Busseola Fusca (Lepidoptera: Noctuidae) Moth and Larval Behaviour in Bt- and Non-Bt Maize: an IRM Perspective

Busseola fusca (Lepidoptera: Noctuidae) moth and larval behaviour in Bt- and non-Bt maize: an IRM perspective

A Visser orcid.org / 0000-0003-3027-702X

Thesis accepted for the degree Doctor of Philosophy in Environmental Sciences at the North-West University

Promoter: Prof J van den Berg Co-promoter: Prof MJ du Plessis Assistant Promoter: Dr A Erasmus

Graduation May 2020 2236614

Draco dormiens nunquam titillandus.

ACKNOWLEDGEMENTS

This study was funded by the National Research Foundation of South Africa.

(UID: 116569)

To Mabel du Toit, Elrine Strydom, and the whole team at the Entomology department at the Agricultural Research Council – Grain Crops Institute (ARC-GCI): thank you for the opportunity to use your facilities and your assistance with the trials that were conducted there. My appreciation, however, does not extend to the mobs of marauding guineafowl who wrecked our field trials by digging up half the maize seeds planted. Twice.

A special word of thanks to Dr. Annemie Erasmus of ARC-GCI for the guidance and encouragement throughout my PhD, but especially during the glasshouse and field trials. Without your expert advice and ingenious solutions to challenges that seemed insurmountable at the time, this study would not have been completed.

To all my fellow post-graduate students at the EcoRehab Centre… it was great sharing a laboratory and a Spur bench with all of you during the last four years. Thanks for all your help – Terése Du Plessis in particular, who also shared the long journeys to fieldwork sites with me.

My hart loop oor van dankbaarheid teenoor my wonderlike familie en vriende, wat my gehelp het om die graadkry met lekkerkry klaar te kry. Aan my ouers: baie dankie vir julle volgehoue ondersteuning en aanmoediging. Niks is ooit te veel gevra nie… Ek is julle ewig dankbaar vir die geleenthede wat julle vir my moontlik gemaak het! En aan my gunsteling mens…dankie dat jy altyd daar was vir my, al het hulle Kathu so vêr gebou.

These people have all made unmissable contributions to this study (either directly or indirectly), but none more so than my two supervisors: Prof Johnnie van den Berg and Prof Hannalene Du Plessis. Prof J and Prof T – thank you for going above and beyond what could ever reasonably be hoped for from a PhD supervisor. More than simply providing expert guidance and advice, you take a genuine interest in the development, future and wellbeing of each and every student you mentor. What an honour and a privilege it has been to work with and learn from you. Thank you for your encouragement, guidance, patience…for all the long (mostly unseen) hours that you devote to the research unit. You’ve had a greater impact on us, your students, than you could ever know.

Laastens – alle eer aan God, ons Skepper en Heer. Psalm 46: 1-12.

ABSTRACT

The African maize stemborer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is one of the most damaging pest species of maize in Africa. Genetically modified (GM) Bt maize that expresses insecticidal Cry proteins could soon be a primary control method for this pest on the continent, since, over the past few years, several African countries have been conducting regulatory field trials that are required for approval of GM crops for commercial release. However, since the sustainability of the Bt maize technology is threatened by the evolution of resistance by pest populations, development of insect resistance management (IRM) strategies such as the high- dose/refuge (HDR) strategy are required. Current HDR strategies require the expression of Bt toxins in a dose high enough to kill heterozygous-resistant individuals, as well as a source of non- Bt host plants (refuge area) near the Bt field, which acts as a source of homozygous susceptible target pest individuals. The refuge area can be structured (e.g. blocks or strips of non-Bt plants within the Bt field) or unstructured, where a blend of non-Bt and Bt plants (seed mixture) is planted within a single field. The functioning of the HDR strategy is based on the validity of several key assumptions about the biology and behaviour of the target pest species. The rapid evolution of resistance to Bt toxins in B. fusca populations in South Africa demonstrated the necessity for deployment of an effective IRM strategy. However, the design of an IRM strategy is complicated by the heterogenous nature of the agricultural systems in Africa, which makes the implementation of a standardized, universal IRM strategy impossible. Although smallholder farmers find it challenging to implement separate refuges due to their limited scale of production, the use of seed mixtures is not an appropriate strategy to delay resistance evolution in pests with highly mobile larval stages. Therefore, an effective IRM strategy must take into consideration both the practical limitations of the agricultural system that it intends to serve, as well as the behaviour of target pest species (especially oviposition and larval migration behaviour). However, when the target pest forms part of a mixed population of pest species, for example B. fusca, Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), the adjustment of the IRM strategy to local conditions becomes complicated. Even closely related pest species generally differ in various biological and behavioural aspects. The aim of this study was therefore to investigate oviposition preference and larval migration behaviour of B. fusca in Bt and non-Bt maize, to review aspects of the biology and ecology of B. fusca, which occur in mixed populations, and develop a synthesis on the possible impact of mixed pest populations on insect resistance management for Bt maize in Africa. Oviposition preferences of moths and feeding preferences of larvae, when offered a choice between Bt and non-Bt maize, were investigated for both resistant and susceptible B. fusca populations in laboratory bioassays. Additionally, larval migration behaviour and the effect of Bt toxin, plant density, and plant age on

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this behaviour were evaluated in laboratory, semi-field (glasshouse), and field experiments. Results indicated a lack of oviposition preference in B. fusca between undamaged Bt and non-Bt maize, which suggests that separate refuges can be implemented in an IRM strategy against this species (assuming a single-species pest population of B. fusca and the presence of ample refuge plants). In contrast, larvae displayed feeding avoidance behaviour on Bt maize, suggesting that the larvae migrate more readily off Bt maize plants. However, the field trials conducted during this study indicated that, although B. fusca larvae do migrate between plants, the migration distance is limited and survival is very low (even in pure stands of non-Bt maize). This contradicts earlier accounts which erroneously reported significant larval movement between plants. This thesis also concluded that the use of separate refuges as an IRM tactic against mixed populations in smallholder Bt maize fields in Africa would be unwise, since the domination of the refuge by a single species will undermine the efficacy of the IRM strategy for another pest species. This synthesis concludes that no single, generic, standardized IRM strategy can be prescribed across the different agroecological zones of Africa. The dissimilarity between the African agricultural regions necessitates a tailored approach to IRM and development of strategies for each region/agro-ecological zone to ensure that it meets the needs of the farmers that are tasked with its implementation. Future research should focus on generating data required for use in models to improve IRM strategies for specific regions and agricultural systems.

Key words: Bt maize, stem borers, insect resistance management

PREFACE

This thesis follows the article style format. In accordance with the prescriptions of the North-West University (NWU), the articles appear in published format and manuscripts were prepared following the instructions to authors of internationally accredited, scientific journals (Table A). Chapters that are not intended to be published were prepared following the general guidelines for theses and dissertations set out by the NWU.

Table A. Publication status of articles contained in this thesis.

Chapter Status Journal

Chapter 1 - NWU general guidelines

Chapter 2 Article 1: Published Entomologia Experimentalis et Applicata (Wiley)

Chapter 3 Article 2: Published Insects (MDPI)

Chapter 4 Article 3: Published Insects (MDPI)

Chapter 5 Article 4: Submitted Integrated Pest Management (Oxford Academic)

Chapter 6 - NWU general guidelines

The instructions to authors of the journals (unpublished articles) are included as Appendix A-B. Permission was obtained from Wiley and Sons to present Article 1 as part of this thesis. The licence and associated terms and conditions are available in Appendix C. The copyright and licensing regulations for Insects are provided in Appendix D.

Table B details the contributions of authors for each article/manuscript and provides consent for use as part of this thesis.

Table B. Contributions of authors and consent for use of article/manuscript in thesis.

Author Contribution Consent

Ms. A. Visser Article 1 - 3 Principle investigator • Study conceptualization • Study design • Execution of experiments • Data analysis • Data interpretation • Writing of manuscripts

Article 4 First author. • Study conceptualization • Review of literature • Writing of manuscript Prof. J. Van den Berg Article 1 - 3 Primary supervisor Contributed to the conceptualization of the study and supervised the design and execution of the experiments. Provided guidance and intellectual input on data analysis and writing of manuscripts.

Article 4 Co-author Contributed to the conceptualization of the study, review of literature and writing of the manuscript. Prof. M.J. Du Plessis

Article 1 - 3 Co-supervisor Contributed to the conceptualization of the study and supervised the design and execution of the experiments. Provided guidance and intellectual input on data analysis and writing of manuscripts. Dr. A. Erasmus Article 1 - 3 Co-supervisor Contributed to the conceptualization of the study and supervised the design and execution of the experiments. Provided guidance and intellectual input on data analysis and writing of manuscripts.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... II

ABSTRACT ...... III

PREFACE ...... V

CHAPTER 1 ...... 11

INTRODUCTION, LITERATURE REVIEW, AND THESIS STRUCTURE...... 11

1.1 Introduction ...... 11

1.2 Research aim and objectives ...... 14

1.2.1 Study aim ...... 14

1.2.2 Study objectives ...... 14

1.2.3 Hypotheses ...... 15

1.3 Literature review ...... 15

1.3.1 The local and global significance of maize ...... 15

1.3.2 Busseola fusca: taxonomy, biology and pest status on maize in SA ...... 21

1.3.3 Bt maize and genetically engineered crops ...... 28

1.3.4 Evolution of resistance to Bt crops ...... 38

1.3.5 Insect resistance management ...... 41

1.3.6 Resistance evolution of Busseola fusca to Bt maize in South Africa ...... 49

1.4 Structure of the thesis ...... 52

1.5 Bibliography...... 53

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CHAPTER 2 ...... 78

PREFERENCE OF BT-RESISTANT AND SUSCEPTIBLE BUSSEOLA FUSCA MOTHS AND LARVAE FOR BT AND NON-BT MAIZE

CHAPTER 3 ...... 98

PLANT ABANDONMENT BY BUSSEOLA FUSCA (LEPIDOPTERA: NOCTUIDAE) LARVAE: DO BT TOXINS HAVE AN EFFECT?

CHAPTER 4 ...... 109

LARVAL MIGRATION BEHAVIOUR OF BUSSEOLA FUSCA (LEPIDOPTERA: NOCTUIDAE) ON BT AND NON-BT MAIZE UNDER SEMI-FIELD AND FIELD CONDITIONS.

CHAPTER 5 ...... 134

BIGGER, FASTER, STRONGER: A REVIEW OF THE IMPLICATIONS OF BIOLOGY, COMPETITION AND INTER-SPECIES INTERACTIONS WITHIN MIXED POPULATIONS OF LEPIDOPTERAN PESTS FOR BT MAIZE IRM IN AFRICA.

4.1 Abstract ...... 135

4.2 Introduction ...... 135

4.3 Part 1: Competition between B. fusca, C. partellus, and S. frugiperda ..... 139

4.4 Factors that affect the competitiveness of a species in mixed populations:

4.4.1 Distribution range ...... 143

4.4.2 General development time and species voltinism ...... 144

4.4.3 Diapause and off-season survival strategies ...... 148

4.4.4 Larval population density ...... 149

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4.4.5 Cannibalism and predatory behaviour ...... 150

4.4.6 Part 1: Summary ...... 151

4.5 Part 2: Validity of IRM assumptions for B. fusca, C. partellus and S. frugiperda ...... 151

4.5.1 Initial resistance allele frequency, fitness cost and incomplete resistance ...... 151

4.5.2 Recessive inheritance of resistance (high-dose requirement of toxin expression) ...... 154

4.5.2.1 Multi-toxin events (pyramids) ...... 154

4.5.3 Abundant refuges of non-Bt host plants ...... 156

4.5.3.1 Naturally occurring refuges ...... 156

4.5.4 Seed mixtures for IRM in smallholder farming systems ...... 158

4.5.5 Part 2: Summary ...... 161

4.6 Part 3: IRM for single and mixed populations of Lepidoptera maize pests in Africa ...... 161

4.6.1 IRM in the context of IPM ...... 163

4.7 Conclusions ...... 163

4.8 References ...... 165

CHAPTER 6 ...... 188

CONCLUSIONS AND RECOMMENDATIONS ...... 188

5.1 Study outcomes & recommendations for future research ...... 188

5.1.1 Hypothesis 1: The oviposition preference and behaviour of gravid B. fusca females will favour the use of separate refuges as an IRM strategy for Bt maize in Africa...... 188

5.1.1.1 Recommendations for future research: ...... 189

5.1.2 Hypothesis 2: The larval migration behaviour of B. fusca will preclude the use of seed mixtures as an IRM strategy for Bt maize in Africa...... 189

5.1.2.1 Recommendations for future research: ...... 190

5.1.3 Hypothesis 3: Mixed populations of pest species will necessitate the tailoring of the IRM strategy to suit the conditions of specific agricultural regions...... 190

5.1.3.1 Recommendations for future research: ...... 190

5.2 Conclusions ...... 191

5.3 References ...... 192

APPENDIX A ...... 194

INSTRUCTIONS TO AUTHORS: INSECTS

APPENDIX B ...... 200

INSTRUCTIONS TO AUTHORS: JOURNAL OF INTEGRATED PEST MANAGEMENT

APPENDIX C ...... 203

JOHN WILEY AND SONS LICENSE

APPENDIX D ...... 208

CHAPTER 1

Introduction, literature review, and thesis structure

1.1 Introduction

Maize, Zea mays (L.) (Poaceae), is a cornerstone of both commercial and subsistence agriculture in Africa (Gouse et al., 2005; Ranum et al., 2014) since it is the staple food of more than 300 million people in the sub-Saharan region (Shiferaw et al., 2011; Okweche et al., 2013). The Food and Agriculture Organization of the United Nations (FAO-ECA, 2018) indicated that agricultural productivity on the continent will have to increase significantly to ensure food security for the growing population (World Bank, 2008; Ray et al.2013, FAO, 2015)

Insect pests are an important limiting factor of maize yields in African agriculture, and the African maize stemborer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is considered one of the most damaging species (Kfir et al., 2002; Tounou et al., 2010). Stemborers such as B. fusca are difficult to control by means of insecticide application, due to their cryptic nature (Wale et al., 2006), and because chemical insecticides and the equipment needed for the safe application thereof, are often lacking in many of the agricultural regions (Sylvain et al., 2015; Tefera et al., 2016). Consequently, biotech crops that are modified to express insecticidal Cry proteins from Bacillus thuringiensis Berliner (Bt) may in future play an important role in sustainable food production on the continent (FAO, 2009; Conceição et al., 2016; ISAAA, 2017; Smyth, 2017; Carzoli et al., 2018). Bt crops have been shown to reduce local pest populations yield losses where these crops are cultivated (Brookes and Barfoot, 2018). Additionally, this technology could lead to area-wide suppression of pest populations (Hutchison et al., 2010; Dively et al., 2018) and a decrease in the use of insecticides that are harmful to both humans and natural enemies of target pests (Naranjo, 2009; Brookes and Barfoot, 2013; Tefera et al., 2016). Bt maize also limits the formation of mycotoxic fungal infections by decreasing the degree of larval feeding damage inflicted to maize ears (Munkvold et al., 1999; Hellmich et al., 2008; Pellegrino et al., 2018).

The foremost threat to the sustainability of Bt maize technology is the evolution of resistance by pest populations (Tabashnik, 1994a; Gould, 1998; Gassmann et al., 2009; Carrière et al., 2010). The widespread use and efficacy of Bt crops result in high and continuous selection pressure for resistance evolution throughout the growing season (Tabashnik, 1994b; Glaser and Matten, 2003; Bourguet et al., 2005; Siegfried and Hellmich, 2012; Siegfried and Jurat-Fuentes, 2016). The threat that insect resistance evolution poses to the sustainability of Bt crops prompted the development of insect resistance management (IRM) strategies and the deployment of these

measures from the first commercial release of these crops (Gould, 1998; Tabashnik and Carrière, 2017). Although several IRM strategies have been put forward, the high-dose/refuge (HDR) strategy is the most widely applied (Gould, 1998; Bourguet et al., 2005; Tabashnik et al., 2013). This strategy requires the expression of Bt toxins in a dose high enough to kill heterozygous- resistant individuals, and a source of non-Bt host plants (refuge area) near the Bt field, which acts as a source of homozygous susceptible target pest individuals (USEPA 1998). The large numbers of homozygous susceptible individuals are then the primary mates for the rare homozygous resistant individuals that survive on the Bt field, giving rise to predominantly heterozygous offspring. This leads to an overall reduction in the frequency of resistance alleles in the pest population (Gould, 2000; Bates et al., 2005; Tabashnik et al., 2009; Tabashnik and Carrière, 2017). The non-Bt refuge area can be structured in several ways, e.g., in blocks or strips of non- Bt plants within the Bt field, as a separate field, or as borders around Bt maize fields (Cullen et al., 2008). An alternative approach is the use of seed mixtures (also known as the refuge-in-a- bag strategy or seed blends), where a bag of seed contains a mixture with a predetermined ratio of non-Bt and Bt seed (Gould, 1998; Carrière et al., 2016). This effectively eliminates the need to plant a separate non-Bt refuge, since the non-Bt plants are included randomly within the Bt field (Onstad et al., 2011, 2018).

Notably, this generic strategy is based on several key assumptions about the biology and behaviour of the target pest species and insect-plant interactions, and violation of these assumptions will lead to rapid evolution of resistance in the pest population (Carrière et al., 2004; Tabashnik et al., 2009; Tabashnik and Carrière, 2017; Onstad et al., 2018, Anderson et al., 2019). This was the case in South Africa, where the first report of B. fusca populations resistant to Bt maize that expresses Cry1Ab protein (event MON810) was made a mere 8 years after commercial release of Bt maize in the country (Van Rensburg, 2007). Subsequent investigations highlighted several factors that contributed to the evolution of resistance in B. fusca, which included the violation of certain assumptions on which the HDR strategy are based (Kruger et al., 2012a; Campagne et al. 2013; Van den Berg et al., 2013; Van den Berg, 2017). By 2010, widespread resistance occurred throughout the maize production areas, despite the implementation of IRM strategies to counter the spread of resistance (Van den Berg et al., 2013). Currently, the producers in South Africa rely on the multi-toxin (pyramided) maize event MON89034, which simultaneously expresses the Bt proteins Cry1A.105 and Cry2Ab2 (Strydom et al., 2018). The latter event was approved for commercial release in South Africa during 2011. This is the only pyramid event released commercially in South Africa and it is still effective at controlling MON810-resistant B. fusca populations after nearly 8 years of commercialization.

At present, the cultivation of Bt maize is limited to only one country on the African continent – South Africa. However, several other African countries are currently conducting trials with the aim of releasing GM crops in their agricultural sector in the near future (ISAAA, 2017).

The rapid evolution of resistance of B. fusca populations in South Africa demonstrated that an effective IRM strategy is crucial to ensure the sustainable use of Bt maize in other African countries (Van den Berg et al., 2013). However, the design of an IRM strategy for the agricultural systems on the continent is complicated by several factors. Agriculture in Africa is practiced mostly on smallholder farming plots, although commercial farming systems are present in some areas (Thompson, 2008; World Bank, 2008; Aheto et al., 2013). This heterogenous nature of the agricultural system makes the implementation of a standardized, universal IRM strategy impossible (Jacobson and Myhr, 2012; MacIntosh, 2009). Unlike commercial-scale farmers in developed countries (for which the IRM strategies were first devised), smallholder farmers find it challenging to implement separate refuges due to their limited scale of production (Bates et al., 2005; Carroll et al.2012; Van den Berg, 2013; Li et al., 2017).

It has been suggested that the use of seed mixtures could be a feasible substitute to planting separate refuges in smallholder agricultural systems (Carroll et al., 2012; IRAC, 2013). However, the use of sed mixtures is limited since this strategy is not appropriate for use to delay resistance evolution by target pests with highly mobile larval stages. Migrating larvae in a field that contains both Bt and non-Bt plants could be exposed to sub-lethal doses of Bt toxins, accelerating resistance evolution (Davis and Onstad, 2000; Heuberger et al., 2011; Ives et al., 2011, Razze and Mason, 2012). An effective IRM strategy must therefore take into consideration both the practical limitations of the agricultural system that it intends to serve, as well as the behaviour and biology of the target pest species (Gould, 1998; Head and Greenplate, 2012; IRAC; 2013). Yet, when the target pest forms part of a mixed population of pest species, the adjustment of the IRM strategy to local conditions becomes difficult, since even closely related pest species generally differ significantly in various biological and behavioural aspects (Roush, 1997; Bates et al., 2005). Busseola fusca forms part of the maize stemborer complex that includes several other species such as Chilo partellus (Swinhoe) (Crambidae), Sesamia calamistis (Hampson) (Noctuidae), and Eldana saccharina (Walker) (Pyralidae) (Kfir et al., 2002; Calatayud et al., 2014; Assefa et al., 2006, 2009, 2015; Ntiri et al., 2019). These stemborers, along with other lepidopteran maize pests such as the invasive Spodoptera frugiperda (J.E. Smith) (Noctuidae), are often found in mixed populations in maize fields, with prevailing climatic conditions in te different agro-ecological zones, largely determining lepidopteran species community that attack maize in the region (Ntiri et al., 2019).

Since B. fusca has already shown its proficiency in evolving resistance to Cry1Ab toxin, an effective IRM strategy is needed to ensure the sustainable use of Bt maize in African countries that plant to adopt this agricultural technology. This IRM strategy will need to be tailored to fit not only the biology and behaviour of the main target pest species (B. fusca) (Gould, 1998; Head and Greenplate, 2012; IRAC; 2013), but also that of other lepidopteran pests that occur in mixed populations with B. fusca (Roush, 1997; Bates et al., 2005). When the heterogeneity of African agricultural systems is considered, it is evident that the IRM strategy for Bt maize in Africa will have to be tailored to the specific receiving environment and local conditions of each agricultural region (MacIntosh, 2009: Head and Greenplate, 2012). For this to be possible, information regarding the biology and behaviour of this pest species on Bt and non-Bt maize, in both single and mixed pest populations, is required. The behavioural traits of B. fusca that are especially relevant to refuge design (and for which data are currently lacking) are the oviposition preferences of moths and feeding preferences of larvae when offered a choice between Bt and non-Bt maize, as well as the subsequent larval migration behaviour. This study will aim to generate relevant information to address this gap in our current knowledge.

1.2 Research aim and objectives

1.2.1 Study aim

To investigate and review how the oviposition preference and larval migration behaviour of B. fusca and other lepidopteran pest species of maize in pure and mixed stemborer populations impact insect resistance management for Bt maize in Africa.

1.2.2 Study objectives

1. Establish breeding colonies of field collected Bt-resistant and Bt-susceptible populations of B. fusca.

2. Compare the oviposition preferences of Bt-resistant and Bt-susceptible populations of B. fusca for Bt and non-Bt maize plants.

3. Determine feeding preferences of Bt-resistant and Bt-susceptible larvae of B. fusca for Bt and non-Bt maize plants.

4. Evaluate the effect of Bt-toxins on plant abandonment behaviour of neonate B. fusca larvae of Bt-resistant and Bt-susceptible populations.

5. Evaluate the effect of plant density and plant age on the larval migration behaviour of Bt- resistant and Bt-susceptible populations of B. fusca. 14

6. Review information on inter- and intra-species interactions and provide a synthesis that compares possible scenarios of mixed populations of lepidopteran maize pests and how the population dynamics in these scenarios influence the implementation of IRM strategies in African smallholder farming systems.

1.2.3 Hypotheses

The following hypotheses were postulated:

1. Oviposition preference and behaviour of gravid B. fusca females will favour the use of separate refuges as an IRM strategy for Bt maize.

2. B. fusca larval migration behaviour will preclude the use of seed mixtures as an IRM strategy for Bt maize.

3. Mixed populations of pest species will necessitate the development of IRM strategies that suit the conditions in the receiving environment.

