vector into a fly strain that lacks P elements allows transposase to catalyze the integration of the P element construct into the genome of the germline cells. The transposase is encoded on a separate plasmid and is not transferred to the next generation. The progeny cannot produce transposase, which leads to stability of the transgene location. Microinjection of embryos is used to deliver the P vector and helper plasmid into the embryo shortly after fertilization and prior to pole cell (germline) formation (Spradling and Rubin 1982). Genetic transformation has revolutionized research on D. melanogaster by allowing the analysis of isolated genes and their effects (reviewed by Wimmer 2003). P-element-mediated transformation has been used in experiments, such as transposon tagging (which facilitates the identification and cloning of genes by inducing new mutations that can be correlated to structure and function), expressing exogenous genes (inserting cloned genes into the genome of D. melanogaster and studying expression), evaluating position effects (adding transposase to a transgenic strain that has an integrated transgene within a P element, which causes movement of the construct to a new genomic site where the effect of the new environment on gene expression can be studied), and targeted gene transfer (adding transposase to a strain that contains a P

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13 element in a known location and replacing it with an extrachromosmal P element containing a gene of interest) (Engels et al. 1990, Gloor et al. 1991). Genetic Transformation of Natural Enemies Although attempts to transform non-drosophilid insects using P-element vectors have been unsuccessful, P-element-mediated transformation has provided the conceptual framework for transforming other insects using different transposable elements (O’Brochta and Handler 1988, Handler et al. 1993, O’Brochta and Atkinson 1997, Atkinson et al. 2001, Handler 2001). TE families from other insect orders have the potential to serve as sources of non-drosophilid insect gene vectors (Beckendorf and Hoy 1985, O’Brochta and Atkinson 1997). As of 2002, four different TEs, Minos, Hermes, piggyBac and Mos1, have been used to transform ten agriculturally important insect species (reviewed by Atkinson 2002 and Hoy 2003). The development of genetic transformation technology with the P element as the paradigm has enabled the identification and isolation of important insect genes involved in pesticide resistance, hormone action, and metabolism (Beckendorf and Hoy 1985, O’Brochta and Atkinson 1997). Even with the availability of various TEs other than P for genetically transforming insects, egg microinjection is not always an option due to egg characteristics such as small size, fragility and difficulty with isolation (endoparasitoids). Therefore, alternative methods for transforming insects are needed. Maternal microinjection has been demonstrated as an effective technique for transformation of the western predatory mite, Metaseiulus occidentalis (Nesbitt) (Presnail and Hoy 1992, Li and Hoy 1996, Presnail et al. 1997), and a braconid endoparasitoid, Cardiochiles diaphaniae Marsh (Presnail and Hoy 1996), and may increase the frequency of stable transformation in insects. The use

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14 of maternal microinjection could be potentially useful in the transformation of Drosophila because a single microinjection event could result in more than one transformed individual. However, an effective maternal microinjection technique has yet to be devised for arthropods other than M. occidentalis and C. diaphaniae. Genetic transformation may provide new strategies for insect pest management by allowing the analysis of gene function in diverse insect species and the use of genetically transformed beneficial and/or pest insects in agriculture (Beckendorf and Hoy 1985, Handler and O’Brochta 1991, Handler 2001, Hoy 2003). Genetic improvement programs for natural enemies currently use artificial selection, which takes a great deal of effort taking many insect generations, and can fail when there are no resistance genes in the natural population. Transgenic technology may increase the effectiveness and applicability of genetic improvement strategies by enabling the rapid spread of resistant natural enemies. In theory, natural enemies would become more practical and less expensive (Beckendorf and Hoy 1985, O’Brochta and Atkinson 1997). For example, transgenic pesticide-resistant natural enemies could be released after the pest population was first treated with the pesticide to drive the resistant gene into the native natural enemy population (Hoy 2000). This could significantly decrease the emergence of insecticide resistance in insect pests and could lead to the compatibility of pesticides and pesticide-resistant predators and parasites. However, the probability that a transgenic natural enemy becomes established in the environment depends on its ability to disperse and the fitness of the insect in the particular environment (Hoy 1985). Fitness is defined as the ability of an organism to persist in the environment as measured by its reproductive success (Pianka 1983). Indirect measures of relative fitness

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15 include differences in survival rate, development rate, fecundity, fertility, adult longevity, sex ratio, and hind tibia length of two or more distinct populations. Fitness costs have been associated with resistance genes and with genetic transformation. D. melanogaster strains artificially selected for heavy-metal resistance exhibited reductions in fecundity and growth rate in unpolluted environments (Shirley and Sibly 1999). Mechanisms proposed that could lead to these fitness costs are a decrease in essential metal uptake leading to micronutrient deficiency (Shirley and Sibly 1999), a detoxification mechanism that requires increased energy and resources, which are no longer available for growth and development (Sibly and Calow 1989), or the inadvertent selection of deleterious genes in laboratory-reared insects (Mackauer 1972, Huettel 1976, Hoy 1987). In addition, inserting exogenous genes into the chromosomes of insects may reduce fitness, possibly due to expression of the gene, mutations caused by its insertion, or laboratory rearing of the insect resulting in genetic bottlenecks, inbreeding, drift and/or laboratory adaptation (Beckendorf and Hoy 1985, Hoy 1985, Lenski and Nguyen 1988, Mackay 1989, Kaiser et al. 1997). Therefore, studies regarding gene expression, stability, silencing, transmission to progeny and fitness of transgenic insects are key steps in determining the feasibility and efficacy of employing transgenic insects in all programs employing transgenic insects (Beckendorf and Hoy 1985, Hoy 1990b). There are potential risks involved with accidental and purposeful releases of transgenic insects that must be addressed (Hoy et al. 1997, 2000). Risk assessment is difficult to quantify and must be evaluated on a case-by-case basis. Before the potential use of transgenic natural enemies in pest management programs can be considered, guidelines on how to assess the potential risk associated with their permanent

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16 establishment in the environment must be established (Hoy 1992b, 1995b). The potential risks that must be studied include the ecological interactions of transgenic insects with their environment to determine if the vector can be mobilized by endogenous transposable elements of the transgenic insect, if its host or prey range has been changed, if the transgenic strains will have unintended effects on other species or environmental processes, and the likelihood of horizontal gene transfer to other organisms (Hoy 1990b, 1992c, Atkinson 2002). Uniform and effective containment procedures should be followed for transgenic insects until all issues involving the safety and efficacy of each transgenic insect line have been resolved (Hoy et al. 1997). Research Objectives With the availability of transposable elements from many different transposon families and the use of dominant selectable markers, transgenic technology may be applied to many important insects (Atkinson 2002). In this project, Drosophila melanogaster will be used as a model insect to study CUP1 as a potentially useful gene for the development of fungicide-resistant beneficial insects and as a dominant selectable marker. The objectives are to transform D. melanogaster with the CUP1 gene regulated by the Drosophila Mtn promoter using P-element-mediated transformation, develop two or more transgenic lines with a single CUP1 insert, confirm the presence of the transgene in the putatively transformed lines by the Polymerase Chain Reaction (PCR) and the number of chromosomal inserts in each by Southern blot analysis. Bioassys for copper tolerance will be conducted to test whether transgenic lines have an increased tolerance to copper, and fitness of the transgenic lines will be evaluated in the absence of copper. We hypothesize that transgenic lines with a single insert of the CUP1 construct will have a

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17 significantly increased tolerance to copper, but that the metal resistance will lead to fitness costs in the absence of copper.

