Studies on resistance and associated fitness costs in Lepidoptera to Bacillus thuringiensis toxins
ASIM GULZAR
B.Sc. (Hons), M.Sc. (Hons)
A thesis submitted for the Degree of Doctor of Philosophy
of Imperial College London
Division of Biology
Silwood Park Campus
Imperial College London
November 2010
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Dedication
This thesis is dedicated to all the members of my family, especially my mother
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Declaration
The work presented in this thesis is entirely my own and has not been submitted anywhere else.
Signed...... Asim Gulzar
Certified...... Professor Denis J. Wright
(PhD Supervisor)
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Acknowledgements
In the first place I would like to record my gratitude to Professor Denis J. Wright for his supervision, advice, and guidance from the very early stages of this research and throughout the work. He provided me unflinching encouragement and support in various ways. His insight has provided a constant source of ideas and passion for science, which have inspired and enriched my growth as a student, a researcher and as the scientist I want to be. I am indebted to him more than he knows. I am also very grateful to Dr. Simon Leather and Professor Jim Hardie for their constructive comments and encouragement during my upgrade exam.
I would also like to thank Professor Juan Ferré (Department of Genetics, University of Valencia, Spain) for giving me a chance to work in his laboratory. I would also like to thank Dr Silvia Caccia for her support and guidance during my stay in Spain. Let me also say „thank you‟ to Dr Ali Sayyed. He was always there to meet and talk about my ideas, and to proof read my chapters. I would also like to thank Dr Graham Moores (Rothamsted) for his help and guidance regarding the esterase work.
Words fail me to express my appreciation to my parents, for educating me, for unconditional support and encouragement to pursue my interests, even when the interests went beyond boundaries of language, field and geography. I especially thanks to my brother Amir and my sister Kanza for listening to my complaints and frustrations, and for believing in me.
I would like to thank the Higher Education Commission of Pakistan for funding, which made this study possible.
Last, but not least, I would like to thank all my friends who were important to the successful submission of thesis, as well as expressing my apology that I could not mention all personally one by one.
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Abstract
Microbial insecticides derived from the common soil bacterium Bacillus thuringiensis (Bt) have become increasingly important for pest management, particularly in Bt (GM) crops. In addition to crystal (Cry) proteins produced during the spore stage, vegetative insecticidal proteins (VIP) produced during vegetative growth are of current interest for use with Cry toxins in GM crops. In this thesis, the effects of Bt toxins on two target lepidopteran pest species, the bollworm, Heliothis virescens, and the diamondback moth, Plutella xylostella, are described. Cross-resistance to non-Bt insecticides was not found in populations of H. virescens (Vip3A-Sel WF06) and P. xylostella (Cry1Ac-Sel Karak) resistant to Vip3A and Cry1Ac respectively. While the synergist piperonyl butoxide (PBO), an inhibitor of microsomal mono-oxygenases and esterases, did not enhance the toxicity of Vip3A in Vip3A-Sel H. virescens and PBO and the esterase inhibitor DEF did not enhance the toxicity of Cry1Ac in P. xylostella Cry1Ac-Sel Karak. Selection studies with PBO on a population of P. xylostella (SERD4) with a history of developing resistance and cross-resistance to insecticides failed to result in resistance to PBO after selection for 20 generations. Electrophoresis studies indicated the presence of an extra esterase band in Vip3A-Sel compared with unselected H. virescens but there is no evidence that it is related to resistance. Binding experiments suggested that the labelling reaction may have inactivated the binding sites of Vip3Aa and evidence of specific binding of Vip3A toxin to midgut epithelial membranes of unselected H. virescens could not be obtained. Treatment of H. virescens (WFO6, unselected) and P. xylostella (Karak, unselected) with a sub-lethal concentration of Vip3A was found to increase larval and pupal development time and pupal weight, reduce survival (neonate larvae to adult emergence) and mating pair success, increase fecundity and reduce egg viability and the intrinsic rate of population increase (rm). Various fitness costs were found to be associated with resistance in Vip3A-Sel H. virescens and some fitness costs were shown to be temperature dependent: survival (egg to adult), mating pair success, production of viable progeny, egg viability and fecundity, and rm of Vip3A resistant insects being lower at sub-optimal culture temperatures compared with unselected insects. Vip3A resistant larvae were also found to be more susceptible to entomopathogenic nematodes, and had a lower flight muscle ratio, and a greater level of fluctuating asymmetry of morphological traits compared with unselected insects. The work is discussed with particular reference to resistance management strategies for Vip toxins.
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CONTENTS
Dedication...... 2 Declaration...... 3 Acknowledgements...... 4 Abstract...... 5 Table of Contents...... 6 CHAPTER 1: Introduction ...... 12
1.1 General background ...... 12
1.2 Aim of present study ...... 14
1.3 Objectives ...... 14
CHAPTER 2: Literature Review ...... 15
2.1 Resistance to insecticides ...... 15
2.2 Bacillus thuringiensis ...... 16
2.2.1 History ...... 16
2.2.2 Virulence factors of Bacillus thuringiensis ...... 17
2.2.2.1 δ-endotoxins ...... 17
2.2.2.2 Mode of action of Cry toxins ...... 18
2.2.2.3 Vegetative insecticidal proteins (Vip) ...... 18
2.2.2.4 Vip classification ...... 18
2.2.2.5 Vip3A ...... 19
2.2.2.6 Mode of action of Vip3A ...... 19
2.2.3 Resistance to Bt ...... 19
2.3 Mechanisms of resistance to Bt ...... 21
2.3.1 Binding site modification ...... 21
2.3.2 Altered proteolytic processing ...... 22
2.3.3 Other resistance mechanisms ...... 22
2.4 Cross-resistance ...... 23
2.5 Fitness costs and insecticide resistance ...... 24
2.6 Sub-lethal effects of insecticides ...... 25
2.7 Fluctuating asymmetry and insecticide resistance ...... 25
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2.8 Piperonyl butoxide (PBO) ...... 26
2.9 Heliothis virescens ...... 26
2.10 Plutella xylostella ...... 28
2.11 Resistance management in Bt crops ...... 30
CHAPTER 3: Materials and Methods ...... 32
3.1 Plants...... 32
3.2 Insects ...... 32
3.2.1 Heliothis virescens ...... 32
3.2.2 Plutella xylostella ...... 32
3.3 Bacillus thuringiensis toxins and other insecticides ...... 33
3.4 Insect cultures ...... 33
3.4.1 Heliothis virescens ...... 33
3.4.2 Plutella xylostella ...... 34
3.5 Bioassays ...... 35
3.5.1 Heliothis virescens ...... 35
3.5.2 Plutella xylostella ...... 35
3.5.2.1 Topical bioassays ...... 35
3.5.2.2 Leaf–dip bioassay ...... 36
3.6 Selection for resistance with PBO ...... 36
3.7 Synergism bioassay ...... 36
3.8 Statistical analysis...... 37
CHAPTER 4: Studies on cross-resistance, synergism and mechanisms of resistance in Bt toxin-selected populations of Plutella xylostella and Heliothis virescens...... 38
4.1 Introduction ...... 38
4.2 Materials and Methods ...... 39
4.2.1 Insect culture and chemicals ...... 39
4.2.2 Cross-resistance and synergism studies with Cry1Ac-Sel and Unsel sub- populations of Plutella xylostella (Karak) ...... 39
th 4.2.3 Toxicity of PBO against early and late 4 instar larvae of the Unsel sub- population of Plutella xylostella (SERD4) ...... 40
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4.2.4 Selection for resistance to PBO in the Unsel sub-population of Plutella xylostella (SERD4) ...... 40
4.2.5 Cross-resistance and synergism studies with Vip3A-Sel and Unsel sub- populations of Heliothis virescens (WF06) ...... 40
4.2.6 Vip3A binding experiment with Heliothis virescens Vip3A-Sel and Unsel populations ...... 41
4.2.6.1 Activation of Vip3Aa protoxin ...... 41
4.2.6.2 Preparation of Brush Border Membrane Vesicles (BBMV) from Heliothis virescens midguts ...... 42
4.2.6.3 Labelling of Vip3Aa with iodine125 ...... 43
4.2.6.4 Binding of 125I-Vip 3Aa to Unsel Heliothis virescens BBMV ...... 44
4.2.6.5 Labelling of Vip3Aa with biotin ...... 45
4.2.6.6 Binding of Biotinylated Vip3Aa to Unsel and Vip3A-Sel Helithis virescens BBMV 46
4.2.7 Polyacrylamide gel electrophoresis of midgut esterases in Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06) ...... 47
4.3 Results ...... 47
4.3.1 Cross-resistance and synergism studies with Cry1Ac-Sel and Unsel sub- populations of Plutella xylostella (Karak) ...... 47
th 4.3.2 Toxicity of PBO with early and late 4 instar larvae of the Unsel sub-population of Plutella xylostella (SERD4) ...... 49
4.3.3 Selection for resistance to PBO in the Unsel sub-population of Plutella xylostella (SERD4) ...... 50
4.3.4 Cross-resistance and synergism studies on Vip3A-Sel and Unsel sub- populations of Heliothis virescens (WF06) ...... 50
4.3.5 Binding of 125l-Vip3Aa to BBMV of Vip3A-Sel and Unsel sub-populations Heliothis virescens (WF06) ...... 52
4.3.6 Binding of biotin labelled Vip3A to BBMV of Vip3A-Sel and Unsel sub- populations Heliothis virescens (WF06) ...... 53
4.3.7 Polyacrylamide gel electrophoresis of midgut esterases in Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06) ...... 54
4.4 Discussion ...... 55
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CHAPTER 5:Sub-lethal effects of Vip3A on survival, development and fecundity of Heliothis virescens and Plutella xylostella ...... 58
5.1 Introduction ...... 58
5.2 Materials and Methods ...... 59
5.2.1 Insect culture and insecticides ...... 59
5.2.2 Initial bioassays with P. xylostella ...... 59
5.2.3 Development and survival of larval stages of Plutella xylostella to adults ...... 59
5.2.4 Adult longevity, mating success, fecundity and egg viability of Plutella xylostella 59
5.2.5 Initial bioassays with Heliothis virescens ...... 60
5.2.6 Development and survival of larval stages of Heliothis virescens to adults .... 60
5.2.7 Adult longevity, mating success, fecundity and egg viability of Heliothis virescens ...... 60
5.2.8 Growth rate of Plutella xylostella and Heliothis virescens ...... 61
5.2.9 Intrinsic rate of population increase ...... 61
5.2.10 Statistical analysis ...... 62
5.3 Results ...... 62
5.3.1 Sub-lethal concentration of Vip3A ...... 62
5.3.2 Development and survival of Heliothis virescens and Plutella xylostella ...... 62
5.3.3 Adult longevity, mating success, fecundity and egg viability of Heliothis virescens and Plutella xylostella ...... 66
5.3.4 Intrinsic rate of population increase ...... 66
5.4 Discussion ...... 67
CHAPTER 6: Effect of temperature on the fitness of the Vip3A resistant sub-population of Heliothis virescens (WF06) ...... 69
6.1 Introduction ...... 69
6.2 Materials and Methods ...... 70
6.2.1 Insects ...... 70
6.2.2 Development and survival of Heliothis virescens ...... 70
6.2.3 Adult longevity, mating success, fecundity and egg viability ...... 70
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6.2.4 Intrinsic rate of population increase ...... 71
6.2.5 Statistical analysis ...... 71
6.3 Results ...... 71
6.3.1 Development and survival of Heliothis virescens ...... 71
6.3.2 Adult longevity, mating success, fecundity and egg viability ...... 76
6.4 Discussion ...... 77
CHAPTER 7: Effect of entomopathogenic nematodes on the fitness of a Vip3A resistant sub- population of Heliothis virescens (WF06) ...... 80
7.1 Introduction ...... 80
7.2 Materials and Methods ...... 81
7.2.1 Insect culture ...... 81
7.2.2 Nematodes ...... 81
7.2.3 Nematode bioassays ...... 81
7.2.3.1 Mortality ...... 81
7.2.3.2 Estimation of nematode penetration ...... 82
7.2.3.3 Nematode reproduction ...... 82
7.2.4 Statistical analysis ...... 82
7.3 Results ...... 82
7.3.1 Effect of entomopathogenic nematodes on mortality of Vip3A-Sel and Unsel sub-populations of Heliothis virescens ...... 82
7.3.2 Nematode reproduction ...... 86
7.3.3 Penetration of nematode infective juveniles (IJ) ...... 89
7.4 Discussion ...... 89
CHAPTER 8: Fluctuating asymmetry, morphological changes and flight muscle ratio in a Vip3A resistant sub-population of Heliothis virescens (WF06) ...... 92
8.1 Introduction ...... 92
8.2 Materials and Methods ...... 93
8.2.1 Insect culture ...... 93
8.2.2 Measurement of fluctuating asymmetry (FA) ...... 93
8.2.3 Flight muscle ratio (FMR) ...... 94
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8.2.4 Analysis of fluctuating asymmetry (FA) ...... 94
8.2.5 Statistical analysis ...... 94
8.3 Results ...... 95
8.3.1 FA data validation ...... 95
8.3.2 Mean values of different morphological traits ...... 96
8.3.3 Overall trend for fluctuating asymmetry (FA) ...... 96
8.3.4 Flight muscle ratio (FMR) ...... 98
8.4 Discussion ...... 99
CHAPTER 9: Summary and General Discussion ...... 101
9.1 Summary of experimental findings ...... 101
9.2 General Discussion ...... 103
9.3 Future work ...... 109
REFERENCES ...... 111
APPENDICES ...... 148
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CHAPTER 1: Introduction
1.1 General background Many insect species damage subsistence and cash crops and other species can act as vectors for diseases of medical and veterinary importance (Metcalf and Metcalf, 1993). Among the insect orders the Lepidoptera contain many pests of agricultural importance, including a number of noctuid moths, e.g. Helicoverpa, Heliothis and Spodoptera spp. (Lepidoptera: Noctuidae) and the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) (Metcalf and Metcalf, 1993).
Various control methods have been used to control insect pests, e.g. physical and mechanical control, and cultural, biological and chemical control (Talekar and Shelton, 1993). Farmers have mostly relied on chemical control for the control of insect and mite pests (Matthews, 1994) but due to frequent use of pesticides, a number of key pest species have developed widespread resistance to these chemical insecticides (Section 2.1).
A recent development (1996) has been the introduction of Bacillus thuringiensis (Bt) endotoxins expressed in genetically modified (GM) crop varieties, which have proved to be effective against a range of major lepidopteran insect pests, particularly on cotton and maize (Jackson et al., 2007; Llewellyn et al., 2007). The Plutella xylostella was the first insect pest to develop resistance to Bt spray formulations in the open field and selection of resistance to Cry toxins has been also reported for greenhouse populations of Trichoplusia ni (Hübner) (Section 2.2.3). Recently, populations of Spodoptera frugiperda (Smith), Busseola fusca (Fuller), Helicoverpa zea (Buddie) and Helicoverpa armigera (Hübner) have also been suggested to have developed some resistance to Bt crops in the field (Section 2.2.3)
Different tactics have been developed to delay insect resistance to Bt crops. These strategies include, moderate toxin dosage, the high dose/ refuge strategy and the pyramided crops (Bates et al., 2005; Section 2.11). The first generation of transgenic cotton plants expressed a single toxin (Cry1Ac) showed very good activity against the bollworms Heliothis virescens (Fabricius), Helicoverpa armigera, Pectinophora gossypiella (Saunders), Earias vitella (Fabricius) and Earias insulana (Boised) but was less effective against H. zea, Spodoptera spp and T. ni. This has led to the development of pyramided transgenic varieties that express two different toxins and show broad spectrum activity against lepidopteran pests. For example, Bollgard II cotton (expressing Cryl1Ac and Cry 2Ab) effectively controls a wider range of lepidopteran pests than
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Bolgard I (Cry1Ac) (Greenplate et al., 1999; Jackson et al., 2007). Recently, a Bt corn has been registered in the USA that expresses five distinct Bt toxins that control all main lepidopteran target pests and rootworm beetles (Tabashnik, 2010). The presence of two and more Bt toxins in pyramided varieties aims to delay the evolution of the resistance in the insects due to lack of cross resistance between the toxins (Section 2.3). GM crops covered 135 million ha worldwide during the 2009 season (James, 2009). In the past few seasons the greatest increase in the area of Bt crops grown has been for crops expressing more than one toxin (Tabashnik, 2010).
Among the various proposed mechanisms for resistance to Bt observed in laboratory-selected insect populations (Section 2.3), loss of binding is the most commonly reported and is usually the major mechanism found, and is termed „Mode 1 resistance‟ (Ferré and van Rie, 2002; Ferré et al., 2008). There is, however, evidence for metabolic mechanisms of Bt resistance in some insect populations (Section 2.3.3). Sayyed et al. (2008) found evidence for reciprocal cross resistance between Cry1Ac and pyrethroids in a laboratory selected population of P. xylostella (SERD4) from Malaysia which was known to possess multiple resistance mechanisms for Cry toxins. Synergism studies suggested that an esterase-based mechanism was present in SERD4 in addition to the loss of binding and that the former mechanism may be linked to cross resistance between Bt toxins and pyrethroids (Sayyed et al., 2008).
Fitness costs are frequently associated with insecticide resistance (Bielza et al., 2008) and different ecological conditions can affect the level of fitness costs (Gassman et al., 2009). In addition to fitness costs associated with development, reproduction and survival, studies have linked Bt resistance with greater susceptibilty to entomopathogenic nematodes and baculoviruses (Gassman et al., 2006; Raymond et al., 2007). Fluctuating asymmetry (Section 2.7), an indicator of environmental stress has also been linked with fitness costs (Ribeiro et al., 2007).
Vegetative insecticidal proteins (Vip) are produced during the vegetative growth of Bt (Estruch et al., 1996; Selvapandiyan et al., 2001) and have been shown to have a broad spectrum of insecticidal activity against lepidopetran and coleopteran pests (Warren, 1997; Yu et al., 1997; Lee et al., 2003) and differ in their protein sequence, receptor binding and pore formation properties from Cry toxins (Lee et al., 2003; Lee et al., 2006). A GM cotton variety expressing Vip3A and CrylAb (VipCot®) has been developed, which has been shown to be very effective against heliothine species (Kurtz et al., 2007). A recent laboratory study has found that high levels of resistance to Vip3A could be selected in a population of H. virescens (WP06) collected from USA (Pickett, 2009) and further studies on this Vip3A-resistant population are of particular
13 interest because of the impending release for commercial production of cotton varieties expressing Vip3A toxin.
1.2 Aim of present study
The aim of the present study was to understand more fully resistance to Bt Cry and Vip toxins in two key lepidopteran species of agricultural importance in order to develop and refine strategies to minimize the development of resistance.
1.3 Objectives i) To investigate the hypothesis that a field-derived population of P. xylostella (SERD 4) with a history of developing multiple and cross-resistance to insecticides, including reciprocal cross- resistance between Cry1Ac and pyrethroids, will develop resistance to the insecticide synergist, piperonyl butoxide (PBO). ii) To test the hypothesis that cross-resistance exists between Vip3A and non-Bt insecticides in a Vip3A-Sel population of H. virescens (WF06) and between Cry1A and non-Bt insecticides in a Cry1A-Sel population P. xylostella. (Karak), where Cry1A resistance is thought to be due to a single major allele associated with loss of binding („mode 1 resistance‟) (Sayyed et al., 2004). iii) To investigate whether the most common type of resistance mechanism associated with Cry toxins in insects (reduced toxin binding) is also involved in resistance in a Vip3A-Sel population of H. virescens (WF06). iv) To investigate the hypothesis that sub-lethal treatments of Vip3A impair the development and reproduction of Bt-susceptible populations of H. virescens and P. xylostella. v) To test the hypothesis that the fitness costs associated with resistance in a Vip3A-Sel population of H. virescens (WF06) will increase when insects are maintained outside their optimal temperature range. vi) To investigate whether increased susceptibility to entomopathogenic nematodes and reduced thoracic (flight) musculature is linked to Vip3A resistance in a H. virescens Vip3A-Sel population of H. virescens (WF06). vii) To investigate the hypothesis that fluctuating asymmetry will be higher in Vip3A-Sel population of H. virescens (WF06) compared with an Unsel population.
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CHAPTER 2: Literature Review
2.1 Resistance to insecticides
Insecticide resistance is very important factor in agriculture because a number of important pest species have a marked ability to develop resistance to insecticides (Cabrera et al., 2003). Resistance can be defined as the microevolutionary process whereby genetic adaption through pesticide selection results in populations of insects which present unique and often more difficult management challenges (Whalon and McGaughey, 1998; Whalon et al., 2008). As defined by the World Health Organization (WHO), resistance is “the development of an ability in a population of insects to tolerate the doses of chemical which would prove lethal to the other individual in a normal population of the same species” (WHO, 1957). A shorter definition proposed by Crow (1960) is that “resistance marks a genetic change in response to selection.” The frequent and indiscriminate use of insecticides to control insect pests has resulted in the development resistance in many insect populations (Estela et al., 2004; Sayyed and Wright, 2006). The first case of insecticide resistance was documented in 1908 in Washington for San Jose Scale to lime sulphur (Melander, 1914). It is reported that approximately 500 agricultural pest insect species have developed resistance to various pesticides in at least one field population (Cabrera et al., 2003; Whalon et al., 2008; Table 1.1). Plutella xylostella has reportedly developed resistance to more than 80 insecticides (Khaliq et al., 2007). Heliothis virescens has also developed resistance to a large number of insecticides (Sparks, 1981; Luttrell et al., 1987; Hardee et al., 2001; Teran–Vergas et al., 2005; Blanco et al., 2008).
Insecticide resistance is now a major problem for the chemical control of a wide range of insect pests (Bisset et al., 1997; Liu and Yue, 2000) and overreliance and overuse of insecticides (often the first response to an undiagnosed resistance problem) has greatly facilitated the development of insecticide resistance in insects (Palumbi, 2008; Xu et al., 2010). Insecticide resistance together with concerns about environmental and toxicological hazards due to the overuse of insecticides have spurred the search for alternatives to conventional, synthetic compounds as well as the development of more integrated methods of insect pest management (Tabashnik, 1994). One group of compounds, which have much less harmful environmental and toxicological side effects are the protein insecticidal toxins derived from the common soil bacterium Bacillus thuringiensis (Bt), and these have become increasingly important for pest management both as conventional sprays and in GM crops (Tabashnik et al., 1998; Bravo and Soberón, 2007).
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Table 1.1 Number of cases and species in which insecticide resistance has been reported.1
Insect Number of cases of resistance
Order Agriculture Medical Parasitoids Others Polinators Total
Coleoptera 860 13 8 3 884 Diptera 291 1937 4 33 2265 Hemiptera 85 77 162 Homoptera 989 3 992 Hymenoptera 6 2 26 3 37 Lepidoptera 1799 1799 Neuroptera 21 21 Siphonaptera 89 89 Thysanoptera 133 133
No of species Coleoptera 68 1 3 2 74 Diptera 26 149 2 10 187 Hemiptera 17 5 22 Homoptera 57 1 58 Hymenoptera 3 1 11 1 16 Lepidoptera 85 85 Neuroptera 1 1 Siphonaptera 9 9 Thysanoptera 8 8
TOTAL 460
1Adapted from Whalon et al. (2008).