1.3 Literature review

1.3.1 The local and global significance of maize

1.3.1.1 Maize as an agricultural crop

Maize is a cereal grain that was first domesticated in current day Mexico between 7 000 and 10 000 years ago (Benz, 2001; Ranum et al., 2014). Since its humble beginnings as domesticated grass, this crop is now a staple food for more than 200 million people (Du Plessis, 2003), many of which live in sub-Saharan Africa (Schimmelpfennig et al., 2012). Maize has a greater annual total production (1.06 billion tonnes) than either that of wheat (749 million tonnes) or rice (741 million tonnes) (UN, 2017a). However, not all maize that are produced are used as food for humans.

Several different varieties of maize exist, each with its distinct agronomical requirements, kernel characteristics and uses. Common examples include dent-, flint-, sweet- and popcorn maize varieties (Abbassian, 2006). Maize can broadly be categorised as either yellow or white. The bulk of maize production and international trade comprises yellow maize, which is traditionally used as animal feed. White maize, on the other hand, is considered a food crop and is produced and consumed in only a handful of countries, i.e. the United States, Mexico and in several countries in east and southern Africa (Abbassian, 2006; Ranum et al., 2014).

In developed countries, maize is mostly consumed as second-cycle produce in the form of meat, eggs, and dairy products, since these products are produced by maize fed animals (Du Plessis, 2003; Ranum, 2014). Maize can also be processed into a variety of industrial products, e.g. starch, sweeteners, oil, beverages, glue, industrial alcohol, and fuel ethanol (Ranum et al., 2014). Currently, approximately 40% of the maize produced in the United States of America (USA) are used to produce fuel ethanol, and this percentage is steadily increasing (Kline et al., 2017).

1.3.1.2 Economic importance of maize

1.3.1.2.1 In the global market

Global maize production in 2017 was estimated at 1.13 billion tonnes, a 7.3% increase from the 2015 season (FAO-ECA, 2018). The three largest producers of maize are the USA (35%), China (23%), and Brazil (7.5%) (Ranum et al., 2014; FAO-ECA, 2018). The strong economic growth in developing economies in recent years has increased maize consumption by an average of over 4% annually since the early 2000’s (OECD-FAO, 2018), a trend that is expected to continue due to the increased use of maize for animal feed in China and the European Union (EU) (OECD- FAO, 2018).

1.3.1.2.2 The South African market

At first glance, South Africa’s (SA) agricultural sector contributes a small percentage (2.5% in 2008 – Goldblatt, 2010) to the GDP of the country. However, when the strong forward and backward linkages into other sectors of the economy (e.g. manufacturing) are considered, the actual contribution is approximately 14% (Goldblatt, 2010). Additionally, the agricultural sector plays a major role in employment and earning of foreign exchange. According to the World Bank (2017a), the agricultural sector accounted for 5.6% of the total employment in SA in 2017. In 2015, Grain SA published an article by Greyling (2015), which indicated that the agriculture sector employed 4.6% of the total labour force in the country, despite accounting for only 2.5% of the country’s GDP. Compared to the mining and manufacturing sectors, which represented 8,5% and 12,5% of the economy while employing only 2,3% and 11,8% respectively of the labour force, agriculture is a labour-intensive sector (Greyling, 2015).

The total value of the maize produced fduring the 2017/2018 cropping season accounted for 9.2% of the total agricultural value for that season (DAFF, 2017). Maize export demands exceed 2.5 million tonnes in high yielding years, as was seen in 2013 and 2014, when maize exports contributed R7.3 billion (2.6 million tonnes) and R6.5 billion (2.1 million tonnes) to the overall value of agricultural exports from South Africa (UN, 2017a). The export values for 2015 reflect the devastating effect of the worse drought to hit SA since 1904 (SAWS, 2017), and only 762 242 16

tonnes of maize were exported to a value of R2.6 billion (UN, 2017a). However, by 2017, the maize exports recovered to pre-drought levels (2.19 million tonnes to the value of R6.29 billion) (Figure 1-1). The annual maize consumption in SA varies between 11 and 12.5 million tonnes and is generally stable (Grobler et al., 2017).

Figure 1-1 Value (in million Rand) and quantity (thousand tonnes) of maize exported from South Africa from 2013 to 2017. (UN Comtrade database, https://comtrade.un.org/)

1.3.1.3 Increasing maize crop yields

In the 2009 publication “Global agriculture towards 2050”, the Food and Agriculture Organization (FAO, 2009) of the United Nations (UN) summarized the challenges facing global agriculture in the foreseeable future:

“Agriculture in the 21st century faces multiple challenges: it has to produce more food and fibre to feed a growing population with a smaller rural labour force, produce more feedstocks for a potentially huge bioenergy market, contribute to overall development in the many agriculture- dependent developing countries, adopt more efficient and sustainable production methods and adapt to climate change.”

At the turn of the century in 1900, the world population was an estimated 1.6 billion people (UN, 1999). Currently, the world population stands at 7.6 billion people, with the last billion added in 17

only during the last 12 years (UN, 2017, 2019). This exponential increase is set to continue, with the world population estimated to reach 9.1 billion by 2050 and 11.2 billion by 2100 (UN, 2017). The FAO expects that developing countries, especially in sub-Saharan Africa, will be responsible for the greatest part of the world population increase (FAO, 2009; Niang et al., 2014).

The continual and rapid growth in the world population will require a 70% increase in the total food production by 2050 (FAO, 2009). The demand for maize and other cereals (as food and animal feed) will have increased by nearly 1 billion tonnes by 2050 (FAO, 2009). An increase in the reliance on biofuels (Kline et al., 2017), and the demand for agricultural products that correlate with higher incomes in developing countries (e.g. meat and dairy products), will also drive the demand higher (Abbassian, 2006) and could alter projected trends. Higher yields and cropping intensity are expected to account for 90% of the expected yield gains, with only 10% expected from agricultural land expansion (FAO, 2009).

South Africa is set to reach a population size of 82 million by 2035. The food supply (production and/or importation) will have to more than double to meet the growing needs of the country (World Bank, 2008; Ray et al.2013; FAO, 2015; FAO-ECA, 2018). The rainfall in SA is extremely variable, both geographically and temporally (Botai et al., 2018). Although nearly a third of SA has an average rainfall that supports crop production, only 12% of these areas have fertile soils. Only 3% of South Africa is considered high-potential land (Figure 1-2).

Therefore, SA, unlike most other developing countries, does not have the option of increasing productivity through land expansion. A large country such as India, for example, is covered by more than 50% fertile land (World Bank, 2017b) and can therefore improve productivity simply by utilizing more land. In order to double the productivity and ensure an adequate food supply by 2050, SA must improve the yields attained on the arable land that is currently in use. Even though the annual production of maize in SA increased from 328 000 tons in 1960 to 12.2 million tons in 2015 (Greyling and Pardey, 2018), maize yields are still among the lowest in the world (Schimmelpfennig et al., 2012, World Bank, 2017c).

1.3.1.4 Challenges facing maize production in South Africa

1.3.1.4.1 Challenges over which producers have little control

SA has highly variable rainfall, both geographically and over scales of time (SAWS, 2017). Climate change is therefore a significant threat for the future of the agricultural sector in SA, as it is predicted that climate change will lead to higher average temperatures and less rainfall across SA (Calzadilla et al., 2014; Niang et al., 2014). The fluctuations in regional water availability could be a severe impediment on crop production in the areas where maize is mostly produced. 18

Figure 1-2 Map illustrating the potential of land to support crop production in South Africa. (Goldblatt, 2010) (Original map created by Institute for Soil, Climate and Water, Agricultural Research Council)

Apart from climatic threats, there are also economic and political challenges that could hamper the increase of maize production. The 2017 report, Agricultural Outlook, compiled by Grobler et al. (2017), summarizes the political challenges faced by the SA agriculture sector as such:

“In principle, an investor only needs two things from a government: First, a friendly policy environment that will support the investment (access to land, water, roads, markets, and disease and pest control). And second, functional departments that deliver these policies with a good record of service delivery, monitoring of the environment and the implementation of measures to ensure market access. It is in this area that the South African government is failing investors, not only in agriculture… The poor policy environment has already led to the downgrading of South Africa’s credit rating.”

From the economic viewpoint, agricultural input costs, such as fuel, fertilizer, seed, and farming equipment are severely impacted by the volatility of the country’s currency. The weakening Rand has increased revenue in some parts of the agricultural sector, especially certain export-oriented divisions, but the net effect has been negative (BFAP, 2018). Increased fuel prices, for example, not only impacts the running costs of farm equipment, but also the transportation of produce. While more than 80% of the grain harvest in 1985 was transported by rail, this decreased to 30% due to unreliable rail services that necessitate the use of road freight transport (Goldblatt, 2010). The combination of global demand outstripping supply, and the increase in price of raw materials and oil has (along with the weaker Rand) led to record prices for fertilizers in recent years (Goldblatt, 2010; DAFF, 2017). This means that the overall profitability per hectare is declining (Grobler et al., 2017; Goldblatt, 2010). Because SA farmers are price-takers (i.e. they are unable to determine the asking price for maize), their only option to keep afloat is to increase production volume and to be early adopters of new technologies (i.e. better seed, fertilizers, equipment etc.) to improve productivity (Grobler et al., 2017).

1.3.1.4.2 Challenges over which producers have some level of control

The maize yield harvested in SA improved from a mean of 1 t/ha in 1960 to 5 t/ha in 2014. This can be attributed to the utilization of new technologies, such as mechanisation, improved genetics, and precision farming (Greyling and Pardey, 2018). Another contributing factor is improved pest control to protect the harvest.

Globally, farmers have to protect their crops against harmful organisms that directly attack their crop (e.g. insects, mites, nematodes, viruses, bacteria, fungi) or compete with their crop (e.g. weeds). These organisms are collectively known as pests (Oerke, 2006). Of the potential crop yield, about a third (35%) is lost to pests even before crops are harvested (Oerke, 2006; Popp et al., 2013). There are many pests in Africa that prevent attaining maximum yield levels, but none more so than the lepidopteran stem borer species. The larvae of these pests can cause 20-40% losses during cultivation, depending on the specific stem borer species, the pest population density and the plant growth stage affected (Sylvain et al., 2015). Apart from stem borers there are also other lepidopteran pests that affect maize production in Africa. In 2016, the leaf feeding Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) invaded several African countries (Goergen et al. 2016), and subsequent surveys reported the presence of the pest in nearly all countries in sub-Saharan Africa (Nagoshi et al. 2019). In South Africa, maize yield losses due to stem borers have reached 25-75% in the past, but averages around 10% annually (Kfir et al., 2002). In maize, the African stem borer (Busseola fusca) (Fuller) (Lepidoptera: Noctuidae) is considered the most injurious, causing estimated yield losses between 10% and total yield loss (Mally, 1920; Matthee, 1974; Barrow, 1987, 1989; Van Rensburg and Bate, 1987). By improving 20

the control of maize stem borers, specifically B. fusca, farmers can make significant gains in maize yields.

1.3.2 Busseola fusca: taxonomy, biology and pest status on maize in SA

1.3.2.1 Taxonomy, distribution and host plant range of Busseola fusca

Busseola fusca, or the African stem borer as it is commonly known, was first described and named by Fuller (Fuller, 1900), with the type designation published by Hampson in 1902 (Hampson, 1902). It was first placed in the Sesamia genus (thus originally named Sesamia fusca). However, in 1953 Tarns and Bowden (1956) revised Sesamia and other related genera and published taxonomic descriptions, diagnoses, and keys for identification. Subsequently, S. fusca was placed in the Busseola genus, and no further taxonomic revisionary work has since been conducted on this species (Harris and Nwanze, 1992).

Busseola fusca occurs throughout sub-Saharan Africa (Kfir et al., 2002), but not on the islands of Zanzibar and Madagascar (Le Rü et al., 2006a). Although it is commonly the predominant pest in cooler highland regions, it occurs across all agroecological zones and its pest status varies between regions (Kumar, 1988; Assefa et al., 2006; Khadioli et al., 2014; Sylvain et al., 2015). In South Africa, B. fusca occurs from coastal regions (Krüger et al., 2008; Assefa, 2015), low- and high-altitude inland areas (Ebenebe et al., 1999, Khadioli et al., 2014) as well as the mountain regions of Lesotho at altitudes of up to 2131 m a.s.l. (Ebenebe et al., 1999).

It is accepted that the cereal stem borers that originated in Africa (including B. fusca) evolved alongside wild grasses, until the introduction of maize into Africa (Bowden, 1976; Félix et al., 2013) from 1550 A.D. onwards (McCann, 2005). Busseola fusca has been associated with maize ever since the start of cultivation of the crop in Africa (Harris and Nwanze, 1992; Félix et al., 2013). As the regions where the crop was cultivated expanded, so did the distribution of B. fusca (Mally 1920, Sylvain et al., 2015).

Busseola fusca is an oligophaguos species, i.e. it has a limited number of suitable hosts, and both larvae and moths are highly selective when choosing a host plant (Bernays and Chapman, 1994; Calatayud et al., 2008; Juma et al., 2008, 2016). Although B. fusca might have originated on wild grasses, molecular studies (Sezonlin et al. 2006) and field sampling (Le Rü et al. 2006a; Mailafiya et al. 2011; Moolman, 2011) showed that B. fusca preferentially colonize cultivated cereals, and rarely attack wild grasses. In total, B. fusca only associates with 5 cultivated cereals (maize and sorghum, and to a smaller extent pearl and finger millet, and sugarcane), and 7 wild grass species, as confirmed by surveys conducted on 197 plant species in 15 African countries (Calatayud et al., 2014; Van den Berg, 2017). 21

1.3.2.2 Biology and lifecycle of Busseola fusca

The biology and ecology of B. fusca has been studied and reviewed extensively during the last century by several authors, as is evident from the list produced by Harris and Nwanze in (1992): Mally (1920), Wahl (1930), Hargreaves (1932, 1939), Lefevre (1935), Du Plessis (1936), Du Plessis and Lea (1943), Bowden (1956), Swaine (1957), Ingram (1958), Nye (1960), Smithers (1960), Walker (1960), Harris (1962, 1964, 1989). Later publications include those of Van Rensburg et al. (1987a), Kfir and Bell (1993), Calatayud et al. (2014), and Glatz (2017).

1.3.2.2.1 Eggs

Busseola fusca eggs are about 1 mm in diameter and hemispherical in shape (Harris and Nwanze, 1992). They are laid in neat rows in batches of between 30-100 eggs on the inner margin of leaf sheaths (Calatayud et al., 2014; Glatz et al., 2017) (Figure 1-3). Rarely, during late infestations, eggs may also be laid underneath the outer husk leaves of maize ears (Mally, 1920). Females preferentially oviposit on plants that are 3-6 weeks old (Van Rensburg et al., 1985; Van Rensburg et al., 1987a). Females prefer to lay egs under leaf sheaths of younger leaves and consequently, eggs are deposited higher up on older maize plants as plants grow and mature (Van Rensburg et al., 1987a). On average, a single female maintained under laborartory conditions produces 7-8 egg batches during her oviposition period (Glatz et al., 2017). The average incubation time for B. fusca eggs are one week (6-7 days) (Kaufmann, 1983, Glatz et al., 2017).

Figure 1-3 a) Busseola fusca eggs underneath a leaf sheath. b) Hatching B. fusca larvae.

1.3.2.2.2 Larvae

The larval stage of B. fusca lacks any distinctive setae or markings. They are usually a creamy- white colour, but this is variable, and they may in some cases display a grey, brown, or pink tint (Figure 1-4). The head capsule of the larvae is dark brown, and the prothorax is yellow-brown. The larvae have prolegs along the abdomen, with crotchets arranged in a semicircle. Along the side of the body, the spiracles are oval with black edges (Harris and Nwanze, 1992). Neonate larvae have been observed to feed on the eggshells immediately after eclosion (Kaufmann, 1983). The larval stage of B. fusca is largely determined by climate and temperature (Glatz et al., 2017), but generally lasts between 31 and 50 days (Ratnadass et al., 2001; Kruger et al., 2012b; Glatz et al., 2017), and consists of 6-8 instars (Unnithan, 1987, Glatz et al., 2017).

Figure 1-4 Examples of colour variations of Busseola fusca larvae.

1.3.2.2.3 Pupae

Final-instar larvae commonly pupate in the stems of the maize plants, but pupae may also be found in maize ear husks, in plant material on the soil surface, or below soil level in the bases of maize stems (Kaufmann, 1983; Van Rensburg et al., 1987a). The colour of the pupae is brown, and both sexes have a single pair of simple spines located on the cremaster (Harris and Nwanze, 1992). The female pupae are generally somewhat larger than the males (about 25 mm in length) and can be distinguished from the males by the lower position of their genital scars (Figure 1-5). Moths typically emerge 13-14 days after pupation (Calatayud et al., 2007). Females are inclined to emerge an hour after sunset (the onset of scotophase), with most males emerging an hour before sunset (Calatayud et al., 2014).

Figure 1-5 Differences in genital scar markings of a) female, and b) male Busseola fusca pupae.

1.3.2.2.4 Moths

Busseola fusca moths are usually light to dark brown, with pale hind wings (Kaufmann, 1983; Harris and Nwanze, 1992). Similar to the larval stage, there exists seasonal and geographical variation in the colouration and prominence of the markings on the wings. The wingspan is 20-40 mm, with the females having a slightly bigger build than their male counterparts (Harris and Nwanze, 1992). Male and female moths can be distinghuished based on the micro-sensillae on their antennae (Figure 1-6) (Calatayud et al., 2006). The sex ratio is generally very close to equal (Kaufmann, 1983). Kruger et al. (2012) found that the sex ratio was biased towards males in some Bt-resistant population and towards females in Bt-susceptible populations. However, in a similar study published by Kruger et al. (2014), the observed sex ratio was 1:1 regardless of where the populations were collected or their status of resistance to Bt maize. On average, both male and female moths live for 8-10 days (Calatayud et al., 2014; Kruger et al., 2014).

The reproductive and oviposition behaviour of B. fusca adults are nocturnal (Calatayud et al., 2007, Calatayud et al., 2014). Only female moths emit pheromones (Calatayud et al., 2014), and they initiate calling behaviour and start mating a few hours after eclosion. Male moths are able to mate the same night of their eclosion, indicating that B. fusca lacks the sexual maturation period (Calatayud et al., 2007) which is a requisite in a number of other stem borer species (Calatayud et al., 2007). Male moths are able to mate multiple times during their lifetime, but only once per night (Unnithan and Paye, 1990). Female moths need to mate only once to fertilize all their eggs. Oviposition commences the night following the successful mating and then lasts largely between 3 and 4 nights (Unnithan, 1987), peaking on the second night (Calatayud et al., 2007).

Figure 1-6 Morphological differences of the sensilla on the antennae of a) male, and b) female Busseola fusca moths.

1.3.2.2.5 Busseola fusca diapause and population dynamics

With the arrival of dry and cold conditions towards the end of the growing season, the 6th instar B. fusca larvae enter a facultative diapause. Generally, a single larva will occupy the last internode at the base of the stem (below the surface of the soil), where it will remain dormant for up to 5 or 6 months (Van Rensburg et al., 1987b; Kfir, 1988, 1991). Larvae can also diapause in internodes higher up in the stem, although this mostly occurs in warmer regions such as Zimbabwe (Smithers, 1960). The ability to conserve water efficiently is one of the main reasons that the larvae are able to survive the extended period of adverse conditions (Usua, 1974, Kfir et al., 2002). The first good rains generally mark the end of the diapause period and the onset of the first moth flight, since the contact with (and consumption of) water are key factors to inducing pupation in a diapausing population (Okuda, 1991). Light and pheromone traps (Van Rensburg et al., 1987b) have made it possible to observe a distinct flight pattern with 2-3 generations for B. fusca populations in South Africa where one rainy season occurs. In areas where maize is cultivated throughout the year, there exists a greater generational overlap, making it difficult to identify a specific flight pattern (Calatayud et al., 2014).

1.3.2.3 Status Busseola fusca as an agricultural pest

Busseola fusca has been considered the most destructive pest of maize and sorghum throughout much of the African continent since the first half of the 20th century (Kfir et al., 2002; Tounou et al., 2010). This species can occur in mixed populations with other cereal stem borers (Van den Berg et al., 1991), but generally dominates in the cooler agroecological zones from mid-altitudes to highland grounds (Calatayud et al., 2008). The reported yield losses caused by B. fusca vary

greatly, between 0-100%. The level of crop loss is dependent on many factors, including the type of cereal crop (sorghum is generally more tolerant of stem borer attacks than maize (Harris and Nwanze, 1992), climatic conditions, crop age at infestation, plant stand (Le Rü et al., 2006b) and whether chemical and/or other control measures are implemented (Kfir et al., 2002). The average annual maize yield loss attributed to stem borers in South Africa was estimated at 10% (Bate and Van Rensburg, 1992), which translated to a million tonnes of maize at a Rand value of approximately R1.2 billion (Schimmelpfennig et al., 2012).

The success of B. fusca can, in part, be attributed to the ability of the larvae to feed on all parts of the host plant (Calatayud et al., 2014). During the early development stages the larvae feed on young leaves in the whorl, causing small holes to appear as leavea grow (a.k.a “window panes”) (Barrow, 1985; Sylvain et al., 2005; Calatayud et al., 2014) (Figure 1-7).

Significant feeding damage during the initial stages of crop development can lead to the formation of “dead hearts”, when the growth points are damaged and the central leaves wither and dry out (Sylvain et al., 2015). As the larvae mature, they migrate towards the base of the host plant, where they bore into the stems (Figure 1-7). Busseola fusca is known for prolific stem tunnelling that inhibits the translocation of water and nutrients in the plant (affecting the development of maize ears), weakens the maize stem (leading to lodging and breakages), and increases susceptibility to pathogens such as maize stalk rot (Sylvain et al., 2005). Busseola fusca larvae also feed on the silk and maize ear itself, causing deformation and increasing the probability of mycotoxin contamination (caused by fungal infections) (Ncube et al., 2017, 2018) (Figure 1-7).

Figure 1-7 Examples of Busseola fusca larval feeding damage to a) maize whorl leaves, b) maize stem, c) maize ear and stem. (Photos b and c by Annemie Erasmus, Grain Crops Institute – Agricultural Research Council)

1.3.2.4 Management of Busseola fusca

1.3.2.4.1 Chemical control

For decades commercial maize farmers in sub-Saharan Africa had to rely on the use of insecticides to control maize stem borers such as B. fusca. However, there are numerous disadvantages associated with this control strategy. Insecticides are expensive, and several applications might be necessary for effective control. Added to this is the cost of a monitoring program, which relies on pheromone-baited traps to alert producers of moth flight peaks (Van Rensburg et al., 1985; Revington, 1987). The correct timing of insecticide application is also crucial, as the spayed chemicals are ineffective once the larvae have bored into the maize stems (Van den Berg and Van Rensburg, 1996; Slabbert and Van den Berg, 2009; Wale et al., 2006). But most importantly, chemical sprays are fundamentally wasteful. The amount of an insecticide that reaches the target pest has been estimated to be less than 0.1% (Pimental and Levitan, 1986). The excess accumulates in the environment, often with detrimental effects on non-target organisms. This could lead to secondary pest outbreaks, since the chemicals often devastate predator and parasitoid populations (Bravo and Soberón, 2008). It has also been shown that, despite the improvement in pesticide formulations and their selectivity, and intensification of spray programs, crop loss to pest infestation remained relatively unchanged since the 1950’s (Oerke, 2006). This is due to proclivity of insect populations to adapt and evolve rapidly, necessitating the stronger and more frequent applications of insecticides (Mallet, 1989). This phenomenon is often referred to as the ‘pesticide treadmill’ (Goldblatt, 2010).

For the majority of smallholder and subsistence farmers in sub-Saharan Africa, there are additional factors that limit the utility of chemical control for their crops. For example, difficulty of application without the proper equipment, a lack of training in safe handling of pesticides, and the high cost and unavailability of agrochemicals in rural areas (Sylvain et al., 2015; Tefera et al., 2016). Thus, even though chemical control has been an integral part of the management of cereal stem borers, it is not a sustainable solution.