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CHAPTER 2 P-ELEMENT-MEDIATED TRANSFORMATION OF Drosophila melanogaster WITH THE YEAST METALLOTHIONEIN GENE CUP1 TO ASSESS THE POTENTIAL FOR GENETIC IMPROVEMENT OF BENEFICIAL INSECTS IN FLORIDA CITRUS Introduction The success of Integrated Pest Management (IPM) programs is strengthened by the compatibility of chemical and biological control methods used in the system (Hoy 1990a, 1992a). IPM is a challenge for complex agricultural systems where it is not possible to control key pests by methods other than chemical control (Hoy 1985, 1995a, 2000). For example, copper-containing fungicides, such as Kocide, are applied to large amounts of citrus acreage in Florida to control several fungal diseases (Childers 1994, McCoy et al. 2004, Lapointe et al. 2004), but may limit the success of biological control management strategies due potentially negative impacts on non-target beneficial insects (Childers et al. 2001). This was shown in both field and laboratory studies where the use of copper fungicides decreased fecundity and survival rate of beneficial insects and increased secondary pest populations (Childers 1994, Villanueva-Jimnez and Hoy 1998, Childers et al. 2001, Michaud 2001). Genetic transformation may provide new strategies for insect pest management (Beckendorf and Hoy 1985). In this system, beneficial natural enemies genetically transformed with a gene that may increase resistance to copper could potentially improve the compatibility of fungal disease control and biological control in Florida citrus. Chemical resistance genes could also serve as effective selectable markers to detect genetic transformation in insects (Beckendorf and Hoy 1985). Transposable elements are 18

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19 being developed as transformation vectors for insects in a variety of orders for which there are no available genetic markers. A chemical resistance gene that could function in a variety of insects may provide a useful selectable marker for transgenic insects, especially in insects that have low transformation rates. Producing beneficial insects transformed with metallothionein genes could facilitate resistance to copper-containing fungicides. Metallothioneins belong to a family of metal-binding proteins found in all eukaryotes and many prokaryotes and function to detoxify metals, including copper (Palmiter 1998). The yeast metallothionein gene CUP1 codes for a 53-residue polypeptide including 12 cysteine sites that can cumulatively bind up to 8 copper ions (Tohoyama et al. 1995). The CUP1 protein functions in yeast to protect cells against toxic levels of copper and to repress the transcription of CUP1 when external levels of copper are low (Winge et al. 1985). In Drosophila melanogaster, copper has been shown to reduce viability and growth rate (Maroni and Watson 1985); however, natural populations of D. melanogaster containing multiple copies of the Drosophila metallothionein gene, Mtn, have a 5to 6-fold increased accumulation of mRNA and exhibit a 1.5-fold increased resistance to the toxic effects of copper (Maroni et al. 1987, Theodore et al. 1991). The CUP1 and MTN proteins have similar structure and function; thus, the CUP1 gene may prove to be effective in increasing copper resistance in this model insect and subsequently in agriculturally important insects. Although D. melanogaster usually are not agriculturally important pests, many of the same genetic and biochemical mechanisms that underlie resistance in pest insects are seen in Drosophila (Wilson 2001). P-element-mediated transformation can be used to transform D. melanogaster (Spradling and Rubin 1982)

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20 and may be an effective technique in determining if a yeast metallothionein gene, CUP1, regulated by the Drosophila metallothionein promoter, Mtn, could increase tolerance to copper in this model insect. There are two methods for inserting exogenous DNA into the germline cells: egg microinjection and maternal microinjection. Egg microinjection involves injecting the posterior end of each individual egg prior to pole cell (germline) formation. Maternal microinjection involves injecting the abdomen of a female with the objective that the DNA will be taken up by multiple oocytes or eggs. Maternal microinjection has been demonstrated as an effective technique for transformation of the western predatory mite, Metaseiulus occidentalis (Nesbitt) (Presnail and Hoy 1992, Li and Hoy 1996, Presnail et al. 1997) and a braconid endoparasitoid, Cardiochiles diaphaniae Marsh (Presnail and Hoy 1996), and may provide an easier method of achieving transformation than egg microinjection. Inserting exogenous genes into the chromosomes of insects may reduce fitness, possibly due to expression of the exogenous gene, mutations caused by its insertion or laboratory rearing of the insect resulting in genetic bottlenecks, inbreeding, drift and laboratory adaptation (Beckendorf and Hoy 1985, Hoy 1985, Levis et al. 1985, Lenski and Nguyen 1988, Mackay 1989, Kaiser et al. 1997). In addition, fitness costs have been associated with resistance genes in unpolluted environments. Drosophila melanogaster strains artificially selected for heavy-metal resistance exhibited reductions in fecundity and growth rate in unpolluted environments (Shirley and Sibly 1999). Therefore, after putatively transformed flies are screened for the selectable marker and assessed for transgene expression, they should be assessed for fitness costs. Fitness has been defined

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21 as the ability of an organism to perpetuate itself as measured by its reproductive success (Pianka 1983). Indirect measures of relative fitness include developmental rate, fecundity, fertility, adult longevity, sex ratio and viability between two or more distinct populations. Studies regarding transgene expression, stability and transmission to progeny, and fitness of transgenic insects are key steps in determining the feasibility and efficacy of employing transgenic insects in pest management programs (Beckendorf and Hoy 1985, Hoy 1990b). Materials and Methods Drosophila melanogaster The w[m] strain, which contains a recessive white-eyed (w) mutant gene in the homozygous condition (ww), was obtained from the Bloomington Stock Center (Bloomington, Indiana). Stocks were reared in 250-ml milk bottles containing a diet of 1:1 Ward's Instant Fly media (Ward’s Natural Science Establishment, Inc., Rochester, NY) to sterile water and maintained at 24C, ~75% RH and 14:10 L:D. The fly stocks were transferred to fresh bottles every two weeks using an air-pump aspirator, and each bottle was kept no longer than one month. P-element Transformation Vectors The P-element vector, pAJ 171 (9.7 kb), constructed by ligating the Drosophila Mtn promoter and the yeast CUP1 open reading frame between the inverted repeats (IRs) of a pCaSpeR-AUG-gal vector, was a gift from Dr. Jerry Rubin (Berkeley, CA) and contained the dominant white+ gene (Ashburner 1989). An Escherichia coli strain, DH5, was transformed with the pAJ 171 and grown overnight. Multiple copies of the construct were extracted and purified using the Qiagen Plasmid Purification kit (Qiagen Inc., Valencia, CA). In an attempt to improve the quality of the plasmid DNA, E. coli

