Report on the Risk Assessment of Bt Maize

Chapter 1 Introduction

Klaus Ammann, Delft, Istanbul and Neuchâtel, 16. 6. 2010

klaus.ammann@ips.unibe.ch

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Fig. 1 Grenade (Haute-Garonne, France). left: Bt maize (MON810) variety PR33P67. right : non Bt variety PR33A46, with fungal infection, source Karine Affaton, EuropaBio, fall 2007, Fotos K.Ammann

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1. Introduction: Bacillus thuringiensis and the make of Bt maize

CHAPTER 1 INTRODUCTION ............................................................................................................................. 1

Klaus Ammann, Delft and Istanbul, 16. 4. 2009 .............................................................................................. 1

1. INTRODUCTION: BACILLUS THURINGIENSIS AND THE MAKE OF BT MAIZE .............................. 3

1.1. BACILLUS THURINGIENSIS AND ITS USE, AN INTRODUCTION ...................................................................................... 8 1.1.1. Taxonomy, genetics and isolation of Bacillus thuringiensis ............................................................... 8

1.1.1.1. Comparison of Bacillus thuringiensis with its close relatives ..................................................................... 8 1.1.1.2. Discovery, natural occurrence and isolation of Bacillus thuringiensis ................................................ 10 1.1.1.3 Unfounded concerns about accumulation of Bt protoxins from GM crops in Water and soil ..... 13

1.1.2.Bacillus thuringiensis external applications.......................................................................................... 14

1.1.2.1. External Bt applications with a long tradition, but remaining marginal in use. ................................. 14 1.1.2.2. Bt sprays cause resistant insects, but not (yet) Bt crops ............................................................................ 15

1.1.3.Transgenic Bt maize ................................................................................................................................ 19

1.1.3.1. Conventional breeding of insect resistant maize not very successful .................................................. 19 1.1.3.2. How it all started, the (re)view of two initiators ................................................................................................ 19 1.1.3.3. First cloning of a Bt gene .......................................................................................................................................... 20 1.1.3.4. Genetically engineered insect resistant Bt maize ........................................................................................... 20

1.1.4. Chemistry, Biology and nomenclature of Bt toxins ............................................................................ 25

1.1.5. Mode of action of Bt proteins and selectivity of the toxins ................................................................ 34

1.1.6. Bt crops: present and future developments, synthetic Bt genes .................................................... 39

1.1.6.1. Present situation in Europe ....................................................................................................................................... 39 1.1.6.2. Recent and future developments of Bt maize breeding ................................................................................ 39

1.2. FUNDAMENTAL STATEMENTS ABOUT RISK ASSESSMENT, PARTICULARLY RELATED TO BT MAIZE. ............... 46 1.2.1.The situation in risk and benefit assessment of Bt maize worldwide and in Europe ..................... 46

1.2.2 The agricultural reality: ............................................................................................................................ 46

1.2.3. And what about Europe ? ...................................................................................................................... 47

1.2.3. There are three major reasons, why we should develop a critical view, strictly based on

scientific data, when we judge risk assessment research and the resulting scientific publications

related to Bt crops. ................................................................................................................................... 49

1.2.3.1..Assessment of the impact of GM crops should also take into account the benefits and balance

it against the risks. ................................................................................................................................................................ 49 1.2.3.2.Assessment of the impact of GM crops should also compare to non GM crops .............................. 50 1.2.3.3.Dealing with complex structures of parameters in risk assessment calls for scientific scrutiny

done by experts ....................................................................................................................................................................... 52 1.2.4.Example of a complex structure of risk assessment, an analysis related to the scientific

questions alone: The British Farm Scale Experiments ....................................................................... 53

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1.2.6.Conclusion: Proposed procedure for future risk assessments .......................................................... 54

1.2.7. Cited Literature ........................................................................................................................................ 57

List of Figures

Fig. 1 Grenade (Haute-Garonne, France). left: Bt maize (MON810) variety PR33P67. right : non Bt

variety PR33A46, with fungal infection, source Karine Affaton, EuropaBio, fall 2007, Fotos

K.Ammann ........................................................................................................................................................... 2 Fig. 2 Schematic overview of the entomopathogenic spore-forming bacteria and their protein toxins.

Localization in crystals or outside the cell (secreted) is depicted as far as known from

literature. Mtx proteins contain signal sequences for secretion, but their localization has not

been experimentally determined. Similar colors for toxins identify members of the same

homology group (yellow for 3-domain Cry proteins; red-orange for Mtx2/3-like proteins; green

for Bin-like proteins). Fig.1 from (de Maagd et al., 2003) ...................................................................... 10 Fig. 3 Above: Primary and tertiary structure of Cry toxins. (a) Relative lengths of Cry protoxins and

position of the five conserved blocks, if present. More details on these conserved blocks, as

well as the identification of three more blocks in the C-terminal ends of the longer protoxins,

can be found in . The positions of the three domains of the activated toxin are indicated for

Cry1 and vary with the positions of blocks 2 and 3 for the other toxins. The remainder of the

protoxin, consisting of short N-terminal part (20–40 amino acids) preceding the first domain

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and the C-terminal part following the third domain in the longer protoxins, is digested away by

gut proteases during the activation process. (b) Three-dimensional structure of an activated

toxin, Cry1Aa (Ref. 7). The toxin has three structural domains. Domain I (blue) is involved in

membrane insertion and pore formation. Domain II (green) and domain III (yellow-red) are

involved in receptor recognition and binding. Conserved block 1 is in the central helix of

domain I, block 2 is at the domain I–II interface, block 3 is at the boundary between domains II

and III, block 4 is in the central β-strand of domain III and block 5 is at the end of domain III.