1.3.2.4.2 Alternative control strategies

There exist alternative strategies apart from the use of pesticides to control B. fusca and other maize stem borers. The most economic strategy arguably involves cultural control practices, which entails altering the habitat to create an unfavourable environment for the survival and reproduction of the pest population (Dent, 2000; Gurmessa et al., 2019). Cultural control includes practices such as intercropping (Reddy and Masyanga, 1988), crop rotation (Harris and Nwanze, 1992), management of crop residues using tillage (Kfir, 1990), removal of volunteer and

alternative host plants (Sylvain et al., 2015), adjusting planting dates and densities (Sithole, 1989; Van Rensburg et al., 1988), and the utilization of stimulo-deterrent (or “push-pull”) tactics (Khan et al., 2000).

Biological control and host plant resistance are other alternative pest control options which could be used in combination with cultural control practices. Several parasitoid and pathogen species have been evaluated for their use in biological control strategies, including entomopathogenic nematodes and parasitic wasps such as Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) (Ngi-Song et al., 1995, 1998; Ngi-Song and Overholt, 1997; Schulthess et al., 1997, 2004; Kfir et al., 2002; Maniania et al., 2011, Ramakuwela et al., 2011).

In the mid 1990’s a new tool emerged which changed the approach to the maize stem borer management, i.e., biotechnology in the form of genetically engineered Bt maize.

1.3.3 Bt maize and genetically engineered crops

1.3.3.1 Biotechnology, genetic modification, and genetic engineering

Biotechnology is a broad term that encompasses a wide range of concepts. In the FAO’s Glossary of Biotechnology and Genetic Engineering (Zaid et al., 1999), biotechnology is defined in two parts:

“The use of biological processes or organisms for the production of materials and services of benefit to humankind. Biotechnology includes the use of techniques for the improvement of the characteristics of economically important plants and animals and for the development of micro- organisms to act on the environment.”

and

“The scientific manipulation of living organisms, especially at the molecular genetic level, to produce new products, such as hormones, vaccines or monoclonal antibodies.”

This means that biotechnology is relevant to a wide range of disciplines such as medicine and vaccines, food sciences, conservation, and agriculture (FAO, 2012a). The focus in this study is on the application of biotechnology in agriculture, especially as it relates to the genetic improvement of crop varieties. This process is referred to as ‘genetic engineering’ (GE), but the term ‘genetic modification’ (with its derivative, genetically modified organism) is also commonly used.

Humans have been genetically modifying animals and plants since the advent of agriculture, using artificial selection and other traditional breeding methods (Figure 1-8) (Harlan, 1976; Barrows et al., 2014). These methods are time-consuming and imprecise and restrict breeders to selecting characteristics that naturally occur in the genetic pool of the species (Gould, 1988; Roush, 1998). However, GE refers specifically to the use of modern genome editing technology to introduce genetic changes (and even foreign genetic material) into an organism with precision and in a fraction of the time (Gould, 1988; Phillips, 2008; Barrows et al., 2014).

1.3.3.2 Genetically engineered crops

Research efforts which focussed on the genetic engineering of crops started in the late 1970’sduring which time the basic techniques were developed (Qaim, 2009). These efforts culminated in the production of the first GE crop: insect resistant tobacco (Hilder et al., 1987; Vaeck et al., 1987; Russo, 2003). However, the first GE crops only became commercially available a decade later. Initially, the number of crops and the GE traits available were limited. However, the past two decades has seen in immense increase in the number of crops and traits available, as well as the global hectarage devoted to the cultivation of GE crops (ISAAA, 2017; Pellegrino et al., 2018). In 1996, the global hectarage of GE crops were estimated at 1.7 million ha, which has increased to 185.1 million ha in 2017 (Figure 1-9).

Figure 1-8 Selective breeding transformed the teosinte ear (Zea mays ssp. mexicana) (left) into the modern-day maize ear (right). Centre is a F1 hybrid of the teosinte and maize plants. (Photo: John Doebley, https://teosinte.wisc.edu/)

Figure 1-9 Total area of genetically engineered (GE) crops cultivated globally over time. (ISAAA, 2017)

The GE traits currently in development are also not limited to benefiting the production of crops in terms of pest control, herbicide resistance and virus resistance. What is referred to as the second- and third-generation GE crops will have traits that enhance crop quality (e.g. nutritional value) and even allow the production of specific pharmaceutical or industrial substances in crop plants (e.g. fruits producing vaccines) (Qaim, 2009; FAO, 2012a). Of the first-generation GE crops (i.e. GE crops with traits that contribute to the production process), varieties engineered to express insecticidal Bt toxins are some of the most popular in the world (ISAAA, 2017).

1.3.3.3 Bt crops

1.3.3.3.1 What are Bt crops?

Bt crops are created by combining genes from a common soil bacterium with the genetic material of the crop plant. The bacterium Bacillus thuringiensis (Berliner) is an aerobic, Gram-positive bacterium (McGaughey and Whalon, 1992; Jouanin et al., 1998), indigenous to a wide variety of environments across the globe (Martin and Travers, 1989; Chaufaux et al., 1997) but typically found in soil (Martin and Travers, 1989; Carozzi et al., 1991; Smith and Couche, 1991). There exist several B. thuringiensis bacterium strains (Crickmore et al., 2007), which are all able to produce a range of different insecticidal Cry proteins (toxins) that are active against numerous insect orders, including Lepidoptera, Coleoptera and Diptera (Herrero et al., 2016). These toxins

are protein crystals (termed Cry proteins or delta-endotoxins) which are formed by the bacterium during sporulation (Hannay and Fitz-James, 1955; Ferré et al., 2008). Bravo et al. (2011) reported that the cry gene sequences that have been identified (more than 500) can be divided into four protein families which may have varying modes of action. These families are the 3D Cry toxins (three domain crystals), Mtx Cry toxins (targeting mosquitos specifically), Bin toxins (binary-like), and Cyt toxin families. Certain B. thuringiensis strains are able to produce an additional group of toxins during the vegetative stage, called VIP toxins (Bravo et al., 2011).

Bt toxins have been used for decades as foliar sprays before they were engineered into the crops themselves (Frisvold and Reeves, 2014). However, these Bt sprays have very low field persistence, which severely limited their utility in large scale commercial farming (Jouanin et al., 1998). However, the inclusion of genes from B. thuringiensis enables the GE Bt crop to produce insecticidal Bt toxins in the plant tissues (De Maagd et al.1999), thereby overcoming the problems of toxin stability when applied externally onto the foliage (McGaughey and Whalon, 1992).

Maize, cotton and potatoes were the first commercially available Bt crops and were released in 1996 (Cohen, 2000; Bravo et al., 2011). Initially, Bt maize cultivars were all single toxin varieties that expressed Cry proteins from the Cry1 or Cry2 group, which is most effective against lepidopteran larvae (Van den Berg et al., 2013; Frisvold and Reeves, 2014). These cultivars were specifically developed to target two maize stem borer species, namely Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) (Ostlie et al., 1997) and Diatraea grandsiosella (Dyar) (Lepidoptera: Crambidae) (Archer et al., 2001), in the USA. Bt maize varieties targeting Coleoptera pests, e.g. Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), contained genes that coded for the production of Cry3 toxins and were released in the USA during 2003 (Jouanin et al., 1998; Frisvold and Reeves, 2014). Soon after, maize cultivars containing multiple transgenes (known as stacked or pyramid varieties) were released, providing insecticidal action against both lepidopteran and coleopteran pests, as well as conferring herbicide tolerance (Frisvold and Reeves, 2014). By 2017, the total area dedicated to Bt maize reached 53.4 million hectares, which represents 32% of all maize produced globally (ISAAA, 2017). In South Africa, during 2017/2018 cropping season, 71% (1.62 million hectares) of the total maize area planted consisted of Bt maize aimed at controlling maize stemborers (ISAAA, 2017).

1.3.3.3.2 Bt maize mode of action

The Bt transgene is inserted into the genetic material of a maize variety accompanied by a promotor gene and a marker gene (Bessin, 1995). The promotor gene regulates the expression of the Bt transgene and can limit its expression to specific growth stages or plant tissues. However, Bt crops generally produce Cry proteins throughout the plant and during all growth

stages. The marker gene makes it possible for breeders to determine whether a plant possesses the transgene material (Bessin, 1995).

The mode of action of 3D Cry toxins are thought to follow one of two hypotheses, known as the ‘pore formation’ or the ‘signal transduction’ hypotheses. However, most studies support the pore formation model in various insect orders, including Lepidoptera (Soberón et al., 2007; Bravo and Soberón, 2008). Consequently, Cry toxins are classified as pore-forming toxins that change conformationally to merge with the midgut cell membrane of the target insect (Bravo et al., 2007; Bravo and Soberón, 2008; Bravo et al., 2011). This means that once the crystal proteins produced by plants are ingested, it is transformed into protoxins in the gut of the larvae. A simplified version of the process described by Bravo and Soberón (2008) is as follows: The protein undergoes a conformational change due to midgut proteases, which gives rise to an activated form of the toxin. The toxin then binds to the cadherin receptor in the microvilli of the midgut cells, which leads to further proteolytic cleavage. The product of the latest cleavage then binds to receptor proteins located in the cell membrane, from where it then inserts into lipid raft membranes. After successful insertion, pore formation and cell lysis follow shortly. This leads to the destruction of the midgut epithelium, followed by paralysis, starvation and septicaemia which eventually causes the death of larvae (Ferré et al., 2008) (Figure 1-10).

The efficacy of Bt crops depend largely on a high level of Cry protein expression in plant tissue where target insects feed (Giband, 1998). To increase the expression levels of Bt genes, the original bacterial Cry gene were truncated and codon-optimized (Schuler et al., 1998). This effectively dealt with a number of factors that hindered gene expression and allowed for an increase in gene expression up to 500 times higher than that of the original bacterial Cry gene (Giband, 1998).

1.3.3.4 Advantages associated with Bt maize

In their 2018 meta-analysis titled “Impact of genetically engineered maize on agronomic, environmental and toxicological traits: a meta-analysis of 21 years of field data.”, Pellegrino and colleagues (Pellegrino et al., 2018) set out to scour the available peer-reviewed literature from 1996 to 2016 and assess the impact of GE maize on yield, grain quality, target and non-target organisms, and soil biomass decomposition. Their initial search found more than 6000 published papers dealing with the abovementioned issues. However, to ensure that the meta-analysis provided rigorous results, strict criteria for papers to be included in the final analysis were used. This strict quality control procedure resulted in only 79 of the papers eventually being included in the meta-analysis. Their findings indicated that GE maize performed better than its near isogenic line, that biomass decomposition was higher in field planted to GE maize, and that the effect on

non-target organisms were negligible (with the exception of Braconidae species (Watanabe, 1967). These results support previous meta-analyses (Finger et al., 2011; Areal et al., 2013; Klümper and Qaim, 2014) and other studies (Ortego et al., 2009; Qaim, 2009; Burachik, 2010; Arthur, 2011; Xu et al., 2013; Nicolia et al., 2014, Wang et al., 2014, ISAAA, 2017; Brookes and Barfoot, 2018) which confirmed the advantages that cultivating GE maize varieties have above conventional maize varieties that require chemical insecticide applications to protect crops against pests. A number of more specific advantages are discussed below.

Figure 1-10 The multi-stage mode of action of Bt Cry proteins. a) A larva ingests Bt spores or Cry proteins. b) In the larval midgut, proteolytic digestion of proteins releases Cry toxins, which bind to epithelial receptors. c) Toxin-binding causes cell lysis, destroying the barrier to the body cavity. (Adapted from figures by Kaitlyn Choi, http://sitn.hms.harvard.edu/flash/2015/insecticidal- plants/)

1.3.3.4.1 Reduced yield loss to pests

The inclusion of Bt genes in maize varieties do not directly increase the yield of the crop, instead, yield gains are due to reduced crop loss to stem borer damage (Bessin, 1995). Thus, the positive effect on yields varies temporally and spatially, since it is correlated to the prevailing pest pressure in the region (Carrière et al., 2010; Van den Berg, 2012). Yield gains obtained by planting Bt maize was observed by both commercial and smallholder farmers in South Africa. A gain of approximately 10% was reported by commercial dryland and irrigation farmers in the two cropping seasons from 1999 to 2001, whereas a yield gain of 32% was observed by smallholder farmers (Gouse et al., 2005; Qaim, 2009). More than 10% of the global arable land is located in sub- Saharan Africa, but yields remain low due to economic and technical constraints precluding the use of fertilizers and pesticides. GE maize could contribute significantly to the food security in this region (Qaim and Zilberman, 2003; Conceição et al., 2016; Carzoli et al., 2018).

1.3.3.4.2 Season-long control of pests

Bt maize provides crop protection against stem borers throughout the growing season. Pesticide sprays (including Bt sprays) often have very low field persistence due to weather conditions such as high temperatures, ultraviolet light, and torrential rain (Losey et al., 2001; Ferré et al., 2008). The presence of Bt toxins throughout all plant parts also means that it is more effective against pests that feed in areas of the plant where sprays are unable to reach (e.g. stem borers that feed chiefly in the stem of the maize plant) (Shelton et al., 2000; Losey et al., 2001; Slabbert and Van den Berg, 2009).

1.3.3.4.3 Decrease in broad-spectrum insecticide use

In 2014, the use of Bt maize lead to a total reduction of 8 million kg of insecticides globally, which translates to 71% of insecticides targeted at stalk boring and rootworm pests. The cumulative pesticide savings since the cultivation of Bt maize commenced during 1996 is estimated at 79.8 million kg (Brookes and Barfoot, 2018). Qaim (2009) reported that South African commercial maize farmers applied 10% less insecticides due to the use of Bt maize. This reduction, although still significant, is less than the global reduction due to higher pest pressure (Qaim, 2009) and the early evolution of resistance to Cry1Ab protein by B. fusca in South Africa (discussed later on in the chapter).

1.3.3.4.4 Lower labour and production costs

Cultivating conventional non-Bt maize varieties often requires rigorous monitoring for pest presence and multiple insecticide applications. This means that higher labour and production

costs are accrued, since the costs of pesticides and fuel to operate farming machinery can culminate to inordinate amounts (FAO, 2012a). A global saving of 898 million ℓ of fuel and 2.396 million kg of carbon dioxide, owed to cultivation of GE crops, was reported by Brookes and Barfoot (2016) for 2014. This saving was due to the use of both Bt maize and cotton, as well as no-tillage practices made possible by GE herbicide tolerant crops.

1.3.3.4.5 Narrow toxicity and use in conjunction with biological control

The use of broad-spectrum insecticides has had severely detrimental effects on non-target species (Bessin, 1995; Sánchez-Bayo, 2011). These insecticides can also cause harm to humans and animals such as birds (McGaughey and Whalon, 1992; Qaim, 2009). Bt toxins target a narrow range of insect species, with no significant effect on other non-target organisms from most insect orders (Carrière et al., 2010; Pellegrino et al., 2018). This specificity makes it an ideal companion to use in combination with biological control in an IPM strategy (Bessin, 1995; Frisvold and Reeves, 2014). It was also reported that Bt maize significantly reduced the level of mycotoxin contamination, which can cause severe health problems in humans (Munkvold and Hellmich, 1999; Wu, 2006; Ncube et al., 2017, 2018).

1.3.3.4.6 Halo effect for nearby non-Bt fields

The Halo Effect (Alstad and Andow, 1996) refers to the phenomenon where pest populations are suppressed on non-Bt crops when they are planted in close proximity to large areas of Bt crops. This occurs when female moths oviposit indiscriminately on Bt and non-Bt plants, which leads to a decrease in regional pest populations because of the greater presence of Bt crops. The benefit of the Halo effect to producers that do not plant Bt maize are described comprehensively by Hutchinson et al. (2010) who showed that even non-adopters of Bt maize benefitted from the technology, due to regional suppression.

1.3.3.4.7 Increased number of cultivars used

After a crop cultivar is developed through conventional breeding and it is shown to produce high yield, it is generally widely adopted, often replacing several other older varieties. This conventional breeding process is time-consuming, and it is expensive to produce large numbers of varieties that all have the desired traits. However, biotechnology makes it possible to backcross GE traits into several other varieties at a much lower cost and over a shorter time period (Trigo and Cap, 2006; Romeis et al., 2008).

1.3.3.5 Disadvantages associated with Bt crops

GE Bt crops are one of the most rapidly adopted technologies in agriculture (Tabashnik, 2010; ISAAA, 2017). The swift adoption of these crops across the globe was accompanied by a rise in concern for the impact of Bt crops on food safety, the environmental at large, as well as the socio- economic implications associated with this technology (FAO, 2012a).

1.3.3.5.1 Unforeseen effects on non-target organisms

Thus far, only non-target Braconidae species (Pellegrino et al., 2018), parasitoids of lepidopteran larvae, have been shown to be adversely affected by Bt maize. This indirect adverse effect (reduced abundance) is however ascribed not to toxicity of Cry proteins for these parasitoids, but to the reduced abundance of their hosts which are lepidopteran pest larvae. Bt maize might primarily target phytophagous insects, but the Bt toxins could indirectly impact negatively on entomophagous insects by reducing prey quantity and quality (Faria et al., 2007)

1.3.3.5.2 Gene flow and escape of GE traits to plants species in natural environments

The unintentional transfer of genetic material from GE crops to related species pose a serious risk to the environment. Once transgenes ‘escape’ and are present in wild species, it would be near impossible to reverse the consequences (Wolfenbarger and Phifer, 2000). The transfer of herbicide tolerance for exmaple, would create weeds that are difficult or impossible to control and Bt genes could have deleterious effects on non-target species, depending on the plant species into which the transgene is introgressed (Wolfenbarger and Phifer, 2000).

1.3.3.5.3 Increase in secondary pest outbreaks

The cultivation of GE Bt maize and other Bt crops resulted a marked decrease in the amount of broad-spectrum insecticides used by producers. Although this change in pest control strategy is positive for the environment, it could also facilitate a surge in secondary pest numbers (Catarino et al., 2015). This phenomenon has been observed in areas in Europe and South America, where the cultivation of Bt maize led to an increase in numbers of aphids, leafhoppers and other secondary pests (Faria et al., 2007; Virla et al., 2010). In China, for example, Hemiptera bugs became important pests after insecticide applications against lepidopteran pests on cotton was reduced on Bt cotton (Wang and Fok, 2017). Along with the absence of broad-spectrum insecticides, the increase in non-target pest populations could also be aided by the decrease in competition for food resources (Bortolotto et al., 2015).

1.3.3.5.4 High cost of GE technology and market access

The seed of Bt maize is generally priced much higher than that of conventional maize varieties, since private companies that develop GE crops need to recover their investment into the research and development of these biotech products (FAO, 2012a). The monopolization of the seed market also contributes to higher seed prices (Sharma, 2004; Fischer et al., 2015). This poses a conundrum for producers when deciding whether to plant Bt maize or not, since GE maize is only beneficial in the presence of high pest pressures (there are no yield gains in the absence of significant pest pressure) (Carrière et al., 2010; Van den Berg, 2012). However, stem borer infestations can vary greatly between seasons, and there is no accurate way of predicting the level of infestation during the planting stage. This could lead to economic losses for the producers (Bessin, 1995; Fischer et al., 2015). The controversy surrounding GE crops and public mistrust of agricultural biotechnology has led to the rejection of Bt maize by a number of markets, most prominently in Europe. Thus, it is also of critical importance that producers establish beforehand whether Bt maize will be accepted by their grain markets (Bessin, 1995; Paarlberg, 2009).

1.3.3.5.5 Socio-economic consequences

The effect that adoption of GE crops will have on tradition knowledge systems and the communal way of life (e.g. seed sharing practices) in developing countries is a concern (Qaim, 2009). The advantageousness of Bt crops for smallholder and subsistence farmers in developing countries have also been brought into question (Aheto et al., 2013). In their 2015 study, Fischer et al. (2015) concluded that there exist cheaper alternatives to Bt maize that are better suited to the needs of smallholder farmers in South Africa.

1.3.3.5.6 Loss of crop diversity

Although GE Bt crops may contribute to increase the range of cultivars used in different countries (as discussed under ‘Advantages’), the diversity of crops is declining due to the use of a limited number of species in monoculture systems (Esquinas-Alcázar, 2005). Goldblatt (2010) reported a loss of 75% of global agricultural crops over the past 50 years. This loss severely limits our capacity to adapt to future challenges of pests, diseases and climatic extremes, since the majority of these crops and their useful traits have become extinct (Esquinas-Alcázar, 2005).

1.3.3.5.7 Resistance evolution by insect pests

The efficacy and ease of deployment of Bt crops has seen their use increase exponentially (ISAAA, 2017). As a result, selection pressure for resistance evolution by the target pest population is very high and is continually applied throughout the growing season (Bourguet et al.,

2005; Siegfried and Jurat-Fuentes, 2016). When considered alongside the evidence that insects are especially apt at evolving resistance to control measures such as pesticides or Bt crops, it is clear that insect resistance is the predominant drawback of Bt crops (Tabashnik, 1994a,b; Gould, 1998; Gassmann et al., 2009; Carrière et al., 2010). Once the target insect pest species become resistant to the Bt toxins expressed in Bt crops, producers will have no choice but to revert back to applying multiple sprays of broad-spectrum insecticides to control pest populations. The numerous advantages (to both producers and the environment) associated with the use of Bt crops (Pellegrino et al., 2018), will then be lost. The phenomenon of insect resistance evolution is discussed in more detail below.

1.3.4 Evolution of resistance to Bt crops

1.3.4.1 What does resistance to Bt toxins mean?

The development of resistance by target pest organisms (whether insect, weed, or pathogen) to chemical control methods has been a thorn in the flesh of many producers and agricultural companies throughout history (Tabashnik et al., 2014). Similarly, Bt crops will only remain useful so long as pest populations remain susceptible to the Bt toxins (Bourguet et al., 2005; Huang, 2006; Tabashnik and Carrière, 2017). In their 2014 article, Tabashnik et al. (2014) set out to improve the dialogue and understanding surrounding the topic of pest resistance by providing definitions for 50 key terms commonly used in the discussion of this topic. They evaluated numerous studies in the field of pest resistance to chemical pesticides as well as GE crops, and assessed the suitability of various commonly used terms and definitions. Their proposed definitions for resistance, laboratory-selected resistance, field-evolved resistance, and practical resistance (among others) are provided in Table 1-1. Following the definitions of Tabashnik et al. (2014), field-evolved resistance of an insect pest population to Bt maize can increase undetected over a period of time, until this resistance causes a marked decrease in the efficacy of the Bt toxins and therefore has practical consequences for producers and seed companies. The insect pest is then said to have practical resistance to a specific Bt toxin.

1.3.4.2 How do pest populations become resistant to Bt toxins?

It was initially thought that the mode of action of Bt toxins were so complex (with multiple toxins, target sites, and stages) that the probability of resistance evolution was low, and that a single mutation would have little to no effect (Burges and Hussey, 1971; Boman, 1981). However, this apparent strength turned out to be the greatest weakness of this technology, as a complex and multistage mode of action only provides more points at which physiological changes in the target pest might render the toxin innocuous (McGaughey and Whalon, 1992). There are several known

mechanisms by which insects could acquire resistance to Bt toxins. Ferré et al. (2008) listed these as: 1) changes in midgut receptor binding (Griffits and Aroian, 2005), altered activation of protoxins (Karumbaiah et al., 2007), rapid toxin degradation (Forcada et al., 1996), rapid repair/regeneration of damaged midgut cells (Castognola and Jurat-Fuentes, 2016), esterase sequestration (Gunning et al., 2005) and heightened immune reactions (Ma et al., 2005). Although a multitude of possible pathways exist, altered binding of receptors in the midgut is by far the most common mechanism by which insects evolve resistance to Bt toxins (Giband, 1998; Ferré and Van Rie, 2002; Soberón et al., 2007; Bravo and Soberón, 2008; Ferré et al., 2008; Bravo et al., 2011; Peterson et al., 2017).

Table 1-1 Insect pest resistance terminology defined by Tabashnik et al. (2014).

