The Genetics of Resistance to Lufenuron in Drosophila Melanogaster
The Genetics of Resistance
to Lufenuron
in Drosophila melanogaster
Michael Bogwitz
Submitted for total fulfilment of the requirements for
the degree of Doctor of Philosophy
February 2005
Centre for Environmental Stress & Adaptation Research
(CESAR)
Department of Genetics
University of Melbourne
Abstract
The rise of large scale agriculture in the 20th century created the need for effective strategies to control insect pests. Treatment with chemical insecticides has been a weapon of choice, but the inevitable evolution of resistance has followed in many insect species. Resistance represents a major challenge, not only for agricultural production, but also for environmental preservation and human health. Two major options for resistance have been identified, and these are target-site based and metabolic-based resistance. Much insecticide resistance research focuses on identifying these mechanisms through genetic and molecular analysis.
The insecticide lufenuron is the focus of this study. It belongs to a novel insecticidal group called the insect growth regulators, which were introduced in 1970s as highly selective insecticides with low vertebrate toxicity. Resistance to lufenuron in the non- pest species Drosophila melanogaster has been observed in field populations, despite the lack of field usage of lufenuron (Wilson & Cain, 1997; O'Keefe, 1997).
This study has taken advantage of this phenomenon to investigate resistance mechanisms in natural populations. At least two detoxification mechanisms were identified.
A resistant isofemale strain isolated in Victoria, Australia was used to identify the detoxification gene, Cyp12a4, that may be involved in resistance. Tissue-specific overexpression of this gene appears to confer resistance to lufenuron, as confirmed through the generation and screening of transgenic overexpressing strains.
ii A second mechanism was identified from an isofemale strain isolated from field populations in Coloardo, USA. Another detoxification gene, Cyp6g1, was found to be overexpressed in this strain. Transgenic screening confirms its involvement in conferring resistance to lufenuron. A partial transposon insertion (Accord) was identified, and implicated as the overexpression conferring mutation. An alternate allele containing a partial P-element within Accord was identified in a strain collected from Australian (Queensland) natural populations. The frequency of these alleles is high.
Mutagenesis was also used in an attempt to identify the lufenuron target. 17 mutants were isolated, and due to an experimental bias, all carried the Cyp6g1 mechanism and another as yet unidentified mechanism on another chromosome.
This study highlights the importance of the need for better understanding of insecticide resistance, and particularly, the arsenal of metabolic detoxification mechanisms available to insect pests, as a method of prolonging the effectiveness of insecticides.
iii Declaration
This is to certify that:
i. This Thesis comprises only my original work
ii. Due Acknowledgement has been made in the text to other material used
iii. The thesis is less than 100,000 words in length, exclusive of tables, figure
legends, bibliographies, and appendices.
Michael Bogwitz
This Copy Printed on Acid-Free paper
iv Acknowledgements
There are so many people who have helped me through these last few years practically, intellectually, and emotionally.
To my supervisor Phil Batterham, my greatest gratitude goes to you for giving me the opportunity to explore the world of insecticide resistance, for guiding me and having faith, and for creating such a great lab in which to work.
To my collaborators and co-workers, thankyou for providing valuable assistance that has mad my task much easier. To Tom Wilson for supplying the WC2 fly strain that became such a large part of my project. To Rene Feyereisen for creating and generously providing results of P450 microarrays. To Phil Daborn for sharing fly strains, data, and ideas, and at one stage, his home. To David Heckel, Charlie Robin, and Adam Williams for intellectual inputs that have helped me immensly. To my fellow Ph.D. student, Trent Perry, for collaboration, discussion, and generous sharing of data on many occasions. To Chris Lumb for performing microinjections. And to
John Damiano and Jayne Lydall for assistance with EMS screens.
To my friends who have been so supportive in so many different ways over the years, thanks especially to – Ben, Tim, and Tom for being such great housmates, to
Affrica, the best friend I could have, to Lisa for getting me through the last year sanely and helping so much with my thesis, to Mel for being great support for over four years, and to Bec, Cameron, Taryn, and many others. And to Mum and Peter for helping me de-stress at Inverloch.
And to all the lab members who have made my time in CESAR such an unforgettable experience. Thanks.
Thankyou all for being a part of this with me.
v Table of Contents
Title------i
Abstract ------ii
Declaration ------iv
Acknowledgements------v
Table of Contents ------vi
List of Figures ------xv
List of Tables ------xxiii
List of Abbreviations ------xx
vi CHAPTER 1
INTRODUCTION...... 1
1.1 THE IMPORTANCE OF INSECTICIDE RESISTANCE...... 2
1.2 THE “EVOLUTION” OF INSECTICIDES ...... 2
1.3 THE EVOLUTION OF RESISTANCE...... 3
1.4 MECHANISMS OF RESISTANCE ...... 6
1.4.1 BEHAVIOURAL AVOIDANCE ...... 8
1.4.2 REDUCED PENETRATION ...... 8
1.4.3 TARGET SITE RESISTANCE...... 9 1.4.3.1 Nervous system targets ...... 9 1.4.3.2 Developmental targets...... 11
1.4.4 METABOLIC RESISTANCE ...... 12 1.4.4.1 Transferases...... 12 1.4.4.2 Hydrolases...... 14
1.5 CYROCHROME P450S...... 16
1.5.1 STRUCTURE AND ORGANISATION...... 17
1.5.2 DIVERSITY AND SUBSTRATE SPECIFICITY ...... 19
1.5.3 P450S AND INSECTICIDE RESISTANCE...... 19
1.5.4 MECHANISMS OF P450-BASED RESISTANCE ...... 20 1.5.4.1 LPR strain of M. domestica...... 21 1.5.4.2 Rutgers strain of M. domestica ...... 22 1.5.4.3 91-R strain of D. melanogaster ...... 24
1.5.5 CHARACTERISATION OF P450-MEDIATED RESISTANCE ...... 25 1.5.5.1 Indicators of P450 involvement...... 26 1.5.5.2 Criteria for demonstrating P450 involvement...... 26 1.5.5.3 Summary of P450s ...... 27
1.5.6 REGULATION OF P450S ...... 28
1.6 MOLECULAR MECHANISMS OF RESISTANCE ...... 31
1.6.1 POINT MUTATIONS ...... 32
1.6.2 GENE AMPLIFICATION...... 33
1.6.3 CHROMOSOMAL ABNORMALITIES...... 35
vii 1.6.3.1 Transposable elements...... 35 1.6.3.1.1 Insertions into coding regions...... 37 1.6.3.1.2 Insertions into introns ...... 38 1.6.3.1.3 Insertions into regulatory regions ...... 39
1.7 TYPES OF INSECTICIDES ...... 41
1.7.1 IGRS ...... 44 1.7.1.1 The cuticle...... 44 1.7.1.2 JHAs ...... 50 1.7.1.3 CSIs ...... 51
1.7.2 LUFENURON ...... 53
1.8 DROSOPHILA AS A MODEL ORGANISM...... 54
1.9 THIS PROJECT ...... 55
viii CHAPTER 2
MATERIALS AND METHODS ...... 60
2.1 FLY MAINTENANCE...... 61
2.1.3 DROSOPHILA MELANOGASTER FOOD MEDIUM...... 61
2.1.4 LUFENURON-CONTAINING FOOD MEDIUM ...... 62
2.1.5 LUFENURON SCREENING METHODS ...... 62
2.2 MOLECULAR TECHNIQUES...... 63
2.2.1 GENOMIC DNA EXTRACTION...... 63
2.2.2 POLYMERASE CHAIN REACTION ...... 63
2.2.3 AGAROSE GEL SEPARATION ...... 64
2.2.4 RESTRICTION ENZYME DIGESTION ...... 64
2.2.5 TOTAL RNA EXTRACTION ...... 65
2.2.6 RNA QUANTIFICATION ...... 65
2.2.7 FIRST STRAND CDNA SYNTHESIS ...... 65
2.2.8 5’ RACE ...... 66
2.2.9 REALTIME POLYMERASE CHAIN REACTION ...... 66
2.2.10 DNA PURIFICATION ...... 67
2.2.11 DNA SEQUENCING ...... 67
2.2.12 PLASMID CLONING AND DNA EXTRACTION ...... 67
2.2.13 MICROINJECTIONS ...... 68
ix CHAPTER 3
MAPPING FIELD RESISTANCE IN AUSTRALIA ...... 68
3.1 INTRODUCTION...... 69
3.1.1 FIELD RESISTANCE STUDIES...... 69
3.1.2 FIELD RESISTANCE TO LUFENURON ...... 70
3.2 MATERIALS AND METHODS ...... 72
3.2.1 FLY STRAINS ...... 72
3.2.2 FLY CROSSES ...... 74 3.2.2.1 Mapping of the resistance locus ...... 74 3.2.2.2 Cross used for sequencing relevant genes...... 76 3.2.2.3 Crosses used to map the site of integration of molecular constructs in transgenic flies...... 77 3.2.2.4 Crosses used to isogenise transgenic flies...... 79 3.2.2.5 Crosses used to analyse transgenic flies...... 80
3.2.3 PCR PRIMERS...... 82
3.2.4 OVEREXPRESSION CONSTRUCT ...... 86
3.3 RESULTS ...... 87
3.3.1 FIELD RESISTANCE STUDY 2001...... 87
3.3.2 GENETIC AND MOLECULAR MAPPING OF RESISTANCE GENES IN NB16 STRAIN....96 3.3.2.1 Fold resistance and dominance ...... 96 3.3.2.2 Genetic mapping...... 98 3.3.2.3 Molecular Mapping...... 100
3.3.3 CANDIDATE GENES ...... 103 3.3.3.1 Sequencing candidates...... 104 3.3.3.2 Expression levels of candidates...... 106
3.3.4 OVEREXPRESSION CONSTRUCT ...... 108
3.4 DISCUSSION...... 110
3.4.1 THIS STUDY...... 110
3.4.2 CANDIDATE GENES ...... 111
3.4.3 RESISTANCE IN NATURAL POPULATIONS ...... 113
x CHAPTER 4
FIELD RESISTANCE IN USA ...... 115
4.1 INTRODUCTION...... 116
4.1.1 INSECTICIDE RESISTANCE FROM A HISTORICAL PERSPECTIVE ...... 116
4.1.2 LUFENURON RESISTANCE IN FIELD POPULATIONS IN USA...... 117
4.1.3 AIMS OF THIS PROJECT ...... 118
4.2 MATERIALS AND METHODS ...... 120
4.2.1 FLY STRAINS ...... 120
4.2.2 FLY CROSSES ...... 120 4.2.2.1 Mapping of the resistance locus ...... 120 4.2.2.2 Crosses used to screen transgenic flies ...... 122
4.2.3 DOSAGE MORTALITY CURVES ...... 122
4.2.4 PCR PRIMERS...... 123
4.3 RESULTS ...... 125
4.3.1 EXAMINING THE EXPRESSION LEVELS OF CYP6G1 IN WC2 ...... 125
4.3.2 GENETIC AND MOLECULAR MAPPING OF RESISTANCE IN WC2...... 127 4.3.2.1 Level of resistance ...... 127 4.3.2.2 Molecular Mapping...... 129 4.3.2.3 Cross-resistance to other insecticides ...... 133
4.3.3 IDENTIFICATION OF A MUTATION IN CYP6G1...... 135
4.3.4 MECHANISMS OF ALTERED TRANSCRIPTION ...... 140 4.3.4.1 Testing run-on transcription ...... 143
4.3.5 RE-EXAMINATION OF OVEREXPRESSION...... 146
4.4 DISCUSSION...... 148
4.4.1 THIS INVESTIGATION: AN OVERVIEW ...... 148
4.4.2 P450S AND RESISTANCE...... 149
4.4.3 ACCORD AND RESISTANCE ...... 150
4.4.4 WHY HAS RESISTANCE EVOLVED? ...... 152
4.4.5 IS THERE MORE TO RESISTANCE THAN JUST CYP6G1? ...... 154
xi CHAPTER 5
OTHER RESISTANCE MECHANISMS IN NATURAL POPULATIONS...... 155
5.1 INTRODUCTION...... 156
5.1.1 MONOGENIC VERSES POLYGENIC RESISTANCE ...... 156
5.1.2 SEVERAL GENES OF MAJOR EFFECT ...... 157
5.1.3 GENES IN ADDITION TO CYP6G1...... 158
5.2 MATERIALS AND METHODS ...... 161
5.2.1 FLY STRAINS USED ...... 161
5.2.2 DOSAGE MORTALITY CURVES ...... 162
5.2.3 MAPPING PROTOCOLS ...... 162 5.2.3.1 Mapping to a chromosome ...... 162 5.2.3.2 Mapping within a chromosome ...... 164
5.2.4 PCR PRIMERS...... 166
5.3 RESULTS ...... 167
5.3.1 LUFENURON RESISTANCE IN INN5...... 167
5.3.2 CYP6G1 EXPRESSION LEVELS IN INN5 ...... 169
5.3.3 A NOVEL ACCORD INSERTION IN THE INN5 STRAIN...... 170
5.3.4 THE SPREAD OF ACCORD VARIANTS IN NATURAL POPULATIONS ...... 172
5.3.5 OTHER FACTORS CONTRIBUTING TO RESISTANCE IN WC2 AND INN5...... 175
5.3.6 RESISTANCE WITHIN CHROMOSOME III IN WC2 AND INN5...... 179 5.3.6.1 Inn5 mapping ...... 179 5.3.6.2 WC2 mapping ...... 182
5.4 DISCUSSION...... 184
5.4.1 ACCORD IN NATURAL POPULATIONS...... 184
5.4.2 CYP6G1 BASED RESISTANCE IN THE INN5 STRAIN ...... 185
5.4.3 CHROMOSOME III-ASSOCIATED RESISTANCE...... 187
5.4.4 THE FUNCTION OF THE CHROMOSOME III RESISTANCE GENES IN WC2 AND INN5 ...... 188
xii CHAPTER 6
SELECTION FOR LUFENURON RESISTANCE FOLLOWING EMS MUTAGENESIS...... 191
6.1 INTRODUCTION...... 192
6.1.1 NATURAL VERSES LAB-BASED SELECTION...... 192
6.1.2 EMS AS A MUTAGEN ...... 193
6.1.3 AIMS OF THIS PROJECT...... 194
6.2 MATERIALS AND METHODS ...... 195
6.2.1 FLY STRAINS AND MAINTENANCE ...... 195
6.2.2 MUTAGENESIS AND ISOLATION OF MUTANTS ...... 197 6.2.2.1 Mutagenesis protocol...... 197 6.2.2.2 Isolation and stabilisation of lufenuron resistant mutants ...... 197
6.2.3 DOSAGE MORTALITY CURVES ...... 199
6.2.4 MAPPING PROTOCOLS ...... 199
6.2.5 DETECTING ACCORD AND MEASURING THE EXPRESSION LEVELS OF CYP6G1.. 199
6.3 RESULTS ...... 200
6.3.1 ISOLATION OF EMS INDUCED MUTANTS ...... 200
6.3.2 ACCORD AND OVEREXPRESSION ...... 200
6.3.3 DOSAGE MORTALITY STUDIES ...... 203
6.3.4 MAPPING TO A CHROMOSOME ...... 206
6.3.5 MAPPING WITHIN A CHROMOSOME...... 214
6.4 DISCUSSION...... 217
6.4.1 MUTAGENESIS AND ISOLATION OF RESISTANT MUTANTS ...... 217
6.4.2 RESISTANCE LEVELS...... 219
6.4.3 INTER-CHROMOSOME MAPPING ...... 219
6.4.4 INTRACHROMOSOMAL MAPPING...... 221
6.4.5 FUTURE WORK ...... 221
xiii CHAPTER 7
DISCUSSION AND CONCLUDING REMARKS ...... 223
7.1 INTRODUCTION...... 224
7.2 THIS INVESTIGATION ...... 226
7.3 OPTIONS FOR RESISTANCE TO LUFENURON...... 228
7.3.1 RESISTANCE ASSOCIATED WITH CYP12A4 ...... 229
7.3.2 RESISTANCE ASSOCIATED WITH CYP6G1...... 231 7.3.2.1 Is Accord causing Cyp6g1 overexpression?...... 232 7.3.2.2 How did Cyp6g1 overexpression evolve?...... 233 7.3.2.3 Why is Accord frequency so high and why has it persisted? ...... 234
7.3.3 OTHER GENES ASSOCIATED WITH RESISTANCE ...... 235
7.4 IMPLICATIONS FOR PEST CONTROL...... 237
7.5 FINAL COMMENTS...... 239
xiv List of Figures
Chapter 1 Figure 1.1: The interactions between an insect and an insecticide leading to the death...... 6
Figure 1.2: The mechanisms of resistance to an insecticide...... 7
Figure 1.3: GSTs in the D. melanogaster genome aligned along polytene chromosomes..... 13
Figure 1.4: Esterases in the D. melanogaster genome, aligned along polytene chromosomes.
...... 15
Figure 1.5: Known cytochrome P450s of D. melanogaster, aligned along polytene maps..... 18
Figure 1.6: A proposed mechanism of diazinon resistance in Rutgers strain of M. domestica.
...... 23
Figure 1.7: Model of regulation of mammalian P450s such as Cyp1a1...... 30
Figure 1.8: The various types of transposable elements...... 36
Figure 1.9: Chemical structures of various insecticides ...... 42
Figure 1.10: Generalised structure of the insect cuticle ...... 45
Figure 1.11: The generalised pathway of chitin formation...... 47
Chapter 2 Figure 2.1: Vector maps of vectors used in PCR product cloning...... 67
Chapter 3 Figure 3.1: Genetic mapping cross showing the generation of gl e+ and gl+ e recombinants. 75
Figure 3.2: Cross used to generate DNA for sequencing candidate genes...... 76
Figure 3.3: Crosses used to determine which chromosome carries Cyp12a4 overexpression construct ...... 78
Figure 3.4: Crosses used to isogenise Cyp12a4 overexpression construct ...... 79
Figure 3.5: The gal4-UAS system...... 80
Figure 3.6: Crosses used to screen transgenic flies...... 81
Figure 3.7: p-UAST expression vector...... 86
Figure 3.8: a) Survival of Natural population strains at 1.6ppm lufenuron ...... 91
xv Figure 3.8: b) Survival of Natural population strains at 1.8ppm lufenuron ...... 92
Figure 3.8: c) Survival of Natural population strains at 2.1ppm lufenuron ...... 93
Figure 3.9: Re-screen of strains kept for further analysis...... 95
Figure 3.10: Dosage mortality curve for NB16 ...... 97
Figure 3.11: Polymorphisms used to map the lufenuron resistance gene in NB16...... 101
Figure 3.12: Molecular map of the region containing the putative resistance locus...... 102
Figure 3.13: The location of the Cyp12a4 sequence differences...... 105
Figure 3.14: The location of the Cyp12a5 sequence differences...... 105
Figure 3.15: Level of Cyp12a5 mRNA in NB16...... 107
Figure 3.16: Level of Cyp12a4 mRNA in NB16...... 107
Figure 3.17: Resistance in Cyp12a4 transgenics...... 109
Chapter 4 Figure 4.1: Genetic mapping cross using cn and vg phenotypic markers...... 121
Figure 4.2: The level of overexpression of Cyp6g1 in the WC2 strain...... 125
Figure 4.3: P450 cDNA microarray for WC2...... 126
Figure 4.3 (cont): P450 cDNA microarray data for Hikone-R...... 127
Figure 4.4: Dosage mortality curve for WC2, CanS, and Celera ...... 128
Figure 4.5: Mapping the lufenuron resistance gene in WC2 ...... 130
Figure 4.6: Recombinants obtained from mapping of the lufenuron resistance locus ...... 131
Figure 4.7: The interval containing a resistance-conferring locus in WC2 and Hikone-R..... 132
Figure 4.8 a) Positions of PCR primers and expected product sizes for Inverse PCR. b) 1.5% agarose gel ...... 136
Figure 4.9: a) Accord diagnostic PCR showing presence/absence of Accord for the strains from around the world...... 139
Figure 4.10: a) Molecular structural analysis of a full-length Accord element...... 141
Figure 4.11: Putative promoter sequences within the Accord element in the WC2 strain .... 142
Figure 4.13: Sequence of the upstream region of Cyp6g1 showing putative transcription factor binding sites...... 145
Figure 4.14: Lufenuron resistance in the progeny of UAS-Cyp6g1 transgenic strains ...... 147
xvi
Chapter 5 Figure 5.1: Chromosome mapping cross using the vg and e phenotypic markers...... 163
Figure 5.2: a) Genetic mapping crosses using ru, h, st, ry, and e phenotypic markers...... 165
Figure 5.3: Dosage mortality curve for the Inn5 strain ...... 168
Figure 5.4: Expression levels if Cyp6g1 ibn Inn5...... 170
Figure 5.5: a) Relative sizes of PCR products generated in the Inn5, WC2, and CanS strains
...... 171
Figure 5.5: b) the location and structure of the partial P-element that is inserted into Accord in the Inn5 strain...... 171
Figure 5.6: Overall Accord / Accord+P-element allele frequencies in natural populations of D. melanogaster from the east coast of Australia...... 172
Figure 5.7: Allele frequencies of Accord variants in natural population strains from the east coast of Australia ...... 174
Figure 5.9: Frequency of each mutant class ...... 180
Figure 5.10: Frequency of each phenotypic marker in WC2...... 182
Chapter 6 Figure 6.1: Genetic mapping crosses used to isolate EMS induced mutants...... 198
Figure 6.2: Cyp6g1 expression levels in EMS-generated lufenuron-resistant mutants ...... 202
Figure 6.3: Dosage mortality curves for EMS generated mutants ...... 204 case, which chromosome(s) contribute the bulk of resistance...... 207
Figure 6.4: a) Proportion of resistance contributed by each chromosome (1.8ppm) ...... 208
Figure 6.4: b) Proportion of resistance contributed by each chromosome (2.1ppm) ...... 209
Figure 6.4: c) Proportion of resistance contributed by each chromosome 2.3ppm) ...... 210
Figure 6.5: Intrachromosomal mapping within chromosome III...... 215
xvii List of Tables
Chapter 1
Table 1.1: Comparisons between mammalian and insect regulatory genes...... 31
Table 1.2: Comparisons between the various classes of transposable elements...... 37
Table 1.3: Mechanisms of resistance for various chemicals...... 43
Table 1.4: Various classes of insect growth regulators...... 49
Chapter 3
Table 3.1: Fly strains used in this investigation...... 73
Table 3.2: Primers and restriction enzymes used in molecular mapping...... 82
Table 3.3: Primers used in sequencing Cyp12a4 and Cyp12a5 ...... 85
Table 3.4: Primers used to generate overexpression construct...... 85
Table 3.5: Natural population strains of D. melanogaster ...... 88
Table 3.6: Lufenuron resistance levels in NB16...... 98
Table 3.7: Number of recombinants of each recombinant class ...... 99
Table 3.8: Candidate resistance genes within in the mapped region...... 103
Chapter 4
Table 4.1: Fly strains used in this study...... 120
Table 4.2: Primers and restriction enzymes used in molecular mapping...... 123
Table 4.3: Accord and other primers ...... 124
Table 4.4: Realtime RT-PCR primers...... 124
Table 4.5: Fold resistance estimates for WC2...... 129
Table 4.6: Cross-resistance levels in WC2 strain...... 133
Table 4.7: Cross-resistance levels in Hikone-R strain...... 134
Table 4.8: Susceptible and resistant strains used to test for the presence of Accord ...... 138
xviii Chapter 5
Table 5.1: Fly strains used in this study ...... 161
Table 5.2 Primers used in PCR and sequencing ...... 166
Table 5.3: Inn5 fold resistance with respect to CanS and Celera ...... 169
Table 5.4: Contributions of chromosome II and III to resistance...... 178
Table 5.5: Indicates the relative contributions of chromosomes III and II ...... 179
Table 5.6: Observed and expected numbers of flies in intrachromosomal mapping ...... 181
Table 5.7: Numbers of observed and expected mutants...... 183
Chapter 6
Table 6.1: Strains used (and generated) in this investigation ...... 196
Table 6.2: Lufenuron resistance in EMS strains...... 205
Table 6.3: Contributions of chromosome II and III to resistance...... 207
Table 6.4: The relative contributions of chromosome II and III for each EMS mutant ...... 212
Table 6.5: χ2 analysis examining the influence of resistant loci on chromosome III ...... 216
xix List of Abbreviations
AchE – Acetylcholine Esterase AHR – Aryl Hydrocarbon Receptor ARNT – Aryl Hydrocarbon Receptor Nuclear Transferase BPU – Benzoylphenyl urea CAPS – Cleaved Amplified Polymorphic Sequence CSI – Chitin Synthesis Inhibitor DDT – dichloro-diphenyl-trichloro-ethane Df - Deficiency EMS – Ethyl Methanesulphonate FB – Foldback element
GABAA – γ Amino Butyric Acid subtype A GST – Glutathione-S-Transferase IGR – Insect Growth Regulator JH – Juvenile Hormone JHA – Juvenile Hormone Analogue LC – Lethal Concentration LINE – Line element LTR – Long Terminal Repeat nAChR – nicotinic Acetylcholine receptor OP – Organophosphorous chemical P450 – Cytochtome P450 PB - Phenobarbitol PBO – Piperonyl Butoxide PTTH – Protoracicotropical hormone SUT - Sulfotransferase TE – Transposable Element TIR – Terminal Inverted Repeat UAS – Upstream Activatin Sequence UGT – UDP-glucuronosyltransferase XRE – Xenobiotic Response Element
xx
Chapter 1
Introduction
1.1 The importance of insecticide resistance
Synthetic chemical insecticides have greatly improved human health, agricultural production, and domestic congeniality in the last century, and they remain the principal weapon used in the control of insect pests. However, the selective pressures ensued on insects through the chemical stresses almost invariably result in the evolution of insecticide resistance. The consequences of resistance include the destruction of agricultural production systems and death of livestock, increased incidence of human diseases vectored by insects, and discomfort for the hosts of pests in the urban environment. The number of resistant species has exploded in the last half of the twentieth century and resistance has now been documented in over
500 species of insects (Hoy, 1998; The-Database-of-Arthropod-Resistance-to-
Pesticides, 2003).
Understanding pesticide resistance at the many different levels enables the formulation of techniques to slow, prevent or reverse resistance in pests and promote it in beneficial insects, enhancing biological control. It can provide the knowledge to reduce the detrimental effects of insecticides and increase their specificity and efficacy. Resistance also represents an example of rapid evolutionary adaptation, which can be understood at genetic, physiological, and ecological levels.
1.2 The “evolution” of insecticides
Early insecticides were often inorganic (such as HCN, lime sulphur.) They had multiple target sites, possibly reducing the probability of resistance evolving.
However they were almost as dangerous to humans as they were to their targets.
2 Environmental and health concerns have since slowed the development of new insecticides by about 6-10 years and drastically increased associated costs to around
$50-100 Million (Broadhurst, 1998). Furthermore, some new insecticides are still developed with very little knowledge of how they exert their lethal effect. Hundreds of thousands of chemicals are randomly screened for their insecticidal properties, and their protein targets are often only discovered as a consequence of the evolution of resistance. Others are variations on structural classes of known action. Their protein targets may have been discovered before resistance, and occasionally even before use in the field. However bad insecticide management practices, the historical lack of research tools or inability to apply research results to the field, the threat of cross- resistance, or the inevitability of resistance may have meant that susceptibility has often been shortlived.
A handful of insecticides are now developed with targets specifically in mind, principally resulting from both pure and applied insecticide research over the preceding years. Although this is a positive development, these insecticides may confront residual cross resistance from previous insecticide usage and its selective effects on the populations that were initially controlled by other chemicals.
1.3 The evolution of resistance
The World Health Organisation (WHO) defines resistance as “the inherited ability of a strain of some organism to survive doses of a toxicant that would kill the majority of individuals in a normal population of the same species” (WHO, 1957). Genetic theory predicts that novel traits such as those that might lead to resistance will evolve fastest when many loci, each of which has a small effect, determine the phenotype.
3 This theory is based on the assumption that a population consists of only susceptible phenotypes when first exposed to the insecticide, and that viability in the population is in a normal distribution and under polygenic control (McKenzie & Batterham, 1994;
McKenzie & Batterham, 1998). In the case where insecticidal concentration encountered by individuals is lower than the lethal concentration for the population
(LC<100), selection acts within this distribution and will predominantly elicit a polygenic response. Resistance may therefore arise through the action of a number of genes acting in concert.
Insecticide dosage is preferably chosen with the aim of killing every individual
(LC>100), such that selection is acting above the normal phenotypic distribution. In this case, a rare mutation that confers a selective advantage over the susceptible distribution cannot be accommodated by a polygenic response, and therefore the selection pressures preferentially screen for single genes of major effect (McKenzie,
1996a). The majority of documented resistance has arisen through this monogenic response (for review see (McKenzie & Batterham, 1998)).
Genetic based research into monogenic insecticide resistance and characterization of resistance genes represents an important method of identifying resistance mechanisms and implementing strategies to prolong the effectiveness of insecticides.
This type of research forms the bulk of this PhD investigation including subsequent results chapters. The remainder of this introduction focuses on the ways in which research is conducted in order to identify resistance mechanisms. Examples are drawn from the literature to highlight specific points that are pertinent to discussion in later chapters, and in some cases assorted aspects of the same example are given in different sections for their relevance to those particular sections.
4 In section 1.4, potential resistance mechanisms are introduced and the two major mechanisms, target site modification and metabolic resistance, are discussed in detail. Section 1.5 is a special subdivision devoted to the cytochrome P450s and their regulation, given that they are later shown to have major roles in the resistance described in chapters 3-6.
The molecular basis of resistance is also a focus of this thesis. Therefore, section 1.6 focuses specifically on the variety of genetic changes leading to resistance, discussing the role of transposable elements in particular. Dialogue in section 1.7 centres on the types of insecticide in use, concentrating mainly on insect growth regulators including lufenuron, the central insecticide in this investigation. What is known about the mode of action of lufenuron is discussed as a background to results and screening techniques. Finally in sections 1.8 and 1.9, the notion of Drosophila melanogaster as a model organism for resistance studies is considered, and the foci of the following chapters are foreshadowed.
5 1.4 Mechanisms of Resistance
Insecticides work through the chosen chemical exerting a lethal effect on the insect.
To do this, they must come into contact with the insect, be ingested by the insect, reach a specific target protein in the insect, and effectively bind to the target, in order to kill the insect. These interactions are shown in figure 1.1 below.
(2)
(1)
(3) (4)
Figure 1.1: Diagrammatic representation of the interactions between an insect (a fly larva having ingested red food dye) and an insecticide that leads to the death of the insect. The insecticide (shown in purple circles) needs to (1) come into contact with the insect, (2) be ingested by the insect, (3) reach its target site (shown in green semi-circles), (4) be able to effectively bind with the target site.
6 Resistance can therefore arise several different ways. The first is through a decreased exposure to the insecticide which could occur from (1) behavioural avoidance, (2) reduced uptake, or (3) increased detoxification of the insecticide.
Alternatively, it can arise through the various means of (4) target insensitivity. These mechanisms are represented in figure 1.2 below.
(2)
(1)
(3) (4)
Figure 1.2: Diagrammatic representation of the mechanisms an insect may use to enable it to become resistant to an insecticide. It may (1) exhibit a behavioural avoidance response allowing it to minimise insecticide exposure, (2) have reduced ingestion of the insecticide through decreased uptake, (3) have increased detoxification of the insecticide limiting the effective concentration of insecticide binding, or (4) have an altered target site such that the insecticide can no longer bind to its target.
7 Molecularly, resistance mutations are classified into those that lead to subsequent alterations in either catalysis or binding of the insecticide. This can occur through upregulation, or downregulation, or structural changes. Increased detoxification resistance is more commonly associated with upregulation and amplification of associated genes, and target insensitivity is most closely associated with structural mutations, but also with upregulation (Mallet, 1989). A more detailed presentation of the mechanisms of resistance is made below, and further discussion of the molecular basis of resistance-conferring mutations is presented in section 1.6.
1.4.1 Behavioural avoidance
Some behavioural characteristics that differ between resistant and susceptible insects have been seen in Anopheles gambiae (Rowland, 1991a; Rowland, 1991b) but the correlation with avoidance as a specific behavioural characteristic could not be made. Conversely avoidance has been documented in field populations of D. melanogaster (Pluthero & Threlkeld, 1981), and in Heliothis virescens (Sparks et al.,
1989) but its correlation with resistance could not be made. This mechanism of resistance appears to be of only minor importance.
1.4.2 Reduced penetration
Insecticides can penetrate an organism via the integument, respiratory system, or the gut. A change to any of these entry points could result in low levels of resistance, but due to the structural complexity of these entry points, a mutation leading to a change in a single biochemical component may rarely be enough to block penetration. Hence few studies have shown reduced penetration in isolation to be a major factor in
8 resistance. One classic example is found in Musca domestica where the pen gene provides 2-3 fold levels of resistance by lowering the penetration rate of insecticides through the cuticle (Plapp & Hoyer, 1968). It is only when combined with other resistance mechanisms that high levels of resistance are seen (Sawicki, 1970).
1.4.3 Target Site Resistance
If an insecticide is ingested by the insect, it then must reach its target to exert its lethal effect. It follows that if the target is altered in such a way that it can still carry out its normal function but can no longer bind the insecticide, the insect will become resistant. There are many documented cases of this type of resistance, but they fall into two main subgroups based on the modes of action of the insecticides. These are nervous system targets, and developmental targets.
1.4.3.1 Nervous system targets
Insecticides that attack the nervous system are desirable because they can produce a fast “knockdown” and can be insect specific. However resistance is common since only a single point mutation is needed in the insecticide’s binding site to render it useless. 60% of reported cases of insecticide resistance are accounted for by cyclodienes which are insecticides that target chloride ion channel receptors in the brain (ffrench-Constant et al., 1991). Several characteristics are almost always seen in association with cyclodiene resistance: a single semi-dominant gene (ffrench-
Constant et al., 1990), cross resistance to the γ-amino butyric acid (GABA) receptor agonist picrotoxinin (Kadous et al., 1983) and, when it has been tested, central nervous system insensitivity to cyclodienes and picrotoxinin (Bloomquist et al., 1992).
9 In D. melanogaster, the Rdl gene confers resistance to the cyclodiene dieldrin.
Dieldrin binds to the γ-amino butyric acid subtype A (GABAA) receptor, and a mutation leading to a single amino acid change (ala302-ser) in this receptor reduces its sensitivity to dieldrin whilst maintaining an effective interaction with the natural ligands (ffrench-Constant et al., 1993). Rdl is found on chromosome IIIL in D. melanogaster and the equivalent positions of chromosomes V and IV in dieldrin- resistant Australian sheep blowfly, Lucilia cuprina, and housefly, Musca domestica, respectively (Oppenoorth, 1985; ffrench-Constant, 1994; Hughes & McKenzie, 1987;
Adcock et al., 1993; McKenzie & Batterham, 1998; Foster et al., 1981). In addition to these species and the closely related Drosophila simulans, the same mutation in Rdl orthologues is also found in the less closely related yellow fever mosquito Aedes aegypti, the American cockroach Periplaneta Americana, the red flour beetle
Tribolium castaneum (ffrench-Constant, 1994) and the cat flea Ctenocephalides felis
(Daborn et al., 2004).
At a population level, 58 dieldrin resistant strains of D. melanogaster from five continents showed the same mutation (Steichen & ffrench-Constant, 1994). Together this evidence of conservation of mutation across populations and species indicates it is the main mechanism of resistance to dieldrin.
For pyrethroid insecticides, a single amino acid substitution (leu1014-phe) in the voltage-gated sodium channel gene (knock down resistance) gene of M. domestica results in target site insensitivity, rendering the insect resistant due to a similar lack of insecticide-target interaction as observed for dieldrin and the Rdl gene (Williamson et al., 1996). In D. melanogaster, mutations in the orthologous gene, para, lead to pyrethroid resistance, again demonstrating a single amino acid change leading to resistance, and the conservation of molecular mechanism across species
(Pittendrigh et al., 1997).