From (de Maagd et al., 2001) ......................................................................................................................... 25 Fig. 4 Transmission electron micrograph of a sporulating Bacillus thuringiensis (Bt) cell. δ-

Endotoxins are produced as regularly shaped crystals (PB; protein body) – hence the name

crystal (Cry) proteins – next to a spore (SP). The vegetative cell wall will eventually break to

release the spore and crystal. The cell shown is approximately 2 μm long. From (de Maagd et

al., 2001) ............................................................................................................................................................. 26 Fig. 5 Spores and crystals of Bacillus thuringiensis serovar morrisoni strain T08025 Microscopy by

Jim Buckman from http://commons.wikimedia.org/wiki/File:Bacillus_thuringiensis.JPG .......... 26 Fig. 6 Scanning electron microscopy of spore (s) and crystals (c) of strains S285 (1); S447 (2); S479

3); S550 (4); S1255 (5); and Bti (6). From (Monnerat et al., 2005) Most of the Bt proteins toxic to

Mosquitos have a round shape, in contrast to the Bt proteins usually inserted in Bt maize. ..... 27 Fig. 7 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains

I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees

were constructed basically as described earlier31, except that toxin alignments were made

using DbClustal45, and updated with Cry protein sequences that were released since 1997.

Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show

similarities with the rest of the Cry protein family, see also Fig. 2(a). Shown are consensus

trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program.

Branches are color-coded according to the insect order specificity of the toxins, as far as is

known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta,

nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001) ...... 29 Fig. 8 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains

I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees

were constructed basically as described earlier31, except that toxin alignments were made

using DbClustal45, and updated with Cry protein sequences that were released since 1997.

Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show

similarities with the rest of the Cry protein family, see also Fig. 2(a). Shown are consensus

trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program.

Branches are color-coded according to the insect order specificity of the toxins, as far as is

known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta,

nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001) ...... 30 Fig. 9 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains

I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees

were constructed basically as described earlier31, except that toxin alignments were made

using DbClustal45, and updated with Cry protein sequences that were released since 1997.

Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show

similarities with the rest of the Cry protein family see also Fig. 2(a). Shown are consensus

trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program.

Branches are color-coded according to the insect order specificity of the toxins, as far as is

known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta,

nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001) ..... 31 Fig. 10 Phylogenetic relationships between the entomocidal toxins. Four distinct homology groups

have been identified within the family of toxins (Crickmore, 2003), three of which are shown

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here. The Cyt group is not shown. The branches are color coded according to insect order

specificity of the toxin where known: red, Coleoptera specific; green, Lepidoptera specific;

blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. Solid and

dashed double-headed arrows indicate associations of proteins in binary toxins or suspected

binary toxins, respectively. Panel A: 3-domain Cry proteins. The C-terminal extension (if

present) was not included for the phylogenetic analysis. Hence, proteins that have the same

primary rank based mainly on sequence similarity between their C-terminal extensions may

sometimes end up on different branches. Panel B: Binary and Bin-like toxins; Panel C: Mtx2,

Mtx3 and Mtx2/3-related proteins. From de Maagd et al. (de Maagd et al., 2003) ........................... 32 Fig. 11 Mode of action of Bacillus thuringiensis crystal proteins. Fig 1 from (de Maagd, 2007) ......... 35 Fig. 12. Primary structure of Cry proteins indicating the variety in length of the protoxin and the

extent of the activated toxin after digestion of the protoxin by gut proteases, as well as the

position of the three structural domains. Bar indicates number of amino acids. B. Tertiary

structure of Cry1Aa toxin. Clearly recognizable are the three structural domains (Roman

numerals). Fig 2 from (de Maagd, 2007) .................................................................................................... 37 Fig. 13 Mode of action of Cry toxins. (a) After ingestion by the insect, crystals dissolve in the gut

juice. (b) Gut proteases subsequently clip off the C-terminal extension in the longer Cry

proteins (purple) as well as a small N-terminal fragment (yellow). (c) The resulting ‘activated’

toxin (i.e. the structure depicted in Fig. 2b) binds to receptors on the epithelial cell membrane,

a process in which both domain II and domain III are involved. (d) Structural rearrangement of

domain I might follow allowing a two-helix hairpin to insert into the membrane. (e) Inserted

toxins form pores probably as oligomers, but the architecture of the pore is still unknown.

From (de Maagd et al., 2001) ......................................................................................................................... 37 Fig. 14 Mechanism of Cry protein toxicity. A: Ingestion of spores or recombinant protein by

phytophagous larva. B: In the midgut, endotoxins are solubilized from Bt spores (s) and

inclusions of crystallized protein. (cp). C: Cry toxins are proteolytically processed to active

toxins in the midgut. Active toxin binds receptors on the surface of columnar epithelial cells.

