A Comparative Study of Arthropod Diversity on Conventional and Bt-Maize at Two Irrigation Schemes in South Africa

A comparative study of arthropod diversity on conventional and Bt-maize at two irrigation schemes in South Africa

Jean-Maré Truter

Thesis submitted in fulfilment of the requirements for the award of the degree Master of Environmental Science at the North-West University (Potchefstroom Campus)

May 2011

Supervisor: J. Van den Berg

Co-supervisor: H. van Hamburg

ACKNOWLEDGEMENTS

There are several people without whom this dissertation and the work it describes would not have been possible. I would like to thank all those people who have contributed towards the successful completion of this work.

God our saviour who held his hand over us with every road trip we made. Without His guidance nothing I have done would have been possible.

My sincere thanks to Prof. Johnnie van den Berg, my supervisor and mentor during this project. His patience, guidance, constructive criticism, sound advice and encouragement throughout the course of the study are greatly appreciated. It is due to Prof. that I have developed an interest in entomology.

Prof. Huib van Hamburg, co-supervisor, thank you for your contribute and patience with all the questions and for assistance in statistical analyses.

Dr. Suria Ellis of the statistical consultation service, which provided assistance with statistical analyses, a warm thanks for the time and patience.

The assistance of Prof. Pieter Theron with identification of Acari is highly appreciated.

Then I am truly grateful to my student colleagues that patiently helped with sampling.

This work forms part of the Environmental Biosafety Cooperation Project between South Africa and Norway coordinated by the South African National Biodiversity Institute and we accordingly give due acknowledgment.

Finally, I express my appreciation and love to my parents and Emil Engelbrecht, for their moral support and encouragement during the whole project period, it is much appreciated.

ABSTRACT

Species assemblages within an agro-ecosystem fulfil a variety of ecosystem functions that may be harmed if changes occur in these niche assemblages. Guild rearrangement due to the elimination of a target pest and subsequent changes in guild structure can lead to development of secondary pests. For this reason it is essential to assess the potential environmental risk the release of a genetically modified (GM) crop may hold and to study its effect on species assemblages within that ecosystem. Assessment of the impact of GM Bt maize on the environment is hampered by the lack of checklists of species present in maize ecosystems. The aims of the study were to compile a checklist of arthropods that occur on maize in South Africa and to determine and compare the diversity and abundance of arthropods and functional groups on Bt and non-Bt maize. An ecological risk assessment model approach was used to identify arthropod species that were most likely to be exposed to Bt protein inside the maize ecosystem, and to prioritise species for further research. Collections of arthropods were done during the 2007/2008 and 2008/2009 growing seasons on Bt and non-Bt maize plants at two localities, i.e. Vaalharts in the Northern Cape- and Tshiombo in the Limpopo province of South Africa. The focus was on collecting arthropods that occured on plants during the reproductive stage of plant growth only. No collections were made of soil arthropods and this study therefore only takes into account above-ground on-plant diversity. Bt and non-Bt maize were sampled at each locality over two seasons. Arthropods collected on these plants were classified to morpho-species level. The Shannon diversity- and Margalef richness indices as well as the total number of species and the total number of individuals were compared between localities, between seasons as well as maize varieties (Bt vs. non-Bt). Rank abundance graphs were also compiled to indicate species richness and evenness at each site. The morpho-species were grouped into functional groups to provide information on the potential exposure of species to Bt toxin in GM maize. These functional groups were: detritivores, herbivores, predators and parasitoids. Priority species for future research was identified following an ecological model approach, using the extensive data base on species richness and abundance of arthropods in the receiving environment. A total of 8771 arthropod individuals, comprising 288 morpho-species from 20 orders of arthropods were collected during this study. Arthropod biodiversity in maize was high and our findings suggest that Bt maize had no effect on total arthropod diversity as well as on the different functional groups. The following non-target functional groups were identified as important for future research: herbivores (Aphididae, Tetranychidae), predators (Anthocoridae, Forficulidae, Coccinellidae, Staphylinidae) and parasitoids (Braconidae and Scelionidae). During this study, non-target species with high abundance that are also exposed to Bt maize were identified. Research gaps were identified and indicated lack of information particularly for the predatory groups, Forficulidae and Staphylinidae as well as for the herbivorous beetles, Nitidulidae. This study provided a start in the study of biodiversity of arthropods in maize in South Africa and generated a basic checklist of these species. For future assessments, this study will be used to determine the possible impact of Bt maize on the environment. This study and reviewed literature therein indicated that many species could be excluded for further testing in South Africa since Bt maize has been reported to have no adverse effect, whereas for others, continued studies are needed.

Key words: Arthropods, biodiversity, diversity indices, GM maize, risk assessment, South Africa

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UITTREKSEL

Spesiesgroeperinge in landbou-ekosisteme vervul ‘n verskeidenheid van ekosisteemfunksies wat benadeel kan word indien veranderings plaasvind in hierdie groeperinge. Die hergroepering van funksionele groepe as gevolg van ‘n teikenplaag wat uitgeskakel word en die daaropvolgende veranderinge in die gilde-struktuur, kan lei tot die ontwikkeling van sekondêre plae. Dit is daarom noodsaaklik om die moontlike omgewingsrisiko’s wat die verbouing van geneties-gemodifiseerde (GM) gewasse kan inhou te ondersoek, en die effek op spesies in ‘n ekosisteem te evalueer. Die impak van GM Bt-mielies op die omgewing en skadebepaling daarvan word belemmer deur die tekort aan inligting rakende athropood diversiteit in mielie-ekostelsels. Die doel van hierdie studie was om ‘n lys op te stel van athropood spesies wat voorkom op mielies in Suid-Afrika, en om die diversiteit en veelheid van Arthropoda en funksionele groepe te bepaal en te vergelyk tussen Bt en nie-Bt mielies. ŉ Ekologiese risiko-assesseringsmodel is gebruik om arthropoodspesies te identifiseer wat waarskynlik die meeste blootgestel word aan die Bt-proteïen in die mielie-ekostelsel en ook om hierdie spesies te prioritiseer vir verdere navorsing. ‘n Opname van Arthropoda is gedoen gedurende die 2007/2008 en 2008/2009 groeiseisoene op Bt en nie-Bt-mielies by twee lokaliteite nl. Vaalharts in die Noord-Kaap- en Tshiombo in die Limpopo provinsie. Slegs Athropoda wat tydens die reproduktiewe stadium van die plant voorgekom het is versamel. Geen grondlewende Arthropoda is versamel nie en hierdie studie neem dus net bo-grondse op-plant diversiteit in ag. Bt en nie-Bt-mielies was versamel by elke lokaliteit oor twee seisoene. Al die athropode wat tydens hierdie opname versamel is, is verder geklassifiseer tot op morfo-spesievlak. Shannon- en Margalef se diversiteitsindekse sowel as die totale aantal spesies en die totale aantal individue is vergelyk tussen lokaliteite, seisoene asook mielievariëteite (Bt vs. nie-Bt). Rangveelheidsdiagramme is opgestel om spesierykheid en gelykheid by elke lokaliteit aan te dui. Die morfo-spesies is gegroepeer in funksionele groepe om inligting te verskaf oor die potensiële blootstelling van spesies aan die Bt-toksien in GM mielies. Die funksionele groepe was: ontbinders, herbivore, predatore en parasitoïde. Prioriteit-spesies vir toekomstige navorsing is geïdentifiseer met behulp van ‘n ekologiese risiko-assesserings-model benadering, deur gebruik te maak van ‘n uitgebreide databasis oor spesierykheid en die veelheid van Arthropoda. 'n Totaal van 8771 arthropood individue, bestaande uit 288 morfo-spesies vanuit 20 orders van Arthropoda is versamel gedurende die studie. Arthropoodbiodiversiteit in mielies was hoog en bevindinge dui daarop dat Bt-mielies geen effek op arthropood diversiteit sowel as op die verskillende funksionele groepe gehad het nie. Die volgende nie-teiken funksionele groepe is geïdentifiseer as belangrik vir vedere navorsing: herbivore (Aphididae, Tetranychidae), predatore (Anthocoridae, Forficulidae, Coccinellidae, Staphylinidae) en parasitoïde (Braconidae en Scelionidae). Tydens hierdie studie is nie-teikenspesies met 'n hoë veelheid, wat ook blootgestel word aan Bt-mielies, geïdentifiseer. Navorsingsvrae is geïdentifiseer en dui opŉ gebrek aan inligting oor sekere predatorgroepe (Forficulidae en Staphylinidae) sowel as die herbivoorkewers, Nitidulidae. Hierdie opname dien as ‘n beginpunt van arthropoodbiodiversiteitstudies in Suid-Afrika, en verskaf ‘n basiese spesielys. Dié studie kan gebruik word om moontlike toekomstige impakte van Bt-mielies op die omgewing te evalueer. Hierdie studie en die literatuur wat bestudeer is dui aan dat baie spesies moontlik uitgeskakel kan word vir verdere navorsing in Suid-Afrika, omdat Bt-mielies geen nadelige effek op hierdie spesies het nie. Vir sekere spesies is voortgesette navorsing egter nodig.

Sleutelwoorde: Arthropoda, biodiversiteit, diversiteitsindekse, GM mielies, risiko assesserings, Suid Afrika.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii ABSTRACT ...... iii UITTREKSEL ...... iv

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ...... 1

1.1 Introduction ...... 1 1.2 What is transgenic Bt maize? ...... 1 1.3 History of Bt maize ...... 2 1.4 Global importance of genetically modified crops ...... 3 1.5 Advantages and disadvantages of Genetically Modified maize ...... 4 1.5.1 Advantages of Genetically Modified crops with insecticidal traits (e.g. Bt maize) ...... 5 1.5.2 Disadvantages of Genetically Modified crops with insecticidal traits (e.g. Bt maize) ...... 5 1.6 Environmental fate of Bt proteins from transgenic maize ...... 6 1.6.1 Effects of Bt protein in aquatic ecosystems ...... 7 1.6.2 Effects of Bt protein in soil ecosystems ...... 9 1.6.2.1 Earthworms ...... 10 1.6.2.2 Isopoda ...... 11 1.6.2.3 Micro-arthropods ...... 11 1.6.3 Effects of Bt protein in plant ecosystems ...... 12 1.6.3.1 Land snail ...... 12 1.6.3.2 Pollinators ...... 12 1.6.3.3 Natural enemies of pests ...... 13 1.6.3.4 Lepidoptera ...... 14 1.6.3.5 Homoptera...... 15 1.6.3.6 Hymenoptera parasitoids ...... 16 1.7 Arthropod diversity on crops ...... 16 1.7.1 Secondary pests ...... 19 1.8 Resistance development and the high-dose/refuge strategy ...... 21

1.9 Environmental risk assessment and management ...... 22 1.9.1 The non-target environmental risk assessment model ...... 25 Step 1: Categorizing and listing potential non-target species and ecological functions and identifying important interactions ...... 25 Step 2: Prioritizing species or functions for pre-release testing according to maximum potential adverse effect ...... 25 Step 3: Conducting exposure pathway analyses ...... 26 Step 4: Describing hazard scenarios and formulating testing hypotheses ...... 26 Step 5: Developing ecologically meaningful testing methods and protocols ...... 26 1.10 Objectives ...... 27 1.11 References ...... 28

CHAPTER 2 COMPARATIVE DIVERSITY OF ARTHROPODS ON BT AND NON-BT MAIZE AT THE VAALHARTS AND TSHIOMBO IRRIGATION SCHEMES IN SOUTH AFRICA ...... 44

2.1 Abstract ...... 44 2.2 Introduction ...... 45 2.3 Material and Methods ...... 47 2.3.1 Study areas ...... 47 2.3.1.1 Tshiombo ...... 47 2.3.1.2 Vaalharts ...... 47 2.3.2 Sampling of arthropods ...... 47 2.3.3 Data analysis ...... 48 2.4 Results ...... 50 2.4.1 Total arthropod diversity ...... 54 2.4.2 Detritivores ...... 56 2.4.3. Chewing herbivores ...... 59 2.4.4. Sucking herbivores ...... 62 2.4.5. Chewing predators ...... 65 2.4.6 Sucking predators ...... 68 2.4.7. Parasitoids ...... 71 2.5. Discussion ...... 74 2.6 Conclusions ...... 76

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2.7 References ...... 77

CHAPTER 3 ARTHROPOD BIODIVERSITY IN MAIZE AND SELECTION OF NON-TARGET ARTHROPODS SPECIES FOR ECOLOGICAL RISK ASSESSMENT OF BT MAIZE IN SOUTH AFRICA ...... 82

3.1 Abstract ...... 82 3.2 Introduction ...... 83 3.3 Material and methods ...... 84 3.4 Environmental risk assessment with reference to Bt maize in South Africa ...... 85 3.4.1 Step 1: Categorizing and listing potential non-target species and ecological function and identification of important interactions ...... 85 3.4.2 Step 2: Prioritizing species or functions for pre-release testing according to maximum potential adverse effect ...... 88 3.4.2.1 Prioritizing functional groups and non-target species ...... 88 3.4.2.1.1 Detritivores ...... 89 3.4.2.1.2 Herbivores ...... 89 3.4.2.1.3 Predators and parasitoids ...... 90 3.4.3 Step 3: Analysing exposure pathways ...... 93 3.4.3.1 Herbivores ...... 96 3.4.3.2 Predators and parasitoids ...... 97 3.4.4 Step 4: Describing hazard scenarios and formulating testing hypotheses ...... 97 3.4.4.1 Herbivores ...... 98 3.4.4.2. Predators and parasitoids ...... 98 3.4.5 Step 5: Developing ecologically meaningful testing methods and protocols ...... 98 3.4.5.1 Herbivores ...... 99 3.4.5.1.1 Aphids ...... 99 3.4.5.1.2 Nitidulidae ...... 100 3.4.5.1.3 Tetranychidae ...... 100 3.4.5.2 Predators ...... 100 3.4.5.2.1 Anthocoridae ...... 100 3.4.5.2.2 Coccinellidae ...... 101 3.4.5.2.3 Staphylinidae and Forficulidae ...... 102 3.4.5.3 Parasitoids ...... 103 3.4.5.3.1 Braconidae and Scelionidae ...... 103 3.5 Conclusions ...... 105

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3.6 References ...... 106

CHAPTER 4 CONCLUSIONS ...... 116

4.1 Discussion and conclusions ...... 116 4.2 References ...... 119

Appendix A ...... 121

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Although the earth provides an enormous amount of food for approximately 6 billion people, nearly 800 million people currently suffer from malnutrition. The food demand of the world population in 2050 is estimated to be twice as much as that of the current level (Koyama, 2000). Sub-Saharan Africa is one of the four largest food consumers which will account for approximately 70% of the world’s food demand by 2020 (Ekboir & Morris, 2004). Maize is one of the most important crops in southern Africa. It is important that production becomes sustainable despite biotic stresses such as insect pests. Because of the susceptibility of maize to insect pests, especially Lepidoptera, genetically modified (GM) maize with insecticidal properties was developed to protect this crop against pest damage.

The target pests of Bt maize in South-Africa are the stem borers Busseola fusca (Lepidoptera: Noctuidae) and Chilo partellus (Lepidoptera: Crambidae) which can cause between 10-100% yield loss depending on planting date and levels of infestation (Kfir et al., 2002).

1.2 What is transgenic Bt maize?

Bacillus thuringiensis (Bt) is a gram-positive bacterium, common in soil, characterised by its ability to produce insecticidal crystal proteins during sporulation (Höfte & Whiteley, 1989). These crystalline proteins have a specific toxic activity against larvae of Lepidoptera, Diptera and Coleoptera (Höfte & Whiteley, 1989; Sharma et al., 2000).

The specificity of Bt to Lepidoptera is due in part to the alkaline midgut environment that is required to solubilize the protoxin into its active form. Solubilized protoxins are activated by midgut proteases and bind to receptors on the epithelial surface. It has been proposed that disruption of the midgut epithelium results in a prolonged cessation of feeding and eventual death by starvation (Broderick et al., 2006).

An alternative proposed mechanism of killing is that extensive cell lysis provides bacterial spores access to the more favourable environment of the hemocoel, where they germinate and reproduce, leading to septicemia and death (Knowles, 1994). The latter hypothesis was

1 supported by Broderick et al. (2006) who indicated that neither of these two models is entirely consistent with experimental observations. For example, the starvation model is not supported by the fact that B. thuringiensis kills insects much more rapidly than does starvation. Bacillus thuringiensis toxin-induced mortality typically takes two to five days, whereas starvation-induced mortality often takes five to seven days (Broderick et al., 2006). The septicemia model is challenged by the observation that the toxin causes mortality when it is separated from the bacterial cells, which has been accomplished with purified toxin and transgenic plants that produce the Bt toxin (Gills et al., 1992).

Broderick et al. (2006) indicated that naturally occurring enteric bacteria are responsible for septicemia associated with B. thuringiensis toxicity. The enteric bacteria alone do not induce mortality, suggesting that B. thuringiensis enables them to reach the hemocoel by permeating the gut epithelium. These results suggest that B. thuringiensis toxicity depends on an interaction with microorganisms of the normal gut community. This finding contrasts sharply with established models that assume that B. thuringiensis itself induces mortality through starvation or direct septicemia (Broderick et al., 2006).

1.3 History of Bt maize

The history of Bacillus thuringiensis as an insecticide goes back 90 years when the Japanese bacteriologist, S. Ishiwata, isolated the bacillus from diseased Bombyx mori (Lepidoptera: Bombycidae) larvae. He named it Sottokin which means ‘sudden death bacillus’ (Beegle, 1992). A decade later, Ernst Berliner isolated a similar organism from diseased granary populations of Anagasta kuehniella (Lepidoptera: Pyralidae) larvae from Thuringia, Germany. Berliner named the bacterium Bacillus thuringiensis and because Ishiwata did not formally describe the organism he found, Berliner is credited with naming it. Nothing further was done with B. thuringiensis for over a decade (± from 1916), but due to a serious problem with the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae) in maize in North America, control measures needed to be developed. Because of the promising nature of some of the B. thuringiensis spray formulations in field trails, commercial production of B. thuringiensis was undertaken (Beegle, 1992). To date, there are 67 registered Bt products with more than 450 different formulations (Sharma et al., 2000). The use of biotechnology and genetic modification done through transfer of genetic material from Bt-bacteria to crops will be discussed in the next section.

1.4 Global importance of genetically modified crops

Following the consistent and substantial economic, environmental and welfare benefits generated from GM crops over the last fifteen years, millions of large, small and resource- poor farmers in both industrial and developing countries continued to plant significantly more hectares of biotech crops in 2010 (James, 2010). In 2010, the global hectarage of biotech crops continued to grow strongly reaching 148 million hectares and the number of farmers benefiting from biotech crops globally in 29 countries reached 15.4 million. Over ninety percent of these were small scale and resource-poor farmers in developing countries (James, 2010). South-Africa is ranked number nine in the world with a total GM crop area of 2.2 million hectares, up from 2.1 million hectares in 2009 (Figure 1.1). Growth in 2009 and 2010 was mainly attributed to an increase in GM maize area, accompanied by an increase in GM soybean of which adoption rate was 85% (James, 2010).

The top nine countries planting biotech crops, plant more than 1 million hectares of GM crops each (Figure 1.1). The growth rate between 1996 and 2010 was an unprecedented 87-fold increase making it the fastest adopted crop technology in recent history. This very high adoption rate by farmers reflects the fact that GM crops have consistently performed well and delivered significant economic, environmental, health and social benefits to both small and large scale farmers in developing and industrial countries (James, 2010).

Figure 1.1. Global map of biotech crop countries and mega-countries in 2010 (Adapted from James, 2010).

1.5 Advantages and disadvantages of Genetically Modified maize

When assessing advantages and disadvantages of the use of modern biotechnology in crops, there are a series of issues to be addressed in order to make informed decisions on the appropriateness of this technology when seeking solutions to current problems in food, agriculture and natural resources management (Cook, 1999). These issues include risk assessment and management within an effective regulatory system as well as the role of intellectual property management. In terms of addressing any risk posed by the cultivation of genetically modified plants in the environment, six safety issues were proposed by the Organization for Economic Cooperation and Development (OECD) that need to be considered. These are gene transfer, weediness, trait effects, genetic and phenotypic variability, expression of genetic material from pathogens and worker safety (Cook, 1999).

When value judgements are made about risk and benefits of the use of biotechnology it is important to distinguish between technology-inherent risks and technology-transcending

4 risks. Technology-inherent risks include assessing any risks associated with food safety and the behaviour of biotechnology-based products in the environment (Persley & Siedow, 1999). This includes possible non-target effects and resistance development. Technology- transcending risks originates from within a political and social context in which the technology is used and how these uses may benefit or harm the interest of different groups in society (Persley & Siedow, 1999).

1.5.1 Advantages of Genetically Modified crops with insecticidal traits (e.g. Bt maize) The advantages that Bt crops could have, can be summarized as follows:

• specificity to target species (Kfir et al., 2002; OECD, 2007). • protection against stem borers throughout the growing season of the plant (Betz et al., 2000; OECD, 2007; Kruger et al., 2009). • transgenic plants overcome the problem of traditional microbial preparations or insecticidal sprays that may not reach insects that burrow through soil or those that bore into and remain inside the plant stem or tissue (OECD, 2007). • reduced environmental impact from pesticides that are no longer used, the frequency of treatments and/or surface area that is treated is reduced (Betz et al., 2000; Wolfenbarger & Phifer, 2000). • increased income due to reduction of production costs through decreased needs for inputs of pesticides (Persley & Siedow, 1999). This may lead to a reduced cultivated area needed to produce the total amount of food required by people. This could result in lower pressure on land not yet under cultivation and could allow more land to be left under protection (Betz et al., 2000; Lövei, 2001).

1.5.2 Disadvantages of Genetically Modified crops with insecticidal traits (e.g. Bt maize) Disadvantages associated with cultivation of crops with insecticidal properties can be summarized as follows:

• the continuous exposure and relative higher amounts of δ-endotoxin may lead to the selection of insects that are resistant to one or more strains of the δ-endotoxin (Bauer, 1995; Persley & Siedow, 1999; OECD, 2007). • there may be non-target organisms that may be affected by the δ-endotoxin (OECD, 2007).