Term Definition

Resistance A genetically based decrease in susceptibility to a pesticide. Susceptibility/ The tendency to be killed or harmed by a pesticide. Sensitivity The process by which a genetically based decrease in Evolution of resistance susceptibility to a pesticide occurs in a population. The genetically based decrease in susceptibility to a pesticide Laboratory-selected in a population caused by exposure of the population to the resistance pesticide in the laboratory. Practical resistance/ Field-evolved resistance that reduces pesticide efficacy and field resistance has practical consequences for pest control. The resistance conferred by enhanced enzymatic Metabolic resistance transformation of a pesticide to make it less toxic. The resistance conferred by changes in behaviour that reduce Behavioural resistance exposure to a pesticide. Resistance to a pesticide caused by exposure of a population Cross-resistance to a different pesticide. The resistance in which fitness is lower for resistant Incomplete resistance individuals exposed to a pesticide, relative to resistant individuals not exposed to the pesticide. A trade-off in which alleles conferring resistance to a pesticide Fitness cost have reduced fitness in environments lacking the pesticide.

Table 1-2 Insect pest species with practical resistance to Bt crops. (Adapted from Tabashnik and Carrière, 2017)

Insect species Crop Toxin Country Years to High resistance dose Diabrotica vigifera Maize Cry3Bb USA 6 No vigifera Maize Cry34/35Ab USA 7 No Maize mCry3A USA 4* No Maize eCry3.1Ab USA 0* No Helicoverpa zea Maize Cry1Ab USA 8 No Maize Cry1A.105 USA 6* No Cotton Cry1Ac USA 6 No Cotton Cry2Ab USA 2* No Pectinophora gossypiella Cotton Cry1Ac India 6 No Cotton Cry2Ab India 8 ? Spodoptera frugiperda Maize Cry1Ab Brazil 2* No Maize Cry1F Brazil 2 No Maize Cry1F USA 4 No Busseola fusca Maize Cry1Ab South Africa 8 No Diatraea saccharalis Maize Cry1A.105 Argentina 4 ? Striacosta albicosta Maize Cry1Fa USA 10 No Years to resistance indicates the number of years from the first commercial planting of a Bt crop in the region to the first sampling of field populations in the region yielding evidence of resistance. High dose indicates if the high dose standard was met, based on direct or indirect evidence. The asterisk (*) indicates that cross-resistance is suspected or known as a factor contributing to resistance.

Up to now we have discussed resistance to Bt toxins as a merely metabolic process. However, insect pests could also adapt their behaviour to avoid exposure to Bt toxins; indeed, a combination of both metabolic and behavioural resistance mechanisms could simultaneously contribute to resistance evolution by target pests (FAO, 2012b; Han et al., 2016). In addition to insect genetic and behavioural aspects there are also factors pertaining specifically to the cultivation practices in the particular farming system (receiving environment) and characteristics of the GE crop that will affect the rate of insect resistance evolution, e.g. crop rotation practices or the level of toxin expression (Head and Greenplate, 2012). These factors will be explored further in the Insect Resistance Management section (p.41).

1.3.4.3 Recent uptick in insect resistance to Bt toxins

During the first decade of commercially available Bt crops (1996-2005), only three cases of insect resistance to Cry proteins were reported. By 2016, the number has risen to 16 (Table 1-2). In their 2017 review article on the status of insect resistance to GE Bt crops, Tabashnik and Carrière (2017) suggested that the increasing global hectarage planted with Bt crops, and the rise in selection pressure that accompanied it, contributed significantly to the surge in pest resistance to Bt toxins. Over the past decade, about two new cases of practical resistance have emerged for every 10 million hectares increase in the area planted to Bt crops globally. Another critical factor that has played a major part in the appearance of practical resistance is cross-resistance between Cry toxins. Of the 16 cases of practical resistance, five can be linked to cross-resistance (Table 1-2). However, Tabashnik and Carrière (2017) also highlighted positive results: no decrease in susceptibility was reported for another 17 target pest species; in 11 of the latter 17 cases, no change in susceptibility has been detected for at least 10 years (Table 1-3). This suggests that it is possible to deploy Bt crops sustainably, since the authors concluded that the data is consistent with the underlying theory of current insect resistance management strategies.

1.3.5 Insect resistance management

1.3.5.1 What is insect resistance management?

The evolution of resistance by pest populations was already identified as a critical pitfall for the sustainability of GE Bt crops before the commercial release of these crops (Tabashnik, 1994a). In order to counter this threat, the implementation of an insect resistance management (IRM) strategy was made mandatory in most countries (USEPA, 2001; Tabashnik et al., 2009; Frisvold and Reeves, 2014).

An IRM strategy aims to delay the evolution of resistance in a pest population to a pest control product (McGaughey and Whalon, 1992). These strategies can ultimately not prevent resistance evolution (Manyangarirwa et al., 2006), but they can increase the timespan of resistance evolution, which allows for the development of new products and technologies to replace previous products that has been rendered obsolete (Manyangarirwa et al., 2006). IRM programs have existed for many years for chemical pesticides (Glaser and Matten, 2003), but participation in these initiatives and compliance to policies has mostly been voluntary (Green et al., 1990). However, the selection pressure exerted by GE crops on pests are of such a magnitude that resistance would evolve rapidly in the absence of effective IRM strategies (Tabashnik, 1994b; Bates et al., 2005), which is why the correct execution of suitable IRM tactics alongside Bt crops have been made mandatory in most GE producing countries.

Mathematical models used to evaluate various IRM strategies indicated that the most effective IRM approach would be a combination of high toxin dose in Bt plants and the provision of a refuge area in the vicinity (Bates et al., 2005), known as the high-dose/refuge (HDR) strategy (Georghiou and Taylor, 1977; Alstad and Andow, 1995). The HDR approach only became a viable option in 1991 when Perlak et al. (1991) demonstrated that the DNA sequence of Bt toxin genes could be specifically altered to induce a dramatic increase in toxin production (dose) within plant tissues. The EPA (2001) therefore defined a high dose Bt expression as 25 times the protein concentration necessary to kill 99% of the susceptible larvae of a species.

1.3.5.2 The high-dose/refuge strategy

1.3.5.2.1 Basic principles of the HDR approach

The basic theory underlying the HDR strategy is based on the premise that resistance is determined by a single-gene locus with two alleles (R= resistant, S= susceptible). By providing a source of non-Bt host plants (known as the ‘refuge’ area), in close proximity to the Bt crop, the survival and proliferation of homozygous susceptible individuals (SS) is guaranteed. The comparatively rare homozygous resistant (RR) individuals that survive on the Bt crop will likely mate with one of the many susceptible individuals arising from the refuge area. The heterozygous (RS) offspring of such matings will then theoretically be controlled by the high-dose of Bt toxins expressed in the tissues of the Bt crop plants. In effect, this strategy decreases the chance that the R-allele is propagated to the next generation, thereby slowing down the rate of evolution of resistance in the pest population (Cohen, 2000; USEPA, 2001; Gould, 2000; Tabashnik et al., 2009).

The refuge area can be structured in several different ways in relation to the Bt crop field. A refuge can be planted as a separate block, as strips within the Bt field or as a perimeter surrounding the Bt field (Figure 1-11) (Cullen et al., 2008). These are referred to as structured refuges. Recently, producers have also been offered the option of using unstructured refuges, also known as seed mixtures, seed blends, refuge-in-a-bag, or mosaic refuge (Onstad et al., 2011, 2018). For this option, the seed company includes a percentage of non-Bt seed within the bag of Bt seed. This results in the non-Bt seeds being planted randomly within a crop field (Figure 1-11) (Onstad et al., 2011, 2018).

Figure 1-11 Examples of different refuge area configurations.

The size of the refuge is calculated as a percentage of the Bt crop field. The guidelines for the size and structure of a refuge differ between crops, target pests, and agricultural environments. In South Africa, for example, an untreated maize refuge area (which receives no pesticide applications) should account for at least 5% of the Bt maize area. If a producer chooses to apply pesticides onto plants in the refuge area, the required refuge size increases to 20% of the Bt maize area (USEPA, 2001; Kruger et al., 2012a; Van den Berg et al., 2013).

1.3.5.2.2 Limiting factors of HDR strategy

Although the theoretical model behind the HDR strategy is sound, the in-field reality seldom reflects the theoretical ideal (Mallet and Porter, 1992). Producers’ compliance with the mandated refuge requirements has been identified as one of the critical weaknesses of the HDR strategy and has been reported as a main reason for resistance evolution and eventual Bt crop product failure (USEPA, 2001; Hurley and Mitchell, 2014). Where the refuge compliance was low, target pests rapidly evolved resistance to Bt crops (Kruger et al., 2012a; Campagne et al., 2016), but in cases where stringent refuge requirements were enforced and producers were educated on the importance of the correct implementation of the HDR strategy, pests have remained susceptible for close to two decades (Tabashnik et al., 2017).

Other pitfalls in the HDR approach include the possibility that, where Bt crops target multiple pest species, the toxin dose might be high for some species but moderate or low for others (Bates et al., 2005; Reisig and Reay-Jones, 2015). Furthermore, expression levels of Bt toxin in the parental line of an event may be high, but expression could show great variability in the subsequent varieties derived from that line (Van den Berg et al., 2013). Pest larvae could also be exposed to sub-lethal doses of Bt toxins (which fast-track resistance evolution) when cross-pollination occurs between the Bt crop and the non-Bt refuge plants (Onstad et al., 2011; Kang et al., 2012), or when 43

the larvae come into contact with non-expressing Bt plants inside the Bt field (Bates et al., 2005). These non-expressing ‘off-types’ could represent up to 3% of the Bt crop, leading to the unintentional formation of seed mixtures (Gould, 1998).

Despite the reality of these limiting factors, the HDR approach is still considered the most effective of the various IRM strategies and it contributes significantly to the delay of resistance development in numerous species (Table 1-3).

Table 1-3 Insect pest species for which no decrease in susceptibility to Bt toxins have been reported since the cultivaton of Bt maize and Bt cotton in different countries. (Adapted from Tabashnik and Carrière, 2017)

Year High- Insect species Crop Toxin Country marketed dose Heliothis virescens Cotton Cry1Ac Mexico 1996 ? Cotton Cry1Ac USA 1996 Yes Cotton Cry2Ab USA 2003 Yes Ostrinia nubilalis Maize Cry1Ab Spain 1998 ? Maize Cry1Ab USA 1996 No Maize Cry1Fa USA 2003 No Pectinophora Cotton Cry1Ac China 2000 Yes gossypiella Cotton Cry1Ac USA 1996 Yes Cotton Cry2Ab USA 2003 Yes Helicoverpa armigera Cotton Cry1Ac Australia 1996 No Cotton Cry2Ab Australia 2004 Yes Helicoverpa punctigera Cotton Cry1Ac Australia 1996 ? Cotton Cry2Ab Australia 2004 Yes Spodoptera furgiperda Maize Vip3Aa Brazil 2010 Yes Sesamia nonagroides Maize Cry1Ab Spain 1998 Yes Chrysodeixis includens Soybean Cry1Ac Brazil 2013 No Diatraea grandiosella Maize Cry1Ab USA 1999 ? Year marketed indicates the first year of commercial planting of a Bt crop in the region monitored. High-dose indicates the test for the high-dose standard based on direct or indirect evidence.

1.3.5.3 Other IRM strategies

Although the HDR strategy is the predominant IRM approach in Bt crops, there exist several additional strategies or variations thereof that can be used in conjunction with the HDR approach to bolster the efficacy of the overall IRM strategy (Bates et al., 2005; Tabashnik and Carrière, 2017).

1.3.5.3.1 Gene pyramiding/stacking

One such strategy is the use of gene pyramiding or gene stacking. First-generation GE crops only expressed a single transgene. However, modern biotechnology has made it possible to engineer crops to express a multitude of transgenes. This is referred to as gene stacking or gene pyramiding (Gould, 1998; Carrière et al., 2015, 2016). Gene stacking denotes the incorporation of two or more insecticidal traits into a sinlge variety, to target unrelated pest species (e.g. Bt toxins targeting lepidopteran and coleopteran pests), and/or herbicide-tolerance traits. Conversely, gene pyramiding only signifies GE crops that express multiple insecticidal traits that all target closely related species of pests (Bravo and Soberón, 2008; Razze and Mason, 2012).

Because the most common resistance mechanism against Bt toxins is the alteration in the binding site in the insect gut (Peterson et al., 2017), the Bt traits incorporated into a pyramid generally target different receptor sites (Ferré and Van Rie, 2002). A pest individual will need to be homozygous resistant (RR) to both Bt traits in order to survive on the pyramid Bt crop. Due to the fitness cost associated with resistance, theory predicts that homozygous resistance to more than one Bt trait will be extremely rare (Manyangarirwa et al., 2006; Carrière et al., 2010; Tabashnik et al., 2013). This could delay the evolution of resistance significantly, so long as the following factors apply: one of the pyramided traits do not confer cross-resistance to the other, the expression of both the Bt traits meet the high-dose requirements, and pyramided cultivars are separated temporally or by distance from single trait cultivars expressing one of the toxins in the pyramid (Tabashnik and Carrière, 2017). Modeling and experimental evidence has shown that cross- resistance will nullify the advantages of multiple pyramided traits (Carrière et al., 2015, 2016).

The pyramiding or stacking of of traits has the advantage that a single crop cultivar can provide the producer with protection against various pest species (Bravo et al., 2011). However, as stacked/pyramid varieties gain market share, the option for producers to opt for GE cultivars containing only specific or single traits they require in their particular farming system, will decline. Since pyramid varieties come at a premium, producers will most likely have to pay for, and deploy unnecessary GE crop traits (Onstad et al., 2011).

1.3.5.3.2 Modified Bt and novel toxins

The modification of existing Cry toxins to bypass primary receptor sites or to act synergistically with other Cry and Cyt toxins could lead to resistant populations being rendered susceptible once again to these Cry toxins (Bravo and Soberón, 2008). Additionally, novel non-Bt toxins derived from other organisms (e.g. other bacteria, plants, insects etc.) that target the digestive functioning and development of phytophagous insects could further diversify the toxins available for incorporation into GE crops, as well as the range of target pests (Jouanin et al., 1998; Shelton et al., 2000; Ferry et al., 2004; Tabashnik and Carrière, 2017).

Furthermore, the possibility of limiting the expression of Bt (or other) toxins to targeted plant parts or exact growth stages could be achieved by coupling the transgene to tissue-specific, wound- specific, or inducible promotors (Gould, 1988; Shelton et al., 2000; Ferré et al., 2008). As a result, the requirement of separate refuge areas could be waived as the portion of the pest population under selection pressure would be significantly reduced.

1.3.5.3.3 Naturally occurring refuge areas

An area of plants can only be considered a refuge if it is characterized by a high percentage of larval survival of the target pest and production of sufficient numbers of good quality adults. These plants should also be abundant in areas where Bt-crops are cultivated (Van den Berg, 2017; Li et al., 2017). Since numerous pest species targeted by Bt crops are polyphagous, it is suggested that areas containing alternative host plants near Bt crops could act as refuge areas (Losey et al., 2002; Huang et al., 2011; Frisvold and Reeves, 2014). This would in effect replace structured refuges, greatly increasing the proportion of productive land that can be devoted to cultivating Bt crops. Natural refuge plants could include both other non-Bt cultivated crops, or wild host plants that are present in adjacent uncultivated areas (Van den Berg, 2017).

Even though the use of natural refuges has been approved by the EPA for management of Heliothis species in Bt cotton (Huang et al., 2011) in the USA, most studies showed that this approach may not be effective to delay resistance evolution by maize stem borer species. It has been reported that maize stem borers show strong ovipositional preference for maize plants compared to other hosts, that very few larvae develop to adults on plants other than maize, and that adult emergence from wild host plants and the Bt-maize crop may not be synchronized (Losey et al., 2001; Van den Berg, 2017; Li et al., 2017). Furthermore, the carrying capacity of wild host plants of stem borers is too low to produce sufficient numbers of moths to mate with those that survive on Bt plants (Van den Berg, 2017).

1.3.5.3.4 Incorporation of IPM principles

The use of GE Bt crops should be nested in an integrated pest management (IPM) program (McGaughey and Whalon, 1992). IPM has been defined by Kogan (1998) as:

“A decision support system for the selection and the use of pest control tactics, singly or harmoniously coordinated into a management strategy, based on cost/benefit analyses that take into account the interests of and impacts on producers, society and the environment.”

In their studies, Bates et al. (2005) and Anderson et al. (2019) emphasized that IRM should include the use of traditional IPM aspects such as cultural and biological control, and persistent monitoring to detect changes in the biology, behaviour, and ecology of pest populations. Cultural control approaches such as narrow planting windows, eradication of diapausing larvae, restrictions on hectarage planted with Bt crops, and temporal/spatial rotations of different Bt crops will reduce the proportion of the pest population subjected to resistance selection (Bates et al., 2005; Manyangarirwa et al., 2006; Midega et al., 2006; Ferré et al., 2008). Furthermore, the cultivation of Bt crops is compatible with the use of biological control since these crops have no or few harmful effects on beneficial predators and parasitoids than insecticide sprays (Frisvold and Reeves, 2014). Unsprayed refuge areas with high pest population densities may allow the population of natural enemies in the Bt crop area to flourish, which could increase the mortality of the resistant individuals in the Bt crop (Gould, 1988; Bates et al., 2005; Midega et al., 2006; Onstad et al., 2011).

Monitoring is a critical part of IRM programs and aims to detect field-evolved resistance in insect pest populations at an early stage. This would allow producers to apply mitigating measures before complete Bt toxin failure (Tabashnik et al., 2013; Kotey et al., 2017). To increase the accuracy of resistance monitoring, samples must be taken from pest populations residing on Bt crop plants, as well as from non-Bt host plants in the vicinity. Since testing for the increase of resistance alleles in the pest population should be conducted regularly, the greatest impediments to resistance monitoring are the financial costs and time-consuming nature of such programs (Huang, 2006).

1.3.5.4 Theoretical assumptions that underpin the HDR strategy

The efficacy of the HDR strategy in delaying the onset of Bt resistance in pest populations are determined by the validity of three key assumptions: 1) the inheritance of resistance is functionally recessive, 2) the initial frequency of the resistance allele is low, and 3) there are abundant refuges in the vicinity of the Bt crop. These assumptions are ubiquitously acknowledged to be critical to the proper functioning of the HDR strategy. Tabashnik et al. (2013) and other authors added to 47

this list, two additional factors that act to delay resistance evolution, namely fitness cost to resistance, and incomplete resistance.

The first assumption holds that if the Bt toxin is not expressed at a high-dose for a particular target species, the mortality of heterozygotes (RS) will decline, which would lead to a rapid increase in resistance alleles in the gene pool of the pest species. However, if the dose is of sufficient magnitude, the resistance allele would become functionally recessive (Tabashnik and Croft, 1982; Giband, 1998; Bourguet et al., 2005; Tabashnik et al., 2009; Glaser and Matten, 2003). Secondly, the low frequency of resistance alleles (suggested frequency of <0.001) (Gould, 1998; Huang et al., 2011; Camargo et al., 2018) lowers the probability that homozygous resistant (RR) individuals will find and mate with each other, advancing the creation of heterozygotes that are controlled by the high toxin dose (Giband, 1998; Glaser and Matten, 2003; Ferré et al., 2008; Van den Berg, 2017). The presence of ample refuge areas makes it more likely that a overflooding ratio of 1:500 resistant to susceptible individuals is achieved, as was recommended by the USEPA (1998) when the initial resistance allele frequency is assumed to be <0.05 (Glaser and Matten, 2003). The added factors of fitness cost of resistance and incomplete resistance both cause the refuge areas to act as a selection pressure against resistance (Gassmann et al., 2009; Carrière et al., 2010; Tabashnik et al., 2013). Fitness cost indicates that susceptible individuals fare better than resistant individuals in the absence of Bt toxins, while incomplete resistance occurs when resistant individuals fare better on non-Bt plants than on Bt plants (Tabashnik et al., 2013; Tabashnik and Carrière, 2017).

The five factors discussed above by no means exhaust the assumptions upon which the HDR strategy is based. For example, the design of the refuge (type of refuge, structure, distance from Bt crop etc.) must take into account the biology and behaviour or the specific target pest (Pannuti et al., 2016). Seed mixtures (refuge-in-a-bag) will not be an appropriate strategy to use with a pest that is highly mobile during the larval stages, since this could lead to larvae being exposed to sub-lethal doses of the Bt toxins, accelerating resistance evolution (Mallet and Porter, 1992; Roush, 1997; Davis and Onstad, 2000; Carrière et al., 2016). However, if the adult stage of a pest is more likely to mate near the location of emergence before migrating, genetically similar pairings will increase in separate refuge areas, leading to a lower frequency of heterozygous offspring (Onstad et al., 2011; Coates, 2016). The HDR strategy also assumes that adults of both resistant and susceptible populations mate randomly; that is, that the adults show no mating preference for either genotype and are not reproductively isolated due to difference in development time (Giband, 1998; Carrière et al., 2004). Once mated, it is assumed that gravid female adults do not preferentially lay their eggs on either Bt or non-Bt plants but select oviposition sites at random (Han et al., 2016). There is a myriad of factors that could influence the efficacy of the HDR strategy

- many of which are related to the biology, behaviour, and/or ecology of the target insect pest and the agro-ecology of the Bt crop.

Tabashnik and Carrière (2017) found that results from theoretical models and experimental evidence supported the theory underlying the HDR strategy. The 16 cases of practical resistance evolution (Table 1-2) can all be traced back to the violation of key assumptions, such as a shortage of refuges, or Bt crops that failed the high-dose requirements (Tabashnik and Carrière, 2017). The evolution of resistance by B. fusca to Bt maize in South Africa is no different.

1.3.6 Resistance evolution of Busseola fusca to Bt maize in South Africa

1.3.6.1 History of Bt maize in South Africa

The widespread cultivation of Bt crops in the world began in 1996. South Africa (SA) was an early adopter (and the first country on the continent) of this new agricultural biotechnology, with the first GE crop (Bt cotton) made commercially available in 1997. A year later, Bt yellow maize (animal feed) became the second Bt crop approved for commercial use, followed by Bt white maize (food crop) in 2001 (Schimmelpfennig et al., 2012). Thus far, SA is the only country in the world to embrace GE varieties of a staple food crop (maize) (Gouse et al., 2005; Keetch et al., 2005; Goldblatt, 2010). The initial adoption of Bt maize was relatively slow (after 2 years Bt maize had a market penetration of only 3%), due to a lack of Bt hybrids adapted to local conditions and the absence of white Bt maize varieties, which comprises between 50 – 60% of cultivated maize in SA (Schimmelpfennig et al., 2012). However, when these obstacles were overcome, the adoption of Bt maize increased exponentially: from a mere 3000 ha in 1998 to 1.96 million ha in 2017 (ISAAA, 2017).

During the nearly two decades of Bt maize production in SA, 42 GE maize events have been approved for food and feed (ISAAA, 2017). Initially Monsanto monopolized the supply of Bt maize, but producers now have a choice of products from several agricultural companies (Ezezika et al., 2012). Bt maize was made available in SA to specifically target the cereal stem borer species B. fusca, C. partellus, and S. calamistis (Van den Berg et al., 2013). Gouse et al. (2008) found that Bt maize conferred a yield advantage of 11% and 10% to commercial irrigated and dry land producers, respectively, and Raney (2006) indicated that smallholder and subsistence farmers also reported higher yields (32%) (Gouse et al., 2005) from adopting Bt maize. Additionally, Gouse et al. (2008) suggested that these yield gains could be substantially higher in areas or periods of high stem borer infestation levels.