Bound toxin inserts into the cellular membrane. D: Cry toxins aggregate to form pores in the

membrane. E: Pore formation leads to osmotic lysis. F: Heavy damage to midgut membranes

leads to starvation or septicemia. From (Whalon & Wingerd, 2003) ................................................. 38 Fig. 15 All the strains and plasmids used or constructed (Xue et al., 2008)are listed in Table fig. 15.

Escherichia coli JM110 was used for plasmid propagation, and SCS110 was used to produce

nonmethylated plasmid DNA for the transformation of B. thuringiensis. Escherichia coli

strains were grown in Luria–Bertani medium (LB) at 37 1C, while B. thuringiensis strains were

grown in peptone-beef extract (PB) medium (0.5% peptone, 0.3% beef extract) at 30 1C. Liquid

cultures were grown in a rotary shaker at 230 r.p.m. Antibiotics were added to autoclaved

media as follows: ampicillin, 100 mgmL-1

(for E. coli); erythromycin 10 mgmL-1

(for B.

thuringiensis), from (Xue et al., 2008) ........................................................................................................ 41 Fig. 16 Engineering specificity in a three-domain Cry toxin; mutagenesis of the toxin-receptor

interaction loop in domain II. Threedimensional structure of Cry3A (1dlc; RCSB) is shown in

ribbon format. Domain I (helices) is at top right, and domain II (sheet structure) is at bottom

left. Domain III (carbohydrate-binding domain; sheet structure) is behind the other domains,

central in this view. Residues mutated (Wu et al., 2000) to increase toxicity toward yellow

mealworm (Tenebrio molitor), Colorado potato beetle, and cottonwood leaf beetle (Chrysomela

scripta) are shown in ball-and stick representation. From (Gatehouse, 2008) ................................ 44 Fig. 17 Engineering specificity in a three-domain Cry toxin; mutagenesis to improve channel-

forming ability. Three-dimensional structure of Cry3Bb (1ji6; RCSB) is shown in ribbon format

in the same view as Figure 1. Residues mutated (English et al., 2003) to increase toxicity

toward corn rootworm are shown in ball-and-stick representation. Mutations are made in

helices of domain I and in the region linking domains I and II. The mutation sites shown are

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taken from the most active toxin produced; a range of other sites for mutation were explored.

From (Gatehouse, 2008) ................................................................................................................................. 45 Fig. 18, Assessment continuum within a tiered scheme of ecological risk assessment. The decision

to reject the risk hypothesis includes consideration of residual uncertainties. With increasing

tiers, the assessment becomes more complex and realistic, with conclusions that are more

specific. The assessment can stop at any stage during the process as soon as sufficient

information has been compiled to address the risk hypothesis. Thus collection of data

irrelevant to the risk assessment is minimized. N, level of risk assessment tier; NTA, nontarget

arthropod. From (Romeis et al., 2008a) ..................................................................................................... 55 Fig. 19, Reconstruction of NTA risk assessment for Bt maize expressing Cry1Ab showing that

different risk hypotheses require different types of data and synthesis at different tiers. From

(US Environmental Protection Agency (USEPA), 1995) taken from (Romeis et al., 2008a) and

see also the fully cited accompanying text below: ................................................................................. 56

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1.1. Bacillus thuringiensis and its use, an introduction

1.1.1. Taxonomy, genetics and isolation of Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a bacterium of great agronomic and scientific interest.

Together, the many subspecies of this bacterium colonize and kill a large variety of

host insects and even nematodes, but each strain does so with a high degree of

specificity (de Maagd, 2007; de Maagd et al., 2003; de Maagd et al., 2001), which makes sense in the

complex web of the ecosystems.

The taxonomy of Bacillus thuringiensis has undergone revisions (Helgason et al., 2000; Lecadet et al.,

1999), which is important for any kind of communication related to risk assessment of Bt pesticide

strategies.

See also a summary of the wider Taxonomy in a recent review by (Swiecicka, 2008):

Bacillus cereus and Bacillus thuringiensis are treated there as members of the Bacillus cereus group, which

includes four additional species, Bacilius anthracis, Bacillus weihenstephanensis, Bacillus mycoides and

Bacillus pseudomycoides, they comprise a clearly distinguishable cluster among facultative anaerobic

endospore-forming bacteria of the genus Bacillus, following (Priest, 1993).

A major comparative genomic study has been published by (Han et al., 2006b). The complete genome

sequence of Bacillus thuringiensis has been uncovered subsequently by the same research group

(Challacombe et al., 2007).

In another recent review of the genetics and taxonomy of B. thuringiensis (Vilas-Boas et al., 2007) show

the full complexity of the matter: Since the first published genome sequence of the B. anthracis A2012

strain (Read et al., 2002) several complete-genome sequencings have been undertaken: 10 strains of B.

anthracis, 8 strains of B. cereus and 3 strains of B. thuringiensis are now available in GenBank, actually

making it the group of closely related bacteria with the highest number of fully sequenced genomes. This

offers an unprecedented opportunity for extensive comparative genomic studies (Vilas-Boas et al., 2007).