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22 was transformed with pAJ 171 and the plasmid DNA was purified using the Qiagen Endotoxin-free Plasmid Maxi Kit (Qiagen Inc.) (Handler 2000). The original P helper, PUChs 2-3, was a gift from Dr. Jerry Rubin (Berkeley, CA). This P helper was extracted from transformed E. coli and purified using the Qiagen Plasmid Purification kit. To ensure high quality P helper plasmid, an endotoxin-free P helper, P25.52-3wc, was obtained from Dr. Alfred Handler (USDA, Gainesville, FL). The original P-element vector and P helper, PUChs 2-3, were used only in the initial maternal microinjection experiment involving 80 females. All subsequent experiments used the endotoxin-free P-element vector and P helper plasmid p25.52-3wc. Maternal Microinjection Abdomens of virgin females were injected with a Narishige microinjector (Narishige USA Inc., Greenvale, NY) according to the protocol described by Presnail and Hoy (1992). Females were chilled on ice for 5 minutes and placed on the injection platform, ventral side up. The platform consisted of two microscope slides, one on top of the other, held in place with Scotch brand double-sided tape, creating a ledge to hold the female in place. Injection needles (1 m fine tip) were made from 25 l Drummond micropipettes pulled on a Narishige automatic needle puller. Needle tips were broken at an angle using a razor blade and were backloaded with the original injection solution, which contained 400:200 g/ml P vector: helper in injection buffer (5 mM KCl, 0.1 M sodium phosphate pH 6.8) mixed with 0.01% green food color. 80 females, 4-8 hours old, were injected and placed into a vial containing Wards instant fly medium and three virgin males that were 24-28 hours old (four replications of 20 females). After five days, adults were removed and the vials were screened daily for red-eyed G0 adults until all

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23 flies had emerged (7 to 28 days). Each day the G0s that emerged were placed in a vial to sibmate for a total of ten vials per injected female. The G0 adults were removed from the vials after five days to prevent errors in identification of G1 progeny. The vials were screened daily and all G1 adults were scored for red eyes until the G1 no longer emerged (7 to 28 days). In an attempt to improve the quality of the microinjection solution and the conditions for transformation, endotoxin-free plasmids were mixed in injection buffer without food coloring. The effect of increasing both the concentration of the injection solution and the age of the virgin females were tested. In the first experiment, two concentrations of the injection solution, 400:200 g/ml and 1200:600 g/ml vector: helper, were injected into 40 females, 4-8 hours old (two replications of 20 females for each solution). In another experiment, 20 females that were 4-8 hours old and 20 females that were 18-24 hours old were injected with the 1200:600 g/ml vector: helper solution (two replications of 10 females per age category). The High-fidelity Polymerase Chain Reaction (PCR) (Barnes 1994) was used to test the duration of the Mtn promoter/CUP1 construct in the females injected with the endotoxin-free 400:200 g/ml vector: helper solution. DNA was isolated from two injected females at each of the following time intervals after injection: 1 minute, 1, 8, 12, 24 and 72 hours, 5 and 7 days, and from five non-injected females. A 533-bp product was expected using the Mtn promoter forward primer (5’-AGT AAG AGT GCC TGC GCA TG-3’) and the CUP1 reverse primer (5’-CAT GAC TTC TTG GTT TCT TCA-3’) using a Perkin-Elmer DNA Thermal Cycler 480 (Perkin-Elmer Corporation, Norwalk, CT).

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24 Egg Microinjection Embryos were injected with the endotoxin-free injection solution consisting of 400:200 g/ml vector: helper in injection buffer without food coloring using the protocol described by Handler (2000). Eggs were collected by placing 200 mature white-eyed adults (ww) into a bottle sealed with a plate containing a yeast-agar Drosophila diet (3.6 L water, 150 g Brewer's yeast, 300 g corn meal, 46g agar, 52 g soy meal, and 400 ml molasses) and inverted for 10 minutes after an initial one hour pre-collection period. Eggs were washed onto a white filter paper in a Buchner funnel (~45 mm inner diameter) attached to a filtering flask while the water was pulled off by a low vacuum. Eggs were dechorionated by soaking them in a 2% hypochlorite solution for 2 minutes, washed three times with distilled water and then washed onto a black filter paper. The filter paper was then transferred to a small plastic tray on ice. Eggs were individually transferred using a fine brush (000) to a thin strip (1 to 2 mm) of Scotch brand double-sided tape placed on a coverslip (30 x 22 mm) with the posterior end of the egg facing the edge of the coverslip. They were then desiccated in room air (~22C and ~50% RH) for 10 minutes and covered with Halocarbon 700 oil, which was contained over the eggs by a wax line that was drawn around the tape ~5 mm from the edge of the coverslip. The injection solution was backfilled into a beveled needle (Sutter Instruments microbevelor BV-10) made from a 25 l Drummond siliconized micropipette drawn out on a Sutter Instrument P-30 needle puller. The coverslip was placed on top of a slide on the mechanical stage of a stereozoom microscope (Olympus SZH) and the injection solution was delivered from the needle to the egg by an electronic air-pulse system (Handler 2000) using a micromanipulator (Narishige MN-151). Eggs were held for 24 hours in a sealed O2 chamber (Billups-Rothenberg, Inc., Del Mar, CA) under oil and crawling larvae were

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25 moved onto Ward’s instant fly medium (40 larvae per vial) where they were allowed to develop as adults. Establishment of Putatively Transformed Lines The injected eggs emerged as G0 adults and were backcrossed to the white-eyed strain (ww) in groups, which consisted of 3 G0 females and 2 ww males or 2 G0 males and 4 ww females. The G1 adults that developed from the crosses were screened for red-eyed flies (ww+) and these were backcrossed in single pairs to ww flies. All red-eyed (ww+) G2 flies were sib-mated in single-pair crosses and selected for red eyes until the lines were pure w+w+. These homozygous lines were reared in a quarantine facility at ~22C, ~50% RH, 14:10 L:D. High-fidelity PCR was used to detect the presence of the CUP1 gene in the putatively transgenic lines. Pooled genomic DNA was prepared by the PUREGENE method (Gentra Systems, Minneapolis, MN) from five G8 adults from each of the putatively transgenic lines. The DNA was screened for a unique 907-bp product using the CUP1 forward primer (5’-CAAAATGAAGGTCATGAGTGCCAAT-3’) and the SV40 reverse (5’-TGTGGTGTAAATAGCAAAGCAAGCA-3’) primers using a Perkin-Elmer DNA Thermal Cycler 480. Southern Blot Analysis EcoR I-digested genomic DNA from putatively transformed flies was analyzed using the method of Southern (1975). Pooled genomic DNA was obtained from 5 flies for each of the putatively transgenic lines and the control strain by the PUREGENE method according to the manufacturer’s recommended methods (Gentra Systems, Inc., Minneapolis, MN). Multiple Displacement Amplification (MDA) was conducted using a GenomiPhi DNA amplification kit (Amersham Bioscience Corp., Piscataway, NJ) to