10
Organophosphorous (OP) and carbamate insecticides bind to acetylcholinesterase
(AchE), leading to hyperexcitation and death (Soderlund & Bloomquist, 1989). In a field strain of D. melanogaster, a single amino acid change (phe368→thr) confers OP resistance (Morton & Singh, 1982; Fournier et al., 1992). It is likely that the amino acid replacement reduces the OP binding capacity of AchE while retaining a relatively normal capacity to hydrolyse the enzyme’s substrate, acetylcholine (Hama,
1983; Oppenoorth, 1985).
Four other mutations were later seen to confer low level resistance by themselves, and high level resistance in combination (Mutero et al., 1994). It was suggested in this case that resistant alleles recombined in natural populations, resulting in the accumulation of mutations leading to higher resistance (Mutero et al., 1994). The existence of several different resistant alleles was seen in other species such as C. pipiens, Leptinotarsa decemlineata, Bemisia tabaci, and M. domestica (Fournier &
Mutero, 1994; Devonshire et al., 1998).
1.4.3.2 Developmental targets
An insecticidal alternative to perturbing neurotransmission is to interrupt the growth and development of the insect. Juvenile hormone analogues (JHAs) such as methoprene mimic endogenous hormones, causing a hormonal imbalance leading to death. Mutations in the Met (Methoprene-tolerant) gene in D. melanogaster lead to a complete loss of Met protein product. Survival of homozygous Met mutants lead
Wilson and Ashok to believe that Met encodes a non-vital Juvenile hormone receptor protein, and its loss reduces the hormone titre, resulting in resistance (Ashok et al.,
1998; Wilson & Ashok, 1998). This is in contrast to other insecticidal targets that
11 encode proteins vital for survival (ffrench-Constant, 1994). The low levels of resistance seen in comparison to other target site resistances may be explained by the presence of multiple methoprene targets whilst only one confers resistance
(Wilson & Ashok, 1998).
1.4.4 Metabolic Resistance
If a sufficient concentration of insecticide is broken down before it reaches its target, resistance will ensue regardless of the nature of the target. Most organisms, including insects, have evolved detoxification genes specifically for the purpose of surviving the onslaught of xenobiotic compounds that they are exposed to. The detoxifying enzymes are encoded by supergene families consisting of the transferases, the hydrolases, and the cytochrome P450s. The transferases and hydrolases are responsible for significant cases of insecticide resistance, but the cytochrome P450s are discussed in more detail because of their relevance to the resistance described in chapters 3-6.
1.4.4.1 Transferases
Transferases are a superfamily of detoxification enzymes whose role is to conjugate glutathione, sulfuric acid, or glucuronic acid to exogenous hydrophilic substrates, like insecticides or their toxic metabolites, facilitating their excretion (Gibson & Skett,
2001). Glutathione S-tranferases (GSTs) are the predominant family but the smaller families of sulfotransferases (SUTs) and UGP-glucuronosyltransferases (UGTs) play similar roles. There are over 25 families of GST or GST-like proteins with one major phylogenetic clade of GSTs conserved across mammalian, arthropod, helminth,
12 nematode, and molluscs (Snyder & Maddison, 1997). They are classified into two main groups, (class I and class II) based on their gene structure and amino acid sequence, although this classification does not extend to their substrate specificities which are wide and varied. They are often found clustered in the genome, as shown in figure 1.3 for the D. melanogaster genome.
Figure 1.3: GSTs in the D. melanogaster genome aligned along polytene chromosomes (C. Robin, Pers comm.)
13 Transferase involvement in insecticide resistance has mainly been associated with increased detoxification of OPs in M. domestica (Ottea & Plapp, 1984; Clark et al.,
1986; Syvanen et al., 1994), although some reports have also implicated GSTs in pyrethroid resistance (Vontas et al., 2001; Vontas et al., 2002). Recently, several
GSTs that were overexpressed in DDT-resistant strains of Anopheles gambiae were shown to be able to metabolise DDT (Ranson et al., 2001; Ortelli et al., 2003). In many cases, upregulation of one or more GSTs in resistant insects appears to be due to a trans-acting regulator (Grant & Hammock, 1992a; Ranson et al., 2000).
The transferase detoxification mechanism often acts as cooperatively a secondary resistance mechanism in conjunction with a P450 or hydrolase-based resistance systems (Hemingway et al., 1991). In a two phase process, P450s introduce a functional group (mainly a hydroxyl group – see section 1.5), and protein-protein interactions allow the metabolite formed by the phase I enzyme to move to the catalytic site of the transferase without being released from the protein complex. In phase II, the transferase catalyses the conjugation of its bulky substituent molecule such as glutathione to the functional group (Gibson & Skett, 2001). This cooperative enzyme system catalyses detoxification reactions consisting of multiple steps more rapidly and efficiently than independent mechanisms and is therefore of importance in insecticide resistance.
1.4.4.2 Hydrolases
Hydrolases are the second superfamily of detoxification enzymes, encompassing thio-, phosphor-, carboxyl- and choline-esterases and various other hydrolases which add their respective functional groups to esters in contributing to detoxification of xenobiotics like insecticides (Ollis et al., 1992). Many hydrolases are believed to use
14 a two-step reaction mechanism based on a ‘‘catalytic triad.” In the first step of the reaction, an alcohol functional group of the substrate is liberated and forms a covalent linkage to the active site of the hydrolase, and the second step cleaves this linkage, resulting in a hydrolysed compound (Ollis et al., 1992; Oakeshott et al.,
1999). There are 37 of the most significant group of hydrolases, the carboxylesterases, in the D. melanogaster genome that are grouped into clusters
(figure 1.4).
Figure 1.4: Esterases in the D. melanogaster genome, aligned along polytene chromosomes (C. Robin, Pers comm.).
15
Pyrethroids, OPs and carbamates are all substrates for carboxylesterases, and esterase-based resistance can be established through either sequestration or metabolism (Karunaratne et al., 1995). The majority of esterases which function by sequestration are elevated through gene amplification in resistant insects
(Devonshire & Field, 1991; Vaughan & Hemingway, 1995); section 1.6.3) and many of those that act through metabolism confer resistance via point mutations. In Myzus persicae for example, gene amplification of esterases E4 and FE4 is responsible for
OP and carbamate resistance (Field & Devonshire, 1998), and in L. cuprina, a single gly137→asp substitution of the carboxylesterase E3 confers broad cross resistance to a range of OP insecticides by altering its activity from a carboxylesterase to an OP hydrolase (Newcomb et al., 1997). The same substitution has been found in the αE7 esterase of resistant M. domestica (Claudianos et al., 1999).
1.5 Cyrochrome P450s
P450s are the third main metabolic system. They are extremely developmentally important because of their involvement in endogenous functions such as the synthesis of hormones, fatty acids, and steroids. They are discussed here as an entirely separate section due to their relevance to results obtained in later chapters.
Induction of one or more P450s can produce changes in development through disruption of hormone titres (Darvas et al., 1992; Fuchs et al., 1993). In fact, they are closely involved in the synthesis and degradation of key developmental hormones juvenile hormone and 20-hydroxyecdysone (Agosin, 1985). They are detected in a wide range of tissues (Hodgson, 1985) and at varying times of development (Agosin,
1985).
16
The other striking feature of P450s is that they are also involved in catabolism and anabolism of many types of xenobiotics such as drugs, pesticides, and plant toxins.
This ability presumably evolved through the plant-animal warfare of the last 800 million years where plants synthesised chemicals that are difficult to metabolise, and animals evolved new P450s to adapt to the altered environment (Nelson & Strobel,
1987; Gonzalez & Nebert, 1990).
1.5.1 Structure and organisation
P450s are characterised by a distinctive absorbance peak at 450nm (Omura & Sato,
1964) and can be identified by a characteristic sequence in the heme-binding region of the gene product (FXXGXXXCXG). They essentially catalyse redox reactions of the kind shown below, requiring several cofactors to do so.
+ + Substrate + NADPH + H + O2 → Substrate-O + NADP + H2O
They have been named based on overall amino acid sequence similarity (Nebert et al., 1987; Nelson et al., 1996). Analysis of the D. melanogaster genome has revealed
90 P450s comprised of 83 functional genes and 7 pseudogenes (see figure 1.5).
These are generally in clusters of genes falling into the same subgroups, a likely result of the duplication from an initial ancestral gene and adaptive diversification resulting from the plant-animal warfare previously mentioned.
17 Figure 1.5: Known cytochrome P450s of D. melanogaster, aligned along polytene maps (adapted from (The-Drosophila-P450-site, 2001)).
18 1.5.2 Diversity and Substrate Specificity
The many different P450s in an organism’s genome have a large variation in substrate specificity, most probably in order to carry out such varied endogenous tasks and to be prepared for exogenous compounds that are encountered. Some heavily studied human P450s such as CYP3A4 have a role in metabolism of over
60% of drugs in current clinical use (Guengerich, 1999; de Wildt et al., 1999). Others have overlapping substrate specificity such that a range of P450s are able to metabolise a single substrate (for example the Cyp2c subfamily in humans (Rendic &
Di Carlo, 1997)). Their nature is such that a single amino acid change can alter their substrate specificity significantly, as seen in the case of the mouse P450coh (Lindberg
& Negishi, 1989). P450 genes are developmentally regulated but nevertheless have malleable expression patterns (Arbeitman et al., 2002). Pressures imposed by compounds such as insecticides make selection for the naturally occurring beneficial mutations that arise to increase in frequency in populations.
1.5.3 P450s and Insecticide Resistance
In susceptible strains, P450-mediated metabolism limits the usefulness of some insecticides such as pyrethrins (Sawicki, 1962), carbaryl (Wilkinson, 1967), and imidacolprid (Wen & Scott, 1997). But more importantly, P450-based resistance forms the most frequent type of metabolic resistance (Scott, 1999) and has had an overwhelmingly devastating effect on the effectiveness of insecticides in general. It is now known, for example, that resistance can occur due to detoxification via a single
P450, and that metabolic attack can be limited to a single site on the insecticide
(Zhang & Scott, 1994).
19 The importance of P450-based resistance first became evident when carbaryl resistance was shown to be abolished by the P450 inhibitor sesamex (Eldefrawi et al., 1960). It was later realised that detoxification by P450s has the potential to confer cross-resistance to other insecticides (Oppenoorth, 1985), and that P450s may also be responsible for the bioactivation of insecticides (Levi et al., 1988).
1.5.4 Mechanisms of P450-based resistance
Whilst their role in exogenous metabolism is a protective one, P450s will metabolise foreign compounds irrespective of the eventual fate of the metabolites. So the required bioactivation of some insecticides enables resistance to develop through inhibition of P450s (Kayser & Eilinger, 2001), but resistance more commonly develops through increased detoxification. This can occur through a change in catalytic activity of the P450 responsible for metabolism of the insecticide such that the resultant effect is a higher turnover of the insecticide (Feyereisen et al., 1995).
Alternatively it can occur through a change in its expression level or its expression profile (Feyereisen, 1999). Increased expression and/or changes in spatial or developmental expression patterns are typically due to increased transcription, gene amplification, mRNA stabilisation, or stabilisation of the protein. A closer examination of these phenomena and how they can be assessed both molecularly and genetically is demonstrated with the following examples.
20 1.5.4.1 LPR strain of M. domestica
The Learn pyrethroid resistant (LPR) and Rutgers strains of M. domestica demonstrate the use of molecular and genetic techniques in identifying P450- associated resistance-conferring mechanisms, and highlight several characteristic features of P450s which will be discussed in following sections.
LPR was isolated from a New York dairy and was found to exhibit P450-mediated resistance to the pyrethroids permethrin (Scott & Georghiou, 1986), deltamethrin
((Wheelock & Scott, 1992), and cypermethrin (Zhang & Scott, 1994). It was shown in vitro that permethrin resistance can be suppressed from 5900 fold to 32 fold by the
P450 inhibitor piperonyl butoxide (PBO) (Scott & Georghiou, 1986), and in vivo that there was enhanced metabolism of deltamethrin and cypermethrin compared to the susceptible strain (Wheelock & Scott, 1992).
The chromosome I P450 Cyp6d1 (initially known as P450lpr) was implicated by the discovery of an overrepresented protein band following HPLC purification of this
P450lpr protein (Scott & Lee, 1993). The gene was sequenced with degenerate primers based on known P450lpr sequences (Tomita & Scott, 1995). Cyp6d1 mRNA was also overrepresented on a Northern Blot (Liu & Scott, 1996). Southern blots of gDNA with a cDNA probe showed similar levels of amplification, indicating that the elevated level of CYP6D1 expression is not from gene amplification (Tomita & Scott,
1995). Similarly, transcription of Cyp6d1 in both the LPR and control strains decreased over time following injection of actinomycin D (a transcription inhibitor), showing that increased expression is not due to increased stability of the mRNA (Liu
& Scott, 1998). The relative transcription rate was measured using an in vitro run-on
21 assay, indicating the increased transcription of Cyp6d1 as a mechanism of resistance in this strain (Liu & Scott, 1998).
1.5.4.2 Rutgers strain of M. domestica
The Rutgers strain of M. domestica was collected after 5 years of diazinon use and exhibits 120 fold resistance to diazinon, and cross resistance to many other insecticides (Forgash et al., 1962). It exhibits about 2.4 fold more total P450 content than a susceptible strain (Folsom et al., 1970).
Cyp6a1 was the first P450 cloned from an insect based Northern blot analysis of this strain, showing that it conferred three fold higher expression (Feyereisen et al.,
1989). Interestingly, the LPR strain also showed higher Cyp6a1 expression levels
(Carino et al., 1992). Heterologous expression showed that it detoxified diazinon
(Feyereisen, 1999) Southern blots indicated that the increased expression of Cyp6a1 was not due to gene amplification (Carino et al., 1994)
Cyp6a1 was mapped to chromosome V (Cohen et al., 1994; Feyereisen et al., 1995).
However crosses to an underproducing strain sbo established that the factor responsible for higher expression in both the Rutgers strain and the LPR strain was linked to chromosome II (Carino et al., 1994; Feyereisen et al., 1995). Induction by the potent P450 inducer phenobarbitol is linked to chromosome II (Liu & Scott, 1997) indicating that a trans-acting factor may be also responsible for induction.
Interestingly, the involvement of the αE7 ali-esterase gene via a single amino acid change was also seen in Rutgers (Claudianos et al., 1999). Diazinon resistance and
CYP6A1 overproduction are genetically linked to this loss-of-function mutation. A
22 speculative model was proposed whereby ali-esterase activity regulates the chromosome II trans-acting factor, which negatively controls Cyp6a1. In resistant flies, the loss of ali –esterase decreases the activity of a repressor leading to overexpression of Cyp6a1 and resistance (Sabourault et al., 2001), figure 1.6. This model also validates the action of the trans-acting factor in upregulating other P450s such as Cyp12a1 in this strain (Guzov et al., 1998).
(1) Mutation
(2) Decreased titre of transctiption factor
(3) Less binding and repression
(4) Overproduction of Cyp6a1 and other p450s
(5) Enhanced diazinon metabolism
Figure 1.6: A proposed mechanism of diazinon resistance in Rutgers strain of M. domestica. (1) A Mutation in the αE7 ali-esterase gene alters its gene product. (2)
This decreases the titre of a negatively-regulating trans-acting factor. (3) Less transcription factor binding occurs. (4) Cyp6a1 is overproduced. (5) Its gene product is able to metabolise more diazinon, making the insect resistant.
23
This model was later hypothesised as unlikely based on non-conservation of the ali- esterase E7 between a range of resistant and susceptible strains that overexpress
P450s (Scott & Zhang, 2003). But in the Rutgers strain, an alternate hypothesis suggests that Cyp6a1 may be responsible for activation of diazinon to its active form diazoxon, and eli-esterase E7 may be involved in detoxification of the diazoxon, thus constituting the major resistance mechanism and explaining the genetic linkage. This ongoing study highlights the complex nature of the regulation of P450s and the interaction between P450s and other detoxification enzymes.
1.5.4.3 91-R strain of D. melanogaster
Following continual selection by DDT between 1952 and 1983, the 91-R strain of D. melanogaster was found to be 70 times more resistant to DDT (Dapkus & Merrell,
1977) and 100 times more resistant to malathion (Sundseth et al., 1989) than its control 91-C. The chromosome II P450s Cyp6a2 and Cyp6a8 have been implicated through high expression of mRNA and/or protein levels (Sundseth et al., 1989;
Waters et al., 1992; Maitra et al., 1996; Dombrowski et al., 1998). Heterologous expression of Cyp6a2 in baculovirus infected cells and accompanying oxidative cleavage of diazinon, aldrin, and heptachlor indicated that the gene product could detoxify these insecticides (Dunkov et al., 1997; Berge et al., 1998). Inducibility (Brun et al., 1996; Maitra et al., 1996), and toxicological tolerance correlating with the level of expression of these genes further implied their involvement (Bride et al., 1997).
The genetic basis of the overexpression patterns was examined by creating F1 hybrids of the 91-R and 91-C (or an alternate low expressing strain ry506) and it was found that expression levels were far less than half that of 91-R, despite having one
24 copy of the overproducing 91-R genomes (Maitra et al., 1996; Dombrowski et al.,
1998; Maitra et al., 2000). This suggested that the 91-C strains carry genes that down-regulated Cyp6a2 and Cyp6a8 expression. Strains homozygous for chromosome II 91-R (and therefore homozygous for Cyp6a2 and Cyp6a8 of 91-R) and chromosome III ry506 were created. Expression levels were significantly lower than those of the original 91-R strain. When a strain carrying homozygous 91-R chromosome III was substituted back in, expression levels increased to the original levels. Taken together, this suggested that the chromosome II genes Cyp6a2 and
Cyp6a8 genes are trans-regulated by genes on chromosome III, and that the normal function of these genes is to repress Cyp6a2 and Cyp6a8. It also suggests that overexpression is likely to be due to a mutation in the repressor genes rather than in cis-regulatory sequences of Cyp6a2 and Cyp6a8 (Maitra et al., 2000).
1.5.5 Characterisation of P450-mediated resistance
The above examples emphasise that there are several important indicators of the involvement of P450s with resistance, and that there are also several criteria for demonstrating their role in mediating resistance. There are different approaches, both molecular and genetic, that can be used to examine more closely the mechanisms, and the new challenges now faced based on the in-depth information that can now be retrieved using these approaches. These will be discussed in turn.
25 1.5.5.1 Indicators of P450 involvement
All of the examples above show a pattern of cross resistance. This is the first important indicator of P450 involvement. It strongly suggests a detoxification-based mechanism because the alteration of a target is unlikely to confer resistance to chemicals of different structures. The second indicator described in the M. domestica examples is the alternative effect of P450 inhibitors and inducers in conferring resistance. Any change in resistance status as a result of their addition is highly indicative of the involvement of P450s. A more specific indicator is the upregulation of specific P450(s). Increased detoxification can be due to a change in catalytic activity of the P450 or, more commonly, to a change in expression level of the P450 as the above examples show. These indicators provide a platform for further investigation, but there are several criteria required to unequivocally demonstrate
P450 involvement in resistance.
1.5.5.2 Criteria for demonstrating P450 involvement
The examples discussed provide two criteria for concluding unequivocally that a
P450 is involved in resistance (Scott, 1999). Firstly, the P450 must be shown to detoxify (or sequester) the compound to which the strain has resistance. Enhanced metabolism of deltamethrin and cypermethrin by CYP6D1 in the LPR strain exemplifies this criterion. Secondly, the resistant strain should have a greater amount of this P450, or the protein encoded by the resistant allele should have a greater catalytic activity, compared to the susceptible allele. In the examples discussed above, both mRNA and protein levels of the respective P450s were shown to be higher in the resistant strains.
26 There are further criteria for demonstrating the “type” of P450 involvement. It was briefly mentioned in section 1.4.3 that increased expression could be due to increased transcription, gene amplification, mRNA stabilisation, or stabilisation of the protein. As the LPR example shows, each of these issues was addressed in turn to show that increased transcription of the Cyp6d1 gene was the source of the increased expression. It is likely that gene amplification plays little role in P450 overexpression since no examples have been described (Berge et al., 1998)
1.5.5.3 Summary of P450s
The D. melanogaster example above shows that whilst molecular biology is the key to showing that resistance is due to P450s, it is the combination of fundamental genetics and molecular biology that increases our understanding of a particular mechanism of resistance and opens new avenues for further study.
It is now clear that the involvement of regulatory changes in P450-mediated resistance is an important discovery in the systems in these examples. Regulation of transcription can occur in cis or in trans (Liu & Scott, 1998) so it is possible that insecticide resistance could map to a location that is different from the p450 gene involved in detoxification because of a mutation in a regulatory gene, rather than a mutation in the P450 structural gene or its 5’ regulatory sequences. In the Rutgers and LPR strains, the transacting factor on chromosome II has been proposed to be a master regulatory factor (Plapp, 1984). The αE7 ali-esterase gene was also seen to play a role, although this role has not been clearly defined (Sabourault et al., 2001;
Scott & Zhang, 2003).
27 A similar phenomenon has also thought to be occurring in the mosquito Anopheles gambiae (Hemingway et al., 1998). Mutations affecting trans regulators may also have an impact on the expression of other p450s not involved in resistance (Scott,
1999). Therefore, some of the p450s overexpressed in a resistant strain may not be involved in resistance. Thus, knowing which subset of P450s is elevated by the same regulatory factors may help understand cross-resistance patterns and mechanisms of resistance.
It is also interesting to note that chromosome II in M. domestica, and chromosome
IIIR in D. melanogaster (containing the putative regulatory factor in the 91-R strain) are syntenic units (Foster et al., 1981). Moreover, the αE7 ali-esterase gene discussed with respect to M. domestica was first cloned in L. cuprina (Newcomb et al., 1997; section 1.4.4.2). Indeed the same amino acid substitution was seen to have a role in resistance to OPs in both species. These types of similarities are important in validating the usefulness of inter-species comparisons and of D. melanogaster as a model organism (discussed further in section 1.8).
1.5.6 Regulation of P450s
A brief review of p450 regulation presented below exemplifies the complex nature of the cis and trans acting factors that may be involved in changes in expression of
P450s in response to insecticides, and the mutations that could result in resistance.
The roles of specific transcription factors involved in P450 regulation, such as the basic–helix–loop–helix-PAS (bHLH–PAS) proteins, have been best studied in mammals (Ramana & Kohli, 1998). bHLH–PAS proteins control a variety of developmental and physiological events including neurogenesis, tracheal and
28 salivary duct formation, circadian rhythms, hormone receptor function and toxin metabolism (hence their role in P450 regulation) (Swanson & Bradfield, 1993;
Hankinson, 1995; Whitlock et al., 1996; Rowlands & Gustafsson, 1997; Mimura &
Fujii-Kuriyama, 2003). The bHLH–PAS regulatory cascade regulates expression of mammalian P450s (rat and mouse CYP1A1, human CYP1A2, hamster CYP2A8) in response to aryl hydrocarbons, a highly toxic class of pollutants produced by the combustion of fossil fuels, and more generally, a structural class containing aromatic groups (Whitlock, 1999).
In the mammalian system, the process works as described below. In the absence of a ligand, the aryl hydrocarbon receptor, (AHR - a member of the bHLH–PAS regulatory cascade), is present in the cytosol in a complex with Hsp90 and other proteins. These proteins are thought to keep the unliganded AHR in a state responsive to ligand binding. Upon binding to a ligand such as a dioxin, the AHR complex translocates into the nucleus and the AHR dissociates from the Hsp90 complex to form a heterodimer with its partner molecule, ARNT (aryl hydrocarbon- receptor nuclear transferase) (Ma, 2001). The formed AHR/ARNT heterodimer enters the nucleus, binding to a xenobiotic response element (XRE) sequence
T(A/T)GCGTG (located at approximately -1100 in the promoter region in the case of the Cyp1a1 gene (Sogawa et al., 1986)), resulting in the enhanced expression of the gene (Denison et al., 1988).
The product of another gene, termed the AHR repressor (AHRR), localises in the nuclei and forms a heterodimer with ARNT constitutively. The AHR/ARNT heterodimer also recognizes the XRE, acting as a negative regulator of AHR by competing with AHR for ARNT heterodimer formation and binding to the XRE sequence. The promoter region of the AhRR gene contains three copies of functional
XREs, making it inducible in an AHR-dependent manner, forming a regulatory
29 feedback loop (Mimura & Fujii-Kuriyama, 2003). A summary of this process is shown in figure 1.7.
LIG
0 9 AHR p cytoplasm s H
0 9 p nucleus s H LIG
T N AHR R A XRE Gene such as Cyp1a1 or AhRR
T N AHRR R A
Figure 1.7: Model of regulation of mammalian P450s such as Cyp1a1, adapted from
(Mimura & Fujii-Kuriyama, 2003).
30 Homologous genes to AhR and Arnt have been found in insects (Duncan et al., 1998;
Crews, 1998; Emmons et al., 1999; Sedaghat et al., 2002); table 1.1), and the high sequence and functional conservation between species studied so far suggests that the mechanisms of transcription factor function in P450 regulation may be generally conserved.
Table 1.1: Comparisons between mammalian and insect regulatory genes
Sequence Functional Mammalian Insect Reference homology homology Spineless (ss) (Duncan et al., AhR (and others) Yes Yes (and others) 1998) (Sonnenfeld et Arnt Tango (tgo) Yes Yes al., 1997) AhR:Arnt ss:tgo (Emmons et Yes heterodimer heterodimer al., 1999) XRE XRE/DRE Yes (T(A/T)GCGTG) (GCGTG)
1.6 Molecular mechanisms of resistance
Moving away from the types of mechanisms that are capable of causing resistance, discussion in this section now turns to how particular types of mutations can manifest changes in those mechanisms. Transposable elements as insertional mutators are emphasised since they have supreme relevance in later chapters.
Prior to the introduction of an insecticide, the frequency of resistant alleles will be determined by the mutation rate towards the resistant alleles and any fitness cost associated with them. Theoretically, there is always a pool of alleles at specific loci that are capable of conferring resistance.
31
In the presence of insecticidal selection, there is a shift in balance. Fitness costs associated with the resistant alleles can be overwhelmed by the fitness advantage conferred through resistance. The mode of action of the insecticide, its chemical similarity to other natural and xenobiotic compounds in the environment that it contacts, the intensity of the selection, and the frequency with which it is encountered all contribute the rate at which its frequency is augmented. The type of mutations seen in resistant field populations of insects in part depends on the mechanisms of resistance available for any given insecticide. These various types are therefore discussed where appropriate with reference to examples in section 1.4.
1.6.1 Point mutations
Target site-based resistance relies on minor changes to the target protein and it is therefore not surprising that the examples discussed in section 1.4.3 (ffrench-
Constant, 1994; Mutero et al., 1994; Williamson et al., 1996) highlight the pivotal role of point mutations in conferring resistance .
The specificity of a point mutation such that biological activity is maintained whilst insecticide-binding is reduced suggests that few mutations are capable of satisfying both of these requirements (Taylor & Feyereisen, 1996). This is seen in Rdl where absolute conservation of the resistance conferring mutation is seen in different populations and even different species (ffrench-Constant, 1994). It is suggested that this mutation not only alters the insecticide binding site but destabilises the insecticide’s preferred receptor conformation, the combination of which is necessary to confer sufficient resistance to be selected in the field (ffrench-Constant et al.,
1998).
32
In the para gene of D. melanogaster, mutations induced elsewhere in the gene conferred some resistance but were not seen in the wild, suggesting that if they have previously arisen in natural populations, the fitness of individuals carrying such alleles is too low relative to the resistance benefit to allow the alleles to approach any appreciable frequency (Pittendrigh et al., 1997; Wilson, 2001). In the case of the
AChE structural gene, various mutations confer resistance but at low levels (Mutero et al., 1994). Some resistant mutants carried combinations of these alleles, but none carried all five as would be expected under selection pressure in the absence of fitness costs. This again implies the need for low fitness costs in order for a point mutation-based resistance mechanism to be successful.
A role for point mutations in conferring resistance is confirmed by induction of resistant mutants from susceptibles using chemical mutagens (Greenleaf et al., 1979;
Wilson & Fabian, 1986; Smyth et al., 1992; McKenzie et al., 1992; Adcock et al.,
1993; McKenzie & Batterham, 1998; Daborn et al., 2000). The use of chemical mutagenesis in predicting and confirming insecticide resistance mechanisms is discussed further in section 6.1.
1.6.2 Gene amplification
The increase in gene copy number leading to overexpression is a well characterised mechanism of carboxylester hydrolase (or esterase; see section 1.4.4) - mediated resistance. In the mosquito Culex pipiens, two closely linked esterase genes are seen to confer resistance to OPs through amplification of one or both genes
(Mouches et al., 1986; Raymond et al., 1989; Poirie et al., 1992; Gullemaud et al.,
1997; Vaughan et al., 1997). The esterase gene B1 is amplified in resistant Culex
33 quinquefasciatus by as much as 250-fold, leading to an esterase protein that comprises as much as 10% of the soluble protein of the insect (Mouches et al., 1986;
Karunaratne et al., 1993).
C. quinquefasciatus mosquitoes also show co-amplification of esterases α2 and β2 of
80-fold in a resistant strain. However there is also differential transcription with a ratio of 10 : 1 for the α2 : β2 transcripts, and a protein level ratio of 3 : 1. This indicates subsequent transcriptional and translational control mechanisms in regulation of the expression of these amplified genes in insecticide-resistant mosquitoes (Paton et al.,
2000).
Similarly in the peach-potato aphid Myzus persicae, gene amplification is accompanied by DNA methylation as a mechanism of transcriptional control for the resistance conferring esterase E4, greatly decreasing the amount of protein relative to gene copy number (Field & Devonshire, 1998; Field et al., 1999). In comparison with the duplicated gene FE4 of the same strain, the two genes are regulated in different ways. FE4 has sequences corresponding to a conventional promoter (TATA box and CAP site) that are not present in E4; on the other hand, FE4 lacks the CpG island present 5' of E4 genes that effect the DNA methylation. The differences are likely to be due to the nature of the duplication event that gave rise to E4 and FE4 leading to different 5' sequences (Field & Devonshire, 1998; Field et al., 1999; Field,
2000).
Thus it is important to note that when an increase in gene copy number is associated with resistance, gene amplification is only part of the pathway leading to overproduction of stable protein. Accompanying regulatory mechanisms are also
34 important in determining the final outcome with respect to protein production and, subsequently, resistance.
1.6.3 Chromosomal abnormalities
Small deletions, insertions, inversions and translocations within a gene usually result in large changes in the structure of a protein and are not usually well-tolerated events in vital genes (Taylor & Feyereisen, 1996). These types of mutations are more likely to be associated with regulatory regions where their mutagenic effect may perturb the spatial and temporal pattern of expression. Most spontaneous mutations seen in D. melanogaster are caused by the insertion of transposable elements (Finnegan &
Fawcett, 1986). This raises the possibility that transposable element insertions may be frequently associated with resistance, at least in this species (Wilson, 1993).
1.6.3.1 Transposable elements
Transposable elements (TEs) are DNA sequences that are capable of movement
(transposition) within the genome. They are found in virtually all eukaryotes and represent 3.86% of the D. melanogaster eukaryotic genome (Kaminker et al., 2002).
They are preferentially found outside genes and are often found as deleted elements or with other elements nestled inside them (Kaminker et al., 2002).
Most TEs are divided into two subtypes that are distinguished by their mode of transposition (Finnegan, 1992). The class I elements, or retrotransposons, replicate via an RNA intermediate, using an internally coded reverse transcriptase to copy
DNA from the RNA intermediate. They are internally categorised depending whether
35 they have characteristic long terminal repeats (the LTR type) or not (LINE type). The
class II elements, or transposons replicate via DNA excision and repair and generally
have terminally inverted repeats (TIRs) or are of the foldback (FB) type. Some of the
general characteristics of these elements are shown in figure 1.8 and table 1.2.
a) R R
TI Transposase TI
b) LIR NR-DNA LIR c)
LTR GAG POL ENV LTR
d) DNA binding Protease Reverse Transcriptase RNAseH Integrase
UTR GAG POL Poly-A
Figure 1.8: Representations of the various types of transposable elements. a) P-
element type transposons have short terminal inverted repeats (TIRs) at each end
surrounding a transposase gene. b) Foldback-type elements have long inverted
repeats (LIRs) at each end and sometimes a central non-repetitive DNA (NR-DNA)
section. c) In LTR-type retrotransposons, the central portion contains gag (coat
protein), pol (the various subunits shown) and in some cases, env (outer envelope)
genes. The LTRs are typically 400-500bp and the central portion is usually 4.5-9kb.
d) Line type retrotransposons have no LTR but a large 5’UTR and variable poly-A tail
at the 3’ end.
36 Table 1.2: similarities and differences between the various classes of transposable elements.
Transposons Retrotransposons Feature P-element FB element LTR-type LINE-type 31bp Repeats None 4-500bp terminal none inverted Duplication of Host 4bp 4bp Usually 8bp sequence DNA DNA Mechanism of RNA RNA excision excision Replication intermediate intermediate & repair & repair
Despite Wilson’s prediction (Wilson, 1993) being soundly based on the D. melanogaster mutation literature, only two examples of transposable element- mediated resistance have been described (Daborn et al., 2002; Schlenke & Begun,
2004); Chapter 4). A more thorough analysis of the potential for insertional mutagenesis by transposable elements is therefore made using other examples from other genes not associated with resistance.
Transposable elements can result in gene expression altered in various ways that are dependent on the location and orientation of the insertion with respect to the different structural and functional domains of the affected gene.
1.6.3.1.1 Insertions into coding regions
In the simplest case scenario, an insertion within the coding region of a gene or at an intron/exon boundary will be manifested by an altered protein structure. These types of insertions are most likely to result in null mutations because of the sensitivity of
37 these regions to frame shifts and the lack of tolerance of highly conserved regions to most mutations of any kind. The insertion of a P-element into the white gene of D. melanogaster in the same transcriptional orientation leads to white transcriptional termination within the P-element (Levis et al., 1984) and hence is a null allele
(whd80k17).
1.6.3.1.2 Insertions into introns
Some insertions into introns are less visible to selection since many can be spliced out during mRNA processing and thus have no or little effect on the function of the gene (Kidwell & Lisch, 1997). The insertion of a partial P-element into the yellow gene of D. melanogaster allow RNA polymerase read-through, producing a larger transcript that is properly spliced, producing only a mild phenotype due to slightly lower mRNA stability (Howes et al., 1988). Alternatively (as in the case of a 412 insertion into the vermillion locus of D. melanogaster), imprecise splicing due to splice sites within the 412 element result in an aberrant mRNA (Searles & Voelker,
1986).
mRNA stability can be drastically affected in some cases by trans-RNA hybridisations. As Wells and Miller (1992) showed, when a transposon is inserted in the opposite transcriptional orientation to that of the host gene, read-through produces a transcript that can hybridise to the transcriptional products derived from the internal promoter of the transposon. These sense-antisense hybridisations significantly decrease mRNA stability and the amount of protein produced (Wells &
Miller, 1992).
38 In addition, introns sometimes contain sites of regulatory sequences, or the insertions contain their own regulatory sequences which can affect gene regulation in varied ways. The insertion of a gypsy element into an intron of the forked gene of D. melanogaster produces a normal sized transcript, but significantly reduces the level of transcript. It was suggested that regulatory sequences within the element play a role in altering the transcript level (Parkhurst & Corces, 1986b). Thus insertions into coding regions or introns are often disguised regulatory mutations.
1.6.3.1.3 Insertions into regulatory regions
Insertions in the regulatory regions can alter the expression pattern of genes, and in fact, in many cases over an evolutionary time frame, insertions added binding sites or other modifications that became effective parts of the regulation system of some genes (Britten, 1996b; Britten, 1996a; Britten, 1997). More often than not though, insertions have deleterious effects on host genes, possessing several ways of exerting those effects (Kidwell & Lisch, 1997).