In essence it is concluded there that classic genomic comparison does not allow for direct conclusions

about horizontal gene flow, it will be necessary to include an ecological species concept (Weisse, 2007).

Analytical chemistry allows to identify and quantify the toxic proteins characterizing the various varieties

of Bacillus thuringiensis (Bt) (Hickle & Fitch, 1998). The large majority of known Bt toxins are classified as

3-domain toxins, according to their homology to a small number of toxins of which the 3-dimensional

structure has been experimentally determined.

1.1.1.1. Comparison of Bacillus thuringiensis with its close relatives

Although it has been suggested that Bacillus thuringiensis, B. cereus and B. anthrax are all members of the

same species (Helgason et al., 2000), and although there is ample evidence that B. thuringiensis and B.

cereus could be taken as members of the same species, this is certainly not the case for B. anthracis, since

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the two plasmids that encode the toxins of B. anthracis do not occur naturally in the other two species,

and they do not have parasporal bodies containing Bt Cry proteins. This implies that there are probably

natural barriers not yet properly understood (Federici & Siegel, 2007).

It must also be stated clearly, that insecticidal crystals formed by Cry and Cyt proteins are the principal

characteristic that differentiates B. thuringiensis from B. cereus, as well as from other species of the B.

cereus group. As far as is known, most if not all Cry and Cyt proteins are encoded on plasmids present in

Bacillus thuringiensis, i.e. not on the bacterial chromosome (Crickmore et al., 1998). Just the other way

around this makes it also clear that any “Bt” lacking the Cry or Cyt plasmids, be it by natural loss or taken

out deliberately - the resulting strain would be identified as B. cereus (Baumann et al., 1984; Hill et al.,

2004; Rasko et al., 2005; Rasko et al., 2007). Despite this, B. thuringiensis is still considered a valid species

due to a combination of tradition and practical value, and this is, according to Federici et al. 2007 (Federici

& Siegel, 2007), unlikely to change, at least in the near future.

There has been a lot of scaremongering in the anti-biotech community about the close relationship of

Bacillus thuringiensis with B. anthracis, here just one example: (Mae, 2001). Such superficial comparisons

on a strict molecular level led to scientifically unfounded safety concerns about the close relationship

between Bacillus thuringiensis and the highly virulent B. anthracis (Heinemann & Traavik, 2004b), which

later had to be rectified (Heinemann & Traavik, 2004a) after having been contradicted (de Maagd et al.,

2005). However, horizontal gene flow seems to be possible between B. cereus and B. thuringiensis under

specific soil conditions: Experimental results (Vilas-Boas et al., 2002) suggest that the rate of gene flow is

higher between strains of the same species; but that exchanges between B. cereus and B. thuringiensis

are nonetheless possible. Overall, these data indicate that it is not important for risk assessment purposes

to determine whether B. cereus and B. thuringiensis belong to a single or to two species. Assessment of

the biosafety of pest control based on B. thuringiensis-toxins requires evaluation of the extent of genetic

exchange between strains in realistic natural conditions. This view has been recently confirmed and

extended (Vilas-Boas et al., 2007): A genomic comparison between B. cereus, B. anthracis and B.

thuringiensis should not barely include sequence data which obviously can lead to wrong superficial

conclusions, but it should follow a much more adequate ecological species concept (Godreuil et al., 2005;

Vanvalen, 1976; Vellai et al., 1999; Weisse, 2007).

A major comparative genomic study has been published by (Han et al., 2006a; Han et al., 2006b). (Hickle

& Fitch, 1998) present the analytical chemistry involved in identifying and quantifying the toxin proteins

present in various varieties of Bacillus thuringiensis.

An instructive figure on related species has been published by (de Maagd et al., 2003):

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Fig. 2 Schematic overview of the entomopathogenic spore-forming bacteria and their protein toxins. Localization in crystals or outside the cell (secreted) is depicted as far as known from literature. Mtx proteins contain signal sequences for secretion, but their localization has not been experimentally determined. Similar colors for toxins identify members of the same homology group (yellow for 3-domain Cry proteins; red-orange for Mtx2/3-like proteins; green for Bin-like proteins). Fig.1 from (de Maagd et al., 2003)

1.1.1.2. Discovery, natural occurrence and isolation of Bacillus thuringiensis

Bacillus thuringiensis (Bt) was isolated from a flour moth collected in the German province of Thuringia

and described by (Berliner, 1915). Ernst Berliner isolated the type species of Bacillus thuringiensis from

the Mediterranean flour moth Ephestia kuehniella.

The same organism had already been described by (Ishiwata, 1901-1902) as Bacillus sotto from Japan

where it causes a wilt disease of silkworm caterpillars, but the description was not known to Berliner.

Bacillus thuringiensis is now the accepted name for a range of aerobic spore-forming bacteria which form

an insecticidal crystal during sporulation.

A still valid and classic account on bacterial insecticides has been published by (Heimpel & Angus, 1960).