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26 amplify 1 l of genomic DNA (Dean et al. 2002, Gorrochotegui-Escalante and Black 2003). DNA amplified by MDA (MDA-DNA) was digested with EcoR I restriction endonuclease and separated on a 0.7 % agarose TBE gel at 40 V for 8 hours. After depurination, denaturation and neutralization, EcoR I-digested MDA-DNA was transferred to a Hybond N Nylon membrane (Amersham Bisciences, Piscataway, NJ.). Probe templates included the Kpn I-EcoR I and Spe I-Pst I DNA fragments isolated from the pAJ171 plasmid. The fragments were gel-purified, nick-translated and labeled with 32P (10 l DNA, 3 l TE Buffer, 3 l Nick-translation buffer, 1 l each ATP, TTP and GTP, 5 l 32PCTP, 3 l Dnase, and 3 l DNA Polymerase I) (Maniatis et al. 1982). The Kpn I-EcoR I probe (405 bp) contained the CUP1 fragment and should produce a 896-bp band, indicating that the CUP1 construct was present; the Spe I-Pst I probe (850 bp) contained the SV40 sequences and should produce a band greater than 1,132 bp but less than 9 kb, indicating that the CUP1 gene was integrated into the nuclear genome. The blot was washed 3 times for 2 hours at 68C in a 2X SSC/0.5% SDS solution and placed on film (Super RX Fujfilm, Tokyo, Japan). The advantage of using MDA to amplify the genomic DNA is that a small number of flies can be used for the Southern blot (Gorrochotegui-Escalante and Black 2003), but the disadvantage is that MDA is an artificial amplification of random fragments of genomic DNA and the sequence of interest may not be sufficiently amplified to allow detection by probes in the Southern blot. Gel purification of small DNA fragments before labeling with 32P, in addition to small size (<1 kb), may make probes less sensitive to the small amount of MDA-DNA on the blot and fail to detect a positive signal. To increase the sensitivity of the Southern blot using DNA amplified by the MDA method, a

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27 more concentrated and larger probe was tested. The CUP1/SV40 fragment was isolated from pAJ171 and cloned into the KpnI-PstI site of pTZ19R plasmid (Amersham Biosciences) to produce plasmid pAJ334. This plasmid (1 g) was used to make a nick-translated 32P-labeled probe that carried the larger 1317-bp Kpn I-Pst I fragment containing the CUP1 and SV40 sequences. The pAJ334 probe was more concentrated because no gel purification was necessary and was larger in size (4 kb); thus, when hybridizing to the MDA-DNA, it was expected to be more sensitive in detecting bands that were 896-bp and >1,132-bp in size. Analysis of Copper Tolerance To obtain concentration-mortality curves for the ww control strain, vials containing 1 7 mM CuOH and a water control were prepared by adding Kocide (53.8% CuOH) to sterile water for a final volume of 3 milliliter (ml) and 3 g of Ward’s instant fly medium. To promote egg laying, females were maintained on a grape-agar plate (68.3 ml Welch’s 100% grape juice concentrate, 81.5 ml water, 8.0 g dextrose, 5.05 g sucrose, 3.30 g agar, 2.70 g yeast) for two days prior to egg collection (Frazier et al. 2001). This was replaced with a fresh grape-agar plate and eggs were collected during a four-hour period, from 10:00 am to 2:00 pm. After 24 hours (10:00 am the following day), 30 first-instar larvae were transferred to a vial containing seven concentrations of CuOH ranging from 1 7 mM CuOH or water (four replications; n=960). The vials were scored for adult emergence every 12 hours starting on the morning of day 7. Adults that emerged were removed from the vial and recorded. To obtain concentration-mortality curves and the lethal concentration to cause 50% (LC50) and 95% mortality (LC95), data were subjected to probit analysis using PoloPlus (LeOra Software 2002-2003, Berkeley, CA).

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28 Copper toxicity tests were performed to determine if two transgenic lines were more resistant to copper than the ww control line. Eggs were collected as described above and 30 first-instar larvae from each transgenic line and the ww control line were placed in vials with 2, 5, 6, 7, and 7.5 mM CuOH or water (12 replications; n=2160 per line). The adult emergence for each replication was obtained as described previously. Concentration-mortality curves and the LC50 and LC95 values were obtained. To analyze significant differences in the concentration-mortality curves of each of the fly lines, the curves were compared for equality and parallelism at the 95% confidence interval (C.I.) (PoloPlus). Four outcomes are possible: the slopes of the curves are not significantly different even though the intercepts differ significantly, the intercepts of the curves are not significantly different even though the slopes differ significantly, the curves do not differ in either slope or intercept, or the slopes and intercepts of the curves differ significantly. To test whether the LC50 and LC95 values for the ww line differed significantly from each of the transgenic lines, the 95% C.I. for the ratios of LC50 and LC95 values were calculated using PoloPlus. If the 95% C.I. for the ratios did not include 1, then the LC values were significantly different (=0.05). This analysis was also used to test whether the LC50 and LC95 values of the transgenic lines were significantly different from each other. Fitness costs of the transgenic lines were determined by analyzing the mean percentage survival and the mean number of days required for larvae to emerge as adults for each line in the water controls of the bioassays (n=360 per line). The percentage survival was estimated as the total number of larvae to reach adulthood per replicate and

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29 was averaged over each replicate within each line. Emergence was calculated as the mean number of days required for the larvae to emerge as adults averaged over each replicate within each line. To test the effect of line on both the mean percentage survival and the mean number of days to emerge, the data were subjected to analysis of variance (ANOVA) using PROC GLM with trial and replicate nested within the trial considered as random effects within the model (SAS Institute 1996). Inducement of the CUP1 Construct To assess whether resistance would be increased if flies (including flies later treated in an ‘induced’ water control) were pretreated with a low concentration of copper, grape/agar plates containing 2.5 mM of CuOH were used to collect eggs from the ww control strain and the transgenic lines 8 and 14. This was considered the induced treatment. For the non-induced treatment, standard grape/agar plates were used to collect eggs from each strain. Eggs were collected for four hours, placed in incubator at 24C, ~75% RH and 14:10 L:D. After 48 hours, 30 larvae from each treatment for each line were transferred to a vial containing 2, 4, 6, 8, 10, or 12 mM CuOH or water control and were held at 24C, ~75% RH and 14:10 L:D (eight replications, n=1680 per line per treatment). After seven days, the vials were reared in the quarantine facility at ~22C, ~50% RH and 14:10 L:D, and adult emergence was observed and recorded every 12 hours until five days after flies stopped emerging. Concentration-mortality curves were obtained for each line in each treatment using PoloPlus. The concentration-mortality curves were analyzed for significant differences between the treatments within each line and between the lines within each treatment. The 95% C.I. for the ratios of the LC50 and LC95 values were calculated to

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30 test for significant differences between the treatments within each line and significant differences between the lines within each treatment. Fitness costs of the transgenic lines in both water controls of the non-induced and induced treatments were determined by analyzing the mean percentage survival and mean number of days to emerge for each line (n=240 per line per treatment). To test the influence of the fixed effects, line and treatment, on both the mean percentage survival and the mean number of days to emerge, a two-way ANOVA was conducted using PROC GLM (SAS Institute 1996). Trial and replicates nested within the trial were considered as random effects within the model. Variances of mean days to emerge were not homogeneous, so the means were log transformed and the least squares means were separated using a probability of a significant difference of P<0.05 (SAS Institute 1996). Results and Discussion Maternal Microinjection The mean percentage survival Standard Deviation (SD) of the 80 females (4-8 hr old) injected with 400:200 g/l vector:helper was 90 (4.08)% and of these an average of 84.6 (3.27)% produced progeny. All G0 adults (~6,000) produced over the five-day mating period were ww indicating that the w+ marker gene had not integrated into the genomic DNA. All G1 adults (~300,000) produced over the five-day ovipositional period of the G0s were screened for red eyes and found to be ww, indicating that no transformants were obtained. The average percentage survival ( SD) of the females that were injected with 400:200 g/ml and 1200:600 g/ml endotoxin-free injection solution were 85.0 (5.8) and 87.5 (3.5)%, respectively, and of these an average of 91 (5.4) and 85.8 (3.5)% produced progeny, respectively. All G0 (~3,000) and G1 (~150,000) adults produced in each group