1.6 Environmental fate of Bt proteins from transgenic maize

Agro-ecosystems consist of organisms that interact in food webs (Mayse & Price, 1978; Janssen et al., 1998; Dicke & Vet, 1999), which are among nature’s most complex creations (Eveleigh et al., 2007). With the increased awareness of the complexity and importance of food webs in agricultural systems, conservation of these ecosystems has been highlighted. Artificial food webs are created in agricultural systems and the interactions between plants, herbivores and natural enemies in these systems may change from simple tri-trophic interactions to more complex food web interactions (Janssen et al., 1998). Crop plants and insect pests form complex agricultural ecosystems that involve interactions between many trophic levels are often referred to as multi-trophic interactions (Poppy & Sutherland, 2004). In studies to determine possible effects of Bt plants on non-target organisms it is important to recognise that delicate food webs exist even in agricultural systems (Groot & Dicke, 2002).

The movement of plant-expressed Bt toxins as well as transgenic DNA in agricultural and natural systems is a growing concern due to the large amounts thereof entering agricultural systems from transgenic crops (Andow & Zwahlen, 2006). Research demonstrated that transgenes can move beyond the intended organism and into the surrounding environment (Marvier & Van Acker, 2005). This movement poses several risks, such as introgression into natural plant communities (Loureiro et al., 2009) and genetic transformation into natural bacterial populations (De Vries & Wackernagel, 2004). Regardless of the mechanism, the movement of transgenes into the environment at large is a real risk, and has serious implications for environmental health, including human safety (Hart et al., 2009).

In order to do reliable risk assessments regarding GM crops, information is needed on biodiversity and the spectrum of possible non-target species in maize based ecosystems. Current risk assessment is hampered by a lack of even a basic checklist of species in these systems. Information on biodiversity is critical in evaluation of the possible impact of Bt maize on non-target organisms at different trophic levels as well as the possible causal pathways of exposure to the GM plant and toxin. The following section reports on some effects of Bt proteins from GM maize in aquatic, soil and terrestrial ecosystems.

1.6.1 Effects of Bt protein in aquatic ecosystems

The possible effects of Bt maize by-products that contain δ-endotoxins on stream organisms have received little attention (Sears et al., 2001; Rosi-Marshall et al., 2007). This is in sharp contrast to numerous studies examining potential effects on non-target organisms in the terrestrial environment (Sears et al., 2001; Oberhauser et al., 2001; Pleasants et al., 2001; O’Callaghan et al., 2005).

The Bt proteins from genetically modified maize enter aquatic systems from agricultural fields through multiple routes. One route that has been examined is the addition of pollen to surface water during flowering of maize plants (USEPA, 2003). During pollen shed, wind can transport maize pollen from 40 to 60 m away from source fields (Raynor et al., 1972), and rain can dislodge and transport pollen away from crops (Pleasants et al., 2001). One potentially important path, however, is the direct entry of crop dust during harvest or crop residues via runoff into aquatic systems (Figure 1.2 A & B). The biomass of a GM crop entering surface water through these routes may be much larger than that of the pollen (Prihoda & Coats, 2008). The Bt proteins adsorb rapidly to clay particles and humic acids in soil and do not readily desorb, thereby resulting in adsorbed proteins being unavailable for degradation by microbial action (Stotzky, 2002). The strong adsorption of Bt proteins to soil particles indicates that they are likely to be transported while bound to surface active particles in sediment eroded either by wind, rain, or snowmelt (Prihoda & Coats, 2008). A study in which soil and surface water were spiked with pure Bt maize endotoxin showed that the endotoxin was degraded more rapidly in water than in soils (Douville et al., 2005). Once in stream channels, possible fates of crop by-products include microbial decomposition, consumption by aquatic invertebrates, burial via sedimentation, or downstream transport (Rosi-Marshall et al., 2007).

A B

Figure 1.2. (A) A typical headwater agricultural stream during pollen shed with a buffer strip of grass and adjacent maize fields, (B) accumulation of maize by-products after harvest (Rosi-Marshall et al., 2007).

A study on the acute effects of root extracts from transgenic maize, event MON863 (Cry3Bb1), on the larvae of the aquatic midge, Chironomus dilutus (Diptera: Chironomidae) was the first assessment of the potential hazard that Bt protein may hold to aquatic invertebrates (Prihoda & Coats, 2008). A significant decrease in C. dilutus survival was demonstrated, but more research is needed concerning both the environmental fate and the effects of transgenic Bt Cry3Bb1 before definitive conclusions about risk can be made (Prihoda & Coats, 2008). This was followed by a study on the hazard of pollen from Bt maize (MON810) to Daphnia (Bøhn et al., 2008; Bøhn et al., 2010). Although acute effects on survival were observed in described laboratory experiments, the study did not realistically replicate the exposure pattern present in an aquatic system. Aquatic invertebrates will likely be exposed to acute concentrations of Bt proteins in crop residues, however, runoff of maize residue could occur at multiple times during the year. Although exposure of aquatic invertebrates to Bt proteins will most likely be short term it may occur at multiple times during the life cycle of the plant or following rain/wind events that occur post harvest (Prihoda & Coats, 2008).

Rosi-Marshall et al. (2007) reported that 50% of filter-feeding trichopterans, collected from streams in Canada during peak pollen shed of maize, had pollen grains in their guts and that detritivorous trichopterans occurred in areas where decomposing maize litter accumulated in streams after harvest. In laboratory feeding trials, the leaf shredding trichopteran, Lepidostoma liba (Trichoptera: Lepidostomatidae), showed more than 50% lower growth rates when they were fed Bt maize litter compared with counterparts feeding on non-Bt maize litter, although mortality of L. liba among litter types did not differ (Rosi-Marshall et al., 2007).

Mortality rates of Helicopsyche borealis (Trichoptera: Helicopsychidae), an algal-scraping trichopteran, were also studied by Rosi-Marshall et al. (2007). Results suggested that pollen adhering to algal biofilms was consumed at high concentrations and that the Bt δ-endotoxin in crop by-products can be harmful to stream dwelling trichopterans (Rosi-Marshall et al., 2007).

1.6.2 Effects of Bt protein in soil ecosystems

Risk assessments relating to soil biota are very complex and no examples of such assessments exist. Many factors may affect the outcome of the risk assessment process. These include heterogeneity of the soil environment, the complexity of the communities present in soil and the aggregation and patterns of movement of soil fauna and flora. These are but some reasons why it is difficult to evaluate the effect of GM plants on non-target organisms in soil systems (Griffiths et al., 2006).

There is a likelihood that soil-dwelling organisms may be influenced by transgene-derived proteins released into the soil as exudates from the roots of GM plants (Borisjuk et al., 1999) and through contact with plant material left in the ground after harvest (O’Callaghan et al., 2005). However, little research has been done on this subject. Soil biota exists within complex soil food webs together with numerous invertebrate species, such as earthworms, Collembola and nematodes. These organisms carry out important soil ecosystem processes such as nutrient cycling and decomposition that have major ecological and agricultural significance (O’Callaghan et al., 2005).

The Cry1Ab protein from transgenic Bt maize can enter the soil via pollen drift, plant residues after harvest, root exudates and decomposition of dead plant material (Zwahlen et al., 2007). During plant growth Cry1Ab protein is released by roots and persists in soil at least until the occurrence of the first frost (Saxena et al., 1999; Saxena & Stotzky, 2000; Saxena & Stotzky, 2001a). The main source of Bt proteins entering the soil is most likely via plant residues, as 2 to 6 tons of residue per hectare can be left on the field after harvest (Zscheischler et al., 1984), and the Cry1Ab protein can persist in the plant matrix for at least two years after sowing (Zwahlen et al., 2003a).

Laboratory and greenhouse studies on the decomposition of Cry1Ab Bt maize indicated that Bt maize either decomposes at a similar rate or more slowly than non-Bt maize (Hopkins & Gregorich, 2003; Flores et al., 2005). Slower rates of residue decomposition can result in nutrient limitations for primary producers and decreased nutrient cycling. The accumulation

9 of plant residues in the soil might cause a longer persistence and subsequent accumulation of Cry1Ab protein, thereby enhancing the probability of exposure for non-target soil organisms (Ferré et al., 1995; Saxena and Stotzky, 2001b). On the other hand, when lower decomposition of the biomass of Bt plants continues for an extended time, it may be beneficial, as the organic matter derived from Bt plants would persist longer and accumulate at higher levels in soil, thereby improving soil structure and reducing erosion (Gisi et al., 1997; James et al., 1998; Flores et al., 2005).

Estimates on the persistence of Bt toxin in soil also vary. A laboratory study showed that much of the Bt endotoxin in crop residues is highly labile and decompose quickly in soil, but that a small fraction may be protected from decay inside residues (Hopkins & Gregorich, 2003). In contrast to the latter study, another reported no degradation of the Cry 1Ab protein after one month that the leaf litter was buried (Zwahlen et al., 2003a).

Examples of effects of Bt proteins on selected non-target organisms in soil ecosystems are provided below.

1.6.2.1 Earthworms Earthworms are considered beneficial organisms in agricultural soils because of their functional roles in the breakdown of dead plant tissue, recycling of plant nutrients and in their influence on soil drainage and aeration (Lee, 1985). Earthworms are particularly important in maintaining or improving soil physical conditions where maize is grown with reduced tillage practices (Lachnicht et al., 1997). However, under field conditions, Bt-protein may persist for up to 200 days (Zwahlen et al., 2003a), implying that earthworms consuming maize residues and soil may be exposed directly to the protein for a significant part of their life-cycle (Vercesi et al., 2006).

Studies on the effect of GM maize on earthworms provided contrasting results. Growth and mortality of Eisenia foetida (Lumbricidae: Haplotaxida) was reported not to be affected when exposed to 120 times the amount of Cry3A toxin present in maize plants (USEPA, 2000). However, in another study, Lumbricus terrestris (Lumbricidae: Haplotaxida) lost 18% of their initial weight after 200 days when fed Bt maize compared with a 4% weight gain when fed non-Bt maize (Zwahlen et al., 2003b). There was, however, no certainty if the loss in weight was caused by the Bt toxin or because of nutritional quality of the plant material ingested by earthworms (O’Callaghan et al., 2005).

Because of its abundance in agricultural soils, Aporrectodea caliginosa (Lumbricidae: Haplotaxida) was also used to evaluate non-target effects of Bt-maize on soil organisms in Denmark. Bt-maize residues had no detrimental effect on growth, development and mass of juveniles. Cocoon production was unaffected after earthworms were fed with Bt-maize residues, but a small statistically significant negative effect was seen on cocoon hatching. Hatching success was reduced from 95 to 75% at one of the highest concentrations of Bt- maize residues. It was, however, not possible to explain why this particular parameter was affected by Bt toxin when other parameters like growth and development were not (Vercesi et al., 2006).

The mechanisms that underlie the apparent insensitivity of A. caliginosa, and other species of earthworms to Bt toxin may be due to the lack of activation of the Bt-protein or the lack of toxin-binding receptors in the cell membranes of the earthworm gut. Bt-protein crystals are activated in insect gut at high pH (>5) and subsequent modification by proteolytic enzymes (Höfte & Whiteley, 1989). The pH in the earthworm gut is between six and seven (Laverack, 1963), suggesting that the toxin is never activated in the earthworm gut (Vercesi et al., 2006).

1.6.2.2 Isopoda No effects of Bt maize were observed on the woodlice, Porcellio scaber (Isopoda: Porcellionidae), in a soil-free laboratory system (Escher et al., 2000). Woodlice did not differentiate between Bt and non-Bt maize in their food preference and their numbers of offspring did not differ. Initial mass of offspring increased when feeding on non-Bt maize leaves, but adults increased in mass when feeding on Bt maize leaves. The reason for the difference in mass gain may, however, have resulted from differences in nutritional quality of Bt and non-Bt leaves (Escher et al., 2000).

1.6.2.3 Micro-arthropods Collembolans are indicator species of soil fertility and health and also play a role in the breakdown and recycling of crop residues (O’Callaghan et al., 2005). They may therefore also be exposed to transgene-derived proteins that remain in crop residues. Laboratory studies showed that there were no adverse effects on selected Collembola species (O’Callaghan et al., 2005). For example, in studies where Bt cotton and Bt potato leaf residues were fed to Folsomia candida (Collembola: Isotomidae) no effect on time of oviposition, egg production or body length was observed (USEPA, 2000).

1.6.3 Effects of Bt protein in plant ecosystems

As mentioned before, an advantage of genetically engineered insect-resistant plants expressing genes encoding δ-endotoxin and of microbial Bt preparations is the greater specificity of δ-endotoxins to target species. This reported specificity may reduce adverse impacts on non-target organisms. In spite of reported target specificity, there may still be insects and other non-target organisms potentially affected by the δ-endotoxin and extended exposure might affect their populations (OECD, 2007). Several studies have demonstrated that susceptibility to the endotoxin in Bt maize varies not only among Lepidoptera species, but also between the various Bt insertion events (NRC, 2002). As a result, studies differ in their conclusion on the estimated impact of Bt maize on non-target species (NRC, 2002). The following are examples of effects of exposure of selected terrestrial non-target organisms to Bt proteins produced by GM crops.

1.6.3.1 Land snail Among soil fauna, land snails are possible non-target organisms that can be exposed through digestive and cutaneous routes to the Bt-protein released in soil by roots or by decomposing plant residues (Kramarz et al., 2009). Snails can eat fresh leaves of Bt maize, which contain high concentrations of Bt-protein (Griffiths et al., 2006) and are therefore exposed to toxins. Research showed that Bt maize influenced the growth of the snail, Cantareus aspersus (Gastropoda: Pulmonata), but only after a long-term exposure (at least 47 weeks), when snails reached sexual maturity. At the end of the growth, snails exposed to Bt-toxin in food and soil had a 25% lower growth coefficient than unexposed snails (Kramarz et al., 2009). This indicated that in the case of long-lived animals, such as snails, the effect of exposure to GM plant material may be demonstrated only in chronic tests. Another study by Ester and Nijënstein (1995) reported a temporary reduction of feeding of the slug Deroceras reticulatum (Gastropoda: Agriolimacidae) fed with seeds of winter wheat treated with the Bt- protein at concentrations as high as 20g active ingredient per kg dry plant material.

1.6.3.2 Pollinators Neither Bt cotton nor Bt maize requires bees for pollination, but cotton nectar is attractive to bees which produce honey, whereas maize pollen may be collected when other pollen is scarce (O’Callaghan et al., 2005). Studies showed that Bt plants have no effect on honeybees (OECD, 2007). However, sprays of B. thuringiensis subsp. aizawai have been shown to be highly toxic to honeybees (USEPA, 1998). Laboratory studies typically expose honey bees to doses of Cry proteins that are ten or more times higher than those

12 encountered in the field, which provide additional reassurance that toxicity in the field is unlikely (Duan et al., 2008).

Pollen is a food source for larvae and young bees after emergence (Haydak, 1970). While bees feed on pollen, the development of physiological structures involved in olfaction and learning performances takes place (Masson & Arnold, 1984; Masson et al., 1993). Exposure of young honey bees to Bt toxins may cause deleterious effects on development and learning performance. Learning is of primary importance in honey bee foraging, therefore any indirect and/or direct impairments of this capability can affect the development of honey bee colonies and their role as pollinators in agricultural crops (Ramirez-Romero et al., 2008). Ramirez- Romero et al. (2008) studied the effect of the Cry1Ab protein on the honey bee, Apis mellifera (Hymenoptera: Apidae) at lethal and sublethal levels. They observed that proboscis extension reflex procedure was a reliable method to quantify effects of chemical pesticides on the learning performance of honey bees (Abramson et al., 1999; Desneux et al., 2007). The experiments evaluated effects on three life traits of young honey bee adults: survival of workers bees during sub-chronic exposure to Cry1Ab, feeding behaviour and learning performances. In the above mentioned studies Cry1Ab protein did not induce lethal effects on honey bees meaning no drastic impact at colony scale. However, results indicated the likelihood of sublethal effects when honey bees are exposed to food containing Cry1Ab protein. Specifically, the Cry1Ab may have an antifeedant effect when present at high concentrations (e.g. 5000 ppb) and could therefore affect learning. In terms of foraging optimality, the extension process is crucial for exploitation of food resources because it enables foraging honey bees to leave depleted food sources (Herrera, 1990). As Cry1Ab may impair behavioural plasticity of honey bees, their foraging may not be optimal. As a result, foragers could spend more time foraging in sub-optimal or depleted food sources instead of exploring new ones (Ramirez-Romero et al., 2008) to the detriment of the hive.

However, the need for additional studies in the field may be warranted if stressors such as heat, pesticides and pathogens are suspected to alter the susceptibility of honey bees to Cry protein toxins (Duan et al., 2008).

1.6.3.3 Natural enemies of pests Predators and parasitoids are significant regulators of insect herbivore numbers, both in integrated pest management systems and in nature itself. It is therefore important that natural enemies should not be adversely affected by GM plants. Natural enemy survival depends upon availability of host insects, therefore reduction in host numbers feeding on GM plants will indirectly affect population densities of natural enemies. GM plants could have a

13 direct effect on individual natural enemies via ingestion of GM plant pollen, other plant tissue or active protein in the bodies of their prey (O’Callaghan et al., 2005).

Lacewings are important predators in most cropping systems. Studies showed that larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae), that feed on prey that had been fed Bt maize had an increased mortality rate and slightly increased developmental times compared to those that fed on non-Bt maize (Hilbeck et al., 1998a). Further studies in which C. carnea were exposed to multiple concentrations of Bt in the prey’s food showed that C. carnea that fed on Cry1Ab (100 mg/g of diet) had a mortality rate of 78% compared to the control mortality rate of 26% (Hilbeck et al., 1999).

Generalist predators and especially spiders belong to the most abundant invertebrate predators in agro-ecosystems and play an important role in biological pest control in many crops (Lang, 2003). This suppressive effect is not only through direct killing of prey, but also through indirect effects such as triggering cessation of feeding on the host plant and superfluous killing (prey dropping from the host plant to escape spiders and as a consequence die of starvation or are captured by other soil-dwelling predators) (Riechert, 1999). Thus, any negative effect on spider populations has potential consequences for biological control. Spiders are likely to be exposed to Bt over the whole season as they are known to prey on herbivores which are likely to ingest and pass the toxin on to predators (Dutton et al., 2002).

In Bavaria, South Germany a study was done on spiders in Bt maize and non-Bt maize fields, with or without pyrethroid insecticide application. The results showed that there was no difference in spider abundance in Bt and non-Bt maize fields and that Bt maize had a reduced effect on spider abundance compared to pyrethroid insecticides (Meissle & Lang, 2005). A similar study done on Bt cotton in Marble Hall, South Africa indicated that the Bt crop often had a marked or persistent negative impact on ground- or plant-dwelling spiders in the field (Mellet et al., 2006).

1.6.3.4 Lepidoptera Bt 176 maize expressing Cry 1Ab toxin was shown to have a negative effect on several Lepidoptera species. Larvae of the butterfly species Danaus plexippus (Lepidoptera: Danaidae), Papilio polyxenes (Lepidoptera: Papilionidae) and Pseudozizeeria maha (Lepidoptera: Lycaenidae) were affected negatively when feeding on pollen of Bt 176 maize which may be deposited on their host plants growing in close association with maize plants

(Losey et al., 1999; Wraight et al., 2000). This pollen affects the survival of larvae, their food consumption rate, body weight and development time (Losey et al., 1999).

Studies on D. plexippus initiated large scale research into non-target effects of GM crops. It was shown that monarch butterfly larvae that were fed milkweed leaves coated with high levels of pollen from Bt maize ate less, grew slower and had a higher mortality rate than larvae that consumed milkweed leaves with pollen from non-Bt maize (Losey et al., 1999). Follow-up research concluded that the majority of pollen moves only a short distance away from maize fields, that maize fields contained a low concentration of host weeds (including milkweeds) and that the exposure of monarchs would be limited only to larvae developing on milkweed plants within or near to maize fields during pollen shed and that exposure was minimal (OECD, 2007).

1.6.3.5 Homoptera Aphids are one of the most common phytophagous insects on maize crops (Dicke & Guthrie, 1988). Rhopalosiphum padi (Homoptera: Aphididae) is an important prey for many predators living in maize which can be indirectly affected by feeding on contaminated prey. Aphids are therefore ideal as a tool for studying the effects of Bt maize on non-target insects (Lumbierres et al., 2004).

The population abundance and age structure of R. padi on Bt and non-Bt maize were studied by Lumbierres et al. (2004). Higher density of aphids, particularly alates and young nymphs, was observed in Bt maize plots at very young development stages. After this period, there were no differences in aphid numbers between Bt and non-Bt maize (Lumbierres et al., 2004). The developmental and pre-reproductive times of the offspring of the first generation of alatae were shorter and the intrinsic rate of natural increase higher, when aphids fed on Bt maize. The opposite occured with the offspring of the first generation of apterous mothers, which had lower nymphal and adult mortality, shorter developmental and pre-reproductive times, a higher effective fecundity rate, and greater intrinsic rate of natural increase, when fed on non-Bt maize. The differences in aphid development observed on the two cultivars may be linked to changes in host-plant quality due to pleiotropic effects of the genetic modification. No differences on aphid mortality, developmental and pre-reproductive times, fecundity, and intrinsic rate of natural increase were observed between the offspring of apterous aphids maintained on Bt or non-Bt maize for several generations (Lumbierres et al., 2004).