Their account makes it clear, that there was ample knowledge on insect pathology of an unknown

bacterium with a very high toxicity, and the authors defined a process with ‘endotoxin’ produced by the

bacterium which is poisonous to many Lepidoptera. The endotoxins of this bacterium, later discovered by

Berliner as the Bt, killed the insects and then it reproduced by spores within the dead insect. This set of

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processes and components evolved in grain-feeding and other pyralid insects, specifically in larvae of

species such as the southern European sunflower moth Homoeosoma nebulella, from which the type

species of Bacillus thuringiensis ssp thuringiensis was discovered by Berliner. So, after all, the toxicity

processes and life cycles of Bt are quite natural and existing in the insect world in abundance.

Since the pioneering work of Steinhaus in California in the early 1950s (Steinhaus, 1951), there has been

considerable commercial interest and products are now sold for external use in most countries of the

world for control of caterpillars (var. kurstaki, entomocidus, galleriae and aizawai), mosquito and blackfly

larvae (var. israelensis) and beetle larvae (var. tenebrionis and var. sandiego) (Milner, 1994).

Bacillus thuringiensis occurs in nature in many different substrates and environments.

(Damgaard et al., 1997) isolated Bacillus thuringiensis from the phylloplane of cabbage foliage. The same

authors have previously shown (Pedersen et al., 1995) that spores of B. thuringiensis serovar kurstaki can

readily be dispersed from soil to the lower leaves of cabbage plants. Therefore they could expect that the

population studied now would not differ from that normally found in soil. Natural occurrence of B.

thuringiensis has also been confirmed by (Kaur & Singh, 2000) in leguminous phylloplanes in the New

Delhi region of India.

The relatively high proportion of isolates from the phylloplane with lepidopteran activity both in the

studies above and in that of (Smith & Couche, 1991) is in contrast to the findings of most surveys on the

natural occurrence of B. thuringiensis in soil, which have shown ‘non-toxic’ strains to be the most

common types (Hastowo et al., 1992; Ohba & Aizawa, 1986a, b; Ohba & Aratake, 1994). The serotyping of

the isolates in (Damgaard et al., 1997) showed that the majority of the isolates belonged to serovar

kurstaki. Isolation of B. thuringiensis from soil has shown to contain a very diverse population of serovars,

but never with a frequency of the insecticidal serovar kurstaki above 50% (Delucca et al., 1981; Ohba &

Aratake, 1994; Rongsen et al., 1990).

The high frequency of lepidopteran-active serovar kurstaki isolates found on foliage in this study indicates

that the (natural!) population of B. thuringiensis on phylloplane is different from that normally found in

soil. It is therefore likely that the phylloplane population is not exclusively the result of transfer of soil

bacteria to the foliage. Apparently some kind of propagation and/or selection of the B. thuringiensis

population takes place on the phylloplane. Bacillus thuringiensis was also discovered on the surface of

clover and other phylloplanes (Bizzarri & Bishop, 2007).

In soils Bacillus thuringiensis is ubiquitous: In a selection approach, (Travers et al., 1987) using a high

acetate medium to isolate Bt semi-selectively from soil and obtained over 8000 isolates. They claimed

that these isolates represented some 73 new biochemically distinct varieties of Bt. (Martin & Travers,

1989) found the insect control agent Bacillus thuringiensis to be a ubiquitous soil microorganism. They

isolated B. thuringiensis in 785 of 1,115 soil samples. These samples were obtained in the United States

and 29 other countries. A total of 48% of the B. thuringiensis isolates (8,916 isolates) fit the biochemical

description of known varieties, while 52% represented undescribed B. thuringiensis types. Over 60%

(1,052 isolates) of the isolates tested for toxicity were toxic to insects in the orders Lepidoptera or

Diptera. This kind of ubiquitous occurrence was again confirmed by (Jouzani et al., 2008) and (Haddad et

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al., 2005) who verified that 77%, 78% and 80.5% of the effective doses (viable spores) remained on the

leaf surface after the first day of external Bt treatment, respectively.

In a recent comprehensive review Swiecicka (Swiecicka, 2008) widens the picture of natural occurrence of

Bacillus thuringiensis and its close relatives: While much is known about the taxonomic properties and

molecular basis for virulence of Bacillus thuringiensis and Bacillus cereus, comparatively less is known

about their ecology in natural environments. Thus, there are limited data regarding their resilience, i.e.

recycling of vegetative and sporulated phases of growth in soil, ecolgical niches including symbiotic

interactions with other organisms, and the impact on ecosystems in which they proliferate. Nevertheless,

based on recent data, apicture is beginning to emerge that B. thuringiensis and B. cereus are capable of

establishing mutual and commensal relationships with both animals and plants. In this regard, these

bacilli can proliferate in the digestive tracts of animals, where upon defecation they form dormant spores

in the soil, and to a lesser extent on the phylloplane and rhizospheres of plants.

Bacillus thuringiensis has been found in many more and diverse habitats (Federici, 1999), such as animal

feces, sludge, etc. (Hwang et al., 1998; Lee et al., 2003a; Mizuki et al., 2001; Okumura et al., 2001; Yu et

al., 1991).