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31 had white eyes, indicating that transformation had not occurred and that increasing the concentration of the plasmids in the injection solution did not improve the effectiveness of this method. The mean percentage survival (SD) of the females injected with 1200:600 g/ml vector: helper solution when they were 4-8 hours old and 18-24 hours old were 85 (7.1)% for both and of these 77 (14.7) and 82.6 (6.9)%, respectively, produced progeny. Approximately 1,300 G0 and 65,000 G1 adults were produced from the females that were 4-8 hours old when injected and 1,400 G0 and 70,000 G1 adults were produced from the females that were 18-24 hours old when injected. All G0 and G1 adults were screened and all were ww, which did not allow a conclusion as to whether the age of the female is a factor in the success of maternal microinjection. Although transformation did not occur in the progeny of the 4-8 hour old females injected with 400:200 g/ml vector: helper, these females did retain the P-element vector for 7 days. A 553-bp product, the expected size of the Mtn promoter/CUP1 fragment, was detected in the injected females using High-fidelity PCR at all eight time intervals tested (1 minute to 7 days) (data not shown). Thus, maternal microinjection was unsuccessful in producing transgenic progeny, for unknown reasons. Egg Microinjection Of the 1,955 eggs that were injected, 450 (23%) survived to adulthood (G0) and these produced 17 red-eyed (ww+) G1 adults. The G1 flies came from 6 group crosses, which could represent as few as six integration events or as many as 17. These 17 adults were used to start the putatively transformed lines, for an estimated transformation rate of 1.3 3.8%, and 16 of the 17 G1 ww+ flies produced pure red-eyed (w+w+) lines after selection, suggesting they were homozygous. The line that was not homozygous produced ww+ females and ww males, and was discarded.

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32 High-fidelity PCR A unique 907-bp product was amplified from each of the 16 putatively transformed lines (data not shown), indicating that the injected sequences were present in the eighth generation. These results provide evidence that transformation occurred, but they do not resolve whether integration occurred or the number of integrations in each line. Southern Blot Analysis Four of the 16 lines were confirmed by two separate Southern blot analyses to contain a single chromosomal integration of the CUP1 construct (Fig. 1), with the integration site of line 8 (Fig. 1A) differing from lines 14, 16 and 17, which did not differ from each other (Fig. 1B and C). In the first blot, line 8 showed a 4-kb band after 4 days of exposure, indicating a single chromosomal integration of the CUP1 construct, but the expected 896-bp band was not detected (Fig. 1A). After 7 days, the background on the film was too strong to see any bands (data not shown). The other 15 lines did not show evidence of hybridization (data not shown), indicating either that the CUP1 construct was not present or that a more sensitive probe was needed. Using the more sensitive probe (pAJ334), the second blot showed that after only 16 hours of exposure, line 14 had the expected 896-bp and 6-kb bands, but lines 16 and 17 had only the 6-kb band (Fig. 1B). However, lines 16 and 17 both had the expected 896-bp and 6-kb bands after a 7-day exposure (Fig. 1C). These results indicate that the pAJ334 probe was more sensitive in detecting the 869-bp band. The 6-kb band in lines 14, 16, and 17 indicated that the three lines likely are from the same integration event of the CUP1 construct into a single location in the nuclear genome. These lines were pooled to form transgenic (pooled) line 14. The other lines were negative for the CUP1

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33 insert (data not shown), possibly due to a failure of the MDA procedure to amplify the transgene. The two transgenic lines, 8 and 14 (pooled), were evaluated for increased resistance to copper and fitness costs. A B C 14 16 17 14 16 17 8 6 kb 6 kb 4 kb 896 bp 896 bp Figure 1. Southern blots of the transgenic lines of D. melanogaster showing genomic integration of the CUP1 construct. A) Southern blot of line 8 using the KpnI-EcoRI and SpeI-PstI 32P-labeled probes after a 4-day exposure. B) Southern blot of lines 14, 16, and 17 using the pAJ334 nick-translated 32P-labeled probe after a 16-hour exposure. C) Southern blot of lines 14, 16, and 17 using the pAJ334 nick-translated 32P-labeled probe after a 7-day exposure. Analysis of Copper Tolerance A bioassay of the ww control was determined after conducting preliminary range tests. The baseline data obtained using the ww control line showed that the CuOH concentrations tested caused mortality ranging from 10 – 95%, as desired (PoloPlus) (Fig. 2). Concentration-mortality curves for the ww control line had an intercept Standard Error (SE) of -1.752 (0.177) and a slope (SE) of 3.688 (0.249). The LC50 value was 2.99 mM CuOH (95% C.I.=2.79-3.21) and LC95 value was 8.59 mM CuOH (7.61-9.9).

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34 01020304050607080901000123456789 mM CuOH% Mortalit y Figure 2. Baseline concentration-mortality curve for the ww control line of D. melanogaster tested with CuOH at 24C, ~75% RH and 14:10 L:D. After the transgenic lines were produced, concentration-mortality lines were obtained for the ww line and lines 8 and 14. The concentration-mortality curve for the ww control line had an intercept of -2.349 (0.631), which differed significantly from the intercepts of the concentration-mortality curves for both lines 8 and 14, which were -3.138 (0.685) and -2.837 (0.467), respectively (95% C.I.; P<0.05) (Fig. 3). However, the slope of the curve for the ww control line, 4.368 (0.754), was not significantly different from the slopes of line 8 (4.052 0.796) and line 14 (3.652 0.541). The concentration-mortality curves for lines 8 and 14 were not significantly different from each other in either intercept or slope. The probit analysis indicates that the transgenic lines require a higher concentration of CuOH than the ww control line to cause similar levels of mortality (Fig.3).

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35 Figure 3. Concentration-mortality curves for the ww control line and transgenic lines 8 and 14 of D. melanogaster when treated with copper at 24C, ~75% RH and 14:10 L:D. Lines 8 and 14 had an approximately 1.5to 2-fold increase in resistance to copper compared to the ww control line, as shown by the LC50 and LC95 values (Table 1), indicating that the yeast CUP1 gene is functioning in D. melanogaster and confers resistance to copper. The LC50 and LC95 values of lines 8 and 14 did not differ Table 1. LC50 and LC95 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at 24C, ~75% RH and 14:10 L:D. Line Concentration of CuOH (mM) (95% C.I.) * LC50 LC95 ww 3.45 (2.37-4.06) b 8.21 (7.57-7.31) b Line 8 5.95 (4.79-6.59) a 15.16 (11.7-31.1) a Line 14 5.98 (5.11-6.56) a 16.9 (13.2-29.3) a *Concentrations with different letters represent a significant difference between the lines within the columns (=0.05; LC ratio test). significantly from each other (95% C.I., =0.05). The approximately 1.5to 2-fold increase in tolerance to copper exhibited by the transgenic lines is similar to that seen in