Bt toxin produced by GM plants is not transported in the phloem sap on which aphids feed, which may explain why no differences between aphid densities on Bt and non-Bt maize is observed in Bt plant / aphid studies (Head et al., 2001). Factors affecting the process of aphid settlement or retention on plants, such as host attraction or plant structure should be considered (Lumbierres et al., 2004). It is known that different colour wave lengths (Cartier & Auclair, 1964; Kring, 1969) and saturations (Moericke, 1969) and plant odours (Bernasconi et al., 1998) influence aphid plant selection of host plants. A study by Faria et al. (2007) reported that aphids actually performed better on Bt maize lines than on near isogenic counterparts, but the generality and cause of the differences remain, as yet, unknown. The aphids feed from the phloem sieve element content and analyses of this sap revealed marginally, but significantly higher amino acid levels in Bt maize, which might partially explain the observed increased aphid performance (Faria et al., 2007). Large colony densities of Rhopalosiphum maidis (Homoptera: Aphididae) on Bt plants resulted in an increased production of honeydew that can be used as food by beneficial insects. Cotesia marginiventris (Hymenoptera: Braconidae), a parasitoid of lepidopteran pests was observed to live longer and parasitized more larvae in the presence of aphid-infested Bt maize than in the presence of aphid-infested isogenic maize lines. Results from the latter feeding experiment imply that this benefit was merely due to the increased honeydew quantity and not to a higher nutritional quality (Faria et al., 2007).

1.6.3.6 Hymenoptera parasitoids In reference to the above, Lövei and Arpaia (2005), observed that hymenopteran parasitoids show adverse effects when parasitizing hosts reared on Bt plants or diets. This was however attributed to a reduced quality of the host. For example, the parasitoid, Cotesia flavipes (Hymenoptera: Braconidae) suffered mortalities on the host C. partellus reared on Bt maize leaf material in laboratory bio-assays and parasitoid weight was reduced and had a lower probability to complete their development (Prütz & Dettner, 2004).

1.7 Arthropod diversity on crops

Several studies related to the impact of Bt crops on non-target organisms have examined the interaction of one or a few species in the laboratory (Sims, 1995; Hilbeck et al., 1998a, 1998b; Dutton et al., 2002). Some results indicated no significant effects on the non-target organisms, whereas some recorded negative effects on the natural enemies. However, translating laboratory results to the field may be problematic because the concentration of toxin doses used in the laboratory may be higher than the doses that arthropods encounter in the field and highly mobile species may spend only a fraction of their life inside GM crop

16 fields. Despite these limitations, laboratory studies can provide valuable insights into the potential effects and the causal mechanisms of patterns in field data (Li et al., 2007). While most field studies assessing potential impacts of Bt crops on biodiversity have focused on limited numbers of species (Wilson et al., 1992; Hardee & Bryan, 1997; Wold et al., 2001; Liu et al., 2003; Schoenly et al., 2003) it is important to study effects on arthropod communities as well.

Arthropod diversity have been studied in China on Bt rice (Li et al., 2007), in the USA on Bt cotton (Torres & Ruberson, 2007) and in Spain on Bt maize (De la Poza et al., 2005). In China 17 706 arthropod individuals from 48 families were recorded over a three year period. These results showed that Bt and non-Bt rice did not differ significantly in terms of diversity or dominance distribution of arthropods (Li et al., 2007). In the cotton survey in the USA a total of 38 980 ground-dwelling arthropod individuals comprising of 65 taxa was recorded over a three-year period. The study concluded that abundance and diversity of ground- dwelling arthropods were not affected by the growing of transgenic Bt cotton (Torres & Ruberson, 2007). No detrimental effect of Bt maize was observed on any predator taxon or on the whole functional group of predators in a farm-scale study in Spain. Abundance and activity of predators varied between years, but no clear tendencies related to Bt maize were found, suggesting that Bt maize could be compatible with the natural enemies that are common in maize fields in Spain and which contribute to reduce insect pest populations (De la Poza et al., 2005).

Species assemblages in agro-ecosystems fulfil a variety of ecosystem functions that may be harmed if changes occur in these assemblages (Dutton et al., 2003). Guild rearrangement due to the elimination of a target pest and subsequent changes in guild structure can lead to development of secondary pests. For this reason it is essential to assess the potential environmental risk which the release of GM plants may hold and to study its effect on species assemblages and ecosystem functions within that ecosystem (Van Wyk et al., 2007a). Predators, omnivores, and parasitoids consume insect pests and weed seeds (Hagler & Naranjo, 2005; Naranjo, 2005), detritivores aid in degrading crop residues and improve soil health (Swift et al., 1979; Bitzer et al., 2005) while herbivores can reduce competition by non-crop plants and serve important roles as prey and hosts for natural enemies (Norris & Kogan, 2005). Because these functional guilds interact differently with crop plants and environments, they are likely to be affected by pest management practices (Wolfenbarger et al., 2008). In order to identify possible secondary pests and non-target effects, knowledge is needed on arthropod species that occur in maize fields. This

17 information will be useful in the evaluation of the possible impact of Bt maize on non-target organisms at different trophic levels (Figure 1.3).

Figure 1.3. Simplified example of different trophic levels and feeding-guilds indicating possible exposure pathways to Bt-proteins in a maize ecosystem (Pictures with green borders indicate non-target organisms. The red border picture indicates a target organism).

1.7.1 Secondary pests

While Bt technology is efficient at reducing infestation levels of target pests, it is not designed to combat other pests that have historically posed less of a threat. The emergence of a secondary pest could prove to be a major problem in countries where GM crops have been widely adopted, particularly in countries where farmers may be poorly informed and/or have unrealistic perceptions of the benefits and performance of these crops. Such may be the case in developing nations, exactly those nations where GM crops have been promoted to help solve poverty and undernourishment (Wang & Just, 2006). Secondary pests can be defined as pest species that are usually present at low levels and whose population numbers are controlled by the actions of natural enemies. Such species can assume full pest status when natural enemies are adversely affected by other pest management tactics (Gordh & Headrick, 2002) such as insecticides or GM crops. Ignoring secondary pests can lead to devastating crop damage that may continue over a considerable period of time. Induced secondary pest infestations, once they arise, may prove difficult to control by chemical means (Harper & Zilberman, 1989).

The widespread adoption of Bt cotton has reduced the need for insecticides for many key pest lepidopterans. However, these insecticides previously afforded excellent control of phytophagous plant bugs and this reduction in insecticide use has resulted in increased population densities of plant bugs (Greene et al., 1999). In a study conducted in northern China to investigate population dynamics of mirids on transgenic cotton, 18 species of mirids were recorded among which Lygus fasciaticollis, Adelphocoris fasciaticollis and Adelphocoris lineolatus (Hemiptera: Miridae) were considered the most important pests (Chu & Meng, 1958; Ting, 1963; Li et al., 1994). There was no significant difference in the density of populations of these mirids between unsprayed non-Bt cotton and unsprayed Bt cotton. However, due to the decrease in insecticidal applications against cotton bollworm on Bt cotton, the damage by the mirids on Bt cotton was more serious than that in sprayed non-Bt cotton fields, suggesting that the mirids have become key insect pests in Bt cotton fields. Therefore their damage to cotton would further increase with the increase of Bt cotton area, if no additional control measures were adopted (Wu et al., 2002).

A survey done in 2004, seven years after the initial commercialization of Bt cotton in China, revealed that through the use of Bt cotton farmers saved 46% on pesticide use for control of Helicoverpa armigera (Lepidoptera: Noctuidae) but that they spend 40% more on pesticides designed to kill emerging secondary pests (Wang & Just, 2006). These secondary pests (one example is Miridae) were rarely found prior to the adoption of Bt cotton, and were

19 presumably kept in check by regular pesticide spraying. The additional expenditure needed to control secondary pests nearly offsets the savings on primary pesticide applications. Farmers clearly understand the benefit of using Bt crops to reduce the use of chemical pesticides to control bollworm, however, farmers may not realize that a secondary pest exists until it has grown to become a significant economic burden. The impact of Bt technology on secondary pests have been ignored in many previous studies of the economic benefits of Bt technology (Wang & Just, 2006).

Another study in China evaluated the effects of pesticide applications based according to an integrated pest management strategy for cotton bollworm and two sap-feeding pests (mirids and leafhoppers) on Bt and non-transgenic cotton. The most important data gained through this experiment was that the leafhopper population was larger on Bt cotton throughout the 3- years study. This could indicate that the cotton variety was more suitable for development of leafhoppers than non-transgenic cotton and that here might be an increased need for insecticide applications on GM varieties (Men et al., 2005). It has also been reported that populations of the leafhopper, Spanagonicus albofasciatus (Hemiptera: Ciccadellidae) were larger on Bt-cotton than those on non-Bt cotton (Wilson et al., 1992). In the latter experiment leafhopper numbers on non-Bt cotton exceeded the pesticide application action threshold (200 individuals/100 plants) only during one year (Men et al., 2005).

The importance of considering non-target organisms that could become secondary pests needs to be considered in risk assessments before release of GM crops. The development of western bean cutworm, Striacosta albicosta (Lepidoptera: Noctuidae) as a secondary pest of transgenic Bt maize in the USA is a good example of secondary pest development after control of the primary pest (Catangui & Berg, 2006). Striacosta albicosta was not considered economically important before release of Bt maize and larvae were apparently not susceptible to the Cry1Ab and Cry9C insecticidal proteins. Thus, although Cry1Ab and Cry9C Bt maize hybrids were almost completely free of the target pest, European corn borer (D. saccaralis), significant damage was caused to Bt maize by S. albicosta larvae. In 2003, Bt maize hybrids were in fact more infested with S. albicosta larvae than conventional maize (Catangui & Berg, 2006). Transgenic crops may be viewed as an introduced or artificial disturbance of the ecosystem that may lead to a rearrangement of niches occupied by maize-associated arthropods. Continuous planting of Cry1Ab Bt maize hybrids over large areas, for example, may favour western bean cutworm and other species by effectively eliminating competition from D. saccaralis (Catangui & Berg, 2006).

1.8 Resistance development and the high-dose/refuge strategy

Insect tolerance to a given insecticide may be expected to develop rapidly when an insect population is characterised by a high rate of development of the immature stages and a quick succession of generations, while being exposed to sub-lethal levels of the toxin (Van Rensburg, 2007). Resistance development can impact directly on biodiversity, ecosystem functions and natural enemies, so it opens up a new exposure pathway towards high trophic levels that are associated with the resistant pest.

Microbial preparations of the entomopathogenic bacterium Bacillus thuringiensis, applied as spray formulations, have been in use without substantial resistance development in field populations (Tabashnik, 1994). The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) was the only insect to eventually develop resistance to Bt applied as a biopesticide (Ferré & Van Rie, 2002). However, five of 10 species of moths representing the families Noctuidae, Plutellidae and Pyralidae, selected with Bt under laboratory conditions, developed more than a 10-fold resistance, suggesting that the ability to evolve resistance to Bt is a common phenomenon among the Lepidoptera (Tabashnik, 1994). Recent laboratory studies have shown that this ability to develop resistance to Bt applies to some major agricultural pest species, including the European corn borer (D. saccaralis) (Chaufaux et al., 2001; Sigueira et al., 2004), the pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) (Tabashnik et al., 2002), the bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) (Tabashnik et al., 2003) and the fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) (Tabashnik et al., 2009).

Busseola fusca and C. partellus are abundant in maize ecosystems and are therefore most likely to develop resistance to Bt maize in South-Africa. The risk of resistance development differs between species based on geographical area and cultivation practices (Van Wyk et al., 2007a). The most likely species to develop resistance in the Highveld region was predicted to be B. fusca, while C. partellus is the species most likely to develop resistance in low-altitude subtropical and arid areas (Van Wyk et al., 2007a). Busseola fusca is the dominant and often the only stem borer species in the South African Highveld region where Bt maize was released in 1998, while C. partellus is the dominant species in low-altitude region where B. fusca does not occur (Bate et al., 1991). The first report of resistance of B. fusca to Cry1Ab maize was made in Christiana, South Africa in 2006 (Van Rensburg, 2007).

The high-dose/refuge strategy which is employed to delay resistance evolution comprises a combination of Bt maize plants producing high dose of toxin and non-Bt plants in close

21 proximity to one another. The purpose of the high dose of toxin is to kill as many individuals of the target pest as possible, whereas the purpose of the refuge is to sustain susceptible pest individuals that survive on that particular crop (Gould, 2000). The goal is to ensure that rare, Bt-resistant individuals that survive on the Bt crop do not produce completely resistant offspring by mating with other toxin-resistant individuals. Instead, susceptible individuals from the refuge can mate with toxin-resistant individuals that survived on the Bt plants. Offspring from the combination of susceptible and resistant individuals are expected to have only a low to moderate level of toxin resistance. These individuals should not be able to survive on plants with high Bt toxin levels (Gould, 2000), lowering the chance of resistant populations developing in a given area (Kruger et al., 2009).

Although the planting of refugia is compulsory to delay resistance development (Monsanto, 2007), the level of compliance in South Africa is low (Kruger et al., 2009). The current refuge requirements are either a 20% refuge planted to conventional maize, which may be sprayed with insecticides, or a 5% refuge area that may not be sprayed (Kruger et al., 2009). The genetically modified organism (GMO) act (Act 15 of 1997) guides the use of Bt maize in South Africa and obligates seed companies to sign “grower agreements” (contracts) with farmers that purchase seed of GM crops. These contracts prescribe the use of the product user guide (Monsanto, 2007) and specify aspects such as the implementation of an insect resistance management program (Kruger et al., 2009).

1.9 Environmental risk assessment and management

A risk assessment process is used to provide information for use in the decision-making process, while risk management is used to provide decision options for consideration in cases where environmental harm could occur (Birch et al., 2004). Risk assessment is the process by which risk is measured. This measurement can be quantitative or qualitative, problematic or deterministic. During the risk management process society determines how to address the risk. This is done either by direct involvement of society or via social representatives or delegated authorities. The main risk management decisions to be made are: whether to tolerate, mitigate or avoid the identified risk (Birch et al., 2004).

The term environmental risk assessment (ERA) can be defined as a systematic process of developing a scientific basis for regulatory decision making (Barnthouse et al., 1992). Risk management seeks measures to limit the risks of GM plants in the environment including, not only ecological, but also socio-economical and political criteria (cost/benefit) (Hilbeck et al., 2008).

Ecological risk assessment generally entails the uncertainty of extrapolating from small, laboratory-based studies to more variable and open natural systems, the possibility of evolution and environmental change altering the circumstances, intrinsic biological time lags, and a high degree of stochastic inputs (Marvier, 2002).

Before GM plants may be marketed in the European Union, an ERA has to be conducted according to EU-Directive 2001/18/EC or the Regulation (EC) No 1829/2003 of the European Parliament and of the Council on genetically modified food and feed. Important elements of the ERA are ecotoxicological tests investigating adverse effects of GM plants on the living environment (Hilbeck et al., 2008). South African regulatory authorities have developed formal ecological risk assessment guidelines, modelled along the lines of the framework used by the Environmental Protection Agency in the USA. This approach includes three formal stages: first the assessment is planned, followed by description and analyses of risk. Lastly this is followed-up with a discussion between risk assessors and risk managers, who, in turn, communicates with interested parties (Claassen et al., 2001a,b). The minimum quality for South African ERAs for GMOs, as well as guidelines and procedures to this effect, currently remain to be specified. Comprehensive integration between the process of ERA under the GMO Act and the risk assessment component of environmental impact assessments (EIAs) performed under National Environment Management Act (NEMA) is necessary (McGeoch & Rhodes, 2006). As the intention (in part) of both the GMO Amendment Bill and legislation requiring an EIA under NEMA (section 78 of NEMBA and section 4 of the GMO Amendment Bill itself) are to limit the possible harmful effects of GMOs to the environment. The requirements of the standard risk assessment process under the GMO Amendment Bill and that required under NEMA should be compatible and compiled concurrently. Furthermore the Department of Agriculture, in fulfilling its duties under the GMO Act, requires information similar to that required should an EIA be requested (McGeoch & Rhodes, 2006).

Current ERA of GM crop plants relies on the ecotoxicological model in which acute toxicity of specified compounds is tested against a set of indicator species. This model is based on data obtained through ecotoxicological testing of environmental chemicals such as pesticides. The reliance of ERA of GM crop plants on the ecotoxicology model has been repeatedly criticized and its shortcomings have been described (Wolfenbarger & Phifer, 2000; Marvier, 2002; Hilbeck & Andow, 2004). One of the main criticisms is that the pre- determined set of test organisms may not occur on the receiving environment where the GM crop will be planted.

Any given cropping system will typically contain between about a 1000 to several thousands of species (Birch et al., 2004). Although it is possible to assess impacts on this biodiversity in its entirety, the pre-release risk assessment of transgenic crops will be in controlled environments such as laboratories, greenhouses or small-scale field evaluations that require selection of a relatively small number of species or species groups for monitoring. Therefore, an important component of a case-specific risk assessment is that the most relevant species are selected for pre-release testing in a scientifically defensible and transparent way (Birch et al., 2004).

As alternative to the ecotoxicological model for risk assessment, an ecological model was developed and suggested for Bt maize in Kenya (Birch et al., 2004) and Bt cotton in Brazil (Hilbeck et al., 2006). This non-target ERA model involves five steps which is graphically presented in Figure 1.4 (Birch et al., 2004).

Figure 1.4. Species and methods selection procedure for ecotoxicity testing of genetically modified plants (Adapted from Hilbeck et al., 2008).

1.9.1 The non-target environmental risk assessment model

The environmental risk assessment (ERA) model makes use of different steps (Figure 1.4) in order to reduce large numbers of species to fewer priority species for use in further testing of possible effects of GM crops. Since the ERA model will be used in this study, it is briefly discussed below.

Step 1: Categorizing and listing potential non-target species and ecological functions and identifying important interactions

A combination of qualitative field expertise and data from biodiversity assessments is crucial for determining the list of possible non-target species, their trophic relationships and relevant functions. The list will be specific to the crop and it’s cropping context and agro-ecosystem. Using ecological function allows one to focus on ecological processes and limits the number of species and functions to be tested. Two types of functional criteria can be used, anthropocentric functions and ecological functions (Birch et al., 2004). Anthropocentric functions are related directly to human goals and include secondary pest species, natural enemies, rare or endangered species, species used to generate income and species of social or cultural value. Ecological functions relate to ecosystem processes independent of human goals and include primary consumption, secondary consumption, pollination, decomposition, nutrient recycling and seed dispersal. These functional groups are not mutually exclusive. A vast number of species found in agricultural fields probably cannot be classified into any one of these functional groups. Hence, it is critical to consider a category of species with unknown function, so that these species are not inadvertently overlooked because of lack of knowledge (Birch et al., 2004).

Step 2: Prioritizing species or functions for pre-release testing according to maximum potential adverse effect

This part of the process involves ranking the non-target species or functions according to ecological principles and prioritizing a number of these for possible assessment, with particular emphasis on those that might be adversely affected by the transgenic crop and are for ecological or anthropogenic reasons (Birch et al., 2004). Criteria for prioritizing non-target species in each functional group are consistent with Annex 111 of the Cartagena protocol. The focus of the Cartagena Protocol on Biosafety is to ensure the safe transboundary movement of living modified organisms including genetically modified crops (CBD, 2000) and provides high-level guidance on risk assessment (Tencalla et al., 2009).

Step 3: Conducting exposure pathway analyses

This step analyses possible causal pathways of exposure to the GM plant, toxin and potential impacts of GM plants for each species or function identified as highest priority in the previous step. The purpose of this evaluation is to differentiate candidate test species likely and unlikely to be exposed to the Bt toxin and for the former to guide the design of the exposure system in the test protocols. Potential likely exposure can occur through many pathways (Birch et al., 2004). Transgenic plant material, transgene products and metabolites may effect non-target species and ecosystem functional dynamics directly via plant residues (Zwahlen et al., 2003a), senescent leaves, sloughed roots and root exudates (Saxena et al., 1999; Saxena & Stotzky, 2000), pollen (Losey et al., 1999) and other plant parts such as seeds, floral and extra-floral nectarines, guttation fluids and phloem sap that express the transgene (Hilbeck, 2002). Any non-target organisms feeding on the transgenic plant or parts of the plant may come in contact with the transgene and its product. In addition, the transgene might interact with existing plant compounds to affect non-target organisms (Birch et al., 2002). Transgenic plant material and transgene products can affect non-target species indirectly through another organism, such as a herbivore (Birch et al., 1999; Hilbeck et al., 1999) or honeydew from Homoptera species such as aphids, scale or whiteflies (Raps et al., 2001; Bernal et al., 2002).

Step 4: Describing hazard scenarios and formulating testing hypotheses

The information from the previous steps guides hazard identification and the development of testable assessment hypotheses. This step in the risk assessment is particularly important when exposure is unlikely, yet indirect effects are still possible. Such indirect effects can arise through changes in the population dynamics of a non-target organism and/or through a shift in the presence or composition of a particular community assemblage (Birch et al., 2004).

Step 5: Developing ecologically meaningful testing methods and protocols

The last step in the ERA is to develop appropriate methodologies and protocols to assess risk. Such methods can be designed once it is evident which life stage of a non-target organism is likely to be exposed and through what route it is likely to receive the genetically modified product, directly and/or mediated indirectly through other non-target prey. Similarly, when and where soil-ecosystem functions may be vulnerable the ecological model can be used to design better assessment methods. Any ecologically meaningful experimental design

26 will mimic identified exposure routes, test identified hazard scenarios and introduce ecological realism as far as possible (Birch et al., 2004).

1.10 Objectives

The use of GM crops, especially Bt maize could directly or indirectly affect non-target organisms. Assessment of the impact of Bt maize crops is hampered by the lack of knowledge on species richness and checklists of species present in most eco-systems (Van Wyk et al., 2007b). This study will provide base-line data on arthropod biodiversity, as well as which arthropods are directly and indirectly exposed to Bt toxin in maize fields. It will also contribute towards future risk assessments for Bt maize in South Africa. The aims of this study were:

• to compile a checklist of arthropods that occurs on maize plants

• to determine and compare the diversity and abundance of arthropods on Bt and non- Bt maize

• to determine possible exposure pathways of arthropods to Cry 1Ab protein expressed in maize

• to use an ecological model to identify priority species for ecological risk assessment

This study will serve as a basis from which to provide early-warning of possible future changes in the abundance and diversity of target and non-target arthropods that may shift over time, resulting in changes in pest status and guild rearrangement of target and non- target arthropods. The study will also provide a list of priority non-target species for future evaluations of the possible effect of Bt-protein on these organisms.