1.3.6.2 Resistance evolution of Busseola fusca to Bt maize

1.3.6.2.1 Timeline of resistance evolution of B. fusca

The first efficacy evaluations for Bt maize in SA, were conducted during the growing seasons of 1994 to 1997 and relied on field and greenhouse evaluations of plants that were artificially infested with first-instar larvae of the target pest species (Van Rensburg, 1999; Strydom et al., 2018). Laboratory trials to determine the baseline susceptibility of the larvae using Bt toxin-incorporated diets could not be carried out, due to difficulties rearing large enough populations of B. fusca on artificial diet (Van Rensburg, 1999). The efficacy evaluations showed that the ability of the experimental Cry1Ab events to control B. fusca varied significantly, and that B. fusca were notably more tolerant to these events than C. partellus (Van Rensburg, 1999; Van den Berg et al., 2013). Although these results indicated that the high-dose requirement was not met for B. fusca, hybrids of the event MON810 was approved for commercial release, since the efficacy was deemed sufficient from a crop protection perspective (Van Rensburg, 2007). Reports of poor control of B. fusca with Bt maize events expressing Cry1Ab and other proteins were also made in East Africa (Tende 2010, Mugo et al. 2011), and even with pyramids of Cry proteins (Mugo et al. 2005).

At harvest time of the second season after commercial release of Bt maize (1998/1999), several localities reported damage and the presence of diapause larvae of late infestations of B. fusca, even though no damage was reported during the early vegetative stages (Van Rensburg et al., 2001). It has been highlighted that that these observations should have prompted the initiation of resistance monitoring programs, since these infestations were a good indication that Bt maize did not meet high-dose standards for B. fusca (Van den Berg, 2012). During the growing seasons of 2004-2006, an increasing number of locations in the Vaalharts irrigation scheme (an irrigated valley of 32 000 ha) reported severe B. fusca damage during the vegetative growth stages of Bt maize. The first official report of B. fusca resistance to maize expressing Cry1Ab was made in 2007 (Van Rensburg, 2007). Subsequent results from fitness evaluations of resistant populations indicated that the larvae could be reared successfully for more than four generations on Cry1Ab Bt maize. By 2011, surveys indicated the presence of resistant populations of B. fusca throughout the maize production area of SA (Van den Berg, 2013).

1.3.6.2.2 Violated assumptions of the IRM strategy

The evolution of resistance to Bt maize by B. fusca cannot be ascribed to any single factor. Instead, there are several key aspects that must be considered. It is clear from the initial efficacy evaluations and subsequent reports of in-field damage that the high-dose requirement of Bt toxin expression was not met for B. fusca (Tabashnik et al., 2009; Campagne et al., 2013). Thus, the

inheritance of resistance alleles was not functionally recessive. Additionally, the resistance mechanism of B. fusca to Cry1Ab protein was shown to be dominant by Campagne et al. (2013). Non-compliance by producers to mandated refuge requirements were also pervasive during the early years after the commercial release of Bt maize. Up until 2004, less than a third of farmers in the Vaalharts area were compliant to refuge requirements (Kruger et al., 2011; Kruger et al., 2012a). The high-dose and refuge requirements are fundamental to the efficacy of the HDR strategy, and the violation of these two assumptions were central to the product failure of Cry1Ab maize in SA (Van den Berg et al., 2013).

Apart from the two assumptions discussed above, other factors that could have contributed to the evolution of resistance have been identified. The initial frequency of resistance alleles in populations of B. fusca were not determined, and it has been suggested that some populations with low susceptibility could already have existed prior to the commercial release of Bt maize (Raybould and Quemada, 2010). Van den Berg et al. (2013) made the case that very little is known about the validity of basic assumptions of the behaviour and ecology of B. fusca in Bt maize fields, and that these factors could have played a significant role in B. fusca resistance evolution. For example, assumptions about the migration behaviour of larvae and adult stages, and the mating and oviposition preference of B. fusca female moths. Moreover, very little is known about how resistant and susceptible populations differ in these characteristics.

1.3.6.3 Future prospects of Bt maize in South Africa

It is generally accepted that the use of pyramided Bt maize events is the only viable option to control the resistant population of B. fusca. However, resistance to toxins in the Cry1 or Cry2 class severely limit the viable toxins for incorporation into a pyramided event, since cross- resistance would soon render the pyramided event obsolete (Mahon et al., 2012; Carrière et al., 2015). Novel Bt toxins (e.g. Vips) would also be an option, but no alternative insecticidal toxin has yet been shown to have high efficacy against stem borers such as O. nubilalis or B. fusca (Lee et al., 2003). The stacked event MON89034 (which expresses the Cry proteins Cry1A.105 and Cry2Ab2) was commercially released in South Africa in 2011 and has successfully controlled resistant populations up until the current growing season (Strydom et al., 2018).

In the aftermath of the resistance evolution of B. fusca to Cry1Ab maize, agricultural companies launched monitoring programs to increase refuge compliance and started campaigns to educate producers of the importance of sound IRM practices (Kruger et al., 2009; Kruger et al., 2011). However, Bt maize events expressing Cry1Ab were not removed from the marketplace, and cultivation thereof continued in parts of the maize production area, even though the product has failed in consecutive seasons in several other areas. Van den Berg et al. (2013) suggested that

it might have been more prudent to withdraw the affected Bt maize events from areas where resistance occurred, and that alternative control measures (e.g. chemical or cultural control) could have been implemented to suppress pest populations in the absence of Bt crops. This remedial protocol was suggested as a way to prevent the simultaneous cultivation of single-trait and pyramided cultivars, since resistance to the single-trait could compromise the sustainability of pyramided cultivars (Zhao et al., 2005; Carrière et al., 2015, 2016).

1.4 Structure of the thesis

Chapter 1 formulates the rational of the study and presents the aims, objectives, and hypotheses. The literature study provides an overview of the importance of maize and impact of B. fusca infestations, the use of Bt maize to control this pest, and the available strategies to delay resistance evolution to Bt toxins. It also highlights the gaps in current knowledge about the effect of pest biology and behaviour on the design and efficacy of IRM strategies, and the challenges to implementing current IRM strategies in African agricultural systems.

Chapter 2 (first article) investigates the assumption that B. fusca adults and larvae (of Bt-resistant and susceptible populations) select and colonize maize plants at random and relates the effect of the findings on the design and implementation of IRM strategies for this pest on Bt maize.

Chapter 3 and 4 (second and third article) focusses on the assumption that B. fusca larvae migrate extensively within maize fields. These articles evaluate the effect of Bt toxins, plant age and plant density on the migration behaviour of B. fusca larvae in laboratory, semi-field and field trials. The results of the three experimental scales are compared and are again discussed in the context of IRM.

Chapter 5 (fourth article) describes how the findings of chapter 2, 3 and 4 might be integrated into IRM strategies for Bt maize in Africa agricultural systems. This chapter emphasizes that the design and implementation of an IRM strategy cannot consider the biological and behavioural parameters identified in the preceding chapters in isolation. Rather, these parameters should form part of a wider analysis that not only considers the characteristics of the target pest species, but also the greater pest complex, the climatic conditions, and the level of organization that is prevalent in a specific agricultural area.

Chapter 6 summarizes the key findings of the thesis by discussing the validity of the hypotheses postulated for this study and provides recommendations for future research on this subject.

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CHAPTER 2

Preference of Bt-resistant and susceptible Busseola fusca moths and larvae for Bt and non-Bt maize

Citation:

Visser, A., Du Plessis, H., Erasmus, A. & Van den Berg, J. 2019. Preference of Bt-resistant and susceptible Busseola fusca moths and larvae for Bt and non-Bt maize. Entomologia Experimentalis et Applicata, 167: 849–867.

CHAPTER 3

Plant abandonment by Busseola fusca (Lepidoptera: Noctuidae) larvae: do Bt toxins have an effect?

Citation:

Visser, A., Du Plessis, H., Erasmus, A. & Van den Berg, J. 2020. Plant abandonment by Busseola fusca (Lepidoptera: Noctuidae) larvae: do Bt toxins have an effect? Insects, 11, 77.

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CHAPTER 4

Larval migration behaviour of Busseola fusca (Lepidoptera: Noctuidae) on Bt and non-Bt maize under semi-field and field conditions.

Citation:

Visser, A., Du Plessis, H., Erasmus, A. & Van den Berg, J. 2020. Larval migration behaviour of Busseola fusca (Lepidoptera: Noctuidae) on Bt and non-Bt maize under semi-field and field conditions. Insects, 11, 16.

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CHAPTER 5

Bigger, faster, stronger: a review of the implications of biology, competition and inter-species interactions within mixed populations of lepidopteran pests for bt maize IRM in Africa.

Prepared for submission to: Journal of Integrated Pest Management

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4.1 Abstract

This review used a hypothetical scenario of mixed populations of Busseola fusca (Fuller) (Lepidoptera: Noctuidae), Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) as a model to investigate the potential effects of mixed populations of lepidopteran pests on the design and implementation of insect resistance management (IRM) strategies for Bt maize on smallholder farms in Africa. To predict the structure of such mixed populations in different agroecological zones, the biological and behavioural characteristics that affect competitiveness of these species were identified and analysed. Additionally, the validity of the assumptions that underlie the high-dose/refuge strategy were compared between the three species. The differences between the species, and the influence thereof on the choice of IRM strategy for a specific receiving environment, were explored through discussion of three hypothetical scenarios. This investigation suggests that the use of separate refuges as component of an IRM strategy against mixed pest populations in smallholder Bt maize fields may be unwise. A seed mixture approach, coupled with an effective integrated pest management strategy would be more sensible, since it could limit the opportunity for a single species to dominate the species complex. The dynamic interactions in a multi-species community and domination of the species complex by a single species may influence moth and larval response to maize plants, which could lead to increased infestation of Bt plants, and subsequent increased selection pressure for resistance evolution. This thought experiment provides insights into the unique challenges that face the deployment of Bt maize in Africa.

Key words: resistance evolution, insect resistance management, integrated pest management, maize

4.2 Introduction

The projected population growth for Africa necessitates an equal or greater increase in agricultural productivity to ensure future food security in the region (World Bank, 2008; Ray et al. 2013; FAO, 2015; 2018). The introduction of agricultural technologies such as Bt maize which has been genetically modified (GM) to express insecticidal proteins from the bacterium Bacillus thuringiensis, could form a key part in the intensification of African agriculture (Conceição et al., 2016; Smyth, 2017; Carzoli et al., 2018). Presently, the commercial use of Bt maize is limited to only one of the 54 countries in Africa (South Africa), though several other African countries are currently conducting research with the aim of commercial release of GM crops in their agricultural sectors (ISAAA, 2017).

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Although the main target pests of Bt maize in Africa is a complex of lepidopteran maize stemborer species (Busseola fusca (Fuller) (Noctuidae), and Chilo partellus (Swinhoe) (Crambidae)) (Van den Berg, 2017), there are several other economically important lepidopteran pests that attack maize that are also susceptible to Cry proteins expressed in Bt maize plants. Sesamia calamistis (Hampson) (Noctuidae), Chilo orichalcociliellus (Strand) (Crambidae) and Eldana saccharina (Walker) (Pyralidae) (Kfir et al., 2002; Calatayud et al., 2014; Mutamiswa et al., 2017a,b; Ntiri et al. 2019) are among these. Out of this multitude of species, the indigenous B. fusca and invasive C. partellus are the two most important stemborer species in many of the agricultural regions on the continent (De Groote, 2002; Kfir et al., 2002; Fitt et al., 2004; Calatayud et al., 2014; Ntiri et al., 2016). The community composition and densities of these stemborers vary by region, as do their injuriousness. Despite the large number of stemborer species, they are not the only lepidopteran pests that affect maize production in Africa. In 2016, the leaf feeding Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) invaded several African countries (Goergen et al. 2016), and subsequent surveys reported the presence of the pest in nearly all countries in sub- Saharan Africa (Nagoshi et al. 2019). Furthermore, Helicoverpa armigera (Hübner) (Noctuidae), which is an indigenous and polyphagous species, also attacks maize and cotton in Africa (Green et al. 2003; Van Wyk et al. 2008).

The main advantages of cultivation of Bt crops are reduced pesticide use, increased pest control and reduced yield losses (Brookes and Barfoot, 2018). This technology could also lead to area- wide suppression of pest populations (Hutchison et al., 2010; Dively et al., 2018) and a decrease in the use of insecticides that are harmful to both humans and the environment (Naranjo, 2009; Brookes and Barfoot, 2013; Tefera et al., 2016). Fewer sprays of broad-spectrum insecticides not only decreases input costs, but also ensures that beneficial insects (e.g. parasitic wasps that attack stem borers) become more abundant, which further contributes to pest control and the aims of integrated pest management (IPM) (Wolfenbarger et al., 2008, Romeis et al., 2019). Reduced larval feeding damage to ears of Bt maize plants, limits the formation of mycotoxic fungal infections (Munkvold et al., 1999; Hellmich et al., 2008; Ncube et al. 2017, 2018; Pellegrino et al., 2018).

Unfortunately, this technology’s greatest strength – the season-long expression of highly effective Bt toxins – is also the source of its greatest weakness, i.e. evolution of resistance in pest populations due to the sustained selection pressure exerted by Bt toxins (Tabashnik, 1994; Glaser and Matten, 2003; Siegfried and Hellmich, 2012; Siegfried and Jurat-Fuentes, 2016). The threat that insect resistance evolution holds to the sustainability of Bt crops, and the associated economic and environmental benefits associated with its use, prompted the development of insect resistance management (IRM) strategies before the commercial release of the first Bt crop

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(Gould, 1998; Tabashnik and Carrière, 2017). The goal of these strategies is to delay resistance evolution in target pest populations (Head and Greenplate, 2012; Onstad et al., 2018). Several IRM strategies have been put forward, but the most popular has been the high-dose/refuge (HDR) strategy (Gould, 1998; Bourguet et al., 2005; Tabashnik et al., 2013). This approach involves planting a refuge area of non-Bt maize plants to accompany Bt maize planting with the aim of sustaining a susceptible population of the target insect pest. The large numbers of homozygous susceptible individuals are then the primary mates for the rare homozygous resistant individuals that survive on Bt fields, giving rise to predominantly heterozygous offspring (USEPA, 1998; Gould, 2000). The expression of the Bt toxin should also be of a high enough dose (referred to as high-dose expression) to control the heterozygous individuals, leading to an overall reduction in the frequency of resistant alleles in the pest population (Gould, 2000; Bates et al., 2005; Tabashnik et al., 2008, 2009; Tabashnik and Carrière, 2017).

It is important to note that the HDR strategy is based on several key assumptions about the target pest population and the GM crop, i.e. 1) initial low frequency of resistance alleles in the pest population, 2) fitness costs are associated with resistance, 3) resistance is incomplete, 4) resistance must be functionally recessive, and 5) presence of abundant refuges of non-Bt host plants (Tabashnik and Carrière, 2017; Onstad et al., 2018, Anderson et al., 2019). Violation of these assumptions leads to rapid evolution of resistance (Tabashnik et al., 2013), for example, B. fusca populations in South Africa (Van den Berg et al., 2013; Campagne et al. 2013, 2016). Several cases of field evolved resistance against Bt crops have been reported worldwide (Tabashnik and Carrière, 2017), mostly due to poor compliance to resistance management strategies (Kruger et al., 2009, 2012a; Tabashnik et al., 2013; Farias et al., 2014a,b).

A mere eight years after the commercial release of the single Bt toxin (Cry1Ab) maize event MON810 in South Africa, the first case of B. fusca resistance was reported (Van Rensburg, 2007; Kruger et al., 2011). Resistance to Cry1Ab has since been documented in many maize production areas in South Africa. Currently, the growers in South Africa use the multi-toxin (pyramided) maize event MON89034, which simultaneously expresses the Bt proteins Cry1A.105 and Cry2Ab2 (Strydom et al., 2018). This is the only pyramid event released commercially in South Africa and it is still effective at controlling Cry1Ab- resistant B. fusca populations (Strydom et al. 2018). The pyramid event started to replace the single-gene event during 2012, with the latter largely phased out during 2018. Strydom et al. (2018) did however report that a shift in susceptibility of B. fusca to the pyramid event was observed.

The rapid evolution of resistance of B. fusca populations in South Africa highlighted the importance of an effective IRM strategy to ensure the sustainable use of Bt maize in other African countries (Van den Berg et al., 2013). However, the design of an IRM strategy for the Bt maize 137

receiving environments on the continent is complicated by several factors. Agriculture in Africa is practiced both on large scale commercial farms and smallholder farming plots, with the latter being by far the most prevalent (Thompson, 2008; World Bank, 2008; Aheto et al., 2013). This heterogeneous nature of the agricultural system makes the implementation of a standardized, structured IRM strategy (as is the case in developed countries) impossible (Jacobson and Myhr, 2012; MacIntosh, 2009), since implementing separate refuges on small holder plots is challenging, due to the limited scale of production (Bates et al., 2005; Carroll et al. 2012; Van den Berg, 2013; Assefa and Van den Berg 2015; Fisher et al., 2015; Kotey et al. 2016; Li et al., 2017).

It has been suggested that the use of seed mixtures, also known as ‘refuge-in-a-bag’ or seed blends, could be a workable alternative to planting separate refuges in smallholder agricultural systems (Carroll et al., 2012; IRAC, 2013; Carrière et al. 2016). This approach involves mixing in a predetermined percentage of non-Bt seed within the Bt seed bag. This means that the refuge plants are incorporated randomly within a field of Bt maize, simplifying compliance to refuge requirements (Gould, 1998; Carrière et al., 2016). Apart from being easier to implement, using seed mixtures and pyramids for IRM have the advantage of being more resilient to certain violated IRM assumptions (Storer et al., 2012). For example, a pyramiding strategy would still delay the evolution of resistance even if only one type of heterozygote has high mortality (Roush, 1998), and seed mixtures could still be successful despite the relatively high frequency of resistance alleles and a lack of recessive inheritance of resistance (Caprio, 1998; Zhao et al., 2003). Unfortunately, the success of the seed mixture strategy is contingent on assumptions of its own, which chiefly relates to the migration behaviour target pest larvae (Mallet and Porter, 1992; Onstad et al., 2018).

An effective IRM strategy must therefore take into consideration both the practical limitations of the agricultural system that it intends to serve, but also the behaviour and biology of the target pest species (Gould, 1998; Head and Greenplate, 2012). This is complicated, however, when the target pest is part of a mixed population of different pest species (Roush, 1997; Bates et al., 2005), for example the occurrence of B. fusca, not only in a maize stemborer complex, but also with other leaf feeding lepidopterans (Ogol et al., 1999; Van Wyk et al., 2008; Mutamiswa et al., 2017b; Ntiri et al., 2016, 2019; Van den Berg et al., 1991a,b; Krüger et al., 2008; Ong’amo et al., 2013; Kuate et al. 2019). Van Wyk et al. (2008) reported 12 lepidopteran species to occur on maize in South Africa, although only a few have pest status. Pest species generally differ significantly with regard to biological and behavioural aspects, even if they are closely related such as stem borers and leaf feeding Lepidoptera. Such differences could complicate the design of Bt maize IRM strategies in areas where mixed pest communities occur (Bates et al., 2005).

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The aim of this study was therefore to review the potential effects of mixed populations of lepidopteran pests on the design and implementation of IRM strategies for Bt maize in smallholder farms in Africa. This thought experiment focussed on mixed populations of the two most important stemborer pest species, B. fusca and C. partellus, and the invasive leaf-feeder, S. frugiperda. First, this study compared the biological and behavioural characteristics that affect competitiveness of these species in order to predict which species may dominate (Figure 4-1–A) in different agroecological zones (Figure 4-2). In the second part, this review addresses the assumptions that underlie the HDR strategy and compares the validity of these assumptions for each of the three above mentioned species (Figure 4-1–B). Finally, the effects that the differences between the species and composition of the pest population could have on the suitability of an IRM strategy for a specific receiving environment is explored through discussion of three hypothetical scenarios (Figure 4-1–C).

4.3 Part 1: Competition between B. fusca, C. partellus, and S. frugiperda

In a mixed population of insect pest species, it is the direct and indirect competitive interactions that determine the structure of the population (Reitz and Trumble, 2002; Wootton and Emmerson, 2005; Kaplan and Denno 2007). Two categories can be used to distinguish between competitive interactions: interference and exploitative competition (Figure 4-3). Interference competition can manifest in direct and indirect interactions, with direct interference involving the infliction of physical harm by one individual on another (through cannibalism, fighting and killing). Indirect interference on the other hand, refers to the use of repellent chemicals, marking of plants, or aggressive behaviour to ward off other individuals from a resource (Figure 4-3–A) (Denno et al. 1995; Duyck et al., 2004; Mutyambai et al. 2015, 2016; Ntiri et al., 2017). Conversely, exploitative competition does not include any direct physical interactions, but rather denotes the deprivation of access to specific resources of one species by the exploitation of the resources by another (Figure 4-3–B) (Denno et al. 1995; Reitz and Trumble, 2002; Preisser and Elkinton, 2008). In a mixed population, the dominance of either B. fusca, C. partellus or S. frugiperda will therefore be determined by how effectively they compete directly and indirectly for resources such as food and space.

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Figure 4-1 Summary of factors that impact the choice of IRM strategy to implement along with the cultivation of a Bt crop. A) Receiving environment, which refers to the agricultural practices of the individual farmers, the level of structure of the wider of the agricultural system, and the agroecological zone where the Bt crop is to be cultivated. B) Validity of the IRM assumptions regarding the genetic and behavioural characteristics for each pest species within the pest complex that will be a target of the Bt crop. C) The competitive interactions that form the basis of the structure of the pest complex. The competitive interactions between pest species will have a significant impact on the behaviour of individual pest species.

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Figure 4-2. Agroecological zones in Africa, categorized by temperature (subtropics and tropics, warm and cool) and by humidity (arid, semiarid, sub-humid, humid) (Sebastian, 2009)

The first report of the presence of C. partellus in Africa was made in Malawi during the early 1900’s (Tams, 1932). This species has since expanded its range throughout east and southern Africa, initially keeping to warmer lowland areas before it also invaded higher altitude regions (Bate et al. 1991; Kfir, 1997; Ebenebe et al., 1999; Yonow et al., 2017). Its expanding distribution has brought it into competition with indigenous stemborer species, with C. partellus often displacing the native stemborers to become the predominant pest of maize and sorghum (Kfir, 1997; Van Rensburg and Van den Berg, 1992; Van den Berg and Van Hamburg, 2015; Mutamiswa et al., 2017a). However, the arrival of the new invasive S. frugiperda could complicate inter-species interactions within stemborer communities, a possibility highlighted by Ntiri et al. (2019). Denno et al. (1995) found that competition was higher when phytophagous species share a feeding niche, especially internal/concealed feeding niches (e.g. stemborers) where they are utilizing the same space- and time-limited resources. The occupation of the whorl as the niche of

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S. frugiperda larvae could mean that there will be minimal direct competitive interference interactions with stemborer larvae, except for the initial developmental stages during which stemborer larvae also feed inside the whorls of maize plants (Slabbert & Van den Berg, 2009; Calatayud et al., 2014; Berger, 1992). However, because the populations of the species still share the resources (maize plants) within a maize field, exploitative competitive interactions will have an impact on the relative dominance of the different species, even if interference competition is low.