In the yellow (y) gene of D. melanogaster, a 7.4kb gypsy insertion 700bp upstream of the transcription start site is manifested in the yellow-bodied (y2) phenotype
(Parkhurst & Corces, 1986a; Geyer et al., 1988). One obvious explanation of the mutant phenotype is that the transcriptional enhancers that regulated yellow transcription are incapable of doing so due to an increase in molecular distance of
7.4kb from the TATA box (Geyer & Corces, 1987). However, the mutant phenotype was seen when just a 430-bp gypsy sequence containing a suppressor of Hairy wing
(suHw) binding site and a wildtype allele of the transcription factor suHw were present (Corces & Geyer, 1991). Thus in this case, the gypsy element per se is not
39 responsible for the mutant phenotype, but is a mediator of the effect of suHw through its binding to gypsy and consequential negative regulatory effect on yellow.
Other studies show transposable elements manifesting changes in tissue specificity
(Kloeckener-Gruissem et al., 1992) or temporal expression (Kidd & Young, 1986) as a result of regulatory element binding sequences on the insertion. Thus the temporal transcription of the transposable element may be responsible for aberrant gene expression. In the Notch gene of D. melanogaster, the pattern of expression of the flea transposon during development correlated with the interruption of gene function
(Kidd & Young, 1986).
Other non-insect examples such as the msl gene in mouse (Stavenhagen & Robins,
1988; Adler et al., 1992), the amylase gene in humans (Samuelson et al., 1988; Ting et al., 1992), and the hcf106 gene in maize (Barkan & Martienssen, 1991) provide cases of transposable elements causing run-on transcription. The associated elements residing in the 5’ regions of these genes provide transcription initiation start sites within their LTRs, creating a transcription product that extends from the element and is therefore subject to the regulatory mechanisms of that element rather than those of the gene.
Having briefly discussed the various ways in which transposable elements and other mutations have negative effects on genes, it is important to note that in the presence of insecticidal selection, normally deleterious alleles can become beneficial for survival of the insect, thus leading to resistance at a high allele frequency within a population. But equally likely is that subsequent removal of the insecticide will lead to negative selection for the mutated allele, decreasing its frequency. In reviewed literature, approximately 50% of resistance alleles cause a fitness deficit and 50% do not (McKenzie, 1996b).
40
1.7 Types of insecticides
Various classes of traditional insecticides and resistance associated with them have already been mentioned in this discussion. These early insecticides, such as DDT, targeted the nervous system but were not insect specific, causing the death of a diverse range of plants and animals (Carlson, 1962). An insecticide revolution took place with the introduction of the insect growth regulators, including lufenuron. The idea of developmental targets increased species specificity and provided a new pool of potential insecticides not realised previously. Concurrently, the development of genetic and molecular tools enabled the assembly of information useful for studying resistance once it evolved, and even with a predictive capacity.
Most recently, insecticides have reverted to targeting the nervous system, but this time, have taken advantage of intelligence both from an insecticidal and an environmental point of view. With the growing body of knowledge of insect development and function in general, this information has been used to create better and more specific insecticides such as the neonicotinoids. These insecticides, including nitenpyram and immidacloprid, target the insect-specific nicotinic acetylcholine receptor.
The structures of some insecticides from each of these categories are shown in figure 1.9, and their main mechanisms of resistance are summarised in table 1.3.
This study focuses on insect growth regulators, and hence forms the centre of discussion in the following sections.
41 DDT cypermethrin
dieldrin
diazinon propoxur (a)
methoprene (b) lufenuron chlorfluazuron
immidacloprid nitenpyram (c)
Figure 1.9: Chemical structures of various insecticides including (a) traditional insecticides, (b) insect growth regulators, and (c) neonicotinoids.
42 Table 1.3: Mechanisms of resistance for various chemicals in the three historically defined types of chemicals.
Type of Selected members Target resistance Metabolic resistance References insecticide of class
(Carino et al., 1994; Devonshire & Field, 1991; Pyke et al., Malathion Organophosphate AchE E4, FE4, Cyp6a1, Cyp6g1 2003; Mutero et al., 1994; Walsh et al., 2001; Le Goff et al., Diazinon 2003); Carbaryl (Devonshire et al., 1998; Walsh et al., 2001; Menozzi et al., Carbamate AchE Propoxur 2004) Permethrin (Pittendrigh et al., 1997; Williamson et al., 1996; Dombrowski et Pyrethroid Na channel (para), Cyp6a2, Cyp6a8, Cyp6d1 Cypermethrin al., 1998) Cyclodiene Dieldrin Cl channel (ffrench-Constant, 1994) DDT (Grant & Hammock, 1992b; Daborn et al., 2002; Amichot et al., Organochlorine Cyp6g1, Gstd1 Paraquat 1998; Tang & Tu, 1994) Lufenuron Diflubenzuron (Daborn et al., 2002; Wilson & Ashok, 1998; Daborn et al., IGRs Cyp6g1 Methoprene 2000) Cyromazine Nitenpyram Neonicotinoids Cyp6g1 (Daborn et al., 2002) Immidacloprid
43 1.7.1 IGRs
Insect growth regulators (IGRs) are a group of insecticides known to adversely interfere with the normal growth and development of insects. At a more specific level, they generally directly or indirectly affect the epidermal cells of the insect such that the cuticle is synthesised or deposited incorrectly, or in an untimely way. A discussion of the mechanisms of resistance to IGRs necessitates a brief description of the formation of the cuticle.
1.7.1.1 The cuticle
The cuticle is a crucial component of the exoskeleton for insects, providing mechanical and structural support and acting as a barrier to the potentially hostile environment. Because of its rigidity, it needs to be periodically replaced by moulting to accommodate growth. The moult cycle consists of an intermoult period where the insect feeds and grows, synthesizing components for the new cuticle, and a moult period where the old cuticle is degraded and a new one synthesised. The entire process is under tight hormonal control to ensure the stringent temporal and spatial pattern of secretion of the new cuticle and degradation of the old. The general structure of the cuticle is shown in figure 1.10.
44 s ne er ll a r r y e e b e s a e l c r l L e cl m a cl l i a i e e t n c Lay i i m l r ut l M e Can ace cu c a f i e d docut t s o ax Lay esocu n u
Ex M E Epi Ba Sur W C Por
e l c i ocut r P e l ic t u ic p E
Figure 1.10: Generalised structure of the insect cuticle indicating the surface layer,
wax layer, and cuticulin layer that make up the epicuticle, and the exocuticle,
mesocuticle, and endocuticle that make up the procuticle.
45
It is comprised mainly of chitin and over 20 associated proteins (Chen & Mayer,
1985). Chitin formation is a complex process starting intracellularly and finishing with exterior macromolecular structures that form the various layers of the cuticle shown in figure 1.10. The cascade of events encompasses various stages. The initial substrate trehalose enters the cytosol and undergoes various reactions, resulting in the chitin precursor substrate UDP-N-acetylglucosamine (UDP-GlcNAc). In the next stage the precursor is transported into the endoplasmic reticulum where it undergoes polymerisation. It then undergoes further processing and orientation in the golgi apparatus. In a final stage it is secreted through the plasma membrane and linked by hydrogen bonds and covalent bonds to proteins to form chitin microfibrils which are then sclerotised and melanised (Cohen, 1991; Palli & Retnakaran, 1999; Cohen,
2001). These steps are shown at a greater level of detail in figure 1.11 (Palli &
Retnakaran, 1999).
46
Figure 1.11: The generalised pathway of chitin formation (taken from (Palli &
Retnakaran, 1999)).
47 The process is orchestrated by several temporally-regulated hormones including prothoracicotrophic hormone (PTTH), 20-hydroxyecdysone (20E), and juvenile hormone (JH). JH is secreted into the haemolymph and transported to epidermal cells (amongst others) where it directs the type of cuticle that is to be synthesised.
High titres yield larval cuticle, whilst lower titres yield pupal cuticle (Rees, 1977). At a critical weight during each instar, signals are sent to neurosecretory cells of the brain to release PTTH which in turn stimulates the release of ecdysteroids (moulting hormones). The most important of these, 20E, directs the down-regulation of intermoult activites such as chitin synthesis, and initiates moulting (Chapman, 1998;
Palli & Retnakaran, 1999).
Based on these complex pathways, it is easy to see why the formation of the cuticle represents a potentially valuable insect control mechanism. The application of 20E or an analogue at a time when 20E is absent can induce inhibition of chitin synthesis
(Retnakaran et al., 1996), the application of UDP-GlcNAc analogues can competitively inhibit chitin synthase in the ER (Nakagawa et al., 1992; Cohen &
Casida, 1990), or epidermal nucleic acid inhibitors can disrupt the synthesis of proteins used in chitin formation (Binnington, 1985). Based on their proposed modes of action, there are several major groups of IGRs. They are the juvenile hormone analogues (JHAs), the chitin synthesis inhibitors (CSIs), and the more general antibiotics or metabolic inhibitors. Some of the more common ones are listed in table
1.4.
48 Table 1.4: Examples of various classes of insect growth regulators and what is known about their effects and proposed modes of action.
Class Inhibitor Mode of Action Effect Reference
Structural analogue of UDP-GlcNAc Antibiotic Polyoxin-D Chitin synthesis inhibited in Chilo suppressalis (Nakagawa et al., 1992) (Competitively inhibits chitin synthase Prevents transfer of GlcNAc to dolichol Antibiotic Tunicamycin Blocks chitin synthesis in Triatoma (Quesada-Allue, 1982) phosphate and prevents glycosylation An S-Triazine inhibitor of dihydrofolate (Binnington & Retnakaren, ?Metabolic Cyromazine Abnormal chitin formation in Lucilia cuprina reductase 1991) Metabolic Amniopterin Inhibits dihydrofolate reductase Inhibits normal chitin deposition in M. domestica (Binnington, 1985) IGR Buprofezin Interferes with mitotic apparatus Inhibits cuticle formation in Nilaparvata lugens (Uchida et al., 1986) IGR Diflubenzuron (Retnakaran & Wright, 1987) IGR Lufenuron Hormone Moulting hormone expresses, 20E Chitin formation is repressed in Manduca sexta (Riddiford, 1991) analogue repressing intermoult activities Hormone Prevents chitin synthesis in intermoult periods in Choristoneura Tebufenozide Agonist of 20E (Retnakaran et al., 1996) analogue fumiferana Hormone Intermoult activities procede Methoprene Moulting does not occur in D. melanogaster (Wilson & Fabian, 1986) analogue indefinitely
49 1.7.1.2 JHAs
The disruption of hormonal control through analogues is a useful way of interfering with normal metamorphic changes in an insect. The possibility of their use in insect control first became apparent when the linden bug (Pyrrhocoris apterus) was inadvertently exposed to a juvenile hormone mimic, later identified as juvabione
(Williams, 1967; Bowers et al., 1966). Instability and rapid decay limited the usefulness of juvabione and other mimics, but prompted attempts to synthesise analogues of juvenile hormone (Staal, 1975). These JHAs have similar chemical structures to the endogenous juvenile hormone, and insects treated with them exhibit problems analogous to those resulting from excessive doses of juvenile hormone such as delays in development, altered cuticle formation, and anomalies in reproductive and sensory organs (Bennett & Reid, 1995). Low vertebrate toxicity was another worthy advantage of JHAs over conventional insecticides (Wright, 1976).
One of the most widely used JHAs, methoprene, disrupts metamorphasis in some insects (Wilson & Fabian, 1986; Restifo & Wilson, 1998). It was initially thought that insects would have difficulty evolving resistance to a compound resembling one of their own hormones (Williams, 1967), but resistance was reported soon after in M. domestica (Cerf & Georghiou, 1972; Plapp & Vinson, 1973), and later in the white fly
Bemisia tabaci (Ishaaya & Horowitz, 1995). Mutagenesis of D. melanogaster resulted in a strain resistant to methoprene and P-element tagging enabled cloning of the gene encoding the methoprene resistance locus (Wilson & Turner, 1992; Ashok et al., 1998). The gene (Met) was found to be a transcriptional regulator, a component of the juvenile hormone signalling pathway that is required early in metamorphosis when endogenous juvenile hormone levels are low (Ashok et al., 1998; Wilson,
2001). Resistance in this case is caused by the absence of the Met gene product, a proposed target site of juvenile hormone and consequently methoprene.
Whilst it is now recognised that the problem of insecticide resistance is a possibility in any form of chemical control, there are two additional features hindering the more widespread use of JHAs. The critical timing of application (shortly before the onset of metamorphosis) and damage caused by additional larval instars (due to inability to moult) make them unfeasible for large scale use and/or crop protection (Chen &
Mayer, 1985).
1.7.1.3 CSIs
Chitin synthesis inhibitors (CSIs) are broadly defined as insecticides that disrupt cuticle formation by specifically inhibiting the synthesis, polymerisation, or deposition of chitin in the eggs or larvae of insects (Cohen, 1987). The discovery of CSIs as a method of insect control was first made in 1972 when the combination of two herbicidal compounds produced an inactive herbicide, but which proved to be a potent insecticide (DU-19111) (Van Daalen et al., 1972). DU-19111 was different to conventional insecticides in that mortality occurred during the process of moulting and the adult stage was not affected. It was suggested that the lack of deposition of chitin as a result of this insecticide was leading to incomplete moulting, subsequently killing the insect (Van Daalen et al., 1972).
Initial work led to the development of the more potent but chemically similar compounds such as diflubenzuron (see figure 1.9 for comparison of structures), which interferes with the deposition of the normal cuticle by affecting some step in chitin synthesis (Post et al., 1974). The exact step that diflubenzuron affects has not
51 been elucidated yet, although several hypotheses point to a role in DNA synthesis inhibition leading to a build-up of a chitin precursor substrate and subsequently diminished cuticular deposition (Mitlin et al., 1977; Meola & Mayer, 1980; O'Brien,
1978; DeLoach et al., 1981; Turnbull & Howells, 1982).
Various other modes of action have been suggested for diflubenzuron and its various derivatives. These include the direct inhibition of the chitin synthase enzyme or inhibition of the protease that activates it (Soltani et al., 1984; Retnakaran, 1986;
Cohen, 1991), inhibition of the formation of UDP-GlcNAc or its transport across the endoplasmic reticulum, the blocking of ecdysone metabolism resulting in excess chitinase production which in turn digests chitin (Retnakaran & Oberlander, 1993;
Palli & Retnakaran, 1999), or blocking the binding of chitin to cuticular proteins (Palli
& Retnakaran, 1999).
These chemicals represented an important advance with respect to insecticidal specificity since organisms without chitin would not be affected. Chitin is the most abundant organic skeletal compound in insects, but it is absent in vertebrates and higher plants (Neville, 1975). Insecticides that disrupt chitin synthesis or deposition therefore have selectivity advantages over other insecticides that have nervous system targets that are found in both insects and mammals.
The particular specificity of the CSI lufenuron (discussed further in the following section) was observed when a study of the Oriental cockroach Blattella orientalis showed no significant effects from lufenuron treatment (Mosson et al., 1995).
However the relatively closely related German cockroach Blattella germanica showed a similar profile to D. melanogaster. Differences were attributed to slightly different biological development, but highlight the potential of CSIs as potent, highly specific insecticides.
52 1.7.2 Lufenuron
Lufenuron is classified as a CSI (Graf, 1993) and is commercially used primarily in the treatment of the cat flea, C. felis. It accumulates in the adipose tissue of cats and is slowly released into the vascular system, providing protection for up to one month
(Blagburn et al., 1994; Blagburn et al., 1995). It is of similar structure to diflubenzuron
(See its structure in figure 1.9) and DU-19111, but is considered more effective, as assessed by dosage-mortality curves in D. melanogaster (Wilson & Cryan, 1997).
In C. felis larvae, lufenuron causes cuticular lesions by either the deposition of randomly oriented chitin microfibrils and protein globules, or by hindering the digestion of the old cuticle (Dean et al., 1998). Degeneration of epidermal cells is also seen, explaining improper chitin formation and moulting fluid necessary for degradation of the old cuticle. It is accompanied by a decrease in cytoplasm in the epidermal cells and changes to cytoplasmic organelles (Dean et al., 1999). Embryos develop fully to the first instar but are unable to perforate the egg case. They contain smaller amounts of ER and their epidermal cells are degrading (Meola et al., 1999).
In D. melanogaster, lufenuron’s physiological effects have been studied in depth and it is found that mortality occurred at ecdysis when interruption of chitin synthesis is expected to be most disruptive, irrespective of the dose. It was seen that the effects varied with stage of development (Wilson & Cryan, 1997). As observed for C. felis, affected embryos were unable to perforate the vitelline membrane of their egg case.
Treated first and second instar larvae die at the next ecdysis, third instar larvae pupariate but then die, and treated adults are largely unaffected. Larvae treated with sublethal doses develop to adulthood but have impaired flight ability owing to the
53 disruption of thoracic morphology through the inhibition of chitin synthesis (Wilson &
Cryan, 1997).
Resistance to lufenuron has been reported in natural populations of D. melanogaster
(Wilson & Cryan, 1996; Wilson & Cain, 1997; Pyke, 2000); Thompson, O’Keefe, &
Batterham, unpubl; this study), and this field resistance is discussed further in later chapters. Resistance has not yet been reported in the target organism, C. felis.
Therefore lufenuron is an excellent candidate for pre-emptive studies into resistance mechanisms with the aim of stalling or preventing the evolution of resistance in the cat flea.
1.8 Drosophila as a model organism
Some of the classic advantages of D. melanogaster for use as a model organism are a rapid life cycle, small number of chromosomes, ease of rearing and maintenance, and its status as a non-pest insect (Wilson, 1988).
Some contemporary advantages are the vast array of mutant stocks available
(Bloomington, 2004), highly detailed cytological maps from polytene chromosomes
(Pardue, 1986), a large body of well described protocols for genetic and molecular analysis (Drosophila protocols, 2000), the ease of germline genetic transformation
(Rubin & Spradling, 1982), the available genome sequence (Adams et al., 2000;
Flybase, 2004), and the availability of large numbers of cDNA clones for microarrays
(White et al., 1999).
D. melanogaster is a particularly good model organism for use in resistance studies.
Resistance to a broad range of chemical insecticides has been found in this species
54 (Feyereisen, 1995; Wilson, 2001; Hemingway et al., 2002). This may be due to incidental exposure to insecticides sprayed to control pest insects.
The resistances discovered in D. melanogaster are particularly useful since the molecular basis of resistance can be readily identified. Furthermore, the evolutionary mutational pathway for the development of resistance alleles can be followed by studying populations in different geographical locations.
The high level of genetic conservation between D. melanogaster and pest species such as M. domestica, L. cuprina, and Anopheles gambiae (Weller & Foster, 1993;
Holt et al., 2002) makes it an even better model since information learned can often be applied to these species based on homology.
1.9 This Project
This introduction has described many different aspects of insecticide resistance and highlighted that the effects of chemicals on organisms can be studied at many levels.
The broadest level is the ecological level, whereby effects of insecticides on predators, parasites, pollinators, and non-target organisms are considered. The population level concerns the effects of general mortality of treated populations, whether they are target or non-target species. At the organismal level, mortality and the symptoms leading to mortality are considered. At the physiological level, functional disruptions related to organs and complex processes of interest are considered. The biochemical level is where the insecticide interacts with specific and definable proteins within the organism, leading to explicit effects in its physiology.
This level is usually the ultimate resolution of the mode of action of an insecticide.
55 Similarly, the mechanisms of resistance to an organism can be studied at each of these levels. But in the case where genetic mutants leading to resistance are seen, an additional level, the molecular-genetic level, encompasses genetic changes that lead to resistance and their manifestation in effects such as specific changes in the transcription-translation process, protein structure or efficacy, temporal or spatial distribution, or regulation.
Studies of mechanisms of resistance are focused on many of these different levels, attempting to answer several questions fundamental to our understanding of insecticide resistance in general. From the most specific to the broadest, these questions can be summarised as follows:
1. What mutation(s) in what gene(s) are causing resistance?
2. How are these specific mutations manifesting themselves physiologically to result
in survival in the face of a mortal threat?
3. Can and do these mutations exist in both natural and lab environments?
4. How conserved are these mutations within and between populations, and what
does this tell us about the evolution of resistance in these populations?
5. How does the absence of insecticide-based selection affect persistence of
resistance?
6. What are the effects in other insect species, both pest and non-pest?
These questions cover a broad array of potential research but it is the aim in the following chapters to examine and discuss them in varying degrees of detail with respect to the insecticide lufenuron and the model organism D. melanogaster.
Chapter 3 investigates the variation in resistance within and between Australian field populations as compared to lab strains. It then concentrates on a particular lufenuron
56 resistant field strain, carrying on from previous work using genetic and molecular mapping to identify potential resistance conferring candidates. The P450s Cyp12a4 and Cyp12a5 are recognized as likely candidates in the mapping region and may be involved based on expression levels, sequence polymorphisms, and creation and study of transgenic overexpressing strains. These propositions are tested through various means. Discussion suggests ways in which these genes might contribute to lufenuron resistance.
Chapter 4 examines a field-derived strain from the the United States of America.
Another detoxification-based mechanism of resistance is identified using mapping and expression analysis. The Cyp6g1 gene is suggested as a candidate and its role in resistance is confirmed through toxicological analysis of overexpressing transgenic flies. A transposon insertion is shown to be associated with overexpression and resistance. Discussion centres on how this transposon might cause overexpression of this gene, leading to resistance.
Chapter 5 expands on work with the USA field strain, comparing it to an Australian field strain, and examining other resistance mechanisms that may be in action in both of these strains. A mechanism on a different chromosome to Cyp6g1 is identified in the two strains studied in depth, and implications for Cyp6g1 and for resistance are discussed in light of these results. In addition, the transposon insertion is surveyed across field populations collected along the east coast of Australia to assess the prevalence of the associated resistance mechanisms. Discourse of the evolution of resistance between and within populations follows.
Chapter 6 examines lab-based resistance, drawing on results discussed in other chapters to make assumptions about mechanisms of resistance in mutagenised strains, and using genetic mapping and expression analysis to corroborate these.
57
Chapter 7 is a more general discussion of lufenuron resistance, discussing the relationships among resistance mechanisms and how knowledge gained from study of D. melanogaster can help in insecticide resistance management.
58
Chapter 2
Materials and Methods
2.1 Fly maintenance
The general fly maintenance reagents and lufenuron resistance screening methods used in this investigation are described here. The Drosophila melanogaster strains and crosses used are described in individual chapters, as are the particular insecticide screening techniques and crosses
2.1.3 Drosophila melanogaster food medium
All strains of D. melanogaster were reared at 25°C under constant light conditions.
When needed, flies were rendered unconscious exposure to CO2. Their diet was a standard food media prepared as shown below and scaled according to the quantities was needed.
80g glucose Combined with 1180mL 40g sugar water and brought to boil 18g yeast
12g potassium tartrate 100g semolina mixed with 8g agar 300mL water added to mixture and returned to boil 0.75g calcium chloride
Tegosept: 100g p-hydroxy benzoic acid methyl ester in Mixture was removed from
100mL ethanol heat and 23mL tegosept and 17.5mL acid mix were in vials and bottles Acid mix: 412mL propionic added acid and 42mL phosphoric
acid in 546mL water
60
The media was poured while still hot into either 25mL glass/plastic scintillation vials or 250mL plastic bottles. Approximately 5mL or 50mL food was added respectively.
Bottles and vials were allowed to cool and stored at 4°C for up to 10 days or until needed.
2.1.4 Lufenuron-containing food medium
Lufenuron (CGA-184699) (N-(2,5-dichloro-4-(1,1,2,3,3,3-hexafluoro-propoxy)- phenylaminocarbonyl)-2,5-difluorobenzamide), was provided by Novartis Animal
Health Australasia Pty. Ltd. A 100ppm stock solution was prepared by dissolving lufenuron powder in 95% ethanol, and stored for up to six months at -20°C.
Appropriate amounts were added to partially cooled fly media (below 65°C) with up to
5Xvolume 95% ethanol (up to 5mL per litre of food medium - in order to facilitate sufficient mixing into the media). Approximately 0.1mL food dye per 100mL food was added in order to distinguish lufenuron-containing food from standard food. Suitable amounts of 95% ethanol were added to no-lufenuron screening controls.
2.1.5 Lufenuron screening methods
Lufenuron food was added to vials as described above. Screening was performed using 1st instar larvae. Controlled crosses were carried out by placing unconscious male and female flies of the appropriate strains into plastic cages (15x15x30cm).
Petri dishes containing standard diet were added. Flies were allowed to lay embryos for 24h and then petri dishes were replaced. 1st instar larvae were then collected 12h later. 100 larvae were collected per vial.
61 2.2 Molecular techniques
The molecular protocols used throughout this thesis are mentioned here. All PCR primers and specific techniques are presented in their respective chapters.
2.2.1 Genomic DNA Extraction
Genomic DNA extractions of multiple flies were performed using the Qiagen
DNeasy® Tissue System kit. Approximately 30 whole third instar larvae or adult flies were used in each preparation. These flies were either fresh or had been frozen in liquid nitrogen and stored at -20 or -70°C. Single fly preparations were made by grinding up flies in 50µL buffer (10mM Tris-Cl pH8.2, 1mL EDTA, 25mM NaCl, 200 ug/mL proteinase K), incubating for 1-3 hours at 37°C and teminating proteinase K activity at 95°C for 2min.
2.2.2 Polymerase Chain Reaction
The components used in a standard 50µL Polymerase Chain Reaction (PCR) are shown below. Reaction components were all halved in instances where 25µL reactions were used.
10x Reaction Buffer 5µL dNTPs (2mM) 5µL MgCl2 (25nM) 3µL Forward Primer (5µM) 5µL Reverse Primer (5µM) 5µL DNA (variable) xµL Promega Taq DNA polymerase 2.5u dH2O up to 50µL
62
PCR was carried out in an Eppendorf Mastercycler or a Biorad iCycler Thermocycler using the below conditions:
One Cycle: 94°C 120 seconds 30-40 cycles: 94°C 15 seconds 60-64°C 30 seconds (temperature variable depending on primer characteristics) 72°C 30-240 seconds (time variable depending on desired product length) One Cycle: 72°C 420 seconds
2.2.3 Agarose gel separation
Agarose gels were prepared by adding the required amount of powdered agarose to
TBE buffer (54g TRIS Base, 20mL 0.5M EDTA, 27.5g Boric acid - adjust to 1L with dH2O and dissolve at 65°C for 30 minutes) and heating until just before boiling. 2µL of 10µg/µL ethidium bromide was added to gels, they were poured and allowed to cool. Samples were separated on 0.7-4.0% agarose gels, depending on the expected size of the product, at 50-250V.
2.2.4 Restriction Enzyme Digestion
Samples were mixed with 5-20U restriction enzyme (Promega or New England
Biolabs) and appropriate buffer and incubated at 37°C or 42°C as appropriate for 2-6 hours. Reactions were terminated at 65°C for 15 minutes.
63 2.2.5 Total RNA Extraction
Total RNA extractions were performed using a modified version of the Qiagen
RNeasy Total RNA Isolation System. 3rd instar larvae or 3 day old adult flies were frozen in liquid N2, ground using a mortar and pestle, and 750µL of Invitrogen Trizol reagent was added. After centrifugation at 12000g for 10 minutes, the supernatant was transferred to a new 1.5mL centrifuge tube. 150µL Chloroform was added and the tube was shaken vigorously for 15sec, then incubated at room temperature for 2 minutes. The sample was then centrifuged at 1000g for 15 minutes. The top phase was pipetted into a new 1.5mL centrifuge tube and a 0.5 x volume of ethanol was added. The mixture was then added to the Qiagen RNeasy column and the RNA isolation procedure completed following the manufacturer’s instructions. Total RNA was stored at -20°C, or at -70°C when longer term storage was required.
2.2.6 RNA quantification
RNA was quantified using a Perkin Elmer Lambda 2 Spectrophotometer at 1/50 dilution. Absorbance at 260nm was read and concentrations calculated using the equation concentration = 40 x dilution factor x absorbance reading.
2.2.7 First strand cDNA synthesis
cDNA synthesis was carried out using the Invitrogen Superscript™ First Strand
Synthesis system using an included “poly A” primer. 1-4µg total RNA was used in each reaction such that final concentration was 100ng/µL.
64 2.2.8 5’ RACE
5’ RACE was performed using Clontech SMART™ RACE cDNA amplification kit
(K1811-system and primer within exon 1 of Cyp6g1 – See chapter 4). Total RNA for use in the RACE reactions was isolated as described in section 2.2.5. 100µg/µL mRNA was used. cDNA amplification products were visualized using 1.8% agarose gels as described in section 2.2.3.
2.2.9 Realtime Polymerase Chain Reaction
Realtime RT-PCR was performed on a Corbett Rotorgene Realtime PCR machine using Applied Biosystems Taqman© probes and Universal mastermix, or with Qiagen
SYBR® green mastermix. The components and conditions were used:
Taqman© Reactions SYBR® green Reactions
Forward Primer (18µM) 2.5µL Forward Primer (5µM) 2.5µL Reverse Primer (18µM) 2.5µL Reverse Primer (5µM) 2.5µL Taqman© Probe(5µM) 2.5µL dH2O 2.5µL cDNA 5.0µL cDNA 5.0µL Universal mastermix 12.5µL SYBR® green mastermix 12.5µL
One Cycle: One Cycle: 94°C 15 minutes 94°C 15 minutes 30-40 cycles: 30-40 cycles: 94°C 60 seconds 94°C 15 seconds 60°C 60 seconds 60°C 60 seconds One Cycle: 72°C 20 seconds 72°C 5 minutes One Cycle: 72°C 5 minutes
65 A six point (three replicates per point) standard curve of cDNA level was created using CanS cDNA of known concentration. 9-15 samples (3-5 different concentrations in triplicate) were placed on that curve to identify expression levels of those samples. The concentrations were adjusted relative to the cDNA levels of housekeeping gene rpL32.
2.2.10 DNA Purification
PCR Products were purified using Promega Wizard® genomic DNA purification kit or the Qiagen QIAquick PCR purification kit. Purified products were eluted in 50-100µL dH2O and stored at -20°C.
2.2.11 DNA Sequencing
DNA sequencing was carried out using Applied Biosystems PRISM® BigDye™ v2.0, v3.0, or v3.1 with standard protocols. Sequences were determined by the gel separation method (performed by Australian Genome Research Facility, Walter and
Eliza Hall Institute).
2.2.12 Plasmid cloning and DNA extraction
PCR products were ligated into appropriate vectors (pGEM-T, PCR-Blunt, (figure 2.1) or pUAST (Chapter 3, figure 3.7)) using T4 DNA ligase and buffer and incubated overnight at 4°C. Ligated products were transformed into competent JM109 cells and plated out onto LB/AMP/IPTG/X-Gal plates as appropriate for the vector, following
66 methods described in Sambrook et al. (Sambrook & Russell, 2001). After incubation, transformants were cultured overnight at 37°C in LB/AMP and DNA was extracted using Wizard® plus minipreps and associated protocols.
Figure 2.1: Vector maps of vectors used in PCR product cloning. pGem®-T easy
(Roche #A1360) and pCR®-Blunt II-TOPO® (K2800-20).
2.2.13 Microinjections
Microinjections of constructs were carried out by C. Lumb (Department of Genetics,
University of Melbourne) using standard protocols (Rubin & Spradling, 1982).
Overexpression construct was injected at 250-500ng/ul, accompanied by 100ng/ul helper plasmid (source of transposase), and 1X injection buffer (10X Injection Buffer,
50mM KCl, 1mM NaPO4, pH 7.8).
67
Chapter 3
Mapping field resistance in
Australia
3.1 Introduction
3.1.1 Field resistance studies
The capacity for a population to respond to the intense selection pressure imposed by insecticides is provided by the available genetic variation. The speed of the change is dependent on the amount of variation and the intensity of selection pressure (Crow, 1957). Analysing the variation in susceptibility of populations can therefore aid in determining when selection occurred, its intensity, and whether it remains active. Such studies can also provide an array of resistant mutants to underpin an investigation of monogenic-based resistance mechanisms.
Insecticide resistance in natural populations of D. melanogaster was reported as early as 1954 (Crow, 1954), despite this species rarely being the target of insecticide exposure. The environment in which D. melanogaster resides, such as orchards, means that it is often inadvertently exposed to insecticidal sprays used to treat insect and plant pest species. When developing in other ecological niches such as rotting fruit or decomposing rubbish, D. melanogaster may be also exposed to a wide variety of other “natural” xenobiotics. Thus resistance presumably evolves from existing in these shared living environments.
Resistance to the cyclodiene insecticide dieldrin was recovered in the field and has been well studied since (refer to Chapter 1, section 1.4.3.1). There are several important universal outcomes of dieldrin studies with respect to field resistance. The insecticide had been used in the field to treat the Australian sheep blowfly L. cuprina, and had also been used to treat the common housefly Musca domestica. Resistance in L. cuprina and M. domestica mapped to chromosomes V and IV respectively.
69 Interestingly, it was found that resistance to dieldrin in D. melanogaster was also seen and mapped to a single locus on chromosome 3L, the homologous chromosome to V and IV in those species (Weller & Foster, 1993).
This result highlights two points with respect to the legitimacy of studying field based resistance in D. melanogaster. The first is that it is likely that D. melanogaster field strains have the ability to evolve resistance to insecticides used to treat other species, simply through sharing their environment. The second is that there appears to be a striking similarity between the resistance mechanisms of different species, particularly where resistance involves target site modification. This validates the utility of D. melanogaster as a model genetic species for studying resistance on a broader scale.
Resistance in D. melanogaster has now been reported to almost all types of chemicals including cyclodienes as mentioned above, DDT, OPs, (Crow, 1954;
Pralavorio & Fournier, 1992); and in 1996, lufenuron (Wilson & Cryan, 1996). The surprising difference between the former insecticides and lufenuron is that lufenuron has had no significant field usage in treatment of any species, and was only marketed as a flea treatment in household pets.
3.1.2 Field resistance to Lufenuron
The initial report of lufenuron resistance was in populations from two widely separated locations in the United States of America (Wilson & Cryan, 1996). The populations were generated from isofemale lines and subjected to a bottleneck to reduce heterozygosity. Several strains were found to show as much as 100 fold resistance to lufenuron when compared with laboratory strains. It was postulated that
70 this resistance resulted from cross-resistance that had evolved to an earlier, widely used insecticide (Wilson & Cryan, 1996).
Field resistance in Australian populations was observed in 1997 (O'Keefe, 1997);
Thompson, O’Keefe & Batterham, Unpubl). A total of 72 isofemale strains isolated from varying locations along the east coast of Australia were surveyed and a large proportion of the strains showed higher lufenuron tolerance when compared to a control strain. Several of the most highly resistant strains were chosen for initial genetic mapping studies. In all cases, resistance to lufenuron mapped predominantly to chromosome III. In 2001, finer scale mapping was conducted with one of these strains, the NB16 strain, isolated from Wandin, Victoria (Magoc, 2001) NB16 is one of the most highly resistant strains from the 1997 survey and therefore warranted further investigation. Genetic mapping was carried out using phenotypic markers.
Resistance was found to map within the 7.1mu interval between the markers glass eye, (gl), chromosome III-63.1, and ebony body, (e), chromosome III 70.7. Given this small interval, it is likely that resistance is conferred by a single gene or a cluster of tightly linked genes.
Cross-resistance testing was also performed (Magoc, 2001) using the insect growth regulators diflubenzuron, dicyclanil, and cyromazine (see section 1.7.1 for discussion of these). The importance of cross resistance testing lies in the possibility of correlating specific patterns with proposed resistance mechanisms. Cross resistance to a range of chemically distinct insecticides would be suggestive of metabolic resistance. Cross resistance limited to chemically similar insecticides would not rule out metabolic resistance, but would suggest target site modification as a viable alternative. The NB16 strain only showed low level cross resistance to the chemically similar diflubenzuron, and no resistance to dicyclanil or cyromazine.