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CHAPTER 2

COMPARATIVE DIVERSITY OF ARTHROPODS ON BT AND NON-BT MAIZE AT THE VAALHARTS AND TSHIOMBO IRRIGATION SCHEMES IN SOUTH AFRICA

2.1 Abstract

The biodiversity of an agro-ecosystem is not only important for its intrinsic value but also because it influences ecological functions that are vital for crop production in sustainable agricultural systems and the surrounding environment. A growing concern about GM crops is the potential negative impact that it could have on diversity and abundance of non-target organisms and subsequently on ecological functions. For this reason it is essential to assess the potential environmental risk the release of a GM crop may hold and to study its effect on species assemblages within that ecosystem. Assessment of the impact of GM Bt maize on the environment is hampered by the lack of even the most basic checklist of species present in maize agro-ecosystems in South Africa. The aims of the study were to compile a checklist of arthropods that occur on maize in South Africa and to compare the diversity and abundance of arthropods and functional groups on Bt and non-Bt maize. Collections of arthropods were done during the 2007/2008 and 2008/2009 growing seasons on Bt and non- Bt maize plants at two localities, i.e. Vaalharts in the Northern-Cape - and Tshiombo in the Limpopo province. The focus was on collecting arthropods that occur on plants during the reproductive stage of plant growth only and this study therefore only takes into account above-ground on-plant diversity. Three maize fields were sampled per locality during each season. Twenty plants, each of Bt and non-Bt maize, were randomly selected from the fields at each site. The arthropods collected during this study were classified to morpho-species level. Morpho-species were grouped into functional groups: detritivores, herbivores, predators and parasitoids. The herbivores and predators were further divided on basis of feeding strategy into sucking herbivores/predators (piercing-sucking mouthparts) and chewing herbivores/predators (chewing mouthparts). A total of 8771 arthropod individuals, comprising 288 morpho-species consisting of 20 orders were collected. Results form this short-term study indicated that abundance and diversity of arthropods in maize, as well as on the different functional guilds, were not significantly affected by Bt maize, either in terms of diversity or abundance.

2.2 Introduction

Agriculture is an important environmental quality driver (Hails, 2002), and its effect is not likely to diminish in the future (Tilman et al., 2001). It has been realised that life on earth depends on the proper functioning of several large-scale ecological processes, many of which provide humanity with irreplaceable benefits, termed ecosystem services (Daily et al., 1996). Loss of biodiversity threatens these benefits, but exactly what type and amount of biodiversity is necessary for unimpaired, sustained ecological functioning and productivity is unknown (Loreau et al., 2002). Considering these future scenarios and uncertainties, agricultural innovations are increasingly scrutinised for their potential environmental impacts (Hails, 2002). This also holds for the growing of genetically modified (GM) crop plants, a fast- spreading agricultural innovation (Shelton et al., 2002; Lövei & Arpaia, 2005).

One concern about growing GM crops is the potential negative impact that it could have on diversity and abundance of non-target organisms (Eckert et al., 2006). Several studies related to the potential impact of Bt crops on non-target organisms have examined the interaction of one or a few species under laboratory conditions (Sims, 1995; Hilbeck et al., 1998a, 1998b; Dutton et al., 2002). Some results indicated no significant effects on the non- target organisms, whereas others reported negative effects. However, extrapolating from laboratory results to the field may be problematic because the concentration of toxin doses used in the laboratory may be higher than the doses that the arthropods encounter in the field and highly mobile species may spend only a fraction of their life inside GM crop fields. Despite these limitations, laboratory studies can provide valuable insights into the potential effects and causal mechanisms that are present under field conditions (Li et al., 2007). While most field studies assessing impacts of Bt crops have focused on limited numbers of species (Wilson et al., 1992; Hardee & Bryan, 1997; Wold et al., 2001; Liu et al., 2003; Schoenly et al., 2003) it is important to also study effects on arthropod communities. The only studies previously conducted on arthropod community diversity were in GM Bt rice in China (Li et al., 2007), in the USA on Bt cotton (Torres & Ruberson, 2007) and in Spain on Bt maize (De la Poza et al., 2005). These three studies concluded that Bt crops did not have adverse effects on arthropod diversity compared to non-Bt crops.

One of the reasons for maintaining and/or encouraging natural biodiversity is that it performs a variety of ecological services (Altieri, 1991). In agricultural systems, biodiversity performs ecosystem services beyond production of food, fibre, fuel and generation income. Examples include recycling of nutrients, control of the local microclimate, regulation of local hydrological

45 processes, regulation of the abundance of undesirable organisms and detoxification of noxious chemicals (Altieri & Nicholls, 1999).

The biodiversity of an agro-ecosystem is not only important for its intrinsic value, but also because it influences ecological functions that are vital for sustainable crop production as well as the surrounding environment (Hilbeck et al., 2006). Species assemblages in agro- ecosystems fulfil a variety of ecosystem functions that may be harmed if changes occur in these assemblages (Dutton et al., 2003). For example, guild rearrangement due to the elimination of a target pest and subsequent changes in guild structure can lead to development of secondary pests. For this reason it is essential to assess the potential environmental risk that the release of a genetically modified organism may hold and to study its effect on species assemblages within that ecosystem (Van Wyk et al., 2007). Predators, omnivores and parasitoids consume insect pests and weed seeds (Hagler & Naranjo, 2005; Naranjo, 2005), detritivores aid in break-down of crop residues and improve soil health (Swift et al., 1979; Bitzer et al., 2005) while herbivores can reduce competition by non-crop plants (weeds) and serve important roles as prey and hosts for natural enemies (Norris & Kogan, 2005). Because these functional guilds interact differently with crop plants and environments, they are likely to be affected by pest management practices (Wolfenbarger et al., 2008). In order to identify possible secondary pests and non-target effects of GM crops with insecticidal properties, knowledge is needed on which arthropod species occur in maize- ecosystems. This information will be useful in evaluation of the possible impact of Bt maize on non-target organisms at different trophic levels. Assessment of the impact of GM Bt maize on the environment is hampered by the lack of even the most basic checklist of species present in maize ecosystems. There is also a need to identify indicator organisms and develop simple methods that combine suitability for ecological risk assessment under field conditions and cost efficiency of assessments (Eckert et al., 2006).

Experimental evidence suggests that biodiversity can be exploited for improved pest management (Altieri & Letourneau, 1984; Andow, 1991). Several studies have shown that it is possible to stabilize insect communities in agro-ecosystems by designing and constructing vegetational architectures that support populations of natural enemies or that have direct deterrent effects on pest herbivores (Perrin, 1980; Risch et al., 1983).

The aims of this study were to compile a checklist of arthropods that occur on maize and to determine and compare the diversity and abundance of arthropods and functional groups on Bt and non-Bt maize.

2.3 Material and Methods

Collections of arthropods were done during the 2008/2009 and 2009/2010 growing seasons in Bt and non-Bt maize fields at two localities, i.e. Vaalharts in the Northern Cape- and Tshiombo in the Limpopo province of South Africa. Sampling was done during April and November at Vaalharts and Tshiombo respectfully.

2.3.1 Study areas

2.3.1.1 Tshiombo This area is characterized by 500-800 mm rainfall per annum with mean minimum and maximum temperatures between 8-10ºC and 32-34ºC respectively. This area is mountainous and altitudes where crop production is practiced range between 500-700 m above sea level. The survey was conducted in two villages at the Tshiombo irrigation scheme (22°48’05’’S, 30°27’07’’E) situated in the Tshiombo valley, on the Mutale River in the Limpopo province. The total area at the irrigation scheme is about 1200 hectares and consists of 930 plots, each approximately 1.2 hectares in size. The single most important crop, in terms of area planted, is maize, the staple food, which accounts for 40-50% of total cultivated area (Lahiff, 1997). Other crops commonly planted in rotation systems with maize are groundnut, brassicas and sweet potato.

2.3.1.2 Vaalharts The Vaalharts irrigation scheme is situated in the semi-arid Northern Cape province of South Africa, between 1050 and 1150 m above sea level (S24˚48’ 693, E27˚38’ 330). This irrigation scheme comprises an area approximately 80 km from north to south and 30 km from east to west. In total there is 30 000 ha of crop production under irrigation (Bornman, 1988; De Jager, 1994) all divided into 25 ha plots (Fourie, 2006). Maize has only been recognised as one of the main crops in this area since 1998, with an annual average of 9000 ha being grown under flood- or centre-pivot irrigation (Kruger et al., 2009).

2.3.2 Sampling of arthropods

Arthropods were collected from Bt maize plants in commercial maize fields at the Vaalharts irrigation scheme and from small fields of Bt maize planted with seed provided to resource- poor farmers in the Tshiombo area of the Limpopo province. The Bt maize sampled during this study were all from the event MON810 expressing Cry1Ab protein for control of the stem borers, Busseola fusca (Lepidoptera: Noctuidae) and Chilo partellus (Lepidoptera:

Crambidae). Pest pressure (target and non-target) is usually very high in the Vaalharts irrigation scheme and provided a unique opportunity for this study. In the Tshiombo area, resource-poor farmers had not been introduced to GM crops prior to this study.

Three maize fields were sampled for each of the two localities, during each season (12 fields in total). Twenty plants, each of Bt and non-Bt maize (from the refuge area of the field), were randomly selected from the fields at each site (480 plants in total). Each plant was bagged (Figure 2.1 A) and all arthropods removed later and placed in 70 % ethanol in 40 ml bottles (Figure 2.1 B). All arthropods were collected and kept such that abundance and diversity could be calculated on a per plant basis.

Arthropods were classified to morpho-species level and into functional groups to provide information on the potential exposure of species to Bt toxin inside GM maize fields. Where possible, morpho-species were further identified to family and species level.

2.3.3 Data analysis

Data were analysed separately for each site and season to compare diversity and abundance between Bt and non-Bt maize. The Shannon diversity index (H1) describes diversity (species richness and evenness), whereas the Margalef richness index (d) describes species richness. The Shannon diversity and Margalef richness indices were calculated using Primer 5 (Version 5.2.9) (Clarke & Gorley, 2001). Rank abundance graphs were also compiled to compare Bt and non-Bt species richness and evenness at each site. Statistical analysis was done with Statisica software (Version 10). Data was not normally distributed and the non-parametric Mann-Whitney-U-test was therefore used. Since the latter test uses a rank of numbers from high to low, a median and not a mean value was calculated to indicate abundance and numbers. Since abundance was generally low, median levels were expressed per 20 plants.

Statistical analyses were done with the total arthropod diversity and for the different functional groups: Detritivores, Sucking herbivores, Chewing herbivores, Sucking predators, Chewing predators and Parasitoids. The Shannon and Margalef indices, the total number of species, as well as the total number of individuals were compared between localities, between seasons and maize variety (Bt vs. non-Bt).

Figure 2.1 A. Maize plants were individually covered with bags.

Figure 2.1 B. Arthropods were sorted to order level and then classified to morpho-species level. Each morpho-species was placed in a separate plastic bottle containing 70% ethanol.

2.4 Results

A total of 8771 arthropod individuals, comprising 288 morpho-species, was collected during this study. In Vaalharts a total of 4154 arthropod individuals (2566 and 1588 in 2008 and 2009 respectively), consisting of 169 morpho-species was collected. In Tshiombo a total of 4617 arthropod individuals (2216 and 2401 in 2008 and 2009 respectively), consisting of 202 morpho-species was collected. The 288 morpho-species consisted of 20 orders. Only 28.8% of these species occurred in both localities.

The Shannon (H1) and Margalef (d) indices, as well as the total number of species and the total number of individuals were compared between the sites, over seasons and maize varieties for the total arthropod diversity and for the different functional guilds. The proportion made up by each functional group was similar for Bt and non-Bt maize with no significant differences between functional groups (Figure 2.2 A & B). More detail regarding each functional group is provided in Appendix A (p. 121).

For the two years, arthropod diversity in Bt and non-Bt maize at the two sites were not significantly different. Results are provided in rank abundance graphs which indicate species richness and evenness. The rank abundance graph for Vaalharts in the first season (Figure 2.3) indicated a high evenness for both Bt and non-Bt maize, with Bt maize having a slightly higher species richness. In season 2 (Figure 2.4) a lower evenness for Bt and non-Bt maize was observed because of the dominant species of Aphididae and Latridiidae that occurred on both Bt and non-Bt maize. Results for the Tshiombo site indicated relatively high species evenness for Bt and non-Bt maize during both seasons (Figures 2.5 & 2.6).

Figure 2.2 A&B. Proportion (%) of total population made up by different functional groups between Bt and non-Bt maize at Vaalharts and Tshiombo between 2008/2009 and 2009/2010 growing seasons.

Species rank Bt Non-Bt

Figure 2.3. Rank abundance graph for arthropod diversity on Bt and non-Bt maize in the Vaalharts area (season 1).

Species rank Bt Non-Bt

Figure 2.4. Rank abundance graph for arthropod diversity on Bt and non-Bt maize in the Vaalharts area (season 2).

Species rank

Bt Non-Bt

Figure 2.5. Rank abundance graph for arthropod diversity on Bt and non-Bt maize in the Tshiombo area (season 1).

Species rank

Bt Non-Bt

Figure 2.6. Rank abundance graph for arthropod diversity on Bt and non-Bt maize in the Tshiombo area (season 2).

2.4.1 Total arthropod diversity

The diversity indices indicated no differences between Bt and non-Bt maize for total arthropod diversity or between the two sites over the two seasons (Table 2.1). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize or between the two sites over the two seasons (Table 2.1).

There was, however, a significant difference for the Shannon index over two seasons at Vaalharts (p=0.03), due to a lower diversity during season 2, but for Tshiombo there was no significant difference (Table 2.2). The low diversity during season 2 can be seen in the rank abundance graph (Figure 2.4).

The Shannon index indicated a significant difference in total arthropod diversity between the two sites (p=0.04) due to a significant difference during the second season (p=0.02) (Table 2.3). This difference is ascribed to lower species richness and evenness during the second season in Vaalharts because of the dominant species of Aphididae and Lathridiidae that occurred on both Bt and non-Bt maize. This was also indicated in the rank abundance graph for season 2 at Vaalharts (Figure 2.4).

Table 2.1. Descriptive statistics and p-value for diversity index values, abundance and number of arthropod species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value non- p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt Bt Shannon index 2.70 2.55 0.44 2.77 2.73 0.66 2.40 1.99 0.38 2.63 3.02 0.66 3.02 2.67 0.38 Margalef index 7.72 8.07 0.93 7.93 7.01 0.66 7.18 6.11 0.66 8.19 8.37 0.66 8.06 9.01 1.00 Number of species / 20 plants 42.50 44.50 0.93 47.00 44.00 0.66 40.00 36.00 0.66 51.00 51.00 1.00 37.00 58.00 1.00 Number of individuals / 20plants 326.00 392.50 0.54 330.00 462.00 1.00 181.00 221.00 0.66 449.00 411.00 0.38 168.00 419.00 0.38

Table 2.2. Descriptive statistics and p-value for diversity index values, abundance and number of arthropod species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2

Shannon index 2.75 2.53 0.40 2.75 2.32 0.03 2.74 2.97 0.47 Margalef index 8.19 7.34 0.44 7.47 6.96 0.30 8.28 8.54 0.94 Number of species / 20 plants 49.00 38.50 0.25 45.50 38.00 0.09 51.00 47.50 0.87 Number of individuals / 20 plants 429.50 209.50 0.06 396.00 209.50 0.09 429.50 275.50 0.30

Table 2.3. Descriptive statistics and p-value for diversity index values, abundance and number of arthropod species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2

Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo Shannon index 2.52 2.89 0.04 2.75 2.74 0.81 2.32 2.97 0.02 Margalef index 7.09 8.28 0.07 7.47 8.28 0.38 6.96 8.54 0.23 Number of species / 20 plants 41.50 51.00 0.25 45.50 51.00 0.69 38.00 47.50 0.52 Number of individuals / 20 plants 326.00 397.00 0.89 396.00 429.50 0.47 209.50 275.50 0.94 55

2.4.2 Detritivores

The rank abundance graph for Vaalharts during both seasons indicated a high evenness for Bt and non-Bt maize, except for non-Bt in the first season (Figure 2.7). The graph for Tshiombo indicated low evenness for Bt and non-Bt maize during both seasons with a much lower species richness during season 2 (Figure 2.8). The low evenness can be ascribed to the dominant species of Oppiidae and Entomobryidae at both localities.

The diversity indices indicated no differences between Bt and non-Bt maize for total detritivore diversity and between the two sites over the two seasons (Table 2.4). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize for detritivore diversity and between the two sites over the two seasons (Table 2.4).

A significant difference was, however, observed between the number of detritivore individuals between the two seasons (p=0.03), as well as in Tshiombo (p=0.02) in comparison to Vaalharts (Table 2.5). The number of detritivore individuals was lower during season 2. There was a significant difference between the Shannon index (0.01) and number of species (0.02) for detritivores between the two seasons at Tshiombo (Table 2.5), with the diversity and abundance being lower in season 2. This was also indicated in the rank abundance graph for Tshiombo over the two seasons (Figure 2.8).

A significant difference in the number of detritivore species was observed between the two sites (p=0.05) (Table 2.6), with the number of species being significantly lower at Tshiombo (Table 2.5). The index values (p=0.01; p=0.01), as well as number of species (p=0.01) and individuals (p=0.05) all indicated highly significant lower detritivore diversity and abundance between Vaalharts and Tshiombo second season, with the diversity and abundance being lower at Tshiombo (Table 2.6). The difference in species richness between the two sites in the second season can be seen in Figures 2.7 and 2.8, with species richness being lower at Tshiombo.

Figure 2.7. Rank abundance graph for detritivore diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.8. Rank abundance graph for detritivore diversity on Bt and non-Bt maize in Tshiombo area over two seasons.

Table 2.4. Descriptive statistics and p-value for diversity index values, abundance and number of detritivore species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 1.58 1.20 0.24 1.66 1.04 0.19 1.66 1.48 0.66 1.77 1.44 0.66 1.00 0.79 1.00 Margalef index 1.85 1.69 0.19 2.25 1.56 0.38 1.85 2.06 0.66 2.42 1.82 0.66 1.59 1.30 0.39 Number of species / 20 plants 6.00 6.00 0.60 9.00 7.00 1.00 6.00 7.00 1.00 8.00 7.00 0.51 3.00 4.00 0.66 Number of individuals / 20 plants 16.50 15.00 0.95 35.00 47.00 1.00 18.00 13.00 0.38 18.00 20.00 0.66 3.00 10.00 0.38

Table 2.5. Descriptive statistics and p-value for diversity index values, abundance and number of detritivore species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2

Shannon index 1.38 1.18 0.34 1.18 1.58 0.38 1.51 0.90 0.01 Margalef index 1.91 1.79 0.64 1.90 2.05 0.58 1.91 1.37 0.17 Number of species / 20 plants 7.00 4.50 0.06 8.00 6.50 0.63 7.00 3.50 0.02 Number of individuals / 20 plants 19.50 11.50 0.03 41.00 16.50 0.26 18.50 6.00 0.02

Table 2.6. Descriptive statistics and p-value for diversity index values, abundance and number of detritivore species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2

Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo

Shannon index 1.49 1.15 0.24 1.18 1.51 0.30 1.58 0.90 0.01 Margalef index 2.05 1.44 0.12 1.90 1.91 1.00 2.05 1.37 0.01 Number of species / 20 plants 7.00 4.00 0.05 8.00 7.00 0.58 6.50 3.50 0.01 Number of individuals / 20 plants 18.50 11.00 0.06 41.00 18.50 0.23 16.50 6.00 0.05 58

2.4.3. Chewing herbivores

The rank abundance graphs for both localities indicated a low evenness for Bt and non-Bt maize during both seasons (Figures 2.9 & 2.10). In Tshiombo a low evenness was observed for non-Bt maize during season 2 (Figure 2.10). The low evenness can be ascribed to the dominant species of Nitidulidae, Lathridiidae and Anthicidae at both localities.

The diversity indices indicated no differences between Bt and non-Bt maize for total chewing herbivore diversity and between the two sites over the two seasons (Table 2.7). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize for herbivore diversity and between the two sites over the two seasons (Table 2.7).

The same applied for season and site for the indices, number of species and number of individuals of chewing herbivores (Tables 2.8 & 2.9).

Figure 2.9. Rank abundance graph for chewing herbivore diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.10. Rank abundance graph for chewing herbivore diversity on Bt and non-Bt maize in Tshiombo area over two seasons. 60

Table 2.7. Descriptive statistics and p-value for diversity index values, abundance and number of chewing herbivore species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 1.72 1.59 0.51 1.71 1.54 1.00 1.76 1.65 0.38 1.77 1.78 1.00 1.60 1.27 0.38 Margalef index 2.06 2.12 0.80 1.88 2.08 0.66 1.98 1.86 0.66 2.28 2.17 1.00 2.33 2.66 0.38 Number of species / 20 plants 8.50 9.50 0.64 8.00 9.00 0.83 8.00 7.00 0.83 11.00 11.00 1.00 8.00 15.00 0.66 Number of individuals / 20 plants 42.50 50.50 0.33 44.00 47.00 1.00 15.00 41.00 0.38 81.00 53.00 1.00 16.00 193.00 0.66

Table2.8. Descriptive statistics and p-value for diversity index values, abundance and number of chewing herbivore species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2

Shannon index 1.72 1.65 0.51 1.62 1.71 0.69 1.77 1.44 0.17 Margalef index 2.10 2.15 0.84 1.98 1.92 1.00 2.22 2.43 0.81 Number of species / 20 plants 9.00 8.00 0.29 8.50 7.50 0.42 11.00 9.50 0.81 Number of individuals / 20 plants 50.50 33.00 0.23 45.50 33.00 0.20 67.00 44.50 0.69

Table 2.9. Descriptive statistics and p-value for diversity index values, abundance and number of chewing herbivore species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2

Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo

Shannon index 1.68 1.65 0.93 1.62 1.77 0.47 1.71 1.44 0.47 Margalef index 1.93 2.30 0.21 1.98 2.22 0.30 1.92 2.43 0.69 Number of species / 20 plants 8.00 11.00 0.16 8.50 11.00 0.26 7.50 9.50 0.58 Number of individuals / 20 plants 42.50 63.00 0.58 45.50 67.00 0.58 33.00 44.50 1.00 61

2.4.4. Sucking herbivores

The rank abundance graphs for Vaalharts indicated a high evenness for Bt and non-Bt maize during the first season, where for the second season a lower evenness occurred (Figure 2.11). Diversity was higher in season 1 than in season 2. The low evenness during season 2 can be ascribed to the dominant species of Aphididae and Phlaeothripidae. The rank abundance graphs for Tshiombo indicated a high evenness for Bt and non-Bt maize during the seasons (Figure 2.12). The most abundant species in Tshiombo belonged to Delphacidae and Thripidae.