Figure 4-3. Underlying mechanisms of competitive interactions between herbivores. A) Interference competition, which can manifest as the infliction of physical harm by one individual on another (through cannibalism, fighting and killing), or the use of repellent chemicals or aggressive behaviour to ward off other individuals from a resource. B) exploitative competition does not include any direct physical interactions, but rather denotes the deprivation of access to specific resources of one species by the exploitation of the resources by another. Direct effects are indicted by solid lines, whereas indirect effects are indicated by dashed lines. (adapted from Kaplan and Denno., 2007)

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4.4 Factors that affect the competitiveness of a species in mixed populations:

4.4.1 Distribution range

There are several biological and behavioural characteristics that could either improve or impair the competitiveness of these pest species during both direct and indirect interactions (Table 4-1). First is the range of optimal climatic conditions needed for development – of which the most important is temperature (Van Rensburg et al., 1985; Honek and Kocourek, 1990; Denno et al., 1995; Ntiri et al., 2016; Yonow, 2017; Babasaheb et al., 2018). Temperature affects nearly all aspects of the growth and development of insects, and most insects have adapted to function optimally only within a specific temperature range (Khadioli et al., 2014; Terblanche et al., 2015; Glatz et al, 2017; Garcia et al. 2018). The broader the range of temperatures that a species can tolerate, the less it will be constrained in its geographical distribution (i.e. the more agroecological zones it could inhabit – Figure 4-2) and the greater its resilience would be to extreme climate conditions (Bale et al., 2002; Battisti and Larsson, 2015; Yonow, 2017; Azrag et al., 2018). Both C. partellus and S. frugiperda have higher optimal temperature ranges than B. fusca (Table 4-1). The composition of stemborer communities have been reported to change along an altitudinal gradient: B. fusca tends to dominate in cooler, higher altitudes, whereas C. partellus dominate in the warmer lowlands (Overholt et al., 2001; Zhou et al. 2003; Mwalusepo et al., 2015; Ntiri et al., 2019). In the midrange altitudes the dominant species is determined by factors other than temperature. For instance, Van Rensburg et al. (1987) found that high levels of humidity (93%) resulted in significantly higher survival rates for B. fusca adults when compared with low levels of humidity (38% humidity) over a 4-day period. Consistent with the results for B. fusca, Tamiru et al. (2012) reported that C. partellus had longer adult longevity at increased levels of relative humidity (80% vs. 40%). However, C. partellus adult females displayed more resilience to changes in humidity, particularly when combined with high temperatures. No significant difference in adult female longevity was observed between the relative humidity levels of 40, 60 and 80% at 30°C (Tamiru et al., 2012). Busseola fusca populations might therefore be more dependent on rainfall (and the accompanying humidity) than C. partellus. With their comparable optimal temperature requirements, S. frugiperda will likely compete with C. partellus for dominance at the lower and mid-range altitudes (Du Plessis et al., 2018).

Mwalusepo et al. (2015, 2018), Ntiri et al. (2019) and Mutamiswa et al. (2017a,b) discussed the possibility that climate change could have a significant effect on the structure of maize stemborer communities. The rising temperatures predicted for much of the African continent (Niang, 2014) could detrimentally affect species such as B. fusca and S. calamistis and lead to the displacement of these once dominant native species by invasive species (Mwalusepo et al., 2015; Mutamiswa et al., 2017a). The effect of climate change on the geographic distribution of insect pest species 143

is a key concern in all agricultural regions across the globe (Björkman and Niemelä, 2015). The altering of climatic conditions could cause insect pest species to invade traditionally cooler agroecological zones that were previously unsuitable for their development, allowing range expansion of pests toward the poles and toward higher elevations (Bale et al., 2002; Forrest, 2016; Reineke and Thiery, 2016; Castex et al., 2018). Concurrently, rising temperatures in the warmer tropics could lead to range contraction, since temperatures could exceed the upper thermal threshold for the development of insect pest species (Gutierrez et al., 2012; Reineke and Thiery, 2016; Castex et al.2018; Deutsch et al., 2018; Lehman et al., 2018). Battisti and Larsson (2015) compiled a summary of the range expansions (ongoing or predicted) of insect pests in agriculture and reported 50 insect pest species of which the range either have been affected by climate change or are predicted to be affected in the future. Included in this list were important lepidopteran maize pests such as Ostinia nubilalis (Hübner) (Crambidae) and Helicoverpa zea (Boddie) (Noctuidae). The range expansion into warming climates of the latter two species in North America were also forecasted by Diffenbaugh et al. (2008). The latter study predicted that the potential range expansion of H. zea was much greater than for the other species included in their study, and that this posed a risk to crops other than maize, since H. zea is a highly polyphagous migratory pest. Diffenbaugh et al. (2008) concluded that insect pest range expansions could lead to severe economic impacts for the newly invaded agricultural systems.

4.4.2 General development time and species voltinism

Another biological trait which affects the indirect competitiveness of species (and which is closely linked to optimal temperature range) is generation time (development time from egg hatch to adult eclosion) and voltinism (number of generations per year) (Honek and Kocourek, 1990; Bale et al., 2002; Altermatt, 2010; Mwalusepo et al., 2015, Azrag et al., 2018). Generation time is closely linked with temperature – the rate of development of an insect species varies with changes in temperature – generally the rate of development will increase with a rise in temperature, but will be fastest at the optimal temperature for development (Honek and Kocourek, 1990; Bale et al., 2002; Mwalusepo et al., 2015; Azrag et al., 2018; Babasaheb et al., 2018).

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Table 4-1. Comparison of biological and behavioural characteristics of Spodoptera frugiperda, Chilo partellus and Busseola fusca.

Temperature range Egg to adult Time to pupation after Response to high larval Species for optimal development development time diapause termination population density (°C) (days) (days)

LD 12:12 at 28 °Cb 25 °C 30 °C LD 16:8 at 26 °Cc

S. frugiperdaa 26 – 32 28.4 19.3 no diapause Cannibalism

Reduced growth rate C. partellusb 25 – 32 42.1 28.9 9.7 and survival

Reduced growth rate B. fuscac 20 – 30 65.4 74.7 34 and survival

Garcia et al. (2018)a Ntiri et al. (2019)a, b, c Prasanna et al. (2018)a Prasanna et al. (2018)a Khadioli et al. (2014)b Ofomata et al. (1999)b Ntiri et al. (2017)b, c Glatz et al. (2017)c Kfir (1993)c

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Longer life cycles are disadvantageous to pest species, since it limits its voltinism and the rate at which the population grows. Thus, the shorter the life cycle of a species, the greater the rate of population growth, and the more dominant the species will be as it relates to exploitative competition with other pest species (Kfir, 1997; Reitz and Trumble, 2002; Mutamiswa et al., 2017a). Busseola fusca has the longest life cycle of the three pest species, with an average generation time of 65.4 days at the optimal temperature of 25 °C (Table 4-1). This limits B. fusca to an average of 3 generations per year, since they generally enter diapause for up to 5 months during winter (Van Rensburg et al., 1985, 1987; Calatayud et al., 2014; Kruger et al. 2012b; Glatz et al., 2017). The life cycle of C. partellus is much shorter than that of B. fusca, but it commonly takes 10 to 12 days longer than the generation time of S. frugiperda (Table 4-1). It is reported that C. partellus can produce up to six generations per year (Table 4-1) (Bate et al. 1990; Mutamiswa et al., 2017a), and under ideal tropical conditions S. frugiperda reproduces continuously throughout the year, up to 8 generations or more (Luginbill, 1928; Andrews, 1988; Day et al., 2017; Du Plessis et al., 2018). The generation time, voltinism, and time of peak moth flight activity of B. fusca have been shown to differ over a temperature gradient ranging from the eastern to the western parts of the maize production areas in South Africa (Van Rensburg et al., 1985). Because the climate more closely approximates the ideal temperatures for B. fusca development in the east, a shortened development time and an additional generation is often observed in these regions compared to the western half of the maize production area (Van Rensburg et al., 1985). Another testament to the advantage that climate change will confer to the invasive species, is the fact that the duration of life cycles of both S. frugiperda and C. partellus decreases with an increase in temperature, while the duration of the life cycle of B. fusca is prolonged even further at increased temperatures (Table 4-1).

Climate change impacts (predominantly the increase of temperatures) on the voltinism of insect pests has been investigated by several authors (Porter et al., 1991; Bale et al. 2002; Steinbauer et al., 2004; Tobin et al., 2008; Jönsson et al., 2009; Altermatt, 2010; Kroschel et al., 2013; Prasad et al., 2012; Fand et al., 2014; Khadioli et al., 2014; Fand et al., 2015; Babasaheb et al., 2018). Altermatt (2010) proposed that, apart from rapid larval development, an increased number of generations of lepidopterans per year could also be explained by an earlier onset of the flight period due to warmer temperatures in spring, and the replacement of univoltine populations by bi- or polyvoltine populations migrating from warmer agroecological zones. Babasaheb et al. (2018) reported on three lepidopteran pests for which an additional 1-3 generations per year are predicted by 2050: C. partellus (Khadioli et al. 2014), Phthorimaea operculella (Kroschel et al. 2013), and Spodoptera litura (Fand et al. 2015). Gagnon et al. (2019) also found that O. nubilalis populations in North America will likely develop an additional generation and have higher densities

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and wider distributions due to climate change, which will increase the pest status of this species in sweet corn.

Climate change could increase the impact of insect pests on crop yield (Bale et al. 2002; Björkman and Niemelä, 2015; Babasaheb et al. 2018), since higher temperatures not only shortens development time of most species, but also elevates their metabolism and population growth rates, which consequently increases their rate of consumption. A study conducted by Deutsch et al. (2018) found that an increase of 2 °C of the average global surface temperature would lead to a median increase of 31% of the global maize yield losses to insect pests. This translates to a loss of 62 metric megatons maize per year. It has been reported that the yields in temperate agricultural regions will be impacted more by insect pests than tropical regions. This is due to the fact that warming in the temperate regions will increase both the size of populations and their associated rates of consumption, whereas, in the tropical regions, further warming would mean that temperatures would exceed the upper limits of optimal conditions for insect pest populations (Deutsch et al. 2018; Lehman et al. 2018). In many of the more temperate agroecological zones in Africa, maize yield losses to insect pests are expected to increase between 20 - 40% should the global surface temperature rise with 2°C (Deutsch et al., 2018) (Figure 4-4).

Figure 4-4. Percentage increase/decrease in maize yield loss due to insect pests, should the average global surface temperature increase with 2°C (adapted from Deutsch et al., 2018)

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4.4.3 Diapause and off-season survival strategies

Because insects are poikilothermic and therefore acutely affected by changes in the ambient temperature, they have evolved several strategies to survive suboptimal conditions. Diapause is a physiological example of such a survival strategy (Bale and Hayward, 2010; Fand et al., 2012; Gill et al., 2017). Throughout Africa, B. fusca enters a facultative diapause when temperatures decrease during the winter months (Usua, 1970; Van Rensburg et al., 1987; Okuda, 1990; Ebenebe et al., 1998; Calatayud et al., 2014). Chilo partellus can also enter a rest phase to escape unfavourable conditions (Kfir, 1991; Kfir, 1993), but S. frugiperda does not have this ability (Johnson, 1987; Goergen et al., 2016; Harrison et al., 2019). This means that S. frugiperda populations cannot overwinter in temperate regions which they invade during the warmer months and they have to recolonize regions when the environmental conditions become favourable during the following summer season (Du Plessis et al., 2018; Early et al., 2018). The temporary absence of S. frugiperda from certain agroecological zones during winter months provides the two stemborer species with a competitive advantage during the off-season, since they are able to overwinter locally after which moths emerge and infest maize plants earlier during spring. However, C. partellus populations generally terminate their rest phase quicker than B. fusca does (~ 10 days vs. 34 days) (Table 4-1). Consequently, where mixed populations occur, C. partellus populations are usually the first to infest maize plants, enabling the first generation to utilize the food and space resources without competition (Kfir, 1997; Dejen et al., 2014; Mutamiswa et al., 2017a). Effectively, C. partellus populations are then able to outcompete the lagging B. fusca and S. frugiperda populations by depriving them of access to shared resources. In agroecological zones which S. frugiperda have to seasonally invade, for example, temperate highlands (Du Plessis et al., 2018; Early et al., 2018), it does not have such a temporal competitive advantage.

Since the onset of diapause is primarily governed by temperature, humidity and photoperiod, a warming climate may affect the life cycles of insects which undergo a winter diapause (Bale and Hayward, 2010; Gill et al., 2017). Warmer seasons might delay the initiation of diapause and cause the early termination thereof, which could result in low mortality in overwintering populations. The large early generations of pests and early crop colonisations would cause significantly greater damage to crops (Harrington et al. 2001; Bale and Hayward 2010; Sharma et al. 2005; Babasaheb et al., 2018). However, should the environmental cues for diapause induction become mismatched with the development of the insect population, the insect population will need to adapt by adjusting their responses to the cues for diapause behaviour or face detrimental consequences (Forrest, 2016). For example, another lepidopteran species, Lasiommata megera (L.) (Lepidoptera: Nymphalidae) (Van Dyck et al., 2015), have been predicted to suffer a reduction in population size from attempting an additional generation in

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regions where summer temperatures are higher, but seasons are not yet long enough to allow completion of the second generation (Chinellato et al., 2014; Van Dyck et al., 2015; Forrest, 2016). This could soon be the fate of O. nubilalis populations in Canada, which could be subjected to high temperatures and long day lengths during critical developmental stages - conditions which are not conducive for diapause (Gagnon et al., 2019). The existence of genetic variability between uni- and bivoltine races of this species could, however, improve the ability of O. nubilalis to successfully adapt to the changing climate (Gagnon et al., 2019). Such changes could also occur in the case of B. fusca in highland regions where it currently has only 2 generations per cropping season, and where additional generations have been reported due to improved environmental conditions associated with cyclical changes in climate (Van Rensburg et al., 1985, 1987).

4.4.4 Larval population density

Another biological and behavioural trait that may enhance or reduce the competitiveness of a pest species, is how it responds to high larval population densities. It has been demonstrated that population density affects the survival, development time, dispersal, choice of oviposition site and adult size of lepidopteran pests (Van Hamburg, 1980; Berger, 1992; Fantinou et al., 2008; Flockhart et al., 2012; Ntiri et al., 2017). In a mixed population scenario, the development of a tolerant species may not be as severely affected by high larval densities than that of other less tolerant species in the population, which may confer to the former, significant size advantages in both direct and indirect competitive interactions (Delong et al., 2014). Ntiri et al. (2017) investigated the effect of larval density on the fitness and competitiveness of C. partellus, B. fusca and S. calamistis and found that the intensity of competitive interactions was density dependant – the higher the density of larvae, the more intense the display of both inter- and intraspecific competition. In their experiments, Ntiri et al. (2017) observed that, although the fitness of all three species of stemborers declined with an increase in larval density, C. partellus larvae were superior in interspecific interactions, since they displayed higher survival and relative growth rates than either B. fusca or S. calamistis when developing at a temperature of 25 °C. In another study, Ntiri et al. (2016) demonstrated that the dominance of a species during interspecific interaction was also dependant on temperature, since B. fusca and S. calamistis both displayed higher survival rates at low temperatures, compared to C. partellus which was dominant at higher temperatures.

Decreased development time, lower larval mass and increased mortality were observed in several studies that evaluated the effect of population density on intra- and interspecific competition in insect species (Goulson and Cory, 1995; Gibbs et al., 2004; Fantinou et al., 2008; Underwood, 2010; Flockhart et al., 2012; Ntiri et al. 2017; Himuro et al., 2018; Pavlushin et al., 2019). Although density dependent mortality among insects is a common phenomenon (Agnew et al., 2002), Pavlushin et al. (2019) reported that high population density did not affect the emergence of 149

baculovirus epizootics in Lymantria dispar (L.) (Lepidoptera, Lymantriidae) populations, they found that larvae that were reared singly had a higher larval mass, increased development time and showed a higher concentration of dopamine in their haemolymph than larvae reared in larger groups.

4.4.5 Cannibalism and predatory behaviour

Spodoptera frugiperda larvae respond to intra-specific competition at high larval densities with the manifestation of cannibalistic behaviour (Luginbill 1928; Chapman et al., 1999). This behaviour generally commences during the 3rd instar and is considered one of the main reasons that no more than three fully grown S. frugiperda larvae are found per plant, despite the high initial numbers of neonate larvae (Du Plessis et al., 2018). Although cannibalism comes with significant costs to fitness (Chapman et al., 1999), it also provides protection from predation via a decrease in the local larval density (Chapman et al., 2000). Additionally, the decrease in larval density also reduces the intra-specific competition for resources. However, S. frugiperda larvae are not merely cannibalistic (attack and devour con-specific larvae only), but they also engage in competitive intraguild interactions, where they attack the larvae of other species sharing the same resource (Luginbill 1928; Bentivenha et al. 2016, 2017). Intraguild interactions have been evaluated between S. frugiperda and Helicoverpa spp. (which also engages in cannibalistic behaviour), and it was found that S. frugiperda had a greater survival when the larvae were the same instar or when S. frugiperda were larger than the competitor (Bentivenha et al., 2017). Thus, S. frugiperda had a competitive advantage over Helicoverpa spp., even though H. zea larvae displayed more aggressive behaviour than S. frugiperda larvae (Bentivenha et al., 2016). It is therefore probable that S. frugiperda larvae would engage in direct interference competition behaviour and attack the larvae of the two stemborer species when the larvae of these species are still feeding in the whorls of plants, prior to them boring into maize stems. Due to the rapid developmental rate of S. frugiperda (Table 4-1), the predacious larvae will often have a size advantage over stemborer larvae which generally feed in the whorl only up to the 3rd or 4th instar (Van Rensburg et al., 1987; Calatayud et al., 2014), after which they commence boring into stems, whereas S. frugiperda larvae feed in the whorl until pupation (Sparks, 1979).

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4.4.6 Part 1: Summary

In this first part of the discussion the biology and behavioural characteristics of the three pest species in question were compared in an attempt to uncover which species would dominate under specific conditions. The following generalisations can be made:

• Busseola fusca will most likely be displaced by both C. partellus and S. frugiperda in the mild to warmer agroecological regions, but it will dominate in cooler areas due to its lower optimal temperature range and ability to diapause. However, this scenario may soon change due to the warmer and drier climate predicted for most of Africa.

• Spodoptera frugiperda has a much shorter life cycle, and is aggressive and dominating in direct competition interactions, which may result in it outcompeting C. partellus in low-land humid agroecological zones where the latter was always dominant. However, the lack of diapause in the life cycle of S. frugiperda will benefit C. partellus in high-altitude areas where winter temperatures do not allow their survival.

4.5 Part 2: Validity of IRM assumptions for B. fusca, C. partellus and S. frugiperda

The biology and behavioural characteristics of B. fusca, C. partellus and S. frugiperda which influence the evolution of resistance in an HDR strategy are discussed in this section. The HDR strategy has proven to be a powerful tool to delay resistance evolution in target pest species if the underlying assumptions are valid (Tabashnik and Carrière et al., 2017) (Table 4-2). The probable validity of the various assumptions regarding the three species of interest are compared below to assess the risk for resistance evolution of each species.

4.5.1 Initial resistance allele frequency, fitness cost and incomplete resistance

One of the five key assumptions for the HDR strategy (Table 4-2) to be effective is that the initial resistance allele frequency in the target population is low. It has often been suggested that a resistance allele frequency of < 0.001 is required to ensure success of the HDR strategy (Gould, 1998; Huang et al., 2011; Camargo et al., 2018). This assumption is suitable as a broad principle but could be too conservative in many cases. Tabashnik et al. (2013) suggested that factors such as the fitness costs associated with resistance could impact the minimum initial resistance allele frequency required for a population. For example, recessive inheritance of resistance together with fitness costs would still significantly delay resistance evolution even at an initial resistant allele frequency as high as 0.3, if refuges are present (Carrière and Tabashnik, 2001; Siegfried and Hellmich, 2012).

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The initial frequency of resistant alleles most likely differs between populations and geographical areas, and, since no information exists on the status of susceptibility of these pests to Bt maize in Africa, it is not possible to validate this assumption for the three species at hand. Strydom et al. (2018), however, indicated that resistant alleles of B. fusca were already present in B. fusca populations in South Africa when initial efficacy evaluations of Cry1Ab maize was done during the mid-1990s. Botha et al. (2019) showed that although Cry1Ab maize provided field-level control of S. frugiperda in South Africa, larval survival on this single-gene Bt maize under laboratory conditions was a high as 35% and that the frequency of resistance alleles against Cry1.104 + Cry2Ab2 was higher than expected (unpublished data). The important effect of high resistant allele frequencies in a population on the rate of resistance evolution was highlighted by Tabashnik et al. (2000, 2005) who showed that unexpectedly high frequencies of resistance alleles were initially present in the populations of Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in Arizona in 1997.

This lack of information on resistant allele frequencies is also true for the assumptions about incomplete resistance and fitness costs associated with resistance (Table 4-2). Both fitness costs and incomplete resistance refers to trade-offs that occur during the evolution of resistance to Bt toxins (García et al., 2015; Paolino and Gassmann, 2017; USEPA, 2018). Incomplete resistance is indicated by reduced fitness of resistant individuals on Bt plants, whereas fitness costs is characterized by the greater fitness of susceptible individuals compared to resistant individuals on non-Bt plants (Tabashnik and Carrière, 2017; USEPA, 2018). These parameters could be key to delaying resistance evolution in pest populations that are under selection pressure from Bt maize.

It is worth noting that a study investigating B. fusca populations resistant to Cry1Ab (MON810), which was conducted six years after the initial report of resistance, could not detect fitness costs when these larvae were reared on Bt maize (Kruger et al., 2014). This suggests that resistance became a stable trait in these B. fusca populations. For S. frugiperda, several studies have indicated the presence of fitness costs to Cry1F, Vip3A and a pyramid maize event (which incorporated Cry1F, Cry1A.105, and Cry2Ab) in populations from the Americas (Jakka et al. 2014; Dangal and Huang, 2015; Horikoshi et al. 2015; Bernardi et al. 2016, 2017). However, the USEPA (2018) indicated that the fitness costs associated with Cry1F were not observed in any field collected strains of S. frugiperda in the USA. Jakka et al. (2014) also found that the lower rate of larval development which was flagged as a fitness cost in Cry1F-resistant S. frugiperda populations in Puerto Rico, did not affect the stability of resistance after 12 generations of selection of a heterogeneous S. frugiperda strain raised on a meridic diet. It would therefore be reasonable to assume that B. fusca and S. frugiperda populations present in the other regions in

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Africa could exhibit a similar lack of fitness costs, incomplete resistance and high frequency of resistant alleles to single-toxin maize events, which will necessitate the use of multi-toxin events from the outset.

Table 4-2. Summary of the key assumptions that underlie the high-dose refuge (HDR) strategy for insect resistance management in Bt crops, as well as the additional assumptions that underpin the use of multi- toxin Bt crops, natural refuges and seed mixtures.

Key assumptions

High-dose/refuge strategya

1. Low initial resistance allele frequency.

2. Fitness cost to resistance.

3. Incomplete resistance.

4. Recessive inheritance of resistance (i.e. meets high-dose standard).

5. Abundant refuges of non-Bt host plants near Bt plants.

Additional assumptions

Multi-toxin (pyramid) cropsb

+ Each toxin meets high-dose standard.

+ No cross-resistance occurs between toxins in the pyramid.

+ Pyramids are not grown simultaneously with single-toxin plants that produce one of the toxins in the pyramid.

Natural refugesc

+ Produce sufficient number of adults.

+ Adults should be of high quality.

+ Must be abundant in the area near Bt crops.

+ Rate of development of pest larvae must be synchronized with larvae developing in the crop.

Seed mixturesd

+ Pest larvae must have low migration potential.

Tabashnik & Carrière (2017)a, b, d Van den Berg et al. (2016)c Carrière et al. (2016)a, b, d Li et al. (2017)c Bates et al. (2005)a, b, d Head (2004)c

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4.5.2 Recessive inheritance of resistance (high-dose requirement of toxin expression)

Another assumption to be considered is also related to the genetics and heritability of the resistance trait. The HDR strategy assumes that the inheritance for resistance is recessive. This means that a heterozygous individual will not be able to survive on Bt plants, which then contributes largely to delay resistance evolution in a population (Tabashnik et al., 2013; Campagne et al., 2016). This assumption is alternatively expressed in terms of the high-dose requirement for a Bt crop, since the toxin expression within Bt plants must ideally be high enough to kill all heterozygous individuals (Tabashnik and Carrière, 2017). Theoretically, a high dose should constitute 25 times the dose needed to kill 99% of susceptible pest individuals (Gould 1998; USEPA, 1998; Glaser and Matten 2003; Campagne et al., 2013). The lack of a high-dose expression of Cry1Ab maize was considered a contributing factor to the rapid evolution of resistance in the South African B. fusca populations (Van Rensburg, 1999; Fitt et al., 2004; Tabashnik et al., 2008, 2009; Campagne et al., 2013; Strydom et al., 2018). Similarly, both Cry1F and Cry1Ab was shown not to meet the high-dose requirements for S. frugiperda and have since been rendered obsolete in Brazil due to resistance development by the target pest populations (Farias et al., 2015; Omoto et al., 2016; Botha et al., 2019). In fact, Tabashnik and Carrière (2017) indicated that, of the 16 cases of practical resistance of insect pests to Bt toxins, not one of the Bt toxins achieved the high-dose standard for the target pest species.