But it should also be mentioned that the widespread use of Bt toxin sprays is the cause of numerous test

results published by (Frederiksen et al., 2006): A total of 128 Bacillus cereus-like strains isolated from

fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39%

(50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by

PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and

plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B.

thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B.

thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates

grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were

primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was

developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the

nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin

genes was higher among the strains indistinguishable from the commercial strains than among the other

B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a

third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B.

thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially

enterotoxigenic.

(Federici & Siegel, 2007) summarize the enormous complexity of more than 70 varieties and subspecies of

Bacillus thuringiensis, there are more than 100’000 isolates that occur among the plasmids and insecticide

protein complements detected in the Bt isolates. 120 different types of genes are encoding Cyt proteins,

and at least 12 different types of genes encode Cyt proteins having been cloned and sequenced up to

now.

Usually, each type of Cry protein has an extremely limited target spectrum (lepidopteran, dipteran,

coleopteran, nematodes), and each specific protein like Cry1Ac is always much narrower than the type as

a whole, and even within a target category such as Lepidoptera (Rosi-Marshall et al., 2007b) there can be

marked differences from species to species: Cry1Ac is highly toxic to Heliothis virescens, but non-sensitive

to Spodoptera exigua.

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1.1.1.3 Unfounded concerns about accumulation of Bt protoxins from GM crops in Water and

soil

Related to the ubiquitous occurrence of Bt toxins and also due to the fact that widespread cultivation of

crops with the endotoxins of Bacillus thuringiensis there is an environmental debate on the impact of Bt

toxins, further chapters will deal with the impact of Bt endotoxins on non-target insects in the agricultural

and non agricultural terrestrial systems, here in advance, included in the general introduction, some

remarks about aquatic and soil systems:

Aquatic systems

(Douville et al., 2007) tested the short time persistence of Bt proteins in aquatic systems. The Cry1Ab

gene persisted for more than 21 and 40 days in surface water and sediment, respectively. The removal of

bacteria by filtration of surface water samples did not significantly increase the half-life of the transgene,

but the levels were fivefold more abundant than those in unfiltered water at the end of the exposure

period. In sediments, the Cry1Ab gene from Bt corn was still detected after 40 days in clay- and sand-rich

sediments. Field surveys revealed that the Cry1Ab gene from transgenic corn and from naturally occurring

Bt was more abundant in the sediment than in the surface water. The Cry1Ab transgene was detected as

far away as the Richelieu and St. Lawrence rivers (82 km downstream from the corn cultivation plot),

suggesting that there were multiple sources of this gene and/or that it undergoes transport by the water

column. Sediment-associated Cry1Ab gene from Bt corn tended to decrease with distance from the Bt

cornfield. Sediment concentrations of the Cry1Ab gene were significantly correlated with those of the

Cry1Ab gene in surface water (R = 0.83; P = 0.04). The data indicate that DNA from Bt corn and Bt were

persistent in aquatic environments and were detected in rivers draining farming areas.

However, the authors also refer to their own previous study (Douville et al., 2005), where the results

showed that Bt-corn endotoxin is degraded more rapidly in water than in soils (t1/2: 4 and 9 days,

respectively), while crystals appeared to be more resilient, as expected. The isotopic patterns of 13C and

15N in Bt-corn endotoxin differed markedly from Bt, making it possible to track the source of Cry1Ab in

the environment. Preliminary field surveys indicate that Cry1Ab is fairly uncommon in aquatic

environments, being found only at trace concentrations when it is detected. This will say that Bt protoxins

are highly unlikely to cause any environmental problems in aquatic systems. As a whole, the publications

of Douville et al. are anyway not convincing, because they lack an important scientific quality: the baseline

comparison is totally lacking. As an example: There are several publications from the same river system,

such as (Tall et al., 2008) and many others which clearly point to metal and phosphorus contamination of

the river sediments, causing negative effects to the fauna and flora.

Critical reference is given to the paper of (Rosi-Marshall et al., 2007a) on the occurrence of Bt protein in

headwater stream ecosystems, written in an unnecessary alarming style and not even confirmed with

hard field data in the chapter on non-target insects of this report. There is not even a hint on the nature

of the Bt toxin (it could well be at least partially of natural origin), and when you compare her own (!)

figure 3 B the graph with realistic concentrations, then you see that Bt shows a clearly lower mortality of

the scraping caddisflies experiment – so what?? And again it shows, like Douville, the deadly sin in science

of a lacking a proper baseline comparison.

14

These comments are extended in a full rebuttal in the ASK-FORCE blog 1 of the major conclusions of (Rosi-

Marshall et al., 2007b), see also the published controversy about the case: (Beachy et al., 2008; Parrott,

2008; Rosi-Marshall et al., 2008).