71 Over the next three chapters, the basis of field resistance to lufenuron will be examined using strains from Australia and the USA. This chapter provides a wide field survey of lufenuron resistance in isofemale strains sampled from natural populations along the east coast of Australia, replicating and complementing the
1997 survey. It then concentrates on the NB16 strain isolated from the original survey, isolating two candidate resistance genes. The aims of this chapter are more specifically:
1. To examine variation in field resistance to lufenuron surveying a transect
running along the east coast of Australia.
2. With respect to NB16, to positionally clone the resistance gene(s) using
molecular markers.
3. To functionally characterize the resistance conferring gene.
4. To molecularly characterize the resistance conferring mutation.
5. To relate this characterization back to other pests specifically targeted by
insecticides.
3.2 Materials and Methods
3.2.1 Fly Strains
Several different strains of D. melanogaster were used in this investigation including the NB16 resistant strain, mapping strains containing visible phenotypic markers that were used in genetic and molecular mapping, and deficiency strains used in sequencing candidate genes. These strains are described in table 3.1. Further, in
2001 a natural population survey was performed by collecting inseminated females from 14 distinct geographical locations from the east coast of Australia. 75 Isofemale lines were established using 1-8 lines per location (A. Hoffman, pers comm.) Details of these locations and strains are shown in table 3.5 in the results section.
72
Table 3.1: Fly strains used in this investigation
Stock Use Fly Strain Genotype/Phenotype Source Chr* Number NB16 Lufenuron resistant field strain Wandin, Vic Resistant Strain WC2 Lufenuron resistant field strain Colorado, US Inn5 Lufenuron resistant field strain Innisfail, Qld
2 4 Mapping gl e gl e Bloomington 507 Strains w1118 w1118; CyO/if; Sb/TM6b Bloomington 507
* 1 Deficiency Df2411 Df(3R)Dl-KX23, e /TM3, Ser Bloomington 2411 strains Df5597 Df(3R)Dl-M2/TM6C, Sb1 Bloomington 5597
CanS Lufenuron susceptible lab strain Lab Std. Susceptible control strains Celera Lufenuron susceptible lab strain Lab Std. Armenia Lufenuron susceptible lab strain Lab Std.
Injection strain w1118 w1118/w1118 Bloomington 3605 Ubiquitous gal4 expression (Tub)gal4 1 1 Bloomington 5138 gal4 Driver y ; P{tubGAL4}/TM3, Sb strains (6g1CS)g Midgut, fatbody gal4 expression P. Daborn al4 y1; P{5’Cyp6g1GAL4}/TM3, Sb1 Overexpression Claire w1118P(UAS-Cyp12a4cDNA) This Study X Strain – X Chr Emily w1118P(UAS-Cyp12a4cDNA) This Study 2
Tony w1118P(UAS-Cyp12a4cDNA) This Study 2
1118 Overexpression Jack w P(UAS-Cyp12a4cDNA) This Study 2 Strains - ChrII Caroline w1118P(UAS-Cyp12a4cDNA) This Study 2
Dave w1118P(UAS-Cyp12a4cDNA) This Study 2
Alyssa w1118P(UAS-Cyp12a4cDNA) This Study 2
Jessica w1118P(UAS-Cyp12a4cDNA) This Study 3
Jennifer w1118P(UAS-Cyp12a4cDNA) This Study 3
Overexpression 1118 Strains - ChrIII Caleb w P(UAS-Cyp12a4cDNA) This Study 3 Max w1118P(UAS-Cyp12a4cDNA) This Study 3H
Jodi w1118P(UAS-Cyp12a4cDNA) This Study 3H * H represents a strain that is homozygous lethal
73 3.2.2 Fly Crosses
There were numerous fly crosses used in this investigation, specifically the crosses used in genetic and molecular mapping, the sequencing of candidate genes and in the creation and isogenising of transgenic lines. These are shown below.
3.2.2.1 Mapping of the resistance locus
A genetic mapping cross was carried out using a strain containing the recessive flanking genetic markers glass (gl) and ebony (e). Unmated F1 females were collected and backcrossed to the mapping strain so that phenotypic evidence of recombination events was carried through to the second generation. Recombination events were then be scored both genetically and molecularly. This cross is summarized in figure 3.1.
74
gl S e gl+ R e+ X gl S e gl+ R e+
Screened on 1.5ppm lufenuron
gl+ R e+ gl S e F1 X gl S e gl S e
Screened on 2.1ppm lufenuron
Backcross gl R e+ gl+ R e progeny gl S e gl S e
Figure 3.1: Genetic mapping cross showing the generation of gl e+ and gl+ e recombinants (note that non-recombinant classes are also generated but are not shown for simplicity). The gl e mapping strain is shown in blue and the NB16 resistant strain is shown in red. S and R refer to the susceptible and resistant alleles of the putative resistance-conferring gene respectively. Strains were maintained by crossing each male recombinant to the gl e strain each generation.
75 3.2.2.2 Cross used for sequencing relevant genes
Genetic mapping placed the resistance locus in a refion known to be deleted in the deficiency strains Df2411 (91C7-D3 – 92A5-8) and Df5597 (91C7-D1 – 92A1). A single cross to either the deficiency strain Df2411 or Df5597 was carried out in order to generate F1 progeny with one copy of the resistant gene(s) over a deficiency.
Given that the NB16 strain was from the field, this would minimize the number of polymorphisms that were only present through random segregation within the population. These progeny were screened on 1.8ppm lufenuron to ensure the resistant allele from NB16 (rather than another allele segregating in the population) was isolated. DNA was then extracted from the progeny (figure 3.2).
Figure 3.2: Cross used to Sb+ S Sb+ R generate DNA for sequencing X candidate genes. S and R refer to Sb S Sb+ R the susceptible and resistant Screened on 1.8ppm lufenuron alleles of the putative resistance- conferring gene respectively. Sb is R a dominant marker on the balancer DNA chromosome (zigzag line) to Sb S distinguish between it and the chromosome with the deficiency.
76 3.2.2.3 Crosses used to map the site of integration of molecular constructs in transgenic flies
When transgenic flies (see section 3.2.2.4 for details) were isolated, they were subjected to several crosses in order to determine where the UAS-Cyp12a4 construct had integrated. Crossing to a balancer strain containing dominant homozygous lethal markers enabled this. Several different chromosomal combinations existed for each possible scenario. One example is shown in figure 3.3, detailing the phenotypes that would be seen if the construct integrates into chromosome II.
77 w CyO Sb P + X w If Ser w + + White eyes Wildtype eyes
w CyO Sb w CyO Sb F1 X w If Ser P +
w If Ser w If Ser P + P Sb F2 All Wildtype eyes w CyO Ser w CyO Ser P + P Sb
Figure 3.3: Crosses used to determine which chromosome carries a stably integrated Cyp12a4 overexpression construct (P). In this case shown, it has integrated into chromosome II, and only the informative phenotypes are shown. In flies carrying the construct (and hence exhibiting wildtype eyes), the If (irregular eye facet) phenotype is never seen in combination with the CyO (curly of oster wing) phenotype, whilst the Sb (stubble bristle) and Ser (serrate wing) phenotypes are seen together in some flies.
78 3.2.2.4 Crosses used to isogenise transgenic flies
When the construct was localised to a particular chromosome in a given transgenic strain, that chromosome was homozgygosed. Again there were several possible scenarios depending on where the construct was integrated. Figure 3.4 shows and example of where it has integrated into chromosome II.
w CyO Sb P + X w If Ser w + + White eyes Wildtype eyes
w CyO Sb w CyO Sb F1 X w P + P +
w P + w P + F2 X w P + P +
Figure 3.4: Crosses used to isogenise the stably integrated Cyp12a4 overexpression construct (P). In this case shown, it has integrated into chromosome II, and only the relevant phenotypes are shown.
79 3.2.2.5 Crosses used to analyse transgenic flies
After the chromosome carrying the construct had been homozygosed, crosses to
gal4 strains were performed. A variety of gal4 strains were used. Each strain had the
gene for the yeast transcription factor GAL-4 (Brand & Perrimon, 1993) cloned
immediately downstream of a particular D. melanogaster enhancer sequence.
Bringing an enhancer/gal4 element together with the upstream activating sequence
(UAS) adjacent to Cyp12a4 drove the expression of Cyp12a4 based on the temporal
and spatial pattern of expression of the GAL-4 protein. This is shown
diagrammatically below in figure 3.5.
CYP GAL-4 12A4 protein Cyp12a4 cDNA CYP UAS 12A4
D. Mel enhancer gal4
Figure 3.5: Diagrammatic representation of the gal4-UAS system. When crossed to
a strain that has specific patterns of gal4 expression determined by a D.
melanogaster enhancer sequence, the GAL-4 protein binds to the upstream
activating sequence (UAS), driving expression of Cyp12a4 to produce CYP12A4
protein in the associated tissues and times.
80
Using this method, two crosses with transgenic strains were performed. The first was to a ubiquitous gal-4 producer that is maintained heterozygously over a balancer containing the Sb marker. This provides an internal control for lufenuron screening since it is assumed that this balancer chromosome cannot confer resistance. The second was a homozygous strain that produces gal4 in the midgut and fatbody only.
100 larvae per vial were screened on various concentrations of lufenuron (three replicates at each concentration). The number of emerging adults was scored 15-18 days later. Results for larval to adult viability on lufenuron were averaged and graphed by dividing by survival on control food for each strain. These crosses are shown in figure 3.6.
(tub) (6g1CS) + + gal4 + P + + + gal4
X X (6g1CS) + + Sb + P + + + gal4 Stubble bristles
Screen on lufenuron Screen on lufenuron
(tub) (6g1CS) + + Sb + + gal4 + + gal4 + P + + P + + P + Stubble bristles
Figure 3.6: Crosses used to screen transgenic flies. The cross on the left is with a gal4 strain carrying a ubiquitously expressing tubulin promoter and is maintained heterozygously (tub)gal4, and the cross on the right is with a promoter expressing gal4 only in the midgut and fatbody of the fliy (6g1CS)gal4. Progeny of these crosses were screened on various concentrations of lufenuron.
81 3.2.3 PCR Primers
CAPS (Cleaved Amplified Polymorphic Sequence) markers were derived for the positional cloning of resistance genes and shown in table 3.2. Sequenced PCR products from the introns of genes within the mapping region were scored for polymorphic differences between the resistant strain and the mapping strain. These differences were used to create Restriction Fragment Length Polymorphisms
(RFLPs) in order to screen recombinant flies for the location of recombination. In one case (labelled microsat in Table 3.2), PCR products were scored for differences in microsatellite repeat sequence length. Also shown in tables 3.3 and 3.4 are primers used for the sequencing of candidate Cyp12a4 and Cyp12a5 genes, and for the creation of the Cyp12a4 overexpression construct.
Table 3.2: Primers and restriction enzymes used in molecular mapping (continues over the next two pages)
Abbrev For/ Length Rest Sequence (5’ – 3’) polymorph Name Rev (bp) Enz
14306 F GCAGGTTTAGGCAGGARGAAG 21
HindIII
R GAGTATGACCGCATCCCAGTAG 22
14301 F CGTCGCAGTAAAAGTTGGTG 20
XmaI
R CAGGATCTGGAGAACGATGAG 21
82 Abbrev For/ Length Rest Sequence (5’ – 3’) polymorph Name Rev (bp) Enz
Cha F CGCAGCATTAGTAGGTGTGTTC 22
RsaI
R GATTACAACAAGGTCCCCA 19
18208 F GCACCAGGATGTACAGGAAGA 21
AvaI
R CCGCCAAGAAAGGAACAATAC 21
Endo F GAACGACATTATCACCCTGTTG 22
HaeIII
R GAAGAACTCAGGTGGAAAGACAA 23
14289 F CACGAAATCGCCAAGTAGGT 20
MfeI
R GGTCCTGCTAGGAAGGTGTAGAG 23
Npi F GTGCGAGTCCGCTCATAATC 20
BstUI
R GATTGCATGGGCGTAGTTAGAC 22
ATP F GCTTCGTTGGTCGTCTGTTC 20
Hsp92II
R GATGGCACTGCTTGAATTCTCT 22
83 Abbrev For/ Length Rest Sequence (5’ – 3’) polymorph Name Rev (bp) Enz
ATP- F CACTTGGCGAGCTCTCAGTA 20 6040
EcoR1
R GCAGATCCTAGTCTTTGTTTGG 22
5835- F GCGGAATGACAAACTCAAGC 20 11779
SspI
R GGTGTCCTGGCCAAATCTATAAC 23
Nos GTACCACTACCACTTGCTGCTC 22
AseI
CCTTGCTGTTGTAACGCTTGT 21
Delta F GTGAACTATTGCATTGGCAGTTC 23
HincII
R CGACAGTCCTGGAGAAGTTATCA 23
Microsat F CCAAACTGAACCGAACCAAC 20
R ATCACAGCCTTCCATCAATC 20
oamb F CATTATGCTTGGGTAGGTGACG 22
DraI
R GCCGATCTTCTGGTGGGTTTG 21
84 Table 3.3: Primers used in sequencing Cyp12a4 and Cyp12a5
Name For/Rev Sequence (5’ – 3’) Length
Cyp12a5 F GGGCTTTAACTATCATGTGTCAG 23
F1 AAACCCATCGTCTTCTCTGC 20
R1 GGACTTTGAGGTGGTTTTCCG 21
F2 CAGGTTAACCAGGAATTTGTGG 22
R2 CACTGCTGAAGAAAATGCTC 20
R3 GGAGAGATGGCTGCGAAACG 20
Cyp12a4 F CAACCTGCCCAATATACCACTC 22
F1 CGTTGTCTGTGGGGTCGCTATC 22
R1 CAGTTTTGTTTGGGCGGTGAGC 22
F2 TGGAGATGTTTGAGGCGATGA 21
R2 ACTTCATAGACACCATCAACCG 22
F3 GCTAATGGCAGGAGTTGATACGG 23
R3 GGTGATGAAGGTGCTGCCCAAC 22
R GCATGGGTCACAAACAGTTG 20
Table 3.4: Primers used to generate overexpression construct
Name For/Rev Sequence (5’ – 3’) Length
12a4 F GTGAGCCGGAAAAGTTCTAATC 22 cDNA
12a4 R TTTGACCATGACTGTATATCGC 22 cDNA
85 3.2.4 Overexpression construct
A Cyp12a4 overexpression construct was created as described below. Total RNA was isolated from the susceptible Celera strain (see section 2.5). Cyp12a4 cDNA was amplified with PCR using the Roche Expand™ high fidelity PCR system
(#11732641001) which generates a blunt ended product. It was cloned into the vector pCR®-Blunt II-TOPO® (Invitrogen) and sequenced to check for errors. The product was then subcloned into the vector p-UAST (Brand & Perrimon, 1993), which contains an upstream activating sequence (UAS) with gal4 binding sites and a constitutive (hsp70) promoter, and a mini-white gene to facilitate screening (figure
3.7). This construct was then injected into w1118 embryos aged 1hr post laying using standard protocols.
Figure 3.7: p-UAST expression
vector showing the mini-white
gene, the UAS, TATA promoter
box, and multi cloning site (Brand
& Perrimon, 1993).
Survivors were scored for presence of wildtype eyes (indicative of the integration of the construct into the genome), and re-crossed to the mapping strain to facilitate homozygosis and indication of the chromosomal location of the insert.
86 3.3 Results
One of the general aims of the next three chapters is to demonstrate the value of insecticide resistance studies in natural populations of D. melanogaster. Such studies allow the isolation of resistant mutants selected for by incidental field exposure to insecticides or other selective agents. With further study, they can lead to the identification of specific mechanisms of resistance. Building on this foundation, specific mechanisms of resistance can be studied within and between populations, highlighting the spread of resistance in general and of particular mutations. This aids the understanding of how, when, and why resistance has arisen. The focus of this chapter encompasses several aspects of this aim.
3.3.1 Field resistance study 2001
Initial work in this investigation was concentrated in identifying strains of D. melanogaster with high levels of resistance to facilitate meaningful future investigation. To this end, 75 isofemale lines of D. melanogaster were collected from along the east coast of Australia as represented in table 3.5. These lines, along with several previously isolated resistant strains, were subjected to larval resistance screening and compared to several susceptible control strains. Screening was performed at three different lufenuron concentrations. The survival of individual strains at various lufenuron concentrations is shown in figures 3.8 a), b), and c).
87 Table 3.5: Natural population strains of D. melanogaster indicating their origin
(grouped from north to south). Also shown are resistance ratings for each strain categorised into H – High resistance (The number of adult flies eclosing on the discriminating dose of 2.1ppm lufenuron was >30% of the number eclosing from the
0ppm control), M – Medium resistance (15-30%), L – Low resistance (5-15%), O –
No resistance (<5%). Table continues over page.
* indicates strains chosen for further analysis.
Resistance Fly Strain Town Latitude Lufenuron
CR1 Cape Tribulation 16 O CR4 Cape Tribulation 16 O CR8 Cape Tribulation 16 O CR11 Cape Tribulation 16 M CR3 Cape Tribulation 16 O CR1-13 Cape Tribulation 16 H CR9 Cape Tribulation 16 O CR1-17 Cape Tribulation 16 O CF1-4* Townsville 19 M CF2-10 Townsville 19 O CF2-13 Townsville 19 O CG1-2* Townsville 19 M BX20 Townsville 19 M BX4 Townsville 19 O BX14 Townsville 19 O BX1-11 Townsville 19 H BX20 Rockhampton 23 L BW2-4 Rockhampton 23 O BW2-5 Rockhampton 23 L BW2-8* Rockhampton 23 H BW2-9 Rockhampton 23 L DC1-17 Gladstone 24 O DC1-24 Gladstone 24 M DC1-22 Gladstone 24 O DC1-25 Gladstone 24 L BS1-5 Maryborough 25 M BN-12 Rainbow Beach 26 L BN2* Rainbow Beach 26 H BN3 Rainbow Beach 26 O BN11 Rainbow Beach 26 H BO2-7 Rainbow Beach 26 O
88 Resistance Fly Strain Town Latitude Lufenuron BO/2-6 Rainbow Beach 26 O BO2-8 Rainbow Beach 26 O BL1 Redland Bay 27 O BL4 Redland Bay 27 M BL9* Redland Bay 27 H BL16 Redland Bay 27 O BL12 Redland Bay 27 O BL3 Redland Bay 27 O BL14 Redland Bay 27 M BL15 Redland Bay 27 O BK2-12 Kingscliff 28 O BK2-13 Kingscliff 28 O BK3-12* Kingscliff 28 H BK4-20 Kingscliff 28 M BI/1-1 Red Rock 30 O BI/1-8 Red Rock 30 O BI/4-18 Red Rock 30 O BI/1-4 Red Rock 30 L BI/1-5* Red Rock 30 H BI/3-1 Red Rock 30 O BI/4-11 Red Rock 30 M COFFS83 Coffs Harbour 30 M COFFS16 Coffs Harbour 30 O COFFS99* Coffs Harbour 30 H COFFS4 Coffs Harbour 30 L COFFS68 Coffs Harbour 30 L COFFS90 Coffs Harbour 30 L TFN1-42 Coffs Harbour 30 L G3 Parkes Forest 33 O G4 Parkes Forest 33 O G5 Parkes Forest 33 O G17 Parkes Forest 33 O CCO-7 Cedar Creek 34 L CCO-11 Cedar Creek 34 L CCO-17 Cedar Creek 34 O CCO-19 Cedar Creek 34 O W1-8 Wandin 37 L W1-16 Wandin 37 L W1-5 Wandin 37 L W1-15 Wandin 37 M DI-6 Franklin 43 O DI-7 Franklin 43 O DI-8 Franklin 43 O DI-15 Franklin 43 L
89 At the outset it should be acknowledged that the sample sizes in this survey was small. There is no obvious geographical trend, but comparisons between collection localities based on these sample sizes are not valid or meaningful. However, the data do reveal a significant amount of variation for the lufenuron resistance phenotype in keeping with the findings of previous studies (Wilson & Cryan, 1996;
O'Keefe, 1997, Thompson, O’Keefe & Batterham, Unpubl). 8 strains showed High resistance, 12 strains showed Medium resistance and 16 strains low resistance.
90 1.6ppm lufenuron 100%
90%
80%
70%
60%
50%
40% Survival wrt 0ppm control Survival wrt 30%
20%
10%
0% 8 0 1 1 8 2 5 6 7 3 5 5 6 3 9 -6 -7 -8 -8 -1 -4 -5 -8 -1 - -8 1 1 1 - -15 G G4 G 1-4 -1 -1 - 1 FF4 I DI DI DI DI BL1 BL3 BL4 BL9 1-42 Arm CR1 CR3 CR4 CR8 CR9 BX4 BN2 BN3 G 2-10 ORC O FF6 FF9 WC DI BL12 BL14 BL15 O NB16 CR11 CR11 BX14 BX20 BX20 BN11 W W1 N BI/1 BI/1 BI/1 BI/1 BI/3 WI-1 W ffs 1 ffs 8 ffs 9 CF BN-12 C CG1-2 BO2-7 BO2-8 O O CCO-7 C BW2-4 BW2-8 BW2-9 BS 1-5 BI/4 BI/4 o o o F CF BK3-12 BK4-20 CR1-13 CR1-17 BX1-11 DC1-17 DC1-22 DC1-24 DC1-25 BK2-12 BK2-13 C CCO-17 CCO-19 C C BO/2-16 T Strain C C C
Figure 3.8: a) Survival of Natural population strains at 1.6ppm lufenuron, scaled according to survival on 0ppm controls.
91 1.8ppm lufenuron 100%
90%
80%
70%
60%
50%
40%
Survival wrt 0ppm control 30%
20%
10%
0% 2 G3 G4 G5 2-4 2-8 2-9 DI-6 DI-7 DI-8 DI-8 BL1 BL3 BL4 BL9 Arm CR1 CR3 CR4 CR8 CR9 BN2 BN3 BX4 G17 ORC WC DI-15 BL12 BL14 BL15 NB16 CR11 CR11 BN11 BX14 BX20 BX20 W1-5 W1-8 BI/1-1 BI/1-4 BI/1-5 BI/1-8 BI/3-1 WI-15 WI-16 CF1-4 BN-12 BO2-7 BO2-8 CG1-2 CCO-7 COFF4 BW BW BW BS 1-5 BI/4-11 BI/4-18 CF2-10 CR1-13 CR1-17 BK4-20 DC1-17 DC1-22 DC1-24 DC1-25 BK2-12 BK2-13 BK3-12 BX1-11 CCO-11 CCO-17 CCO-19 COFF68 COFF90 BO/2-16 TFN1-42 Strain Coffs 16 Coffs 83 Coffs 99
Figure 3.8: b) Survival of Natural population strains at 1.8ppm lufenuron, scaled according to survival on 0ppm controls.
92 2.1ppm lufenuron 100%
90%
80%
70%
60%
50%
40%
Survival wrt 0ppm control Survival wrt 30%
20%
10%
0% 2 2 3 11 16 G3 G4 G5 -12 DI-6 DI-7 DI-8 DI-8 BL1 BL3 BL4 BL9 CR3 CR4 CR8 CR9 Arm CR1 BX4 BN BN G17 ORC WC DI-15 BL12 BL14 BL15 CR11 CR11 NB BX14 BX20 BX20 BN W1-5 W1-8 WI-15 WI-16 BI/1-1 BI/1-4 BI/1-5 BI/1-8 BI/3-1 CF1-4 BN CG1-2 BO2-7 BO2-8 CCO-7 BW2-4 BW2-8 BW2-9 BS 1-5 COFF4 BI/4-11 BI/4-18 CF2-10 CR1-13 CR1-17 BX1-11 DC1-17 DC1-22 DC1-24 DC1-25 BK4-20 BK2-12 BK2-13 BK3-12 CCO-11 CCO-17 CCO-19 COFF68 COFF90 BO/2-16 TFN1-42 Strain Coffs 16 Coffs 83 Coffs 99
Figure 3.8: c) Survival of Natural population strains at 2.1ppm lufenuron, scaled according to survival on 0ppm controls.
93 The observed differences in resistance between strains observed could be due to the presence or absence of resistant allele(s), but also due to their frequency. The use of isofemale strains in this assay meant that there were up to four alleles segregating at any one locus, and as such, much of the observed variation may be due to variations in allele frequency at loci of interest. In accordance with such a hypothesis, variation between strains was high even when strains were reared on control media (data not shown). Thus no statistical tests were performed on these data, but rather, highly resistant strains were kept for future examination (marked in table 3.5). The results of re- screening of some of these strains is shown in figure 3.9. The viability of these strains on concentrations above 2.1ppm indicates that they are genuinely resistant compared to the Armenia control.
There were no strains that showed larval to adult viability levels that were significantly higher than resistant strains previously isolated. Therefore, since the NB16 strain had been previously investigated, the remainder of this chapter concentrates on NB16.
94 90 %
80 %
70 %
60 % BW2- 8 t n
o CF 1 - 4 c
m 50 % CO FF S 9 9 p 0p
t CG 1 - 2 r
w BK3 - 1 2
val 40 % i v
r BL 9 u S BI / 1 - 5 30 % BN 2 ARM
20 %
10 %
0% 1.6 1.8 2.1 2.2 2.3 2.5 Conc e ntr ation lufe nur on (ppm )
Figure 3.9: Re-screen of strains kept for further analysis. Survival is measured as a percentage with respect to survival on 0ppm lufenuron.
95
3.3.2 Genetic and molecular mapping of resistance genes in
NB16 strain
3.3.2.1 Fold resistance and dominance
It had been determined previously that resistance in the NB16 strain was dominant, since heterozygotes and homozygotes were seen to be equally resistant (Magoc,
2001). It was also established that resistance was high, but it was beneficial to know the level of resistance on a more quantitative basis. This would allow more appropriate choice of screening concentrations and act as a measure to make a reliable comparison to other resistant strains. To this end dosage mortality curves were created for the NB16 strain and compared to those of the control strains CanS and Celera. The results are shown below in figure 3.10 and table 3.6.
96 3
y = 4 . 7897x + 1. 01 84 2 R = 0.963 y = 5 . 087 4x + 0. 8516 2 2 R = 0. 9653
1 y = 3 . 6146x - 1. 15 11 R2 = 0. 9024
Probi t Mortality 0 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
-1
NB 16 -2 Ce l era Ca nS
-3 Log D os e (ppm l ufenuron)
Figure 3.10: Dosage mortality curve for NB16 compared to CanS and Celera including trendline and associated equation, error bars (standard error), and regression coefficient.
97 Table 3.6: Lufenuron resistance in NB16 compared to CanS and Celera at various lethal concentrations (LCs)
LC(ppm) Fold resistance Fold resistance Strain (95% CL) wrt CanS wrt Celera 2.08 NB16 LC50 3.06 3.40 (1.98-2.19) 5.94 NB16 LC95 4.15 4.39 (5.64-6.23) 9.16 NB16 LC99 4.70 4.89 (8.71-9.62)
Based on these data, working screening concentrations of 2-2.3ppm lufenuron were chosen as they kill >99% of both CanS and Celera individuals, and <50% of NB16 individuals. It was also determined that NB16 showed approximately 3-fold resistance with respect to CanS.
3.3.2.2 Genetic mapping
A genetic cross (figure 3.1) was undertaken using a mapping strain containing the flanking phenotypic markers glass (gl) and ebony (e). Assuming random recombination within the region, a genetic map position of the resistance locus can be determined by comparing the number of emergent individuals of each recombinant phenotypic class. A smaller proportion of recombinants of one class would suggest the resistance locus is closer to the associated genetic marker since there is less chance for recombination to occur over a smaller distance. The larger the number of recombinants scored, the more precisely the location of the resistance locus can be determined.
98 322 recombinants were scored, comprising 62 gl e+ and 260 gl+ e phenotypes. Data from this cross is summarized in table 3.7 below. Based on the phenotypes, the resistant locus is approximately 28% or 1.98mu or 803kb from the gl phenotypic marker.
Table 3.7: Number of recombinants of each recombinant class and associated genetic mapping information
Survival on 2.1ppm lufenuron Distance from gl % of map Viability Corrected 3 Phenotype 1 2 distance 4 5 number Ratio number Genetic Molecular gl e+ 62 1.279 79.2 28.0 gl+ e 260 0.782 203.3 72.0
Total 322 282.5 1.98mu 803kb
1 Represents the % survival on 0ppm lufenuron. With no insecticide, gl e+ and gl+ e recombinants should emerge in equal numbers; Only 61% of gl e+ recombinants survived and 78% of gl+ e recombinants survived. A correction was made for these viability differences in estimating the map position of the resistance locus. The correction was calculated by dividing the percentages of viability values by each other. Thus the gl e+ ratio is 78/61, and the gl+ e ratio is 61/78. 2 Represents the adjusted number of recombinants expected accounting for the differences in fitness. 3 Represents the percentage of the gl – e map distance accounted for by each recombinant class. 4 Represents the predicted map position of the resistance locus within the 7.1mu region with respect to the gl locus; the locus is 1.98 mu from gl. 5 Represents the predicted molecular map position of the resistance locus based on the 2868kb molecular distance between the phenotypic markers; the locus is 803kb from gl.
99 3.3.2.3 Molecular Mapping
264 resistant recombinants from the above genetic cross comprising 62 gl e+ and
204 gl+ e were scored with the molecular markers as described in table 3.2. The remaining 56 gl+ e recombinants were not scored since the number of gl e+ recombinants was likely to be the limiting factor in refining the position of the resistance locus. Random recombination events would allow determination of its position since an individual must contain the resistant phenotype in order to survive.
Since gl+ e recombinants refine its location from one side, and gl e+ recombinants refine its location from the other side, fewer gl e+ recombinants means a lesser chance of a recombination event occurring near the resistance locus. Each individual used in mapping was heterozygous for the resistant locus since lines were maintained by re-crossing individual males to the gl e mapping strain in each generation.
Mapping became finer through successive rounds of molecular marker scoring. Initial recombinants isolated were mapped using just three broadly spaced markers, and after a position of the resistant locus was assigned within those markers, further markers were developed within the region of interest, and the remaining recombinants were scored with respect to the new markers. This process is shown diagrammatically in figure 3.11.
100 2868kb glass cha microsat oamb ebony 338kb 1040kb 967kb 523kb
1040kb
18208 del 142kb 477kb 421kb
477kb endo 14289 npi atp nos 58.2kb 61.9kb 58.9kb 67.6kb 62.2kb 169kb
55 ggeenneess 26.8kb 30.7kb 4.7kb
Figure 3.11: : map of polymorphisms used to molecularly map the lufenuron resistance gene in NB16.
101
When markers internal to these were used, all remaining recombinants were eliminated from being able to provide additional position information. Therefore the narrowest region that encompasses the resistant locus was seen to be 30kb.
Inspection of the annotated D. melanogaster genome sequence (Flybase, 2004) indicated that this region contains 5 genes. The arrangement of these genes is shown below in figure 3.12.
Figure 3.12: Molecular map of the region containing the putative resistance locus with positions of molecular markers highlighted in red. Also shown is a cytological map and molecular coordinates within chromosome III (Flybase, 2004).
102 3.3.3 Candidate genes
Two of the five genes located within the 30kb interval to which resistance maps are
cytochrome P450 genes. The remaining genes are of unknown function (table 3.8).
Given the literature (reviewed in section 1.5) implicating P450s in insecticide
resistance, Cyp12a4 and Cyp12a5 were adopted as the best candidates for the
resistance gene(s).
Table 3.8: Candidate resistance genes within in the mapped region (Flybase, 2004).
Note that CG11779 is included as an additional candidate (in parentheses) since the
polymorphism used was found within the 5’ untranslated region of the gene.
Gene Function Gene structure
Cell CG6040 communi- cation
Cyp12a5 Detoxification
Cyp12a4 Detoxification
CG5629 Unknown
CG5835 Unknown
Protein (CG11779) translocation
103 3.3.3.1 Sequencing candidates
An individual male from the NB16 strain was crossed to virgin females of either
Df5597 or Df2411, and the progeny screened on food containing 1.8ppm lufenuron.
DNA of the progeny was then isolated. This essentially allowed the provision of DNA from one genome, enabling sequencing of the genes in the region without the possible presence of polymorphisms that may confound the identification of a resistance-conferring mutation.
The entire region encompassing Cyp12a5 and Cyp12a4 was sequenced. It has been seen previously that the mutations in cytochrome P450 genes which cause resistance predominantly do so through a mechanism causing overexpression of the
P450 (Taylor & Feyereisen, 1996). There is therefore a high probability that a resistance-conferring mutation could be in an upstream or downstream regulatory element, and thus these were sequenced as well as the genes themselves.
Cyp12a4 sequencing revealed two coding region polymorphisms as detailed in figure
3.13, but no inferences could be drawn from these. Sequencing of the surrounding regions revealed a 33bp deletion 358-391bp downstream of the 3’UTR, however this deletion was found in the cn vg mapping strain which does not show resistance or
Cyp12a4 overexpression.
104 NB16 1178 TGATGC 1835 CGGAAT Celera 1178 TGGTGC 1835 CGCAAT
NB16 358 3’ ------Celera 358 3’ TAAAATGCCCAAAACAAAAGAGTTAACAGTTGA
Figure 3.13: Graphical representation of the location of the Cyp12a4 sequence differences between Celera and NB16, indicating the most significant difference in a
33bp deletion 3’ of the gene.
The Cyp12a5 gene sequencing revealed several polymorphisms between the NB16 strain and Celera, the most significant of these being a 2bp deletion in exon 2 causing a frameshift and subsequent premature termination codon (figure 3.14).
a) NB16 358 ATGGA–- Celera 358 TTTGAAA
1 11 21 31 41 Celera MLKGRIALNILQSQKPIVFSASQQRWQTNVPTAEIRNDPEWLQAKPFEEI NB16 MLKGRIALNILQSQKPIVFSASQQRWQTNVPTAEIRNDPEWLQAKPFEEI b) 51 61 71 81 Celera PKANILSLFAKSALPGGKYKNLEMME...MIDALRQDYG~~~(Continued) NB16 PKANILSLFAKSALPGGKYKNLEMFDD*
Figure 3.14: a) Graphical representation of the location of the Cyp12a5 sequence differences between Celera and NB16, and b) comparison of the amino acid sequences, indicating the frameshift bringing about a stop codon (*).
105
Primers were designed for allele-specific PCR reactions to detect this polymorphism in other natural population strains. Genomic DNA from 64 strains from various locations throughout Australia was provided by Claire Milton (Department of
Genetics, University of Melbourne, Australia). These strains had been made isogenic for chromosome III (C. Milton, Pers. Comm.). None of the strains contained the polymorphism (data not shown) and so resistance testing was not performed.
3.3.3.2 Expression levels of candidates
Extraction of mRNA, quantitation, and reverse transcription was performed using the control strains CanS and Celera, and the resistant strains WC2, Inn5 (refer to chapter
5 for more details of these strains), and the NB16 strain in question. Transcription levels of Cyp12a4 and Cyp12a5 were tested using realtime RT-PCR (section 2.2.9).
The results of these tests are encapsulated in figure 3.15 and figure 3.16.
There is no significant difference in Cyp12a5 transcript levels when comparing NB16,
CanS and Celera (figure 3.15). However Cyp12a4 has a 4.2-fold higher transcription level in the NB16 strain in comparison to these susceptible lab-strains (figure 3.16).
This specific over-transcription suggests that it may be the resistance conferring mechanism based on the examples of P450-mediated resistance in the literature.