Interestingly, no cases of field-evolved resistance to Bt maize of event MON810 (or any other event) have been observed for C. partellus or S. calamistis, even though these species have been subjected to the same selection pressure as B. fusca populations over the two decades that this maize event has been cultivated in South Africa. Similarly, Tabashnik and Carrière (2019) reported on 19 cases where no significant decrease in susceptibility to Bt crops have been recorded after a mean of 10 years of exposure to the Bt toxin. The lack of resistance evolution in C. partellus and S. calamistis may indicate that event MON810 is a high-dose event for these species, that the frequency of resistant alleles is very low, and that fitness costs are associated with resistance evolution in the latter two species. Van Wyk and Van den Berg (2008), Van den Berg and Van Wyk (2007) reported that S. calamistis was highly susceptible to Cry1Ab maize and Van Rensburg (1999, 2001) reported similarly for C. partellus.

4.5.2.1 Multi-toxin events (pyramids)

Considering the history of resistance evolution of B. fusca and S. frugiperda against single-toxin maize events (Cry1Ab and Cry1F), it would be sensible to immediately employ a pyramided Bt maize event to proactively address invalid IRM assumptions for populations of these two priority species in Africa (Head and Greenplate, 2012; Storer et al., 2012, Carrière et al., 2015). Important

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to note, however, is that the use of multi-toxin crops brings with it an additional three assumptions that must be satisfied to ensure the sustainability of a pyramid event (Table 4-2) (Carrière et al., 2015). For the purpose of this discussion, we focus on a pyramid event, MON89034, which expresses Cry1A.105 and Cry2Ab2 proteins. Maize plants with this event are cultivated in South Africa and has been shown to be effective against both B. fusca and S. frugiperda populations (Strydom et al., 2018; Botha et al., 2019).

The first of the pyramid-specific assumptions related to IRM is that each toxin in the pyramid should meet the high-dose standard for the target pest. MON89034 expresses Cry1A.105 and Cry2Ab2 toxin proteins. Cry1A.105 is a chimeric gene, containing domains I and II of Cry1Ab and Cry1Ac, respectively, and domain III of Cry1F (Siegfried and Hellmich, 2012; USEPA, 2018). Recent findings confirmed that cross-resistance is highly likely between toxins with similar amino acid sequences in domain II (Hernandez-Rodriguez et al., 2013; Bernardi et al., 2015; Welch et al. 2015, Carrière et al., 2015). This could compromise the efficacy of Cry1A.105 against both S. frugiperda and B. fusca and indicates that this protein does not meet the high-dose requirements for both these target species. Although Strydom et al. (2018) confirmed that MON89034 is still highly effective against B. fusca, they highlighted that the inherent low susceptibility of this species to Cry proteins, combined with the low-dose expression of Cry1Ab in MON810 maize, create an environment for resistance evolution of B. fusca to pyramid maize in South Africa. Similarly, Botha et al. (2019) found only moderate susceptibility of S. frugiperda to Cry1Ab, which they ascribed to MON810 being low-dose event for this pest, and to the fact that the population that has invaded Africa could already have contained alleles with resistance to this protein. Because Cry1- and Cry2-proteins do not share binding sites (Hernandez-Rodriguez et al., 2013), it is safe to assume that MON89034 fulfils the requirement of the second assumption for pyramided crops (Table 4- 2).

Finally, an important IRM-requirement in farming systems where pyramid events are cultivated, is that multi-toxin plants are not grown simultaneously with single-toxin plants that produce one of the toxins in expressed in plants of the pyramid event (Table 4-2). This is because the single- toxin event would compromise the efficacy of using two toxins against a pest population, since it would allow the stepwise evolution of resistance against the pyramid variety (Zhao et al., 2003; Bates 2005; USEPA, 2018). This assumption will conceivably only be violated in South Africa (Van den Berg et al., 2013, Strydom et al., 2018), if other African countries decide to completely forego the first generation single-toxin events, and only commercialize pyramid events. Even though cultivating MON810 maize alongside MON89034 could compromise the efficacy of the pyramid event, this was done for several years in South Africa. This is in contrast to countries such as Australia, where the simultaneous cultivation of both single toxin Cry1Ac and pyramid

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Cry1Ac + Cry2Ab cotton has been prohibited, and where the replacement of the single toxin event with the pyramid was accomplished in a single year (Downes and Mahon, 2012; Carrière et al., 2016).

4.5.3 Abundant refuges of non-Bt host plants

Any plant species in a particular agroecosystem can serve as a refuge plant, provided it is a suitable host of the target species. Abundant and appropriate non-Bt refuges near Bt maize fields is a critical component of the HDR strategy. Without these refuges, the selection pressure for the evolution of resistance would be severe, since very few susceptible alleles would remain in the population after even a single exposure to the Bt crop (USEPA, 1998; Gould et al., 1998; Bates et al., 2005). The lack of sufficient refuges has contributed to the evolution of resistance in several pest populations, including B. fusca in South Africa (Van den Berg et al., 2013), P. gossypiella in India (Mohan et al., 2016), S. frugiperda in Brazil (Monnerat et al., 2015), and Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae) in the USA (Tabashnik and Gould, 2012). The planting of separate, dedicated areas of non-Bt maize near Bt maize fields is considered the best option for IRM in most situations, but not all (Carroll et al., 2012). Smallholder farms is the norm in Africa and mandating the planting of a separate refuge by each farmer that cultivates Bt maize would be impractical (Bates et al., 2005; Carroll et al. 2012; Van den Berg et al. 2013; Li et al., 2017). It has been suggested that if the adoption rate for Bt maize in these farming systems are will be low, that the non-Bt maize fields of non-adopters in the area would suffice as refugia (Van den Berg and Campagne, 2015). It is also conceivable that several farmers could plant a communal refuge area to service all their Bt fields (Head, 2004). Still, the management of these refuges would require training of farmers and ongoing support from government or industry extension personnel, both services which are generally inefficient or lacking in smallholder agricultural areas (Jacobson and Myhr, 2012; Kotey et al., 2016; Kotey et al., 2017).

4.5.3.1 Naturally occurring refuges

It has been suggested that the implementation of structured refuges might not be necessary for smallholder farmers if sufficient wild host plants are present in the uncultivated areas near smallholder plots (Head, 2004; Onstad and Carrière, 2014; Li et al., 2017). While investigating this claim for maize stem borers in Africa, Van den Berg (2016) compiled a list of conditions that must be met if wild host plants are to be used as a refuge in an HDR strategy (Table 4-2), and concluded that wild host plants are unsuitable for use as refuges for African maize stemborers. Instead of serving as refuges for the survival and production of susceptible individuals of the target pest species, wild grasses were found to fulfil the role of dead-end-trap plants. Wild host plants were found to be less attractive to the stemborers as oviposition sites, and did not allow a high

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enough development or survival rate of the larvae, which resulted in the emergence of a limited number of adults that could compete for mating opportunities with resistant individuals (Van den Berg et al., 2016; Li et al., 2017). Consequently, even though both B. fusca and C. partellus are able to survive and propagate to a very limited degree in these uncultivated habitats (C. partellus more so than B. fusca) (Ofomata et al., 2000; Songa et al., 2002; Mutamiswa et al., 2017a), unstructured refuges in the form of wild host plants will not be an IRM option for these species (Van den Berg et al., 2016; Li et al., 2017; Sokame et al., 2019a).

Unlike the oligophagous nature of the two stemborer species, S. frugiperda is a polyphagous species with approximately 350 host plant species (Montezano et al., 2018). It is primarily a pest of maize, but also shows preference for other poaceous plants, including crops such as millet, sorghum and rice. However, feeding damage has been observed on crops as diverse as cowpea, groundnut, potato, soybean and cotton (Goergen et al., 2016; Day et al., 2017, Assefa and Ayalew, 2019). The richness of alternative host plants in diverse agricultural landscapes (characteristic of smallholder farming systems in Africa) (Van den Berg et al., 2015) could mean that the implementation of structured refuges for S. frugiperda would not be necessary (Head, 2004; Onstad and Carrière, 2014). Although, this would necessitate that the abundance and reliability of alternative hosts are determined before the release of Bt maize and would also need to be monitored to ensure their constant availability (Head, 2004; Van den Berg, 2016). At this stage, however, the polyphagous nature of S. frugiperda has not materialised in Africa and host plants other than maize that are attacked are sorghum and millet. This limited host association is ascribed to S. frugiperda in Africa being an inter-strain hybrid with limited affinity for crops other than maize and sorghum (Nagoshi, 2019; Nagoshi et al. 2019).

In a case similar to S. frugiperda, the possibility of using other crops as refuges for Bt cotton seems viable for a highly polyphagous pest such as Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). A study of the population dynamics of H. armigera in China showed that other crops can be used as a refuge for H. armigera in Bt cotton-growing areas (Ye et al. 2015). This was also done with Heliothis spp. infesting cotton in the United States, or other crop species refuges such as tobacco for Bt cotton in United States, and pigeon pea for Bt cotton in Australia (Andow 2008). Indigenous plants and weedy hosts of H. armigera were also indicated to provide appropriate refuges in Bt cotton production areas in South Africa (Green et al. 2003). The legume pod borer, Maruca vitrata (Fabricius) (Lepidoptera: Crambidae), the target pest of Bt cowpea in West Africa, is an olyphagous pest of grain legumes that has a wide distribution throughout tropical and subtropical regions worldwide (Agunbiade et al., 2014). The populations of M. vitrata that are sustained on alternative host plants can contribute to IRM by sustaining individuals that have not been subjected to selection pressure on Bt cowpea (Onstad et al., 2012).

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4.5.4 Seed mixtures for IRM in smallholder farming systems

An alternative approach to structured refuges is the use of seed mixtures. This approach has been identified as a possible solution to the challenges facing IRM for Bt maize in smallholder farming systems, since it does not require the farmer to plant a separate structured refuge (Carroll et al., 2012; IRAC, 2013; Erasmus et al., 2016; Carzoli et al., 2018). Not only will this approach be more practical to implement at smaller scales of production, but it also ensures farmer compliance to refuge requirements, since the seed is premixed (Head, 2004; Head and Greenplate, 2012). The spatial integration of Bt maize and refuge areas also improves the likelihood that resistant and susceptible adult individuals will mate, compared to the separate structured refuges (Onstad et al., 2011; Spencer et al., 2013).

Seed mixtures could also prevent gravid female moths from preferentially ovipositing on Bt plants. This phenomenon was observed by Téllez-Rodríguez et al. (2014) with S. frugiperda in Cuba. The preference for Bt maize was most likely due to damage avoidance behaviour (favouring undamaged Bt plants over the heavily damaged refuge plants), and not due to an outright preference for Bt maize, since females of Lepidoptera pests generally do not show any oviposition preference for either Bt or non-Bt plants that are of the same age and quality (Visser et al., 2019). Additionally, several studies have found no significant differences in the volatile profile of Bt and non-Bt maize and rice plants when the amount of damage to the plants were controlled for (Dean and De Moraes, 2006; Sun et al., 2013; Liu et al., 2015). Although Turlings et al. (2005) and Xu et al. (2019) found differences in the amount of specific volatiles released by Bt and non-Bt maize, the differences in the volatile profiles of plants from the same species that arise from conventional breeding programs are far greater than the changes caused by genetic engineering (Turlings et al., 2005; Vogler et al., 2009, 2010; Xu et al., 2019), and therefore should not greatly impact the preference of gravid female moths between Bt and non-Bt plants.

The damage avoidance behaviour observed by Téllez-Rodríguez et al. (2014) may increase the selection pressure exerted on the pest population, increasing the rate of resistance development (Téllez-Rodríguez et al., 2014; Visser et al., 2019). Seed mixtures could prevent the build-up of high pest population densities, thereby preventing the total decimation of refuge plants in the case of S. frugiperda. Contrary to the damage avoidance oviposition behaviour observed for S. frugiperda, Sokame et al. (2019b) observed that adults of the stemborer species B. fusca, C. partellus and S. calamistis significantly preferred volatile organic compounds emitted by plants infested by larvae (either conspecific or heterospecific) over those from uninfested plants, and that these inclinations also translated to the preferential selection of oviposition sites on infested plants vs. uninfested plants (a phenomenon also observed by Ntiri et al. (2018)). Using gas chromatography/mass spectrometry and chemical analysis, Sokame et al. (2019b) discovered 158

that the volatiles emitted from infested plants, were compositionally richer than those released by uninfested plants.

The biggest threat to the sustainability of the seed mixture approach is the propensity for migration of pest larvae. Several studies have indicated that if the larvae of the target pest are prone to migrate between Bt and non-Bt plants, seed mixtures may lead to accelerated evolution of resistance (Mallet and Porter, 1992; Roush, 1997; Carrière et al., 2016). This would occur due to two processes: firstly, reduced survival of susceptible insects (and effectively the refuge size), and secondly, increased survival of heterozygotes, which leads to a rise in the frequency of resistant alleles in the population (Head and Greenplate, 2012; Carrière et al., 2016; Onstad et al., 2018). It is therefore important to take into account the larval migration behaviour of the target pest when considering the use of a seed mixture as IRM strategy. Pannuti et al. (2016) investigated the plant-to-plant movement of S. frugiperda in non-Bt maize plots and found that 92% of S. frugiperda larvae were recovered within a 1.1 m radius from the inoculated plant, and more than half of these were found within the same row as the natal plant, two weeks after inoculation. Given the inter- and intra-row spacing at 0.76 m and 0.15 m, respectively, this radius roughly translates to an average migration across 7 to 8 maize plants within the same row. Visser et al. (2020a) reported that, on average, B. fusca larvae were found no further than 2 plants away from the inoculated plant in a non-Bt maize field after three weeks in the field (0.75 m inter- and 0.20 m intra-row spacing). Although few similar studies have been conducted for C. partellus on maize, it has been reported that the larvae of this species have a higher propensity for inter-plant movement than B. fusca, when these species were compared on sorghum (Van den Berg et al., 1991b). Chapman et al. (1983) found that, within a week after inoculation, C. partellus larvae migrated from a single inoculated sorghum plant to infest a total of seven sorghum plants in a stand with an inter- and intra-row spacing of 0.75 m and 0.10 m, respectively. In a study of C. partellus larval dispersal on maize, Päts and Ekbom (1992) observed that the mean number of plants infested from a single egg batch after a 7-day period ranged between 2.3 to 4.2 (inter-row spacing = 0.7 m, and intra-row spacing = 0.3 m). The higher level of larval migration observed by Chapman et al. (1983) compared to that of Päts and Ekbom (1992) can, in part, be attributed to the lower plant density used in the latter study. All the studies discussed for the three species reported a low larval survival rate, which is common for lepidopteran species under field conditions (Zalucki et al., 2002).

The propensity of all three species to migrate between host plants should, in theory, preclude the use of seed mixtures as IRM strategy (Heuberger et al., 2011; Carrière et al., 2016; Visser et al., 2020a,b). However, Visser et al. (2020a) suggested that, in practice, there are ways to mitigate the successful migration of the pest larvae. A decrease in plant density would significantly affect

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the ability of larvae to successfully migrate between plants, since they are more prone to desiccation in heat and wind, drowning in rain and falling victim to predators and parasitoids (Bonhof, 2000; Zalucki et al., 2002; Midega et al., 2006, Visser et al., 2020a). Intercropping with non-host plants, which is a common practice in African small holder farming systems, could further contribute to decreased survival of migrating larvae, since the presence of non-host crop plants would not only decrease the density of host plants and serve as obstructions to migrating larvae, but a higher plant diversity increases the presence of natural enemies within the maize field (Khan et al., 1997; Landis et al., 2000, Midega et al., 2006). Khan et al. (2014) and Midega et al. (2018) have also indicated the utility of push-pull systems in controlling both stemborer species and S. frugiperda (Figure 4-5). This technology uses the drought tolerant greenleaf desmodium, Desmodium intortum (Miller) Urban (Leguminosae), and Brachiaria grass (cv Mulato II) (Poaceae) as the ‘push’ and ‘pull’ crops respectively (Figure 4-5) (Khan et al., 2014; Midega et al., 2018). In their study Midega et al. (2018) found that, compared to pure stands of maize, reductions of 82.7% in average number of S. frugiperda larvae per plant and 86.7% in plant damage per plot were observed in the push-pull stands.

Figure 4-5. Description of the mechanism of the push-pull technology. Volatile organic compounds facilitate the aerial responses of the stemborer (B. fusca and C. partellus) and S. furgiperda female moths, whereas the chemicals released into the rhizosphere by the roots of desmodium inhibit the development of striga weed (Khan et al., 2017).

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4.5.5 Part 2: Summary

Validity of HDR strategy assumptions for managing insect resistance evolution by C. partellus, B. fusca and S. frugiperda to Bt maize in Africa:

• Both B. fusca and S. frugiperda have high inherent risk of evolving resistance to single-toxin Bt maize due to pre-existing high resistance allele frequencies within populations and a lack of fitness costs. After more than two decades of selection pressure on Bt maize in South Africa, no resistance has yet been detected for C. partellus, suggesting that single-toxin events would be effective to manage this pest successfully throughout the continent.

• The MON89034 event is high-dose for all three species, ensuring recessive inheritance of resistance. However, since one of the toxins produced in the pyramid is not high-dose for both B. fusca and S. frugiperda, care must be taken that this pyramid is not cultivated in tandem with single-toxin events of the weaker toxin.

• Due to the oligophagous nature of B. fusca and C. partellus, and extremely low carrying capacity of their wild host plants these wild hosts will not suffice as refuges in IRM programs, necessitating the planting of non-Bt maize refuges for both these species. The conservative feeding strategy of the inter-hybrid strain of S. frugiperda which occurs in Africa, also limits its effective host range, with only cultivated sorghum that could be considered as an alternative to non-Bt maize refuges.

• In spite of the propensity for larval migration between maize plants, seed mixtures could be a viable approach to refuge implementation for all three species when combined with IPM practices such as intercropping and push-pull systems.

4.6 Part 3: IRM for single and mixed populations of Lepidoptera maize pests in Africa

The information provided in the sections above and the implications thereof on IRM strategies against these pests are discussed below.

Firstly, it would be most advantageous for countries in Africa to immediately opt for the use of the pyramided event MON89034 and forego the use of single-toxin events (e.g. MON810), especially in regions where the prevalence of mixed populations of the species in question are high. Single- toxin events should only be considered in cases where it is targeted against a pure population of C. partellus.

The information discussed in the study suggests that S. frugiperda will most likely dominate the Lepidoptera species complex in non-Bt maize fields in moderate to warm agroecological zones, if it occurs in mixed populations with B. fusca and C. partellus. Due to the limited sizes of fields in smallholder farming systems, only a very small area can be devoted to planting a separate refuge of non-Bt maize, if structured refuges are enforced. If such structured refuges are the recommended IRM strategy, inter-species interactions could influence refuge efficacy. The

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biology and behaviour of S. frugiperda enables it to outcompete the other two species, displacing them from the refuge plants. This would increase the selection pressure on stemborers, since fewer adults will emerge from the refuge to act as mates for the resistant individuals that might survive on the Bt maize. High population densities of S. frugiperda, coupled with its short development time, could also lead to the complete decimation of plants in small refuge areas, which would limit the available resources and compel gravid females of both C. partellus and S. frugiperda to preferentially oviposition on plants in the Bt field, since the females of both these species oviposit on the leaves of maize plants. Busseola fusca might still oviposit in the highly damaged refuge area, since gravid female moths show preference for damaged maize plants. However, the resulting B. fusca larvae would need to compete with the more dominant and aggressive S. frugiperda and C. partellus larvae that remain in the refuge, and could suffer high mortality as a result. This would decrease the number of susceptible B. fusca individuals available to mate with the resistant individuals. Spodoptera frugiperda populations could escape the increase in selection pressure on Bt maize if alternative host plants are available that it can utilize as refuge plants. However, alternative host plants will not suffice as refuge for the B. fusca and C. partellus populations. The use of separate refuges might therefore not be advisable in warmer agroecological zones where mixed populations of lepidopteran pests are common.

The use of seed mixtures together with farming practices that reduces colonisation, establishment and survival of these lepidopteran pests may be a viable strategy to manage resistance evolution of pests to Bt maize in Africa. For example, intercropping practices that disrupts larval migration and enhances mortality within maize fields would be a preferable approach, since it would afford the females of each of the species an equal opportunity of randomly selecting a non-Bt maize plant for oviposition, allowing the survival of susceptible individuals of all species.

In more temperate climates, where winter temperatures prohibit the overwintering of S. frugiperda, the effect of this pest on plants grown in small refuges could be delayed until later in the summer season when S. frugiperda re-establishes itself in an area. In the case of C. partellus, on the other hand, its early termination of diapause, higher rate of development, and proclivity to migrate would result in it becoming the dominant species in the refuge area, possibly displacing B. fusca from the refuge in the case of high population densities. Since B. fusca is less susceptible to Bt toxins and one of the toxins within the MON89034 event does not meet high-dose requirements, evolution of resistance against the pyramided crop could be accelerated. Therefore, even in the absence of S. frugiperda, mixed seed refuges could be a more sustainable IRM strategy for mixed stemborer populations in smallholder farming systems.

In temperate regions where all three species occur in mixed populations, the rate of development of C. partellus and S. frugiperda would be slower than in sub-tropical and tropical regions, which 162

may have a negative effect on their competitiveness. Under such environmental conditions, no species may dominate, which will allow similar infestation of refuge areas, followed by production of sufficient numbers of susceptible adults, to delay resistance evolution in all these species.

4.6.1 IRM in the context of IPM

The use of IPM strategies in lieu of the reliance on a single pest control tactic (e.g. Bt crops, or chemical insecticides) is the most sustainable approach to insect pest management (Gassmann et al. 2011, Downes et al. 2017, Anderson et al. 2019, Hurley and Sun 2019; Tabashnik and Carrière, 2019). Combining IPM and IRM strategies could also be the most sustainable approach to preventing resistance evolution in insect pest populations, especially in developing countries where the implementation of the standard IRM practices are complicated by several factors. The case of P. gossypiella in India supports this claim (reference). The extensive nature of the resistance of P. gossypiella to Bt cotton in India necessitates the return to IPM programs that also employ control tactics other than Bt toxins (Kranthi, 2015; Mohan, 2017; Tabashnik and Carrière, 2019). For P. gossypiella, these approaches include planting of early to medium maturing cotton hybrids, removal of crop residues and ratoon cotton, use of insecticides, crop rotation, mass trapping and mating disruption, and biological control with natural enemies (Kranthi, 2015; Mohan, 2017; Tabashnik and Carrière, 2019). The implementation of most IPM practices are generally impractical at large scales of production, whereas several of these IPM measures (e.g. intercropping) are already in widespread use among smallholder farmers (Tefera et al., 2016). By combining seed mixtures with these habitat management practices, the risk that larval migration poses to resistance management can be mitigated (Midega et al., 2006).

4.7 Conclusions

This review indicates that the use of structured separate refuges as components of IRM strategy against mixed pest populations in smallholder Bt maize fields would be unwise. A seed mixture approach, coupled with IPM practices (e.g. intercropping), would be more sensible, since it may limit the opportunity for a single species to dominate the species complex and cause structured refuges to be unsuitable for other species, which may lead to increased infestation of Bt plants, and subsequent increased selection pressure. This will ensure that all species in the complex have sufficient access to space and resources, to allow production of susceptible adults to dilute the frequency of resistant alleles in the population. Large commercial scale fields - for which the implementation of IPM strategies such as intercropping would be impractical – could opt for separate refuges but would possibly need to increase the relative size of the refuge area when confronted with high pest pressures and mixed populations of lepidopteran pests.