Soil systems

The whole question on persistence of Bt toxins in soil is treated in a separate chapter in this report – there

are again, after a first wistle blower phase (Saxena et al., 1999), enough long term studies to demonstrate

that accumulation does not take place to a degree that it could harm soil organisms, here just as an

example two papers: (Head et al., 2002; Saxena & Stotzky, 2001):

1.1.2.Bacillus thuringiensis external applications

1.1.2.1. External Bt applications with a long tradition, but remaining marginal in use.

Bacillus thuringiensis toxins have a long tradition in organic and conventional agriculture for over a 100

years as external sprays (Nester et al., 2002) and (Croft, 1990; Federici & Siegel, 2007; Flexner et al., 1986;

Glare & O'Callaghan, 2000; Hickle & Fitch, 1998; Krieg, 1968; Meher et al., 2002; Metz, 2004; Punja, 1997).

Bt toxins were also recommended as pesticide spray by Rachel Carson in her classic book The Silent Spring

(Carson, 1962 - 2002). It can be assumed that Rachel Carson would have welcomed the new Bt crops as a

more elegant solution to fight the European corn borer within the maize stems.

According to (Federici & Siegel, 2007) there are four major subspecies/serovarieties (kurstacki, aizawai,

morrisoni and israelensis with a total of 13 Bt proteins in use for bacterial insecticides.

In numerous risk assessments those sprays have revealed to be unproblematic related to environmental

impact, and provided the prescriptions are respected: The effects of the commercial Bt spray Dipel on

green lacewings and its prey herbivores (aphids, spider mites, and lepidopteran larvae) were compared to

those of transgenic Bt maize in a study (Dutton et al., 2003). The field studies reveal small differences, on

an agronomic scale they are negligible. However, another safety assessment of Bt spray applications of

Bishop et al. concludes with some critical remarks (Bishop et al., 1999):

“commercial insecticide containing B. thuringiensis was sprayed onto spinach leaves. After normal food

preparation regimes some leaves retained residual spore loads sufficient for a strongly enterotoxic strain to

cause food poisoning in humans. These findings suggest that the agricultural use of some, previously

unvalidated, strains of B. thuringiensis could give rise to cases of food poisoning and that rodents are

unsuitable for testing the safety to humans of oral exposure to this organism.”

The same authors dismiss for external Bt applications food safety concerns after experimenting with rats:

1 Ammann, K. ASK-FORCE blog on the impact of Bt toxins on aquatic organisms,

http://www.botanischergarten.ch/AF-3-Aquatic-Bt/AF-3-Aquatic-Bt-toxins-20100423-opensource.pdf

15

“Six strains of Bacillus thuringiensis were tested with two commercially available kits for their ability to produce

Bacillus cereus-type enterotoxin and by dipteran bioassay for the production of beta-exotoxin. All of the strains were

positive for enterotoxin production including three which have been used world-wide for many years to control pest

insects. Rats given oral doses totaling 1 x 10(12) spores ( +crystals), over three weeks, or a single subcutaneous dose

of 1 x 10(6) spores ( +crystals) showed no ill-effects in terms of their condition or in the pathology of their internal

organs: this was in spite of the strain of B. thuringiensis used (13B) being an active producer of both beta-exotoxin

and enterotoxin”

In a comparative study with 20 kinds of pesticides, the microbial formulations containing Bacillus

thuringiensis revealed to be harmless after having been tested against 19 beneficial arthropodes (Sterk et

al., 1999).

Overall, concerns about safety of Bt insecticides for humans can be dismissed. (Glare & O'Callaghan, 2000;

McClintock et al., 1995; Siegel, 2001). (Federici & Siegel, 2007) produce extensive results in table 3.6 p. 67,

results show, despite of the very large amounts of test materials used in these studies, which are 100- to

more than 1000-fold the amount of material used to control insect pests: as a result most assessments of

the safety of Bt to humans are based on a lack of reported effects, i.e., the overall lack of reported

infections or other documented cases of disease, especially in areas where human populations numbering

in the tens of thousands have been exposed to Bt applications during aerial spray programs to eliminate

lepidopteran forest pests (de Amorim et al., 2001; Pearce et al., 2002; Petrie et al., 2003; Teschke et al.,

2001).

1.1.2.2. Bt sprays cause resistant insects, but not (yet) Bt crops

There are several cases documented already in the early nineties and later, where insects developed

resistance against Bt formulations sprayed in the fields and in greenhouses: (Li et al., 2005; Tanaka &

Kimura, 1991). Already in 1994 and 1995 there was growing and well documented concern about evolving

pest resistance against Bt toxins used in sprays (Bauer, 1995; McGaughey, 1994). On the other hand,

despite massive use of Bt crops, resistance to the Bt endo-toxin remains rare up to now, although it can

be detected occasionally in the field with rather costly analysis, it does not pose a problem up to now

(Bates et al., 2005; Bourguet et al., 2005; Shelton et al., 1993; Tabashnik et al., 2006; Tabashnik et al.,

2008). The most recent paper of Tabashnik et al. 2008 (Tabashnik et al., 2008) is often cited as now having

provided the ‘ultimate proof’ with field data that Bt resistance for some Bt cotton pests has been

detected, which is fact, but only in exceptional cases and (not yet) causing any agronomic problems. Here

the authors own comments:

“Nonetheless, resistance of H. zea to Cry1Ac [endotoxin] has not caused widespread control failures for

several reasons. First, even in the few states with documented resistance, most populations tested were

not resistant to Cry1Ac. Second, insecticides have been used from the outset to augment control of H. zea

on Bt cotton because Cry1Ac alone is not sufficient to control high-density populations of the pest (EPA