106 1.6 Figure 3.15: Level of Cyp12a5 1.4 mRNA in NB16 compared to 1.24 1.18 nS
a 1.2 mRNA levels in susceptible C t 1 r 1 w strains CanS and Celera. l e v e 0.8
on l i
pt 0.6 i r c s
n 0.4 a Tr 0.2
0 NB16 CanS Celera Strain
4.5 4.195
4
3.5
nS 3 a C t r
w 2.5 vel e l on i
t 2 p i scr
an 1.5
Tr 1.203 1.251
1 1 0.85
0.5
0 NB16 WC2 Inn5 CanS Celera Strain
Figure 3.16: Level of Cyp12a4 mRNA in NB16 compared to susceptible strains
CanS and Celera. Also shown are the mRNA levels of other resistant strains WC2 and Inn5 (see chapter 4 and chapter 5 for further discussion of these strains).
107
3.3.4 Overexpression construct
In order to test the hypothesis that Cyp12a4 overexpression is responsible for resistance, the Cyp12a4 gene from the susceptible Celera strain was overexpressed using the Gal-4/UAS system. 15 transgenic strains containing the Celera Cyp12a4 cDNA attached to an upstream activating sequence (UAS) were created. Two of these were crossed to a ubiquitously expressing gal4 strain (carrying a tubulin promoter – labeled as (tub)gal4 in table 3.1) and scored for survival on lufenuron
(figure 3.17). This strain is maintained over a balancer chromosome containing the
Sb (Stubble bristle) marker. The surprising result was that only adults containing the
Sb marker emerged, even on a 0ppm control. It appeared that the temporal and spatial overexpression pattern of Cyp12a4 driven by the tubulin promoter caused lethality. Appropriately under screening concentrations of lufenuron, no resistance was seen since only adults carrying the Sb balancer chromosome (and no overexpression) emerged (as shown on figure 3.17).
A different outcome was observed when Cyp12a4 expression was driven by a different enhancer. The transgenic strains were crossed to a strain driving gal-4 expression in the midgut and fatbody of the fly (courtesy P. Daborn, Department of
Genetics, University of Melbourne – labeled (6g1CS)gal4 in table 3.1), where most
P450-mediated detoxification takes place. These crosses showed comparable resistance to positive controls carrying an NB16 chromosome, suggesting that overexpression of Cyp12a4 in specific metabolic tissues may be the cause of lufenuron resistance.
108 0.9
0.8
0.7 1ppm 1.3ppm
l 0.6 o
r 1.6ppm t n 1.9ppm co
m 0.5 p 0p t r w 0.4 val i v r u S
% 0.3
0.2
0.1
0 Caroline x w1114 Jennifer x w1114 Caroline x Jennifer x Caroline x Jennifer x NB16 x Caroline NB16 x (Tub)Gal4 (Sb) (Tub)Gal4 (Sb) (6g1CS)Gal4 (6g1CS)Gal4 (6g1CS)Gal4
Figure 3.17: Resistance in Cyp12a4 transgenics Caroline and Emily. Negative controls – strains crossed to w (white), positive controls – strains crossed to NB16, and experimental – strains crossed to (Tubulin)Gal4 where only strains carrying balancer chromosome emerged, and strains crossed to (5’6g1CS)Gal4.
109 3.4 Discussion
3.4.1 This study
Resistance to lufenuron has been reported to be widespread in both USA and
Australia (Wilson & Cryan, 1996; Wilson & Cain, 1997; Thompson, O’Keefe &
Batterham, Unpubl). Results of a field resistance survey described here corroborated the indications of widespread lufenuron resistance. The nature of the surveys conducted in these investigations meant that a statistical comparison of the significance of resistance levels between strains was inappropriate. The proportion of strains considered “resistant” could be influenced by variation within each strain, and as such, is quite subjective. Instead, individual strains were highlighted for further analysis. These strains may become particularly useful in identifying new alleles of resistance-conferring genes, or in fact identifying new resistance mechanisms.
The strain chosen for further analysis in this study is the NB16 strain from Wandin in
Victoria. It showed high levels of resistance which appeared to be linked to a single genetic locus on chromosome III, (O’Keefe, 1997) although a cluster of tightly linked genes could not be excluded. Using genetic and molecular mapping techniques, the position of the resistance locus in this strain was determined. It lies on chromosome
III between the phenotypic marker genes glassy eye (gl) and ebony body (e). Genetic mapping placed it approximately 800kb (1.98mu) from gl (table 3.7). This correlates well with the position calculated using a more precise molecular mapping method.
The gene(s) is located in a 30kb region approximately 755-785kb from gl (table 3.1, figure 3.11). It remains to be seen whether the same gene region is associated with resistance in other lufenuron-resistant strains.
110 3.4.2 Candidate genes
There are 5 genes within this region (Table 3.8), but two stood out as obvious candidates - Cyp12a4 and Cyp12a5. Both are cytochrome P450s, members of a detoxification gene super-family previously implicated in resistance to insecticides
(Feyereisen, 1999). Cross resistance to diflubenzuron was seen in the NB16 strain, further support for a detoxification based mechanism of resistance. However since the structure of diflubenzuron is so similar to that of lufenuron (see section 1.7), it is possible that the protein target of these two insecticides is the same, and that resistance is due to target site modification.
Since only Cyp12a4 and Cyp12a5 stood out as potential resistance-conferring genes, efforts were focused on these genes. The presence of a structural mutation in the Cyp12a5 gene (figure 3.14) has the effect of causing a premature stop codon in the transcribed RNA. The transcript may be broken down by cell machinery in a process called nonsense-mediated decay (NMD) which, as the name suggests, destroys RNA species that harbour nonsense mutations (Hentze & Kulozik, 1999;
Maquat, 2004). Such a mutation could confer resistance if Cyp12a5 has a role in activating lufenuron, increasing its toxicity. In the absence of Cyp12a5 activity, the
NB16 strain would be more resistant than strains lacking this mutation. It should be noted that recessive resistance phenotype would be expected if a “knockout” of the gene (based on the homozygous knockout mutation that is effectively seen in NB16 under this hypothesis) results in resistance. But resistance in this strain is dominant, and the levels of transcript as analysed through real-time RT-PCR were no different in NB16 compared to susceptible control strains, suggesting that the polymorphism in
Cyp12a5 is not involved.
111 Cyp12a4 was also examined. Cyp12a4’s closest known orthologue is Cyp12a1 from
Musca domestica based on blast searches for sequence similarity. Given how rapidly
P450 genes evolve, the two genes have a high protein and sequence similarity.
Furthermore, there is evidence that the Cyp12a1 gene is involved in insecticide resistance in M. domestica. In the Rutgers strain, it is 5-fold overexpressed and maps to chromosome II (which is syntenic to IIIR in D. melanogaster) (Guzov et al., 1998).
It was also shown to be able to detoxify Aldrin, Heptachlor, and Diazinon, thus satisfying the two criteria for demonstrating P450 based resistance in the Rutgers strain. However, this strain has not been tested for resistance to diflubenzuron or lufenuron.
Cyp12a4 mRNA was seen to be present at a 4-fold excess in NB16 compared to other susceptible and resistant strains (figure 3.16). This apparent overexpression is consistent with a role in resistance (Feyereisen, 1995). Whilst there were some DNA sequence changes observed, these changes were silent, not affecting the amino acid sequence of the enzyme. Regulation of P450s (section 1.5.6) is an important contributor to P450-mediated resistance, and so the regulatory regions of this gene, which are not specifically defined, may contain key resistance conferring mutation(s).
Analysis of transgenic Cyp12a4 overexpressing strains (figure 3.17) suggests that differences in spatial and temporal regulation of this gene may be responsible for the observed resistance. When overexpressed in digestive tissues such as the midgut and fatbodies, a level of resistance comparable to NB16 was seen in these strains.
However when also overexpressed in other tissues, a lethal phenotype was observed. Based on these data, it is likely that it is the overexpression of Cyp12a4 in specific tissues in NB16 that is causing resistance. While not the focus of this study, the lethality observed here is intriguing. It appears that, when expressed in inappropriate cell types, Cyp12a4 may produce toxic metabolites.
112
3.4.3 Resistance in Natural populations
Resistance to insecticides in D. melanogaster has been reported many times to a wide variety of insecticidal classes over the years following insecticide usage
(Wilson, 2001). It is generally presumed that being genetically and physiologically similar to insect pests, D. melanogaster evolves resistance via similar mechanisms.
This has been shown in some cases to be true (as discussed in the introduction).
Thus conservation of resistance-conferring mechanism between species is a notable prospect and validates the study of D. melanogaster, as well as providing a possible explanation for the widespread resistance seen.
Given this assumption, resistance in Australian D. melanogaster field strains would have presumably resulted through incidental exposure to another previously used insecticide that is used to treat pest species. Only insecticides chemically related to lufenuron and with the same target could confer target-site resistance to lufenuron.
Several other IGR-type insecticides with similar structures are registered for commercial use in Australia. These are flufenoxuron and chlorfluazuron (see chapter
1, figure 1.9 for structures). It is possible that resistance has evolved to these chemicals due to a mutation in a target, and the similarity in chemical structure has allowed lufenuron resistance to develop. Comparing cross resistance to flufenoxuron and chlorfluazuron using lufenuron resistant strains would either help support or disprove this hypothesis.
Another possible reason for lufenuron resistance in field populations is that D. melanogaster feeds on rotting fruit that is generally high in nitrogen. Furthermore, nitrogen based fertilizers are commonly used in food crops. These two properties
113 permit speculation that high nitrogen contents might trigger an insensitivity to the nitrogen (urea) based insecticides like lufenuron, in turn providing a reason why resistance may have evolved despite lack of direct selection by lufenuron. It is likely to be extremely widespread under this assumption, given that this diet is endemic to virtually all D. melanogaster populations. This theory could be tested in lufenuron resistant and susceptible strains by altering nitrogen content of their food, or again comparing cross resistance patterns of lufenuron resistant strains using other nitrogen-based insecticides. In the NB16 strain, the cross resistance to another nitrogen-based insecticide, diflubenzuron, does support this reasoning.
Considering the lack of field usage of lufenuron or lufenuron-like insecticides, and the implausibility of the natural diet selecting for resistance, it is more feasible to assume that resistance is metabolic based: that it has developed through the broad specificity of detoxifying enzymes like P450s conferring cross resistance to intensively used insecticides such as pyrethroids, carbamates, and organophosphates. Thus the mapping of resistance to a small region containing two P450s is unlikely to be coincidental. It remains to be shown how one or both of these P450s plays a role in lufenuron resistance, but the creation of a targeted knockout of Cyp12a5 may shed light on the mechanism.
Once further knowledge of the molecular basis of resistance has come to light, it would be possible to test whether the same mechanism is present in all of the previously isolated mutants that mapped to chromosome III. The following two chapters therefore further examine field-based lufenuron resistance, again highlighting the role of P450s in resistance, how it might have evolved, and its spread in other natural populations.
114
Chapter 4
Field Resistance in USA
4.1 Introduction
4.1.1 Insecticide resistance from a historical perspective
The evolution of resistance in insect species became a global issue after the widespread introduction of DDT to control malaria transmitting mosquito species
(Brown, 1967). The ubiquitous nature of the DDT target meant that the evolution of resistance in other species that were exposed was inevitable. Accordingly, reports of resistance in natural population strains of D. melanogaster were seen as early as
1954 (Bochnig, 1954; King, 1954; Crow, 1954). Several studies concluded that laboratory-selected resistance was polygenic, with significant contributions coming from chromosomes II and III (Crow, 1954; Dapkus & Merrell, 1977). Some studies using field-derived resistant strains mapped a single dominant locus at 65cM on chromosome IIR (Ogita, 1960; Ogita, 1961; King & Somme, 1958).
Whilst genetic mapping resolution was insufficient to clone the gene responsible, a
P450-based mechanism was postulated following the discovery of negative cross resistance to phenylthiourea (PTU) (Ogita, 1960). It was suggested that the increased amount of P450 allows the metabolism of PTU into the toxic phenylurea.
Furthermore, increased P450 enzymatic activity was seen to be linked to the locus at
65cM on chromosome II (Hallstrom, 1985).
In more recent studies, molecular markers and recombinational mapping were used to precisely localize the DDT resistance locus in two field strains of diverse geographical origin - Hikone-R (Japan) and Wis1 (Wisconsin). In both cases resistance was mapped within the cytological interval 48D5-6 to 48F3-6 on
116 chromosome IIR which, in agreement with the earlier studies, corresponds to a map position of 65cM (Daborn et al., 2001). This interval contains a cluster of P450 genes including Cyp6g1, Cyp6g2, and Cyp6t3. 13 P450 genes were examined for expression levels using northern blot analysis. These were chosen on the basis of their documented overexpression in other resistant strains of D. melanogaster (or putative orthologues in resistant strains of other species), or the D. melanogaster
P450s (or their putative orthologues) mapping near map position 65 on chromosome
II. Of these, Cyp6g1 was the only P450 overexpressed in Wis1 and Hikone-R. In addition to this, cross resistance to the neonicotinoid insecticide immidacloprid was seen (Daborn et al., 2001).
4.1.2 Lufenuron resistance in field populations in USA
Wilson and colleagues screened for lufenuron resistance in natural populations of D. melanogaster as a way of addressing the genetic and molecular basis of resistance to IGRs in this ideal model organism (Wilson & Cryan, 1996; Wilson, 1988); section
1.7.1).
Isofemale lines were collected from Vermont, USA (1991), and inbred to minimize heterozygosity. They were then screened on lufenuron 4-6 generations later. Several strains had high level resistance compared to standard laboratory strains (Wilson &
Cryan, 1996). To determine whether resistance was a local population effect, more strains were collected from Colorado, USA (1994), and subjected to the same tests.
Similar results were seen, indicating that resistance is widespread and not a single season phenomenon in a given population (Wilson & Cryan, 1996). One of the strains, WC2, showed an unchanged level of resistance after four years of unselected maintenance in the laboratory. This suggested that the strain was
117 homozygous for the lufenuron resistant gene. Given that the strain was quite viable, it also appeared that there were no significant negative fitness effects associated with the resistance-conferring mutation(s) (Wilson & Cryan, 1996).
Wilson postulated that lufenuron resistance had evolved as a result of cross resistance to other insecticides that had been widely used for the preceding three decades. On the basis of similarity of chemical structure, and hence the potential for similar metabolic degradation, carbamates were suggested to be the insecticides to which resistance had originally evolved (Wilson & Cain, 1997). To this end, cross resistance to the carbamate propoxur was tested in a variety of geographically separated field strains. Some strains were resistant to propoxur but not lufenuron, and others to lufenuron but not propoxur. Some, such as WC2, were resistant to both insecticides. Overall no specific correlation between propoxur and lufenuron resistance was found (Wilson & Cain, 1997).
Further work with the most resistant strain, WC2, indicated P450 involvement in lufenuron resistance. Resistance was partially suppressible by the P450 inhibitor piperonyl butoxide (PBO) and mapped to chromosome III and 65cM on chromosome
II. Wilson observed DDT cross resistance in WC2 (Wilson, T. G., pers. comm.).
4.1.3 Aims of this project
Given that the WC2 strain exhibited DDT resistance, it was considered likely that this resistance would map to the ‘65cM locus’ on chromosome 2, since DDT resistance in
Hikone-R and Wis1 had already been mapped there (Daborn et al., 2001). Studies with other strains had mapped resistance to other insecticides to this locus (Ogita,
1960; Daborn et al., 2001), presenting the possibilty that resistance to lufenuron
118 would also map to this locus in the WC2 strain. The observation that Cyp6g1 was overexpressed in Hikone-R and Wis1 raised the interesting possibility that this gene might be responsible for cross resistance to a range of chemically distinct insecticides in these strains and WC2. Having been provided with the WC2 strain the aims of this study were to:
1. Analyse the expression levels of Cyp6g1 and other P450s
2. Precisely map the lufenuron resistance locus using molecular markers
3. Identify the resistance-conferring mutation and investigate the molecular
basis of resistance
4. Determine if Cyp6g1 expression levels are correlated with lufenuron
resistance
5. Examine the cross resistance patterns to investigate whether a single gene
might be responsible
Research from this chapter is published in (Daborn et al., 2002).
119 4.2 Materials and methods
4.2.1 Fly strains
Fly strains in use in this investigation are listed in table 4.1
Table 4.1: Fly strains used in this study. Stock numbers, where provided are those used by the Bloomington Drosophila Stock Center, the source of these stocks.
Fly Stock Use Genotype Source Strain Number
Lufenuron resistant field Vermont, Resistant strain WC2 strain USA Mapping cn vg bw cn1 vg21-3 bw1 Bloomington 3984 strains w1118 w1118; CyO/if; Sb/TM6b Bloomington 6897 Injection strain w1118 w1118/w1118 Bloomington 3605 Overexpression 3a w1118, P{UAS-Cyp6g1} Transgenic strains for 1118 Cyp6g1 8a w , P{UAS-Cyp6g1} Transgenic gal4 driver (Tub)gal4 y1; P{tubGAL4}/TM3, Sb1 Bloomington 5138 strain Chromosome III balancer TM3, Sb1/TM6B, red1 Tb1 Bloomington 1792 strain Lufenuron susceptible Celera Lab Std. Susceptible lab strain control strains Lufenuron susceptible CanS Lab Std. lab strain
4.2.2 Fly Crosses
4.2.2.1 Mapping of the resistance locus
A genetic mapping cross was carried out using a strain containing the recessive flanking genetic markers cinnibar (cn, chromosome II-57.5cM) and vestigal (vg, chromosome II-67.0cM). By collecting unmated F1 females and backcrossing them
120 to the mapping strain, phenotypic evidence of recombination events is carried through to the backcross progeny. Recombination events can then be scored both genetically and molecularly. This cross is summarized in figure 4.1.
cn S vg cn+ R vg+ X cn S vg cn+ R vg+
Screened on 1.5ppm lufenuron
cn+ R vg+ cn S vg F1 X cn S vg cn S vg
Not Screened on lufenuron F2 cn S vg+ cn R vg+ cn+ R vg cn+ Svg cn S vg cn S vg cn S vg cn S vg
Figure 4.1: Genetic mapping cross using cinnibar (cn) and vestigal (vg) phenotypic markers. Recombination occurs in the F1 females, allowing for the scoring of recombinants. The WC2 resistant strain is shown in red and the cn vg mapping strain in blue. A representation of the general position of recombination with respect to the phenotypic markers and the resistance locus is shown for each recombinant class.
The stocks of recombinants were maintained whilst in use by re-crossing males to females of the mapping strain each generation.
121
4.2.2.2 Crosses used to screen transgenic flies
Two UAS-Cyp6g1 transgenic strains were independently crossed to a ubiquitously expressing gal4 strain, (Tub)gal4 (Table 4.1). The untransformed progenitor strain w1118 was also crossed to (Tub)gal4 to act as a negative control. 100 larvae per vial were then screened on various concentrations of lufenuron (three replicates at each concentration). The number of emerging adults was scored 15-18 days later. Results for larval to adult viability on lufenuron were averaged and graphed by dividing by survival on control food for each strain.
4.2.3 Dosage mortality curves
The dosage mortality relationship was established using 15 different lufenuron concentrations to ensure that the analysis was comprehensive. Three replicates, each with 100 first instar larvae in a vial, were set up for each concentration. The number of emerging adults was scored and an average was calculated for the three replicates initiated at a given concentration. The data were converted into probit form
(using Microsoft Excel). Standard errors were calculated for each point. LC50, LC95, and LC99 were calculated using the straight line equations produced. Fold resistances were calculated as the product resultant from dividing the LC50, 95, 99 of the resistant strain by the LC50, 95, 99 of the CanS or Celera strains.
122 4.2.4 PCR Primers
The PCR Primers used in this investigation are shown below in tables 4.2, 4.3 and
4.4.
Table 4.2: Primers and restriction enzymes used in molecular mapping
For/ Cytol Rest Name Sequence (5’ – 3’) bp polymorph Rev pos Enz
Microsatellite F GAAAGTGATCTTTCCTGCGAACT 22 WBF
48B1
Microsatellite R CACTTAAGCAACTGAGATGGAGAG 24 WBR
Microsatellite F GCTAGCTGCTGAAATGGACTT 21 AC11F
48E1
Microsatellite R CTTTGTCAGTATTTGAGTTG 20 AC11R
CCT5F F GAATATGGTCGGCCTTTCATC 21
48E4 HpaII
CCT5R R AGAGAGGTCTTCAGGGTGGAC 21
CG8857F F CCTGAGTTACCGCCTTCATC 20
Bsp1 48E8-9 286I
CG8857R R CAGCAGCTTCTTCTTGGTGATAC 23
123 Table 4.3: Accord and other primers
Abbrev For/ Full Name Sequence (5’ – 3’) bp Use Name Rev
AccordForward AccF F CTTGACGAGAAAGCCGGTTG 20 Accord Sequencing AccordReverse AccR R CACAGCAAATCCAGAGGGACTC 22
Accord internal AccintF F GGGTGCAACAGAGTTTCAGGTA 22 Accord Diagnostic AccordReverse AccR R CACAGCAAATCCAGAGGGACTC 22
Cyp6g1inverseF 6g1invF F TATGGAATTGAGGGCAACTGTG 22 Inverse PCR Cyp6g1inverseR 6g1invR R CAACTGGCCTAATTCGCAAC 20
Cyp6g1RACE RACER R CAGTGCGGCGACCACCAAAAGAG 24 5’ RACE
Table 4.4: Realtime RT-PCR primers
Abbrev For/ Full Name Sequence (5’ – 3’) bp Name Rev
Cyp6g1 Realtime 6g1TaqF F CCTTCTGACAGCTGGCATTTG 21 taqman F
Cyp6g1 Realtime 6g1TaqR R CGCTTCTAACACACGGATTATC 25 taqman R
Cyp6g1 Realtime 6g1TaqProbe ACTTATGATGCAGATTATA 19 taqman probe
124 4.3 Results
4.3.1 Examining the expression levels of Cyp6g1 in WC2
Based on the previous studies discussed in section 4.1.2 and the past history of
P450-based insecticide resistance (section 1.5) the P450 gene Cyp6g1 became an excellent candidate for the lufenuron resistant gene in the WC2 strain. Cyp6g1 expression levels in this strain were examined using real-time RT-PCR. The results shown in figure 4.2 There is clearly an elevated level of Cyp6g1 mRNA in the WC2 strain compared to the CanS control, correlating with the results seen for other resistant strains (Daborn et al., 2001). The apparent overexpression seems to be constitutive, since mRNA levels were similar in selected and unselected flies (figure
4.2).
14 Figure 4.2: 12.28232312 Realtime RT-PCR 12 11.05 results showing
10 the level of nS a C overexpression of t r 8 w
l e
Cyp6g1 in the v e on l WC2 strain as i 6 s s e pr
compared to x E 4 CanS.
2 1
0
WC2 Screened WC2 Unscreened CanS Strain
125 Gene expression in the WC2 strain was also analysed on a cDNA microarrays carrying DNA sequences corresponding to all known D. melanogaster P450 genes
(The-Drosophila-P450-site, 2001). These data were provided by Rene Feyereisen and published in Daborn et al., (2002). The data are shown below in figure 4.3 (a).
Again it can be seen that Cyp6g1 mRNA levels were significantly elevated in WC2 compared to the susceptible control strain, Oregon-RC. This effect was specific to
Cyp6g1. All other P450 genes showed similar expression levels in WC2 and
Oregon-RC. For comparison purposes, the resistant strain Hikone-R was also tested.
Strikingly similar results were seen (figure 4.3(b)).
4 Cyp6g1 3.5 a)
-RC 3 n o
g 2.5 e Or
2 /
2 1.5 C
W 1 o
ti all other Cyp genes Cyp4g1
a 0.5 r 0
0 10000 20000 30000 40000 Absolute Intensity
Figure 4.3: P450 cDNA microarray data provided by R. Feyereisen (see (Daborn et al., 2002)). The ratios indicate the level of intensity of each particular P450 in
(a)WC2, and (b) Hikone-R (over page), compared to the susceptible Oregon-RC control strain.
126 3 -S n
o Cyp6g1 b)
t 2.5
Can 2
-R / 1.5 e n
ko 1 Cyp4g1 Hi
o all other Cyp genes ti 0.5 a r
0 0 10000 20000 30000 40000 Absolute Intensity
Figure 4.3 (cont): P450 cDNA microarray data provided by R. Feyereisen (see
(Daborn et al., 2002)). The ratios indicate the level of intensity of each particular
P450 in (a) WC2 (previous page), and (b) Hikone-R, compared to the susceptible
Oregon-RC control strain.
4.3.2 Genetic and molecular mapping of resistance in WC2
An important way of identifying a resistance-conferring mechanism is to locate the resistance locus within the genome, or if a candidate is known, as in the case of the
WC2 strain, to ensure that resistance maps to the candidate. Accordingly, here lufenuron resistance was mapped with respect to molecular markers flanking the
Cyp6g1 gene.
4.3.2.1 Level of resistance
A dosage mortality curve was created for WC2 to enable the most appropriate lufenuron concentrations to be used in subsequent experiments, including the genetic mapping cross. The dosage mortality curve is shown in figure 4.4, and estimates of the fold resistances are shown in table 4.5.
127 3.000
2.500 y = 4.7897x + 1.0184 y = 5.0874x + 0.8516 R2 = 0.963 R2 = 0.9653 2.000
y = 3.5837x - 0.5356 1.500 R2 = 0.9431
1.000 y t i l a t r o 0.500 M t obi r P 0.000 -0.400 -0.300 -0.200 -0.100 0.000 0.100 0.200 0.300 0.400 0.500 0.600
-0.500
CanS -1.000 Celera WC2
-1.500
-2.000 Log Dose (ppm lufenuron)
Figure 4.4: Dosage mortality curve for WC2, CanS, and Celera with line of best fit equation and standard error bars.
128 Table 4.5: Fold resistance estimates for WC2 compared to CanS and Celera at lethal concentrations (LCs) LC50, LC95, LC99.
Fold resistance Fold resistance LC(ppm) Strain compared to compared to (95% CL) CanS Celera 1.41 WC2 LC50 2.07 2.30 (1.34-1.48) 4.06 WC2 LC95 2.83 3.00 (3.86-4.26) 6.29 WC2 LC99 3.23 3.35 (5.97-6.60)
4.3.2.2 Molecular Mapping
In contrast to the mapping cross used in chapter 3, a candidate gene was already known in the case of the WC2 strain. The Cyp6g1 gene has a high probability of contributing to resistance in WC2 based on the generalized location of the resistance locus on chromosome II, its overexpression and the cross-resistance pattern, and the response to PBO. Given this and parallel evidence for the Hikone-R strain, the mapping strategy concentrated on confirming or disproving the involvement of
Cyp6g1 as a resistance-conferring locus in the WC2 strain.
To this end, a cross was designed with no insecticide screening (figure 4.1). This would enable a greater quantity of recombinants to be generated, narrowing the interval containing the putative resistance-conferring mutation. It would also allow generation of recombinants for testing on both lufenuron and nitenpyram (performed by T. Perry (Daborn et al., 2002)). All recombinants could be scored with molecular markers. Only the recombinants with crossover events in the appropriate region would need to be screened on lufenuron to determine their resistance status and hence, the map position of resistance.
129
292 recombinants (104 cn+ vg and 188 cn vg+) were scored with the molecular
markers described in table 4.2. Each individual used in mapping was either
heterozygous for the resistant allele or homozygous for the susceptible allele since
lines were maintained by re-crossing individual males to the cn vg mapping strain in
each generation. The mapping data are shown diagrammatically in figure 4.5.
4068kb
cn MicrosatWb AC11 Cct5 Cyp6g1 CG8875 vg
3200kb 113k 46.2kb 10.4kb14.3kb 684kb
cn vg+ recombinants 57 29 4 1 3 10
cn+ vg recombinants 80 59 5 0 1 43
Figure 4.5: Mapping the lufenuron resistance gene in WC2. The molecular distance
between the genes cn and vg is 4068 kb. The position and the name of the
molecular markers scored in the recombinants are shown above the red line. The
number and location of the recombination events identified in the recombinants are
shown in the table.
130 The recombinants between markers AC11 and CG8875 (highlighted) were the most
informative in terms of locating the resistance locus when combined with lufenuron
screening. The location of the recombination events and the resistance status of
these recombinants is therefore further broken down in figure 4.6. Three cn vg+
recombinants with recombination events between Cyp6g1 and the CG8857 were
susceptible for lufenuron. This suggested that resistance maps somewhere to the left
of the CG8857 marker. Similarly, the four cn vg+ recombinants with recombination
events occurring somewhere between the AC11 and the Cct5 were resistant to
lufenuron, indicating that resistance is to the right of AC11. The same logic was
applied to the other recombinant classes shown.
WC2 allele AC11 Cct5 Cyp6g1 CG8857 cn vg allele
Number Resistance recomb. status 5 S cn+ vg
1 R cn+ vg
3 S cn vg+
1 R cn vg+
4 R cn vg+
Figure 4.6: Representation of recombinants obtained from fine scale mapping of the
lufenuron resistance locus within chromosome II in the WC2 strain. WC2 DNA is
represented in red and cn vg in blue. Shown are approximate positions of
recombination for each of the groups of recombinants with respect to the molecular
markers, and their lufenuron resistance status.
131
In summary, these results imply that resistance maps at least between the markers
AC11 and CG8875. On the tenuous basis of a single recombinant, resistance can be suggested to map between Cct5 and CG8857.
The same set of recombinants from WC2 crosses were screened with the neonicotinoid insecticide nitenpyram. From Hikone-R, crosses using both DDT and the neonicotinoid, imidacloprid were performed (Daborn et al., 2002). In each case resistance mapped to a region that included the gene Cyp6g1. This evidence strengthened the argument that the resistance-conferring mechanism was the same for multiple insecticides in both WC2 and Hikone-R. A summary of the intervals resistance mapped to in each of these strains is shown in figure 4.7.
CG8857 polymorphism
Cct5 EP(2)2051 polymorphism EP(2)0502 Cyp6g1
Figure 4.7: The interval containing a resistance-conferring locus in both WC2 (red) and Hikone-R (green) (Flybase, 2004). Also shown are the molecular and cytological locations of the various polymorphic sequences used in mapping.
132
4.3.2.3 Cross-resistance to other insecticides
Cross-resistance is an important indicator of the mechanism of resistance. Target site-based mechanisms are likely to confer resistance to a limited range of chemically related insecticides. Metabolic mechanisms can generate resistance to chemically distinct insecticides (see section 1.5.4 for examples). For WC2, levels of cross resistance to a variety of insecticides chemically distinct from lufenuron have been tested by other researchers. The results are shown in table 4.6.
Table 4.6: Insecticides tested for resistance levels in WC2 strain, and approximate levels of cross resistance observed.
Fold Insecticide Class Source resistance
(Wilson & Cain, Lufenuron Acyl-urea chemical 2.8 1997);This investigation
Nitenpyram Neonicotinoid 3 (Daborn et al., 2002)
Thomas Wilson (pers DDT Organochlorine 8 comm)
Propoxur Carbamate 8 (Wilson & Cain, 1997)
Organophosphrus Thomas Wilson (pers Dichlorvos 13 chemical comm)
133 The investigation into the strain Hikone-R continued in parallel, and again cross resistance was examined. Several insecticides were tested, and these included chemically distinct insecticides of several classes. Some of these insecticides were also tested on WC2. Results are shown in table 4.7.
Table 4.7: Insecticides tested for resistance levels in Hikone-R strain, and approximate levels of cross resistance observed.
Fold Insecticide Class Source resistance
(Ogita, 1961), (Daborn DDT Organochlorine 13 et al., 2001)
Nitenpyram Neonicotinoid 3 (Daborn et al., 2002)
(Daborn et al., 2001); Imidacloprid Neonicotinoid 9 (Daborn et al., 2002)
Acetamiprid Neonicotinoid 7 (Daborn et al., 2002)
Organophosphorus Parathion 4 (Ogita, 1961) chemical
Organophosphorus Malathion 2-5 (Ogita, 1961) chemical
134 4.3.3 Identification of a mutation in Cyp6g1
Since Cyp6g1 was shown to be overexpressed in WC2 and resistance mapped to a small interval containing this gene in WC2, the obvious next step was to look for the mutation responsible for the overexpression. The types of mutations that may be involved in causing overexpression and conferring resistance were discussed in section 1.6.4. Their involvement in causing P450 overexpression was discussed using several examples in section 1.5.3.
In this study the entire region including and surrounding Cyp6g1 was sequenced from the WC2 strain. No coding region differences were seen in comparing WC2 to the Celera sequences, but several differences were seen in introns (data not shown).
However, one region of approximately 300bp upstream of the gene could not be amplified using the primers chosen. Therefore an inverse PCR approach was taken to amplify this region.
Genomic DNA was cut with the restriction enzyme EcoR1 (figure 4.8 (a)), chosen because of the appropriately positioned cut sites according to previously obtained sequence information from WC2 and predicted sequence from Celera (Flybase,
2004). After ligation and amplification, ligation 2 showed the expected sized product based on the Celera genomic sequence whilst ligation 1 yielded a larger product, suggesting that an insertion was present (figure 4.8 (b)). Sequencing indeed revealed an insertion containing an internal EcoR1 site. The insertion in WC2 occurs -291bp from the start site of Cyp6g1 transcription. PCR primers were designed to specifically flank the insertion, and the full insertion was subsequently sequenced.
135
Inverse PCR Inverse PCR ligation product ligation product 1 2
Accord Cyp6g1
Normal Accord EcoR1 site EcoR1 site EcoR1 site EcoR1 site Expected Size 170bp Expected Size 360bp
Actual Size 472bp Actual Size 360bp
Figure 4.8 a) Positions of PCR primers Primer set 1 Primer set 2 and expected product sizes for Inverse WC2 WC2 Cel WC2 WC2 Cel
PCR. b) 1.5% agarose gel showing for primer set 1, the expected 170bp product only in Celera, and for primer set 2, the expected 360bp product in both Celera and WC2.
Database analysis indicated the insertion was a fragment of a transposable element called Accord. There is at least one full copy of this element in the Celera genome
(Figure 4.10). Accord has the characteristics of an LTR-type retrotransposon (see section 1.6.2 for discussion) meaning that it has an internal promoter and putative regulatory factor binding sites.
136
Comparative analysis of the Hikone-R strain showed that this strain contained the same Accord fragment both in terms of sequence and the insertion location with respect to the Cyp6g1 gene. It was apparent that the presence of Accord may be related to Cyp6g1 overexpression and that it may be a widespread phenomenon among insecticide-resistant strains.
To test whether this was the case, resistant strains from around the world were subjected to PCR diagnostics by a collaborator, Phillip Daborn, to test for the presence or absence of Accord (Daborn et al., 2002). The first intron of Cyp6g1 was also sequenced in these strains to indicate whether the presence of Accord was associated with specific sequence conservation, (indicating selection for the Accord element). The results are shown below in Table 4.8 and figure 4.9.
137 Table 4.8: Susceptible and resistant strains used to test for the presence of Accord.
A tree based on Cyp6g1 intron 1 sequence was constructed (Daborn et al., 2002).