2.2 Bacillus thuringiensis
2.2.1 History
Bacillus thuringiensis (Bt) is a spore forming bacterium that produces protein toxins each of which have a narrow spectrum of activity against members of several insect orders (Shelton et
16 al., 2002). Bt is widely used a microbial pesticide spray product and the genes encoding Bt crystal (Cry) toxins have been engineered into insect resistant genetically modified crops
(Shelton et al., 2002). Bt was used for the first time as a biopesticide in the late 1920s and early 1930s for the control of European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae) in South East Europe (Glare and O‟Callaghan, 2000). In 1938, the first commercial Bt spray product, Sporeine®, was marketed for the control of Plodia interpunctella in France (Weiser, 1986). Dulmage (1970) discovered B. thuringiensis var. kurstaki (Btk) isolate HD-1, which was 20-200 times more potent against various lepidopteran pests than existing commercial products, and was widely adopted for commercial use (e.g. Dipel®). HD-1 was also used in the establishment of an international system for standardizing the potency of commercial products (International units (IU) per unit product (Beegle and Yamamoto, 1992). The discovery of B. thuringiensis var. israelensis (Bti), for the control of dipteran pests, and B. thuringiensis var. tenebrionis (Btt), for coleopteran pests (Goldberg and Margalit, 1977; Beegle and Yamamoto, 1992; Schnepf et al., 1998; Glare and O‟Callaghan, 2000) added considerably to the spectrum of activity of Bt products.
2.2.2 Virulence factors of Bacillus thuringiensis
Bacillus thuringiensis produces several types of virulence factors including α-exotoxin, β- exotoxin, enterotoxins, δ-endotoxins (crystal or Cry toxins), chitinases, hemolysins, phospolipases, proteinase and vegetative insecticidal proteins (Vip toxins) (Hansen and Salamitou, 2000). Cry toxins are currently by far the most important Bt toxins for controlling agricultural pests.
2.2.2.1 δ-endotoxins Bacillus thuringiensis produces parasporal inclusion bodies that contain crystal proteins during the sporulation phase. These crystals are mainly comprised of one or more protein crystal (Cry) and cytolytic (Cyt) toxin. Crystal proteins can be defined as “a parasporal inclusion (Cry) protein from Bt that exhibit experimentally verifiable toxic effect to a target organism or have significant sequence similarity to a known Cry protein” (Bravo et al., 2007). Cry proteins collectively are broad spectrum, being active against Lepidoptera, Coleoptera, Hymenoptera, Diptera and some nematodes. Cyt proteins are reportedly active only against Diptera (Bravo et al., 2007).
In 1989, Bt was classified on the bases of combination of their deduced amino acid sequence and host range but due to the limitations of this system the current nomenclature system was introduced in the late 1990‟s (Crickmore et al., 1998, 2010), based on the degree of homology of amino acid sequences between the toxins. The three dimensional structures of six crystal
17 toxins, Cryl1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa and Cry4Ba have been determined by X-ray crystallography (Bravo et al., 2007).
2.2.2.2 Mode of action of Cry toxins The parasporal crystals contain Cry proteins in the form of protoxins. When ingested by an insect these crystals dissolve and the protoxin is activated through partial proteolysis (Estada and Ferré, 1994). The activated toxin crosses the peritrophic membrane and binds to target sites in the epithelial (brush border) membrane of the midget. After the binding, pores are formed in the cell membrane and this eventually leads to colloid-osmotic lyses of the cell (Estada and Ferré, 1994; de Maagd et al., 2001; Fig. 2.1).
Fig 2.1 Mode of action of Bt Cry toxins: a) Solubilisation of protoxin in insect midgut (pH 9.5); (b) Proteolytic activation by trypsin-like gut enzymes, which remove a small N-terminal and a large C-terminal peptide. (c) Membrane receptor binding (d) Conformational change and membrane insertion (e) Pore formation; adapted from de Maagd et al. (2001).
2.2.2.3 Vegetative insecticidal proteins (Vip) In addition to crystal protein, several strains of both Bt and B. cereus secrete toxic proteins during vegetative growth that do not form crystals and are called vegetative insecticidal proteins (VIP) (Estruch et al., 1996; Selvapandiyan et al., 2001). Vip do not share sequence homology with Cry proteins (Estruch et al., 1996; Lee et al., 2003).
2.2.2.4 Vip classification Vip proteins are grouped into three classes: Vip1, Vip2, Vip3 (Crickmore et al., 2010) using the same system to the one used to classify Cry toxins (Crickmore et al., 2010). Vip1 and Vip2 (100
18 and 52kDa respectively; Hernnάndez-Rodríguez et al., 2009) form a binary toxin, as they are only active when used in combination. These are active against some species of Coleoptera, e.g. western corn rootworm (Fang et al., 2007) but do not show any insecticidal activity against Lepidoptera.
2.2.2.5 Vip3A The Vip3A gene encodes an 88-kDa protein that is secreted into the supernatant by cultures of Bt (Estruch et al., 1996; Liu et al., 2007). The first –identified Vip3 toxin, Vip3Aa1, is highly toxic to several agriculturally important lepidopteran pests, including the noctuids Agrotis ipsilon (Hufnagel), Spodoptera frugiperda (J.E Smith), Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Heliothis virescens (Fabricius), Helicoverpa zea (Buddie), Trichoplusia ni (Hübner), Helicoverpa armigera (Hübner), Helicoverpa punctata (Wallengren), Pseudoplusia includens (Walker) and some other lepididopteran species, including Phthorimea opercullela (Zeller) (Lepidoptera: Gelechiidae), Plutella xylostella L. (Lepidoptera: Plutellidae) and Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae). Vip3A is reported to show no activity against the European corn borer Ostrinia nubilalis (Hübner) (Leipdoptera: Crambidae) (Estruch et al., 1996; Yu et al., 1997; Selvapandiyan et al., 2001; Liano et al., 2002; Bommireddy and Leonard, 2008; Fang et al., 2007).
2.2.2.6 Mode of action of Vip3A
The mode of action of Vip3A has been investigated in A. ipsilon, S. frugiperda, S. exigua, Manduca sexta L (Lepidoptera: Sphingidae) and H. zea (Yu et al., 1997; Lee et al., 2003, 2006) and appears similar to Cry toxins overall. The symptoms that develop following Vip3A ingestion in susceptible insects are similar to Cry1 toxins, with cessation of feeding, loss of gut peristalsis, paralysis and death but in Vip3A these symptoms develop over a period of 48 to 72 h compared with 16 to 24 h for Cry1 toxins (Yu et al., 1997). Studies have showed that Vip3A in addition to a unique protein sequence (Section 2.2.2.3) has independent receptors on midgut epithelium and different pore formation properties compared with Cry1 toxins (Estruch et al., 1996; Yu et al., 1997; Sena et al., 2009).
2.2.3 Resistance to Bt
Insect resistance to Bt toxins has widespread significance because of increasing reliance on Bt toxins in genetically engineered crops and and as conventional sprays in insect pest
19 management (Tabashnik et al., 1998, 2008). McGaughey (I985) was the first to report Bt resistance in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) selected with Dipel® after 15 generations of laboratory selection. Resistance to several Bt toxins has been selected in the laboratory in a number of a range of lepidopteran and one coleopteran species. For example, S. exigua and S. littoralis have been selected for >500-fold resistance to Cry1C (Moar et al., 1995; Muller-Cohn et al., 1996) and Cry1Ab (Hernández-Martínez et al., 2009) and populations of P. gossypiella have been selected for resistance to Cry1Ac (Tabashnik et al., 2002) and Cry2Ab (Tabashnik et al., 2009). Other lepidopteran species selected for Bt resistance include, O. nubilalis (Cry1Ab; Siqueira et al., 2004 and Cry1F; Pereira et al., 2008), Ostrinia furnacalis Guenée (Cry1Ab; Xu et al., 2010), H. zea (Cry1Ac; Anilkumar et al., 2008) and H. armigera (Cry1Ac; Akhurst et al., 2003; Gunning et al., 2005 and Cry2Ab; Mahon et al., 2007). The western corn rootworm, Diabrotica virgifera (virgifera) (Coleoptera: Chrysomelidae) reared on transgenic corn expressing Cry3Bb1 in the greenhouse has also developed resistance (Meihls, et al., 2009).
Several populations of H. virescens have selected for Cry1A toxin resistance in the laboratory (Stone et al., 1989; Gould et al., 1992, 1995; Forcada et al., 1999; Jurat-Fuentes et al., 2003), with one population (YHD2) showing extremely high (>10000-fold) levels of resistance to Cry1Ac (Gould et al., 1995). Most recently a field population (WF06) selected with Vip3A toxin in the laboratory showed high levels of resistance (> 2000-fold) after selection for 13 generations (Pickett, 2009).
Bt resistance has been particularly well studied in laboratory and field populations of P. xylostella, the first insect pest to be reported to have evolved resistance to Bt in the field due to intensive use of Bt sprays on crucifer crops (Tabashnik et al., 1990; Ferré et al., 1991; Tabashnik et al., 1994; Sayyed et al., 2000; Ferré and Van Rie, 2002; Gong et al., 2010). A field collected population of P. xylostella (BCS-Cry1C-2) selected with Cry1Ca for ten generations has been shown to have developed sufficient resistance (> 740-fold) to allow its neonate can survive on transgenic broccoli expressing Cry1Ca toxin (Zhao et al., 2001).
Resistance to Bt sprays has also been reported to have developed in a several greenhouse populations of T. ni in British Columbia (Janmaat and Myers, 2005) and there have been isolated reports of various other insects developing Bt resistance in the field: Busseola fusca (Fuller) (Lepidoptera: Noctuidae) to Bt corn (Cry1Ab) in South Africa (van Rensburg, 2007; Kruger et al., 2009), H. zea to Bt cotton (Cry1 Ac) in the USA (Tabashnik et al., 2009) and S. frugiperda to Bt Corn (Cry1F) in Puerto Rico (Storer et al., 2010).
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2.3 Mechanisms of resistance to Bt
Understanding the genetic basis and mechanisms of resistance to Bt toxins is important for developing and implementing strategies to delay and monitor pest resistance (Tabashnik et al., 2006). Insects have been shown to become resistance to Cry1 toxins due to mutation in genes encoding proteins involved in different steps in the mode of action (Heckel et al., 2007; Section 2.2.2.2). Several mechanisms of resistance have been proposed for laboratory-selected insect populations: altered binding to midgut receptors (Ferré and Van Rie, 2002); altered protoxin activation (Forcada et al., 1996; Li et al., 2004; Karumbaiah et al., 2007); toxin degradation (Forcada et al. 1996); replacement of damaged midgut cells ( Loeb et al., 2001); esterase sequestration (Gunning et al., 2005; Sayyed and Wright 2006); elevated immune system ( Ma et al., 2005).
2.3.1 Binding site modification
The most common mechanism of Cry1 resistance found to date is reduced binding of toxins to target sites in the brush border membrane of the larval midgut (Van Rie et al., 1990; Tabashnik et al., 1997; Cabrera et al., 2003; Gong et al., 2010). Ferré and Van Rie (2002) have modelled the Cry1 toxin binding sites in P. xylostella. In their model, Site 1 is only recognized only by Cry1Ac, Site 2 is shared between Cry1Aa, Cry1Ab, Cry1Ac, Cry1F and Cry1J, Site 3 binds Cry1Ba and Site 4 binds Cry1Ca (Fig. 3.2).
Fig. 3.2 Proposed model for Cry protein binding sites in the midgut brush border membrane of Plutella xylostella (after Ferré and Van Rie, 2002; Ferré et al., 2008).
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In resistant P. xylostella, selecting with Bt products containing Cry1A altered Site 2, explaining cross resistance to Cry1F and Cry1J, while Sites 3 and 4 appeared to be unaltered, explaining their susceptibility to Cry1B and Cry1C (Ferré et al., 2008).
In H. virescens three Cry1 binding sites have been detected: Site A, with a cadherin-like protein as a key binding protein (Jurat-Fuentes et al., 2004), which is recognized by Cry1Aa, Cry1Ab, Cry1Ac, Cry1F and Cry1J; Site B, which is recognized by Cry1Ab, Cry1Ac; and Site C, which is recognized by Cry1Ac (Van Rie et al., 1989; Jurat-Fuentes and Adang, 2001). Cry2Aa does not compete with these sites (Jurat-Fuentes and Adang, 2001). In the YHD2 strain of H. virescens, which was selected with Cry1Ac and cross resistant to Cry1Aa, Cry1Ab and Cry1Fa but not resistant to Cry2Aa, the binding of Cry1Aa was completely reduced but the binding of Cry1Ab and Cry1Ac was not reduced (Lee et al., 1995). Studies on strains of H. virescens selected with Cry1Ac and with Cry2Aa have indicated that resistance to these toxins is due to the presence of two or more distinct resistance mechanisms rather than a single resistance gene (Jurat- Fuentes et al., 2003; Gahan et al., 2005; Karumbaiah et al., 2007).
2.3.2 Altered proteolytic processing
In this mechanism of resistance, the protoxin is not completely reduced to its active form, causing a loss of toxicity (Ferré and Van Rie., 2002). For example, midgut extracts from a strain of P. interpunctella (198r) selected for resistance to Bt var. entomocidus (Bte) were found to have significantly lower proteolytic activity compared with extracts from the susceptible insects and had a reduced capacity to activate Cry1Ac toxin (Oppert et al., 1996). In a H virescens strain (CP73-3) selected with Cry1Ab, slower activation of Cry1Ab protoxin and a faster degradation of Cry1Ab in midgut juices was observed in resistant compared with the susceptible larvae (Forcada et al., 1996).
2.3.3 Other resistance mechanisms
In an Australian population of H. armigera, a Cry1Ac resistance mechanism associated with elevated esterase levels has been proposed (Gunning et al., 2005). While Ma et al. (2005) have reported that an elevated immune response is associated with Bt resistance in the flour moth, Ephestia kuehniella (Keller) (Lepidoptera: Pyralidae).
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2.4 Cross-resistance
Previous selection with insecticides can confer resistance to other insecticides through cross- resistance and have a major impact on the control of insect pests by reducing the effectiveness of new insecticides (Bisset et al., 1997; Liu and Yue, 2000).
There is evidence for cross-resistance between some Cry toxins, although this varied considerably between selected populations. Zhao et al. (2001) reported that strains of P. xylostella selected with Cry1C showed high levels of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F and Cry1J. While Cry1Ab-selected strains of O. nubilalis showed a high level of cross-resistance to Cryl 1Ac but only a low level of cross-resistance to Cryl1F (Siqueira et al., 2004). Cross-resistance between Cry1A toxins (Akhurst et al., 2003) and between Cry2A toxins (Mahon et al., 2007) has also been found in populations of H. armigera, and between Cry1A toxins (Tabashnik et al., 2002) and between Cry2A and Cry1A toxins (Tabashnik et al., 2009) in populations of P. gossypiella. Cross-resistance with Cry1 and Cry2 toxins has be observed in two H. virescens populations selected with Cry1Ac (Gould et al., 1992, 1995) and another H. virescens population showed cross-resistance to various Cry1 toxins following selecttion with Cry2Aa (Jurat- Fuentes et al., 2003). Populations of S. exigua (Hernández-Martínez et al., 2009) and O. furnacalis (Xu et al., 2010) have also been shown to have cross-resistance between various Cry1 toxins. In contrast, one population of H. armigera selected with Cry1Ac did not exhibit cross-resistance to Cry2Aa or Cry2Ab (Akhurst et al., 2003), while another population selected with Cry2Ab was not cross-resistant to Cry1Ac (Mahon et al., 2007). Similarly, cross-resistance was not found in two populations of P. xylostella selected with Cry1Ac to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa and Cry9Ca (Tabashnik et al., 2000). Jackson et al., (2007) reported that three populations of H. virescens selected with Cry1 and Cry2 did not show cross-resistance to Vip3A.
Lack of cross-resistance has also been reported between Vip3A and Cry1Ac in a Cry1Ac- selected population of H. zea (Anilkumar et al., 2008). While the WFO6 population of H. virescens selected with Vip3A toxin (Vip3A-Sel) has been shown to have little or no cross- resistance to Cry1Ab and Cry1Ac (Pickett, 2009).
There has been very little work on cross-resistance between Bt toxins and other insecticides although there is evidence for cross-resistance in a Cry1Ac-selected population of P. xylostella (SERD4) between Cry1Ac and deltamethrin, esfenvalerate, lambdacyhalothrin and that cross- resistance was reciprocal for a deltamethrin-selected SERD4 for Cry1Ac and Cry1Ca (Sayyed et al., 2008). 23
2.5 Fitness costs and insecticide resistance
Reduced relative fitness of resistant genotypes in insecticide free environment is characteristic for many insect species (Alyokhin and Ferro, 1999; Carrière et al., 2001; Li et al., 2007; Bielza et al., 2008). Knowledge of fitness costs associated with resistance is essential for understanding the evolution of resistance in the field and for designing effective resistance management strategies (Li et al., 2007). However, measurement of fitness is not always straightforward. Fitness costs can vary with environmental conditions, e.g. host plant quality, and may not be always be detected under the laboratory conditions where fitness costs are assessed (Li et al., 2007; Raymond et al., 2007).
Fitness costs affect a wide range of characters, including life history traits (Alyokhin and Ferro, 1999; Li et al., 2007), diapause induction (Carrière et al., 1994) and mating ability (Alyokhin and Ferro, 1999). For example, a tebufenozide-resistant population of P. xylostella was shown to have decreased survival rates and reproduction rates compared with unselected populations (Cao and Han, 2006).
Studies have identified various fitness costs associated with resistance to Cry toxins. A population of H. zea (AR) selected with Cry1Ac showed greater mortality during development, longer larval and pupal development times and lower larval and pupal weights compared with unselected insects, when reared on artificial diet in the absence of toxin (Anilkumar et al., 2008). Similarly, O. nubilalis selected with Cry1Ab had reduced pupal weight and increased development time compared with a susceptible population (Crespo et al., 2010). Other fitness costs observed include a P. gossypiella population selected with Cry1Ac, where transfer of fertile sperm was lower than in a susceptible population and resistant males weighed less than susceptible males (Carrière et al., 2009) and delayed and reduced female calling behaviour in a Cry1Ac resistant population of H. armigera (Zhao et al., 2010).
There have also been some laboratory studies, where no fitness factors have been detected in Bt resistant populations. Wu et al. (2009) reported that no significant differences between Cry1A-resistant sugarcane borer (Diatraea saccharalis L.) and a susceptible population. While a population of H. armigera selected with Cry2Ab did not exhibit fitness costs (Mahon and Young, 2010).
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2.6 Sub-lethal effects of insecticides
Studies on sub-lethal effects of insecticides are necessary to determine their full impacts on life history parameters that may affect population dynamics (Stark and Banks, 2003; Galvan et al., 2005; Storch et al., 2007). Sub-lethal effects of insects on insect development and reproduction are common. For example, fenvalerate has been reported to reduce the size and viability of P. xylostella eggs (Fujiwara et al., 2002; Chen and Nakasuji, 2004). Effects can vary substantially between compounds and target species. A population of P. xylostella treated with cypermethrin showed reduced pupal weight and fecundity compared with untreated insects but no such effects were found with abamectin (Abro et al., 1993). Sub-lethal doses of abamectin have been shown to reduce fecundity and viability in Deraeocoris brevis (Uhler) (Hemiptera: Miridae) but acetamaprid had no effect on development or reproduction (Kim et al., 2006).
2.7 Fluctuating asymmetry and insecticide resistance
Fluctuating asymmetry can be defined as a measure of developmental stability and refers to minor random deviations from perfect symmetry in bilaterally symmetrical organs (Palmer, 1996). Living organisms in a stressed environment require more energy for somatic maintenance, which can cause a diversion of resources from growth and reproduction and thus deterioration in fitness (Parsons, 1992; Hoffman and Parsons, 1991; Polak et al., 2002). It has been proposed that the extent of deviation from bilateral symmetry may reflect the fitness of a individual organism (Swaddle et al., 1994). Environmental stresses, such as food quality, temperature and pesticides may thus cause fluctuating asymmetry in insects during their developmental stage (Sciulli, et al., 1979; Hoffman and Parson, 1989; Parsons, 1990). Fluctuating asymmetry was studied in insects e.g Lucilia cuprina (Wiedemann) (Clarke and McKenzie, 1987; McKenzie and Yen, 1995), Drosophila melanogaster (Meigen), (Hoffmann et al., 2005), Copera annulata (Selys) (Odonata: Zygoptera) (Chang et al., 2007).
The differences between the right and left side of bilaterally symmetrical organsisms are usually very small (Palmer 1996; Van Dongen, 2006) and the very small values therefore obtained for fluctuating asymmetry can create considerable potential for measurement error (Swaddle 1994; Palmer and Strobeck 1997; Van Dongen, 2006). In addition to the need for very accurate measurement, several traits need to be taken in to account to maximise the probability of detecting fluctuating asymmetry (Leung et al., 2000; Servia et al., 2004).
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Different patterns of asymmetry have been described: (i) ideal fluctuating asymmetry; (ii) directional asymmetry, where there is a consistence bias in asymmetry toward the one side of the organism, but the distribution of asymmetry remain normal; (iii) anti-symmetry, in which signed asymmetry scores display a platykurtic distribution (distribution in which statistical value is negative) in which there are consistent gross asymmetries but the direction in individuals is random (Palmer and Strobeck, 1986). Fluctuating asymmetry was the only accepted measure of developmental stability, and anti-symmetry and directional asymmetry were believed to contain some unknown genetic component and were considered incorrect measures of environmental stress (Palmer and Strobeck, 1986, 1992), however, some studies have indicated that different pattens of asymmetry are interchangeable (Graham et al., 1993; McKenzie and O‟ Farrell, 1994; Freebairn et al., 1996).
2.8 Piperonyl butoxide (PBO)
Pipronyl butoxide (PBO) belongs to the methylenedioxyphenyl group of compounds and is the most commonly used insecticide synergist, typically for pyrethrin and pyrethroid insecticides (Amweg et al., 2006). PBO inhibits metabolic mechanisms of resistance associated with cytochrome-P450 microsomal monoxygenases (mixed function oxidases) and esterases (Gunning et al., 2005). PBO can also increase penetration of insecticides through the insect cuticle (Winteringham et al., 1955; Farnham, 1973; Devine and Denholm, 1998). PBO can itself affect the development of some dipteran and homopteran insects (Bowers, 1968; Kotze and Sales, 1994; Satoh et al., 1995; Devine and Denholm, 1998).
2.9 Heliothis virescens
Heliothis virescens is an important, highly polyphagous pest (Neunzig, 1969; Sudbrink and Grant, 1995; Abney et al., 2007), whichin North and South America, with a permanent population between 40º N and 40º S (Neunzig, 1969; Fitt, 1989; King, 1994; McCaffery, 1998). It attacks a wide range of important food, fiber, oil crops and is one of the key pests of cotton in the Americas (Fitt, 1989; Teran-Vergas et al., 2005; Blanco et al., 2008).