The diversity indices indicated no differences between Bt and non-Bt maize for total sucking herbivore diversity and between the two sites over the two seasons (Table 2.10). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize for herbivore diversity and between the two sites over the two seasons (Table 2.11).

The Shannon index indicated a significant difference in diversity of the sucking herbivore complex between the two sites during the second season (p=0.03), with the diversity being lower at Vaalharts (Table 2.12). The difference in diversity between the two sites for the second season can be seen in Figures 2.11 and 2.12, especially at Vaalharts where the species richness was lower.

Figure 2.11. Rank abundance graph for sucking herbivore diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.12. Rank abundance graph for sucking herbivore diversity on Bt and non-Bt maize in Tshiombo area over two seasons. 63

Table 2.10. Descriptive statistics and p-value for diversity index values, abundance and number of sucking herbivore species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 1.40 1.19 0.40 1.57 1.62 1.00 1.00 0.97 0.66 1.23 1.45 1.00 1.77 1.30 0.66 Margalef index 2.40 2.25 0.26 2.48 1.81 0.19 1.86 1.78 1.00 3.13 2.39 0.66 2.91 2.26 1.00 Number of species / 20 plants 12.00 12.00 0.58 15.00 11.00 0.13 10.00 10.00 1.00 17.00 12.00 0.66 10.00 0.70 1.00 Number of individuals / 20 plants 132.50 143.50 0.62 210.00 253.00 1.00 126.00 156.00 0.66 163.00 100.00 0.66 52.00 4.40 0.66

Table 2.11. Descriptive statistics and p-value for diversity index values, abundance and number of sucking herbivore species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2 Shannon index 1.51 1.13 0.67 1.59 0.98 0.09 1.34 1.71 0.38 Margalef index 2.38 1.97 0.26 2.34 1.82 0.09 2.54 2.59 0.94 Number of species / 20 plants 12.00 10.00 0.09 12.00 10.00 0.07 12.50 12.00 0.58 Number of individuals / 20 plants 186.50 128.50 0.34 231.50 143.50 0.69 131.50 70.00 0.30

Table 2.12. Descriptive statistics and p-value for diversity index values, abundance and number of sucking herbivore species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2 Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo Shannon index 1.04 1.55 0.19 1.59 1.34 0.58 0.98 1.71 0.03 Margalef index 1.89 2.54 0.06 2.34 2.54 0.47 1.82 2.59 0.07 Number of species / 20 plants 11.50 12.50 0.34 12.00 12.50 0.58 10.00 12.00 0.47 Number of individuals / 20 plants 183.00 94.00 0.06 231.50 131.50 0.13 143.50 70.00 0.23 64

2.4.5. Chewing predators

The rank abundance graphs for Vaalharts and Tshiombo during both seasons indicated a high evenness for Bt and non-Bt maize (Figures 2.13 & 2.14). Species richness was higher at Tshiombo than at Vaalharts. The most abundant species belonged to Coccinelidae; Formicidae and Forficulidae at Vaalharts and Formicidae at Tshiombo.

The diversity indices indicated no differences between Bt and non-Bt maize for total chewing predator diversity and between the two sites over the two seasons (Table 2.13). There was also no significant difference between the number of species and number of individuals between Bt and non-Bt maize between predator diversity and between the two sites over the two seasons (Table 2.13).

There was a significant difference in the Margalef richness index (p=0.04) for chewing predators over the two seasons in Tshiombo with species richness being lower in season 1 (Table 2.14). This was also indicated in the rank abundance graph for Tshiombo over the two seasons (Figure 2.14).

A significant difference was observed between the two sites for number of chewing predator individuals (p=0.03) with the number of predators being higher at Tshiombo. This difference was ascribed to the significant difference observed during season 1 (p=0.05) between the two sites (Table 2.15). The difference in the number of individuals between the two sites can be seen in Figures 2.13 and 2.14, with species richness being higher at Tshiombo.

Figure 2.13. Rank abundance graph for chewing predator diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.14. Rank abundance graph for chewing predator diversity on Bt and non-Bt maize in Tshiombo area over two seasons. 66

Table 2.13. Descriptive statistics and p-value for diversity index values, abundance and number of chewing predator species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 1.93 1.93 0.62 1.91 1.97 1.000 2.01 2.12 0.66 1.30 1.70 0.38 2.12 1.86 0.66 Margalef index 2.48 3.16 0.14 2.47 2.86 0.663 2.49 3.27 0.38 2.32 2.86 0.08 3.74 3.40 0.66 Number of species / 20 plants 11.00 11.50 0.26 10.00 11.00 0.663 10.00 12.00 1.00 11.00 14.00 0.28 12.00 16.00 1.00 Number of individuals / 20 plants 50.00 31.00 0.71 31.00 33.00 0.663 47.00 28.00 0.51 113.00 99.00 1.00 53.00 105.00 0.83

Table 2.14. Descriptive statistics and p-value for diversity index values, abundance and number of chewing predator species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2

Shannon index 1.86 2.03 0.07 1.94 2.03 0.47 1.66 2.02 0.07 Margalef index 2.74 3.26 0.07 2.76 2.86 0.94 2.59 3.41 0.04 Number of species / 20 plants 11.00 12.00 0.33 11.00 11.00 0.87 11.50 14.00 0.26 Number of individuals / 20 plants 40.50 38.00 0.80 32.00 28.50 0.94 106.00 79.00 0.81

Table 2.15. Descriptive statistics and p-value for diversity index values, abundance and number of chewing predator species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2

Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo

Shannon index 1.99 1.85 0.40 1.94 1.66 0.17 2.03 2.02 0.94 Margalef index 2.76 3.04 0.54 2.76 2.59 0.47 2.86 3.41 0.09 Number of species / 20 plants 11.00 12.00 0.06 11.00 11.50 0.30 11.00 14.00 0.13 Number of individuals / 20 plants 30.00 102.00 0.03 32.00 106.00 0.05 28.50 79.00 0.47 67

2.4.6 Sucking predators

The rank abundance graphs for Vaalharts and Tshiombo during both seasons indicated a low evenness for Bt and non-Bt maize (Figures 2.15 & 2.16). Bt maize species richness was especially low in Vaalharts during the second season. The low evenness can be ascribed to the dominant species of Anthocoridae and Miridae for Vaalharts and Anthocoridae and Phytoseiidae for Tshiombo.

The diversity indices indicated no differences between Bt and non-Bt maize for total sucking predator diversity and between the two sites over the two seasons (Table 2.16). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize for predators diversity and between the two sites over the two seasons (Table 2.16).

There was, however, a significant difference between the number of sucking predator individuals over the two seasons in Vaalharts (p=0.01) with the numbers being significantly lower in season 2 (Table 2.17). This was also indicated in the rank abundance graph for Vaalharts over the two seasons (Figure 2.15).

The Shannon index-value indicated that diversity was significantly (p=0.05) lower during season 2, with diversity being lowest at the Tshiombo site. The number of individuals (p=0.01) was also lower during season 2, with number of individuals being lowest at the Vaalharts site during the second season (Table 2.18). The low number of individuals in Vaalharts is illustrated in Figure 2.15 and the low diversity in Tshiombo in Figure 2.16.

Figure 2.15. Rank abundance graph for sucking predator diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.16. Rank abundance graph for sucking predator diversity on Bt and non-Bt maize in Tshiombo area over two seasons. 69

Table 2.16. Descriptive statistics and p-value for diversity index values, abundance and number of sucking predator species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 1.01 0.99 0.93 0.96 1.13 0.38 1.03 1.16 1.00 1.15 0.82 0.83 0.62 0.39 0.51 Margalef index 1.03 1.12 0.89 1.04 1.13 1.00 1.02 1.15 0.66 1.41 0.98 0.51 0.84 0.84 1.00 Number of species / 20 plants 4.00 4.50 0.86 5.00 5.00 1.00 4.00 4.00 0.83 5.00 5.00 1.00 4.00 4.00 1.00 Number of individuals / 20 plants 33.50 33.00 0.08 47.00 26.00 0.08 10.00 15.00 1.00 17.00 53.00 0.51 35.00 38.00 0.51

Table 2.17. Descriptive statistics and p-value for diversity index values, abundance and number of sucking predator species over two seasons at Vaalharts and Tshiombo irrigations schemes over.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2 Shannon index 0.99 1.01 0.75 0.99 1.10 0.23 0.99 0.50 0.07 Margalef index 1.09 1.05 0.71 1.09 1.13 0.58 1.12 0.84 0.30 Number of species / 20 plants 5.00 4.00 0.13 5.00 4.00 0.17 5.00 4.00 0.42 Number of individuals / 20 plants 35.50 32.00 0.20 35.50 12.50 0.01 34.50 36.50 1.00

Table 2.18. Descriptive statistics and p-value for diversity index values, abundance and number of sucking predator species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2 Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo

Shannon index 1.03 0.82 0.11 0.99 0.99 0.94 1.10 0.50 0.05 Margalef index 1.12 0.91 0.40 1.09 1.12 0.58 1.13 0.84 0.09 Number of species / 20 plants 4.00 4.50 0.82 5.00 5.00 0.87 4.00 4.00 0.87 Number of individuals / 20 plants 25.50 36.50 0.11 35.50 34.50 0.81 12.50 36.50 0.01

2.4.7. Parasitoids

The rank abundance graphs for Vaalharts and Tshiombo during both seasons indicated a low evenness for Bt and non-Bt maize (Figures 2.17 & 2.18). Species richness was especially low in Vaalharts during both seasons, whereas species richness was low only during the first season in Tshiombo. The low evenness can be ascribed to the dominant species of Braconidae and Scelionidae for Vaalharts and Tshiombo respectively.

The diversity indices indicated no differences between Bt and non-Bt maize for total parasitoid diversity and between the two sites over the two seasons (Table 2.19). There was also no significant difference between number of species and number of individuals between Bt and non-Bt maize for parasitoid diversity and between the two sites over the two seasons (Table 2.19).

The Shannon index-value indicated a significant difference (p=0.02) in diversity, as well as number of parasitoid species (p=0.04), over the two seasons in Tshiombo (Table 2.20). The index-value and number of species was lower in season 1 (Table 2.20). This was also indicated in the rank abundance graph for Tshiombo, with species richness being especially low during season 1 (Figure 2.18).

There was a significant (p=0.02) difference in the Shannon index-value for parasitoids between the two sites with the diversity being lower at Vaalharts. This was ascribed to a significant (p=0.01) difference between the two sites during the second season (Table 2.21). The difference between Vaalharts and Tshiombo second season can be seen in Figures 2.17 and 2.18, with the diversity being lower at Vaalharts. The number of parasitoid species also differed significantly (p=0.01) between the two sites due to a significant difference at the Vaalharts site during the second season (p=0.01) (Table 2.21). A significant difference was observed in the number of individuals during the second season between the two sites (p=0.01), with abundance being lower at Vaalharts (Table 2.21). The low number of parasitoid species and individuals was also indicated in the rank abundance graphs for Vaalharts (Figure 2.17) and Tshiombo (Figure 2.18), where the species richness was lower in Vaalharts during the second season.

Figure 2.17. Rank abundance graph for parasitoid diversity on Bt and non-Bt maize in the Vaalharts area over two seasons.

Figure 2.18. Rank abundance graph for parasitoid diversity on Bt and non-Bt maize in Tshiombo area over two seasons.

Table 2.19. Descriptive statistics and p-value for diversity index values, abundance and number of parasitoid species in Bt and non-Bt maize at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Vaalharts Tshiombo Season 1 Season 2 Season 1 Season 2 Median Median Median Median Median p-value p-value p-value p-value p-value Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt Bt non-Bt

Shannon index 0.28 0.31 1.00 0.00 0.00 1.00 0.00 0.00 0.66 0.56 0.00 1.00 1.28 1.10 0.51 Margalef index 0.91 0.62 0.46 0.46 0.31 1.00 - 0.46 1.00 0.79 0.91 1.00 1.82 1.82 0.83 Number of species / 20 plants 1.50 1.50 0.80 1.00 1.00 1.00 0.00 1.00 0.19 2.00 1.00 1.00 4.00 3.00 0.51 Number of individuals / 20 plants 3.00 3.00 0.56 3.00 5.00 0.83 0.00 2.00 0.13 4.00 3.00 0.66 7.00 11.00 1.00

Table 2.20. Descriptive statistics and p-value for diversity index values, abundance and number of parasitoid species over two seasons at Vaalharts and Tshiombo irrigations schemes.

Combined data Vaalharts Tshiombo Median Median Median p-value p-value p-value Season 1 Season 2 Season 1 Season 2 Season 1 Season 2

Shannon index 0.00 0.63 0.24 0.00 0.00 0.63 0.28 1.19 0.02 Margalef index 0.67 1.68 0.08 0.31 0.46 1.00 0.79 1.82 0.17 Number of species / 20 plants 1.00 2.00 0.24 1.00 1.00 0.81 1.50 3.50 0.04 Number of individuals / 20 plants 3.00 3.00 1.00 4.00 1.00 0.26 3.00 9.00 0.09

Table 2.21. Descriptive statistics and p-value for diversity index values, abundance and number of parasitoid species at Vaalharts and Tshiombo irrigations schemes over two seasons.

Combined data Season 1 Season 2

Median Median Median p-value p-value p-value Vaalharts Tshiombo Vaalharts Tshiombo Vaalharts Tshiombo

Shannon index 0.00 0.87 0.02 0.00 0.28 0.69 0.00 1.19 0.01 Margalef index 0.31 1.68 0.05 0.31 0.79 0.47 0.46 1.82 0.13 Number of species / 20 plants 1.00 3.00 0.01 1.00 1.50 0.38 1.00 3.50 0.01 Number of individuals / 20 plants 1.50 3.50 0.09 4.00 3.00 0.75 1.00 9.00 0.01 73

2.5. Discussion

The diversity of arthropods collected during this study was high. A total 8771 arthropod individuals were collected that consisted of 288 morpho species. Although arthropod diversity described in this study was higher compared to studies on other crops, the total number of arthropod individuals found in this study was low relative to other studies (Li et al., 2007; Torres & Ruberson, 2007). A study of arthropod abundance and diversity in Bt and non-Bt rice fields recorded 17 706 arthropod individuals (Li et al., 2007) and another diversity study on ground-dwelling arthropods in Bt cotton and non-Bt cotton collected 38 980 individuals, consisting of only 65 taxa (Torres & Ruberson, 2007). An arthropod diversity study on Bt maize ears recorded 48 521 individuals that consist of 23 taxa (Eckert et al., 2006). We realise that data from this study cannot be compared to studies that used different sampling methods. However, the mentioned studies on cotton, rice and maize are the only studies that provide comparative data on arthropod abundance and diversity in agro-ecosystems in the world.

The purpose of this study was not to determine the arthropod diversity between the two sites, but important site-specific observations were made during this survey. The farming system at the Vaalharts site is a high-input system, while that at Tshiombo is a low-input system managed by subsistence farmers. A possible reason for the difference in diversity between the sites is that at Vaalharts insecticides such as pyrethroids are used regularly, whereas in Tshiombo no insecticides are sprayed onto maize. At Vaalharts, sampling was done before the farmer’s sprayed insecticides in order to limit the effect that this could have had on sampling. The reduced species diversity at Vaalharts could therefore possibly be ascribed to the long term effect of insecticides on arthropods at the site.

Diversity and abundance of detritivores were lower in Tshiombo especially during the second season. A possible explanation is that at Vaalharts the plant population per hectare is denser, creating an optimum environment for outbreak species, such as Acari, to occur.

The low diversity of sucking herbivores during the second season between the sites, due to a low diversity in Vaalharts, can be ascribed to the frequent use of insecticides and the practise of monoculture, whereas in Tshiombo no insecticides are used and polyculture is practised.

Chewing predator richness was lower during the first season in Tshiombo, as well as the diversity and number of parasitoid species. A possible reason for the latter, during the first season in Tshiombo, could be ascribed to poorer plant growth and because plants did not

74 reach the height they normally do because of drought-stress. Since maize cropping at the Tshiombo site is done in rotation with groundnut and sweet potatoes the reduced diversity on maize could, however, also be associated with the previous crop which could have hosted a different arthropod species complex. The lower abundance of chewing predators at Vaalharts can be ascribed to the frequent use of insecticides and the practise of monoculture.

The significant difference between the numbers of sucking predator individuals over the two seasons in Vaalharts, with the numbers being lower in season 2, can possibly be ascribed to the extend of insecticide use during the previous season or crop. The diversity of sucking predators was low but the abundance was higher in Tshiombo than in Vaalharts. Since no data were collected on insecticide use patterns, no conclusions can be drawn.

The difference in diversity and abundance of parasitoids between the two sites could be due to the different farming methods that are used by the farmers and because habitat diversity is higher at Tshiombo. At Vaalharts insecticides such as pyrethroids are used regularly, whereas in Tshiombo no insecticides are sprayed onto maize.

In this study, we did not find a significant difference in abundance and diversity of the different functional groups (detritivores, herbivores, predators and parasitoids) between Bt and non-Bt maize. However, a study on Bt cotton showed a decrease in diversity of natural enemy sub-communities (Men et al., 2003). A long-term study showed essentially no effects of Bt cotton on natural enemy function in Arizona cotton and minor reductions in density of several predator taxa in Bt cotton were observed (Naranjo, 2005). Similarly no detrimental effect of Bt maize was observed on any predator taxa or on the whole functional group of predators in a farm-scale study in Spain (De la Poza et al., 2005). Li et al. (2007) also found no significant differences in sub-communities phytophages, parasitoids, predators, detritivores between Bt and non-Bt rice. The most abundant species of each functional group are listed in Table 3.1 (Chapter 3; p 87).

There was, however, a significant difference in total arthropod diversity between the two localities, Vaalharts and Tshiombo. The same applied for number of detritivores species, number of chewing predator individuals and the Shannon index-value and number of species for parasitoids between the two sites. Results further indicated that diversity and abundance of parasitoids in the high-input farming system at Vaalharts were significantly lower than at the Tshiombo site where subsistence farming is practiced. This could be due to the two different farming methods that are used by the farmers at the different sites and the fact that commercial farmers make use of insecticides. A study by Whitehouse et al. (2005) found that

75 the diversity and species richness of the beneficial arthropod communities was reduced in sprayed cotton crops. Insecticide applications reduced the abundance of total arthropods, pest and natural enemies in both the non-transgenic/insecticides and Bt cotton/insecticides plots (Men et al., 2003).

In this study, abundance and diversity of the arthropod complex in maize were not significantly affected by Bt maize. Other studies on the effect of transgenic crops on arthropods also recorded similar results. Torres and Ruberson (2007) found that abundance and diversity of ground-dwelling arthropods were not significantly different between Bt cotton and non-Bt cotton. A study on diversity and dominance distribution of arthropods in Bt rice and non-Bt rice also found no significant difference (Li et al., 2007). Men et al. (2003) indicated that Bt cotton increased the diversity of arthropod communities and pest sub- communities. A slight difference between total arthropod communities was found in unsprayed conventional and Bt cotton in Australia (Whitehouse et al., 2005). No effects of Bt maize on the communities of soil-dwelling and non-target plant-dwelling arthropods were observed by Candolfi et al. (2004). Dively (2005) also found that the densities of non-target taxa exposed to Bt and non-Bt maize showed no difference, whilst a multi-year study showed that Bt cotton has no significant adverse impact on non-target arthropod populations (Head et al., 2005). In China, the diversity of arthropod communities in transgenic cotton was similar to that in conventional cotton fields without spraying (Li et al., 2004).

2.6 Conclusions

It can be concluded that this short-term study on only two localities indicated that abundance and diversity of arthropods in maize were not significantly affected by Bt maize. This study provided a start in the study of biodiversity of arthropods on maize in South Africa and generated a basic checklist of these species. This study can be used in the future assessment of the possible impact of Bt maize on the environment. Our findings suggest that Bt maize has no effect on total arthropod diversity, as well as the different functional groups.

We realize that this study was limited in its extent and that the contribution of soil arthropods was not recognized at all. Larger and long-term studies and surveys of biodiversity, both inside and adjacent to maize fields, should be conducted and other sampling techniques should be included to provide improved assessment of total biodiversity.

2.7 References

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CHAPTER 3

ARTHROPOD BIODIVERSITY IN MAIZE AND SELECTION OF NON-TARGET ARTHROPODS SPECIES FOR ECOLOGICAL RISK ASSESSMENT OF BT MAIZE IN SOUTH AFRICA

3.1 Abstract

In order to perform risk assessments that are relevant to the receiving environment, information is needed on biodiversity and the spectrum of possible non-target arthropod species of Bt maize in maize-based ecosystems. Current risk assessment is hampered by a lack of checklists of species in these systems and information regarding species richness is largely limited to that of economically important pests and the well-studied complex of natural enemies of the lepidopteran stem borer complex. Information on biodiversity is essential for the future evaluation of the possible impact of Bt maize on non-target organisms at different trophic levels, as well as identification of the causal pathways of exposure to insecticidal proteins produced by genetically modified plants. In this study, priority species for future research was identified following an ecological model approach, using an extensive data base on species richness and diversity of arthropods in the receiving environment. A series of selection matrixes were developed in which each species was ranked for its maximum potential exposure to Bt toxin by assessing its occurrence, abundance, presence and linkage in the maize ecosystem. The following non-target functional groups were identified as important: herbivores (Aphididae, Tetranychidae); predators (Anthocoridae, Forficulidae, Coccinellidae, Staphylinidae) and parasitoids (Braconidae and Scelionidae). During this study, non-target species with high abundance that are also exposed to Bt maize were identified. Through the use of these selection matrixes, knowledge gaps were also identified for future research. This study and reviewed literature therein indicated that many species could be excluded for further testing in South Africa since Bt maize has been reported to have no adverse effect on them, whereas for others, continued studies are needed.