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36 D. melanogaster populations that have duplications of the Drosophila Mtn gene, either by P-element mediated transformation or naturally (Maroni et al. 1987, 1995). Fitness assessment. Transgenic lines 8 and 14 did have fitness costs in the water controls of this bioassay (Table 2). The mean percentage survival of the ww control line was significantly greater than that of the two transgenic lines, which did not differ significantly from each other (F=5.97; df=2, 22; P=0.0085). In addition, the mean number of days to emerge as adults was significantly shorter in the ww control line than in either of the transgenic lines (F=7.86; df=2, 22; P=0.0027), which did not differ significantly from each other. Reduced survivorship and delayed development in the absence of copper of lines 8 and 14 are indications of fitness costs, which could be due to the insertion site of the transgene, the extra resources required to produce additional proteins, or from inbreeding of these lines (Beckendorf and Hoy 1985, Hoy 1985, Levis et al. 1985, Lenski and Nguyen 1988, Mackay 1989, Sibly and Calow 1989, Kaiser et al. 1997). Table 2. The mean percentage survival and the mean number of days to emerge as adults for each D. melanogaster line in the water controls at 24C, ~75% RH and 14:10 L:D. Line Mean % survival (SEM) * Mean days to emerge (SEM) * ww 95.8 (1.05) a 11.6 (0.14) b 8 79.9 (3.97) b 14.1 (0.47) a 14 76.9 (5.53) b 14.6 (0.81) a *Means with different letters represent significant differences between the lines within the columns (P<0.05). Inducement of the CUP1 Construct All lines in the induced treatment exhibited an increase in tolerance to copper, indicating that pretreatment with a low CuOH concentration does induce higher levels of resistance (Fig. 4A, B and C). In the ww control line, the concentration-mortality curves of the non-induced and induced treatments were significantly different in their intercepts

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37 (SE), -5.730 (0.517) and -4.620 (0.580), respectively, but not in their slopes (SE), 9.012 (0.727) and 7.199 (0.739), respectively, (95% C.I.; P<0.05) (Fig. 4A). The concentration-mortality curves of the non-induced and induced treatments for transgenic line 8 were also significantly different in their intercepts (SE), -5.155 (0.489) and -4.946 (0.546), respectively, but not in their slopes (SE), 7.332 (0.589) and 6.657 (0.610), respectively (95% C.I.; P<0.05) (Fig. 4B). Similarly, the concentration-mortality curves of the non-induced and induced treatments for line 14 were significantly different in their intercepts (SE), 5.388 (0.453) and -6.931 (0.658), respectively, but not in their slopes (SE), 7.740 (0.563) and 8.360 (0.711), respectively (95% C.I.; P<0.05) (Fig. 4C). The increase in tolerance to copper after pretreatment appears to be greater in line 14 than in line 8 (Tables 3 and 4). In line 8, the LC50 values of the non-induced and induced treatments were not significantly different (Table 3), but the LC95 values differed significantly (Table 4). In line 14, both the LC50 and LC95 values of the non-induced and induced treatments were significantly different (Tables 3 and 4). The increase in copper tolerance of the pretreated transgenic lines could be a result of induced transcription of CUP1, as was found in a transgenic yeast strain with 11-15 copies of CUP1 when grown in 50 mM of copper (Jeyaprakash et al. 1991) and the induced transcription of native metallothionein genes, such as Mtn and Mto (Mokdad et al. 1987). The LC50 values of the ww control line in the non-induced and induced treatments were not significantly different (Table 3); however, the LC95 values were significantly different, indicating a slight increase in tolerance to copper when pre-treated. This increase in copper tolerance of the ww control line could possibly be due to the increased transcription of native metallothionein genes.

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38 Figure 4. Concentration-mortality curves of the non-induced and induced treatments for the ww control line, and lines 8 and 14 of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D. A) Concentration-mortality curves for the ww control line in the non-induced and induced treatments. B) Concentration-mortality curves for line 8 in the non-induced and induced treatments. C) Concentration-mortality curves for line 14 in the non-induced and induced treatments.

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39 The increase in tolerance to copper after pretreatment appears to be greater in line 14 than in line 8 (Tables 3 and 4). In line 8, the LC50 values of the non-induced and induced treatments were not significantly different (Table 3), but the LC95 values differed significantly (Table 4). In line 14, both the LC50 and LC95 values of the non-induced and induced treatments were significantly different (Tables 3 and 4). The increase in copper tolerance of the pretreated transgenic lines could be a result of induced transcription of CUP1, as was found in a transgenic yeast strain with 11-15 copies of CUP1 when grown in 50 mM of copper (Jeyaprakash et al. 1991) and the induced transcription of native metallothionein genes, such as Mtn and Mto (Mokdad et al. 1987). The LC50 values of the ww control line in the non-induced and induced treatments were not significantly different (Table 3); however, the LC95 values were significantly different, indicating a slight increase in tolerance to copper when pre-treated. This increase in copper tolerance of the ww control line could possibly be due to the increased transcription of native metallothionein genes. Table 3. LC50 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D in the non-induced and induced treatments. Line LC50 * (95% C.I.) Non-induced Induced ww 4.32 a, B (4.11-4.52) 4.38 a, C (3.84-4.81) Line 8 5.05 a, A (4.69-5.36) 5.53 a, B (5.04 to 5.95) Line 14 4.97 a, A (4.60-5.29) 6.75 b, A (6.35-7.09) *Concentrations with different lower case letters represent significant differences between the treatments within the rows and concentrations with different upper case letters represent significant differences between the lines within each column.

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40 Table 4. LC95 values in mM CuOH for the ww control and each transgenic line of D. melanogaster when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D in the non-induced and induced treatments. Line LC95 * (95% C.I.) Non-induced Induced ww 6.58 b, B (6.24-7.03) 7.42 a, C (6.81 to 8.31) Line 8 8.46 b, A (7.98-9.08) 9.78 a, B (9.17-10.6) Line 14 8.10 b, A (7.59-8.80) 10.6 a, A (10.1 to 11.3) *Concentrations with different lower case letters represent significant differences between the treatments within the rows and concentrations with different upper case letters represent significant differences between the lines within each column. The data were then analyzed to determine how the level of copper tolerance of the lines differed from each other (Tables 3 and 4). In the non-induced treatment, the LC50 and LC95 values of the transgenic lines were significantly different from the ww control line, but were not different from each other (95%CI; =0.05) (Tables 3 and 4), as expected from the first bioassay. In the induced treatment, the LC50 and LC95 values of line 14 were significantly higher than the LC50 and LC95 values of line 8, and both lines were significantly higher than the values for the ww control line (95%CI; P=0.05) (Tables 3 and 4). The concentration-mortality curves for the ww control line, and the transgenic lines 8 and 14 in the non-induced treatments differed significantly in shape from the induced treatments (95% CI; P<0.05) (Figs. 5A and B). The concentration-mortality curves for the ww control line in the non-induced and induced treatments were significantly different from the non-induced and induced treatments of lines 8 and 14 (Fig. 5A). The concentration-mortality curves for the transgenic lines 8 and 14 were not significantly different from each other in the non-induced treatments; however, in the induced