The eggs of H. virescens are subspherical with a flattened base. Freshly laid eggs are whitish or cream coloured, and develop a reddish brown band as the embryo develops and become grey before hatching. Eggs hatch after 2-5 days depending upon the temperature (Neunzig,
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1969; King, 1994). Newly hatched larvae have a brown to dark brown head and a mostly translucent yellowish body. Older 1st instar larvae become light yellow or green, usually with yellowish orange longitudinal lines. The body of 2nd instar is mostly yellowish green. The colour of 3rd instar larvae through to the final instar larvae is variable, ranging from blue-green to yellow, pink and reddish brown. These colour variation depends on light, temperature, food and heredity (Neunzig, 1969; King, 1994).
On hatching, neonate larvae usually eat some or all of the empty egg shell and may wander for some distance before feeding on the host plant (King 1994: Pickett, 2009). On cotton, young larvae feed on the flower buds or flowers, while older larvae prefer buds and tender bolls. Larvae also feed on leaves on young plants (King, 1994). Larval development goes through four to six instars depending upon the climate and nutritional conditions, although most larvae have six instars (Abney et al., 2007).
Larval development depends on temperatures, e.g. 32 days at 20 ºC and 17 days at 25 ºC (Neunzig, 1969; Fye and McAda, 1972; King, 1994). On completing its development, the larva drops to the ground and enters the soil to pupate; newly formed pupae are shiny and reddish brown and become dark brown near eclosion (Nuenzig, 1969; King, 1994). Male and female pupae can be differentiated. Male pupae were identified by their genetelia; two joined circular shaped gonads at the partition of the 9th and 10th abdominal segments on the ventral side. The female pupa has a V-shaped pattern between the 8th and 9th and the 9th and 10th abdominal segments, in the direction of the anterior end (Pickett, 2009). The pupae can undergo a facultative diapause, which is induced by environmental factors, e.g. low temperatures and short day length (King, 1994). The adults are light olive to brownish olive colour, with three oblique darker bands with an adjacent whitish border on the forewing. The hind wings are pearly white with dark bands along the margin (King, 1994).
The complete life cycle ranges from 33 to 38 days depending upon the temperature. Adult females lay eggs singly on the reproductive and vegetative structures of the host plants. One adult female can produce 300-500 eggs (Abney, 2007). The preoviposition period varies from 1 to 8 days and it is greatly influenced by temperature (King, 1994). Adult females oviposit throughout the night and the greatest number of eggs is laid at 21 and 27ºC (Haneberry and Clayton, 1991; King, 1994).
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2.10 Plutella xylostella
Plutella xylostella is an important pest species worldwide on brassica and other crucifer crops, including broccoli, Brussels sprout, cabbage, cauliflower, Chinese cabbage, collard, mustard, rapeseed, radish and turnip (Talekar and Shelton, 1993; Yin et al., 2008). In the absence of such crops, it can feed on crucifers which are considered as weeds, including Arabis glabra, Bararea stricta, Brassica caulorapha and Brassica kaber (Talekar and Shelton, 1993).
It is generally believed that P. xylostella originated in the Meditterranian region but it now occurs wherever crucifers are grown and is the most universally distributed of all the Lepidoptera (Talekar and Shelton, 1993; Saeed et al., 2010). In the tropics and sub-tropics, P. xylostella causes damage throughout the year (Talekar and Lee, 1985). Losses due to P. xylostella on crucifers such as cauliflower and cabbage frequently reach 90% without the use of insecticides (CIMBAA, 2008).
Plutella xylostella mainly attacks crucifers that contain mustard oils and their glucosides (Hillyer and Thorsteinson, 1971; Talekar, 1992; Nofemale, 2010; Zhou et al., 2010). Most glucosinolates can stimulate the feeding of P. xylostella, an exception being 3-butenyl and 2- phenylethyl glucosinolates, which are toxic at high concentrations (Nayar and Thorsteinson 1963; van Loon et al., 2002). The glucosinolates sinigrin, sinalbin and glucocheirolin are specific feeding stimulants for P. xylostella, and crucifer crops containing one or more of these compounds serve as hosts for this species. Non-host plants may also contain these compounds but also contain feeding inhibitors (Gupta and Thorsteinson, 1960; Nayar and Thorsteinson 1963; Sarfraz et al., 2010)
Adult P. xylostella emerge during photophase. Adults rest on host plants during the day, becoming active at dusk (Hartcourt, 1957; Talekar and Shelton, 1993). Adults mate during the first 4 to 15 h following emergence and mating reaches at the peak between 1 and 2 h of scotophase (Pivinic et al., 1990). Males can mate with more than 10 females and mating success may be influenced by age, size, production of sex pheromones and exposure to host plants (Pivinic et al., 1990). Females of P. xylostella are largely monandrous, with only 20% of females remate (Wang et al., 2005; Davie et al., 2010). Egg laying starts soon after mating and the oviposition period usually lasts 4 days. During this period female moth lay up to nearly 200 eggs, with the majority of eggs laid before midnight; peak oviposition occur between 1900 and 2000 h (Pivnick et al., 1990).
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The eggs are yellowish white to yellowish green, cylindrical to oblong, with average dimensions of 0.48 x 0.25 mm (Bhalla and Dabey, 1986; Chelliah and Srinivasan, 1986; Yang et al., 2009). The majority of eggs are laid on the upper and lower leaf surfaces, with few eggs are laid on stems and petioles (Gupta and Thorsteinson, 1960). The oviposition period is influenced by plant volatiles, temperature, and trichomes and waxes on the leaf surface (Bhalla and Dabey, 1986; Tabashnik, 1985). The egg incubation period ranges between 2 to 11 days, depending on the temperature (Talekar and Shelton, 1993).
On hatching, 1st instar larvae start feeding and mine into the spongy mesophyl tissue (Talekar and Shelton, 1993). The 1st instar larvae are light green in colour with a dark head (Ho, 1965). The early 2nd instar larvae feed from the lower leaf surface and consume all tissues except the upper wax layer, creating a „window‟ in the leaf, a characteristic feature of a P. xylostella infestation (Hartcourt, 1957). The greatest damage to the host plant occurs during the 3rd and 4th larval instar stages due to their much greater feeding rates compared with early instars (Salinas, 1972). Fourth instar male larvae can be differentiated by the fifth abdominal segments, which are lighter in colour than the adjacent segments due to presence of gonads (Liu and Tabashnik, 1997). In the prepupal phase, 4th instar larvae stop feeding and construct a cocoon on the leaf surface and spend two days in it before pupating.
Pupation lasts from 4 to 15 days, depending on the temperature (Talekar and Shelton, 1993). The pupa is initially green but gradually becomes light brown with dark markings and becomes almost black before emergence. Adult moths are small with a wing expansion of 13 to 17 mm, narrow bodied and greyish-brown. Each wing has three lobe-like yellow-white stripes that form a diamond shaped pattern. Male and female moths can be differentiated on the basis of these yellow stripes. The male has brilliant stripes, in the female these are less distinct (Iqbal, 1996; Sayyed, et al., 2000).
The duration of the life cycle is greatly influence by the temperature and in the lowland tropics with continuous crucifer cultivation there can be more than 20 generations p.a. (Talekar and Shelton, 1993).
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2.11 Resistance management in Bt crops
Resistance to Bt can be determined by discriminating dose and dose-response bioassays, and by F2 screening (Bates et al., 2005; Haung, 2006) and is an important part of resistance management
The most common strategy for managing resistance in the most important Bt crops, cotton and maize, is based on two components: the use of crops with high expression of cry gene(s) and the use of refugia. The refuge strategy has been suggested to be the surest way to slow the development of resistance in insects (Tabashnik, 1994). In this strategy (Fig. 3.3) non-Bt plants are planted with Bt plants on the assumption that Bt resistant individuals mate with the susceptible individuals from the refugia and produce heterozygous progeny and that the heterozygous progeny will die on the Bt crops (Ferré et al., 2008; Gassman et al., 2009).
Fig. 3.3 Schematic representation of the high dose / refuge strategy under two assumptions: (A) resistance is recessive; (B) resistance is dominant; where RR are homozygous resistant; RS are heterozygous; SS are homozygous susceptible (From Ferré et al., 2008).
The prerequisites for this high dose/refuge strategy are thus: (1) resistance should be recessive, heterozygotes having a „susceptible‟ phenotype; (2) the frequency of resistance alleles in the insect population should be very low; (3) refuges should be close to the Bt plants; (4) resistant and susceptible individuals should be synchronized to allow cross-mating, (5)
30 mating between the individual and susceptible should be random; (6) the concentration of expressed toxin should be high enough to kill the all the insects heterozygous for the resistance allele (Andow , 2000; Ferré et al., 2008).
Refuge (non-Bt) plants can be planted surrounding the Bt fields, intercalated as rows or blocks within Bt fields, or sown as mixtures of Bt and non-Bt seeds (Ferré et al., 2008). Cohen et al. (2008) have pointed out that using a seed mixture is not suitable for pests whose larvae can move from one plant to other, where this could favour the selection of resistance if larvae move from Bt plants to non-Bt plants before ingesting a lethal dose.
Another approach to delay the development of resistance is the combination of two insecticidal proteins in the same plant (pyramided plants), which is based on the concept that resistance to two or more toxins will be very rare. As in the high-dose / refuge strategy, a key requirement of this strategy is that the initial frequency of the resistance alleles should be very low. It is also imperative that there is no cross-resistance between the toxins (Roush, 1997; Gould, 1998 Bates et al., 2005; Ferré et al., 2008). Models indicate that the refuge size could be reduced by up to 40 % for pyramided plants (Roush, 1997).
Rotation of Bt crops containing different insecticidal proteins is another possible strategy. The key requirement of this strategy is that there should be no cross-resistance between the toxins (Ferré et al., 2008).
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CHAPTER 3: Materials and Methods
3.1 Plants
Chinese cabbage, Brassica chinensis L. var pekinensis cv. Wonk Bok (Kingseeds, Colchester, UK) was grown under greenhouse conditions with supplementary lighting (20 ± 5 ºC, 16 h : 8 h L : D cycle). Cabbage seeds were sown every week in John Innes No 2 compost (Fargo Ltd, Littlehampton, UK) in plastic pots (5 cm diameter). Potted plants were placed on capillary matting and watered regularly. Four to five-week-old plants were used for experiments and to culture P. xylostella (Section 3.2).
3.2 Insects
3.2.1 Heliothis virescens
The Heliothis virescens population (designated WF06) was collected from Leland, Washington County, Miississippi, in November 2006 and maintained in the laboratory at Imperial College, Silwood Park. In the laboratory, the population was divided in two sub- populations; one was selected with Vip3A for more than 15 generations (WF06-Vip3A-sel) and one was left unselected (WF06-unsel) (Pickett, 2009). Previous studies indicate that inheritance of resistance to Vip3A in the Vip3A-Sel sub-population was completely recessive to incompletely dominant with possible paternal influence. It was also reported that resistance was polygenic (Pickett, 2009).
3.2.2 Plutella xylostella
Several populations of P. xylostella were used in the present study. The SERD 4 population was collected from Serdang, Malaysia in December 1998; the Karak population was collected from the Karak area, near Kuala Lumpur, Malaysia in November 2001. An insecticide- susceptible population (LAB-UK) of P. xylostella was obtained from Rothamsted Research (Harpenden, Hertfordshire, United Kingdom), where it had been maintained in the laboratory for more than 150 generations.
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3.3 Bacillus thuringiensis toxins and other insecticides
Cry1Ac (4 mg / ml) was provided by Dr Neil Crickmore, (University of Sussex, UK) and stored at -80°C. Vip3Aa19 (hereafter written as Vip3A) (14.8 mg/ml) was obtained from Syngenta (Research Triangle Park, NC, USA) and stored at -80°C. Lamda-cyhalothrin (Karate®, 50 g a.i./l EC) and cypermethrin (Cyperator®, 100 g a.i./l EC) were supplied by Syngenta (Jealott‟s Hill, UK); chloropyrifos (Lorsban® 400 g a.i./l EC) and spinosad (Tracer®, 480 g a.i./l SC) were supplied by Dow Agro Sciences (Hitchin, UK); indoxacarb (Steward®, 300 g a.i./l WG) was supplied by DuPont (Stevenage, UK); deltamethrin (Decis®, 50 g a.i./l EC) was supplied by Syngenta (Basel, Switzerland). All of the above insecticide formulations were stored at room temperature. The synergists PBO (94% a.i., Sigma Aldrich, Poole, UK), an inhibitor of cytochrome P450 monoxygenases (microsomal oxidase) and esterases, and S, S, S, tributyl phosphorotrithioate (DEF; Sigma Aldrich, Poole, UK), an esterase inhibitor, were stored at 4°C.
3.4 Insect cultures
Hygiene protocols were followed to maintain healthy cultures, including cleaning equipment with 1 % (w/v) Virkon solution (Virkon®, Antec International, Suffolk, UK) and rinsing with distilled water. Laboratory and controlled environment room surfaces were cleaned weekly with 10 % (v/v) bleach solution.
3.4.1 Heliothis virescens
Larvae of H. virescens were reared at 25 ± 5 ºC and 75 ± 5% RH under 16 h : 8 h L : D cycle on artificial diet (Table 3.1). To prepare the diet, agar was added to 1360 ml distilled water, stirred and heated in a microwave until the agar had dissolved, and the solution became clear and started to boil. The remaining ingredients (excluding ampicillin) were mixed thoroughly with 900 ml of distilled water in a separate container. The heated agar solution was then added to the above mixture and stirred until a smooth, even consistency was achieved. Ampicillin was added to 0.5 ml distilled water and mixed into the rest of the diet mixture before solidification.
Adults of H. virescens were kept in open plastic containers (34 x 27 x 22 cm) covered with white netting, and a cotton wool pad soaked in 10% (v/v) honey solution was placed on the top of the netting as a food source. The placement of honey soaked cotton pads on top of the netting helped encourage mated females to lay eggs on the netting. The egg laden netting was replaced every 48 h and the process repeated until egg production decreased or
33 until no further eggs were required. The netting was cut into smaller squares and placed in 250 ml round plastic cups sealed with a plastic lid and left in a controlled environment room until the eggs hatched. A single 1st instar larva was placed in each cell of a rearing tray (2mil PET 27HT-1 32 cells 6‟‟ x 11‟‟, Bio-Serv, NJ, USA) using a fine paint brush (size: 00). Larvae were reared individually to avoid competition and cannibalism. The trays were sealed with breathable polyester film (10 mp / 2mil PET, Bio-Serv, NJ, USA). The larvae were left undisturbed to complete their development. The pupae were collected after 18 to 20 days. Each sub-population was split into groups of approximately 100-150 pupae with each group being placed into a separate container and emerging adults were transferred to a new container for egg laying.
Table 3.1 Composition of artificial diet for culture of Heliothis virescens (Pickett, 2009).
Ingredient Quantity
Agar 30 g Distilled water 1360 ml Ascorbic acid 8 g Sorbic acid 4 g Methyl Paraben 6.6 g Vitamin mixture 13.3 g Pinto bean 166 g Wheatgerm 133 g Soyabean flour 67 g Casein 49 g Yeast 85 g Ampicillin sodium salt 0.28 g Distilled water 900 ml
3.4.2 Plutella xylostella
Cultures of P. xylostella were maintained in a controlled environment room at 25 ± 5 ºC and 75 ± 5% RH under 16 h : 8 h light : dark cycle. The larvae were fed on 4 to 5-week-old Chinese cabbage plants (Section 3.1) until they pupated. Plants were changed when necessary. On emergence the adult were transferred to a wooden cage (280 X 550 X 450 mm) with a cotton
34 wool pad soaked in 10% (v/v) honey solution in a Petri dish supplied as a food source. Chinese cabbage plants (4 to 5-week-old) were placed in the cage for ovipostion.
3.5 Bioassays
3.5.1 Heliothis virescens
Bioassays were conducted using a diet incorporation method where the toxin preparation is mixed into the diet (Dulmage et al., 1970; Beegle, 1990; Liaon et al., 2002). The main advantage of this method is the even distribution of the toxin throughout the diet, thus reflecting expression of the toxin in Bt crops. Five to six concentrations of Vip3A toxin were used, plus a control with distilled water. Each test concentration used 100 ml of artificial diet solution including the toxin solution. The artificial diet preparation was similar to that used in culture maintenance (Section 3.4.1) except for the exclusion of ampicillin and a 10 % reduction in the distilled water content to allow the addition of the toxin solution to the diet preparation at a ratio of 1 : 9 (toxin : diet). The artificial diet was allowed to cool to a temperature between 40 - 45 °C before addition of the toxin solution to avoid thermal degradation of the toxin. For each toxin concentration, a 10 ml toxin solution (10 ml distilled water for control) was prepared., which was added to a 250 ml glass beaker, along with 90 ml of artificial diet solution and mixed thoroughly with a glass stirring rod. The diet incorporated toxin solution was poured evenly into 48 wells (approximately 1.5 - 2 ml per well) of 24-well bioassay plates (BD Falcon™ culture plate, Becton Dickinson Labware, suppled by VWR international Ltd, West Sussex, UK) and allowed to solidify and cool to room temperature. One 1st instar larva (<24h-old) was transferred using a fine brush to each well. For 3rd instar larvae, the method was similar to that for the 1st instar larvae with the following exceptions. The artificial diet incorporated toxin solution for each concentration was 200 ml instead of 100 ml (20 ml toxin solution + 180 ml diet solution), with approximately 3 - 4 ml per well. The bioassay plates were replaced by 32-well rearing trays (24 wells used) and heat sealed with breathable polyester film. Mortality was assessed after seven days.
3.5.2 Plutella xylostella
3.5.2.1 Topical bioassays
Different concentrations of PBO were topically applied (0.5 µl) using an Arnold micro- applicator (Buckard Manufacturing Co. Ltd, Rickmansworth, UK) microapplicator fitted with a glass syringe to the dorsal side of 4th instar larvae. A sharpened metal hole punch was used
35 to cut leaf discs, which were transferred to individual plastic Petri dishes (5 cm dia.) containing a single filter paper (4.5 cm dia. Whatmans No.1, Maidstone, UK) moistened with distilled water. Treated larvae (3 larvae per leaf disc) were then transferred to freshly cut leaf discs (4.8 cm dia.) Ten replicates per treatment were used including the control. Insects were maintained in a controlled environment room for 3 days. The remains of each leaf disc were then removed and a fresh leaf disc was placed in each Petri dish. Mortality was assessed after 3 and 5 days and at eclosion.
3.5.2.2 Leaf–dip bioassay
Bioassays were conducted with 3rd instar larvae of P. xylostella on Chinese cabbage leaf discs (Section 3.1). Test solutions were prepared in distilled water with Triton X-100 (50 μg/ml) as an additional surfactant. Each leaf disc (4.8 cm dia.) was immersed in a test solution for 10 s and allowed to dry on a rack made of aluminium foil at room temperature. The leaf discs were placed in individual Petri dishes (5 cm dia.) containing a moistened filter paper. Five larvae were placed in each dish, and each treatment was repeated 6-7 times. The mortality was assessed after 120 h for Bt toxin and after 48 h for other insecticides.
3.6 Selection for resistance with PBO
The SERD4 population was divided into two sub-populations. One sub-population was left unselected (Unsel) and other was selected with PBO (PBO-Sel). Selection was conducted by exposing 4th instar larvae to PBO on Chinese cabbage leaf discs as described above. The number of larvae selected per generation used ranged between 200 and 300. The initial PBO concentration used for the selection experiments was 100 μg / ml.
3.7 Synergism bioassay
To test for synergism of PBO on the toxicity of Vip3A against 3rd instar larvae of H. virescens, and of PBO and DEF on the toxicity of Cry1Ac against 3rd instar larvae of P. xylostella, the synergist was applied topically in acetone (0.5 µl) to the dorsal side of third instars larvae using a micro-applicator (Section 3.5.2.1). Treated larvae then transferred to leaf discs (P. xylostella) or diet (H. virescens) treated with the toxin (Section 3.5.2.2).
36
3.8 Statistical analysis
All analyses were conducted in R version 2.9.0 (R Development Core Team, 2009). LC50 values were determined by specifying a generalised linear model in with binomial errors (or quasibinomial if data was overdispersed) to estimate the slope and its standard error, with significance tested at the 5 % level (Crawley, 2007). Mortality data used to estimate LC50 values were corrected for control mortality by substracting the number of dead larvae in the control treatment from toxicant treatments. Abbott‟s formula (1925) for correction for control mortality was not used to estimate the lethal concentration as it is not compatible with analysis of generalised linear models with binomial errors used in R. A function called
“dose.p” from the MASS library that used logit regession analysis calculated estimated LC50 values and their standard errors (s.e.). Using these s.e. values, 95 % Confidence Intervals
[CI; (LC50 ± (1.96 x se))] were calculated. Pairwise comparisons of LC50 values were significant at the 1 % level if their respective 95 % CI‟s did not overlap (Crawley, 2007).
Data for larval duration, pupal duration and pupal weight at each temperature were transformed to normalise (See chapter 5 and 6) and were subject to ANOVA. Mating pair success and the number of pairs producing viable progeny was modelled using the binomial proportions test (prop.test) for both sub-populations at each temperature. The mean number of eggs and the mean number of fertile eggs was analysed using a generalised linear model with a poisson error structure corrected for overdispersion.
37
CHAPTER 4: Studies on cross-resistance, synergism and mechanisms of resistance in Bt toxin-selected populations of Plutella xylostella and Heliothis virescens.
4.1 Introduction
Insecticide resistance to Bt toxins (Section 2.2.3) has widespread significance because of the use of Bt toxins in genetically engineered crops and in conventional sprays, where they are particularly useful in IPM and organic systems (Tabashnik et al., 1998; Zhao et al., 2001; Gong et al., 2010; Xu et al., 2010; Tabashnik and Carrière, 2010). In order to delay the development of resistance to an insecticide it is necessary to know whether cross-resistance can occur with others group of insecticides (Cao and Han, 2006; Section 2.4).
Synergists have long been exploited as tools for the laboratory diagnosis of particular insecticide resistance mechanism, based on their ability to inhibit specific metabolic pathways (Young et al., 2006). The synergists PBO (Section 2.8) and S,S,S-tributyl phosphorotrithioate (DEF) are often used in insect bioassays to provide a preliminary assessment of the contribution of detoxification enzymes in insecticide resistant insects (Sanchez-Arroyo et al., 2001; Abd El- Latif and Subrahmanyam, 2010). PBO is known primarily as an inhibitor of mixed function oxidases (Devine and Denholm, 1998) but in some insects it also known to inhibit esterases (Young et al., 2005). Biochemical studies have shown that pyrethroid resistance associated esterases in H. armigera are inhibited by the PBO (Young et al., 2005). DEF is a selective inhibitor of esterases in insects (Ninsin and Tanaka, 2005).
Reciprocal cross-resistance between Cry1Ac and the pyrethroid deltamethrin has been observed in laboratory-selected sub-populations of P. xylostella SERD4 (Sayyed et al., 2008), a population which has high level of resistance to Cry1Ac and Cry1Ab, with an incompletely dominant mode of inheritance of resistance controlled by more than one gene (Sayyed and Wright, 2001). There is also evidence that cross-resistance between Cry1Ac and the pyrethroid deltamethrin is in part esterase-mediated since a PBO analogue, which is a more selective inhibitor of esterases than mixed function oxidases, synergised both Cry1Ac and deltamethrin activity against SERD4 resistant sub-populations (Sayyed et al., 2008).