3.2 Introduction

Crop plants and insect pests form complex agricultural ecosystems that involve interactions between many trophic levels (Poppy & Sutherland, 2004). In studies to determine possible effects of genetically modified (GM) Bt maize plants on non-target organisms it is important to recognise that delicate food webs exist even in agricultural systems (Groot & Dicke, 2002). With the realisation of the complexity and importance of food webs in agricultural systems, conservation of these ecosystems has been highlighted. Food webs exist in agricultural systems and the interactions between plants, herbivores and natural enemies in these systems may change from simple tri-trophic interactions to more complex food web interactions (Janssen et al., 1998).

Any given cropping system will typically contain more or less 1000 to several thousands of species (Birch et al., 2004). Although it is possible to assess impacts on this biodiversity in its entirety, the pre-release risk assessment of transgenic crops is done in controlled environments such as laboratories, greenhouses or small-scale field evaluations that require selection of a relatively small number of species, species groups or surrogate species to be studied or monitored. Therefore, an important component of case-specific risk assessment of a GM crop is the selection of the most relevant species in the particular receiving environment for pre-release testing (Andow & Hilbeck, 2004; Birch et al., 2004).

In order to perform reliable risk assessments for Bt maize, information is needed on biodiversity and the spectrum of possible non-target species in maize-based ecosystems. Bt maize (MON810), expressing a Cry1Ab protein, has been planted in South Africa since 1998 (James, 2010), with the target pests being the stem borers Busseola fusca (Lepidoptera: Noctuidae) and Chilo partellus (Lepidoptera: Crambidae). Current risk assessment is hampered by a lack of even a basic checklist of species in these systems and in South Africa knowledge is largely limited to that of economically important pests (Annecke & Moran, 1982), Lepidoptera diversity (Van Wyk et al., 2007a) and the well-studied complex of Hymenoptera parasitoids of the stem borer complex (Kfir et al., 2002). Increased knowledge of biodiversity in the receiving environment of, for example, Bt maize, will lead to improved risk assessment and evaluation of the possible impacts, of Bt maize on non-target organisms at different trophic levels. It could also lead to the identification of possible causal pathways of exposure to insecticidal proteins produced by GM plants.

The term environmental risk assessment (ERA) can be defined as a systematic process of developing a scientific basis for regulatory decision making (Barnthouse et al., 1992). Risk

83 assessment is a process during which risks are identified and the seriousness of the risks characterized so that decisions can be made on whether or how to proceed with the technology (NRC, 1996). Risk management seeks measures to limit the risks of GM plants in the environment including not only ecological, but also socio-economical and political criteria (cost/benefit) (Hilbeck et al., 2008). Risk assessment has gained increasing prominence as a methodology for evaluating the environmental risks of new technologies. The value and relevance of risk assessments is improved if information is available on biodiversity, and importance and functions of particular groups or species in the receiving environment (Andow & Hilbeck, 2004).

The aims of this study were to use an ecological risk assessment model approach to identify arthropod species from different functional groups that are most likely to be exposed to Bt protein inside the maize ecosystem, and to prioritise species for further research.

3.3 Material and methods

Collections of arthropods were made during two growing seasons (2008/09 and 2009/10) in Bt and non-Bt maize fields at two localities, i.e. the Vaalharts area in the Northern-Cape province and the Tshiombo area in the Limpopo province of South Africa.

During this study the focus was on collecting arthropods that occur on plants during the reproductive stage of plant growth only. No collections were made of soil arthropods and this study therefore only takes into account above-ground on-plant diversity. The sampling method of arthropods is discussed in detail in Chapter 2 (2.3.2; p. 47).

A total of 8771 arthropod individuals, comprising 288 morpho-species, were collected over the two seasons during the study period. In the Vaalharts area a total of 4154 arthropod individuals consisting of 169 morpho-species were collected and in Tshiombo a total of 4617 individuals consisting of 202 morpho-species. The overall 288 morpho-species consisted of 20 orders of Arthropoda. Only 28.8% of these species occurred in both localities. Where possible and where taxonomical expertise existed morpho-species were identified to species level. In the case of ants and mites this was not possible in South-Africa.

The 288 morpho-species were grouped into the following functional groups: detritivores, herbivores, predators and parasitoids. The herbivores and predators were further divided on basis of feeding strategy into sucking herbivores/predators (piercing-sucking mouthparts) and chewing herbivores/predators (chewing mouthparts).

The Andow and Hilbeck (2004) ERA model, which was also used by Birch et al. (2004), was used in this study to reduce the number of species from 288 to a more realistic number for further evaluation. This model consists of the following steps: • Categorizing and listing potential non-target species and their ecological function. • Prioritizing species or functions for pre-release testing according to maximum potential adverse effects. • Analysis of exposure pathways. • Describing hazard scenarios and formulating testing hypotheses. • Developing ecologically meaningful testing methods and protocols.

A literature search was done to identify sources of information on the effects of Bt-toxin on the species identified as important by the ERA model. The relevance of this information to the South African receiving environment was assessed and knowledge gaps identified with regard to which local species should be studied further and for which species sufficient information existed to possibly exclude them from future non-target effects research.

3.4 Environmental risk assessment with reference to Bt maize in South Africa

The ERA model makes use of different steps in order to reduce the very large numbers of species to a smaller number of priority species for use in further testing of possible effects of GM crops. These steps are discussed below with reference to Bt maize and abundance of arthropods in this crop in South Africa.

3.4.1 Step 1: Categorizing and listing potential non-target species and their ecological function

This step involves the identification of important ecological functions of particular arthropod groups that could be considered in risk assessment for a given case. This list is specific to the crop, it’s cropping context and agro-ecosystem (Hilbeck et al., 2006; Van Wyk et al., 2007a). Using ecological function such as detritivory, herbivory, predation and parasitism, allows focus on ecological processes and limits the number of species and functions that need to be tested and considered in the risk assessment process (Birch et al., 2004).

A functional group is a group of organisms that carries out the same broad ecological function. In spite of belonging to different taxa, organisms may belong to the same feeding guild, have the same impact mode on ecosystem structure or have the same pest control

85 function (Hilbeck et al., 2008). Two types of functional criteria, anthropocentric and ecological, can be used to categorize different functions. Groups, whose function is anthropocentric or related directly to human goals, include secondary pest species, natural enemies, rare or endangered species, species used to generate income, and species of social or cultural value. Ecological functions relate to ecosystem processes and are independent of human goals. Ecological functions include non-target primary consumers of maize, secondary consumers, pollinators, decomposers, and seed dispersers. These functional groups are not mutually exclusive, for example, a species may simultaneously both be a less important pest, a pollinator and a non-target primary consumer. Such species may be key species because of their multiple functions and can be involved in more than one anticipated environmental risk (Andow & Hilbeck, 2004).

While some non-target herbivores are also minor or sporadic pests of maize, others are potential secondary pests of the crop. Since Bt maize reduces target pest numbers to insignificant levels, other herbivore species may increase in numbers and become damaging pests. This is, however, not always the case since the numbers of other herbivores may also decrease or remain similar. These herbivores may also be vectors of diseases, food for natural enemies or seed dispersers, making them important species to consider in the risk assessment process.

Natural enemies (parasitoids) and generalist predators (sucking and chewing predators) are beneficial organisms that help reduce pest populations and in so doing, may be indirectly exposed to Cry proteins produced by Bt plants by feeding on herbivorous prey that consumed Bt Cry-toxin produced by plants (Birch et al., 2004). If this indirect exposure leads to adverse effects on beneficial organisms it may have a negative effect on ecosystem functions. The risk assessment process should identify these risks.

The most abundant non-target organisms found on maize and which should be considered in a risk assessment are listed in Table 3.1. The species indicated in Table 3.1 made up 9.03% of the total number of 288 morpho-species. The most abundant species of each functional group was selected for further consideration in the ERA. The proportion of the number of species selected in each functional group was as follows: detritivores (1.1% of n=56 spp.), sucking herbivores (8.8% of n=57 spp.), chewing herbivores (10.4% of n=48 spp.), sucking predators (23.5% of n=17 spp.), chewing predators (8.5% of n=82 spp.) and parasitoids (0.7% of n=23 spp.). A more comprehensive list is provided in Tables 3.2-3.4.

Table 3.1 Functional groups and abundance of non-target organisms used in this environmental risk assessment of Bt maize in South Africa.

Mean Total number Standard Order Family Genus (N) / plant deviation (n=480) Detritivores Acari Oppiidae Oppiidae sp. 1 166 0.35 1.89 Collembola Entomobryidae Entomobryidae sp. 1 151 0.31 1.05 Sucking herbivores Homoptera Aphididae Several genera 1552 3.23 7.68 Hemiptera Delphacidae Peregrinus maidis 822 1.71 6.08 Thysanoptera Thripidae Chirothrips sp. 1 187 0.39 1.20 Thysanoptera Phlaeothripidae Haplothrips sp. 2 174 0.36 1.01 Acari Tetranychidae Tetranychidae sp. 1 127 0.26 1.55 Chewing herbivores Coleoptera Nitidulidae Carpophilus sp.1 514 1.07 5.39 Coleoptera Nitidulidae Carpophilus sp.3 352 0.73 7.36 Coleoptera Nitidulidae Carpophilus larva sp. 2 169 0.35 1.54 Coleoptera Anthicidae Notoxus monoceros 167 0.35 5.14 Coleoptera Lathridiidae Carticaria japonica 147 0.31 6.75 Sucking predators Hemiptera Anthocoridae Orius sp. 1 401 0.84 1.33 Hemiptera Berytidae Metacantus sp. 1 78 0.16 0.07 Hemiptera Miridae Miridae sp. 5 71 0.15 2.35 Acari Phytoseiidae Phytoseiidae sp. 1 70 0.15 0.75 Chewing predators Hymenoptera Formicidae Formicidae sp. 1 316 0.66 2.69 Dermaptera Forficulidae Forficulidae larva sp. 1 253 0.53 3.40 Dermaptera Forficulidae Forficulidae sp. 1 164 0.34 1.10 Hymenoptera Formicidae Formicidae sp. 2 133 0.28 1.12 Coleoptera Coccinelidae Scymnus nubilus 71 0.15 0.51 Coleoptera Coccinelidae Cheilomenes sp.3 54 0.11 0.40 Coleoptera Staphylinidae Oxytelus sp. 1 53 0.11 0.44 Parasitoids Hymenoptera Braconidae Braconidae sp. 1 36 0.08 0.63 Hymenoptera Scelionidae Scelionidae sp. 1 15 0.03 0.25 Hymenoptera Scelionidae Scelionidae sp. 2 15 0.03 0.29

3.4.2 Step 2: Prioritizing species or functions for pre-release testing according to maximum potential adverse effects

This part of the process involves ranking non-target species or functions according to ecological principles and prioritizing these for possible assessment. Particular emphasis is placed on those organisms that have a high likelihood of being adversely affected by the transgenic crop or are important for ecological or anthropogenic reasons (Birch et al., 2004).

To prioritize species, the arthropods collected during the 2-year study were grouped into functional groups and sorted according to mean abundance per plant. The species with the highest abundance from each functional group were selected for use in the species prioritization process. Selection matrices developed by Birch et al. (2004) were used to rank these species for its maximum potential exposure and for the potential adverse effect that exposure to Bt-toxin may have. This was done by assessing its occurrence, abundance, presence and linkage in the maize ecosystem, as well as assessing for potential adverse effects that exposure may have on the non-target species. Species, groups or ecological functions given the highest priority (rank 1) are therefore the ones that have the maximum potential exposure and those that have a known important ecosystem function. Species were only assigned one of three possible ranks to simplify the ranking procedure and since only rank-1 species or functions are likely to be considered for further assessment. This approach overcomes the simplistic assumption of species abundance as a direct measure of ecological significance. To ensure a precautionary approach, species with a high standing biomass or those that are found in frequent association with the crop should be prioritized for testing even if their significance is unknown (Birch et al., 2004).

In the context of the ecological model, “occurrence” refers to the presence of a non-target species in the agro-ecosystem, its range and prevalence. “Abundance” refers to local abundance and prevalence, while “presence” involves temporal association with the crop. “Linkage” refers to habitat specificity and the degree of specialization of the non-target species on maize (Andow & Hilbeck, 2004).

3.4.2.1 Prioritizing functional groups and non-target species

The detritivore-, herbivore-, predator- and parasitoid functional groups, of which the most abundant species were listed in Table 3.1, were further prioritized in this step of the ERA.

3.4.2.1.1 Detritivores The following two taxa, Opiidae (Acari) and Entomobryidae (Collembola) were grouped as detritivores, because of their known biology. Collembolans are recognized as key indicator species of soil fertility and health, as they play a vital role in the breakdown and recycling of crop residues (O’Callaghan et al., 2005).

Since the Cry-toxin may persist associated with GM plant residues, its presence and availability in soils could continue long after the crop has been grown and harvested. Non- target species such as these detritivores could therefore be exposed to the toxin for a prolonged period of time. Because of their relatively low abundance, the detritivores species listed in Table 3.2 were ranked medium. However, considering the importance of their ecological function, and the fact that they were collected on plants, although they are known to be soil organisms, detritivores should be considered important for future studies. There is a likelihood that soil-dwelling organisms may be influenced by transgene-derived proteins released into the soil as exudates from the roots of GM plants (Borisjuk et al., 1999) and through contact with plant material left in the ground after harvest (O’Callaghan et al., 2005). The main source of Bt proteins entering the soil is most likely via plant residues, as two to six tons of residue per hectare are left on the field after harvest (Zscheischler et al., 1984), and the Cry1Ab protein can persist in the plant matrix for at least two years after sowing (Zwahlen et al., 2003).

3.4.2.1.2 Herbivores The herbivores considered as important during step 1 (Table 3.1) consisted of five orders and eight families that were divided on the basis of their feeding strategy into either sucking or chewing herbivores (Table 3.3). The Aphididae were ranked highly as they occurred in high abundance and can transmit viruses such as maize dwarf mosaic virus to maize plants (Table 3.3). Tetranychidae are usually low in abundance but may occur in high numbers any time during the growth cycle of maize. Tetranychidae have a high damage potential especially under irrigation conditions and were therefore ranked highly (Table 3.3). The spider mite, T. urticae, compared to other organisms that are exposed directly or indirectly, is also known to contain a high concentration of Cry proteins after feeding on Bt maize (Dutton et al., 2002; Obrist et al., 2006; Meissle & Romeis, 2009; Li & Romeis, 2010). For these reasons it could be considered an important non-target organism for future research.

Carpophilus spp. (Nitidulidae) which are common saprovores (Table 3.3), are indirectly exposed to Bt toxin as beetles feed on the frass of lepidopterans that feed on Bt maize. They are, however, also directly exposed to Bt protein, since they actively feed on maize kernels

89 and ingest plant sap produced by damaged kernels. Since large numbers of nitidulid larvae occur on maize ears and because this group was one of those with the highest biomass on maize, it received a high rank. These nitidulid beetles may also transmit Aspergillus flavus (Eurotiales: Trichocomaceae), an ear rot fungal infection of maize, between plants. Although A. flavus is not economically important in terms of yield loss, it produces aflatoxin. Fungal toxins reduce the value of grains to animal feed and devalue it as an export commodity (Nichols, 1983). Lussenhop and Wicklow (1990) demonstrated the role of nitidulid beetles in transmitting A. flavus infective inoculum to both silks and wounded-kernel tissue in release and recapture experiments, as well as when caged with ears in wire-mesh sleeves. These beetles also consume the conidia and mycelium of A. flavus and subsequent observations have revealed that conidia can germinate after passage through the nitidulid gut (Wicklow et al., 1988). Since nitidulid beetles seem to play an important role as potential vectors of disease and also causes damage to maize, it is considered important for future research. If these beetles are adversely affected by exposure to Bt-toxin, it could have a positive knock- on effect in that the between-plant spread of A. flavus in maize could be reduced.

Notoxus spp. have been recorded on maize in South Africa, but seems to be rare and therefore considered to be a highly sporadic pest (Drinkwater, in press). No information exists on Carticaria japonica (Coleoptera: Lathridiidae) in any agro-ecosystem (Table 3.3).

3.4.2.1.3 Predators and Parasitoids The most important predators were Orius sp. (Hemiptera), earwigs (Forficulidae spp.), ladybird beetles (Coccinelidae) and rove beetles (Staphylinidae) (Table 3.4). These species are indirectly exposed to Bt-toxin as they in turn feed on various herbivores species that feed on Bt maize, for example Tetranychidae, Hemiptera nymphs and Lepidoptera and Coleoptera larvae. For this reason these species received high rankings.

Parasitoid species also received the highest possible ranking because of their important ecological function (Table 3.4). The Braconidae are parasitoids of economically important lepidopteran pests of maize, while, Scelionidae are egg parasitoids, especially on species of the family Acrididae (short-horned grasshoppers) (G. Prinsloo, pers comm., March 2011) Larval and pupal parasitoids fulfil an important ecological function in that they suppress stem borer numbers (Van den Berg et al., 1998; Birch et al., 2004)

Table 3.2 Selection matrix for prioritizing non-target detritivore species associated with maize.

Maximum potential exposure Family Occurrence Abundance Presence Linkage Rank Oppiidae Certain Low Anytime Weak 2 Entomobryidae Certain Low Anytime Weak 2

Table 3.3. Selection matrix for prioritizing non-target herbivore species associated with maize.

Maximum potential exposure Possible adverse effect Functional Family Genus Disease groups Occurrence Abundance Presence Linkage Significance Damage Rank vector Maize dwarf Aphididae Several genera Certain Medium Anytime Weak Low Sometimes 1 mozaic virus Sucking Delphacidae Peregrinus maidis Certain Medium Anytime Weak Low None 3 herbivores Thripidae Chirothrips sp. 1 Certain Low Anytime Weak None None 3 Phlaeothripidae Haplothrips sp. 2 Certain Low Anytime Weak None None 3 Tetranychidae Tetranychidae Certain Low Anytime Weak Low Sometimes 1 sp. 1

Aspergillus Nitidulidae Carpophilus sp. 1 Certain High Reproductive Weak Low Seldom 1 flavus Aspergillus Nitidulidae Carpophilus sp. 3 Certain Medium Reproductive Weak Low Seldom 1 flavus Chewing Nitidulidae larva Carpophilus sp.2 Certain Low Reproductive Weak Low None 1 herbivores Notoxus Anthicidae Occasional Low Anytime Weak None Seldom 3 monoceros Carticaria Lathridiidae Occasional Low Reproductive Weak None None 3 japonica

Table 3.4. Selection matrix for prioritizing non-target predator and parasitoid species associated with maize.

Maximum potential exposure Functional Rank Family Genus Occurrence Abundance Presence Linkage groups Anthocoridae Orius sp. 1 Certain Medium Anytime Weak 1

Berytidae Metacantus sp 1. Certain Low Anytime Weak 3 Sucking predators Miridae Miridae sp. 5 Certain Low Anytime Weak 2

Phytoseiidae Phytoseiidae sp. 1 Certain Low Anytime Weak 3

Formicidae Formicidae sp. 1 Occasional Medium Anytime Weak 3 Foficulidae larva Foficulidae Certain Medium Anytime Weak 1 sp. 1

Forficulidae Forficulidae sp. 1 Certain Low Anytime Weak 1 Chewing predators Formicidae Formicidae sp. 2 Occasional Low Anytime Weak 3

Coccinelidae Scymnus nubilus Certain Low Anytime Weak 1 Coccinelidae Cheilomenes sp.1 Certain Low Anytime Weak 1 Staphylinidae Oxytelus sp. 1 Certain Low Anytime Weak 1

Braconidae Braconidae sp. 1 Occasional Low Anytime Weak 1 Parasitoids Scelionidae Scelionidae sp. 4 Occasional Low Anytime Weak 1 Scelionidae Scelionidae sp. 7 Occasional Low Anytime Weak 1

3.4.3 Step 3: Analysis of exposure pathways

This step analyses possible causal pathways of exposure to the GM plant, toxin and potential impacts of GM plants for high ranking species or ecological functions identified during the previous prioritization step. The purpose of this evaluation is to differentiate candidate test species likely and unlikely to be exposed to the Bt toxin, as well as to guide the design of the exposure system to be used in future test protocols (Birch et al., 2004).

Potential likely exposure to Bt toxin can occur through many pathways (Birch et al., 2004). It has been estimated that there are over 250 different exposure pathways by which a transgene

93 product or its metabolites could affect a secondary consumer, of which only a few are direct effects of the transgene product (Andow & Hilbeck, 2004). Non-target species could therefore be affected by transgene products in the plant, plant secretions, herbivores, herbivore excretions or species containing transgene products. Metabolites of the transgene products or interaction effects of these products with other plant or herbivore compounds that alter plant, herbivore composition or physiology, might also affect non-target species (Saxena & Stotzky, 2001; Birch et al., 2002).

Exposure pathways and eventual concentration of Bt protein inside herbivores or beneficial organisms may be influenced by many factors. Herbivore exposure to Bt protein depends on mode of feeding, site and time of toxin expression, amount of plant material ingested and the rate of proteolytic degradation and excretion. Exact levels of exposure are, however, too complex to calculate (Meissle & Romeis, 2010). The major route of exposure for many predators in Bt crops is through the prey that they consume (Romeis et al., 2009).

A study conducted under European field conditions provided a comprehensive data set of the temporal distribution of Cry1Ab in transgenic maize (MON810). Cry1Ab expression was the lowest in pollen, very low in the stalks, low in roots and highest in the leaves. Levels also varied in different plant tissues at different growth stages (Nguyen & Jehle, 2007). Székács et al. (2010) showed that the Cry1Ab in MON810 was the highest in the leaves followed by the roots, stem, anther wall, pollen and grain.