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41 treatment, line 14 was more resistant to copper than line 8, as shown by the significantly different intercepts (95% CI; P<0.05). Although lines 8 and 14 each have a single insert of the CUP1 construct, the lines may respond to copper differently due to differences in chromosomal location, which can lead to variations in gene expression (Hazelrigg et al. 1984, Levis et al. 1985). Fitness assessment of the non-induced and induced water controls . The mean percentage survival and the mean number of days required to emerge as adults were analyzed for each of the lines in the water controls of the non-induced and induced (pretreated with a low concentration of CuOH, but no subsequent treatment with CuOH) treatments to determine if there were significant effects of line, treatment or an interaction between line and treatment (Table 5). The mean percentage survival was affected significantly by line, treatment and the interaction between the line and the treatment. The effect of line on mean percentage survival was analyzed by averaging the means of flies in the water controls of the non-induced and induced treatments within each line (Table 5). The mean percentage survival of line 14 was significantly greater than line 8 (F=3.67; df=1, 35; P=0.0358); however, neither of the transgenic lines differed significantly from the ww control line. To analyze the effect of pretreatment on mean percentage survival, the means in the water controls of ww and lines 8 and 14 were averaged and compared to the survival of flies not pretreated with copper (Table 6). The survival of the flies that were pretreated was significantly lower than flies experiencing no pretreatment (F=16.76; df=1, 35; P=0.0002). The induced treatment appears to have a negative impact on the survivorship of all fly lines tested.

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42 Figure 5. Concentration-mortality curves for the ww control line, and lines 8 and 14 of D. melanogaster in the non-induced and induced treatments when treated with copper at ~22-24C, ~50-75% RH, and 14:10 L:D. A) Concentration-mortality curves for each line in the non-induced treatment. B) Concentration-mortality curves for each line in the induced treatment.

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43 Table 5. The mean percentage survival and the mean number of days to emerge as adults in the water controls of each D. melanogaster line averaged over the non-induced and induced treatments when held at ~22-25C, ~50-75% RH, and 14:10 L:D. Line Mean % survival (SEM)* Mean days to emerge (SEM) * ww 77.1 (3.71) a b 12.2 (0.23) b Line 8 72.1 (2.58) b 13.5 (0.32) a Line 14 81.9 (3.23) a 13.6 (0.51) a * Means with different letters represent significant difference between the lines within the columns (P<0.05). Table 6. The mean percentage survival and the mean number of days to emerge as adults of D. melanogaster pretreated with copper and then treated with water compared to flies treated only with water when held at ~22-24C, ~50-75% RH, and 14:10 L:D. Treatment Mean % survival (SEM) * Mean of days to emerge (SEM) * Non-induced 83.1 (2.43) a 13.5 (0.37) a Induced 71.0 (2.39) b 12.8 (0.27) a *Means with different letters represent significant difference between the lines within the columns (P<0.05). To determine how survivorship varied by line, the interaction between line and treatment (non-induced and induced) on the mean percentage survival was analyzed (Table 7). There was no significant difference in mean percentage survival of the ww control line and lines 8 and 14 in the non-induced treatments; however, in the induced treatment the mean percentage survival of line 14 was significantly higher than that of both the ww control and line 8 (F=5.44; df=2, 35; P=0.0088), which were not significantly different from each other. Furthermore, the mean percentage survival of the induced treatment was significantly lower than the non-induced treatment for both the ww control line and line 8, whereas there was no difference between the non-induced and induced treatments of line 14 (Table 7).

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44 Table 7. The mean percentage survival for each D. melanogaster line averaged over the non-induced and induced treatment in the water controls at ~22-24C, ~50-75% RH, and 14:10 L:D. Line Mean % survival (SEM) * Non-induced Induced ww 88.8 (2.74) a, A 65.4 (3.56) b, B Line 8 78.8 (3.50) a, A 65.4 (1.88) b, B Line 14 81.7 (5.56) a, A 82.0 (3.72) a, A *Means with different lower case letters represent significant differences between the treatments within the rows and percentages with different upper case letters represent significant differences between the lines within each column. The mean number of days to emerge did not differ significantly by treatment (Table 6), nor was there a significant interaction between treatment and line (data not shown), but there was a significant effect of line (F=4.73; df= 2, 35; P=0.0152) (Table 5). The mean number of days to emerge was significantly lower in the ww control line than in lines 8 or 14, which did not differ significantly from each other (Table 5). The transgenic lines take significantly longer to emerge as adults than the ww control regardless of whether they have been subjected to a pretreatment with copper. The results of this fitness assessment indicate that line 14 is as fit in the non-induced treatment as in the induced treatment and has a greater level of fitness than the ww control and line 8 when pretreated with copper (Tables 6 and 7). Although there was no difference in the number of days to emerge between the non-induced and induced treatments of both the ww control line and line 8, the significant decrease in survival to adulthood exhibited in the induced treatments indicates a fitness cost (Table 7). Lines 8 and 14 may have different fitness levels in the induced treatment due the chromosomal site of integration of the Mtn/CUP1 construct, which may have an affect on the expression of the transgene and/or of native genes (Hazelrigg et al. 1984, Levis et al. 1985, Mackay 1989). In the absence of copper, the higher percentage survival and

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45 shorter emergence time of the ww control compared to the transgenic lines 8 and 14 in the non-induced treatments (Tables 5, 6, and 7) indicate that the ww control has a greater level of fitness than lines 8 and 14. The decreased fitness of the transgenic lines in the non-induced treatment reduces the probability that they could persist in an environment in the absence of copper and thus, could reduce the potential risks associated with the release of these lines. Conclusions The reduced survival and the longer time to emerge as adults in the absence of copper exhibited by lines 8 and 14 may be attributed to the presence of the CUP1 gene, the insertion site, or the extra resources required in producing additional proteins. The fitness costs attributed to CUP1 in these lines may be beneficial in reducing potential risks if they are observed in transformed natural enemies, because, in the absence of copper, the transgenic populations should not persist. For example, a transgenic strain of M. occidentalis was suitable for evaluating risks associated with the release of transgenic natural enemies in a short-term field release due to the fact that the eggs of this strain (and the nontransgenic strain) would not hatch at temperatures above 33C at 100% RH, making it unlikely to survive Florida summers (Li and Hoy 1996, McDermott and Hoy 1997). The first bioassay of the D. melanogaster lines differed in LC50 and LC95 values from the second bioassay in absolute values, but not in relative values (Tables 1, 3 and 4). Environmental conditions may affect bioassays and temperature and relative humidity were lower in the second bioassay than in the first, which are factors that have been shown to affect the toxicity of chemicals to insects (Reichenbach and Collins 1984). Insects develop more slowly at lower temperatures, which may lead to longer exposure to

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46 toxins. Temperature and humidity also affect the viability of D. melanogaster, with optimal viability and development rate of D. melanogaster at 25C and a relative humidity of >70% (Roberts 1986, Greenspan 1997), which is similar to the conditions in the first bioassay. In each bioassay, the transgenic lines were significantly more resistant to copper than the ww control line. If the maternal microinjection method could be developed for D. melanogaster or other insects, then genetic transformation may be more cost and time effective because multiple transgenic lines could be produced from a single injection. The age of the female, the concentration of the injection solution and the plasmid delivery system may be factors that affect the rate of success in transforming the oocytes or eggs within the female. This Mtn/CUP1 construct may be useful in future experiments for developing a maternal microinjection method for D. melanogaster. This appears to be the first time that the yeast metallothionein gene, CUP1, has been inserted and expressed in an insect. This work shows that CUP1 confers resistance to copper in D. melanogaster, and suggests that CUP1 may be a useful gene for increasing the resistance of copper-sensitive beneficial insects where copper is applied in pest management programs. CUP1 also might be a useful selectable marker in the genetic transformation of agriculturally important insects using recombinant DNA methods provided that the Mtn promoter functions in other insects or that another useful promoter is isolated.