Heliothis virescens is the first insect species to have been selected for resistance against a Vip toxin (Vip3A; Section 2.2.2.5) in the laboratory (Pickett, 2009). There is little or no
38 evidence of cross-resistance to date between Vip3A and Cry toxins (Jackson et al., 2007; Anilkumar et al., 2008; Pickett, 2009). There has been no work to date on synergism or resistance mechanisms for Vip toxins.
The objectives of the work described in this Chapter were to determine:
1) whether a Cry1Ac-sel sub-population of P. xylostella (Karak), where resistance to Cry1Ac is believed to be due to loss of toxin binding (Sayyed et al., 2004), exhibits cross-resistance to non-Bt insecticides or synergism with PBO and/or the specific esterase inhibitor, DEF; 2) the activity of PBO against early and late 4th instar larvae of Unsel P. xylostella (SERD4); 3) if the Unsel sub-population of P. xylostella (SERD4), a population with a history of resistance and cross-reistance to Bt and other insecticides, can be selected for resistance to PBO; 4) whether there is cross-resistance between Vip3A and non-Bt insecticides in the Vip3A-Sel sub-population of H. virescens (WF06) and if Vip3A activity is synergised by PBO. 5) whether loss of binding is involved in resistance to Vip3A in the Vip3A-Sel sub- population of H. virescens (WF06).
4.2 Materials and Methods
4.2.1 Insect culture and chemicals
See Sections 3.3 and 3.4.
4.2.2 Cross-resistance and synergism studies with Cry1Ac-Sel and Unsel sub- populations of Plutella xylostella (Karak)
Cry1Ac, chloropyriphos, spinosad, deltamethrin (Section 3.3) were used in bioassays with 3rd instar larvae of P. xylostella on Chinese cabbage leaf discs as described in Section 3.5.2.2.
To test if an esterase-specific inhibitor (DEF) or a mixed function oxidase and esterase inhibitor (PBO) can synergise the activity of Cry1Ac in Cry1Ac-Sel, Unsel Karak sub-
39 populations and the Lab-UK susceptible population of P. xylostella, PBO or DEF in acetone (0.5 µl) were applied topically to the dorsal side of 3rd instar larvae (Section 3.5.2.1) 1 h before transfer to Cry1Ac-treated Chinese cabbage leaf discs. Control larvae were treated topically with acetone alone and then transferred to leaf discs that had been dipped in water with Triton X-100 (50 ppm). Mortality was recorded after 120 h exposure on leaf discs.
th 4.2.3 Toxicity of PBO against early and late 4 instar larvae of the Unsel sub- population of Plutella xylostella (SERD4)
To determine the LD50 for PBO, five concentration of PBO (100, 300, 1000, 3000 and 10,000 ppm) plus control were tested against batches of 30 early and late 4th instar larvae by topical application (Section 3.5.2.1). Mortality was assessed after 3 and 5 days and at eclosion.
4.2.4 Selection for resistance to PBO in the Unsel sub-population of Plutella xylostella (SERD4)
The Unsel sub-population was divided into two further sub-populations. One sub-population was left unselected (Unsel) and other was selected (PBO-Sel). Fourth instar larvae were treated topically with PBO (Section 3.2.2.1) and then transferred on Chinese cabbage leaf discs (5 cm dia.; Section 3.5). Three leaf discs, with five larvae per disc) were then transferred to plastic Petri dishes (9 cm dia.) containing a single filter paper (9 cm. dia.) moistened with distilled water. Mortality was assessed after 3 and 5 days. The number of larvae selected per generation ranged between 200 and 300. Dose ranges of 300 to 1000 ppm of PBO were used during the selection experiment, which continued for 20 generations.
4.2.5 Cross-resistance and synergism studies with Vip3A-Sel and Unsel sub- populations of Heliothis virescens (WF06)
Diet incorporation bioassays (Section 3.5.1) were used for cross-resistance studies with 1st instar larvae of both Unsel and Vip3A-Sel sub-populations. For synergism studies with PBO, 3rd instar larvae were used. A preliminary test was conducted to determine the maximum concentration of PBO that caused no mortality in both Vip3A-Sel and Unsel populations. Different concentrations of PBO: 0, 6250, 12500, 25000, 50000 and 100000 ppm in acetone were topically applied (Section 3.5.2.1). Forty eight larvae per treatment were used. After
40 topical application, larvae were transferred to individual plastic cups with artificial diet and placed in controlled environment room (Section 3.4.1). Mortality was assessed after 7 days (Appendix 1). The concentration selected for the experiment was 12500 ppm. The following treatments were used to assess synergism of PBO on the toxicity of Vip3A:
i) PBO alone; ii) PBO + Vip3A (exposed at the same time) iii) Treatment with PBO 4 h prior to treatment with Vip3A iv) Vip3A alone v) Control
4.2.6 Vip3A binding experiment with Heliothis virescens Vip3A-Sel and Unsel populations
4.2.6.1 Activation of Vip3Aa protoxin The concentration of Vip3Aa was determined by Bradford‟s (1976) method, using Bovine Serum Albumin (BSA) (Sigma Aldrich) as a standard. Readings were made at 595 nm using a Spectronic Genesys 5 Spectrophotometer. Vip3Aa protoxin was proteolytically activated in vitro with trypsin (Trypsin type XI from bovine pancreas; Sigma Aldrich) in PBS buffer pH 7.4 at 30 °C (Fig. 4.1). Proteolysis was stopped by adding Sample Buffer 2X for SDS-PAGE electrophoresis (150 mM Tris-HCl at pH 6.8, 750 mM sucrose, 0.075 % bromophenol blue, 3.8 mM EDTA, 2.5 % SDS, 1% β-mercaptoethanol). The samples were then heated for 10 min at 99 °C and run in a 12% SDS PAGE gel to check the proteolysis profiles.
Fig. 4.1 Proteolytic activation of Vip3Aa protoxin (88 KDa) by trypsin produced a major fragment of 62 KDa. Incubations were performed at 30 °C with mild agitation. The protoxin and the major bands (62 KDa) of activated Vip3Aa were quantified (Table 4.1) by densitometry (1D Manager para Windows 95/98/NT [T.D.I. 1999] version 2.0)
41
Table 4.1 Protein concentration of Vip3Aa protoxin and the 62 KDa fragments obtained by different trypsin treatments.
Protein Conditions of activation Protein concentration (mg/ml)
Vip3Aa Protoxin - 0.245
Activated Vip3Aa 10% w/w Trypsin/protoxin, 1 h 0.092
10% w/w Trypsin/protoxin, 2 h 0.057
1% w/w Trypsin/protoxin, 1 h 0.235
1% w/w Trypsin/protoxin, 2 h 0.156
The analysis of the proteolysis profiles together with the protein concentration of the active fragments showed that the best conditions for in vitro activation were incubation with 1% (w/w) trypsin/protoxin for 1 h (Table 4.1).
4.2.6.2 Preparation of Brush Border Membrane Vesicles (BBMV) from Heliothis virescens midguts Midguts from 3rd instar larvae of Vip3A-Sel and Unsel H. virescens were used to prepare BBMV according to the method of Wolfersberger et al. (1987). The midguts were isolated, frozen immediately in liquid nitrogen and stored at -80 °C. The samples were then shipped by air to Spain in dry ice. In Spain, the binding studies were carried out by the author in collaboration with Prof. Juan Ferré (Department of Genetics, University of Valencia).
Midguts were homogenized with a Potter homogenizer in 10 ml MET buffer (17 mM Tris HCl, pH 7.2, 300 mM mannitol, 5 mM EGTA). The homogenate was then filtered through a cloth gauze, the volume of homogenate measured and an equal volume of 24 mM of MgCl2 added. The homogenate was mixed and kept on ice for 15 min, then centrifuged at 4500 rpm for 15 min at 4 °C. The supernatant was then centrifuged at 16000 rpm for 20 min at 4 °C. The pellet obtained was resuspended in MET buffer in a by Potter homogenizer and 24 mM
MgCl2 added to the suspension to give a concentration of 12 mM MgCl2. The mixture was mixed and kept on ice for 15 min and then centrifuged at 4500 rpm for 15 min at 4 °C. The supernatant was centrifuged at 16000 rpm for 20 min at 4 °C and the pellet resuspended by syringe with 0.5 x by volume MET buffer. The protein concentration of the BBMV preparation
42 was determined (Section 4.2.6.1) and the BBMV (20 to 50 µl) were transferred to Eppendorf tubes, frozen in liquid nitrogen and stored at -80 °C until used.
4.2.6.3 Labelling of Vip3Aa with iodine125 Trypsin–activated Vip3Aa was iodinated by the chloramine–T method (Van Rie et al., 1990). Bio-gel P-30 gel filtration resin (3.4 g; BioRad No. 150-4154) was mixed with 50 ml of 20 mM Tris-HCl buffer (pH 8.6), 200 mM NaCl and 0.1% BSA in a beaker and left overnight at room temperature. The matrix was then poured into a 30 ml glass column and packed by passing 10 column volumes of buffer through the column, which was then left overnight at 4 °C. The column was then put at room temperature and 10 column volumes of buffer run through the column. (Exclusion limit 5000 Mr) (1.45 x 5.0 cm) (GE Healthcare, Little Chalfont, UK)
One µl NaI125 (0.3 mCi), 34 µl of 5 mg/ml chloramine-T (as an oxidising agent) in PBS (8 mM
Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4) and 25 µg of activated Vip3Aa was added to an Eppendorf tube. After shaking, the tube was incubated for 45 s at room temperature and the labelling reaction was stopped by adding 34.3 µl of 23 mM Na metabisulphite and 45.8 µl of 1M NaI. The mixture was then loaded in the gel filtration column in order to remove free iodine and degradation products. The elution was performed with 30 ml of 20 mM Tris-HCl, pH 8.6, 200 mM NaCl and 0.1% BSA. Fifty x 500 µl fractions from the column were collected in individual Eppendorf tubes and the radioactivity of 4 µl aliquots from each fraction measured (1282 Compugamma CS Gamma-Counter; LKB, Uppsala, Sweden) to detect the fractions containing labelled toxin (Fig. 4.2).
43
2.0×105
1.5×105
l
1.0×105 CPM/4 5.0×104
0 10 12 14 16 18 20 22 24 26 28 30
Fractions
Fig. 4.2 Elution peak of Vip3Aa labelled with 125I; radioactivity expressed in counts per minute (CPM) in 4 µl aliquots of fractions 10-30.
Aliquots of fractions 15 to 23 were separated by SDS-PAGE electrophoresis (10%). The gel was dried and exposed to an autoradiography film overnight at -20 °C (Fig. 4.3).
Fig. 4.3 Autoradiography of the SDS-PAGE gel for column fractions 15 to 23. Each lane of the gel was loaded with an aliquot containing c. 30000 CPM.
4.2.6.4 Binding of 125I-Vip 3Aa to Unsel Heliothis virescens BBMV
The BBMV were thawed and centrifuged at 16000 rpm for 10 min at 4 °C. The pellet was resuspended in PBS 1X, 0.1% BSA to obtain a concentration of 1mg/ml BBMV protein. Table 4.2 shows the components of the reaction mixture. The buffer was added first, then the labelled toxin, then unlabeled toxin (non-specific binding only), and finally the BBMV. The initial radioactivity in each tube was counted (Section 4.2.6.3) and the tubes then incubated for 1 h at room temperature. After 1 h the tubes were centrifuged at 16000 rpm for 7 min at 4°C. The supernatant was discarded and the pellet washed by resuspending in 500 µl of 44
PBS 1X, 0.1% BSA using a vortex mixer. The resuspension was centrifuged at 16000 rpm for 7 min at 4°C, the supernatant was discarded and the pellet counted for radioactivity.
Table 4.2 Composition of the binding reaction mixture for increasing concentrations of Unsel Heliothis virescens BBMV. Total binding was obtained by incubating BBMV in the presence of 125I-Vip3Aa at pH 7.4 and 0.1 % BSA. Non-specific binding was obtained by the addition in the reaction mixture of an excess of unlabeled Vip3Aa. BBMV (1 mg/ml) 125I-Vip3Aa Unlabeled Vip3Aa Buffer (PBS1X, Total (0.2 mg/ml) 0.1% BSA) Volume l g l l l l
2 2 10 - 88 100 4 4 10 - 86 100 6 6 10 - 84 100 8 8 10 - 82 100 10 10 10 - 80 100 15 15 10 - 75 100 20 20 10 - 70 100 2 2 10 10 78 100 6 6 10 10 74 100 8 8 10 10 72 100 15 15 10 10 65 100 20 20 10 10 60 100
4.2.6.5 Labelling of Vip3Aa with biotin
The activated toxin was dialysed in 40 mM carbonate buffer (pH 8.6) overnight at 4 °C and the protein concentration was determined (Section 4.2.6.1). Fifteen µl of Biotinylation Reagent (RPN 2202; Amersham International, Little Chalfont, UK) were added to 80 µg of Vip3Aa and incubated for 2.5 h at room temperature. Thirty min before the reaction was ended a 10 ml pre-packed gel filtration column (Section 4.2.6.3) was equilibrated with 20 ml of 40 mM carbonate buffer and 0.1% BSA. After 2.5 h, the sample was loaded onto the column and eluted with 12 to 13 ml of 40 mM carbonate buffer. The eluted solution was collected in 16 fractions of 500 µl each. A piece of nitrocellulose membrane (Hybard ECL, Amersham International) was divided by pencil marks into 16 squares in two rows of eight as shown in Fig. 4.4 to perform a Dot Blot. One µl of each fraction was transferred to an individual square and the membrane was dried at room temperature for 10 min. The membrane was then blocked by incubating with mild shaking for 5 min at room temperature in PBST (PBS 1x, 0.1% Tween 20) and 3% Blocking reagent (Membrane blocking reagent RPN2125V, GE Healthcare, Little Chalfont, UK). The membrane was then rinsed twice for 2.5 min with PBST and then incubated with alkaline phosphatase conjugated streptavidin (Streptavidin-AP conjugated 11089161001, Roche, Mannheim, Germany) at 1: 5000 in
45
PBST for 10 min. The membrane was then rinsed three times for 5 min in PBST and transferred face down in a developing solution (100 µl of reactive NBT/BCIP stock solution (code number 11681451001, Roche) in 5 ml of 100 mM Tris–HCl buffer, pH 9.5, with 100 mM NaCl, 50 mM MgCl2) for 1 to 5 min in the dark. The fractions with biotin-labelled Vip3Aa were noted (Fig. 4.4).
Fig. 4.4 Dot Blot of the biotinylated toxin Vip3Aa. The dark circle indicate the fraction that contain biotinylated toxin.
4.2.6.6 Binding of Biotinylated Vip3Aa to Unsel and Vip3A-Sel Helithis virescens BBMV
The BBMV of both the sub-populations were thawed before use and centrifuged at 16000 rpm for 10 min at 4 °C. The pellet was re-suspended in PBS 1 x and 0.1% BSA to obtain a final concentration of 1 mg/ml BBMV proteins. Samples of BBMV from both sub-populations were incubated as described in Table 4.3 for 1 h at room temperature, and then processed as described for the binding of 125I-Vip3Aa (Section 4.2.6.4). The pellets were resuspended in 8 µl of PBS1x and 4 µl of sample buffer 2x was added. The samples were boiled for 5 min and then loaded onto a 12 % SDS PAGE gel. After SDS PAGE electrophoresis, the proteins in the gel were blotted onto a nitrocellolose membrane (Section 4.2.6.5).
Table 4.3 Composition of the binding reaction for increasing concentrations of Heliothis virescens BBMV. The total binding was obtained by incubating BBMV in the presence of 50 ng of biotinylated Vip3Aa at pH 7.4 and in the presence of 0.1 % BSA. Non-specific binding was obtained by the addition in the reaction mixture of a 100x excess of unlabeled Vip3Aa (5 g).
BBMV(1 Biotinylated Unlabeled Vip3Aa PBS1X Buffer (PBS1X, Total mg/ml) Vip3Aa(Fraction 6 (0.2 mg/ml in 0.2% BSA) Volume Diluted 1:10 in PBS1X) PBS1X)
l g l ng l l l l
20 20 7 50 - 24 49 100 20 20 7 50 24 - 49 100
46
The protocol for the Blot was: i) block overnight at 4 °C in PBST and blocking reagent 3 % (w/v); ii) rinse 3 times for 10 min with PBST at room temperature; iii) incubate with streptavidin-AP conjugated 1: 5000 in PBST; iv) rinse as in ii above; v) reveal with BCIP/NBT developing solution (Section 4.2.6.5).
4.2.7 Polyacrylamide gel electrophoresis of midgut esterases in Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06)
Native polyacrylamide gel electrophoresis (PAGE) was performed to determine esterase banding patterns (Devonshire and Moores, 1982). Third instar larvae of each sub-population of H. virescens were homogenised in 5 µl of phosphate buffer (pH 7.5, Na2HPO4, KH2PO4, Triton-X100). Fifteen µl of dilution solution (Sucrose, Triton-X100 and a pinch of bromocresol purple) was then added to the sample. The samples were then centrifuged at 14000 rpm for 15 min and 8 µl supernatant were added to the each well of a Mini-gel. Gels were run at 250 V and 50 mA for 1 h at 5 ºC. Esterase bands were stained with 0.1mM 1-naphthyl acetate for 15 min at room temperature. The stained gels were rinsed in distilled water and then incubated in 7% acetic acid until the stained bands were clearly visualised (c. 3 days). Twenty one individual (one midgut / lane) and twenty four (3 midgut / lane) were tested.
4.3 Results
4.3.1 Cross-resistance and synergism studies with Cry1Ac-Sel and Unsel sub- populations of Plutella xylostella (Karak)
The toxicity of deltamethrin, chlorpyrifos and spinosad was significantly greater (P < 0.01) than Cry1Ac. The resistance ratio for Cry1Ac in the Cry1Ac-Sel Karak population was > 600- fold compared with Lab-UK but 10-fold compared with Unsel (Table 4.4). The toxicity of chlorpyrifos to Cry1Ac-Sel Karak was similar to chlorpyrifos toxicity to Lab-UK. The LC50 and slope of deltamethrin, chlorpyrifos and spinosad for Cry1Ac-Sel Karak was similar to Unsel Karak.
47
Table 4.4 Response of laboratory susceptible (Lab-UK), Unsel and Cry1Ac-Sel sub- populations of Plutella xylostella (Karak) to Cry1Ac, deltamethrin, chlorpyrifos and spinosad; mortality assessed after 120 h for Bt toxin or 48 h for other insecticides; n = 180.1
Insecticide Strain LC50 (ppm) (95% CI) Slope ± SE RR2 RR3 ------Cry1Ac Lab-UK 0.017 (0.012-0.066) 1.50 ± 0.29 deltamethrin Lab-UK 0.007 (0.003-0.011) 1.95 ± 0.37 chloropyrifos Lab-UK 0.008 (0.002-0.015) 1.39 ± 0.30 spinosad Lab-UK 0.013 (0.006-0.023) 1.69 ± 0.32
Cry1Ac Unsel 1.19 (0.76-1.68) 2.55 ± 0.47 70 deltamethrin Unsel 0.16 (0.09-0.20) 2.19 ± 0.42 14 chloropyrifos Unsel 0.09 (0.06-0.13) 2.72 ± 0.50 11 spinosad Unsel 0.21 (0.13-0.30) 2.71 ± 0.50 16
Cry1Ac Cryl 1Ac-Sel 11.3 (8.67-14.5) 3.77 ± 0.59 663 10 deltamethrin Cryl 1Ac-Sel 0.06 (0.04-0.08) 2.42 ± 0.49 8 0.37 chloropyrifos Cryl 1Ac-Sel 0.013 (0.001-0.03) 1.14 ± 0.53 2 0.14 spinosad Cryl 1Ac-Sel 0.11 (0.07-0.15) 2.88 ± 0.54 8 0.52 ------1 Number of larvae exposed to insecticides including control.
2 Response ratio = LC50 of Cry1Ac-Sel LC50 of Lab-UK.
3 Response ratio = LC50 of Cry1Ac-Sel LC50 of Unsel.
A comparison of the LC50 values indicated that neither PBO nor DEF significantly (P > 0.05; overlapping 95% Cl) increased the insecticidal activity of Cry1Ac in Lab-UK and Cry1Ac-Sel populations (Table 4.5).
48
Table 4.5 Response of Lab-UK and Cry1Ac-Sel Karak population of Plutella xylostella to Cry1Ac, Cry1Ac + PBO and Cry1Ac + DEF; mortality assessed after 5 days; n = 180.1.
Strain Insecticide LC50 (ppm) (95% CI) Slope ± SE SR2
------Lab-UK Cry1Ac 0.017 (0.012-0.066) 1.50 ± 0.29
Lab-UK Cry1Ac + PBO 0.013 (0.006-0.023) 1.75 ± 0.32 1.30
Lab-Uk Cry1Ac + DEF 0.015 (0.007-0.11) 1.82 ± 0.33 1.13
Cry1Ac-Sel Cry1Ac 11.27 (8.67-14.5) 3.77 ± 0.59 --
Cry1Ac-Sel Cry1Ac + PBO 9.27 (7.44-11.5) 4.65 ± 0.73 1.21
Cryl Ac-Sel Cry1Ac + DEF 9.43 (7.24-12.2) 3.65 ± 0.61 1.19 ------1 Number of larvae exposed to insecticides including control.
2 Synergism ratio = LC50 of insecticide alone LC50 of insecticide + synergist.
th 4.3.2 Toxicity of PBO with early and late 4 instar larvae of the Unsel sub- population of Plutella xylostella (SERD4)
th The LD50 for PBO was 3.8-fold greater (P < 0.01) for late compared with early 4 instar larvae (Table 4.6).
th Table 4.6 Toxicity of PBO against early and late 4 instar larvae of the Unsel SERD4 population of Plutella xylostella after 5 days exposure.
------Instar LC50 (ppm) (95% CI) Slope ± SE n
Early 4th 491 (267-900) 0.71 ± 0.15 180
Late 4th 1859 (1044-3307) 0.70 ± 0.14 180 ------
At the highest concentration of PBO tested (100000 ppm), the mortality of early 4th instar larvae was 66 %, 90 % and 93 % after 3 days, 5 days and at emergence respectively (Appendix 2). At the lowest concentration of PBO tested against late 4th instar SERD4 (100
49 ppm) mortality was 13 %, 23 % and 43 % after 3 days, 5 days and at emergence respectively (Appendix 3).
4.3.3 Selection for resistance to PBO in the Unsel sub-population of Plutella xylostella (SERD4)
In the selection experiment with PBO only the successfully emerged adult moth was recorded. After 20 generations the resistance did not develop in Unsel population of P. xylostella (SERD4) against PBO. For insects treated with PBO, the number of moths emerging after each generation is shown in Fig. 4.5.
Fig. 4.5 Survival (%) to adult stage of the Unsel sub-population of Plutella xylostella (SERD4) after 20 generations treated topically with PBO.