Strain Name Origin R Name Origin
S1 Canton-S Lab standard R1 Hikone-R Japan
S2 CA 1 South Africa R2 BOG 2 Columbia
S3 KSA 4 South Africa R3 PYR 2 Spain
S4 BOG 1 Columbia R4 Hikone A-W Japan
S5 BOG 3 Columbia R5 Hikone A-S Japan
S6 PVM Portugal R6 EV New York, USA
S7 M 2 Australia R7 RVC 2 California, USA
S8 VAG 1 Greece R8 RVC 3 California, USA
S9 QI 2 Israel R9 Pi[2]
Wisconsin, USA
S10 Reids 1 Portugal R10 CO 4 New York, USA
S11 Reids 3 Portugal R11 CO 7 New York, USA
S12 Wild 3B Ohio, USA R12 MR-Haifa 12 Israel
S13 Swedish-C Sweden R13 Wild 1B New York, USA
S14 MWA 1 Wisconsin, USA R14 Wild 2A Ohio, USA
S15 BV 1 Virginia, USA R15 Wild 5A Georgia, USA
S16 BS 1 Spain R16 Wild 5B Georgia, USA
S17 BER 1 Bermuda R17 Wild 5C Georgia, USA
S18 Florida 9 Florida, USA R18 Wild 10E South Carolina, USA
S19 Harwich Conneticut, USA R19 Wild 11C North Carolina, USA
S20 Oregon R-C Oregon, USA R20 Wild 11D North Carolina, USA
R21 WC2 Vermont, USA
138 n S 16 oge S * r 17 * P R11 R S R1 S1 R2 S2 R3 S3 R4 S4 5 5 R6 S6 R7 S7 R4 R13 R17 e R14 ad R10 l R16 R t c 99 5 n R a 2 t R9 s R
R8 S8 R9 S9 R10 S10 R11 S11 R12 S12 R13 S13 R14 S14 20 esi
R1 R R8 R3 R18 R15 R12 R7 R6 R19 R15 S15 R16 S16 R17 S17 R18 S18 R19 S19 R20 S20 S15 71 S 64 20 S19 s e S3 S11 ad
S10 cl S9 S13 S14 eptible S5 S7 sc u
S8 S S6 S12 S18 S 4 s
S1 n S2 la mu
0.06 0.05 0.04 0.03 0.02 0.01 0.00 si D.
Figure 4.9: a) Accord diagnostic PCR showing presence/absence of Accord for the strains from around the world. b) Tree of the strains showing the single cluster representing the resistant strains (with two susceptible strains that may be progenitors), and several clusters of susceptible strains (Daborn et al., 2002).
139 Results from this molecular and phylogenetic study showed that resistance was absolutely correlated with the presence of Accord in the worldwide sample. Resistant alleles showed no sequence variation within the Cyp6g1 intron tested, whilst susceptible alleles fell into several different clusters that are phylogenetically distinct from resistant clusters. This evidence suggests that the Accord allele spread from a single source that was strongly favoured by selection.
Similar data sets at many levels for WC2 and Hikone-R strongly suggest that the same mechanism is contributing to resistance. The concordance in overexpression, mutation, and cross resistance patterns results indicate that Cyp6g1 overexpression mediated by the Accord insertion is likely to be the mechanism of resistance in these strains. In further developing this hypothesis, the mechanism by which the presence of the Accord insertion might mediate Cyp6g1 overexpression was investigated.
4.3.4 Mechanisms of altered transcription
Some potential methods of transposon-mediated gene disruption are discussed in section 1.6.3. If the portion of Accord inserted into WC2 (and Hikone-R) contains its own regulatory binding sequences, it is possible that Accord regulatory mechanisms are influencing the transcription of Cyp6g1. If it contains a promoter, run-on transcription may cause the overexpression pattern seen. Alternatively, if it disrupts a binding site for a repressor, the overexpression could eventuate through lack of binding. These two hypotheses were therefore tested bioinformatically and experimentally.
140 The entire Accord insertion sequence (Flybase, 2004) was used as an input into a
GENSCAN (Burge & Karlin, 1997; GENSCAN, 2004) search to detect the exonic
structure of the native element including open reading frames and translated regions.
This allowed the identification of the relevant functional domains of the partial
insertion in WC2. Figure 4.10 shows these results diagrammatically.
a) 2256- 1495- 6098 6847- 1-558 2604 7404
LTR GAG POL LTR 7404bp CDS 2901-5575
DNA binding Protease Reverse RNAseH Transcriptase b)
1-421 493- 558
1-66 67-490 c)
Figure 4.10: a) Molecular structural analysis of a full-length Accord element
indicating Long Terminal Repeats (LTRs), gag DNA binding gene, and the
overlapping pol gene with associated domains. b) Enlargement of the native Accord
LTR reversed in its orientation. c) The Accord element in WC2 showing that it is in
the opposite direction to the native element and contains a 70bp deletion within the
element.
141 As shown in this figure, the portion of Accord in WC2 is in reverse orientation with respect to the Cyp6g1 gene, so the Accord promoter is unlikely to be causing run-on transcription. An analysis of putative promoter sequences was nevertheless performed using NNPP (Neural-Network-Promoter-Prediction, 2001) in order to explore this hypothesis further. As indicated below in figure 4.11, there are several putative promoters within the element that may cause run-on transcription.
Promoter predictions for positive strand : Start End Score 61 111 0.87 61 – CCCAGGTTTAAATAAATCAACTGCTCGGTTTGGTGTCAAGATCAAGAATG – 111
Start End Score 380 430 0.95 380 – GATCAATATATATATATACAGCGTTGGCACTTTGCTGATGTCGCCTACCG – 430
Promoter predictions for the reverse strand : Start End Score 452 402 0.95 452 – CACCTCCGCCTATGAAGCCCGCCCGGTAGGCGACATCAGCAAAGTGCCAA – 402
Start End Score 404 354 1.00 404 – AACGCTGTATATATATATATTGATCACGAGCTACCATGCCAGCATAGCCT – 354
Start End Score 82 32 0.85 82 – AGTTGATTTATTTAAACCTGGGCAGCAACTAACTAAGAGTAGAGAAAGGA – 32
Figure 4.11: Putative promoter sequences within the Accord element in the WC2 strain (Neural-Network-Promoter-Prediction, 2001). The highlighted nucleotide represents the starting point of transcription.
It is therefore possible that Cyp6g1 overexpression results from run-on transcription initiating at the promoter of the Accord element. To test this hypothesis, 5’ RACE was performed.
142 4.3.4.1 Testing run-on transcription
5’ RACE using a primer within the
Cyp6g1 gene showed that Figure 4.12: Products of transcription initiated at the predicted 5’ RACE for both WC2 start site in WC2 (figure 4.12), (left) and CanS (right) disproving the run-on transcription showing that both hypothesis. Indeed the 5' RACE products were the same products were sequenced and shown size. to be identical. While this experiment indicated that transcription is not initiated within the Accord sequence in WC2, it does not provide any evidence as to whether regulatory binding sites on the Accord fragment are involved in causing
Cyp6g1 overexpression.
To further examine this possibility, the Accord sequence and the Cyp6g1 upstream sequence were entered into Transfac (Wingender et al., 2000; Transfac), a database containing known transcription factor binding site sequences. Several transcription factors thought to be associated with P450 expression were considered in particular.
These were Oct-1, Barbie, CHOP, and Ahr/Arnt. Oct-1 is the vertebrate Octamer protein binding site. The binding of the Octamer protein to Oct-1 sequences leads to the repression of rat Cyp1a genes (Sterling & Bresnick, 1996). Barbie boxes are so- called (Shaw & Fulco, 1993) due to their presence in the 5’ flanking region of virtually all eukaryotic and prokaryotic P450s known to be inducible by barbiturates (Liang et al., 1995). CHOP is a mammalian nuclear protein that is activated by starvation and by ingestion of some toxins. It can act both as a direct activator of its target genes and as an indirect inhibitor by dimerizing with other members of the C/EBP
143 transcription factor family, inhibiting their ability to activate some genes (Ubeda et al.,
1996). AHR/ARNT binding sites are described in detail in section 1.6.1.
Several putative binding sites for these proteins were identified (Transfac) surrounding the Accord element insertion, within the insertion itself, and most importantly, at a site disrupted by the Accord insertion (figure 4.13). Evidence for the similarity between the mammalian and insect ahr/arnt regulatory cascades was discussed in section 1.6.1. If a binding site for this regulatory heterodimer is disrupted due to the presence of the Accord element, constitutive overexpression of Cyp6g1 could easily result.
144 Oct-1 AHR/ARNT Accord Barbie Box CHOP Insertion
-359 AATAATGAAA TTCACAAATG CATCAAAAGC TTGACGAGAA AGCCGGTTGT GTTTAATTAT
-299 TTATAGATTA TAGCGTGCAA TACTTTTCAT ATCGTATATG TATTGCGTTA ACGCTTTTAA
-239 AAATCTAACT AAACCATAGC ACACAAAAAG TAAATAAGGT TGTTAAAACT AAGAATCATT
-179 ATAATAAATG TAATCATGAC TTGTAATTAT CTTAGAGTCC CTCTGGATTT GCTGTGGTTT
-119 GTTTGTCGTA TTTTAAAGCT TTTTCCACCA CACAGGTGAA TTTATAAGTA TGCACTTGAA
-59 ATTGCTATCT CAGAACTTTT GAGACTTTCG AGTATAAAAA CGCAAACAAC ATTTCAAATC
Cyp6g1 transcription initiation
1 GCCCCAAGTG
Figure 4.13: Sequence of the upstream region of Cyp6g1 showing putative
transcription factor binding sites with respect to the position of the Accord insertion
and Cyp6g1 transcription initiation start site (Note that Accord and binding sites
within it are not to scale).
145
4.3.5 Re-examination of overexpression
While a significant body of evidence presented links between Cyp6g1 overexpression and resistance, none of it indicates that the link is causal. Such a link would be demonstrated if controlled overexpression of Cyp6g1 yielded resistance.
Accordingly, two transgenic UAS-Cyp6g1 overexpression strains (3a and 8a supplied courtesy of Phillip Daborn, (Daborn et al., 2002)) were crossed to a constitutive gal4- expressing strain (see chapter 3, figure 3.5). The progeny of these crosses showed lufenuron resistance, albeit at lower levels than WC2 (figure 4.14).
Resistance to DDT was also tested in these strains with results confirming the involvement of the overexpression of Cyp6g1 in DDT resistance (Daborn et al., 2002;
S. Rigby, Pers. Comm.).
146 40%
35%
30% 1.3ppm l
o 1.6ppm r 25% 1.9ppm ont c ppm 0 t 20% r w l a v i v r u 15% s %
10%
5%
0% 3a x 3a x 8a x 8a x w1114 x w1114 x (Tub)gal4 (Tub)gal4 (Tub)gal4 (Tub)gal4 (Tub)gal4 (Tub)gal4 (+) (Sb) (+) (Sb) (+) (Sb)
Figure 4.14: Lufenuron resistance in the progeny of UAS-Cyp6g1 transgenic strains
(3a and 8a) crossed to the ubiquitously expressing gal4 strain ((Tub)gal4). (+) refers
to the wildtype phenotype carrying gal4, and (Sb) refers to the stubble bristle
phenotype carrying the balancer TM3. Also shown are the progeny of crosses where
3a and 8a have been replaced by the untransformed w1118 strain.
147 4.4 Discussion
4.4.1 This investigation: An overview
This investigation has used WC2, a lufenuron resistant strain from the USA, to uncover a significant detoxification-based mechanism of resistance that has been subsequently detected in many populations of D. melanogaster collected from around the world. Parallel investigations were carried out by Phillip Daborn on the
Hikone-R strain.
Resistance to lufenuron in the WC2 strain was mapped using molecular and genetic techniques to an interval of approximately 25kb within chromosome II, which contained three P450 genes including Cyp6g1 (figure 4.7). This gene was selected as the resistance gene candidate because it was overexpressed in WC2 as shown by real-time RT-PCR (figure 4.2). More significantly it was confirmed as the only overexpressed P450 in the WC2 strain by microarray analysis (figure 4.3). This evidence strongly suggested that Cyp6g1 had a role in conferring resistance in this strain. The role was confirmed by the creation of transgenic Cyp6g1 overexpressing strains that also showed some resistance.
WC2 was initially isolated because it conferred resistance to lufenuron and propoxur
(Wilson & Cain, 1997), but it was later shown to be resistant to nitenpyram (Daborn et al., 2002). This nitenpyram resistance mapped to the same location as lufenuron resistance (Daborn et al., 2002). Further cross-resistance was seen towards DDT and dichlorvos (T. Wilson, Pers. Comm.), indicating that a single P450 may be capable of metabolizing a wide range of chemical structures (table 4.7).
148 The transposable element Accord was isolated upstream of the transcription start site of Cyp6g1 in the WC2 strain (figure 4.9), the Hikone-R strain, and a variety of field- derived strains from around the world (Daborn et al., 2002). It contains putative transcription factor binding sites and disrupts two such sites in the native DNA (figure
4.13). Its presence was perfectly correlated with resistance to DDT in natural population strains (Daborn et al., 2002), suggesting that some aspect associated with the existence of this single allele may be causative of Cyp6g1 overexpression and subsequent resistance.
The way in which Accord may interact with Cyp6g1 to produce overexpression was investigated using a bioinformatic and experimental approach, indicating that overexpression may be the result of the action of altered transcription factor binding.
4.4.2 P450s and resistance
The overexpression of a variety of P450 alleles has been previously shown to cause insecticide resistance, as discussed in chapter 1, section 1.5.3. Given the number of
P450s and the complexity of their interactions, it was somewhat surprising that a single P450 could confer resistance to such a wide range of chemistries. However parallel phenomena have been seen before.
The human P450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing enzymes, and collectively contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use (Hodgson, 2001).
Cyp3A4 alone metabolises 60% of all drugs in clinical use (Guengerich, 1999; de
Wildt et al., 1999). The Cyp6g1 gene product may be a D. melanogaster equivalent.
Blast searches and phylogeny trees (data not shown) indicate Cyp6g1’s closest non-
Drosophila relative is in fact the human Cyp3a4 gene.
149
It has been established that only a limited number of amino acid replacements are required for marked changes in specificity of P450s (Feyereisen, 1999), and as such, similarities between P450s may not necessarily be indicative of function. However, it should still be noted that this gene has extremely broad substrate specificity and potent detoxative ability (Lewis et al., 2002) and is therefore functionally as well as molecularly similar.
4.4.3 Accord and resistance
The possibility of transposable elements being involved in insecticide resistance has been raised in the literature (Wilson, 1993). Only one study (Waters et al., 1992) has suggested the causal role of a transposon in P450-based resistance. However this hypothesis was subsequently disproven (Delpuech et al., 1993).
The ways in which a transposable element could mediate P450 overexpression were described in Section 1.6.3.1. The hypothesis of run-on transcription of Cyp6g1 via an
Accord promoter was disproven (figure 4.12), but this does not eliminate the possibility that Cyp6g1 may be subject to regulation via enhancer sequences within
Accord. Nor does it eliminate the possibility that the insertion acts simply by increasing the distance between the 5’ transcription factor binding sites of the
Cyp6g1 gene and the startpoint of transcription for Cyp6g1.
The Accord element itself contains transcription factor binding sites for Oct-1, CHOP, and Ahr/Arnt, and the Cyp6g1 upstream sequence contains binding sites for Oct-1,
Barbie, CHOP, and Ahr/Arnt, the latter two being specifically disrupted by the presence of the Accord element (figure 4.13). Recent investigations using site-
150 directed mutagenesis have shown that specific sequences in the upstream region of the Cyp6b1 gene are important in its regulation in lepidopteran SF9 cells (Li et al.,
2002; Petersen et al., 2003). The region contains Oct-1, Ahr/Arnt, and EcRe binding sites, and mutagenesis of as little as 4bp of the consensus region significantly alters expression. Several studies also show that regulatory elements within transposon insertions can upregulate the transcription of a nearby gene (Ackerman et al., 2002;
Moran et al., 1999; Willoughby et al., 2000).
The ligand-Ahr/Arnt transcription factors have a demonstrated role in P450 regulation
(e.g. mammalian Cyp1a1, see section 1.5.6), although this role is one of positive regulation. In applying a model such as this to Cyp6g1 in WC2, it would be expected that the disruption of the putative Ahr/Arnt binding site by the Accord element would lead to a reduction in Cyp6g1 expression. To date there has been no evidence of a negative regulatory role for this complex. However, considering the interactions with the repressor AhRR (Mimura & Fujii-Kuriyama, 2003), there may be another
(negative) mode of regulation.
Studies on the Cyp1a1 gene revealed that the ligand-Ahr/Arnt complex binds within the major groove of the DNA double helix, distorting it somewhat (Carrier et al.,
1992). It has been proposed that the ligand-Ahr/Arnt complex in the Cyp1a1 gene mediates the loss of the nucleosomal structure around the promoter, thus allowing other transcription factors to bind and enhance the expression of the gene (Elferink &
Whitlock, 1990); (Ramana & Kohli, 1998). In the absence of an inducer, the transcription factors are not able to bind the DNA. It is speculated here that the absence of such a site, due to the Accord element, may influence the binding of negatively regulating transcription factors, thus effecting an overexpression expression of Cyp6g1.
151 Further work is underway to investigate the ability of Accord to drive overexpression using transgenic strains that contain the Accord element (or another piece of DNA) inserted into a susceptible strain. Also being investigated are the important components of the Cyp6g1 upstream region, using transgenic strains containing partial deletions of this region (P. Daborn, Pers comm.) It is clear though that further understanding of the regulation of P450s such as Cyp6g1 is needed in order to determine definitively how Cyp6g1 is overexpressed in this strain.
4.4.4 Why has resistance evolved?
Insecticide resistant D. melanogaster strains from several different continents consistently showed both the presence of the Accord insertion and Cyp6g1 overexpression. Conversely, susceptible strains contained no Accord or Cyp6g1 overexpression. Phylogenetic data separated these strains into two main clades, distinguished most conspicuously by these factors (Daborn et al., 2002). This evidence supports the hypothesis that there was selection of this single resistant allele which has since spread worldwide.
In most parts of the world D. melanogaster is not targeted with insecticides.
However, as a cosmopolitan species that is found across the planet, its distribution overlaps with that of many insect pests that are controlled with insecticides. It could therefore be argued that resistance to DDT, propoxur, and dichlorvos amounts to
‘collateral damage’, that it is the result of incidental exposure to insecticide intended for the control of an insect pest. Under this model, incidental exposure to one of these insecticides selected for an increase in the frequency of the Cyp6g1 Accord allele. Daborn et al (2002) have argued that DDT provided the selective agent because the Accord insertion was not detectable in field strains collected prior to its
152 introduction. No resistance was found in strains that were established in the 1930s
(Oregon-R-C, Swedish-C, Canton-S in table 4.4) but 28/75 strains established in the
1960s were Accord positive and resistant. These data are consistent with the DDT hypothesis but do not rule out some other xenobiotic being the selective agent e.g. a plant toxin, another insecticide or agricultural chemical.
Whatever the source of the initial selection, the apparent broad substrate specificity of CYP6G1, means that a range of chemically distinct insecticides used in D. melanogaster’s environment will favour the Cyp6g1 Accord allele. The relative fitness of the Cyp6g1 Accord genotype is yet to be thoroughly assessed, particularly in the absence of insecticides. The high global Accord frequencies suggest that there is either minimal fitness cost in the absence of insecticide, or that the Accord allele is maintained at these frequencies by ongoing selection.
It is rather disturbing that overexpression of Cyp6g1 confers resistance to lufenuron and nitenpyram, since these chemicals have not been used in environments frequented by D. me lanogaster. The possibility that overexpression of P450s of broad substrate specificity could lead to resistance to insecticides that have not yet been developed should concern primary producers and the agri-chemical industry. If similar broad spectrum resistance was to arise in a pest, the organism would be difficult to control with the current arsenal of insecticides.
153 4.4.5 Is there more to resistance than just Cyp6g1?
The evidence presented here confirms a role for Cyp6g1 in mediating resistance to a large range of insecticides. But a recent study implicates another P450 gene in the region contributing to DDT resistance in a field strain (Wis1) – the Cyp12d1 strain is constitutively overexpressed and is induced further by DDT exposure (Brandt et al.,
2002). A different gene in the region, Cyp6a8, is the only P450 overexpressed in the
Wis1lab strain, a strain derived from Wis1, but with the region encompassing Cyp6g1 removed via recombination, and selected further on DDT. It shows a slightly different cross resistance profile to Wis1 in addition to the different P450 overexpression profile (Le Goff et al., 2003). It is clear that there may be several resistance factors operating in different strains.
Considering lufenuron, two different P450s have been associated with resistance in the NB16 and WC2 strains. Therefore at least two mechanisms of resistance are possible. The next chapter further examines D. melanogaster strains and identifies an additional factor that may contribute to lufenuron resistance in some strains.
154
Chapter 5
Other resistance mechanisms in
natural populations
5.1 Introduction
5.1.1 Monogenic verses polygenic resistance
The evolution of the insecticide resistance phenotype in the field is affected by factors including the intensity of the insecticidal selection, the specific characteristics of the insecticides used, and gene flow. Individual differences create variation that forms a normal distribution of phenotypes within a population. As a general evolutionary phenomenon, the adaptation can therefore be either polygenic or monogenic depending on the nature of the effective selection pressure that is applied
(McKenzie & Batterham, 1994; McKenzie, 2000). If the applied insecticide selection pressure lies within the normal phenotypic distribution of the population, allowing a proportion of the population to survive, then the response is likely to be polygenically based. Since the individual characteristics of an insect (such as weight, size, and fitness) are all assumed to be influenced by many genes, these differences are maintained when selecting a certain proportion to survive. Alternatively, if the selective pressure occurs beyond the normal phenotypic distribution of a population, then a monogenic response is expected. In this case, no individuals would theoretically survive selection. So if the majority of individuals were exposed to this lethal dose of insecticide, a rapid change needs to occur for a population to persist. A single genetic change leading to a single resistant allele is likely to be the most efficient way of creating the adapted phenotype.
Insecticide application in the field usually aims to kill every individual. This generates immense selection pressure, so it is not surprising that in examples where the
156 genetic basis of field resistance has been determined, it has typically been attributed to allelic variants of single genes (McKenzie, 1996). Under the monogenic resistance model, the resistance conferring allele is likely to increase in frequency within the population under continued selection, and spread across populations where similar selective pressures are acting (for review see (ffrench-Constant et al., 2004)).
Cyp6g1 overexpression confers resistance to lufenuron and other insecticides
(Chapter 4; Daborn et al., 2002). Accord is highly associated with resistant strain and
Cyp6g1 overexpression. Therefore, the presence of Accord alleles can act as a screening tool for the examination of Cyp6g1 expression levels and the associated resistance phenotype in other strains. Thus the use of an Accord diagnostic test to examine a variety of natural population strains is one of the focal points of this chapter.
5.1.2 Several genes of major effect
What happens if the resistant allele increases to such a frequency that the insecticide is no longer useful at the current dose? An obvious answer to the problem is to increase the dose of the insecticide such that it again kills every individual. Should there be other resistance mechanisms available to the insect, again a single genetic change conferring a higher level of resistance can be selected for. Thus an augmented monogenic response, or a “major effect” polygenic response (as distinct from the traditional polygenic response previously discussed), ensues.
An example of this is evident in the so-called “knockdown” resistance to pyrethroids, seen in Musca domestica (Miyazaki et al., 1996; Williamson et al., 1996), Blatella germanica (Miyazaki et al., 1996), and Myzus persicae (Martinez-Torres et al., 1998)
157 through a single amino acid change in a para-type sodium channel gene. Some strains subsequently isolated showed a second amino acid change resulting in heightened “super-knockdown” resistance (Williamson et al., 1996). It is likely that continued selection led to the evolution of the second mutation in the same gene. In comparison, the P450s Cyp12a1, Cyp6a1, and Cyp6a2 are all able to detoxify diazinon and were found to be overexpressed in the Rutgers strain of Musca domestica (Guzov et al., 1998; Sabourault et al., 2001; Dunkov et al., 1997). In this case, several different genes of major effect may be involved in conferring the high level of resistance observed.
5.1.3 Genes in addition to Cyp6g1
Additional genes have been associated with resistance in strains known to overexpress Cyp6g1. The WIS1 strain of D. melanogaster shows overexpression of
Cyp6g1 and resistance to DDT (Daborn et al., 2001). Further DDT selection of this strain co-selects for overexpression of the nearby Cyp12d1 gene (Le Goff et al.,
2003) which is also thought to have a role in conferring DDT resistance based on its induction in the presence of DDT, and the reduction of resistance in the presence of the P450 inhibitor PBO (Brandt et al., 2002). If the Cyp6g1 overexpression phenotype is removed from this strain through recombination, Cyp6a8 overexpression is selected for (Le Goff et al., 2003). A full genome microarray of the
WIS1 strain also indicates that several other detoxification enzymes are over- expressed (Pedra et al., 2004). The lab-selected 91-R strain shows a different overexpression profile under microarray analysis including Cyp6a2 overexpression
(Pedra et al., 2004), but again Cyp6g1 overexpression is present.
158 While Cyp6g1 overexpression seems to be the constant in many resistant strains, it is not the only candidate resistant gene that is overexpressed. For WC2 and Hikone-
R, P450 microarrays show overexpression of Cyp6g1 (Daborn et al., 2002; chapter
4). Full genome microarrays on these strains may ultimately reveal expression differences in other detoxification genes.
From an evolutionary point of view, it is likely that the continued application and increased concentration of DDT (or other insecticides) could select for several resistance genes of major effect. Thus resistance to DDT has been seen to map to combinations of all three autosomes in different resistant strains (Crow, 1954; Ogita,
1960; Dapkus & Merrell, 1977). The presence of Cyp6g1 overexpression in several strains (chapter 4, this chapter) and its association with the Accord allele suggests that it may have been the original resistance mechanism, and combinations of other resistance mechanisms evolved after continued selection.
The genetic basis of resistance to five insecticides (including three different OP insecticides) was also examined recently (Miyo et al., 2002). Continuous variation in resistance was found using many isofemale lines of D. melanogaster from the same locations over different seasons (Miyo et al., 2000), and from different locations in the same season (Miyo et al., 2001). The correlations in resistance seen between the insecticides indicated that there may be common resistance factors, but also unique factors. Resistance in the strains tested was controlled by a minimum of two genes mapping to chromosome II-62 (in the region of Cyp6g1) and chromosome III-50.
Variations in cross resistance patterns between different strains were explained by differences in the frequencies of these factors.
A similar phenomenon was seen with another OP insecticide, diazinon (Pyke, 2000).
Three Australian strains from different locations showed contributions from similar
159 regions of chromosomes II and III (Pyke, 2000). Given the broad substrate specificity of Cyp6g1 and its association with OP resistance (chapter 4, tables 4.6 and 4.7), it is likely that Cyp6g1 is the chromosome II gene. The chromosome III gene(s) remains undefined.
The purpose of this investigation is to:
1. Examine other factors that may contribute to lufenuron resistance in the WC2
strain,
2. Examine further the genetic basis of resistance and cross-resistance in the
diazinon resistant Innisfail (Inn5) strain studied by Pyke (Pyke, 2000),
3. Use Accord and allelic variants as a diagnostic to examine the spread of
resistance in natural populations.
Material from this chapter is published in Pyke et al., (2003).
160 5.2 Materials and methods
5.2.1 Fly strains used
Fly strains used are marked below in table 5.1. In addition, 96 males from each of 6 distinct geographical locations from the east coast of Australia were collected during a 2002 natural population survey. Genomic DNA was prepared from these males (S.
McKechnie, pers comm.) and this DNA was generously donated for use in Accord diagnostic PCRs. The locations sampled are shown in figure 5.7.
Table 5.1: Fly strains used in this study. Stock numbers, where provided are those used by the Bloomington Drosophila Stock Center, the source of these stocks.
Stock Use Fly Strain Genotype Source Number Lufenuron resistant Vermont, WC2 Resistant field strain USA strains Lufenuron resistant Inn5 Innisfail, Qld field strain 21-3 21-3 4 4 Mapping vg e +/+ ; vg / vg ; e / e Bloomington 4070 ru1 h1 st1 ry506 e1/ strains ru h st ry e 1 1 1 506 1 Bloomington 93 ru h st ry e Lufenuron susceptible CanS Lab Std. Susceptible lab strain control strains Lufenuron susceptible Armenia Lab Std. lab strain
161 5.2.2 Dosage mortality curves
15 different lufenuron concentrations were used to establish a dosage mortality curve for this insecticide. For each concentration three repetitions of 100 first instar larvae were established in individual vials. Results were averaged for each concentration and converted into probit form. Standard errors were applied for each point. LC50,
LC95, and LC99 were calculated using the straight line equations produced. Fold resistances were calculated as the product resultant from dividing the LC50, 95, 99 of the resistant strain by the LC50, 95, 99 of the standard susceptible strains, CanS or
Celera.
5.2.3 Mapping protocols
5.2.3.1 Mapping to a chromosome
Resistance was mapped to chromosomes with the cross described in figure 5.1. In each of these crosses, 25-50 virgin females were crossed to 25 males. 25-50 F1 males were mated to 75 virgin females and screened in bottles containing 1.8, 2.1, or
2.3ppm lufenuron. These concentrations were chosen to allow discrimination of the contributions of genes of the different chromosomes at both low and high doses. F2 progeny were scored for each of the phenotypic markers.
162
+ vg e + vg+ e+ X + vg e + vg+ e+
+ vg e + vg e F1 X + vg e + vg+ e+
Screened on 2.1ppm lufenuron
+ vg e + vg e + vg e + vg e + vg+ e+ + vg+ e + vg e+ + vg+ e+ No Information R not on Chr III R not on Chr II No Information F2 + vg e + vg e + vg e + vg e + vg e+ + vg+ e + vg e + vg e R not on Chr II R not on Chr III R not on Chr II Lufenuron or Chr III conc. too small
Figure 5.1: Chromosome mapping cross using the vestigial (vg) and ebony (e)
phenotypic markers. Resistant chromosomes are marked in red and susceptible
chromosomes in blue. The information provided by each phenotypic class is also
shown.
163 5.2.3.2 Mapping within a chromosome
Resistance was mapped within chromosome III via the cross shown in figure 5.2 (a).
In each of these crosses, 25-50 virgin females were crossed to 25 males. 75-100 F1 virgin females were mated to 40 males (in a total of 3-5 bottles each) and screened at 2.1ppm lufenuron. The flies were transferred every 5 days to produce 3 replicates of each bottle. The F2 progeny were scored for each of the phenotypic markers. The relative positions of these markers within chromosome III are shown in figure 5.2 (b).
164 + + + + + a) ru h st ry e ru h st ry e X ru h st ry e ru+ h+ st+ ry+ e+
ru+ h+ st+ ry+ e+ ru h st ry e F1 X ru h st ry e ru h st ry e
Screened on 2.1ppm lufenuron
F2 Various recombinant classes
ru h st ry e
b) 0.0 26.5 44.0 52.0 70.7
Figure 5.2: a) Genetic mapping crosses using roughoid (ru), hairy (h), scarlet (st),
rosy (r), and ebony (e) phenotypic markers. Recombination occurs in the F1 females,
allowing for the scoring of resistant recombinants after screening. b) The relative
positions of the markers within chromosome III on the recombination map.
Recombinational map positions are also shown.
165 5.2.4 PCR Primers
Primers were used for various purposes in this investigation, including the scoring of
the presence/absence of the Accord insertion, DNA sequencing, and realtime RT-
PCR. These primers are shown below (except for realtime RT-PCR primers that are
shown in table 5.2).
Table 5.2 Primers used in PCR and sequencing
Abbrev For/ Full Name Sequence (5’ – 3’) bp Use Name Rev
AccordForward AccF F CTTGACGAGAAAGCCGGTTG 20 Accord/P- element PCR AccordReverse AccR R CACAGCAAATCCAGAGGGACTC 22
Accord+P PintFOR F CTGCAAAGCTGTGACTGGAGTA 22 Accord/P- InternalFor element Accord+P sequencing PintREV R ATTAACCCTTAGCATGTCCGTG 22 InternalRev
Accord+P PintFORB F CGTTTGCTTGTTGAGAGGAAAG 22 Accord/P- InternalForB element Accord+P sequencing PintREVB R GATTAACCCTTAGCATGTCCGTG 23 InternalRevB
166 5.3 Results
5.3.1 Lufenuron resistance in Inn5
Pyke (2000) identified a major diazinon resistance gene in the Inn5 strain. This gene was mapped to chromosome II in the interval between 62cM and 66.7cM. Given that Cyp6g1 is within this interval, the relationship between diazinon resistance, lufenuron resistance, and Cyp6g1 was examined in Inn5. To this end a dosage mortality analysis of lufenuron resistance was conducted for the Inn5 strain. This analysis, shown in figure 5.3 and table 5.3, shows that there is a high level of cross resistance to lufenuron in this strain.
167 3
2.5 y = 4.7897x + 1.0184 R2 = 0.963 y = 5.0874x + 0.8516 R2 = 0.9653 2
y = 3.5837x - 0.5356 2 1.5 R = 0.9431
1 y t i
0.5 y = 2.9375x - 0.5977 t mortal R2 = 0.9286 obi r P 0 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
-0.5 Celera CanS -1 Inn5 WC2 -1.5
-2 Log dose (ppm lufenuron)
Figure 5.3: Dosage mortality curve for the Inn5 strain in comparison to WC2, and susceptible controls CanS and Celera. Shown are
the lines of best fit (and equations), correlation coefficients, and error bars.
168 Table 5.3: Inn5 fold resistance with respect to CanS and Celera at Lethal concentrations (LCs) LC50, LC95, LC99.
Fold resistance Fold resistance LC(ppm) Strain compared to compared to (95% CL) CanS Celera 1.63 Inn5 LC50 2.39 2.66 (1.55-1.71) 6.10 Inn5 LC95 4.06 4.51 (5.79-6.41) 10.55 Inn5 LC99 5.41 5.62 (10.01-11.07)
The pattern of resistance seen is similar to that of WC2 although at higher doses, the
Inn5 strain appears to be more resistant than WC2, suggesting that different mechanisms of lufenuron resistance may be in action in these two strains.
5.3.2 Cyp6g1 expression levels in Inn5
The Inn5 strain was cross-resistant to lufenuron and diazinon and Cyp6g1 overexpression could explain resistance to these insecticides (Daborn et al.,2002),
Therefore Cyp6g1 expression levels were examined using realtime RT-PCR (figure
5.4). Cyp6g1 overexpression was seen in the Inn5 strain, but at a reduced level to that observed in WC2.
169 Figure 5.4: 12.00 Realtime RT-PCR 11.05 results showing 10.00 the level of overexpression of 8.00 Cyp6g1 in the Inn5 7.11 strain as compared 6.00 to WC2 and CanS.
The expression 4.00 Expression level wrt CanS wrt level Expression level for CanS was assigned an 2.00
1.00 arbritary level of
1.00. - Inn5 WC2 CanS Strain
5.3.3 A novel Accord insertion in the Inn5 strain
Since the Accord insertion had been associated with Cyp6g1 overexpression and had been detected in various resistant strains of D. melanogaster collected from around the world (Daborn et al., 2002), the Inn5 strain was tested for its presence by
PCR and sequencing. As shown in figure 5.5 (a) a surprising result was seen. An
Accord insertion was present but it was of a different size to the insertion characterized in Chapter 4. Sequencing revealed the same 490 bp Accord sequence with a fragment of the P-element (Flybase, 2004) inserted within it (figure 5.5 (b).
170
Inn5 WC2 CanS a)
1-490
b) Accord in WC2
2907-2717 2637-2326 1-361
P-element within Accord
Figure 5.5: Representation of a) the relative sizes of PCR products generated in the
Inn5, WC2, and CanS strains, and b) the location and structure of the partial P- element that is inserted into Accord in the Inn5 strain. The Accord element (including nucleotide numbers) is the same element as described in chapter 4, figure 4.10. The nucleotide numbers of the partial P-element according to the published sequence
(Flybase, 2004), are also shown.
171 5.3.4 The spread of Accord variants in natural populations
With the discovery of two different Accord variants associated with Cyp6g1 overexpression and resistance, the prevalence of these variants was surveyed. In
2002, a survey of seven natural populations of D. melanogaster along the east coast of Australia was performed. DNA from 96 males from each population was extracted
(by S. McKechnie, Monash University) and used in diagnostic analysis for the presence of Accord variants. As shown in figure 5.6, only 4% of flies carried an allele that did not contain an insertion upstream of Cyp6g1. The Accord and Accord+P- element alleles were in roughly equal proportions although Accord+P-element appeared to be slightly more common in these Australian populations.
P-element/Accord overall allele frequencies Figure 5.6: Overall Accord / in natural populations Accord +P Accord+P-element allele 663 57% frequencies in natural populations of D. melanogaster from the east Accord 455 coast of Australia. 39%
No Insert 49 4%
172 As well as examining the overall frequencies of Accord and variants in natural populations, an examination of the variation between populations was undertaken.