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This thought experiment was limited to the effect that scale of production, climatic conditions and pest population dynamics could have on the choice and design of an IRM strategy for Bt maize in Africa. Several other important factors that affect the sustainable cultivation of Bt maize also exist, but are ultimately out of the scope of this chapter. This includes considerations such as the availability of training and extension support for farmers, which also plays a role in determining the appropriate IRM approach under different farming systems. Future studies should aim to test the hypotheses laid out in this text by producing resistance evolution models for specific areas. These models should consider agroecological zones as well as population dynamics of the pest complex regarded the target of Bt maize in a particular region. This study provides insights into the unique challenges that face the deployment of Bt maize in Africa.

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CHAPTER 6

Conclusions and recommendations

5.1 Study outcomes & recommendations for future research

The aim of this study was to investigate B. fusca larval migration behaviour and oviposition preference for Bt and non-Bt maize and how this behaviour could impact the success of certain IRM strategies. Furthermore, a review was provided regarding principles and assumptions that relate to IRM strategies and how it may affect B. fusca resistance evolution when it occurs either in pure and mixed pest species populations with C. partellus and S. frugiperda. These aims were achieved by successfully addressing all the associated objectives.

The outcome of the study and recommendations for future research will be discussed per each of the three hypotheses set out in the first chapter of this thesis (Heading 1.2.3).

5.1.1 Hypothesis 1: The oviposition preference and behaviour of gravid B. fusca females will favour the use of separate refuges as an IRM strategy for Bt maize in Africa.

Separate refuges are widely considered to be the ideal application of the HDR strategy (Carroll et al., 2012; Tabashnik and Carrière et al., 2017). However, when the adult females of the target pest species do not select oviposition sites randomly and display a preference for Bt maize, the separate refuge approach could undermine the goal of IRM, which is to produce large numbers of moths in the non-Bt refuges and very low numbers in Bt fields (Ives and Andow, 2002). This is because oviposition preference for Bt maize will lead to an increase in the proportion of the pest population that is under selection for resistance (Obonyo et al., 2008; Téllez-Rodríguez et al., 2014).

In this thesis, chapter 2 reported that gravid female B. fusca moths of both the resistant and susceptible populations did not preferentially oviposit on either Bt or non-Bt plants, but that they select plants at random when other physical characteristics (including feeding damage) are controlled for. This lack of discretion has also been observed for several other maize stem borer species as well as other lepidopteran pest species (Pilcher and Rice, 2001; Kumar, 2004; Van den Berg and Van Wyk, 2007; Luong et al., 2016; Visser et al., 2019). It has also been reported that the volatile compounds released by Bt and non-Bt maize are similar and that it did not influence attractiveness of different cultivars for parasitic wasps that attack stem borers (Turlings et al., 2005). It is therefore likely that gravid adult moths are unable to detect the Bt toxins within

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plants and therefore select oviposition sites at random. Consequently, the lack of oviposition preference in B. fusca for Bt and non-Bt maize indicates that separate refuges can be implemented in an IRM strategy against this species. The first hypothesis is therefore accepted. However, this hypothesis is contingent on single-species pest populations of B. fusca and the presence of ample refuge plants. Chapter 5 demonstrated that, should B. fusca occur in a mixed population together with more dominant lepidopteran species such as Spodoptera frugiperda, or a species that has a competitive advantage due to its tolerance for high and low temperatures and rapid development, a separate refuge would not be the ideal IRM strategy, due to the possibility of damage avoidance oviposition behaviour.

5.1.1.1 Recommendations for future research:

Previous investigations showed that B. fusca females avoided ovipositing on plants that were already infested (Kfir, 1997; Sétamou and Schulthess, 1995; Chabi-Olaye et al., 2005; Calatayud et al., 2007), yet other studies did not observe this avoidance behaviour in B. fusca (Ntiri et al., 2018; Sokame et al., 2019). This ambiguity in results makes it unclear if B. fusca moths is likely to avoid the refuge areas (which purpose is to harbour higher infestation levels than in the main Bt crop), which would increase the rate of resistance evolution. Future studies should therefore aim to determine if B. fusca populations display damage avoidance oviposition behaviour under both laboratory and field conditions. Furthermore, not only should the effect on conspecific infestation and damage to refuge plants on B. fusca oviposition behaviour be investigated, but also the influence that the severe levels of leaf damage caused by S. frugiperda larval feeding have on B. fusca moths

5.1.2 Hypothesis 2: The larval migration behaviour of B. fusca will preclude the use of seed mixtures as an IRM strategy for Bt maize in Africa.

The results generated on B. fusca larval feeding preferences (Chapter 2) and plant abandonment behaviour (Chapter 3) suggested that both Bt-resistant and susceptible neonate larvae migrate more readily off Bt plants. This would lead to an increase in larval migration between plants, which could exclude the use of seed mixtures as an IRM strategy for B. fusca on Bt maize, since several studies reported that a mixed seed refuge was not suitable for target pest species that migrate extensively between plants (Mallet and Porter, 1992; Gould, 2000; Onstad et al., 2011). However, the field trials conducted during this study (Chapter 4) indicated that, although B. fusca larvae do migrate between plants, the migration distance is limited and survival is very low, even in pure stands of non-Bt maize. These observations contradict earlier reports which erroneously reported significant larval movement between plants. Chapter 4 also highlights the fact that farming practices which limit the successful migration and survival of larvae on maize in mixed farming

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systems where for example intercropping is practiced, could contribute to IRM. The second hypothesis is therefore rejected.

5.1.2.1 Recommendations for future research:

Future research should aim to incorporate the migration parameters quantified in this study in models that predict resistance evolution of pest populations. This would reveal whether the limited migration of larvae and the use of cultural practices restrict larval migration enough to allow the use of mixed seed IRM strategies against B. fusca.

5.1.3 Hypothesis 3: Mixed populations of pest species will necessitate the tailoring of the IRM strategy to suit the conditions of specific agricultural regions.

Chapter 5 of this thesis concluded that the use of separate refuges as an IRM tactic against mixed populations in smallholder Bt maize fields in Africa would be unwise, since the domination of the refuge by one species will undermine the efficacy of the IRM strategy for another pest species. A seed mixture approach, coupled with other IPM practices to minimize the effect of larval migration (Chapter 4, Chapter 5), would be a more logical approach, since it ensures that all species in pest complex have equal access to refuge plants. However, this endorsement is subject to the characteristics of the receiving environment, including scale of production, the climatic conditions, and the availability of support for the farmers in the form of extension services and training. The implementation of IPM strategies such as intercropping would be impractical on large commercial scale fields. Consequently, farmers in these agricultural systems could alternatively opt for the use of larger (greater percentage of Bt maize field), separate refuges when confronted with high pest pressures and mixed populations of lepidopteran pests.

Therefore, the third hypothesis is accepted, since it is apparent that the design of an effective IRM strategy will need to be based on specific local conditions.

5.1.3.1 Recommendations for future research:

Future studies should aim to develop resistance evolution models for specific agroecological zones to take into consideration climatic conditions, the scale of production, as well as the population dynamics of the target pest complex of Bt maize in that zone. The latter will be critical to the final design of IRM strategies for specific agricultural systems in Africa.

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5.2 Conclusions

This study found that the biology and behaviour of B. fusca would favour the use of mixed seed refuges, particularly when this species forms part of a mixed population of lepidopteran maize pests. However, this study also suggests that no single, generic, standardized IRM strategy can be prescribed for all of Africa, or even for entire countries on the continent. The dissimilarity between the African agricultural regions necessitates a tailored approach to IRM and development of strategies for each region/zone to ensure that it meets the needs of the farmers that are tasked with its implementation. The findings also indicate that the commercialization of single-toxin maize events in African countries should be waived in favour of pyramid events, to ensure the maximum longevity of the Bt toxins. Future research should focus on generating more data for use in models to improve IRM strategies for specific regions and agricultural systems.

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Kumar, H. 2004. Orientation, feeding and ovipositional behaviour of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae), on transgenic cabbage expressing Cry1Ab toxin of Bacillus thuringiensis (Berliner). Environmental Entomology, 33: 1025–1031.

Luong, T.T.A., Downes, S.J., Cribb, B., Perkins, L.E. & Zalucki, M.P. 2016. Oviposition site selection and survival of susceptible and resistant larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) on Bt and non-Bt cotton. Bulletin of Entomological Research, 106: 710–717.

Ntiri, E. S., Calatayud, P.A., Musyoka, B., Van den Berg, J. & Le Rü, B.P. 2018. Influence of feeding-damaged plants on the oviposition responses within a community of female moths. Phytoparasitica, 46: 607–615.

Obonyo, D.N., Songa, J.M., Oyieke, F.A., Nyamasyo, G.H.N. & Mugo, S.N. 2008. Bt-transgenic maize does not deter oviposition by two important African cereal stem borers, Chilo partellus Swinhoe (Lepidoptera: Crambidae) and Sesamia calamistis Hampson (Lepidoptera: Noctuidae). Journal of Applied Biosciences, 10: 424-433.

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Pilcher, C.D. & Rice, M.E. 2001. Effect of planting date and Bacillus thuringiensis corn on the population dynamics of European corn borer (Lepidoptera: Crambidae). Journal of Economic Entomology, 94: 730–742.

Sétamou, M. & Schulthess, F. 1995. The influence of egg parasitoids belonging to Telenomus busseolae (Hymenoptera: Scelionidae) species complex on Sesamia calamistis (Lepidoptera: Noctuidae) populations in maize fields in southern Benin. Biocontrol Science and Technology, 5: 69–81.

Sokame, B.M., Ntiri E.S., Ahuya, P., Torto, B., Le Rü, B. et al. 2019. Caterpillar-induced plant volatiles attract conspecific and heterospecific adults for oviposition within a community of lepidopteran stemborers on maize plant. Chemoecology, 29(3): 89-101.

Tabashnik, B.E. & Carrière, Y. 2017. Surge in insect resistance to transgenic crops and prospects for sustainability. Nature Biotechnology, 35: 926-935.

Turlings, T.C.J., Jeanbourquin, P.M., Held, M. & Degen, T. 2005. Evaluating the induced-odour emission of a Bt maize and its attractiveness to parasitic wasps. Transgenic Research, 14:807– 816.

Van den Berg, J. & Van Wyk, A. 2007. The effect of Bt maize on Sesamia calamistis in South Africa. Entomologia Experimentalis et Applicata, 122: 45–51.

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APPENDIX A

Instructions to authors: Insects

6.1 Manuscript Preparation

6.1.1 General considerations

1. Research manuscripts should comprise: a. Front matter: Title, Author list, Affiliations, Abstract, Keywords b. Research manuscript sections: Introduction, Materials and Methods, Results, Discussion, Conclusions (optional). c. Back matter: Supplementary Materials, Acknowledgments, Author Contributions, Conflicts of Interest, References. 2. Review manuscripts should comprise the front matter, literature review sections and the back matter. The template file can also be used to prepare the front and back matter of your review manuscript. It is not necessary to follow the remaining structure. Structured reviews and meta-analyses should use the same structure as research articles and ensure they conform to the PRISMA guidelines. 3. Graphical abstract: Authors are encouraged to provide a graphical abstract as a self- explanatory image to appear alongside with the text abstract in the Table of Contents. Figures should be a high-quality image in any common image format. Note that images displayed online will be up to 11 by 9 cm on screen and the figure should be clear at this size. 4. Abbreviations should be defined in parentheses the first time they appear in the abstract, main text, and in figure or table captions and used consistently thereafter. 5. SI Units (International System of Units) should be used. Imperial, US customary and other units should be converted to SI units whenever possible 6. Accession numbers of RNA, DNA and protein sequences used in the manuscript should be provided in the Materials and Methods section. Also see the section on Deposition of Sequences and of Expression Data. 7. Equations: If you are using Word, please use either the Microsoft Equation Editor or the MathType add-on. Equations should be editable by the editorial office and not appear in a picture format. 8. Research Data and supplementary materials: Note that publication of your manuscript implies that you must make all materials, data, and protocols associated with the publication available to readers. Disclose at the submission stage any restrictions on the

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availability of materials or information. Read the information about Supplementary Materials and Data Deposit for additional guidelines. 9. Preregistration: Where authors have preregistered studies or analysis plans, links to the preregistration must be provided in the manuscript. 10. Guidelines and standards: MDPI follows standards and guidelines for certain types of research. See https://www.mdpi.com/editorial_process for further information. 6.1.2 Front Matter

These sections should appear in all manuscript types

1. Title: The title of your manuscript should be concise, specific and relevant. It should identify if the study reports (human or animal) trial data, or is a systematic review, meta- analysis or replication study. When gene or protein names are included, the abbreviated name rather than full name should be used. 2. Author List and Affiliations: Authors' full first and last names must be provided. The initials of any middle names can be added. The PubMed/MEDLINE standard format is used for affiliations: complete address information including city, zip code, state/province, and country. At least one author should be designated as corresponding author, and his or her email address and other details should be included at the end of the affiliation section. Please read the criteria to qualify for authorship. 3. Abstract: The abstract should be a total of about 200 words maximum. The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used. 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations. The abstract should be an objective representation of the article: it must not contain results which are not presented and substantiated in the main text and should not exaggerate the main conclusions. 4. Keywords: Three to ten pertinent keywords need to be added after the abstract. We recommend that the keywords are specific to the article, yet reasonably common within the subject discipline. 6.1.3 Research Manuscript Sections

1. Introduction: The introduction should briefly place the study in a broad context and highlight why it is important. It should define the purpose of the work and its significance, including specific hypotheses being tested. The current state of the research field should be reviewed carefully and key publications cited. Please highlight controversial and

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diverging hypotheses when necessary. Finally, briefly mention the main aim of the workand highlight the main conclusions. Keep the introduction comprehensible to scientists working outside the topic of the paper. 2. Materials and Methods: They should be described with sufficient detail to allow others to replicate and build on published results. New methods and protocols should be described in detail while well-established methods can be briefly described and appropriately cited. Give the name and version of any software used and make clear whether computer code used is available. Include any pre-registration codes. 3. Results: Provide a concise and precise description of the experimental results, their interpretation as well as the experimental conclusions that can be drawn. 4. Discussion: Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible and limitations of the work highlighted. Future research directions may also be mentioned. This section may be combined with Results. 5. Conclusions: This section is mandatory, and should provide readers with a brief summary of the main achievements/results of your work. 6. Patents: This section is not mandatory, but may be added if there are patents resulting from the work reported in this manuscript. 6.1.4 Back Matter

1. Supplementary Materials: Describe any supplementary material published online alongside the manuscript (figure, tables, video, spreadsheets, etc.). Please indicate the name and title of each element as follows Figure S1: title, Table S1: title, etc. 2. Acknowledgments: All sources of funding of the study should be disclosed. Clearly indicate grants that you have received in support of your research work and if you received funds to cover publication costs. Note that some funders will not refund article processing charges (APC) if the funder and grant number are not clearly and correctly identified in the paper. Funding information can be entered separately into the submission system by the authors during submission of their manuscript. Such funding information, if available, will be deposited to FundRef if the manuscript is finally published. 3. Author Contributions: Each author is expected to have made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; or have drafted the work or substantively revised it; AND has approved the submitted version (and version substantially edited by journal staff that involves the author’s contribution to the study);

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AND agrees to be personally accountable for the author’s own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used "Conceptualization, X.X. and Y.Y.; Methodology, X.X.; Software, X.X.; Validation, X.X., Y.Y. and Z.Z.; Formal Analysis, X.X.; Investigation, X.X.; Resources, X.X.; Data Curation, X.X.; Writing – Original Draft Preparation, X.X.; Writing – Review & Editing, X.X.; Visualization, X.X.; Supervision, X.X.; Project Administration, X.X.; Funding Acquisition, Y.Y.”, please turn to the CRediT taxonomy for the term explanation. For more background on CRediT, see here. "Authorship must include and be limited to those who have contributed substantially to the work. Please read the section concerning the criteria to qualify for authorship carefully". 4. Conflicts of Interest: Authors must identify and declare any personal circumstances or interest that may be perceived as inappropriately influencing the representation or interpretation of reported research results. If there is no conflict of interest, please state "The authors declare no conflict of interest." Any role of the funding sponsors in the choice of research project; design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results must be declared in this section. Insects does not publish studies funded by the tobacco industry. Any projects funded by pharmaceutical or food industries must pay special attention to the full declaration of funder involvement. If there is no role, please state “The sponsors had no role in the design, execution, interpretation, or writing of the study”. 5. References: References must be numbered in order of appearance in the text (including table captions and figure legends) and listed individually at the end of the manuscript. We recommend preparing the references with a bibliography software package, such as EndNote, ReferenceManager or Zotero to avoid typing mistakes and duplicated references. We encourage citations to data, computer code and other citable research material. If available online, you may use reference style 9. below. 6. Citations and References in Supplementary files are permitted provided that they also appear in the main text and in the reference list.

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6.1.5 References should be described as follows, depending on the type of work:

1. Journal Articles: Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range. 2. Books and Book Chapters: Author 1, A.; Author 2, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, Year; pp. 154–196. Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, Year; Volume 3, pp. 154–196. 3. Unpublished work, submitted work, personal communication: Author 1, A.B.; Author 2, C. Title of Unpublished Work. status (unpublished; manuscript in preparation). Author 1, A.B.; Author 2, C. Title of Unpublished Work. Abbreviated Journal Name stage of publication (under review; accepted; in press). Author 1, A.B. (University, City, State, Country); Author 2, C. (Institute, City, State, Country). Personal communication, Year. 4. Conference Proceedings: Author 1, A.B.; Author 2, C.D.; Author 3, E.F. Title of Presentation. In Title of the Collected Work (if available), Proceedings of the Name of the Conference, Location of Conference, Country, Date of Conference; Editor 1, Editor 2, Eds. (if available); Publisher: City, Country, Year (if available); Abstract Number (optional), Pagination (optional). 5. Thesis: Author 1, A.B. Title of Thesis. Level of Thesis, Degree-Granting University, Location of University, Date of Completion. 6. Websites: Title of Site. Available online: URL (accessed on Day Month Year). Unlike published works, websites may change over time or disappear, so we encourage you create an archive of the cited website using a service such as WebCite. Archived websites should be cited using the link provided as follows: Title of Site. URL (archived on Day Month Year). 6.1.6 Preparing Figures, Schemes and Tables

1. File for Figures and Schemes must be provided during submission in a single zip archive and at a sufficiently high resolution (minimum 1000 pixels width/height, or a resolution of 300 dpi or higher). Common formats are accepted, however, TIFF, JPEG, EPS and PDF are preferred. 198

2. Insects can publish multimedia files in articles or as supplementary materials. Please contact the editorial office for further information. 3. All Figures, Schemes and Tables should be inserted into the main text close to their first citation and must be numbered following their number of appearance (Figure 1, Scheme I, Figure 2, Scheme II, Table 1, etc.). 4. All Figures, Schemes and Tables should have a short explanatory title and caption. 5. All table columns should have an explanatory heading. To facilitate the copy-editing of larger tables, smaller fonts may be used, but no less than 8 pt. in size. Authors should use the Table option of Microsoft Word to create tables. 6. Authors are encouraged to prepare figures and schemes in color (RGB at 8-bit per channel). There is no additional cost for publishing full color graphics.

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APPENDIX B

Instructions to authors: Journal of Integrated Pest Management

6.2 Manuscript preparation: new submissions

6.2.1 Formatting

For new submissions, our formatting requirements are simple—just make sure your paper has the following items:

1. Continuous line numbers 2. Double-spaced lines 3. A title page and abstract in the main document 4. A main document in a doc, docx, PDF (if prepared in LaTeX), or rtf file type 5. Tables in a Word document (we cannot accept Excel files, unless they are supplementary files) 6. Figure and table legends in the main document 7. All coauthors entered into the online review system (email addresses required) 8. References listed in alphabetical order, cited by author and year in the text (not numbered) 9. Figures and tables at the end of the main document after the references, or uploaded as separate files. Figure legends should be included at the end of the main text after the references, and table legends should be next to their corresponding tables 10. Text is single-column 11. Please note there are more formatting guidelines for revised versions, as those are closer to being accepted (see the Revised Versions section of these author instructions). The title page should include:

1. Corresponding author: Include full name, mailing address, telephone number, and email address. 2. Title: Should be as short as possible. Only include common names that are listed in the ESA Common Names of Insects & Related Organisms. Do not include authors of scientific names. Insert “([Order]: [Family])” immediately after the name of the organism. 3. Author list: Include all authors in the order the names should be published. 4. Affiliation line: Include full addresses of all authors. If there are multiple affiliations, designate through numbered footnotes. 5. Abstract 250 words or less. 200

6. Give scientific name and authority at first mention of each organism. 7. Do not cite references, figures, tables, probability levels, or results. 8. Refer to results only in the general sense. 9. A second abstract in a second language is permitted. 10. Keywords 11. Below the abstract, provide three to five keywords, separated by commas. 6.2.2 Article types

JIPM publishes original extension- and outreach-focused articles, rather than articles based on original research. Because of this focus, articles submitted for publication in JIPM will appear and read very differently from articles found in research journals.

Articles published in JIPM are written primarily for professionals engaged in the practice of integrated pest management, including but not limited to crop producers, individuals working in crop protection, retailers, manufacturers and suppliers of pest management products, educators, and pest control operators. Although academics and other scientists will read, appreciate, and cite the articles, the intended readership will be people who typically do not belong to scientific societies and do not have ready access to scientific journals. Because of this difference in readership, the style of writing for articles submitted to JIPM should be less formulaic and more narrative than much of the scientific writing in research-focused journals.

JIPM considers the following article types

1. Profiles 2. Issues 3. Recommendations 4. Case Studies 5. Surveys and Needs Assessments 6. Brief Communications Issues: These articles will focus on emerging integrated pest management issues such as "Transgenic Bt Cotton and Insect Management" or "Prevention and Management of Bed Bugs in Commercial Buildings." Articles will include information on the issue’s relevance, why the issue developed, balanced perspectives on the issue, and possible solutions.

6.2.3 Acceptable file types

1. Main document: doc, docx, rtf, PDF (only if prepared in LaTeX) 2. Tables: Editable tables at the end of the main document. xls and xlsx files are not accepted (except as supplementary files)

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3. Figures: tif (preferred), eps (preferred), rtf, doc/docx, ppt/pptx, pdf, ps, psd, ai, gif, png 4. Supplementary files: Most file types accessible to users. Extremely large files should be uploaded in a third-party repository, such as Dryad, Harvard’s Dataverse, figshare, etc. 6.2.4 Authorship

The list of authors of the work should accurately reflect who carried out the work, who wrote the article, and others who made substantive contributions to the work. Only one author can be designated as the corresponding author. Authors are welcome to include a footnote designating that multiple authors contributed equally to the work. Authors are encouraged to include a statement of author contribution and are welcome to use the CRediT taxonomy of roles.

6.2.5 Theses, dissertations, and pre-prints

JIPM accepts papers that have been submitted to a pre-print server or that were part of a thesis or dissertation. If your paper (or a previous version of it) was posted on a pre-print server or is part of a thesis or dissertation that has been published online or in an institutional repository, please note this in your cover letter so that it won’t be flagged for plagiarism.

6.2.6 Personal communications

Personal communication citations should be accompanied by a letter from the person being cited giving permission to use him or her as a citation and verifying the claim being cited. This letter should be uploaded as a supplementary file.

6.2.7 Abbreviations

Abbreviations should be used sparingly. Standard abbreviations for measurements according to Scientific Style and Format, 8th edition, are acceptable, as well as common abbreviations that improve the readability of a manuscript (e.g., DNA, PCR). All other abbreviations used should be defined at the first use. Sentences should never begin with an abbreviation.

6.2.8 Measurements

Use metric units. English units may follow within parentheses if needed.

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APPENDIX C

John Wiley and Sons license

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APPENDIX D

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