4.3.4 Cross-resistance and synergism studies on Vip3A-Sel and Unsel sub- populations of Heliothis virescens (WF06)
The resistance ratio for Vip3A in the Vip3A-Sel sub-population was > 200-fold compared with the Unsel sub-population (Table 4.6). The toxicity of spinosad, indoxacarb, λ-cyhalothrin and cypermethrin was not significantly different (P >0.05 in both Unsel and Vip3A-Sel sub- populations of H. virescens (Table 4.7).
50
Table 4.7 Response of Unsel and Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06) to spinosad, indoxacarb, λ-cyhalothrin and cypermethrin; mortality assessed after 168 h for Bt toxin and after 48 h for other insecticides.
Insecticide LC50 (ppm) (95% CI) Slope ±SE No.1 RR2
Vip3A Vip-Sel 103 (46-226) 0.89 ± 0.15 288 234
Vip3A Unsel 0.44 (0.30-0.64) 0.21 ± 0.21 240 ------
spinosad Vip-Sel 1.26 (0.73-2.18) 0.56 ± 0.08 288 0.947
spinosad Unsel 1.33 (0.75-2.33) 0.53 ± 0.07 288 ------
indoxacarb Vip-Sel 3.01 (1.57-5.79) 0.48 ± 0.07 288 2.18
indoxacarb Unsel 1.38 (0.79-2.42) 0.54 ± 0.07 288 ------
λ-cyhalothrin Vip-Sel 0.93 (0.58-1.48) 0.71± 0.09 288 1.38
λ-cyhalothrin Unsel 0.67 (0.42-1.08) 0.71± 0.09 288 ------
cypermethrin Vip-Sel 5.13 (2.84-9.25) 0.59 ± 0.09 288 1.30
cypermethrin Unsel 3.93 (2.33-6.62) 0.64 ± 0.10 288 ------
1 Number of larvae exposed to insecticides including control
2 Response ratio = LC50 of Vip3A-Sel population LC50 of Unsel population
A comparison of the LC50 values indicated that PBO did not significantly increase the insecticidal activity of Vip3A in the Vip3A-Sel sub-population compared with the Unsel sub- population applied either same time or 4h before toxin application (Table 4.8).
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Table 4.8 Response of Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06) to Vip3A and PBO; mortality assessed after 168 h. ______WF06 Treatment LC50 (ppm) (95% CI) Slope ± SE No 1 SR2 ------
Unsel Toxin 1.80 (1.31-2.47) 1.60 ± 0.160 150
Unsel T+ PBO 1.96 (1.45-2.64) 1.76 ± 0.154 150 0 .90
Unsel T - 4 h PBO 1.96 (1.43-2.68) 1.66 ± 0.16 150 0.90
Vipsel Toxin 112 (31.8-392) 0.54 ± 0.64 120
Vipsel T+ PBO 440 (264-732) 1.44 ± 0.26 120 0.25
Vipsel T - 4 h PBO 322 (179-578) 0.83 ± 0.30 120 0.34 - ______
1 Number of larvae exposed to insecticides including control.
2 Synergism ratio = LC50 of insecticide alone LC50 of insecticide + Synergist.
4.3.5 Binding of 125l-Vip3Aa to BBMV of Vip3A-Sel and Unsel sub-populations Heliothis virescens (WF06)
125I-Vip3Aa Total binding and the non-specific binding 125I-Vip3Aa was similar (Fig. 4.6).
52
6
5 I-Vip3Aa
125 4
3 Total binding
Non specific binding % % Binding 2 0.00 0.05 0.10 0.15 0.20 0.25
BBMV concentration (mg/ml)
Fig. 4.6 Binding of 125I-Vip3Aa to BBMV from the Unsel sub-population of Heliothis virescens. Data points represent a single replicate.
4.3.6 Binding of biotin labelled Vip3A to BBMV of Vip3A-Sel and Unsel sub- populations Heliothis virescens (WF06)
The total binding and non-specific binding bands in both sub-populations have very similar intensity (Fig. 4.7), indicating that there was no detectable specific binding of biotin-labelled Vip3A.
Fig. 4.7 Binding of biotinylated Vip3A to BBMV from Vip3A-Sel and Unsel sub-populations of Heliothis virescens. Biotinylated Vip3A as incubated with BBMV in the absence (lanes 1 and 3) or in the presence (lanes 2 and 4) of an excess of unlabelled Vip3Aa, and the final pellet obtained in the binding experiment was subjected to SDS-PAGE and the proteins blotted and revealed on nitrocellulose membrane. Lane 5 = a sample of the biotinylated Vip3Aa used in the binding assays.
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4.3.7 Polyacrylamide gel electrophoresis of midgut esterases in Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06)
Electrophoresis showed that the Vip3A-Sel midguts had an extra esterase band compared with Unsel midguts (Fig. 4.8; extra band indicated).
Lane 1 Molecular weight markers Lanes 2, 3, 4 and 5 = midguts of Vip3A-Sel Lanes 6, 7, 8 and 9 = midguts of Unsel
Fig. 4.8 Polyacrylamide gel electrophoresis of Vip3A-Sel and Unsel sub-populations of Heliothis virescens (WF06).
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4.4 Discussion
Bacillus thuringiensis transgenic plants reduce substantially the need for conventional insecticides against insect pests, most notably on cotton (Liu et al., 2008). In the absence of resistance management systems, the technology is, however, likely to short lived (Liu et al., 2008). Various strategies have been proposed to delay onset of resistance and the US Environmental Protection Agency (EPA) has mandated the high dose–refuge system (Section 2.11). For example, in cotton, non-Bt plants are deployed as a separate refuge, in which 20% of the field is planted with non-transgenic plants that can be treated with a non-Bt foliar insecticide or 4% non-Bt cotton when untreated with a non-Bt insecticide (Shelton et al., 2000). The strategies have been successful so far since the introduction of Bt crops in 1996 (Tabashnik et al., 2009). Unlike Bt transgenic crops, Bt sprays have been unregulated on vegetable crops resulting in development of resistance to Bt formulation and toxin P. xylostella in the open field (Tabashnik et al., 1990; Shelton et al., 1993; Sayyed et al., 2000), T. ni in greenhouses (Janmat and Myers, 2005), and S. frugiperda in Puerto Rico (Strorer et al., 2010).
There is evidence of cross-resistance between the Bt toxins (Siqueira et al., 2004; Section 2.4) but little is known about cross-resistance between the Bt toxins and non-Bt insecticides. In the present study the Unsel population of Karak was significantly (P<0.01) less susceptible to Cry1Ac or the other insecticides tested compared with the Lab-UK population. No evidence for cross-resistance between Cry1Ac and non-Bt insecticides was found in the Karak population. Selection with Cry1Ac significantly reduced the susceptibility of the Cry1Ac-Sel sub-population of Karak to Cry1Ac compared with Unsel and Lab-UK but had no significant effect on the response of Cry1Ac-Sel to spinosad and significantly increased its susceptibility to deltamethrin and chlorpyriphos. Similarly the Vip3A-Sel sub-population of H. virescens (WF06) showed no indication of cross-resistance to spinosad, indoxacarb, λ- cyhalothrin and cypermethrin.
Resistance to pyrethroids, carbamates and organophosphates is commonly associated with metabolic mechanisms of resistance in insects (Gunning et al., 1999). Non-specific esterases, a class of serine hydrolases that are found in the insect haemolymph and gut, have been implicated as a broad-spectrum insecticide-resistance mechanism to organophosphate, carbamate and pyrethroid insecticides in numerous insect pests owing to their ability to hydrolyse insecticidal esters and to sequester insecticides (Gunning et al., 1999) and esterase sequestration of Cry1Ac has been reported to be a mechanism of resistance in H. armigera (Gunning et al., 2005). Resistance to spinosad has also been
55 shown to be associated with esterases in S. exigua (Wang et al., 2006) and inhibitors of esterases synergize the activity of a number of insecticides, including pyrethroids, chlorpyrifos, spinosad and Cry1Ac against a number of insecticide resistant insect populations (Gunning et al., 1999; Young et al., 2006; Sayyed et al., 2008).
In contrast to Cry1Ac-Sel P. xylostella SERD4 (Sayyed et al., 2008), PBO was not found to synergise the activity of Cry1Ac in the Cry1Ac-Sel Karak population of P. xylostella, nor was synergism observed with the more specific esterase inhibitor, DEF. Since Karak has been reported to be a single major factor Bt-resistant population, with a high level of resistance to Cry1A and no cross-resistance to Cry1Ca (Sayyed et al., 2004), that is, characteristic of „mode one‟ resistance associated with loss of toxin binding to midgut receptors (Tabashnik et al., 1998; Section 2.3.1), this finding was not particularly unexpected.
The failure of PBO treatment, either before or at the same time as toxin treatment, to effect the activity of Vip3A also suggests that esterases or cytochrome P450 mononoxygenases are not associated with Vip3A resistance in H. virescens (WF06). Electrophoresis did, however, indicate the presence of an extra esterase band in the Vip3A-sel sub-population of H. virescens (WF06). This suggests that there may be a correlation between Vip3A selection and esterases and it remains to be investigated whether specific esterases may be implicated in resistance, for example, through sequestration or degradation of toxin.
Resistance to Bt toxins is most commonly linked to changes to midgut membrane receptors (Van Rie et al., 1990; Pigott and Ellar, 2007; Ferré et al., 2008; Gong et al., 2010; Caccia et al., 2010; Section 2.3.1). Previous binding studies have shown that Vip3A binds specifically to BBMV prepared from H. virescens and H. zea (Lee et al., 2006) and S. frugiperda (Sena et al., 2009), In the present study, however, a binding experiment conducted with the Unsel sub-population of H. virescens (WF06) showed no specific binding. Since the Unsel sub- population was susceptible to the toxin in vivo, these results could be due to unproper experimental conditions rather than a lack of binding capacity of Vip3Aa protein. This suggests that the labelling reaction may have inactivated the binding sites of Vip3Aa, that is, Vip3Aa may not be able to be labelled without losing the epitope for binding (J. Ferré, personal communication). Studies are being conducted to optimize the experimental conditions prior to further experiments on the mechanisms of resistance to Vip3A in H. virescens.
The selection of P. xylostella with PBO indicated that even after 20 generations of selection there was no sign that resistance was developing. A similar study conducted in
56 parallel at Rothamsted, where another population of P. xylostella was selected with PBO, also failed to show resistance to PBO (Philippou, D., unpublished data). This suggests that the frequency of alleles conferring resistance to PBO in the populations of P. xylostella examined was very low, possibly because possible resistance mechanisms are limited for this type of compound. There is no evidence that the widespread use of PBO as a synergist with pyrethroids and other insecticides has led to a significant loss in its efficacy.
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CHAPTER 5:Sub-lethal effects of Vip3A on survival, development and fecundity of Heliothis virescens and Plutella xylostella
5.1 Introduction
Toxicological studies on pesticides can be divided in to two major categories: (1) acute and (2) chronic exposure. Acute toxicological studies investigate the end point mortality while chronic studies deal with the effect of repeated exposure to pesticides (Stark and Bank, 2003; Martinez-Villar et al., 2005). Demographic toxicology studies have been considered more appropriate and reliable estimates than acute toxicity in predicting the impact of pesticides in the field. They provide an accurate measure for the total effect of a pesticide on the growth rate, and particularly on the intrinsic rate of increase, of a target or non-target organism (Stark and Bank, 2003; Teodoro et al., 2005).
Not all individual target insects will be exposed to a lethal dose of an insecticide under field conditions due to variations in susceptibility, variations in the efficiency of insecticide application and in the behaviour of insecticides in the environment (Copping and Menn, 2000; Desneux et al., 2007; Yin et al., 2008). Sub-lethal effects of insecticides can affect the behaviour of insects and reduce their rate of development and reproduction, and behaviour (Stark and Bank, 2003; Desneux et al., 2004, 2007; Teodoro et al., 2005; Section 2.6). For example, Yin et al. (2008) evaluated the effect of spinosad on susceptible and resistant populations of P. xylostella and reported significant differences between susceptible treated and control insects in their life table traits but that such differences were non-significant between in resistant insects. To the best of my knowledge there is no documented report of sub-lethal effects of Vip toxins in insects.
The present study investigated the effects of sub-lethal concentrations of Vip3A on a susceptible species, H. virescens, and a species that has relatively low susceptibility to this toxin, P. xylostella (A. Gulzar, preliminary observations).
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5.2 Materials and Methods
5.2.1 Insect culture and insecticides
Unsel P. xylostella (Karak) and Unsel H. virescens (WF06) were used. See Section 3.1.
5.2.2 Initial bioassays with P. xylostella
Bioassays were conducted with 1st instar larvae of P. xylostella on Chinese cabbage leaf discs (Section 3.1). Five concentration of Vip3A were prepared in distilled water with Triton X-100 (50 μg / ml) as an additional surfactant. A control containing only water was also used. Each leaf disc (4.8 cm dia.) was immersed in a test solution for 10 s and allowed to dry at ambient temperature (Section 3.5.2.2). The leaf discs were placed in each individual Petri dishes (5 cm dia.) containing moistened filter paper. Five larvae were placed in each dish, and each treatment was repeated 6 times. Mortality was assessed after 120 h. The mortality data was analysis in R. Based on the bioassay results the LC5 was selected as the concentration to use in subsequent experiments.
5.2.3 Development and survival of larval stages of Plutella xylostella to adults
Fresh leaves leaf discs of Chinese cabbage (Section 3.1) were immersed in a freshly prepared st LC5 concentration of Vip3A (Section 5.2.2). Control leaves were dipped in water only. Five 1 instar larvae were placed in a Petri dish (9 cm dia.) containing three treated leaves. Each treatment was replicated seven times. The treated leaves were maintained at 25 °C, 70 % RH and 16:8 (L: D); surviving larvae were fed with fresh leaves after 120 h. The pupae were placed individually into Eppendrof tubes (1.5 ml). The developmental time from larva to pupa was noted. Pupal weight was recorded one day after pupation. The number of adults that emerged was recorded daily. The total developmental time from neonate larva to adult was recorded.
5.2.4 Adult longevity, mating success, fecundity and egg viability of Plutella xylostella
Adults from the development study (Section 5.2.3) were used to estimate fecundity and adult longevity. Following emergence (< 24 h), adults were paired as follows: Untreated control Karak male x Untreated control Karak female (10 pairs) and Vip3A-treated Karak male x treated karak female (10 pairs). Parafilm® sheets (Alpha laboratories Ltd, Eastleigh, Hampshire, UK) were soaked in cabbage juice and used to line a transparent plastic cup (250 ml). Individual pair was placed in this cup and covered with white netting, and a cotton wool pad soaked in
59
10% honey solution was placed in the cup as a food source. When a female started laying eggs, the insect pair was transferred to a new cup every 48 h until they both died. Mating success, the number of eggs laid per female, the proportion of eggs that hatched, and adult longevity was recorded.
5.2.5 Initial bioassays with Heliothis virescens
Bioassays were conducted with five concentrations of Vip3A prepared in distilled water and a control with distilled water only. Each test concentration used 100 ml of artificial diet containing toxin and bioassays conducted with 1st instar larvae (<24 h-old) of H. virescens as described in Section 3.5.1. Mortality was assessed after 7 days; larvae that failed to respond to gentle contact with a fine brush was considered as dead. Based on the bioassay results the estimated LC5 was used in subsequent experiments.
5.2.6 Development and survival of larval stages of Heliothis virescens to adults
st One hundred 1 instar larvae were placed on artificial diet incorporated with the LC5 dose of Vip3A in individual 1 oz plastic cups (9051, Bio-Serv, Frenchtown, NJ, USA) and sealed with a snap-on lid (9053, Bio-Serv). One hundred larvae were set up on artificial diet without toxin as controls. Larvae were examined daily and the development time from larva to pupa was noted together with the proportion that pupated. Pupal weight was recorded one day after pupation, when the sex of each insect was also determined. The pupae were placed individually in 250 ml plastic cups covered with white netting secured by a plastic band. The number of adults that emerged was recorded daily and the proportion that emerged successfully was also recorded, together with the total developmental time from neonate to adult.
5.2.7 Adult longevity, mating success, fecundity and egg viability of Heliothis virescens
Adults that emerged from the development study were used to estimate fecundity and adult longevity. On emergence, adults (< 24 h-old) were paired as follows: untreated WF06-Unsel male x untreated WF06-Unsel female (20 pairs) and Vip3A-treated WF06-Unsel male x Vip3A- treated WF06-Unsel female (20 pairs). Each pair was placed in transparent plastic cup coved with white netting, and a cotton wool pad soaked in 10% honey solution was placed on the top of the netting as a food source. When a female started egg laying the insect pair was
60 transferred to a new cup every 48 h until they died. Mating success, the number of eggs laid per female, the proportion of eggs that hatched and adult longevity was recorded.
5.2.8 Growth rate of Plutella xylostella and Heliothis virescens
For P. xylostella, 30 randomly selected 3rd instar larvae from untreated controls and 30 Vip3A- treated larvae (see Section 5.2.3) were weighed individually and put back on Chinese cabbage leaves (Section 5.2.3). Pupae were removed, weighed (after 1 day) and placed in separate Eppendorph tubes (1.5 ml). For H. virescens, 200 1st instar larvae were weighed collectively and 100 larvae placed on diet contain toxin and 100 placed on diet without toxin (Section 5.2.6).
The mean relative growth rate (MRGR) was calculated using the formula:
MRGR = [In W2 (mg) – In W1 (mg)] / T (Radford, 1967, Saeed et al., 2010)
W1= Initial larval weight
W2= Pupal weight
T = Time to pupal stage
5.2.9 Intrinsic rate of population increase
The net replacement rate (Ro), the average number of female offspring per female during its entire lifetime, was calculated for P. xylostella and H. virescens as follows:
Ro = (n x l ex la)/2
n= mean number of eggs per female
le= fraction of fertile eggs
la= fraction of eclosing adults
Where 2 is the sex ratio coefficient as H. virescens and P. xylostella have 50:50 sex ratio (Sayyed and Wright, 2001; Pickett, 2009)
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Ro was then used to calculate the intrinsic rate of population increase rm (Birch, 1948; Sayyed and Wright, 2001).
rm = (ln Ro)/ T
Where T= total developmental time from neonate to adult.
5.2.10 Statistical analysis
All analyses were conducted in R version 2.9.0 (R Development Core Team, 2009). Data for developmental time, larval duration, pupal duration and pupal weight were subject to analysis of variance (ANOVA). Mating pair success and the number of pairs producing viable progeny was modelled using the binomial proportions test (prop.test). The mean number of eggs and the mean number of fertile eggs was analysed using a generalised linear model with a poisson error structure corrected for overdispersion.
5.3 Results
5.3.1 Sub-lethal concentration of Vip3A
The susceptibility of 1st instar larvae of P. xylostella and H. virescens was determined after 120 h and 168 h respectively (Table 5.1). LC5 were selected as an approximation to a sub-lethal concentration.
st Table 5.1 LC50 and LC5 values of Vip3A to 1 instar larvae of Heliothis virescens and Plutella xylostella.
Strain LC50 (µg / ml) LC5 (µg / ml) Slope ± SE
H. virescens WF06 1.35 0.26 1.11 ± 0.20 P. xylostella Karak 2236 2.82 0.44 ± 0.19
5.3.2 Development and survival of Heliothis virescens and Plutella xylostella
The mean larval duration (neonate to pupation) of H. virescens treated with the LC5 concentration of Vip3A was significantly longer compared with untreated control larvae
62
(Table 5.1; F= 24.80 df 1, 157 P< 0.001). The mean larval duration of P. xylostella on treated leaves was significantly longer compared with untreated controls (Table 5.1; F= 73.77 df 1, 175 P< 0.001)
The mean pupal development time of Vip3A-treated H. virescens and P. xylostella were significantly longer compared with untreated larvae (Table 5.2; F= 33.31 df 1, 138 P<0.001 and F= 101.22 df 1, 160 P<0.001, respectively). The mean total developmental time from neonate to adult of Vip3A-treated H. virescens and P. xylostella was also significantly longer than controls (Table 5.2; F=48.40 df 1, 138 P<0.001 and F=163.4 df 1, 160 P<0.001, respectively). The mean pupal weight of Vip3A-treated H. virescens and P. xylostella was significantly greater than controls (Table 5.2; F= 3.19 df 1, 157 P<0.001 and F= 36.36 df 1, 175 P<0.001, respectively).
The percent pupation (Fig. 5.1), percent adult emergence (Fig. 5.2) of H. virescens and the percent pupation (Fig. 5.3) and percent adult emergence (Fig. 5.4) of P. xylostella were lower in Vip3A-treated H. virescens and P. xylostella.
Table 5.2 Mean development time (days ± SE) and pupal weight (mg ± SE) of Vip-treated
(LC5) and control Heliothis virescens and Plutella xylostella.
Treatment LTDa PDb Totalc Adult longd PWe
H. virescens control 14.7 ± 0.12 12.6 ± 0.29 26.9 ± 0.22 11.2 ± 0.40 246 ± 2
H. virescens treated 15.8 ± .21 13.3 ± .20 28.6 ± 0.45 11.7 ± 0.29 253 ± 3
P. xylostella control 8.68 ± 0.07 3.80 ± 0.06 13.4 ± 0.16 7.05 ± 0.16 5.52 ± 0.03
P. xylostella treated 9.16 ± 0.05 4.74 ± 0.03 14.4 ± 0.07 6.95 ± 0.16 6.35 ± 0.13
a. Larval developmental time b. Pupal duration c. Development time from neonate to adult d. Adult longevity e. Pupal weight
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Fig. 5.1 Percentage pupation (± SE) for Heliothis virescens treated with the LC5 concentration of Vip3A compared with untreated control insects.
Fig. 5.2 Percentage adult emergence (± SE) for Heliothis virescens treated with the LC5 concentration of Vip3A compared with untreated control insects.
64
Fig. 5.3 Percentage pupation (± SE) for Plutella xylostella treated with the LC5 concentration of Vip3A compared with untreated control insects.
Fig. 5.4 Percentage emergence (± SE) for Plutella xylostella treated with the LC5 concentration of Vip3A compared with untreated control insects.
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5.3.3 Adult longevity, mating success, fecundity and egg viability of Heliothis virescens and Plutella xylostella
The mean adult longevity of the H. virescens or P. xylostella was not significantly different between the Vip3A-treated and control treatments (Table 5.3; F=0.985 df 1, 62 P>0.05 and F=0.177 df 1, 38 P>0.05, respectively).
There was no significant difference in the proportion pairs of H. virescens or P. xylostella that produced eggs between treatments (Table 5.3; α2= 0.989 df 1 P>0.05 and α2= 0 df 1 P>0.05, respectively). Similarly there was no significant difference in proportion of pairs of H. virescens or P. xylostella that produced viable progeny (α2=0 df 1 P>0.05 and α2=0 df 1 P>0.05, respectively) between the treated and control treatments.
The mean number of eggs of H. virescens or P. xylostella treated with Vip3A was significantly greater than control treatments (Table 5.3; df 38, P<0.05, n=40 and df 18, P<0.05, n=20, respectively). The mean egg viability of H. virescens or P. xylostella treated with Vip3A was significantly lower than control treatments (df 38, P<0.05, n=40 and df 18, P<0.05, n=20, respetively).