This was aimed at highlighting whether there was any temperate/latitudinal variation contributing to the frequencies seen. The results are shown in figure 5.7.
173 Cape Tribulation I
Accord+P Accord Cape Tribulation II None
Gladstone
Maryborough
Coffs Harbour
Wollongong
Bega
Figure 5.7: Allele frequencies of Accord variants in natural population strains from the east coast of Australia, indicating their origin to show clinal variation.
174 It is clear that significant variation exists between the populations tested. At lower latitudes (northern populations), the frequency of the Accord+P-element allele is highest. Its frequency decreases in southern populations whilst the frequency of the
Accord element increases. A larger proportion of alleles with no insertion is also seen in southern populations.
5.3.5 Other factors contributing to resistance in WC2 and Inn5
The Inn5 had been shown to have a variant Accord insertion (figure 5.5), to overexpress Cyp6g1 (figure 5.4), and to be resistant to diazinon (Pyke, 2000), nitenpyram (T. Perry, pers. comm.) and lufenuron (table 5.3). While these data implicated one gene, Cyp6g1, Pyke (2000) mapped diazinon resistance to chromosomes II and III. It was therefore decided that an examination concerning the overall contributions of chromosomes II and III should be undertaken in relation to lufenuron resistance. Since chromosome III had been observed to contribute to resistance in various strains where Cyp6g1 also plays a role (see section 5.1), this analysis was extended to the WC2 strain. (In chapter 4 mapping in WC2 was limited to chromosome II).
A cross was set up that would allow the contributions of various chromosomes to lufenuron resistance to be detected (detailed in figure 5.1). Random assortment would produce a 1 : 1 : 1 : 1 ratio of each of the marker phenotypes, signifying equal contributions in each of the four phenotypic classes +;+, vg;+, +;e, vg;e. A deviation from this ratio under the selection pressure of lufenuron screening would be due to the contribution of one or more of the chromosomes towards resistance. Some of the possible outcomes and the underlying genetic interpretations are summarised in table 5.4.
175
Table 5.4: Summary of various scenarios resulting from chromosome II and III screens, indicating in each case, which chromosome(s) contribute the bulk of resistance. Note that sex-linked inheritance was not directly examined here, but the chromosome mapping scheme used nevertheless detects X-linked resistance. Since males were used in the F1 (see, figure 5.1), all F1 X-chromosomes originate from the mapping strain, and so extremely low levels of survival would be seen in the F2 under lufenuron selection (scenario A).
Important Ratio of phenotypes seen when screened Outcome Chromosomes +;+ vg;+ +;e vg;e
A Neither II or III 0 0 0 0
B Both II and III 4 0 0 0
C II only 2 0 2 0
D III only 2 2 0 0
E II or III equally 2 1 1 0
176 Three different lufenuron concentrations were used such that the relative contributions of each chromosome could be assessed under the lower and higher insecticide stress conditions. A semi-quantitative profile was developed for each mutant in an effort to dissect different resistance mechanisms that may have been operating amongst the WC2 and Inn5 resistant strains. These data are shown in figure 5.8. The relative contribution of the chromosomes can be estimated by examining the ratio of vg;+ to +;e phenotypes. The ratio would be one if the chromosomes contributed equally, greater than one if chromosome III makes a greater contribution, and less than one if chromosome II is more significant. The results of this analysis are shown in table 5.5.
The results indicate several features of resistance. 0ppm lufenuron controls shows the emergence of approximately equal proportions of each phenotypic class, suggesting that there are few marker-phenotype related effects. In Inn5, both chromosomes II and III contribute to resistance; chromosome II is slightly more important at lower concentrations and becomes relatively less so at higher concentrations (also represented in table 5.5). In WC2, a different pattern is seen.
Once again both chromosomes contribute to resistance, but at each concentration chromosome III plays a more prominent part than chromosome II. Similar results to these were seen in WC2 when resistance to the neonicotinoid insecticide, nitenpyram, was mapped (T. Perry, Pers. Comm).
177 300
280
260 Neither II or III (vg;e) 240 Chr II only (+;e) Chr III only (vg;+) 220 Chr II & III (+;+)
200 y t ili b
a 18 0 i v
dult 16 0 a o t l 14 0 a v r a
L 12 0 l a
Tot 10 0
80
60
40
20
0 WC2 WC2 WC2 WC2 Inn5 Inn5 Inn5 Inn5 0ppm 1.8ppm 2.1ppm 2.3ppm 0ppm 1.8ppm 2.1ppm 2.3ppm Strain and concentration lufenuron
Figure 5.8: Contribution to resistance by each chromosome based on the frequency
of emergence of each phenotypic class. Note that 0ppm controls emerged in
approximately equal proportions as expected.
178 Table 5.5: Indicates the relative contributions of chromosomes III and II calculated by dividing % emergence of the “chromosome III only” (vg;+) class by “chromosome II only” (+;e) class.
Ratio chromosome III/chromosome II at each concentration
Strain 0ppm 1.8ppm 2.1ppm 2.3ppm
WC2 1.29 4.25 4.00 6.00
Inn5 1.41 0.53 0.80 1.17
The similarities and differences between Inn5 and WC2 were intriguing. While
Cyp6g1 overexpression was found in both strains, the contribution of chromosome III appeared to differ. While other explanations could be offered, one possibility was that different chromosome III genes contributed to resistance in these strains.
Therefore resistance was mapped within chromosome III in Inn5 and WC2.
5.3.6 Resistance within chromosome III in WC2 and Inn5
5.3.6.1 Inn5 mapping
A mapping cross was undertaken to examine the location(s) of resistance loci within chromosome III. The frequency of each phenotype was calculated and shown on figure 5.9, and the observed and expected numbers of recombinants is shown in table 5.6, along with associated χ2 values.
179
0.70
0ppm 2.1ppm
0.60
0.50
0.40
0.30 Frequency of marker
0.20
0.10
- ruhstrye Phenotypic Marker
Figure 5.9: Frequency of each mutant class for both the 0ppm control and 2.1ppm
screen in the Inn5 strain.
Table 5.6: Observed and expected numbers of flies showing each phenotypic
mapping marker for both the 0ppm control (n=78) and the 2.1ppm screen (n=156).
180 The χ2 values and probability for the null hypothesis of no difference between observed and expected numbers (df=4) are also shown.
ru h st ry e χ2 Probability
Observed 48.75 36.79 41.29 48.75 48.75 at 0ppm
Expected 39 39 39 39 39 at 0ppm n=78 7.57 0.2 Observed 61.29 22.94 5.17 1.01 15.38 at 2.1ppm Expected 78 78 78 78 78 at 2.1ppm n=156 236.7 P<0.001 No significant difference was seen between the observed and the expected numbers for the 0ppm control, indicating that there were no significant viability affects associated with any of the phenotypic markers in the absence of insecticide. At 2.1ppm lufenuron, there were significant differences between observed and expected numbers for the markers. The data indicate a major resistance locus between the markers st and e, but closer to st. The locus appears tightly linked to the marker ry, situated between st and e. Similar mapping data were reported for diazinon resistance in this strain (Pyke et al., 2003). It is possible that the same chromosome III gene may be conferring resistance to both lufenuron and diazinon in this strain. 181 5.3.6.2 WC2 mapping The mapping methods used for Inn5 were also applied to WC2. Again the frequency of each phenotypic marker was calculated (figure 5.10). The observed and expected numbers of recombinants, along with associated χ2 values, are shown in table 5.7. 0.8 0.7 0ppm 2.1ppm 0.6 0.5 0.4 Frequency of Marker 0.3 0.2 0.1 0 ruhstrye Phenotypic Marker Figure 5.10: Frequency of each phenotypic marker in WC2 for both the 0ppm control and the 2.1ppm lufenuron screen in the WC2 strain. 182 Table 5.7: Numbers of observed and expected mutants for both the 0ppm control (n=73) and the 2.1ppm screen (n=119). The χ2 values and probability for the null hypothesis of no difference between observed and expected (df=4) are also shown. ru h st ry e χ2 Probability Observed 53.88 45.42 33.58 45.42 50.93 at 0ppm Expected 36.5 36.5 36.5 36.5 36.5 at 0ppm n=73 3.27 0.7 Observed 70.25 32.57 4.07 0.99 5.13 at 2.1ppm Expected 59.5 59.5 59.5 59.5 59.5 at 2.1ppm n=119 176.95 P<0.001 Again, no significant difference was seen between the observed and the expected numbers for the 0ppm control, indicating that there were no significant viability affects associated with any of the phenotypic markers in the absence of insecticide. At 2.1ppm lufenuron, there were significant differences between observed and expected numbers for the markers. The data indicate a major resistance locus between the markers st and e, very close to ry. Based on these data, the resistance locus in WC2 and Inn5 may be the same, but further fine-scale mapping would be required to determine this with any degree of certainty. 183 5.4 Discussion 5.4.1 Accord in natural populations Given that Cyp6g1 overexpression is prevalent in natural population strains and that Accord is highly associated with it, an examination of the spread of this mechanism can be performed by examining Accord presence. A survey of 96 individuals from seven different geographical locations showed that the frequency of Accord or Accord+P-element is high. With only 4% of alleles not carrying an insertion, Cyp6g1 overexpression and its associated cross-resistance phenotypes appears to be wildtype in natural population strains (figure 5.6). As to the clinal variation in insertion frequency, there does appear to be a trend such that Accord+P-element frequency is highest at lowest latitudes, and gradually decreases as latitude increases (figure 5.7). Correspondingly Accord frequency increases significantly, whilst the frequency of the no insert allele shows a small increase. There are many speculative explanations for these patterns. Variation based on another environmental stress may vary clinally, and carry an insertion and Cyp6g1 overexpression patterns with it. This explanation still supports the lack of clinal variation in patterns of resistance since both the Accord and Accord+P-element alleles are associated with resistance, however the selection effects based on this model are not likely to impart frequency of insertion of 96%. Selection by previously used insecticides is the favoured explanation for the high allele frequency and for the clinal variation. Under this reasoning, insecticide usage increased the frequency of an initially low frequency Accord allele. The emergence of the Accord+P-element allele may have been in northern Australia, followed by its 184 spread south, accounting for the lower frequency in southern populations. Alternatively, some as yet unidentified differences in insecticide usage patterns may have altered the relative frequencies of the alleles. However, it must be acknowledged that the high frequency of the Accord insertion may not be completely dependent upon insecticide selection. It is often assumed that D. melanogaster would not be exposed to insecticides at either the concentration or frequency that an insect pest species would be. If this assumption is correct it would be at least surprising if insecticide resistant alleles achieved a frequency of 96%. 5.4.2 Cyp6g1 based resistance in the Inn5 strain The Inn5 strain was initially established as an isofemale strain, and found to be resistant to the OP insecticide diazinon (Pyke, 2000). Resistance in this strain mapped to both chromosomes II and III, indicating at least two genes of major effect (figure 5.8). Within chromosome II, it mapped between phenotypic markers en (II-62) and sca (II-66.7), the region encompassing Cyp6g1 (Pyke, 2000). This study has revealed that Cyp6g1 is overexpressed in the Inn5 strain (figure 5.4). In other Cyp6g1 overexpressing strains, WC2 and Hikone-R, cross-resistance to OPs has been seen; dichlorvos in WC2 (T. Wilson, Pers. Comm.), malathion and parathion in Hikone-R (Ogita, 1960). This suggests that Cyp6g1 overexpression may confer diazinon resistance. Cyp6g1 overexpression also confers resistance to lufenuron (chapter 4, figure 4.14), so the lufenuron cross resistance observed in the Inn5 strain is not surprising. 185 The discovery of an insertion upstream of Cyp6g1 strengthens the case for its role in conferring overexpression, and the nature of the mutation (an insertion of a P- element within an identical Accord element) suggests a possible evolutionary pathway with regard to the spread of this element in populations. The chimeric Accord/P-element allele also provides an opportunity to generate deletions at the 5’ end of the Cyp6g1 gene via induced P-element excision (Voelker et al., 1984; Daniels et al., 1985) in order to study its regulation. It is intriguing to note that there is a lower level of Cyp6g1 expression in the Inn5 than to the WC2 strain (figure 5.4). If, in fact, the location of the Accord insertion in WC2 is the major factor influencing Cyp6g1 overexpression, then one would expect the presence of the internal P-element in Inn5 to have no or little effect on Cyp6g1 expression levels. Thus it is likely that, as discussed in Chapter 4. section 4.4.3, regulatory factors within the accord element may be influencing expression levels. Inter-chromosome mapping showed lufenuron resistance was contributed by both chromosome II and III in this strain, and that the contributions were similar. In comparison with WC2, the proportion of resistance explained by chromosome II in Inn5 is high. In addition, the Inn5 strain is slightly more lufenuron-resistant than WC2 (figure 5.3) despite its lower level of Cyp6g1 expression. These two pieces of evidence combined suggest that there may be another mechanism on chromosome II that is contributing to resistance in Inn5. No data presented here is able to confirm the existence of another chromosome II lufenuron-resistance locus, however, such a locus is quite possible given the variety of P450 and other genes capable of mediating resistance to insecticides (see section 1.5 and below). 186 Overexpression of the chromosome II (42c8) linked P450 Cyp6a2 has been previously implicated in conferring DDT resistance (Waters et al., 1992; Maitra et al., 1996), and has been shown to metabolize OP insecticides (Dunkov et al., 1997). It was confirmed to be overexpressed in the 91-R strain, but not in the WIS-1 strain (Pedra et al., 2004). Accordingly 91-R had higher DDT resistance than WIS-1. Variation in Cyp6a2 levels could possibly explain the different chromosome II contributions to lufenuron resistance in Inn5 and WC2 strains. There are also many other candidates. Other detoxification genes such as Cyp12d1, Cyp6a14, Cyp6w1, and CG17530, (a GST), have also been implicated in conferring resistance through overexpression in strains where Cyp6g1 overexpression is already acting (Pedra et al., 2004). It should be noted, however, that since both Inn5 and WC2 strains were derived from isofemale lines, differences may also be explained by variation within the strains contributing to deceptive screening results in the limited numbers tested. 5.4.3 Chromosome III-associated resistance In the Inn5 strain, intra-chromosomal mapping on chromosome III indicated a resistance locus of major effect between st (III-44.0) and e (III-70.7), but closer to st. The locus appears to be tightly linked to ry (III-52.0) which lies between st and e. This compares remarkably to the loci reported in Inn5 for diazinon resistance (Pyke et al., 2003), implying that the same loci may be operating for both insecticides. In a recent study resistance to OP insecticides was mapped to the same regions of chromosomes II and III to which resistance was mapped in Inn5 and WC2 (Miyo et al., 2002). Given the diverse geographical origins of the strains involved, the resistance described here may represent a global polymorphism. 187 For the WC2 strain, resistance was most significantly associated with chromosome III. This extended the evidence presented in chapter 4, suggesting that Cyp6g1 overexpression did not completely explain resistance in this strain. Within chromosome III, resistance maps close to the ry locus and in slight distinction to the Inn5 strain, appears completely unlinked to the ru locus. The differences in the contribution of chromosome III towards resistance in these two strains may be suggestive of different mechanisms acting. However this conjecture is made tentatively due to the background of the strains and the limited numbers tested. 5.4.4 The function of the chromosome III resistance genes in WC2 and Inn5 In chapter 4, evidence was presented to support the hypothesis that resistance in WC2 is due to the overexpression of the Cyp6g1 gene. Other genes that contributed to the overexpression of Cyp6g1 would also increase the level of resistance. Following this logic the resistance gene(s) on chromosome III may function as a regulator of Cyp6g1. While this hypothesis is attractive, it is not supported by the available evidence. First, resistance is not simply correlated with levels of Cyp6g1 expression. The Inn5 strain has lower Cyp6g1 expression levels than WC2 (figure 5.4) but has resistance levels that are equal or higher (figure 5.3). The overexpression of Cyp6g1 using the GAL4-UAS system produces higher levels of Cyp6g1 expression than is observed in either Inn5 or WC2, but the observed levels of resistance are lower (chapter 4, figure 4.14). Secondly, the second chromosome from the WC2 strain has been placed into different genetic backgrounds with respect to chromosome III (T. Perry, unpubl). The chromosome III background does not 188 make a significant difference to the observed Cyp6g1 expression levels (T. Perry, unpubl). The alternate hypothesis is that the chromosome III gene encodes a detoxification enzyme. In various strains, resistance has mapped to chromosome III for DDT (Ogita, 1961), four different OPs (Miyo et al., 2002; Pyke et al., 2003), nitenpyram (T. Perry, unpubl), and lufenuron (figure 5.8). The possibility exists that Cyp6g1 partially detoxifies these many different insecticides, generating substrates for secondary metabolism by the product of the chromosome III gene(s). Candidates for this suggested secondary metabolism exist within the UDP-glucuronosyltransferase (UGT) gene cluster that maps to cytological position 86d5 (map position 48.5cM). UGTs, like GSTs, are often involved in two-phase detoxification pathways as the phase II mechanism (see chapter 1, section 1.4). The transfer of the glucuronic acid moiety to a hydroxyl-, amino-, thiol-, or carboxyl group generated in a P450 phase I detoxification results in a more polar water soluble metabolite that is easily excreted (McGurk et al., 1998). Interactions between mammalian CYP1A1 and several UGTs have been demonstrated (Taura et al., 2000). The recent co-expression of CYP1A1 and UGT1A6 has facilitated the sequential detoxification of insecticides to be simulated (Ikushiro et al., 2004). UGTs have also been shown to have increased activity in the presence of DDT in mammalian systems (Okazaki & Katayama, 2003). The three members of the UGT cluster mapping to chromosome III-48.5 are overexpressed in one (Ugt86Dd, Ugt86Dh) or both (Ugt35b) DDT resistant strains tested (Pedra et al., 2004), suggesting that DDT may be a substrate for this gene product, or that a partially detoxified insecticidal metabolite could be subject to further detoxification by this gene product. 189 It is possible that a number of genes on chromosome III contribute to the resistance reported in Inn5 and WC2. Whereas Cyp6g1 would be viewed as an enzyme that could utilize a wide variety of substrates (e.g. diazinon, lufenuron and nitenpyram), the phase II metabolism would be carried out by enzymes with a more limited substrate specificity. Hence, there could be different, albeit tightly-linked chromosome III genes with a role in diazinon, lufenuron and nitenpyram resistance. It is also possible based on the different resistance profiles of WC2 and Inn5, that chromosome III genes are different in these strains, or that they are alleles of the same gene with different functional effect. Some methods that could be employed to investigate these possibilities further are fine-scale mapping within chromosome III in order to positionally clone the genes, microarray studies in order to identify candidate genes with an altered expression profile, and chromosome substitution studies to tease apart the mechanisms of resistance in these strains and examine their associations. These approaches are currently being employed in the Batterham laboratory. Whilst it has proven beneficial to scrutinize individual strains of D. melanogaster for resistance characteristics, assessment at the population level can also provide insights into the evolution and spread of particular resistance mechanisms. Chapter 6 examines the extent of lufenuron resistance and associated Cyp6g1 overexpression between and within populations from the east coast of Australia. 190 Chapter 6 Selection for Lufenuron resistance following EMS Mutagenesis 6.1 Introduction 6.1.1 Natural verses lab-based selection There are many benefits to identifying the genes responsible for resistance in natural populations of D. melanogaster. This study has shown that two different P450s (Cyp6g1 and Cyp12a4) can confer resistance to lufenuron in D. melanogaster. While this suggests the potential for P450 based resistance in other species, it is not clear whether the orthologues of these genes (Cyp6g1 and Cyp12a4) in pest species would have the capacity to metabolize lufenuron. The P450s are a rapidly evolving family of genes and at this point in time the consequences of sequence divergence for function are ill defined. If target site resistance could be found in D. melanogaster, the gene for the target could be isolated using positional cloning strategies. In identifying the target, the mode of action of the insecticide lufenuron would be to some extent understood. Further, it is likely that lufenuron exerts its toxic effect in different species by binding to an orthologous target. Therefore the identification of the lufenuron target in D. melanogaster would allow the target to be identified in pest species. D. melanogaster has been shown to have the same target as pest species for OPs and carbamates (Walsh et al., 2001; Weill et al., 2003; Nabeshima et al., 2003), pyrethroids (Taylor et al., 1993; Williamson et al., 1996; Ranson et al., 2000) and cyclodienes (Thompson et al., 1993; ffrench-Constant, 1994). Target site mutations can be found in natural populations of D. melanogaster. For example, resistant mutations at the Ace and Rdl loci are found at significant frequencies (ffrench-Constant et al., 1998; ffrench-Constant et al., 2004; Menozzi et 192 al., 2004). The possibility that lufenuron target site resistance exists in natural populations of D. melanogaster cannot be excluded, however, to date such mutations have not been detected. Therefore, in this chapter, the focus switches to mutagenesis and selection for resistance in the laboratory. Based on precedents (McKenzie & Batterham, 1998), this approach can yield both target site and detoxification based resistance. 6.1.2 EMS as a mutagen In most cases where the molecular basis of target site resistance is known, it is conferred through minor genetic lesions such as single base changes. Ethyl methanesulphate (EMS) has a bias towards point mutations and specifically GC-AT transitions (Pastink et al., 1991). It has been an effective mutagen in other studies (Wilson & Fabian, 1986; Smyth et al., 1992; McKenzie et al., 1992; Adcock et al., 1993; Daborn et al., 2000), and was therefore thought to be useful in generating a broad spectrum of lufenuron-resistant mutants. In conjunction with an inter- and intra- chromosome mapping strategy and analysis of previously identified candidate genes, it provides a powerful option in the investigation of lufenuron resistance. 193 6.1.3 Aims of this Project The aims of this project are therefore: 1. To isolate EMS-induced lufenuron-resistant mutants 2. To characterize these mutants through examination of: a. Fold resistance with respect to standard controls b. Mapping resistance to chromosomes c. Mapping resistance within chromosomes d. Comparison between mutants e. Assessment of any candidate genes 3. To make any conclusions on the mode of action of lufenuron and mechanisms of resistance 194 6.2 Materials and methods 6.2.1 Fly strains and maintenance The isofemale strain, Armenia, was chosen for EMS mutagenesis due to its high level of reproductive fitness compared with other isofemale lines tested and its significant genetic divergence (measured through the analysis of microsatellites) from the sequenced Celera strain (J. Damiano Pers.Comm). Armenia males and virgin females were collected at 10-14 hour intervals and stored at 10°C to prevent mating. They were stored for 1-6 days before being exposed to EMS and mated. Other strains used in this project including mapping, wildtype, and resistant strains are listed in table 6.1. 195 Table 6.1: Strains used (and generated) in this investigation. EMS-generated strains were named using the notation “EMS trial number, mutant number” such that the tenth potential mutant from the fifth screen would be named EMS#5-10. Fly Strain Background genotype Source Use Armenia Wildtype (Armenia) Bloomington EMS strain EMS#2-2 Wildtype (Armenia) This project EMS Mutant EMS#4-7 Wildtype (Armenia) This project EMS Mutant EMS#4-10 Wildtype (Armenia) This project EMS Mutant EMS#5-5 Wildtype (Armenia) This project EMS Mutant EMS#5-7 Wildtype (Armenia) This project EMS Mutant EMS#5-8 Wildtype (Armenia) This project EMS Mutant EMS#8-6 Wildtype (Armenia) This project EMS Mutant EMS#11-4 Wildtype (Armenia) This project EMS Mutant EMS#17-2 Wildtype (Armenia) This project EMS Mutant EMS#17-4 Wildtype (Armenia) This project EMS Mutant EMS#17-8 Wildtype (Armenia) This project EMS Mutant EMS#18-11 Wildtype (Armenia) This project EMS Mutant EMS#19-7 Wildtype (Armenia) This project EMS Mutant EMS#20-3 Wildtype (Armenia) This project EMS Mutant EMS#20-4 Wildtype (Armenia) This project EMS Mutant EMS#20-10 Wildtype (Armenia) This project EMS Mutant EMS#20-12 Wildtype (Armenia) This project EMS Mutant vg e +/+ ; vg21-3 / vg21-3 ; e4/ e4 Bloomington Mapping ru1 h1 st1 ry506 e1/ ru h st ry e Bloomington Mapping ru1 h1 st1 ry506 e1 CanS Wildtype Standard lab Susceptible Celera Wildtype Standard lab Susceptible WC2 Wildtype Field population Resistant strain Inn5 Wildtype Field population Resistant strain 196 6.2.2 Mutagenesis and isolation of mutants 6.2.2.1 Mutagenesis protocol Armenia males and virgin females (4-6 days post-eclosion) were exposed to 100µL of EMS under a vacuum as described in (Sega & Lee, 1970) with modifications described in (Smyth et al., 1992). The flies were then mass-mated in perspex containers also containing standard food media. A nylon mesh covered the media. Embryos collected on the mesh were washed into a fine cotton sheet. F1 embryos were collected every 12-14 hours for the next 7-10 days and spread onto 250mL bottles with 50mL of fly food containing 2.3ppm lufenuron (approximately 1000 embryos per bottle). This mutagenesis protocol was repeated approximately 22 times over a period of 8 months. Non-EMS treated Armenia flies maintained under the same conditions were used as controls in several of the mutagenesis trials. 6.2.2.2 Isolation and stabilisation of lufenuron resistant mutants Flies emerging from the initial lufenuron screen were mated with previously unmated Armenia flies of the opposite sex and embryos screened on 2.0ppm lufenuron to check for stable transmission of lufenuron resistance. This screen was then repeated in the following generation at 2.3ppm lufenuron. Mutants that survived the initial re- screen were then re-screened for several successive generations in order to increase and maintain the frequency of the resistant allele. After this strains were periodically re-screened to maintain the resistant allele. A summary of this process is shown in figure 6.1. 197 EMS Armenia Armenia X Armenia Armenia Embryos screened on 2.3ppm lufenuron Armenia Armenia* F1 X Armenia Armenia Survivors rescreened on 2.0ppm lufenuron F2 Initial and periodic rescreening on 2.3ppm lufenuron Figure 6.1: Genetic mapping crosses used to isolate EMS induced mutants. Lines were re-screened several times to ensure the mutation was heritable and that there were no susceptible escapers. Stocks were also periodically re-screened to ensure maintenance of the resistant allele in the strain. * Signifies mutations due to EMS. 198 6.2.3 Dosage mortality curves The dosage mortality relationship for lufenuron was analysed using up to 15 different lufenuron concentrations. At each concentration three repetitions of 100 first instar larvae were tested in individual vials. Results were averaged for each concentration and converted into probit form. Standard errors were applied for each point. LC50, LC95, and LC99 were calculated using the straight line equations produced. Fold resistances were calculated by dividing the LC50 of the resistant strain by the LC50 of the susceptible CanS strain. 6.2.4 Mapping protocols Genetic mapping to a chromosome and within chromosome III was undertaken as described in chapter 5, section 5.2.3. 6.2.5 Detecting Accord and measuring the expression levels of Cyp6g1 Cyp6g1 expression levels and the presence of Accord were examined using techniques as described in chapter 2, section 2.2.9, and chapter 4, section 4.2.3, respectively. 199 6.3 Results 6.3.1 Isolation of EMS induced mutants An estimated 10 million embryos were screened over 22 screens. Several hundred flies emerged from initial screening but after successive screens, 17 stably transmitted lufenuron-resistant mutants were isolated. These were shown in table 6.1. 6.3.2 Accord and overexpression The isolation of these mutants preceded the discovery of the Accord element at the Cyp6g1 locus described in Chapter 4. Therefore, once the Accord polymorphism had been discovered, the Armenia base strain and all EMS-generated mutants were genotyped for it. PCR-based tests revealed that the Armenia strain was segregating the same Accord insertion (and associated Cyp6g1 overexpression) that had been characterized in the WC2 strain (data not shown). Subsequently, all of the lufenuron- resistant mutants isolated were shown to contain the same Accord insertion (data not shown). Cyp6g1 expression levels were therefore tested in these mutants using realtime RT-PCR, and all showed increased but varying levels of Cyp6g1 overexpression with respect to the CanS susceptible strain (figure 6.2). The frequency of the Accord insertion in the Armenia strain was found to be approximately 90% (data not shown). Given that only 17 highly resistant mutants were recovered from 10 million embryos screened, it seemed unlikely that the 200 resistance could be completely explained by Accord/Cyp6g1. However, the fact that all resistant mutants contained Accord suggested that the insertion was either required, or at least beneficial for resistance. Presumably, regardless of new mutations generated through EMS mutagenesis, exposure to lufenuron in initial screening selected for pre-existing putative resistance conferring mutations such as Accord and the highly associated Cyp6g1 overexpression. 201 22 20 18 16 14 l wrt CanS e 12 lev n o ssi 10 expre 8 g1 6 p Cy 6 4 2 0 CanSArmenia WC2 Inn5 EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS #2-2 #4-7 #4-10 #5-5 #5-7 #5-8 #8-6 #11-4 #17-2 #17-4 #17-8 #18-11 #19-7 #20-3 #20-4 #20-10#20-12 Strain Figure 6.2: Cyp6g1 expression levels in EMS-generated lufenuron-resistant mutants, scaled with respect to the CanS susceptible strain and against RpL32 housekeeping control gene. Note that the Armenia base strain also shows a higher level of expression of Cyp6g1 than CanS, attributable to the segregation of the accord allele within this population and a high frequency (approximately 90%). 202 6.3.3 Dosage mortality studies It was apparent from initial screening that all of the mutants isolated were highly resistant to lufenuron, but the exact resistance levels were not known. Four of the 17 lines were chosen for more detailed analysis.The lines EMS#5-5, EMS#5-7, EMS#5- 8, EMS#8-6 and the standard control strains CanS and Celera were rigorously tested for resistance (figure 6.3. and table 6.2). 203 3 y = 4.7897x + 1.0184 R2 = 0.963 2 y = 5.0874x + 0.8516 R2 = 0.9653 y = 4.4192x - 1.3597 R2 = 0.9202 1 y y = 2.818x - 1.0472 t i l 2 a R = 0.8606 t r o 0 M t -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 obi r P y = 2.7742x - 1.1867 2 -1 R = 0.8073 y = 2.6864x - 1.0229 R2 = 0.8264 CanS Celera -2 EMS#5-5 EMS#5-7 EMS#5-8 EMS#8-6 -3 Log Dose (ppm lufenuron) Figure 6.3: Dosage mortality curves for EMS generated mutants in comparison to susceptible controls CanS and Celera. Shown are the lines of best fit (and equations), correlation coefficients, and error bars. 204 Table 6.2: Lufenuron resistance in EMS strains compared to CanS and Celera at various lethal concentrations (LCs). Strain LC(ppm) Fold resistance Fold resistance (95% CL) tC S tC l EMS#5-5 LC50 2.68 3.94 4.37 EMS#5-5 LC95 10.49 7.32 7.76 EMS#5-5 LC99 18.46 9.47 9.85 EMS#5-7 LC50 2.403 3.53 3.92 EMS#5-7 LC95 9.841 6.87 7.28 EMS#5-7 LC99 17.650 9.05 9.41 EMS#5-8 LC50 2.353 3.46 3.84 EMS#5-8 LC95 9.022 6.30 6.68 EMS#5-8 LC99 15.745 8.08 8.40 EMS#8-6 LC50 2.031 2.99 3.31 EMS#8-6 LC95 4.785 3.34 3.54 EMS#8-6 LC99 6.825 3.50 3.64 205 It can be seen that three of the strains (EMS#5-5, EMS#5-7, and EMS#5-8) had extremely similar resistance profiles. They may carry the same mutation(s) based on these profiles, and the fact that they are from the same EMS trial. EMS#8-6 has a significantly different resistance profile, showing a similar LC50 to the other mutants but comparatively lower levels of resistance at higher dose s, reflected in the significantl y lower LC95 and LC99. This difference in resistance profile may suggest a different resistance conferring mutation or locus may be acting. The resistance of these strains was not directly compared to WC2. However, relating the data from tables 4.5 & 6.2, it seems that the four mutant lines are all more resistant than WC2. At this stage in the analysis it was not known with any certainty that the mutant lines were homozygous for the resistant gene(s). The maintenance of the lines on lufenuron would have selected for homozygosity. The linear probit curves observed for the lines tested suggest that it might have been achieved. However, this could only be verified once the resistant genes had been mapped. 6.3.4 Mapping to a chromosome Some EMS lines were subjected to chromosome mapping using the cross shown in Chapter 5, figure 5.1. Three different lufenuron concentrations were used so that the relative contributions of each chromosome could be assessed under the lower and higher stress conditions. A semi-quantitative profile was developed for each EMS mutant in an effort to dissect different resistance mechanisms that may have been operating amongst the mutants. The lines WC2 and Inn5 are also shown for comparison. A table of the more extreme possible scenarios is reproduced from chapter 5 (table 6.3). The results are shown in figure 6.4 (a), (b), (c), and table 6.4. 206 Table 6.3: Summary of various scenarios of chromosome II and III screens indicating in each case, which chromosome(s) contribute the bulk of resistance. Important Ratio or phenotypes seen when screened Scenario Chromosomes ++ vg+ +e vge A Neither II or III 0 0 0 0 B Both II and III 4 0 0 0 C II only 2 0 2 0 D III only 2 2 0 0 E II or III equally 2 1 1 0 207 1.8ppm lufenuron 100% 90% 80% 70% 60% on i t Neither II or III bu i r 50% Chr II only nt o Chr III only c % 40% Chr II & III 30% 20% 10% 0% 2 7 5 7 8 6 4 8 7 3 4 4 2 0 2 ------10 11 1 12 C 2 4 5 5 5 8 7 9 0 0 1 7 - nn5 I # # # # # # 1 1 2 2 1 1 0 W # #17 # # # #4- # # 2 S S S S S S #18- # #20- S S S S S S S S M M S S S M M M M M M M M E E EM EM EM EM M M M E E E E E E E E E E E Figure 6.4: a) Proportion of resistance contributed by each chromosome based on the frequency of emergence of each phenotypic class. 208 2.1ppm lufenuron 100% 90% 80% 70% n 60% o i t Neither II or III bu i 50% Chr II only ontr Chr III only c % 40% Chr II & III 30% 20% 10% 0% 2 7 5 7 8 6 8 7 3 4 4 2 4 2 2 10 11 10 1 - nn5 I #2- #4- #5- #5- #5- #8- 0 WC #17- #19- #20- #20- #4- #11- #17- #17- 18- 20- 2 S S S S S S # # # S S S S S S S S M M M M M M S S S M M M M M M M M E E E E E E M M M E E E E E E E E E E E Figure 6.4: b) Proportion of resistance contributed by each chromosome based on the frequency of emergence of each phenotypic class. 209 2.3ppm lufenuron 100% 90% 80% 70% 60% on ti u Neither II or III b i 50% Chr II only ntr o Chr III only c % 40% Chr II & III 30% 20% 10% 0% 2 7 5 7 8 6 4 8 7 3 4 4 2 10 11 10 12 nn5 I #2- #4- #5- #5- #5- #8- WC2 #17- #17- #19- #20- #20- #4- #11- #17- S S S S S S #18- #20- #20- S S S S S S S S M M M M M M S S S M M M M M M M M E E E E E E M M M E E E E E E E E E E E Figure 6.4: c) Proportion of resistance contributed by each chromosome based on the frequency of emergence of each phenotypic class. 210 There are several points to note from these figures. Firstly, at the doses tested none of the mutants gave a profile suggesting that both chromosomes II and III are essential for resistance (see scenario B in table 6.3), because representatives of at least one of the vg+ or +e classes was seen. In most cases, the percentage of the ++ class seen was as expected for scenarios C, D, of E to be occurring (50%). Secondly, it can be seen that in the majority of cases, resistance mapped overwhelmingly to chromosome III (Scenario D in table 6.3), especially at the higher lufenuron concentrations. There were some occasions where chromosome II and chromosome III explained similar levels of resistance (Scenario E, table 6.3). As in Chapter 5, a ratio of survival of the vg+ to +e classes was created as an analysis of the relative contributions of each of chromosomes II and III. The mutants were then grouped into specific categories based on their resistance ratio profiles. The results of these calculations for each concentration used are shown in table 6.4. 211 Table 6.4: Ratios of the contributions to resistance of chromosome II and chromosome III for each EMS mutant at each of the lufenuron concentrations tested, and grouping based on these patterns. Group 1: Chromosome III explained a large proportion of resistance at all lufenuron concentrations, Group 2: Chromosomes II and III had similar contributions, Group 3: Similar contributions at lower concentrations and more of an influence of chromosome III at higher concentrations. Also shown are strains WC2, and Inn5 for comparison. 1.8ppm 2.1ppm 2.3ppm Group WC2 4.25 4.00 6.00 1 Inn5 0.53 0.80 1.17 2 EMS#2-2 1.27 1.67 22.00 3 EMS#4-7 11.33 9.50 7.50 1 EMS#4-10 1.40 1.43 5.67 3 EMS#5-5 5.00 8.50 8.00 1 EMS#5-7 6.33 8.00 10.00 1 EMS#5-8 4.20 9.00 11.00 1 EMS#8-6 2.80 1.00 11.00 3 EMS#11-4 5.00 4.00 4.75 1 EMS#17-2 15.00 2.08 2.75 EMS#17-4 2.00 1.50 9.00 3 EMS#17-8 1.50 9.25 4.50 EMS#18-11 8.40 8.00 7.50 1 EMS#19-7 0.91 1.33 14.50 3 EMS#20-3 1.20 3.25 2.29 2 EMS#20-4 4.50 13.50 14.00 1 EMS#20-10 1.54 1.60 1.63 2 EMS#20-12 1.00 1.50 1.33 2 212 It is clear from table 6.4 that several groups exist based on their chromosome ratio profiles over the different concentrations chosen. The members of group 1 include WC2, the three mutants isolated from EMS trial 5 (EMS#5-5, EMS#5-7, EMS#5-8) and other mutants isolated from trials 11, 18. and 20 (EMS# 11-4, EMS#18-11, and EMS#20-4). While chromosome II contributes to resistance, chromosome III has the overwhelmingly larger effect. It is possible that the same mutations or mutations of similar function effect underpin this group 1 type resistance. In this context it should be emphasised that mutants arising from the one mutagenesis could be derived from a single mutational event. Pre-meotic mutations can lead to clusters of identical sperm being produced by a single mutagenized male (McKenzie & Batterham, 1998). This may provide an explanation for the similarity between the EMS 5 mutants. Similarly, three of the four mutants isolated from trial 20 (EMS#20-3, EMS#20-10, EMS#20-12) had the same profile as Inn5, again suggesting that they have the same mechanism of resistance and originated from an EMS-generated mutation in a single male. Members of this group showed reasonably equal contributions from each chromosome at all concentrations tested, defining group 2. Members of group 3 are less distinguishable. Group 3 members appear to show equal contributions at the two lower lufenuron concentrations and it is only when they are particularly stressed at 2.3ppm that the effect of chromosome III becomes more significant. 