Several studies have been done on selected arthropod species to detect the Cry protein concentration in these species. No Bt protein was detected in aphids that feed on plants before anthesis, but low concentrations were detected in Rhopalosiphum padi (Homoptera: Aphididae) collected after flowering (Meissle & Romeis, 2009). Rhopalosiphum padi was probably contaminated with pollen, plant remains or arthropod faeces, because no Cry3Bb1 was detected in R. padi in a climate chamber experiment under clean conditions with the same maize variety. Aphids feed on the phloem sap of plants (Douglas, 2003) which has been shown to contain no or very small amounts of Bt protein of Cry1Ab-expressing Bt maize (Raps et al., 2001), with the consequence that aphids do not ingest the Bt protein (Head et al., 2001; Raps et al., 2001; Dutton et al., 2002, 2004).

Leaf-hoppers, Psammotettix alienus (Hemiptera: Cicadellidae), feeding on phloem sap, have been reported to ingest no or very little Cry3Bb1 protein. In contrast, the leaf-hoppers Empoasca pteridis, Eupteryx atropunctata and Zyginidia scutellaris (Hemiptera: Cicadellidae) which feed on mesophyll cells (Nickel, 2003) do ingest the Bt protein. This has also been shown for Z. scutellaris feeding on Cry1Ab-expressing maize (Dutton et al., 2004; Obrist et al., 2006).

Mesophyll-feeding thrips, some mirid bugs and tissue feeding chrysomelid beetles have been reported to contain considerably higher concentration of Cry3Bb1 protein, almost reaching the level detected inside maize tissue. It has been shown that Cry1Ab protein levels in herbivores can reach the concentrations at which they are present in maize leaves (Obrist et al., 2006). In another study of Bt maize in the USA, mesophyll-feeding herbivores were reported to be scarce, but the chrysomelid beetles present contained large amounts of Cry1Ab protein (Harwood et al., 2005). The latter authors also indicated that predators such as thrips (Aeolothrips spp.), bugs (Orius spp. and Nabis spp.), beetles (Cantharis lateralis and Coccinellidae), fly larvae (Syrphidae) and lacewing larvae (Chrysoperla spp.) generally contained less Cry3Bb1 than the herbivores feeding directly on maize. Bt protein concentrations are often one order of magnitude lower in predators than in their prey, because the prey’s gut, in which the Bt protein is located, represents only part of the consumable prey and because some Bt protein is digested and excreted (Romeis et al., 2009).

In addition to prey, many predators also feed on maize pollen (Romeis et al., 2009). This can explain why Orius spp., Chrysoperla spp. and coccinellid beetles contained relatively high Cry3Bb1 concentrations during anthesis relative to before or after pollen shed (Meissle & Romeis, 2009). Similar observations were made in Cry1Ab-expressing maize in Spain (Obrist et al., 2006). Harwood et al. (2005) also reported considerable amounts of Cry1Ab in Orius insidiosus (Hemiptera: Anthocoridae) and in some coccinellid beetles collected in Bt maize fields, but it remains unclear whether pollen-feeding played a role.

Environmental exposure concentrations may occasionally approach the expression level present inside Bt maize tissue, but the average exposure level is usually considerably lower (Meissle & Romeis, 2009). For this reason, Raybould et al. (2007) suggested that the Bt protein concentration in plant tissue divided by five should be used as an estimated realistic environmental exposure concentration for generalist predators in risk assessment.

3.4.3.1 Herbivores Differential exposure of herbivores depends on mode of feeding, i.e. sucking or chewing. Aphids are one of the most common phytophagous insects on maize crops (Dicke & Guthrie, 1988). Aphids feed on any part of the maize plant and spend their entire life cycle on maize, which leads to continuous exposure. However, aphids are not affected by Bt toxin as it is not transported in the phloem sap on which aphids feed (Head et al., 2001; Raps et al., 2001; Dutton et al., 2002). Although expression of Cry1Ab protein in the maize plant phloem is probably unlikely, it should be verified in each transgenic Bt crop event in case new events express toxin in this tissue, intentionally or unintentionally (Birch et al., 2004).

Tetranychidae mites, which have pierce-sucking mouthparts, ingest cell contents of mainly maize leaves and husk leaves covering the ears, and therefore ingests Bt toxin directly. Tetranychidae spend their entire life cycle on maize, which leads to continuous exposure of these organisms to Cry-toxins.

Sap-feeding arthropods such as Carpophilus spp. beetles and larvae, which have chewing mouthparts, feed primarily on Bt maize kernels and plant sap on tips of maize ears, but also on the frass of lepidopteran herbivores such as Helicoverpa armigera (Lepidoptera: Noctuidae) that survive on Bt maize (Birch et al., 2004). Although Birch et al. (2004) reported that Carpophilus spp. feed on frass of H. armigera, they will also be exposed to frass of stem borer species that survive on Bt maize in South Africa (Van Wyk et al., 2007b). Development of resistance of target pests of Bt maize, opens new exposure pathways to Cry-toxins since higher trophic level organisms may depend on, or utilize by-products of resistant target pests. Interesting interactions may develop in Bt maize production regions where target pests develop resistance to Bt proteins. Although several cases of resistance have been reported in the world, the most significant and wide spread so far is that of B. fusca (Van Rensburg, 2007; Kruger et al., 2011). It has been reported that the frass of a non-target lepidopteran pest, Spodoptera littoralis (Lepidoptera: Noctuidae) contained high concentrations of Bt toxin when fed on Bt maize (Raps et al., 2001). It is therefore assumed that Nitidulidae beetles may be exposed to high toxin concentrations via the frass of these lepidopteran herbivores (Birch et al., 2004). Personal observations indicated that Carpophilus spp. adults and larvae do indeed feed on maize kernels and stem sap.

3.4.3.2 Predators and Parasitoids Natural enemies can be exposed to the effects of Bt toxin through multiple pathways. It is important to consider not only the primary tritrophic exposure route through the prey or host but also bi-trophic exposure through direct feeding on plant tissues or feeding on herbivore excretions such as honeydew or frass (Birch et al., 2004). Orius sp., Forficulidae, Coccinelidae and Staphylinidae are indirectly affected as they feed on herbivore species that feed on Bt maize. Bt-resistant B. fusca larvae that feed on Bt maize and ingest Bt toxin are very likely to result in exposure of Braconidae at the tritrophic level (Birch et al., 2004). It has not yet been determined whether the ingested Bt toxin is passed on to the lepidopteran adult stage and subsequently to the eggs that the lepidopteran adults produce (Birch et al., 2004). This would constitute the most likely tritrophic route through which Scelionidae species could be exposed. Further research on this exposure pathway should be conducted.

3.4.4 Step 4: Describing hazard scenarios and formulating testing hypotheses

Hazards relate to the effects of the Bt toxin, its metabolites or any combination of effects with the plant secondary metabolites on the life history and fitness parameters of non-target organisms. These parameters include development time, survival, fecundity and fertility. Behavioural parameters, such as host plant preference and oviposition are also of great ecological importance (Birch et al., 2004). This step is of particular importance when exposure to Bt toxin is unlikely, yet, indirect effects are still possible. Such indirect effects can arise through changes in the population dynamics of a non-target organism and/or through a shift in the species composition of a particular community assemblage (Birch et al., 2004). Andow and Hilbeck (2004) suggested that of the species with unknown ecological function, those with a high standing biomass or those that are found in frequent association with the transgenic crop habitat should also be selected for testing. By explicitly considering such species for initial non- target testing, a scientifically justified precautionary approach is introduced into risk assessment. Those species that are given the highest priority should be candidates for testing. In this study a number of species other than those listed in Table 3.1, occurred at a high standing biomass or in frequent association with the maize crop (Chapter 2). These species could, however, be ranked higher in the future if their numbers on plants change or when the value of their contribution to specific ecological functions are realised.

3.4.4.1 Herbivores On the basis of standing biomass of herbivores with pierce-sucking feeding strategies, two non- target herbivore species complexes, aphids and Tetranychidae (red spider mites) could be recommended in a pre-release non-target effects testing programme on Bt maize in South Africa (Table 3.3). The hazard associated with these species is that they might become secondary pests of maize (Birch et al., 2004). Outbreaks of aphids and mites would lead to increased use of broad-spectrum chemical insecticides on maize crops, which could cause a reduction of natural enemy populations and therefore less natural biological control (Sujii et al., 2006).

3.4.4.2. Predators and parasitoids Six predator and three parasitoid species are ranked highly and are therefore recommended in a pre-release effects testing programme on Bt maize in South Africa (Table 3.4). All the selected predators feed on herbivores that are also ranked highest and on lepidopteran larvae and eggs and could therefore be tri-trophically exposed.

3.4.5 Step 5: Developing ecologically meaningful testing methods and protocols

The final step of the ERA is to develop appropriate methodologies and protocols to assess risk. Such methods can be designed once it is evident which life stage of a non-target organism is likely to be exposed and through what route it is likely to receive the genetically modified product, directly and/or mediated indirectly through other non-target prey (Birch et al., 2004). Surrogate species for species identified through the ERA in the receiving environment can be used for testing and assessment of risk. These species are chosen because of their supposed sensitivity to chemical toxins, their wide availability, their ease of culture, and their genetic uniformity (Chapman, 2002). Such species are supposed to provide information on the likely effects of the chemical on a wider range of species (Kitano, 1992; Elmegaard & Jagers op Akkerhuis, 2000).

Research methods and experiments have already been described in the literature for most of the potential hazards that have been identified. Examples of these experiments are briefly discussed below:

3.4.5.1 Herbivores

3.4.5.1.1 Aphids It has been concluded by several authors that aphids are not affected by Bt toxin inside plants, as it is not transported in the phloem sap on which aphids feed (Head et al., 2001; Raps et al., 2001; Dutton et al., 2002) or the aphids may break down the Cry1Ab protein after ingestion (Head et al., 2001). Aphids are important prey for many predators living on maize which can be indirectly affected by feeding on contaminated prey (Lumbierres et al., 2004). Although aphids were ranked highly in this study and should therefore be evaluated, this may not be necessary because conclusive results have been reported in other studies.

Two independent experiments were performed by Lumbierres et al. (2004), in which aphid density was evaluated by visual counting and the performance studied of the offspring of alate forms and the offspring of different generations of apterous forms of R. padi fed with Bt maize. Lumbierres et al. (2004) observed that a higher density of aphids, particularly alates and young nymphs, occurred in Bt plots at very young maize development stages, corresponding to the settlement period. After this period, there were no differences between Bt and non-Bt maize. The developmental and pre-reproductive times of the offspring of the first generation of alataes were shorter and the intrinsic rate of natural increase higher, when aphids fed on Bt maize. The opposite occurred with the offspring of the first generation of apterous mothers, which had lower nymphal and adult mortality, shorter developmental and pre-reproductive times, a higher effective fecundity rate, and greater intrinsic rate of natural increase, when fed on non-Bt maize. The differences in aphid development on the two cultivars may be linked to changes in hostplant quality due to pleiotropic effects of the genetic modification. No differences on aphid mortality, developmental and pre-reproductive times, fecundity, and intrinsic rate of natural increase were found between the offspring of apterous aphids maintained on Bt or non-Bt maize for several generations (Lumbierres et al., 2004).

A study on performance of cotton aphids on three Indian Bt cotton varieties was also done by Lawo et al. (2009), which concluded that Bt cotton poses a negligible risk for aphid antagonists and that aphids would probably remain under natural control in Bt cotton fields (Lawo et al., 2009).

3.4.5.1.2 Nitidulidae A lack of research on Bt adverse effects on Nitidulidae beetles exists, therefore further research is necessary. These insects are a particular problem because larvae may be found hidden in kernels throughout the ear (Connell, 1956; Harrison 1960).

A study by Dowd (2000) determined the incidence of sap beetles between Bt and non-Bt maize over two seasons. The incidence of nitidulid beetle adults was higher, and larvae, lower for the Bt versus non-Bt maize in 1999. The incidence of ears with more than five kernels damaged by sap beetles was higher for the Bt compared with non-Bt maize in one season. Ears with husks flush with the ear tip or with ear tips exposed had significantly higher sap beetle damage for both hybrids, and the Bt maize had signifcantly higher incidence of exposed ear tips in both years (Dowd, 2000).

3.4.5.1.3 Tetranychidae A worst-case exposure study by Li and Romeis (2010) in which Stethorus punctillum (Coleoptera: Coccinellidae) were fed Tetranychus urticae (Acari: Tetranychidae) individuals reared either on Bt maize (insecticidal protein Cry3Bb1) or non-Bt maize plants, showed that the developmental and reproduction life-history parameters were not affected by Bt maize.

A study conducted over three generations showed that the Bt cotton did not affect the development or survival of immature stages and reproductive outputs of either T. urticae or the predator, Phytoseiulus macropilis (Acari: Phytoseiidae). The preference for feeding and oviposition for both mites were similar on both cotton types (Esteves et al., 2010), indicating that Bt-toxin did not affect mites. This indicates that further experiments in which the effects of Bt- toxin on mites may not be a priority on Bt maize and cotton in South Africa.

3.4.5.2 Predators

3.4.5.2.1 Anthocoridae In a laboratory study to determine the effects of Bt-toxin on O. insidiosus nymphs, feeding on one-day old Ostrina nubilalis (Lepidoptera: Crambidae) larvae that ingested Bt toxins, no adverse effects on this predatory species was observed. Body weight, body length and number of days to adulthood of O. insidiosus were not affected significantly (Al-Deeb et al., 2001).

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In a study to determine the acute toxic effect of Bt maize pollen (CrylAb protein) on O. insidiosus and other predators no acute detrimental effects on pre-imaginal development and survival of O. insidiosus were observed (Pilcher et al., 1997). Due to these results and several other studies done on predator species (Pilcher et al., 1997; Zhang et al., 2006a) it is not a priority to conduct further research on the effects of Cry1Ab-toxin on Orius spp. in South Africa.

3.4.5.2.2 Coccinellidae The effect of Bt toxin on predatory coccinelid beetles have been addressed in several studies. The effects of Bt toxin on Propylaea japonica (Coleoptera: Coccinellidae) were examined after being fed Bt-treated Bt-resistant cotton bollworm (H. armigera) during the third and fourth instars (Zhang et al., 2006a). After ingesting Bt-treated Bt-resistant H. armigera larvae, the body mass and body length of adult P. japonica decreased. These observations were ascribed to both a combined effect of poor prey quality and Cry1Ac Bt-toxin, however, larval survival and development, pupal mortality, fecundity and adult longevity of P. japonica were not affected. These results suggested that preying on individuals that consumed Cry1Ac-toxin might have no significant effect on the fitness of the predatory beetle, P. japonica (Zhang et al., 2006a).

However, a study in which P. japonica was fed two different Bt cotton treated (expressing the fused Cr1Ab/Ac and Cry1Ac toxin respectively) Spodoptera litura (Lepidoptera: Noctuidae) larvae showed that P. japonica fed with Cry1Ac S. litura larvae suffered increased larval mortality and decreased body mass. This result was ascribed to a combined interaction of poor- prey quality and presence of Cry1Ac toxin. Results suggested that the Cr1Ab/Ac fusion toxin had no direct effect on P. japonica. The reasons for these observations were, however, not clear since it was ascribed to higher Cry1Ac protein levels compared to the Cr1Ab/Ac fusion protein, or to the plant background of both Bt cotton cultivars (Zhang et al., 2006b).

Studies by Álvarez-Alfageme et al. (2010) to determine effects of Cry1Ab and Cry3Bb1 on larvae of Adalia bipunctata (Coleoptera: Coccinellidae), using T. urticae as carriers, showed no adverse effects on the predator. Ingestion of the two Cry proteins by A. bipunctata did not affect its larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately ten times higher than measured in Bt maize- fed spider mites. It was therefore concluded that larvae of A. bipunctata were not sensitive to

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Cry1Ab and Cry3Bb1 and that Bt maize expressing these proteins would not adversely affect this predator (Álvarez-Alfageme et al., 2010).

A study by Li and Romeis (2010) in which S. punctillum were fed T. urticae reared either on Bt maize (insecticidal protein Cry3Bb1) or the non-Bt maize plants, indicated that larval survival, development and adult survival, as well as adult dry weight was not affected in this predator. However, female beetles that were fed T. urticae from Bt maize had a shorter pre-oviposition period, increased fecundity, and increased fertility relative to females fed spider mites from non- Bt maize. The reasons for these effects is unclear but were ascribed to possible unidentified differences in plant characteristics. Detrimental effects on this predator when preying in Cry3Bb1-expressing Bt maize fields are unlikely in the field (Li & Romeis, 2010).

Several studies in which ladybird beetles were exposed to Bt plant products or Bt exposed herbivores indicated no adverse effects. Studies in which ladybird beetles were fed Cry1Ab- or Cry3Bb1-expressing maize pollen or Bt maize-fed herbivores have revealed no negative effect on Coleomegilla maculata (Coleoptera: Coccinellidae) (Pilcher et al., 1997; Lundgren & Wiedenmann, 2002; Ahmad et al., 2006) or S. punctillum (Álvarez-Alfageme et al., 2008). Results indicated no negative effects on different life-history parameters of these species and further research with these specific cry-toxins is not a priority in South Africa.

3.4.5.2.3 Staphylinidae and Forficulidae The Staphylinidae is one of the largest beetle families with more than 47 000 species and is distributed worldwide in almost all types of ecosystems (Bohac, 1999; Markgraf & Basedow, 2002). However, a lack of information on species ecology and prey preferences exists because of taxonomic constraints (Balog et al., 2008a,b,c; Balog & Markó, 2008).

Staphylinidae beetles are rarely tested under laboratory conditions (with Cry proteins) because of difficulties in rearing and maintenance of beetle colonies (Stacey et al., 2006; Raybould et al., 2007). Although exposure of these predatory beetles to Cry proteins under field conditions is likely to be low, the additive and unforeseen effects of the transgene can only be assessed in field trials (Balog et al., 2010). Further research on the adverse effect of Bt on Staphylinidae is therefore necessary.

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The only study done specifically to assess the effect of Cry1Ab toxin on Staphylinidae was by Balog et al. (2010). No difference in the activity–density of these beetles in Bt maize (MON810) and near isogenic maize, as well as the effect of the prey R. padi on Staphylinidae beetle populations were observed (Balog et al., 2010).

A similar knowledge gap exists for Forficulidae species, as no studies have been conducted of the possible effects of Bt toxin on these species. Further research is necessary on the possible effects of Bt-proteins on Forficulidae and Staphylinidae in South Africa.

3.4.5.3 Parasitoids

3.4.5.3.1 Braconidae and Scelionidae It is important to aid the conservation of hymenopteran parasitoids, as these parasitoids are among the most successful biological control agents of pests (Van Driesche & Bellows, 1996). Many synthetic pesticides have reduced the numbers of natural enemies in crop fields and it has become evident that sustainable agricultural production methods need to conserve natural enemies (Birch et al., 2004). Parasitoids usually complete their development on a single host individual and are likely to be adversely affected if their Bt-susceptible hosts are affected with Bt toxin and are weakened or killed (Salama & Zaki, 1983), a phenomenon referred to as host- quality mediated effects (Chen et al., 2008).

A study by Baur and Boethel (2003), determined whether Bt protein (Cry1Ac) produced by cotton plants (Event 531) could affect survival and fecundity of two hymenopteran parasitoids, Cotesia marginiventris (Hymenoptera: Braconidae) and Copidosoma floridanum (Hymenoptera: Encyrtidae), using a Bt-exposed host, the soybean looper, Pseudoplusia includens (Lepidoptera: Noctuidae). Cotesia marginiventris is a solitary larval endoparasitoid that commonly attacks early instars (Daigle et al., 1990), where C. floridanum is a poly-embryonic egg-prepupal parasitoid (Burleigh, 1971; Daigle et al., 1990). It was concluded that reduced fecundity may be expected from C. marginiventris or C. floridanum developing in P. includens fed on Bt-cotton. Results indicated that immature stages of parasitoids required more time to fully develop, thus potentially exposing host and parasitoid to increased predation. However, some evidence indicated that predator numbers may be lower in Bt cotton fields compared to non-Bt cotton because of reduced host density (Hagerty et al., 2000), and therefore the effect of increased development time may not lead to increased predation (Baur & Boethel, 2003).

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In a study on the comparative susceptibility of S. litura and H. armigera and two of their larval parasitoids, Cotesia kazak and Meteorus pulchricornis (Hymenoptera: Braconidae) to Bt sprays and Cry1Ac-containing artificial diet, no significant effects were observed. Survival of M. pulchricornis was unaffected even when the survival of S. litura larvae was significantly reduced. In H. armigera, Bt-containing artificial diets resulting in severely reduced survival of the host were accompanied by reductions in survival of M. pulchricornis. Similar or increased levels of survival of C. kazak were noted in H. armigera that were fed less toxic Bt-amended diets. Poor survival of parasitoids on reduced-nutrition diets suggested that reduced nutrition had a comparatively greater effect on parasitoid survival than Cry1Ac toxins. Overall, when Bt was ingested at concentrations that had minor effects on development or survival of host larvae, there was no impact on either parasitoid species (Walker et al., 2007).

A meta-analysis by Wolfenbarger et al. (2008) corroborates the strong negative effect of Bt maize on specialist parasitoids reported in the literature (Pilcher et al., 2005). However, a closer examination suggests that most of the studies on parasitoids in GM cropping systems focuses on the abundance of Macrocentrus grandii (Hymenoptera: Braconidae), which specializes on the target pest. From the limited number of studies on other parasitoids, there was no detectable effect on parasitoids. However, more studies will be needed to resolve whether there is a general effect on parasitoids in all receiving environments. Choosing other species that account for larger parasitism rates in agricultural systems will improve our understanding of how Bt crops affect parasitoids and whether any effects are due to the direct effect of Bt toxin or to poor prey quality due to feeding on Bt toxin (Wolfenbarger et al., 2008).

A study by Chen et al. (2008), was the first to compare and to confirm that the negative impact of Bt plants on parasitoids was due to the poor host-quality because of ingestion and susceptibility to Bt toxin, rather than the direct toxicity to the parasitoid. As only a few studies have come to this conclusion, it is necessary that further research should be done in South Africa.

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3.5 Conclusions

This chapter described the use of the ecological model to identify priority species and non-target species which could be affected by Bt maize in South Africa. The selection matrix together with the information on species mentioned above can be used to decide which species to use as test species for the future and which could possibly be eliminated.