Table 5.3 Mean reproductive parameters for Heliothis virescens and Plutella xylostella in
LC5 Vip3A-and control treatments. Treatment Pairsa MPSb (%) PVPc (%) Eggs/female % viabilty of
(± SE) eggs (± SE)
H. virescens control 20 75 40 659 ± 60 72 ± 3.4 H. virescens treated 20 60 30 742 ± 97 62 ± 3.1 P. xlostella control 10 70 50 312 ± 18 78 ± 2.3 P. xylostella treated 10 60 40 402 ± 13 66 ± 2.4
a. Total number of pairs b. Mating pair success (produced eggs) c. Pairs producing viable progeny
5.3.4 Intrinsic rate of population increase
Heliothis virescens treated with Vip3A showed an 11% decrease in its rm as compared with the control treatment (Table 5.4). Plutella xylostella treated with Vip3A showed a 17% decrease in its rm compared with the control treatment.
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Table 5.4 Life table parameters Heliothis virescens and Plutella xylostella treated with LC5 Vip3A and control treatments.
a b c d Pop Treatment Ro rm RF MRGR
H. virescens control 384 0.19 1 0.27
H. virescens treated 138 0.17 0.89 0.26
P. xylostella control 101 0.37 1 0.56
treated 9292 93 0.83 P. xylostella 0.31 0.48
a. Net replacement rate b. Intrinsic rate of population increase c. Relative fitness = rm treated insect / rm of control d. Mean relative growth rate
5.4 Discussion
In the present study, while taking into account that different bioassays were used, larvae of the H. virescens appeared to be much more susceptible to Vip3A compared with larvae of P. xylostella. Differences in susceptibility between the two species were particularly marked at the LC50 level and much less so at the LC5 concentration due to the lower slope (= greater heterogeneity) for the log-dose response regression for P. xylostella compared with H. virescens. The basis for the much greater heterogeneity of response to Vip3A observed for the Karak population of P. xylostella (field collected in 2001) compared with the WFO6 population of H. virescens (field collected in 2006) is unknown but could reflect a more variable binding pattern for Vip3A to midgut receptors between individual insects in the former species.
In the present study, exposure to the LC5 concentration of Vip3A increased larval and pupal developmental times and reduced the survival rate for larvae and the rate of adult emergence for both H. virescens and P. xylostella. Similar effects have been reported with sub-lethal treatments of other types of insecticides in a range of insect species (e.g. Fujiwara et al., 2002; Adamski, et al, 2007, 2009). More surprisingly, the average pupal weight for H.
67 virescens and P. xylostella was greater for insects treated with Vip3A compared with untreated controls. These results are in contrast, for example, with the finding that the pupae of P. xylostella treated with a sub-lethal concentration of spinosad were lighter than the pupae of control insects (Yin et al., 2008).
It is known that insecticides can cause resurgence of insect pests, due to suppression of natural enemies or through physiological resurgence, a process where insect reproduction can be stimulated by sub-lethal doses of insecticides (Kern and Stewart, 2000; Lashkari et al., 2007; Wang et al., 2008; Antonio et al., 2009). This effect on reproduction has been documented in P. xylostella (Sota et al., 1998). The greater fecundity of Vip3A-treated insects compared with control insects for H. virescens on artificial diet and P. xylostella on leaf discs may be an example of this phenomenon. However, the egg viability of both H. virescens and P. xylostella was reduced when treated with the Vip3A toxin. Fujiwara et al. (2002) also reported that fecundity of P. xylostella treated with sub-lethal concentrations of the pyrethroid fenvalerate was greater compared with control insects but observed that the eggs laid by the treated female were significantly smaller and had reduced viability compared with eggs laid by untreated insects.
No significant difference was observed in adult longevity or mating success for H. virescens and P. xylostella treated with Vip3A compared with control insects. A similar lack of effect on adult longevity has been reported for sub-lethal doses of other insecticides in a range of insect species (e.g. Antonio et al., 2009; Cutler et al., 2009). The intrinsic rate of population increase is a useful parameter for assessing the population growth of insects (Birch, 1948; Hamedi et al., 2010; Saeed et al., 2010). In the present study the Vip3A-treated populations of H. virescens and P. xylostella showed lower rm values compared with untreated populations. This is in agreement with several previous studies on insects (e.g. Martínez- Villar et al., 2005; Yin et al., 2008; Wang et al., 2008).
In conclusion, the present study has shown that sub-lethal concentrations of Vip3A are likely to reduce population growth of H. virescens and P. xylostella, although experiments over several generations under conditions more applicable to field conditions would be required to confirm this.
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CHAPTER 6: Effect of temperature on the fitness of the Vip3A resistant sub-population of Heliothis virescens (WF06)
6.1 Introduction
Fitness can be defined as the ability of an individual to survive and reproduce relative to other individuals of the same species (Roush and McKenzie, 1987; Gassmann et al., 2009). Fitness costs occur when individuals of a resistant population reared without exposure to a toxicant show lower fitness than susceptible individuals (Crowder et al., 2009). Reduced fitness of resistant insects in an insecticide free environment is charactersistic for many insect pest species and it has been documented both for insect resistance to synthetic insecticides, as well as for insects resistance to Bt (Alkyokhin and Ferro, 1999). As discussed in Section 2.5 various life history traits of a resistant insect have been associated with fitness costs (Li et al., 2007), including development rate, mating behaviour, fecundity and diapause (Carriére et al., 1994; Alyokhin and Ferro, 1999; Sayyed and Wright, 2001). For example, a population of aphids showing broad resistance to phosphate, carbamates and pyrethroids showed lower survival during cold and windy weather compared with a susceptible aphid population of the same species (Foster et al., 1996). While Alkyokhin and Ferro (1999) reported that in Colorado potato beetle resistant to Cry3A toxin, net reproduction rate and intrinsic rate of population increase was lower compared with the susceptible strain. Most fitness studies with Bt resistant insect populations have shown high levels of fitness costs (e.g. Groeters et al., 1994; Alyokhin and Ferro, 1999; Pickett, 2009). However, in some cases Bt resistant insect populations have been reported to have a fitness advantage over their susceptible counterparts (e.g. Gould and Anderson, 1991; Sayyed and Wright, 2001; Bielza et al., 2008).
Various environmental factors can affect fitness costs, most notably temperature and relative humidity (Li et al., 2007; Pappas et al., 2008; Rwomushana et al., 2008; Mironidis and Savopoulou-Soultani, 2008). Previous work on the Vip3A-Sel population of H. virescens (WF06) (Section 3.2.1) selected for a high level of resistance to Vip3A has shown that resistance was unstable in the absence of exposure to Vip3A, suggesting that resistance was linked to fitness costs (Pickett, 2009).
The aim of this study was to investigate whether fitness costs in Vip3A-Sel H. virescens (WFO6) varied at different temperatures.
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6.2 Materials and Methods
6.2.1 Insects
Vip3A-Sel and Unsel sub-populations of H. virescens (WF06) were cultured on artificial diet under constant environmental conditions as described in Section 3.2.1. The Vip3A-Sel sub- population was left unselected for 6 generations to avoid possible maternal effects. The LC50 value of the Vip3A-Sel population was >2000 ug/ml when used in the fitness experiments.
6.2.2 Development and survival of Heliothis virescens
Eggs laid on the same day (< 24 h-old) were collected on white netting (Section 3.4.1) and kept in 250 ml round cups sealed with a lid. Eggs of Vip3A-Sel and Unsel sub-populations were divided into four groups and kept at 20, 25, 30 and 35 °C respectively under standard environmental conditions (Section 3.4.1). A total of 500-600 eggs were used per treatment for both Vip3A-Sel and Unsel sub-populations. The number of eggs that hatched at each temperature was recorded daily. The first 200 neonates (<24 h-old) hatched from egg batches at each temperature were used for fitness studies, with each 1st instar larvae placed on artificial diet in individual 1 oz plastic cups (9051, Bio-Serv, Frenchtown, NJ, USA) and sealed with a snap-on lid (9053, Bio-Serv). Larvae were examined daily and the developmental time from larva to pupa was noted together with the proportion that pupated. Pupal weight was recorded one day after pupation, when the sex of each insect was also determined. The pupae were placed individually in 250 ml plastic cups covered with white netting secured by a plastic band. The number of adults that emerged was recorded daily and the proportion that emerged successfully was also recorded, together with the total developmental time from egg to adult.
6.2.3 Adult longevity, mating success, fecundity and egg viability
Adults that emerged from the development study (Section 6.2.2) were used to estimate fecundity and adult longevity. On emergence, adults (< 24 h-old) from the two sub-populations were paired as follows: Vip3A-Sel male x Vip3A-Sel female (15, 22, 24 pairs at 20 ºC, 25 ºC and 30 ºC respectively) and Unsel male x Unsel female (17, 21, 21 pairs at 20 ºC, 25 ºC and 30 ºC respectively). Each pair was placed in transparent plastic cup coved with white netting, and a cotton wool pad soaked in 10% honey solution was placed on the top of the netting as a food source. When a female started egg laying the insect pair was transferred to a new cup every 48 h until they died. Mating success, the number of eggs laid per female, the proportion of eggs that hatched, the oviposition period and adult longevity was recorded.
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6.2.4 Intrinsic rate of population increase
The net replacement rate (Ro), the average number of female offspring per female during its entire lifetime was calculated as described in Section 5.2.9.
6.2.5 Statistical analysis
All analyses were conducted in R version 2.9.0 (R Development Core Team, 2009). Data for developmental time, larval duration, pupal duration and pupal weight at each temperature were normalised and subject to ANOVA. Mating pair success and the number of pairs producing viable progeny was modelled using the binomial proportions test (prop.test) for both sub- populations at each temperature. The mean number of eggs and the mean number of fertile eggs was analysed using a generalised linear model with a poisson error structure corrected for overdispersion.
6.3 Results
6.3.1 Development and survival of Heliothis virescens
Vip3A-Sel and Unsel sub-populations of H. virescens completed their development from egg to adult eclosion at all of the temperatures tested (Table 6.1).
Table 6.1 Mean development time (days ± SE) and pupal weight (mg ± SE) of Vip3A-Sel and
Unsel sub-populations of Heliothis virescens at different temperatures.f
Temp WF06 Eggs LDTa PDUb Totalc Ad Longd PWe ºC
20 Uunsel 5.84 ± 0.15Aa 32.1 ± 0.19Aa 28.7 ± 0.17Aa 66.7 ± 0.36Aa 14.64 ± 1.00Aa 274 ± 0.85 Aa 20 Sel 5.72 ± 0.24Aa 32.3 ± 0.24 Aa 26.6 ± 0.50Bb 65.5 ± 1.00Aa 15.14 ± 0.74Aa 272 ± 0.87 Aa 25 Unsel 3.95 ± 0.04Bb 16.9 ± 0.27Bb 14.8 ± 018Cc 35.7 ± 0.58Bb 11.03 ± 0.32Bb 270 ± 0.34 Bb 25 Sel 3.83 ± 0.24Bb 15.6 ± 0.14Cc 13.4 ± 0.07Dd 33.3 ± 0.33Cc 10.16 ± 0.34Bb 257 ± 1.00 Cc 30 Unsel 2.87 ± 0.13Cc 11.7 ± 0.03Dd 9.28 ± 0.20Ee 23.9 ± 0.16Dd 9.40 ± 0.45Cc 264 ± 0.72 Dd 30 Sel 2.78 ± 0.21Cc 10.3 ± 0.05Ee 9.19 ± 0.21Ee 22.2 ± 0.19Ee 8.54 ± 0.32Cc 251 ± 1.50 Ee 35 Unsel 2.76 ± 0.23Cc 10.1 ± 0.12Ef 9.03 ± 0.27Ee 21.7 ± 0.15Ff ------234 ± 1.95Ff 35 Sel 2.58 ± 0.25Cc 9.31 ± 0.07 Fg 9.00 ± 0.00Ee 21.1 ± 0.53Ff ------225 ± 0.44Gg
a. Larval developmental time b. Pupal duration c. Development time from egg to adult d. Adult longevity e. Pupal weight f. For the Unsel and Vip3A-Sel sub-populations, values sharing a common upper case letter are not significantly different (P>0.05) between temperatures. At each temperature, values sharing a common lower case letter are not significantly different (P>0.05) between Unsel and Vip3A-Sel sub-populations.
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The mean development time for eggs (Fig. 6.1) from the two sub-populations was not significantly different at all the test temperatures: 20 °C (F = 1.015, df 1,6, P>0.05); 25 °C (F= 1.598 df 1,6, P>0.05); 30 °C (F= 0.149, df 1,6, P> 0.05) and 35 °C (F= 0.269, df 1,6, P>0.05). The larval duration (neonate to pupation) (Fig. 6.2) of Vip3A-Sel was significantly shorter than Unsel at 25 °C (F = 45.739, df 1,6, P<0.001), 30 ºC (F = 647.83, df 1,6 P< 0.001) and 35 °C (F = 37.474 df 1,6, P<0.05). At 20 °C, larval duration of the two sub-populations was not significantly different (F = 0.591, df 1,6, P>0.05). The mean pupal development time (Fig. 6.3) of Vip3A-Sel was significantly shorter compared with Unsel at 20ºC (F = 14.627 df 1,6, P<0.01) and 25 ºC (F = 50.413, df 1,6, P<0.001). Pupal duration of the two sub-populations was not significantly different at 30ºC (F = 0.088, df 1, 6, P>0.05) and 35ºC (F = 0.11 df 1, 6, P>0.05). The mean total developmental time from egg to adult (Fig. 6.4) of Vip3A-Sel was significantly faster at 25ºC (F = 12.462, df 1,6, P<0.05) and 30ºC (F = 12.462, df 1,6, P<0.05) than Unsel, but there was no significant difference between the two sub-populations at 20 ºC (F = 0.838, df 1,6, P>0.05) and 35ºC (F = 11.05, df 1,42, P>0.05).
Fig. 6.1 Duration of egg stage (days ± SE) of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures. 20 °C (F = 1.015, df 1,6, P>0.05); 25 °C (F= 1.598 df 1,6, P>0.05); 30 °C (F= 0.149, df 1,6, P> 0.05); 35 °C (F= 0.269, df 1,6, P>0.05).
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Fig. 6.2 Development of larvae of (days ± SE) Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures. 20 °C, (F = 0.591, df 1,6, P>0.05); 25 °C (F = 45.739, df 1,6, P<0.001); 30 ºC (F = 647.83, df 1,6 P< 0.001);35 °C (F = 37.474 df 1,6, P<0.05).
Fig. 6.3 Development of pupal stage (days ± SE) of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures. 20ºC (F = 14.627 df 1,6, P<0.01); 25 ºC (F = 50.413, df 1,6, P<0.001); 30ºC (F = 0.088, df 1, 6, P>0.05); 35ºC (F = 0.11 df 1, 6, P>0.05).
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Fig. 6.4 Total development time (days egg to eclosion ± SE) of Vip3A-Sel and Unsel sub- populations of Heliothis virescens at different temperatures. 20 ºC (F = 0.838, df 1,6, P>0.05); 25ºC (F = 12.462, df 1,6, P<0.05); 30ºC (F = 12.462, df 1,6, P<0.05); 35ºC (F = 11.05, df 1,42, P>0.05).
The mean pupal weight (Fig. 6.5) of Vip3A-Sel was significantly less at 25 ºC (F = 142.32, df 1, 6, P<0.001), 30ºC (F = 65.317, df 1,6, P<0.001) and 35 ºC (F = 165.20, df 1,6, P<0.001). At 20ºC, there was no significant difference between the two sub-populations (F = 3.026, df 1,6, P>0.05). The Vip3A-Sel sub-population had a lower pupation percentage (Fig. 6.6) and adult emergence (Fig. 6.7) than the Unsel sub-population at 25, 30 and 35 ºC.
Fig. 6.5 Pupal weight (mg ± SE) of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures. 20ºC (F = 3.026, df 1,6, P>0.05); 25 ºC (F = 142.32, df 1, 6, P<0.001); 30ºC (F = 65.317, df 1,6, P<0.001); 35 ºC (F = 165.20, df 1,6, P<0.001).
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Fig. 6.6 Percentage pupation (± SE) of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures.
Fig. 6.7 Percentage eclosion of adults ± SE from pupae of Vip3A-Sel and Unsel sub- populations of Heliothis virescens at different temperatures.
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6.3.2 Adult longevity, mating success, fecundity and egg viability
The mean adult longevity of the Vip3A-Sel and Unsel sub-populations (Table 6.2) was not significantly different at 20ºC (F=0.308 df 1,60, P>0.05), 25ºC (F,=,3.365, df 1,80, P>0.05) and 30ºC(F,=,1.769, df 1,84, P>0.05). Adults that emerged at 35ºC were deformed in Vip3A- Sel sub-populations therefore further studies were not possible to carry out at this temperature.
There was no significant difference between Vip3A-Sel and Unsel in the proportion of pairs that produced eggs at 20 ºC (α2=0, df 1, P>0.05), 25 ºC (α2=2.96, df 1, P>0.05) and 30ºC (α2=0.904, df 1, P>0.05) or in the proportion of pairs that produced viable progeny at 20 ºC (x2=0.021, df 1, P>0.05), 25 ºC (α2=0, df 1, P>0.05) and 30ºC (α2=0, df 1, P>0.05).
The mean number of eggs produced by Vip3A-Sel was significantly less at 30ºC (df 43, P<0.05, n=45) and 20ºC (df 30, P<0.05, n=32) compared with Unsel. At 25 ºC, the mean number of eggs produced by the two sub-populations was not significantly different (df 41, P>0.05, n=43). The mean egg viability of Vip3A-Sel was significantly lower compared with Unsel at all test temperatures.
Table 6.2 Mean (± SE) reproductive parameters for Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures.d
Temp WF06 Pairsa MPSb (%) PVPc (%) Eggs/female Egg Viability ºC (%)
20 Unsel 17 76 29 492 ± 98Aa 55 ± 5.98 Aa 20 Sel 15 73 20 242 ± 39Bb 38 ± 1.47 Bb 25 Unsel 21 90 47 523 ± 90Ac 67 ± 7.71 Aa 25 Sel 22 63 31 550 ± 66Ac 40 ± 5.41 Bb 30 Unsel 21 76 58 486 ± 78Ad 59 ± 5.62 Aa 30 Sel 24 58 20 234 ± 55Bb 33 ± 3.51 Bb
a. Total number of pairs b. Mating pair success (produce eggs) c. Pairs producing viable progeny d. For the Unsel and Vip3A-Sel sub-populations, values sharing an upper case letter are not significantly different (P>0.05) between temperatures. At each temperature, values sharing a common lower case letter are not significantly different (P>0.05) between Unsel and Vip3A-Sel sub-populations.
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The Vip3A-Sel sub-population showed a 29%, 9% and 25% decrease in its rm compared with Unsel at 20, 25 and 30 ºC, respectively (Table 6.3).
Table 6.3 Life table parameters of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different temperatures. a b c Temp WF06 Ro rm RF oC
20 unsel 104.79 0.07 1 20 sel 26.03 0.05 0.71 25 unsel 185.96 0.14 1 25 sel 71.58 0.13 0.92 30 unsel 144.96 0.2 1 30 sel 33.06 0.15 0.75
a. Net replacement rate b. Intrinsic rate of population increase c. Relative fitness = rm Vip3A-Sel / rm of Unsel
6.4 Discussion
It has previously been shown that the magnitude of fitness costs associated with the insecticide resistance can be influenced by the environment (Carrière and Tabashnik, 2001; Bird and Akhurst, 2007) and that such costs are usually greater under stressful conditions (Raymond et al., 2005, 2010).
Several studies have demonstrated that the fitness costs in resistance insects are associated with low temperatures during overwintering (e.g. Foster et al., 2000; Gazave et al., 2001; Carrière et al., 2001). In the present study, while the effect of temperature on the rate of developmental was similar in both the Vip3A-Sel and Unsel sub-populations, however, fitness costs associated with pupation, pupal weight, adult eclosion and fecundity differed between the Vip3A-Sel and Unsel sub-populations at particular temperatures.
Using the intrinsic rate of increase as an approximation to an overall measure of fitness, fitness costs associated with Vip3A resistance were relatively greater below and above the optimal culture temperature for the Vip3A-Sel sub-population. Plutella xylostella selected with spinosad, have also been reported to have significantly greater fitness costs at unfavourably low or high temperatures (Li et al., 2007). The intrinsic rate of increase is closely related to the mean relative growth rate for insects (Leather and Dixon 1984) and this
77 has been demonstrated, for example, in spinosad and Cry1Ac-resistant P. xylostella (Sayyed and Wright, 2001, Sayyed et al., 2008). In the present study, this relationship was also significant with an increase in the mean relative growth rate associated with a corresponding increase in the intrinsic rate of population increase (Table 6.3). The lower rate of population increase for the Vip3A-Sel sub-population of H. virescens seemed to be due largely to the number of viable eggs produced, developmental time and adult eclosion.
Several insect species have been shown to have insecticide resistance-related fitness costs associated with low temperatures. In the mosquito Culex pipiens L (Diptera: Culicidae ), a 7% reduction was reported in the daily survival rate of homozygous organophosphate resistant C. pipiens compared with susceptible C. pipiens during winter (Gazave et al., 2001). While a P. xylostella population resistant to spinosad had a significantly lower level of survival compared with a susceptible population for both egg and larval stages at lower temperatures (Li et al., 2007), and a Btk-resistant population of T. ni had a lower fecundity at 10 ºC compared with susceptible insects (Caron and Myers, 2008). The results of the present study are similar to the above studies, with the fecundity and viability of eggs of the Vip3A-Sel sub-population being lower compared with the Unsel sub-population at low temperatures.
Li et al. (2007) reported that fitness costs in P. xylostella selected with spinosad were less marked at high temperatures compared with low temperatures. In the present study of Vip3A-Sel H. virescens the opposite was observed. The greater fitness costs observed in Vip3A-Sel H. virescens at higher temperatures suggests the presence of a trade-off in the distribution of resources between resistance and development, survival and reproduction. The greater proportion of non-viable eggs produced at higher temperatures in the Vip3A-Sel sub-population may be due to elevated abdominal temperatures damaging the eggs, adversely effecting fertilization by killing sperm, or impairing the transport of sperm from the spermatheca. Several studies have shown that a temperature of 30 oC or similar can incapacitate insect sperm (Laug and Masner, 1974) or hamper egg development (Gerber and Howlader, 1987).
Most species have stable ecological niches, and negative pleiotropic effects of mutations conferring the necessary adaptation to expand ecological niches may be one of the elements which impose restrictions on evolution (Bradshaw, 1991). In the present study, the interaction between resistance and temperature on life history traits seemed to be very strong. Such effects, if found under field conditions in insect populations with non- overlapping generations, could lead to developmental asynchrony and non-random
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(assortative) mating between resistant individuals and thus increase the rate of development of resistance (Liu et al., 1999).
It is more likely, however, that the temperature-related fitness costs observed for the Vip3A- Sel population of H. virescens may, if replicable under field conditions and in other insect populations that may develop resistance to Vip toxins, prove useful tool for the management of Vip toxin resistance.
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CHAPTER 7: Effect of entomopathogenic nematodes on the fitness of a Vip3A resistant sub-population of Heliothis virescens (WF06)
7.1 Introduction
In Chapter 6 the effect of temperature on fitness costs related to development and reproduction were discussed for the Vip3A-Sel sub-population of H. virescens. In this chapter fitness costs associated with the response to entomopathogenic nematodes in Vip3A-Sel insects will be considered.