213 6.3.5 Mapping within a chromosome Genetic mapping crosses as described in chapter 5 figure 5.2 were carried out with several of the EMS mutants in order to map resistance loci that may be segregating within chromosome III. Varying numbers of flies were scored for each mutant examined, and due to the examination of different strains and therefore many individuals, control screens (0ppm lufenuron) were pooled. Results are shown on figure 6.5, and tabulated in table 6.5. It appears that in all of the EMS mutant strains examined, resistance maps to the same location on chromosome III. It is also the location to where resistance maps in Inn5 and WC2 (chapter 5, figures 5.9 and 5.10). It is unlikely that a chromosome III resistance locus was segregating in the Armenia base strain on the basis of the lack of high level resistance in unmutagenised controls, and the number small number of mutants isolated. Chromosome III-associated resistance is therefore likely to have arisen as an EMS-induced mutation in each of the strains. The levels of resistance and relative contributions of each of chromosomes II and III vary between the EMS strains, and between these strains and WC2 and Inn5. If the same loci are involved, as mapping data would suggest, different alleles of the resistance loci are likely to be present in the EMS strains. 214 EMS#5- 5 EMS#5-7 EMS#5-8 0.6 0.6 0.6 0.5 0.5 0.5 r e er er k k k r r r 0.4 0.4 a 0.4 a a m m f o of m of y y y 0.3 0.3 0.3 c n e u uenc q quenc 0.2 0.2 0.2 e e eq r r r F F F 0.1 0.1 0.1 0 0 0.0 ru h st ry e ru h st ry e ru h st ry e Phenotype of marker Phenotype of marker Phenotype of marker EMS#4-10 EMS#8-6 EMS#18-11 0.6 0.6 0.6 0.5 0.5 0.5 r r r e e e k k k 0.4 0.4 r 0.4 a mar mar m f o of of y y 0.3 0.3 y 0.3 c n e u 0.2 0.2 q 0.2 equenc equenc e r r r F F F 0.1 0.1 0.1 0 0 0 ru h st ry e ru h st ry e ru h st ry e Phenotype of marker Phenotype of marker Phenotype of marker Figure 6.5: Intrachromosomal mapping within chromosome III. Frequency of each mutant class for both the pooled 0ppm control and 2.1ppm screen is shown for each EMS mutant tested. 215 Table 6.5: χ2 analysis examining the influence of resistant loci on chromosome III for each of the EMS mutants screened (df=4). The χ2 values is the probability based on the probability of accepting the hypothesis that Observed (Obs) = Expected (Exp) is also shown P<0.05 leads to a rejection of the hypothesis. ru h st ry e χ2 Probability Pooled control Obs 0ppm 333.2 283.6 299.3 358.3 315.6 Exp 0ppm 320 320 320 320 320 n=640 10.67 0.1 n=335 477.4 P<0.001 EMS#5-7 Obs 2.1ppm 67.0 27.0 21.4 6.7 9.7 Exp 2.1ppm 118.5 118.5 118.5 118.5 118.5 n=237 377.9 P<0.001 EMS#5-8 Obs 2.1ppm 96.1 18.3 6.2 2.0 23.0 Exp 2.1ppm 122.5 122.5 122.5 122.5 122.5 n=245 404.3 P<0.001 EMS#8-6 Obs 2.1ppm 51.6 20.8 6.4 0.0 5.1 Exp 2.1ppm 43 43 43 43 43 n=86 120.8 P<0.001 EMS#4-10 Obs 2.1ppm 130.8 52.6 16.3 0.0 12.8 Exp 2.1ppm 109.0 109.0 109.0 109.0 109.0 n=218 306.3 P<0.001 EMS#18-11 Obs 2.1ppm 70.6 14.4 1.0 1.0 8.5 Exp 2.1ppm 66.0 66.0 66.0 66.0 66.0 n=132 218.7 P<0.001 216 6.4 Discussion 6.4.1 Mutagenesis and isolation of resistant mutants The frequency of a resistance allele without selection is estimated to be 10-6 to 10-13 (Anderson, 1995). Therefore, new spontaneous resistant alleles will arise very rarely in susceptible strains maintained in the laboratory. The exposure to a mutagen such as EMS which is estimated to produce a mutation every 2000bp, depending on the dose applied, increasing the likelihood of isolating a resistant mutant dramatically. This study was successful in isolating 17 lufenuron-resistant mutants in D. melanogaster after mutagenesis and selection in the strain Armenia. The selection procedure used had several biases. The different outcomes of selecting above and below the LC100 were discussed in Chapter 5, section 5.1. EMS mutagenesis, as applied in this study, offered a bias towards single-gene based resistance since the screening concentrations used were above the LC100 of Armenia. This most closely mimics the selection occurring in natural populations exposed to insecticides (Roush & McKenzie, 1987; McKenzie et al., 1992; McKenzie, 1996) but offers it in a more highly defined background and affords the potential for a variety of mutants to be isolated, allowing for a more comprehensive assessment of resistance mechanisms to be made. There is a bias towards dominant, partially dominant, or sex-linked recessive phenotypes since an autosomal recessive mutant would not survive the screening as a heterozygote in the F1 generation (see figure 6.1). 217 There was an unforeseen and unforeseeable bias. EMS mutagenesis commenced before the Accord polymorphism was discovered. However, the high frequency of Accord and Cyp6g1 overexpression in the Armenia strain meant that all of the mutants isolated would have these elements in their genetic background. Since mutagenesis was not also carried out in an Accord minus background, it is not clear whether the presence of Accord biased the nature of the mutants recovered. In theory genetic background will create constraints. The outcomes that are possible in mutagenesis will be determined by the nucleotide sequences of the relevant genes in the genome. There are limits to what can be achieved in a single mutational step. It is possible, therefore, that the mutations identified are in genes that functionally interact with Cyp6g1, their products acting as either trans-acting regulators that further boost Cyp6g1 expression or as detoxification enzymes. These possibilities will be discussed further in Chapter 7. If this mutagenesis were to be repeated it would be done in an Accord minus genetic background (e.g. Celera), opening up the possibility of isolating a different spectrum of mutants. However, it should be emphasized that the Armenia background mimics the common genetic background found in natural populations (chapters 4 and 5). The possibility of evolution of Cyp6g1-based resistance through DDT selection and increased resistance through further selection by DDT or other insecticides was discussed in the previous chapters. The lab-selection regime used in this investigation may therefore inadvertently mimic the evolutionary phenomenon seen in natural populations. 218 6.4.2 Resistance levels The lufenuron resistance level of several of the EMS mutants was tested through dosage mortality analysis. Resistance to other insecticides can be in the order of tens of thousands (see, for example (Kranthi et al., 2001)), and so it is significant that levels in these mutants are under 10-fold in relation to susceptible controls. By this comparison, resistance was reasonably similar to field resistant strains discussed in previous chapters (NB16, WC2, and Inn5). It is suggestive that the capability for high levels of resistance to lufenuron is low, a good indicator for the continued efficacy of the product in commercial use, however the pre-existing Cyp6g1 resistance may again be a prejudicial factor. Target site resistance may yet offer a significantly higher fold resistance. Comparing just the strains examined in this investigation, resistance appeared generally higher in lab-generated mutants than the field strains, and patterns emerged between the strains tested. The three strains tested from the same EMS trial (EMS#5-5, EMS#5-7, EMS#5-8) all showed similar resistance profiles, indicating the same mutagenised male may have inseminated several females. Other patterns discussed in the following sections provide corroborating evidence this. The higher levels of resistance may be indicative of different resistance mechanisms, but may also indicate alleles of the same locus having different functional effects. 6.4.3 Inter-chromosome mapping Mapping resistance to chromosomes in the EMS lines has confirmed the involvement of both chromosome II and chromosome III in conferring resistance to lufenuron. 219 Sex-linked inheritance is uncommon in resistance studies (McDonald. & Schmidt., 1957; Heather, 1986), and was therefore not directly examined in these EMS mutants, but the chromosome mapping scheme used nevertheless detects high levels of X-linked resistance. Since males were used in the F1 (see chapter 5, figure 5.1), all F1 X-chromosomes originate from the mapping strain, and so extremely low levels of survival would be seen in the F2 under lufenuron selection (scenario A in table 6.3). All of the EMS mutants showed high resistance at the doses tested, indicating little or no contribution by the X-chromosome. Chromosome III appeared to contribute to resistance overwhelmingly, despite the contribution due to Cyp6g1 overexpression and any other resistance loci on chromosome II. In group 1 strains (table 6.5), a major resistance locus exists on chromosome III. In group 2 strains, the relative influence of chromosome III is less (although still important), and group 3 strains show a peculiar pattern where chromosome III appears to become significantly more important at higher doses. It may be notable that the WC2 and Inn5 strains discussed in chapter 5 fall into groups 1 and 2 respectively. Resistance loci in these strains may be equivalent to those in the relevant groups of EMS mutants. The strains where a full dosage mortality analysis was undertaken did show correlation between the curve generated (as discussed in the previous section) and the relative contribution of chromosomes - EMS#5-5, EMS#5-7, EMS#5-8 fell into group 1 whilst EMS#8-6 fell into group 3. Hence the combination of chromosome mapping and full dosage mortality analysis may be a reliable method of delineating between resistance loci in these strains. 220 6.4.4 Intrachromosomal mapping Mapping within chromosome III was performed with six EMS mutants, and all showed resistance mapping to the same general region as in Inn5 and WC2 (chapter 5, figures 5.9 and 5.10) closest to the ry marker. There was no discernable pattern between each of the mutants, however two mutants showed close linkage to the st marker as well as ry, suggesting that a resistance locus may lie somewhere in between (only 6.0 mu separates these two markers). As in chapter 5, only speculative candidates for a chromosome III resistance locus can be suggested without further testing, but they may well be the same candidates as in WC2 and/or Inn5. Inter- and Intra- chromosomal mapping corroborate evidence of this, however resistance levels appeared generally higher in EMS strains. This may be indicative of different alleles of the same loci that have more pronounced effects. 6.4.5 Future work Identification of the resistance conferring loci in field resistant strains would allow relatively fast comparisons to be made to the EMS mutants. If the same mechanisms are in action, comparison of the resistance alleles in the EMS strains would be beneficial. The presence of the same allele in each case suggests that very few mutations are capable of causing resistance, and presence of multiple alleles gives clues as to the function of the associated gene and how the various mutations are affecting it. The combination of evidence presented here suggests that several different mechanisms are acting amongst the strains but some commonality exists. 221 Whilst lab-based selection in this investigation has been biased by a pre-existing mutation (Accord) in the strain used for mutagenesis, it serves as an excellent model of the evolution of resistance in the field. If there is a specific genetic or functional link between the activity of CYP6G1 and the factor(s) seen present on chromosome III, mutagenesis is likely to select for that mutation based on the high resistance levels of those strains that carry it and the pre-existing resistance conferred by Cyp6g1. Thus the isolation of strains containing mutated target sites, the original aim of the investigation is biased against. If Cyp6g1 overexpression was the original selective mechanism in the field following insecticide use, the creation and spread of the chromosome III allele following continued selection is mimicked well in this lab mutagenesis study. Further comparisons between resistance loci isolated in these strains and natural population strains may provide valuable information as to resistance mechanisms in the field. 222 Chapter 7 Discussion and concluding remarks 7.1 Introduction The large scale use of insecticides in since the 20th century has enabled the increase in agricultural production for an ever-growing human population, the moderation of household pests in increasingly dense urban environments, and the lessening and in some cases eradication of disease carrying vectors. Resistance is the natural evolutionary response to environmental stress and is inevitable over a short or long period, unless there is total extinction of the species in its local and subsequently broader geographical environment. Complete elimination of a pest species is unlikely (and has not been demonstrated, other than in confined local areas), and so resistance research aims not at finding an impossible solution to the resistance problem, but at prolonging the effectiveness of insecticides by the mitigation of the factors leading to resistance. One of the ways of accomplishing this goal is to use powerful biological tools in order to dissect the genetic and molecular makeup of resistant strains. More specifically, by isolating resistance-conferring genes and the mutations leading to the specific alterations in those genes, the biochemical pathways connected to the associated proteins can be assessed and conclusions drawn as to the functional effects of the insecticide and the ensuing resistance caused by allelic changes. Examination of resistant mutants isolated from the field is the instinctive method of understanding resistance. The broad sampling of populations from different geographical, temperate, vegetated, and pesticide-exposed locations can provide an array of mutants with which to begin these genetic examinations. 224 In comparison, the generation and analysis of lab-induced mutant strains has the potential to expose a range of resistance mechanisms that may or may not have arisen in the field, and can provide clues to the target of the insecticide in the cases where field-based studies have failed. It has the additional advantage of the generation of mutations within strains of more defined background, allowing more rapid genetic analysis. Further, mutations can be induced in the laboratory before resistance evolves in the field (McKenzie & Batterham, 1998) D. melanogaster is not a pest species, but has become an indispensable tool for the examination of almost every aspect of biology, providing a model even for distantly related species. The availability of the entire genomic sequence of D. melanogaster has revealed striking similarities between insect species in particular, and corroborates evidence of the developmental, neurological, physiological, structural and genomic conservation between species. It is even greater an asset in the study of insecticide resistance since collateral exposure to insecticides used to treat pest species has allowed many of the same mechanisms of resistance to evolve (Wilson, 2001). In this thesis, the powerful tools of D. melanogaster genetics are used to investigate mechanisms of resistance to the insect growth regulator insecticide, lufenuron. At the time this study was commenced, neither the mode of action of the chemical nor mechanisms of resistance were understood. This research was conducted with the goals of providing a knowledge base for the better management of insect pests, as well as comprehension of the evolutionary pathways used by insect species in responding to chemical stress. 225 7.2 This investigation This thesis begins an examination in this light with an overview of the current literature associated with insecticides including what is known about their modes of action, the wide variety of resistance mechanisms observed in both the field and the lab, how they are identified using genetic tools, and how their elucidation fits into the context of resistance management. This thesis investigates resistance to the insect growth regulator insecticide, lufenuron. After a discourse on the materials and methods used, each of four results chapters is presented. Chapter 3 examined the broad extent of lufenuron resistance in isofemale strains of D. melanogaster from the east coast of Australia. One such strain, NB16, was chosen for further study due to its high level of resistance to lufenuron. A positional cloning approach allowed the identification of a P450, Cyp12a4 (table 3.8), that has elevated expression in this strain (figure 3.16). The creation and screening of transgenic Cyp12a4 overexpressing strains revealed an intriguing dichotomy of larval to adult viability outcomes. Depending on the temporal and spatial pattern of Cyp12a4 overexpression, either resistance or death of the insect was observed (figure 3.17). Cyp12a4 was suggested to be the resistance-conferring gene, although the presence of a loss of function mutation in the adjacent gene, Cyp12a5 (figure 3.14), leaves this gene as another possible candidate. Chapter 4 began an examination of a field-derived lufenuron resistant strain from USA (WC2), and identified another P450 associated with lufenuron resistance. The Cyp6g1 gene was found to be overexpressed in this strain (figure 4.2). Indeed, 226 Cyp6g1 was the only P450 overexpressed in this strain (R. Feyereisen, INRA, France). The Accord transposable element insertion was identified in this study (figures 4.8, 4.10). Phillip Daborn (University of Bath) subsequently found that the presence of this insertion was perfectly correlated with DDT resistance in resistant strains from around the world (Daborn et al., 2002). Transgenic Cyp6g1 overexpressing strains showed increased larval to adult viability on lufenuron (figure 4.14), confirming its involvement with resistance. Chapter 5 demonstrated that a gene or genes other than Cyp6g1 contribute to lufenuron resistance in WC2. The differences between WC2 and another strain (Inn5) were noted, including the discovery of a partial P element insertion within Accord in Inn5 (figure 5.5). Preliminary mapping in both of these strains indicated a region of interest for a potential candidate resistance gene. The gene(s) map to chromosome 3, near ry (52.0cM) which is between the markers st (44.0cM) and e (77.0cM) (figures 5.9 and 5.10). Finally, a survey of the frequency of the Accord insertion variants was undertaken on a transect taken along the east coast of Australia. Accord insertion frequencies were high (figures 5.6 and 5.7). Although the data set was limited, frequencies appeared to vary clinally. Chapter 6 introduced the notion of lab-based resistance with the aim of identifying the target of lufenuron. Biases in the experimental protocol meant that Cyp6g1-based resistance was selected for in the 17 mutants isolated. Resistance in all of these mutants was mapped to both chromosome II, and predominantly, chromosome III (figure 6.4, table 6.4). Varied levels of resistance (figure 6.2), chromosomal contributions (figure 6.4), and locations of resistance loci (figure 6.5) suggested that different closely linked mechanisms may be acting, or that different alleles of the same resistance conferring gene were present. 227 Finally, this discussion aims to link the results obtained in the previous chapters in order to draw more general conclusions of the lufenuron-resistance options that are available to D. melanogaster, and their implications in pest species. 7.3 Options for resistance to lufenuron The physiological effects of lufenuron were discussed in chapter 1, section 1.6. The necessity for better understanding of the complex physiology of the insect epidermis and the complicated hormonal regulation of the moulting process has been the goal of many pure research studies (for example (Bennett & Reid, 1995; Palli & Retnakaran, 1999; Cohen, 2001), and some insecticide resistance studies (for example (Retnakaran & Wright, 1987; Retnakaran & Oberlander, 1993; Wilson & Cryan, 1997). Given the complexity of cuticle biology, these studies have revealed little about the target for lufenuron or mechanisms of resistance. This thesis aimed to use D. melanogaster genetics to address both of these issues. Success has been limited to resistance mechanisms, specifically detoxification mechanisms based on the activity of P450s. The discovery of widespread field resistance to lufenuron in D. melanoagaster was unexpected (Wilson & Cryan, 1996; Wilson & Cain, 1997; O'Keefe, 1997). Not so much because D. melanogaster is not a pest and therefore not intentionally treated, but because lufenuron has not seen significant field usage. The most obvious reason for the resistance was cross-resistance resulting from exposure to a previously used insecticide. Given that detoxification enzymes can have remarkably broad substrate specificities (chapter 1, section 1.5), it is not suprising that at least two of the three lufenuron resistant genes identified in this project encode detoxification enzymes. 228 7.3.1 Resistance associated with Cyp12a4 In the NB16 strain, resistance mapped to a region encompassing two P450 genes (chapter 3, figure 3.12). These became obvious candidates as for conferring resistance. The absence of gene product from Cyp12a5 (which contains a nonsense mutation and may be subject to nonsense-mediated decay) has not yet been tested. The lack of such a product is theoretically capable of causing resistance if its role as a native protein is to activate lufenuron by creating a more toxic metabolite. This hypothesis forms an intriguing and unique possibility worth further examination, and is currently being undertaken in the Batterham laboratory through the creation of a targeted gene knockout. Most documented cases of P450-associated resistance are associated with overexpression and detoxification (chapter 1, section 1.5). Thus the overexpression of Cyp12a4 is considered the more likely explanation for the resistance observed. Due to the functional redundancy of some P450s, it had been a possibility that the overexpression of this gene could be an artefact related to the lack of Cyp12a5 product. That the endogenous overexpression of Cyp12a4 is capable of causing lethality suggests that, when overexpressed in one or more specific tissues, CYP12A4 is capable of producing toxic concentrations of metabolites from endogenous substrates. However, the overexpression of the gene in other tissues including the midgut and fatbody leads to resistance. Given that different patterns of tissue specific regulation of Cyp12a4 can generate the extremes of lethality in the absence of insecticide, and resistance in the presence of insecticide, the cis-acting regulatory control sequences for this gene are of intense interest. The comparison of DNA sequences around Cyp12a4 for NB16 and Celera 229 revealed no obvious differences that would account for the observed overexpression. One formal possibility is that the mutation in the Cyp12a5 gene leads to a compensatory upregulation of Cyp12a4. This will be easily tested once the Cyp12a5 knockout strain becomes available. While Cyp12a4 may mediate resistance in NB16, its importance as a resistance mechanism more broadly in D. melanogaster populations is not known. If the molecular basis of the overexpression were known, a PCR-based molecular diagnostic would allow this question to be quickly addressed. In the absence of such a diagnotic, microarrays or realtime PCR to look for overexpression in a large sample of field strains needs to be employed. A survey of 85 field strains taken from along the east coast transect has failed to find any strains with the Cyp12a5 mutation identitied in NB16 (data not shown). In spite of the fact that the overexpression of P450s is a major insecticide resistance mechanism, the transcriptional regulation of these genes in insects is poorly understood. The further study of the Cyp12a4 overexpression in the NB16 strain has a contribution to make. Realtime PCR on Cyp12a4 levels in various tissues in NB16 and Celera should be used to identify the tissues that may contribute to resistance. The gal4-UAS system can then be used to drive overexpression in those tissues ,testing for an association with resistance. Nested DNA fragments from around the Cyp12a14 gene should be cloned in front of a reporter gene such as GFP for use in transformation studies to identify the sequences responsible for the overexpression in the particular tissue(s). 230 7.3.2 Resistance associated with Cyp6g1 The overexpression of Cyp6g1 appeared to confer resistance to insecticides in many natural population strains of D. melanogaster (Daborn et al., 2002; Pyke et al., 2003) chapters 4 and 5). As described in this investigation, resistance can be mapped to Cyp6g1 (chapter 4, figure 4.7), and the overexpression of this gene driven in vivo using the gal4-UAS system undoubtedly causes a low level of lufenuron resistance (chapter 4, figure 4.14). Based on other studies, it also confers high level cross resistance to DDT, and medium level resistance to imidacloprid, and nitenpyram (Daborn et al., 2002); T. Perry, Pers. Comm.). The overexpression is highly associated with the presence of an Accord element or other Accord variants upstream of the gene (chapter 4 figures 4.9, 4,10, chapter 5, figure 5.5). This association is complete from strains collected in Australia and around the world chapter 4, figure 4.9). It should be briefly noted that to location of the insertion is within the 3’ UTR of an adjacent gene, CG33154, although this gene can be ruled out as the resistance-conferring gene through results of the Cyp6g1 transgenic screening (figure 4.14). The frequency of the Accord element is so high within natural population strains, that the associated Cyp6g1 overexpression is now the wild-type phenotype (chapter 5, figure 5.7). Recent work (C. Robin, and T. Perry, Pers. Comm.) has identified additional Accord variants, including variants where an internal P-element (such as in Inn5) has remobilized and removed most of Accord. Overexpression seems to be associated with all of these variants although controlled experiments have not been conducted to determine if particular Accord variant types have characteristic levels of Cyp6g1 expression. The frequency of these variant elements also needs to be assessed in natural populations. 231 The Cyp6g1/Accord story presents both unresolved questions and some implications for insecticide resistance and management. These are discussed below. 7.3.2.1 Is Accord causing Cyp6g1 overexpression? The correlation between the Accord element insertion and Cyp6g1 overexpression is complete in the 40 lines surveyed from diverse geographical locations (Daborn et al., 2002), but the mechanism that would provide irrefutable evidence for causality has not been identified. In the sibling species, D. simulans, a recent study has implicated the insertion of a complete Doc retrotransposon in the upstream region of the Cyp6g1 to its overexpression (Schlenke & Begun, 2004). This Doc insertion also contains small portions of an unknown gene (CG9137) and the 5’ flanking region of the Cyp12c1 gene (Schlenke & Begun, 2004). Analysis of the Cyp6g1 upstream region and of the insertion in this species also reveals several putative transcription factor binding sites that could potentially have a role in regulation changes, although once again the results of this type of analysis must be considered speculative. The 5’ end of the D. melanogaster gene contains several putative transcription factor binding sites, including the putative Ahr/Arnt binding site that is disrupted by Accord. However, these sequences are not conserved in D. simulans, and not interrupted by the Doc insertion (A. Williams, Pers. Comm.). Therefore, the Accord insertion may not disrupt sequences essential for the negative regulation of Cyp6g1. The possibility that Accord insertion causes upregulation by increasing the spacing between the gene and enhancer sequences 5’ to Accord cannot be discounted. However, given that retrotransposons carry tissue specific enhancers (Britten, 1996), it seems more likely that the Accord sequences insertion carries enhancers that cause the overexpression of Cyp6g1 in specific metabolic tissues. 232 The further investigation of the Accord insertion and its contribution to Cyp6g1 overexpression is currently being undertaken in the Batterham laboratory through the creation of transgenic strains (P. Daborn, Pers. Comm). These will include strains with the deletion of various parts of the Cyp6g1 upstream region, strains with an Accord element replaced with other DNA, and strains where Accord is removed entirely. 7.3.2.2 How did Cyp6g1 overexpression evolve? A way of understanding the evolution of Cyp6g1-based resistance is to identify the initial selective mechanism. Since all lab strains that were collected before 1930s such as CantonS, Celera, and Oregon-RC did not show Cyp6g1 overexpression (Daborn et al., 2002), it is likely that Cyp6g1 overexpression has evolved since then. It then begs the question as to what has caused the overexpression phenotype to evolve. The 1940s and 50s saw widespread usage of DDT throughout the world, and whilst D. melanogaster is not a pest species, incidental exposure would have been unavoidable. The selective pressure exerted on this species would have required a response. Whether the Accord insertion already existed at low frequency or arose as spontanenous mutation, it would have rapidly increased in frequency given the strength of selection as a result of global DDT use. It is not surprising then that the Accord mutation appears to be of a single origin based on phylogenetic analysis (Daborn et al., 2002; ffrench-Constant et al., 2004). Thus the most likely reason for the evolution of this Cyp6g1 overexpression and its geographical spread over a short period of time is the worldwide usage of DDT. 233 7.3.2.3 Why is Accord frequency so high and why has it persisted? The second conclusion relating to the Cyp6g1 resistance phenomenon involves the persistence of the Accord allele in natural population strains. Approximately half of resistance-conferring mutations have deleterious effects in the absence of a selective force, and it is only in its presence of insecticide selection that they become beneficial (McKenzie, 1996b; Taylor & Feyereisen, 1996). Recent laboratory population cage experiments have shown a decrease in Accord frequency over several generations in the absence of insecticide, indicating that Accord may have an associated fitness cost in the absence of selection (Robin et al., unpubl.). Thus under a hypothesis of the DDT-associated origins of Cyp6g1 overexpression, a decrease in frequency of Accord would be expected given the withdrawal of DDT. Clearly this is not the case; Accord insertion frequencies are high. Several factors may signify why this is the case. Since the abolition of DDT use, many other insecticides have taken its place, and although no one insecticide is as widely used, the broad cross resistance profile associated with Cyp6g1 overexpression may allow its continued selection in natural populations. Under this same argument, there may be many other selective forces in the environments where natural populations reside, including other toxicants and natural factors that could select for Accord/Cyp6g1 overexpression. Although these compounds may have been present before insecticide use, the DDT hypothesis is favoured because the available data suggest that the Accord insertion was not at high frequency before DDT was used. 234 7.3.3 Other genes associated with resistance Apart from Cyp12a4 and Cyp6g1, there appears to be at least one other gene associated with lufenuron resistance. Interchromosomal mapping in WC2 and Inn5 revealed contributions of chromosome III towards resistance in both of these strains (chapter 5, table 5.5). Intrachromosomal mapping identified a resistance-associated region that was closely linked to the rosy locus (figures 5.9 and 5.10). A similar location was identified in each of the 6 EMS mutants examined (chapter 6, figure 6.5). Fine-scale mapping is needed to identify the resistance-conferring gene(s), however several possibilities can be put forward as to its relationship with Cyp6g1- based resistance. The gene could represent a completely independent mechanism of resistance to lufenuron. The isolation of chromosome substitution lines (currently being untertaken by T. Perry) would shed light on this possibility by providing an Accord minus, Cyp6g1 overexpression-free background with which to assess independently assess the lufenuron resistance contributed by chromosome III. An independent chromosome III resistance mechanism would confer resistance in this background. An interaction between Cyp6g1 and the chromosome III mechanism is an alternative and probable explanation, and would be indicated by susceptibility. Such an interaction could occur in two ways. Cyp6g1 may act as a primary detoxification mechanism, providing a substrate for the secondary mechanism on chromosome III. Such two-phase mechanisms are common in detoxification systems (Hemingway et al., 1991; chapter 1, section 1.4.4.1). Alternatively, the chromosome III gene may form part of a regulatory mechanism, altering the special or temporal specificity of 235 Cyp6g1 overexpression, enhancing its detoxification effect. Positional cloning is the most direct route to resolving this issue. If there is a connection between Cyp6g1 and chromosome III resistance, the generation of resistance in EMS screens was biased towards this mechanism. Mapping located the major resistance locus on chromosome III to the same region identified in WC2 and Inn5 (chapter 6, figure 6.5). However the resistance levels appeared to be higher in most EMS mutants (chapter 6, figure 6.2) than in WC2 (chapter 4, figure 4.5) or Inn5 (chapter 5, figure 5.3). These higher levels may represent different genes, different alleles of the same gene as in WC2 or Inn5, or some general genetic background effect. The goal of the lab-based study was to identify the target of lufenuron. The recovery of just 17 highly resistant mutants from approximately 10 million embryos screened, all of which exhibit the Cyp6g1 overexpression mechanism, suggests there was a particularly strong bias in this experiment. Future target-focussed experiments may take advantage of the known sequence and little variation of the Celera strain to avoid some of these unforseen biases. Further, target site resistance is often recessive (McKenzie, 1996a). Future experiments may focus on the Zuker lines, a collection of over 12,000 strains of heavily treated with EMS and balanced to the F3 generation (Koundakjiana et al., 2004). Due to mosaic mutations induced in the initial exposure, many of the strains are fixed for lethal mutations on the mutagenised chromosome, enabling the isolation of recessive resistance-conferring mutations which may include a lufenuron target. 236 7.4 Implications for pest control One of the reasons that field strains of D. melanogaster are used as a model species in many insecticide resistance studies is that they share the environment with pest species. The similarity between insect genomes would suggest that D. melanogaster and pest species would have similar capacities to respond to the same insecticide selection pressures. There are excellent examples of this for target site resistances e.g. Rdl, kdr and Ace (see (ffrench-Constant, 1994; ffrench-Constant et al., 2004) for review) different species show similar or the same mutations in the orthologous genes. The extent to which D. melanogaster will be a good model for detoxification based resistance is not so clear. The Cyp6g1-based resistance mechanism has so far been uncovered only in Drosophila species. Even in this case it is not known whether the overexpression of Cyp6g1 in D. simulans provides resistance to the broad spectrum of resistances, observed to be associated with Cyp6g1 overexpression in D. melanogaster. The P450s are a rapidly evolving gene family. There is a lack of information on how differences in amino acid sequences impact upon the substrate specificity of insect P450s. This point is underlined by the fact that this study suggests that two P450s from distinct families (and therefore having limited amino acid sequence similarity), CYP6G1 and CYP12A4, may both detoxify lufenuron. Building on the current study, two types of experiments would be valuable. The first of these would be to overexpress other D. melanogaster P450 genes in D. melanogaster. These overexpression strains could be tested for resistance to a range of insecticides. The second class of experiments would be to overexpress Cyp6g1 orthologues isolated from different species in D. melanogaster. Since the 237 genomes of several Drosophila species have been sequenced, a number of orthologues are readily available, and recently, the Cyp6g1 orthologue from the sheep blowfly, Lucilia cuprina was isolated (Chen and Batterham, unpubl.). The overexpression of these genes in live insects has the advantage over heterologous expression in that it is simpler to achieve and it is possible to assay for resistance to a wide range of chemicals without the need to develop biochemical assays. That the Cyp6g1 resistance mechanism is seen to act with respect to lufenuron, an insecticide used for the treatment of fleas in household pets, has important implications for pest control in general. Remembering that it is extremely unlikely D. melanogaster or any pests found in the field have been exposed to lufenuron, the observation of such widespread lufenuron resistance is alarming at least. It suggests in a more general case that this mechanism of resistance may operate for compounds that an organism has never previously been exposed to. Cyp6g1 may confer resistance to many insecticides that have yet to be discovered. The huge time and cost associated with the release of insecticides means that such information is pivotal for assessing the viability of new products at early stages of development. The development of a Cyp6g1 heterologous expression system would be a beneficial investment for chemical companies. It could be used as a diagnostic test to observe the metabolic ability of CYP6G1 towards new insecticides. More generally, the defence systems of insects must be understood, starting with D. melanogaster where the genome can be manipulated, and then moving on to pest systems. 238 7.5 Final Comments Insecticide resistance is an intriguing biological phenomenon and a seemingly inevitable evolutionary outcome for insect species. Resistance research focuses not on the unattainable goal, the complete eradication of pest species, but rather on the management of resistance and the prolonging of susceptibility. The generation of a strong knowledge base is the most intelligent way of better understanding the methods that insects employ to overcome susceptibility, ultimately enabling the design of better-acting, more specific insecticides. In the past, chemical insecticides of unknown mode of action have been deployed against insects with unknown defence systems, with disastrous repercussions in terms of resistance. 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