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CHAPTER 3 REFLECTIONS As I reflect on the many lessons learned throughout the course of this project, I realize how far I have come. I have learned the process of experimental design, the importance of critically analyzing the literature, and how to write scientifically. This experience has taught me that although I have persistence and determination, I still need to further strengthen my time management skills, my ability to think critically about what I read and my independence as a researcher. Though I see the importance of being independent, I have found that interacting with those far more experienced than I was extremely valuable in getting hands on experience, and thus avoided mistakes and loss of time. One goal of this project was to develop a maternal microinjection method for Drosophila melanogaster. I feel the way that I approached the problem led to many unanswered questions regarding whether this method could be devised for this insect. In retrospect, I would have used egg microinjection first to develop microinjection skills, test the P element vector and helper plasmid, and developed lines to test the CUP1 gene. As I established the putatively transgenic lines, I could have then devoted my time developing a maternal microinjection method for D. melanogaster by experimenting with the concentration of plasmid DNA, age of the female, and methods for DNA delivery, such as lipofection, which are modified forms of cationic lipids that encapsulate DNA (Handler 2000). 47

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48 I have learned how valuable the literature is in planning and implementing a research project and I would like to further develop these skills. Initially, I had a tendency to read from one source and as a result I did not acquire all the knowledge necessary to complete my research in an efficient amount of time. I have learned how vital it is to analyze what I read. Developing these skills will now allow me to be more efficient in planning, developing, and implementing a research project. I feel that I still have a great deal to learn, but this experience has given me a greater appreciation of the scientific process, attention to detail, admitting to and learning from my mistakes.

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LIST OF REFERENCES Aerts, M.J., Nesheim, O.N., 2000. Florida Crop/Pest Management Profiles: Citrus. Circ. 1241. Florida Cooperative Extension Service, IFAS, University of Florida, Gainesville, FL. http://edis.ifas.ufl.edu. Anxolabhre, D., Kidwell, M., Periquet, G., 1988. Molecular characteristics of diverse populations are consistent with the hypothesis of a recent invasion of Drosophila melanogaster by mobile P elements. Mol. Biol. Evol. 5, 252-269. Ashburner, M., 1989. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, pp. 1017-1063. Ashburner, M., Hoy, M.A., Peloquin, J., 1998. Transformation of arthropods: research needs and long term prospects. Insect Mol. Biol. 7, 201-213. Atkinson, P.W., 2002. Genetic engineering in insects of agricultural importance. Insect Biochem. Molec. Biol. 32, 1237-1242. Atkinson, P.W., Pinkerton, A.C., O’Brochta, D.A., 2001. Genetic transformation systems in insects. Annu. Rev. Entomol. 46, 317-346. Barnes W.M., 1994. PCR amplification of up to 35 kb DNA with high fidelity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. Beach, L.R., Palmiter, R.D., 1981. Amplification of the metallothionein-I gene in cadmium-resistant mouse cells. Proc. Natl. Acad. Sci. USA 78, 2110-2114. Beckendorf, S.K., Hoy, M.A., 1985. Genetic improvement of arthropod natural enemies through selection, hybridization or genetic engineering techniques. In: Hoy, M.A., Herzog, D.C. (Eds.), Biological Control in Agricultural IPM Systems. Academic Press, Orlando, pp. 167-187. Bingham, P.M., Kidwell, M.G., Rubin, G.M., 1982. The molecular basis of P-M hybrid dysgenesis: the role of the P element, a P-strain-specific transposon family. Cell 29, 995-1004. Bonneton, D.M., Boissonneau, F., Andre, E., Wegnez M., 1995. Expression of metallothionein genes during the post-embryonic development of Drosophila melanogaster. Biometals 8, 339-51. 49

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50 Brenes-Pomales, A., Lindegren, G., Lindegren, C.C., 1955. Gene control of coppersensitivity in Saccharomyces. Nature 176, 841-842. Cha, J.S., Cooksey, D.A., 1991. Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proc. Natl. Acad. Sci. USA 88, 8915-8919. Childers, C.C., 1994. Effect of different copper formulations tank-mixed with fenbutatin-oxide for control of citrus rust mites (Acari: Eriophyidae) on Florida citrus. Fla. Entomol. 77, 349-365. Childers, C.C., Villanueva, R., Aguilar, H., Chewning, R., Michaud, J.P., 2001. Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida. Exp. Appl. Acarol. 25, 461-474. Cherian, M.G., Goyer, R.A., 1978. Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci. 23, 1-10. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: Roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159-182. Cousins, R.J., 1994. Metal elements and gene expression. Annu. Rev. Nutr. 14, 449-69. Daniels, S.B., Chovnick, A., 1993. P element transposition in Drosophila melanogaster: an analysis of sister-chromatid pairs and the formation of intragenic secondary insertions during meiosis. Genetics 133, 623-636. Daniels, S.B., Peterson, K.R., Strausbaugh, L.D., Kidwell, M.G., Chovnick, A., 1990. Evidence for horizontal transmission of the P transposable element between Drosophila species. Genetics 124, 339-355. Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du,Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., and Lasken, R.S., 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA 99, 5261-5266. Ecker, D.J., Butt, R.T., Sternberg, E.J., Neeper, M.P., Debouck, C., Gorman, J.A., Crooke, S.T., 1986. Yeast metallothionein function in metal ion detoxification. J. Biol. Chem. 261,16895-16900. Eger, J. E., Jr., Ferguson, V. M., Townsend, K. G., 1985. Efficacy of selected miticides and spray tank mixtures used to control rust mite in Florida citrus. Proc. Fl. St. Hortic. Soc. 98, 11-14. Engels, W.R., 1992. The origin of P elements in Drosophila melanogaster. BioEssays 14, 681-686.

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BIOGRAPHICAL SKETCH Jennifer Lee Meyer was born Jennifer Lee Steill on January 15, 1979, in Lafayette, Indiana, moved to Akron, Ohio, at the age of 11 and returned once again to Lafayette at the age of 17. She graduated from high school in 1997 and went on to attend Purdue University. While getting her undergraduate degree in biology, Jennifer worked as a laboratory technician for Dr. Jonathan Neal in the Department of Entomology. It was there that she developed a love for using molecular biology techniques to research agriculturally important problems and also met Jason M. Meyer, whom she married on June 21, 2003. She obtained her Bachelor of Science degree with a major in biology and minors in chemistry and psychology in August 2001 and continued to work on developing selectable genetic markers for crop rotation resistant corn rootworm in Dr. Neal’s laboratory. To strengthen her education and skills in molecular biology and entomology, Jennifer pursued her master’s degree in the Department of Entomology and Nematology at the University of Florida in August of 2002. Jennifer is a member of the Florida Entomological Society and the Entomological Society of America. 58