Entomopathogenic nematodes (EPN) of the families Steinernematidae and Heterorhabditidae are used to control many insect pests (Ehlers, 1996; Gouge et al., 1999; Fitters et al., 2001; Susurluk et al., 2009). Infective nematode juveniles (IJ) may enter in insect‟s haemocoel directly via thin parts of the cuticle, through the midgut epithelium via the mouth or anus, or through the tracheae via the spiracles (Koppenhofer et al., 2000). IJ release symbiotic bacteria into the insect haemocoel; the bacteria start to grow and release toxins that kill the host insect, usually within 24 to 48 h (Burnell and Stock, 2000).
As discussed in Section 2.5, insecticide resistance is associated with fitness costs in many insects (Carriére and Tabashnik, 2001). Bt resistance has been reported to be variously recessive, as in a P. interpunctella population (McGaughey, 1985), partly to completely recessive, as in the YHD2 population of H. virescens (Gould et al, 1995), or may be non recessive or incompletely dominant as in O. nubilalis. In the Vip3A-sel sub- population of H. virescens, resistance ranged from almost completely recessive to incompletely dominant with an associated fitness cost (Pickett, 2009). Carrière et al. (2004) reported that ecological conditions can increase non-recessive types of fitness costs in some insects and may be important in delaying the evolution of resistance
Studies on the effects of entomopathogenic nematodes and baculoviruses on the fitness of Cry1Ac-selected P. gossypiella and P. xylostella have shown that both types of entomopathogen increase the fitness cost of resistance (Gassmann et al., 2006; Raymond et al., 2007) and it has been suggested that the use of entomopathogens in Bt
80 resistance management (Section 2.11) could increase the effectiveness of the use of refuges (Gassmann et al., 2008).
7.2 Materials and Methods
7.2.1 Insect culture
Unsel and Vip3A-Sel sub-populations of H. virescens (WF06) were used (Section 3.2).
7.2.2 Nematodes
Commercially-produced entomopathogenic nematodes, Steinernema carpocapsae (Weiser) (Nematoda: Rhaditida) and Heterorhabditis bacteriophora (Poinar) (Nematoda: Rhabditida) were obtained from Becker Underwood Ltd (Littlehampton, West Sussex, UK) and stored at 4 ºC prior to use. Nematodes were used within one week of storage.
7.2.3 Nematode bioassays
7.2.3.1 Mortality
Nematode bioassays were conducted on 2nd, 3rd, 4th, 5th and 6th instar larvae of Vip3A-Sel and Unsel sub-populations of H. virescens.
A preliminary experiment was conducted to check the effect of starvation on the insect larvae. The results indicated that there was no mortality for any instar of either sub- population after 24 and 48 h periods of starvation. Subsequent bioassays were conducted in Petri dishes (5 cm dia.) containing a Whatman No. 1 filter paper (4.5 cm dia.). Four hundred µl of water applied to the filter paper to moisten it. A single insect larva was put in each Petri dish. Five nematode concentrations: 0, 10, 20, 50, 100 and 150 IJ in distilled water were applied directly to the individual larvae by pipette. Insect mortality was noted after 24 and 48 h. Surviving insect larvae were transferred to individual plastic cups (1 oz plastic cups, No. 9051, Bio-Serv, Frenchtown, NJ, USA) containing diet (Section 3.4.1). Larvae that pupated were transferred to new containers and the emergence of adults was recorded. Twelve larvae per treatment were used in bioassays and the bioassay was replicated 4 times.
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7.2.3.2 Estimation of nematode penetration
Only 6th instar larvae were used in this part of the experiment. Five dead larvae per treatment from the above bioassay (Section 7.2.3.2) were washed thoroughly to remove any nematodes attached to the insect cuticle. The larvae were then homogenised in pepsin solution (Sigma Aldrich, Poole, UK) and the number of nematodes that had entered each larva was counted under a stereo microscope.
7.2.3.3 Nematode reproduction
Reproduction of nematodes was determined in 6th instar larvae of both sub-populations using the White Trap method (Kaya and Stock, 1997). Five dead larvae per treatment from the above bioassay (Section 7.2.3.1) were placed in individual round plastic cups (250 ml) and kept at 25 °C. After approximately 10 days, the number of nematodes that emerged from the insect cadaver in the water was counted under a stereo microscope.
7.2.4 Statistical analysis
All analyses were conducted in R version 2.9.0 (R Development Core Team, 2009). Mortality data for both nematode species were corrected for control mortality using Abbott‟s correction (Abbott, 1925) and subjected to analysis of covariance (ANCOVA). Nematode reproduction data for both nematodes species were also analyzed by ANCOVA. Nematode penetration data for S. carpocapsae was log transformed and then analysed by ANOVA.
7.3 Results
7.3.1 Effect of entomopathogenic nematodes on mortality of Vip3A-Sel and Unsel sub-populations of Heliothis virescens
There was no survival in the Vip3A-Sel or Unsel sub-populations of H. virescens for 2nd, 3rd, and 4th instar larvae after 48 h in any of the nematode treatments. This data was therefore excluded from the analysis. For 5th instar larvae, mortality of the Unsel sub- population after exposure to either nematode species for 48 h was significantly lower than
82 the Vip3A-Sel sub-population (Fig. 7.1; t = -2.223; df 7,72; P <0.05). The overall mortality of both sub-populations increased with increasing nematode concentration (t = 5.30; df, 7,72; P< 0.001). The overall mortality caused by the S. carpocapsae was significantly higher than H. bacteriophora in both Unsel and Vip3A-Sel sub-populations of H. virescens (t = 4.97; df, 7,72; P< 0.001).
Fig. 7.1 Mortality (% ± SE) of 5th instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different concentrations of Steinernema carpocapsae and Heterorhabditis bacteriophora infective juveniles after 48h.
For 5th instar larvae, mortality of the Unsel sub-population after exposure to either nematode species at eclosion was significantly lower than the Vip3A-Sel sub-population (Fig. 7.2; t = -2.318; df 7, 72; P <0.05).
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Fig. 7.2 Mortality (% ± SE) at eclosion following exposure of 5th instar larvae of Vip3A-Sel and Unsel sub-populations of Helithis virescens to different concentration of Steinernema carpocapsae and Heterorhabditis bacteriophora infective juveniles.
With 6th instar larvae of H. virescens, mortality in the Unsel sub-population at 48h and eclosion following exposure to either nematode species, was significantly lower than in the Vip3A-sel sub-population (Figs 7.3 and 7.4; t = -2.32: df, 7,72; P < 0.05 and t = -3.045; df, 7,72; P<0.05, respectively). Mortality for both sub-populatons and nematode species showed a general trend to increase with increasing nematode concentration (t = 4.33, df, 7 72; P< 0.001 and t = 3.90, df, 7,72; P< 0.001, respectively).
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Fig. 7.3 Mortality (%) ± SE at 48 h of 6th instar larvae of Vip3A-sel and Unsel sub- populations of Heliothis virescens at different concentrations of Steinernema carpocapsae and Heterorhabditis bacteriophora infective juveniles.
Fig. 7.4 Mortality (%) ± SE at eclosion of 6th instar larvae of Vip3A-Sel and Unsel sub- populations of Heliothis virescens at different concentrations of Steinernema carpocapsae and Heterorhabditis.. bacteriophora infective juveniles..
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7.3.2 Nematode reproduction
The reproduction of of S. carpocapsae and H. bacteriophora was significantly greater in cadavers of the Unsel compared with the Vip3A-Sel sub-population of H. virescens for all larval instars (Fig. 7.5a-e; t = 3.207; df, 6,493; P< 0.001). There was positive correlation between nematode reproduction and the larval instar infected with nematodes (t = 7.77: df, 6,493; P<0.001).
Fig. 7.5a Reproduction ± SE of Steinernema carpocapsae and Heterorhabditis bacteriophora in cadavers of 2nd instar larvae of Vip3A-Sel and Unsel sub-populationsof
H. virescens at different nematode doses (IJ / Petri dish).
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Fig. 7.5b Reproduction ± SE of Steinernema carpocapsae and Heterorhabditis bacteriophora in cadavers of 3nd instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different nematode doses (IJ / Petri dish).
Fig. 7.5c Reproduction ± SE of Steinernema carpocapsae and Heterorhabditis bacteriophora in cadavers of 4th instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different nematode doses (IJ / Petri dish).
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Fig. 7.5d Reproduction ± SE of Steinernema carpocapsae and Heterorhabditis bacteriophora in cadavers of 5th instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different nematode doses (IJ / Petri dish).
Fig. 7.5e Reproduction ± SE of Steinernema carpocapsae and Heterorhabditis bacteriophora in cadavers of 6th instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different nematode doses (IJ / Petri dish).
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7.3.3 Penetration of nematode infective juveniles (IJ)
The penetration of IJ of S. carpocapsae (Fig. 7.6) was significantly different between Vip3A-sel sub-population and Unsel sub-population of H. virescens (F= 7.45, df, 1 40; P>0.001).
Fig 7.6 Penetration ± SE of Steinernema carpocapsae IJ in 6th instar larvae of Vip3A-Sel and Unsel sub-populations of Heliothis virescens at different nematode doses (IJ / Petri dish).
7.4 Discussion
The present study showed that 5th and 6th instar larvae of the Vip3A-Sel sub-population of H. virescens were significantly more susceptible to both S. carpocapsae and H. bacteriophora compared with the Unsel sub-population. These results, which suggest a fitness cost, are in accordance with a previous studies, where S. carpocapsae marginally increased the fitness costs of Bt resistance in P. xylostella (Baur et al., 1998) and S. riobrave and H. bacteriophora increased the fitness costs of Cryl1Ac resistance in P. gossypiella (Gassmann et al, 2006, 2009a). Raymond et al. (2007) have reported that baculoviruses (NPV) also increase the fitness costs of a Cryl1Ac resistant population of P. xylostella.
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The mechanisms by which the entomopathogenic nematodes cause greater mortality in
Bt resistant insect populations remain unclear. The study shown that, CO2 is involved in the long distance attraction of plants parasitic and entomopathogenic nematodes (Klingler, 1965; Lewis et al, 1993; Robinson, 1995; Susurluk et al, 2009). One hypothesis is that the Bt toxin make insects more debilitated and subsequently more susceptible to entomopathogens and these debilitated insects respire more and release more CO2 ultimately the selected population attract more the EPNs. One hypothesis is that Bt resistant insects show a reduced capacity to fend off pathogen infections compared with susceptible insects (Gassmann et al., 2006). For example, organophosphate resistant mosquitoes have been reported to have higher levels of Wolbachia infections than susceptible mosquitoes and to show a clear interaction between the presence resistance alleles and Wolbachia load (Berticat et al., 2002).
Stress can reduce the general immunocompetence in insects against natural enemies. The major immune defence against endoparasitoids are encapsulation and melanisation (Karimzadeh and Wright, 2008). Insects may fend off infection from nematodes through encapsulation or melanisation of nematode infective juveniles before they release bacteria (Li et al., 2007a). Melanization occurs by the action of the prophenoloxidase pathway and difference in phenoloxidase activity could contribute to differences in susceptibility to pathogens between Bt resistant and susceptible insects (Gassmann et al., 2009). Bt resistance has been associated with higher phenoloxidase activity in E. kuehniella and H. armigera (Rahman et al., 2004; Ma et al., 2005) but it is not known whether these insects are susceptible to the entomopathogenic nematodes (Gassmann et al., 2009a).
The present results indicate that S. carpocapsae imposed greater fitness costs compared with H. bacteriophora by causing greater mortality in the Vip3A resistant than in the susceptible population of H. virescens. This difference could be due to differences in the behaviour of the nematodes (Campbell and Gaugler, 1993; Lewis et al, 1993; Boff et al., 2001; Susurluk et al., 2009) or to the different bacterial symbionts found in Steinernema Xenorhabdus spp) and Heterorhabitis (Photorhabdus spp) species (Kaya and Gaguler, 1993).
In the present study, penetration of IJ was greater in the Vip3A-Sel sub-population than in the Unsel sub-population of H. virescens. While the reason for this is unknown, it could be due to differences in insect behaviour in the Petri bioassay, the greater number of IJ in Vip3A-Sel insects is the simplest explanation for the greater mortality in resistant insects
90 rather than a reduced immune response. Further studies are required to determine whether Vip3A-resistant insects are more susceptible to entomopathogenic nematodes under conditions more relevant to the field. The potential use of entompathogens in Bt resistance management is discussed in Chapter 9.
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CHAPTER 8: Fluctuating asymmetry, morphological changes and flight muscle ratio in a Vip3A resistant sub- population of Heliothis virescens (WF06)
8.1 Introduction
The measurement of field evolved insecticide resistance can be a useful biomonitoring strategy due to its quick evolutionary response to insecticide use; the rate of insecticide use being in general directly proportional to the level of insecticide resistance expected (Walker et al., 2005). Fluctuating asymmetry (FA) increases when an organism develops under a stressful environment (Hardersen, 2000; Hoffman et al., 2005; Section 2.7) and insecticide resistant insect populations have been reported to show higher levels of FA compared with susceptible populations (Clarke and Mckenzie, 1987; Clarke et al., 2000; Ribeiro et al., 2007).
FA is considered to be a measure of developmental instability (Palmer and Strobeck, 1986; McKenzie and Yen, 1995; Section 2.7) The assumption behind FA analysis is that the development of both sides of a bilateral symmetrical organism is influenced by identical genes, and thus any deviation between the two sides results from developmental perturbation (Clarke, 1998; Jones et al., 2005). FA has been shown to increase with genetic and environmental stress (Jones et al., 2005; Vishalakshi and Sing, 2006; Vishalakshi and Sing, 2008) and factors such as increased homozygosity, hybridization, inbreeding, extreme temperatures, chemical exposure and food quality have been linked to FA in morphological traits (Leary and Al-Lendorf, 1989; Mpho et al., 2001; Liu et al., 2005; Jones et al., 2005). It has been demonstrated that in populations that persist under stressful conditions mutations can occur in different morphological traits related to stress resistance that could lead to modifications in the trait directly related to stress resistance and in traits genetically correlated with stress resistance in a population (Van Straalen and Timmermans, 2002; Kurbalija et al., 2010).
Most insects rely on flight to find food sources, mate or interact socially. When flight performance is compromised these fitness related traits will be impaired (Frazier et al., 2008). Flight performance is thus an important factor for behavioural success in most insects (Samejima and Tsubaki, 2010) and is greatly influenced by body morphology and the ratio of thorax mass to total body mass, the flight muscle ratio (FMR) (Marden, 2000; Almbro and
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Kullberg, 2008). The FMR of insects represents a major allocation of energy and constitutes as much as 55 to 65% of body mass (Marden, 2000).
This chapter examines different morphological characteristics (length of forewing, hindwing and tibia), FA and FMR of Vip3A-Sel and Unsel sub-populations of H. virescens.
8.2 Materials and Methods
8.2.1 Insect culture
The Vip3A-Sel and Unsel sub-populations of H. virescens (WF06) were used (Sections 3.2.1 and 3.4.1).
8.2.2 Measurement of fluctuating asymmetry (FA)
One hundred 1st instar larvae of each sub-population were placed on artificial diet (Section 3.4.1). On pupation, pupae were transferred to individual 250 ml plastic cups covered with netting (Section 3.4.1). On emergence, sixty individual adults (30 male and 30 female) of each population were randomly selected for the measurement of FA. The insects were frozen at -20 ºC and wings and legs detached from the thorax at the point of their attachment and mounted on glass slides. The following traits were measured (data from males and females were analysed together):
(1) Forewing length of right side (FWLR) (2) Forewing length of left side (FWLL) (3) Hindwing length of right side (HWLR) (4) Hindwing length of left side (HWLL) (5) Tibia length of meta leg of right side (TBLR) (6) Tibia length of meta leg of left side (TBLL)
A dissecting stereo microscope with eyepiece graticule (units 1-100 µm) was used; measurements were taken through the right eyepiece only to avoid distortion from change of angle (Peck, 2009).
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8.2.3 Flight muscle ratio (FMR)
The head, thorax and abdomen from 100 adults (50 male and 50 female) selected from each sub-population (Section 8.2.2) were separated and dried for 24 h at 55 ºC, then weighed using a microbalance. The FMR was calculated as follows:
FMR = Dry thorax mass / Dry body mass
8.2.4 Analysis of fluctuating asymmetry (FA)
Measurement error (ME) can account for variation in asymmetry, and repeat measurements are required to ensure that FA is detectable and that there is no effect associated with the directional asymmetry (Palmer, 1986; Vishalakshi and Singh, 2008). In order to estimate the ME, 20 adult insects were randomly taken from the culture and each insect trait (Section 8.2.2) measured twice. The significance of ME was assessed using a two-way mixed model ANOVA, where sides were entered as fixed factors and individuals as random factors (Vishalakshi and Singh, 2008). FA was calculated for each trait of every individual using the indexes;
FA = [R-L]
Where R is the value of trait on right side and L is the value of trait on the left side.
The resulting measurement was added to the data set.
8.2.5 Statistical analysis
All analyses were conducted in R version 2.9.0 (R Development Core Team, 2009). For FA, a two-way ANOVA test of side (fixed) × individuals (random) with repeated measurements of each side was used to assess the magnitude of measurement error. The Shapiro-Wilk test was applied to check the normality of the trait where the error mean square is larger than Side x individual mean square (Peck, 2009). FA values, FMR and length of morphological traits were analysed by ANOVA. There was no significant difference (P>0.05) between the male and female data for Vip3A-Sel and Unsel sub-populations and the data of male and female insects was combined for all the analyses shown.
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8.3 Results
8.3.1 FA data validation
A two-way ANOVA with replicated measurements of each side, indicated that the mean square for error was not significantly smaller than side × individual (non-directional asymmetry), with two out of the six traits having larger errors than side × individual, (highlighted in grey, Table 8.1). This meant the ME for these two traits was of concern for the analysis of FA in the full data set as it appeared ME could be larger than the calculated FA. However, despite the implications of high ME in these traits, directional asymmetry could be ruled out as there was no significant difference between right and left measurement „sides‟ for any of the traits (ANOVA P>0.05, df = 1, n = 20) and anti-symmetry could be ruled out as the data did not depart from normality (Shapiro-Wilk test, P>0.05). It can therefore be assumed that the asymmetry occurring is due to FA.
Table 8.1 Two-way ANOVA (side x individual) performed for each trait to assess measurement error (ME) for a sub-sample of 20 specimens from Vip3A-Sel and Unsel sub- populations of Heliothis virescens measured twice.
Trait Mean Square (ANOVA) Side x Sides Individual ME* individual* Forewing Unsel 0.006 4.93 0.01 0.0 1 Forewing Vip3A-Sel 0.124 5.298 0.004 1.20 Hindwing Unsel 0.180 2.25 0.15 0.11 Hindwing Vip3A-Sel 0.080 7.100 0.20 0.10 Tibia Unsel 0.122 .508 0.01 0.01 Tibia Vip3A-Sel 0.028 0.321 0.001 0.04
*Figures highlighted in grey are where the ME for these two traits is of concern (see above text).
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8.3.2 Mean values of different morphological traits
The mean ± SE of different morphological traits of Vip3A-Sel and Unsel adults are shown in Table 8.2. Mean FWLR (F = 11.51, df 1,57, P< 0.01) and FWLL (F = 19.57, df 1,57, P< 0.001) were significantly smaller in the Vip3A-Sel compared with the Unsel sub-population. The mean HWLR (F = 10.78, df 1, 74, P< 0.01) and HWLL (F = 20.93, df 1,74, P< 0.001) was significantly smaller in Vip3A-Sel compared with Unsel. The mean tibia length of right side (F = 7.82, df 1, 67, P< 0.01) and left side (F = 14.50, df 1,74, P< 0.001) were significantly smaller in Vip3A-Sel compared with Unsel.
Table 8.2 Mean (mm ± SE) morphological traits of Vip3A-Sel and Unsel sub-populations of Heliothis virescens. FWLR = forewing length of right side; FWLL = forewing length of left side; HWLR= hindwing length of right side; HWLL= hindwing length of left side, TBLR= tibia length of right side; TBLL= tibia length of left side
FWLR FWLL HWLR HWLL TBLR TBLL
Unsel 14.61 ± 0.09a 14.41 ± 0.10a 10.67 ± 0.07a 10.50 ± 0.08a 3.43 ± 0.03a 3.36 ± 0.03a
Vip3A-Sel 14.28 ± 0.06b 13.91 ± 0.08b 10.34 ± 0.06b 10.02 ± 0.06b 3.32 ± 0.02b 3.21 ± 0.02b
Mean values (±) followed by a different letter are significantly different (P< 0.01) between the two sub- populations.
8.3.3 Overall trend for fluctuating asymmetry (FA)
The ANOVA for FA of H. virescens indicated significance differences between the two sub- populations. The mean FA value of forewing in Vip3A-Sel was significantly greater compared with Unsel (Fig. 8.1; F= 7.17, df 1, 59, P <0.001). The mean FA value of hindwing in Vip-Sel was significantly greater compared with Unsel (Fig. 8.2; F = 32.411, df 1, 74, P <0.001). The mean FA value for tibia in Vip3A-Selwas significantly greater compared with Unsel (Fig. 8.3; F = 16.42, df 1, 67, P <0.001).
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Fig. 8.1 Fluctuating asymmetry for forewing length in Unsel and Vip3A-Sel sub-populations of Heliothis virescens. (F= 7.17, df 1, 59, P <0.001)
Fig. 8.2 Fluctuating asymmetry for hindwing length in Unsel and Vip3A-Sel sub-populations of Heliothis virescens. (F = 32.411, df 1, 74, P <0.001)
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Fig. 8.3 Fluctuating asymmetry for tibia length in Unsel and Vip3A-Sel sub-populations of Heliothis virescens. (F = 16.42, df 1, 67, P <0.001)
8.3.4 Flight muscle ratio (FMR)
The FMR of the Vip3A-Sel sub-population was significantly less compared with Unsel (Fig. 8.4; F = 17.78, df 1,198, P < 0.001).
Fig. 8.4 Flight muscle ratio (FMR) of Unsel and Vip3A-Sel sub-populations of Heliothis virescens.
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8.4 Discussion
The value of FA for a trait depends on its functional importance (Vishalakshi and Singh, 2009) and it has been argued that the wing characters are more closely associated with the fitness due to their role in locomotion (Clarke et al., 2000). Insect wing lengths are mostly used in FA studies due to ease with which they can be measured accurately (Mpho et al., 2002). Stress can disrupt the developmental stability of a trait and increase the FA level but traits may be vary in their sensitivity to specific stress factors (Polak et al., 2002). It is also reported that the magnitude of the stress over the time may have different effect on the FA value as well as on the fitness (Van Dongen, 2006).