Research gaps were identified and indicated lack of information particularly for the predatory groups, Forficulidae and Staphylinidae. The high abundance of Nitidulidae adults and larvae also necessitates further study. There is a need to evaluate possible non-target species for risks for each new GM crop or GM event that is developed.

Bt crops are one tool in the integrated pest management toolbox and their comparative environmental impact on non-target organisms will depend on how these tools are integrated and applied within agricultural production systems (Wolfenbarger et al., 2008).

The importance of soil organisms in the maize ecosystem has not been addressed in this study. Many species of herbivores and detritivores, as well as predators, occur in the soil. Different collection methods should be used for sampling soil organisms and a risk assessment on soil organisms is needed for future research. As studies on agro-ecosystems and biodiversity surveys are done, priorities assigned to species in this study may change. It will therefore be necessary to re-evaluate research priorities in future.

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CHAPTER 4

CONCLUSIONS

4.1 Discussion and conclusions

The advent of genetically modified (GM) maize with insecticidal properties and the wide-scale planting thereof all over the world necessitates assessment of the risk that this technology may hold for the environment. In order to do risk assessments and study the possible effects of Bt maize on the environment, knowledge is needed on species which occur in these systems.

Prior to this study hardly any knowledge existed with regard to arthropod diversity and food webs in maize in South African environments. Knowledge was limited to that of economically important pests (Annecke & Moran, 1982), Lepidoptera diversity (Van Wyk et al., 2007) and the well studied complex of Hymenoptera parasitoids of the stem borer complex (Kfir et al., 2002). Information on biodiversity is critical in evaluation of the possible impact of Bt maize on non- target organisms at different trophic levels, as well as the possible causal pathways of exposure to the genetically modified plant and toxin. In order to do reliable risk assessments regarding GM crops, information is needed on biodiversity and the spectrum of possible non-target species in maize based ecosystems. The biodiversity of an agro-ecosystem is not only important for its intrinsic value, but also because it influences ecological functions that are vital for sustainable crop production, as well as the surrounding environment (Hilbeck et al., 2006).

Results from field studies indicated no significant effects on the non-target organisms, whereas some others recorded negative effects on the natural enemies. While most field/laboratory studies assessing potential impacts of Bt crops on biodiversity have focused on a limited number of species (Wilson et al., 1992; Hardee & Bryan, 1997; Wold et al., 2001; Liu et al., 2003; Schoenly et al., 2003) it is important to study effects on arthropod communities as well. Arthropod diversity in agro-ecosystems has been studied in China on Bt rice (Li et al., 2007), in the USA on Bt cotton (Torres & Ruberson, 2007) and in Spain on Bt maize (De la Poza et al., 2005). These studies concluded that abundance and diversity of arthropods were not affected by the growing of transgenic Bt crops compared to non-Bt crops.

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The aims of this study were to compile a checklist of arthropods that occur on maize in South Africa and to determine and compare the diversity and abundance of arthropods and functional groups on Bt and non-Bt maize. An ecological risk assessment model approach was used to identify arthropod species from different functional groups that were most likely to be exposed to Bt protein inside the maize ecosystem, and to prioritise species for further research.

This study provided a basis for the future study of biodiversity of arthropods in maize in South Africa and generated a basic checklist of arthropod species. This study can be used in the future assessment of the possible impact of Bt maize on the environment. Our findings suggest that Bt maize has no adverse effect on total arthropod diversity or on the different functional groups. Results and species identifications done by means of the selection matrix, used in this study, together with analyses of other published information on these species can be used in future to decide which species to use as test species. This process will also indicate which species can be disregarded for future research since the availability of sufficient, reliable and relevant information of the effect of Cry1Ab maize on that particular species may indicate no adverse effects. Species that can be disregarded are Aphididae, Coccinellidae and Anthocoridae. Research gaps were identified and indicated a lack of information particularly for certain predatory groups (i.e. Forficulidae and Staphylinidae) parasitoids, as well as for the herbivore beetles, Nitidulidae. Future studies should investigate specific aspects regarding the effect of Bt-toxin on life history and fitness of larvae and adults of these groups. The effect of the Cry1Ab protein on parasitoids should be confirmed whether it is due to poor host-quality or the direct toxicity to the parasitoid. Parasitoids in all receiving environments should be studied.

The importance of soil organisms in the maize ecosystem has not been addressed in this study, but is essential for future research. Many species of herbivores and detritivores as well as predators occur in the soil and serve as key indicator species of soil fertility and health, as they play a vital role in the breakdown and recycling of crop residues (O’Callaghan et al., 2005). This study only investigated arthropod abundance and diversity during the reproductive stage of plant development. Future research should address all stages from seedling up to harvest. To gain a better understanding of arthropod biodiversity on maize, sampling should be done at more localities and more maize plants should be sampled.

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Bt crops are only one tool in the integrated pest management toolbox and their comparative environmental impact on non-target organisms will depend on how these tools are integrated and applied within agricultural production systems (Wolfenbarger et al., 2008).

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4.2 References

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Van Wyk, A., Van den Berg, J & Van Hamburg, H. 2007. Diversity and comparative phenology of Lepidoptera on Bt- and non-Bt maize in South Africa. International Journal of Pest Management 54: 77-87.

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Appendix A

List of arthropod species and their abundance on maize plants

Legend

Functional groups: SH = Sucking herbivores CH = Chewing herbivores SP = Sucking predators CP = Chewing predators P = Predators D = Detritivores

? = Identifications were not done because it consists of unknown species.

?* = Because identifications were not done to species-level, it was not possible to distinguish between predatory and herbivorous species.

- = Species was not included in functional groups.

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List of arthropod species and their abundance sampled during this study on Bt and non-Bt maize plants in South Africa. Functional Mean / Order/Family Species name Total groups plant Hemiptera Pentatomidae Gynenica marginella SH 2 0.00 Veterna sp. 2 SH 1 0.00

Nezara sp. 3 SH 4 0.01

Anthocoridae Orius sp. 1 SP 401 0.84 Hemiptera nymph Nymph sp.1 SH 237 0.49 Nymph sp. 2 SH 1 0.00

Cicadellidae Cicadellidae sp. 1 SH 41 0.09 Cicadellidae nymph sp. 1 SH 14 0.03

Miridae Miridae sp. 1 SH/SP 8 0.02 Miridae sp. 2 SH/SP 20 0.04

Miridae sp. 3 SH/SP 2 0.00

Miridae sp. 4 SH/SP 1 0.00

Miridae sp. 5 SH/SP 71 0.15

Miridae sp. 6 SH/SP 2 0.00

Miridae sp. 7 SH/SP 2 0.00

Lygaeidae Geocorus sp. 1 SP 5 0.01 Lygaeidae sp. 2 SH 17 0.04

Lygaeidae sp. 3 SH 2 0.00

Lygaeidae sp. 4 SH 7 0.01

Lygaeidae sp. 5 SH 34 0.07

Delphacidae Peregrinus maidis SH 822 1.71 Corixidae Corixidae sp. 1 - 6 0.01 Berytidae Metacanthus sp. 1 SH 78 0.16 Berytidae sp. 2 SH/SP 1 0.00

Tingidae Tingidae sp. 1 SH 1 0.00 Reduviidae Reduviidae sp. 1 SP 1 0.00 Psyllidae Psyllidae sp. 1 SH 1 0.00 Coleoptera Coleoptera larva Larva sp. 1 SH 2 0.00 Larva sp. 2 SH 11 0.02

Anthicidae Formicomus caeruleus D 20 0.04 Notoxus monoceros CH 167 0.35

Scydmaenidae Scydmaenidae sp. 1 CH 6 0.01 Lathridiidae Carticaria japonica CH 147 0.31 Chrysomelidae Monolepta bioculata CH 5 0.01 Chrysomelidae sp. 2 CH 6 0.01

Chrysomelidae sp. 3 CH 28 0.06

Chrysomelidae sp. 4 CH 6 0.01

Chrysomelidae sp. 5 CH 7 0.01

Chrysomelidae sp. 6 CH 2 0.00

Chrysomelidae sp. 7 CH 1 0.00

Chrysomelidae sp. 8 CH 1 0.00

Chrysomelidae sp. 9 CH 1 0.00

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Functional Mean / Order/Family Species name groups Total plant Coleoptera (cont.) Chrysomelidae sp. 10 CH 17 0.04

Chrysomelidae sp. 11 CH 1 0.00

Chrysomelidae sp. 12 CH 1 0.00

Chrysomelidae sp. 13 CH 3 0.01

Chrysomelidae sp. 14 CH 1 0.00

Chrysomelidae sp. 16 CH 27 0.06

Chrysomelidae sp. 17 CH 15 0.03

Chrysomelidae sp. 18 CH 8 0.02

Chrysomelidae sp. 19 CH 1 0.00

Hispinae larva sp. 20 SH 25 0.05

Chrysomelidae larva sp. 1 CH 5 0.01

Chrysomeloidea Chrysomeloidea sp. 1 SH 15 0.03 Chrysomeloidea sp. 2 SH 12 0.03

Coccinellidae Scymnus nubilus CP 71 0.15 Coccinellidae sp. 2 CP 20 0.04

Cheilomenes sp. 3 CP 54 0.11

Coccinellidae sp. 4 CP 2 0.00

Epilachna sp. 5 CH 1 0.00

Coccinellidae sp. 6 CP 39 0.08

Coccinellidae sp. 7 CP 21 0.04

Coccinellidae sp. 8 CP 4 0.01

Coccinelidae sp. 9 CP 1 0.00

Coccinellidae sp. 10 CP 1 0.00

Coccinellidae larvae sp. 1 CP 36 0.08

Bruchidae Bruchidae sp. 1 SH 26 0.05 Nitidulidae Carpophilus sp. 1 CH 514 1.07 Carpophilus sp. 2 CH 83 0.17

Carpophilus sp. 3 CH 352 0.73

Carpophilus larva sp. 1 CH 15 0.03

Carpophilus larva sp. 2 CH 169 0.35

Melyridae Astylus atromaculatus SH 16 0.03 Curculionidae Sitophilus sp. 1 SH 11 0.02 Curculionidae sp. 2 SH 89 0.19

Curculionidae sp. 3 SH 2 0.00

Curculionidae sp. 4 SH 30 0.06

Curculionidae sp. 5 SH 1 0.00

Buprestidae Buprestidae sp. 1 SH 1 0.00 Elateridae Elateridae sp. 1 SH 2 0.00 Elateridae sp. 2 SH 1 0.00

Staphylinidae Oxytelus sp. 1 CP 53 0.11 Staphylinidae sp. 2 CP 1 0.00

Bostrichidae Bostrichidae sp. 1 CH 1 0.00 Bostrichidae sp. 2 CH 1 0.00

Silvanidae Silvanidae sp. 1 CP 1 0.00

123

Functional Mean / Order/Family Species name groups Total plant Coleoptera (cont.) Carabidae Carabidae sp. 1 CP 1 0.00 Carabidae sp. 2 CP 1 0.00

Apionidae Apionidae sp. 1 SH 3 0.01 Cucujidae Cucujidae sp. 1 CP 1 0.00 Tenebrionidae Tenebrionidae sp. 1 D 1 0.00 Unknown ? 18 0.04

Thysanoptera Thripidae Chirothrips sp. 1 SH 187 0.39 Phlaeothripidae Haplothrips gowdeyi SH 117 0.24 Haplothrips sp. 2 SH 174 0.36

Thysanoptera sp. 3 ?* 66 0.14

Thysanoptera sp. 4 ?* 1 0.00

Thysanoptera sp. 5 ?* 3 0.01

Thysanoptera sp. 6 ?* 15 0.03

Thysanoptera sp. 7 ?* 75 0.16

Thysanoptera sp. 8 ?* 55 0.11

Homoptera Aphididae Aphididae sp. 1 SH 1552 3.23 Aphididae sp. 2 SH 17 0.04

Aphididae sp. 3 SH 65 0.14

Aphididae sp. 4 SH 17 0.04

Aphididae sp. 5 SH 8 0.02

Aphididae sp. 6 SH 2 0.00

Aphididae sp. 7 SH 2 0.00

Pseudococcidae Pseudococcidae sp. 1 SH 1 0.00 Lepidoptera Crambidae Chilo partellus CH 13 0.03 Noctuidae Busseola fusca CH 47 0.10 Helicoverpa armigera CH 37 0.08

Sesamia calamistis CH 15 0.03

Leucania loreyi CH 5 0.01

Geometridae Geometridae sp. 1 CH 5 0.01 Lepidoptera larvae Larva sp. 1 CH 1 0.00 Larva sp. 2 CH 12 0.03

Larva sp. 3 CH 6 0.01

Larva sp. 4 CH 5 0.01

Busseola fusca pupa - 6 0.01

Helicoverpa armigera pupa - 1 0.00

Busseola fusca moth - 2 0.00

Micro-Lepidoptera Larva sp. 1 CH 2 0.00 Unknown ? 6 0.01

Hymenoptera Braconidae Braconidae sp. 1 P 36 0.08 Braconidae sp. 2 P 4 0.01

Cotesia sp. 3 P 4 0.01

124

Functional Mean / Order/Family Species name groups Total plant Hymenoptera (cont.) Formicidae Formicidae sp. 1 CP 316 0.66 Formicidae sp. 2 CP 133 0.28

Formicidae sp. 3 CP 16 0.03

Formicidae sp. 4 CP 5 0.01

Formicidae sp. 5 CP 2 0.00

Formicidae sp. 6 CP 6 0.01

Formicidae sp. 7 CP 2 0.00

Vespidae Vespidae sp. 1 CP 1 0.00 Vespidae sp. 2 CP 1 0.00

Apidae Apidae sp. 1 P 1 0.00 Ichneumonidae Ichneumonidae sp. 1 P 1 0.00 Ichneumonidae sp. 2 P 1 0.00

Eulophidae Eulophidae sp. 1 P 2 0.00 Scelionidae Scelionidae sp. 1 P 15 0.03 Scelionidae sp. 2 P 15 0.03

Ceraphronidae Ceraphronidae sp. 1 P 2 0.00 Hymenoptera Hymenoptera sp. 1 P 1 0.00 Hymenoptera sp. 2 P 7 0.01

Hymenoptera sp. 3 P 1 0.00

Hymenoptera sp. 4 P 2 0.00

Hymenoptera sp. 5 P 1 0.00

Hymenoptera sp. 6 P 11 0.02

Hymenoptera sp. 7 P 6 0.01

Hymenoptera sp. 8 P 2 0.00

Hymenoptera sp. 9 P 2 0.00

Hymenoptera sp. 10 P 2 0.00

Hymenoptera sp. 11 P 1 0.00

Hymenoptera sp. 12 P 2 0.00

Hymenoptera sp. 13 P 1 0.00

Diptera Diptera Diptera sp. 1 D 16 0.03 Diptera sp. 2 D 3 0.01

Diptera sp. 3 D 1 0.00

Diptera sp. 4 D 4 0.01

Diptera sp. 5 D 1 0.00

Diptera sp. 6 D 4 0.01

Diptera sp. 7 D 1 0.00

Diptera sp. 8 D 2 0.00

Diptera sp. 9 D 5 0.01

Diptera sp. 10 D 8 0.02

Diptera sp. 11 D 1 0.00

Diptera sp. 12 D 2 0.00

Diptera sp. 13 D 1 0.00

Diptera sp. 14 D 4 0.01

125

Functional Mean / Order/Family Species name groups Total plant Diptera (cont.) Diptera sp. 15 D 1 0.00

Diptera sp. 16 D 2 0.00

Diptera sp. 17 D 1 0.00

Diptera sp. 18 D 5 0.01

Diptera sp. 19 D 2 0.00

Diptera sp. 20 D 1 0.00

Diptera sp. 21 D 1 0.00

Diptera sp. 22 D 1 0.00

Syrphidae Syrphidae sp. 1 CP 5 0.01 Syrphidae sp. 2 CP 4 0.01

Chloropidae Anatrichus erinaceus D 2 0.00 Psychodidae Psychodidae sp. 1 D 2 0.00 Sciaridae Sciaridae sp. 1 D 19 0.04 Muscidae Antherigona sp. 1 D 2 0.00 Muscidae sp. 2 D 3 0.01

Muscidae sp. 3 D 1 0.00

Dolichopodidae Dolichopodidae sp. 1 CP 21 0.04 Culicidae Culicidae sp. 1 D/SH 6 0.01 Culicidae sp. 2 D/SH 1 0.00

Diptera larva Larva sp. 1 D/SH 55 0.11 Tabanidae Tabanidae larva sp. 1 D 2 0.00 Tabanidae pupa - 3 0.01

Stratiomyidae Stratiomyidae sp. 1 D/SH 2 0.00 Unknown ? 1 0.00

Orthoptera Acrididae Acrididae sp. 1 CH 1 0.00 Acrididae sp. 2 CH 1 0.00

Acrididae sp. 3 CH 1 0.00

Bradyporidae Acanthoplus armiventris CH 1 0.00 Gryllotalpidae Gryllotalpidae sp. 1 CP 1 0.00 Gryllotalpidae sp. 2 CP 1 0.00

Gryllidae Gryllidae sp. 1 CP 1 0.00 Tettigoniidae Tettigoniidae sp. 1 CH 4 0.01 Neuroptera Chrysopidae Chrysoperla larva sp. 1 SP 14 0.03 Chrysoperla adult sp. 1 CH 1 0.00

Hemerobiidae Hemerobiidae larva sp. 1 SP 4 0.01 Collembola Entomobryoidea Entomobryoidea sp. 1 D 151 0.31 Entomobryoidea sp. 2 D 1 0.00

Sminthuridae Sminthuridae sp. 1 D 1 0.00 Poduroidea Poduroidea sp. 1 D 14 0.03 Phthiraptera Thaumastocoridae Thaumastocoris peregrinus - 3 0.01 Phthiraptera Phthiraptera sp. 1 - 3 0.01

126

Functional Mean / Order/Family Species name groups Total plant Dermaptera Labiduridae Labiduridae sp. 1 CP 5 0.01 Forficulidae Forficulidae sp. 1 CP 164 0.34 Forficulidae larva sp. 1 CP 253 0.53

Psocoptera Psocoptera sp. 1 D 31 0.06

Crustacea Crustacea sp. 1 - 6 0.01

Blattodea Blatellidae Blatellidae sp.1 D 2 0.00 Blattodea Blattodea sp. 1 D 1 0.00 Blattodea sp. 2 D 5 0.01

Blattodea sp. 3 D 3 0.01

Blattodea sp. 4 D 1 0.00

Araneae Salticidae Thyene sp. 1 CP 2 0.00 Salticidae Heliophanus debilis CP 1 0.00 Salticidae Enoplognatha sp. 3 CP 10 0.02 Clubionidae Clubiona sp. 4 CP 21 0.04 Theridiidae Enoplognatha sp. 5 CP 10 0.02 Theridiidae Theridion sp. 6 CP 3 0.01 Linyphiidae Meioneta sp. 7 CP 6 0.01 Oonopidae Gamasomorpha sp. 8 CP 2 0.00 Sparassidae Olios correvoni CP 5 0.01 Miturgidae Cheiracanthium sp. 10 CP 1 0.00 Arachnida sp. 11 CP 3 0.01

Arachnida sp. 12 CP 1 0.00

Arachnida sp. 13 CP 1 0.00

Arachnida sp. 14 CP 25 0.05

Arachnida sp. 15 CP 2 0.00

Arachnida sp. 16 CP 18 0.04

Arachnida sp. 17 CP 1 0.00

Arachnida sp. 18 CP 7 0.01

Arachnida sp. 19 CP 2 0.00

Arachnida sp. 20 CP 1 0.00

Arachnida sp. 21 CP 7 0.01

Arachnida sp. 22 CP 7 0.01

Arachnida sp. 23 CP 1 0.00

Arachnida sp. 24 CP 6 0.01

Arachnida sp. 25 CP 2 0.00

Arachnida sp. 26 CP 1 0.00

Arachnida sp. 27 CP 1 0.00

Arachnida sp. 28 CP 2 0.00

Arachnida sp. 29 CP 1 0.00

Arachnida sp. 30 CP 1 0.00

127

Functional Mean / Order/Family Species name groups Total plant Araneae Arachnida sp. 31 CP 1 0.00

Arachnida sp. 32 CP 1 0.00

Arachnida sp. 33 CP 1 0.00

Arachnida sp. 34 CP 1 0.00

Arachnida sp. 35 CP 1 0.00

Arachnida sp. 36 CP 4 0.01

Arachnida sp. 37 CP 1 0.00

Arachnida sp. 38 CP 1 0.00

Arachnida sp. 39 CP 1 0.00

Arachnida sp. 40 CP 1 0.00

Arachnida sp. 41 CP 2 0.00

Arachnida sp. 42 CP 3 0.01

Arachnida sp. 43 CP 1 0.00

Arachnida sp. 44 CP 1 0.00

Arachnida sp. 45 CP 1 0.00

Unknown ? 2 0.00

Acari Phytoseiidae Phytoseiidae sp. 1 SP 70 0.15 Phytoseiidae sp. 2 SP 51 0.11

Phytoseiidae sp. 3 SP 11 0.02

Phytoseiidae sp. 4 SP 3 0.01

Tetranychidae Tetranychidae sp. 1 SH 127 0.26 Anystidae Anystidae sp. 1 CP 23 0.05 Eupodidae Eupodidae sp. 1 D 1 0.00 Ascidae Ascidae sp. 1 CP 17 0.04 Ascidae sp. 2 CP 1 0.00

Acaridae Caloglyphus sp. 1 D 4 0.01 Rhizoglyphus sp. 1 D 11 0.02

Tyrophagus sp. 1 D 5 0.01

Tydeidae Tydeidae sp. 1 D 2 0.00 Oppiidae Oppiidae sp. 1 D 166 0.35 Rhodacaridae Rhodacaridae sp. 1 D 5 0.01 Eremobelbidae Eremobelbidae sp. 1 D 11 0.02 Stigmaeidae Stigmaeidae sp. 1 D 2 0.00 Tarsonemini Tarsonemini sp. 1 D 1 0.00 Myriapoda Myriapoda sp. 1 D 1 0.00

Stylommatophora Agriolimax sp. 1 CH 35 0.07

Unknown Unknown ? 73 0.15

128