The present study suggests that FA can be linked with resistance to Vip3A in H. virescens, with FA values for forewing, hindwing and tibia length being greater in the Vip3A-Sel sub- population than the Unsel sub-population. Despite the potential of FA as a method of studying fitness effects (Section 2.7), evidence linking FA with insecticide resistance has been limited to two species, the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) (Clarke and McKenzie, 1987; Clarke et al., 2000; Ribeiro et al., 2007). and the maize weevil, Sitophilus zeamais (Motsch) (Coleoptera: Curculionidae) (Ribeiro et al., 2007). The present results are similar to studies with L. cuprina, where insects resistant to organophosphates, had higher FA values and reduced fitness compared with susceptible insects (Clarke et al., 2000). Studies have also shown higher FA in the mosquitoes treated with the organophosphate insecticide temephos compared with control insects (Mpho et al., 2001). While studies showed that laboratory and field populations of H. armigera reared on Bt cotton displayed increased FA levels compared with populations reared on non-Bt crops (Li et al., 2004a) and Liu et al. (2005) reported similar findings for the aphid Aphis gossypii (Glover) (Hemiptera; Aphididae) feeding on Bt compared with non-Bt cotton.
FA can be affected by continuous selection pressure. In L. cuprina, initial selection with organophosphates led to higher levels of FA compared with unselected populations (McKenzie and Clarke, 1988) but continued selection led to a return to the FA levels found in susceptible populations (McKenzie and O'Farrell, 1994). Ribeiro et al. (2007) have reported that in the maize weevil, Sitophilus zeamais (Motsch) (Coleoptera: Curculionidae) insects resistant to deltamethrin had a lower FA value compared with susceptible insects. This may have been due to the evolution of a modifier allele that enhanced the fitness of resistant insects (Ribeiro et al., 2007). Investigations of FA in L. cuprina have shown a series of distinctive qualitative and quantitative shifts in FA between resistant and susceptible strains. 99
McKenzie and Clarke (1988) found that wild resistant populations that had been exposed to the organophosphate insecticide diazinon for many generations had no significant difference in FA compared with susceptible populations. Backcrossing indicated the presence of a modifier allele that both ameliorated the fitness costs of resistance and returned FA levels to those comparable with susceptible populations (McKenzie and O'Farrell 1994).These experiments revealed that the same modifier allele was responsible for the amelioration of fitness costs and FA in L. cuprina independently selected for malathion resistance (McKenzie and O'Farrell 1994). In addition, the modifier allele was specific to modifying the asymmetry repercussions from insecticide exposure, and analysis showed the allele to be dominant and independent acting against other stressors such as temperature and population densities (Freebairn et al., 1996).
Flight performance can affect the fitness of individual insects by influencing feeding, predation avoidance, dispersal, inter-male fighting outcomes and mate acquisition (Marden, 1989; Chai and Srygley, 1990; Coelho and Holliday, 2001; Marden and Cobb, 2004; Almbro and Kullberg, 2008; Samejima and Tsubaki, 2010). A decrease in the flight activity in butterflies (Colias spp and Pontia spp) was reported by Kingsolver and Srygley (2000) after an experimental reduction of FMR by 10-17%. Interspecific studies of tropical butterflies revealed that species with higher FMR showed faster flight and better manoeuvrability than the species with lower FMR (Pinheiro, 1996; Kingsolver and Srygley, 2000; Almbro and Kullberg, 2008). The lower FMR observed in the Vip3A-Sel sub-population of H. virescens provides indirect evidence that Vip3A resistant insects are poorer fliers compared with susceptible individual.
In the present study, the length of right and left forewing, right and left hindwing, and right and left tibia was smaller in each case for the Vip3A-Sel sub-population compared with the Unsel sub-population. These results were similar with previous studies in which the length of first, second and third instar was significantly smaller in a population of the whitefly Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae) selected with thiamethoxam compared with an unselected population (Feng et al., 2009).
Further studies are required to investigate the genetic changes responsible for the observed variations in morphological traits, FA value and FMR in Vip3A-Sel H. virescens, and their stability and significance in relation to the performance (fitness) of resistant compared with susceptible insects.
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CHAPTER 9: Summary and General Discussion
9.1 Summary of experimental findings
Chapter 4: Studies on cross-resistance, synergism and mechanisms of resistance
Cross-resistance to non-Bt insecticides was not found in a Cry1Ac-Sel sub- population of P. xylostella (Karak), in contrast to previous studies on Cry1Ac-Sel and deltamethrin-Sel sub-populations of another P. xylostella population (SERD4).
Cross-resistance to non-Bt insecticides was not found in the Vip3A-Sel sub- population of H. virescens (WF06).
PBO and DEF did not synergise the toxicity of Cry1Ac in a Cry1Ac-Sel sub- population of P. xylostella (Karak).
PBO did not synergise the toxicity of Vip3A in a Vip3A-Sel sub-population of H. virescens.
Resistance to PBO did not develop in P. xylostella (SERD4) when selected with PBO for 20 generations; a population where resistance to Cry1Ac and deltamethrin has been reported to be synergised by PBO.
Electrophoresis indicated that extracts of midguts of Vip3A-Sel H. virescens had an extra esterase band compared with Unsel midguts but there is no evidence either way regarding a link with Vip3A resistance.
Binding experiments with biotinylated Vip3Aa and BBMV from Unsel H. virescens WF06 suggested that the labelling reaction had inactivated the toxin‟s binding sites and studies are underway to optimize toxin labeling, prior to experiments to determine whether loss of binding is linked to Vip3A resistance in Vip3A-Sel H. virescens.
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Chapter 5: Sub-lethal effects of Vip3A on survival, development and fecundity of Heliothis virescens and Plutella xylostella
A sub-lethal concentration of Vip3A had the following effects on insecticide susceptible insects compared with untreated susceptible insects:
increased larval and pupal development time, pupal weight and reduced survival (neonate to adult emergence);
reduced mating pair success and reduced egg viability;
lower rm values;
increased fecundity (hormoligosis).
Chapter 6: Effect of temperature on the fitness of a Vip3A resistant sub- population of Heliothis virescens (WF06)
The fitness costs of resistance to Vip3A were temperature dependent; being greater at sub-optimal temperatures (high and low).
The survival (egg to adult), mating pair success, production of viable progeny, egg viability and fecundity of Vip3A resistant H. virescens was lower than susceptible insects at sub-optimal temperatures.
Chapter 7: Effect of entomopathogenic nematodes on the fitness of a Vip3A resistant sub-population of Heliothis virescens (WF06)
Bioassays with Steinernema carpocapsae and Heterorhabditis bacteriophora on 5th and 6th instar larvae of H. virescens increased the fitness costs of Vip3A resistance.
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Chapter 8: Fluctuating asymmetry, morphological changes and flight muscle ratio in a Vip3A resistant sub-population of Heliothis virescens (WF06)
Fluctuating asymmetry of morphological traits was greater in Vip3A resistant compared with unselected insects.
The flight muscle ratio was lower in Vip3A resistant compared with unselected insects.
9.2 General Discussion
The success of Bt spray products has been limited by their narrow spectrum (specificity), low persistence and higher production costs in comparison to synthetic chemical insecticides. The surface deposit achieved by Bt applied as sprays has to be ingested. While this can occur with foliar pests such as P.xylostella, it is less likely with bollworms such as H. virescens feeding on buds that are not sprayed directly due to the presence of bracts. The development and utilization of expression of Bt toxin genes in the plants has greatly increased the use of Bt. GM plants have the advantage over the microbial insecticide as the toxin is inside the plant and provides more persistent control and is effective against the first instar larvae. This has reduced the number of insecticide applications required, particularly on cotton (Ferré et al., 2008).
The high dose/refugia strategy for resistance management in Bt cotton and Bt maize has been largely successful in preventing or reducing the development of resistance to Cry toxins (Tabashnik, 2010; Section 2.11). Previous studies have suggested that there is little or no cross-resistance between Vip3A and Cry1Ab, Cry1Ac and Cry2A, including studies on Cry1Ac and Cry1Ab with Vip3A-Sel H. virescens WF06 (Jackson et al., 2007; Anilkumar et al., 2008; Pickett, 2009; Section 2.4). This lack of cross-resistance is probably due to the lack of sequence homology and different mode(s) of action for Vip and Cry toxins (Estruch et al., 1996; Yu et al., 1997; Lee et al., 2006). The present study found no evidence for cross-resistance between Vip3A and non-Bt insecticides in Vip3A- Sel H. virescens.
If the above findings are substantiated, including, when selected, for other Vip3A-resistant insect populations, then:
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1) Bt plants expressing a mixture of Cry and Vip toxins (e.g. VipCot®; Section 1.1) may prove effective in preventing the development of resistance to either toxin, and
2) Bt cotton expressing both types of toxin should be compatible with the use of non-Bt insecticides in refugia, as recommended in some Bt resistance management strategies by the US Environmental Protection Agency (Shelton et al., 2000; Tabashnik et al., 2009).
Synergists such as PBO are useful tools to help identify metabolic mechanisms of resistance in insects (Section 2.8). PBO has also been used widely in insect public health control as a synergist for insecticides (Section 2..8) and its use in microencapsulated formulations with insecticides has been proposed for field applications against agricultural pests (Bingham et al., 2007). The lack of effect of PBO observed on the toxicity of Cry1Ac in P. xylostella Karak in the present study is in contrast to work on the SERD4 population of this species (Sayyed et al., 2008). Similarly, the present studies with Vip3A-Sel H. virescens found that PBO applied either before or at the same time as Vip3A did not synergise toxin activity. This suggested that metabolic enzymes were not involved in Vip3A resistance. Whether loss of binding or other resistance mechanisms (Section 2.3) are involved in Vip3A resistance in H. virescens WFO6 is the subject of ongoing studies (Section 9.3). Crossing studies with this population have suggested that resistance is polygenic and thus could involve multiple resistance mechanisms (Pickett, 2009).
Individual insects that survive pesticide exposure may still be significantly affected by sub-lethal effects on their physiology and behaviour (Yin et al., 2008; Wang et al., 2008; Hamedi et al., 2010) and this can have a negative impact on insect population dynamics (Pineda et al., 2007, 2009; Knight and Flexner, 2007). The sub-lethal effects of such compounds are therefore an important consideration for estimating the total effect of a pesticide (Hamedi et al., 2010). In the present study, sub-lethal doses of Vip3A increased the developmental time (neonate to adult) of treated H. virescens and P. xylostela and reduced the rm compared with untreated controls. This suggests that sub-lethal doses of this toxin could exert selection pressure for resistance and this possibility should be tested experimentally.
Resistance management to insecticides is widely believed to depend in part on associated fitness costs; with the frequency of resistance alleles within a population declining in the absence of selection pressure (Tabashnik, 1994; Section 2.5), although some consider that even strong fitness costs have only a minimal impact on the evolution of resistance (Roush, 1998).
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Fitness costs can vary between different ecological and environmental conditions (Carrière and Tabashnik, 2001; Bird and Akhrust, 2004, 2007; Raymond et al., 2005, 2010; Gassmann et al., 2009). Host plant cultivar and specific plant allellochemicals have been shown to influence the magnitude and dominance of fitness costs associated with resistance to Bt (Carrière et al., 2005; Bird and Akhrust, 2007). High concentrations of secondary metabolites can, for example, affect insect survival and development. Tannin, a plant phenolic compound, has been shown to effect survival and larval development of H. virescens, and gossypol, a terpenoid aldehyde in cotton, can reduce survival and inhibit development in larvae of H. zea (Shaver et al., 1970; Chan et al., 1978). High concentrations of gossypol have shown to increase the fitness costs of Cry1Ac resistance in the pink bollworm, P. gossypiella (Carrière et al., 2004). The use of cultivars with high level expression of allelochemicals could therefore enhance the dominance of fitness costs and, provided they had no significant adverse effects, could be used in resistance management (Bird and Akhrust, 2007).
Temperature has been shown to influence the fitness costs associated with insecticide resistance in various insect populations. For example, in Nilaparvata lugens (Stal) (Homoptera: Delphacidae), resistant to imidachlorpid (Liu et al., 2004), Culex quinquefasciatus (Say) (Diptera: Culicidae) resistant to malathion (Swain et al., 2008), and P. xylostella resistant to spinosad (Yin et al., 2008). The lower rate of development, survival, egg viability and rm in the Vip3A-Sel sub-population of H. virescens compared with Unsel sub-population at sub-optimal temperatures was a particularly interesting finding in the present study. Further studies are, however, required (Section 9.3) to determine whether temperature-dependent fitness costs observed under laboratory conditions occur and if so whether they can be exploited for resistance management under field conditions; for example, by relaxation of selection pressure when fitness cost are greatest. The developmental asynchrony observed in the Vip3A-Sel and Unsel populations of H. virescens in the laboratory could, on the other hand, lead to assortative mating and a greater rate of development of resistance (Liu et al., 1999; Section 6.4). However, to best of my knowledge there are no documented reports that such asynchrony occurs under field conditions.
Expression of Bt toxins in plants should be at a sufficient level (e.g. for Cry toxins > 0.2% of total soluble protein in the appropriate tissue) to kill the heterozygous insects (Gatehouse, 2008). Factors that affect the expression of toxin in the Bt crops include plant age, reproductive stage and a variety of environmental factors (Benedicate et al., 1993, 1996; Wu et al., 1997), of which high temperature is probably the most important
105 factor that can reduce the efficacy of Bt crops (Chen et al., 2005) and is associated with a reduction in insecticidal proteins (Benedicate et al., 1996). Chen et al. (2005) reported that high temperature stress had a significant effect on the reduction in Cry toxin content in the leaf during boll development in Bt cotton. They related this to changes in nitrogen metabolism in cotton leading to reduced toxin production due to increased resource allocation for boll formation within the plant. For example, temperature and irradiance can trigger changes in the production of secondary metabolites that could interfere with the production of Bt toxin towards the end of the growing season (Llewellyn et al., 2007). Similarly, waterlogging and drought have been shown to reduce the expression of Cry toxin in leaves and squares of Bt cotton (Wu et al., 1997; Wang et al., 2001). The present work suggests that (seasonal) temperature effects on enhancing the fitness costs of Vip3A resistance may help counteract any negative correlation between temperature and the efficacy of Vip3A crops.
The use of fluctuating asymmetry (FA) estimates in research is increasing, but this has focused mostly on birds and amphibians. Swaddle and Witter (1994) reported that increased nutritional and energetic stress caused larger developmental asymmetries in the European starling. In great tits, tail feathers grown under high lead concentrations showed no change in FA (Talloen et al., 2008). Combined FA values of toe, leg, wing and feather in eleven breeds of chicken were greater in chickens kept under continous light compared with control chickens (Campo et al., 2007).
The greater fluctuating asymmetry of morphological traits observed in Vip3A-Sel sub- population of H. virescens compared with Unsel sub-population suggests greater developmental instability in the Vip-Sel sub-population; an observation in keeping with the developmental fitness costs found in the Vip3A-Sel sub-population. While the lower flight muscle ratio in the Vip3A-Sel sub-population compared with the Unsel sub-population may be reflected in significant differences in flight performance, although this possibility has not yet been tested directly (Section 9.3).
Various strategies have been deployed for delaying the development of resistance to Bt crops in insects. One strategy is the use of moderate toxin doses that ensure the survival of a proportion of susceptible insects. Two disadvantages of this strategy are that modelling suggests that it only results in a small delay in resistance compared with other strategies and that the efficacy of Bt crops at lower dose levels is variable (Roush, 1997). The principal strategy adopted since Bt crops were first grown commercially in 1996
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(Section 2.11) has a very different approach: the high dose / refuge strategy (Tabashnik, 1996; Ferré et al., 2008).
The high dose / refuge strategy has been used successfully in the USA and some countries but suffers from several disadvantages that make it far from ideal for long-term preservation of Bt crops (Bates et al., 2005). Studies have shown that one or more assumptions of the high dose / refuge strategy (Section 2.11) may be violated in some strains of P. gossypiella, O. nubilalis, H. virescens H. zea, P. xylostella and L. decemlineata (Frutos et al., 1999; Li et al., 2001; Akhurst et al., 2003; Burd et al., 2003). In addition to this, the debate on the refuge size and the grower‟s compliance may also affect the efficacy of this strategy, and in developing countries farmers with small holdings cannot devote some of their land to a refuge.
An alternative approach, therefore, has seen an increase in GM crops expressing two or more Cry genes (= pyramidid genes; Chen et al., 2010; ISAAA, 2010). It is suggested that this is best way to delay the resistance provided the different toxins expressed have different binding sites and do not exhibit cross-resistance to the other toxin(s) (Roush, 1998; Bates et al., 2005). However, studies have suggested that this multiple toxin strategy may not be a cure all solution due to the potential development of multiple toxin resistance in the field. For example, a population of H. virescens has been selected in the laboratory for resistance to both Cry1Ac and Cry2Aa (Gahan, et al., 2005; Gatehouse, 2008). The development of pyramiding a Cry protein with a non-Bt insecticidal protein is a relatively new approach to delay the resistance. For example, the co-expression of Cry1A genes and the cowpea trypson inhibitor gene in cotton (Ferré et al., 2008).
Plant volatiles offer other possibilities for new methods of crop protection (Schnee et al., 2006). For example, the volatile composition of the tobacco plant has been altered by RNA interference-mediated suppression of a cytochrome P450 oxidase gene expressed in trichomes (Wang et al., 2001a; Gatehouse, 2008). The developments of such new technologies could provide a better choice to growers and relieve the pressure on the existing strategies (Llewellyn et al., 2007).
In the high dose/refugia strategy, heterozygous recessive or partially recessive resistant individuals are expected to die if they feed on Bt crops expressing a high dose of toxin (Tabashnik et al., 2004). With time, however, the production of homozygous resistance individuals that can survive a high toxin dose may increase (Sisterson et al., 2004; Gassmann et al., 2006; Franklin and Myers, 2008) and resistance management may start
107 to fail. This possibility has spurred the search for other techniques to be used in conjunction with the high dose/refugia system, including the use of entomopathogens such as baculoviruses, fungi and nematodes.
The application of pathogens as a biopesticides alone or in combination with Bt plants to control pests has been examined in various studies (Johnson et al., 1997; Baur et al., 1998; Nahar et al., 2004; Raymond et al., 2007). Insect pathogens are known to enhance the fitness costs of Bt resistance (Section 7.4). For example, Raymond et al. (2007) reported that a mixture of NPV and Bt reduced the fitness of Bt-resistant P. xylostella compared with non-Bt insects. While Gassmann et al. (2006) and the present study have shown that entomopathogenic nematodes can increase the fitness costs of Bt resistance in P. gossypiella and H. virescens, respectively. However, entomopathogenic nematodes are normally found in the soil and require particularly favourable environmental conditions and application methods to be effective against above-ground pests (Wright et al., 2005; Georgis et al., 2006). Studies on the effectiveness of entomopathogenic nematodes have highlighted their limitations in the field as they require special conditions to be effective. Desiccation and ultraviolet light can readily kill entomopathogenic nematodes and they are also often only effective in a narrow temperature range (Georgis et al., 2006). Further studies are required to determine if nematodes (and other pathogens) can be optimised sufficiently for use under field conditions with Bt crops.
Tabashnik et al. (2010a) have reported a new approach that replaces the refuge strategy with the release of sterile insects throughout season for the control of pink bollworm in Arizona. According to this approach, resistant insects mate with sterile insects rather than the resistance and susceptible fertile insects and do not reproduce. This strategy has two key advantages over the refuge strategy: (i) farmers can reduce or eliminate planting of refuges and thus increase yield; (ii) mating with the sterile insects will delay the development of resistance whether resistance is recessive or dominant (Tabashnik et al., 2010a). According to computer simulation models, a sterile insect release strategy of 0.62 sterile insects per ha per week in Bt cotton without refugia resistance would prevent the development of resistance over a 20 year period.
Bates et al. (2005) reported the different factors (Fig 9.1) that affect the efficacy of integrated resistance management for genetically modified crops and studies suggest that traditional aspects of IPM, including pest monitoring, cultural and biological control can be important for delaying the development of resistance to Bt crops (Fitt, 1989, 2000).
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Figure 9.1 Factors that affect the efficacy of IRM in GM crops (Bates et al., 2005).
9.3 Future work
The present work has provided information on cross-resistance, synergism and fitness costs in a Vip3A-Sel sub-population of H. virescens and the effects of sub-lethal concentrations of Vip3A toxin on H. virescens and P. xylostella. Future work includes studies on the molecular and biochemical basis for the mechanism(s) conferring resistance to the Vip3A, further studies on cross-resistance and an evaluation of fitness costs associated with resistance under field-related conditions.
1. Serial analysis of gene expression (SAGE) in the midgut tissues of toxin-exposed and unexposed Vip3-Sel and Unsel sub-populations of H. virescens larvae). [In progress - with C. Ayra-Pardo, Havana]
2. Bioassays with RNA interference-mediated suppression of genes identified by SuperSAGE (1) to have have a regulatory pattern that correlates with interactions
109
between Vip3A and the larval midgut in resistant insects. [To follow SAGE studies]
3. Cross-resistance studies between Vip3A and other Bt toxins that are currently used in Bt crops.
4. Further binding studies with biotinylated Vip3A toxin to determine the role (if any) of binding in resistance to Vip3A in H. virescens WF06 and to look at interactions between Vip3A and other Bt toxins. [In progress - with J. Ferré, Valencia]
5. Studies on the effects of Bt-cotton expressing Vip3A and Cry1Ab (VipCot®) and Cry1Ac and Cry2Ab (Bollargard2®) on the survival of Vip3A resistant H. virescens over multiple generations.
6. Studies on the sub-lethal effects of Bt toxins on the selection and maintenance of resistance.
7. Investigate fitness costs linked to development and reproduction in the Vip3A resistant sub-population of H. virescens over several generations in large scale cage experiments and investigate possible effects on flight behavior directly in wind tunnels and in flight mills.
8. Investigate fitness costs in Vip3A resistant H. virescens on different plant hosts and varieties and the effects of specific plant secondary compounds, including gossypol and phytic acid. [Preliminary studies in progress]
9. Further studies on the effects of entomopathogens (nematodes, NPV and fungi) on fitness costs of Vip3A resistance, conducted under conditions more applicable to the field, and the potential for such interactions in resistance management investigated.
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APPENDICES
Appendix 1 Toxicity of PBO against Unsel and Vip3A-Sel sub-populations of H.virescens: topical bioassay; n = 48.
PBO % (µg / ml) Mortality
Unsel 0 0
Unsel 6250 2
Unsel 12500 2
Unsel 25000 8
Unsel 50000 12
Unsel 100000 22
Vip3A-Sel 0 0
Vip3A-Sel 6250 2
Vip3A-Sel 12500 2
Vip3A-Sel 25000 6
Vip3A-Sel 50000 16
Vip3A-Sel 100000 20
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Appendix 2 Toxicity of PBO against SERD4 Unsel population of P. xylostella (early 4th larval instar): topical bioassay; n = 30.
% Mortality PBO (µg/ml) Day 3 Day 5 Eclosion ------0 10 13 20 100 30 43.3 46.7 300 30 46.7 46.7 1000 36.6 53.3 63.3 3000 53.3 76.7 86.7 10000 66.6 90 93.3
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Appendix 3 Toxicity of PBO against SERD4 Unsel population of P. xylostella (Late 4th larval instar): topical bioassay; n = 30.
% Mortality PBO (µg/ml) Day 3 Day 5 Eclosion ------0 10 23.3 10 100 13.3 23.3 43.3 300 23.3 33 43.3 1000 26.7 36.7 50 3000 36.7 66.7 70 10000 56.7 83.3 86
150