Environmental Protection Agency, 1998; Jackson et al., 2004b). Insecticide sprays decrease any problems

associated with reduced control of H. zea by Bt cotton. Third, against strains with 44- to 100-fold

resistance to Cry1Ac, the Cry1Ac in Bt cotton still caused 48–60% larval mortality (Ali et al., 2006; Jackson

et al., 2004a; Luttrell et al., 2006),. Finally, ‘pyramided’ transgenic cotton producing Bt toxins Cry2Ab and

16

Cry1Ac was registered in December 2002 and planted on more than 1 million ha in the United States in

2006 and 2007 (Monsanto Co., 2002). Control of Cry1Acresistant H. zea by Cry2Ab also limits problems

associated with resistance to Cry1Ac19.”

This is also due to good agricultural practice with refuge areas, and, as (Jackson et al., 2008) reports, due

to ecological factors revealed in the latest field assessment:

“These data demonstrate that Helicoverpa. zea adults move extensively from their natal host origins. Therefore,

non-cotton crop hosts, and even relatively distant hosts, contribute significantly to effective refuge for H. zea on Bt

cotton. The results presented here demonstrate that substantial natural refuge is present for Bt-resistance

management of H. zea throughout the mid-South and Southeast portions of the US cotton belt.”

As (Moar et al., 2008) rightly mention, the two publications of (Ali et al., 2006; Luttrell et al., 1999) cannot

be used (as (Tabashnik et al., 2008) did), as an argument of raising resistance of cotton pests:

“We emphatically disagree with the conclusions of (Tabashnik et al., 2008) that the data published in

these two articles demonstrate field-evolved resistance in H. zea for four reasons: first, the definition of Bt

resistance used by Tabashnik et al. is purely laboratory based, whereas field efficacy and larval survival on

plant tissues are the ultimate criteria for contextualizing laboratory-based estimates of resistance, and no

change in Bt cotton efficacy has been documented during the past decade; second, larval samples should

not be collected from Bt crops because they will not be representative of the population as a whole,

especially for highly mobile insects such as H. zea; third, the data from Luttrell's laboratory on which

Tabashnik et al. base their conclusions have been evaluated using LC50 (median lethal dose; 50%) values to

measure resistance, which introduces artifacts into the analysis; and fourth, the baseline comparator used

to assess variability in these laboratory assays is not representative of field susceptibility; when a more

appropriate comparator colony is employed, results from Luttrell's laboratory bioassays indicate no

change in susceptibility. We discuss each of these aspects in turn below.”

In a recent paper, (Tabashnik et al., 2009b) points to the fact, that Asymmetrical cross-resistance between

Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in pink bollworm has been detected, again not in the field,

but with laboratory experiments.

From the abstract:

“We show here, however, that laboratory selection of pink bollworm with Cry2Ab caused up to 420-fold

crossresistance to Cry1Ac as well as 240-fold resistance to Cry2Ab. Inheritance of resistance to high

concentrations of Cry2Ab was recessive. Larvae from a laboratory strain resistant to Cry1Ac and Cry2Ab in

diet bioassays survived on cotton bolls producing only Cry1Ac, but not on cotton bolls producing both

toxins. Thus, the asymmetrical cross-resistance seen here does not threaten the efficacy of pyramided Bt

cotton against pink bollworm. Nonetheless, the results here and previous evidence indicate that

crossresistance occurs between Cry1Ac and Cry2Ab in some key cotton pests. Incorporating the potential

effects of such cross-resistance in resistance management plans may help to sustain the efficacy of

pyramided Bt crops.”

In the most recent paper, Tabashnik et al. (Tabashnik et al., 2009a) come to the following conclusions,

summed up in a press release of the Entomological Society of America

http://www.entsoc.org/resources/press_releases/2009_btcrops.htm

17

“According to lead author Dr. Bruce E. Tabashnik, “Resistance is not something to be afraid of, but

something that we expect and can manage if we understand it. Dozens of studies monitoring how pests

have responded to Bt crops have created a treasure trove of data showing that resistance has emerged in

a few pest populations, but not in most others. By systematically analyzing the extensive data, we can

learn what accelerates resistance and what delays it. With this knowledge, we can more effectively predict

and thwart pest resistance.”

Among the authors’ conclusions are:

The refuge strategy (growing non-Bt crops near the Bt crops) can slow the evolution of insect

resistance by increasing the chances of resistant insects mating with non-resistant ones, resulting in non-

resistant offspring.

Crops that are “pyramided” to incorporate two or more Bt toxins are more effective at controlling

insect resistance when they are used independently from crops that contain only one Bt toxin.

Resistance monitoring can be especially effective when insects collected from the field include

survivors from Bt crops.

DNA screening can complement traditional methods for monitoring resistance, such as exposing

insects to toxins in the lab.

Despite a few documented cases of field-evolved resistance to the Bt toxins in transgenic crops, most

insect pest populations are still susceptible.

With Bt crop acreage increasing worldwide, incorporating enhanced understanding of observed patterns

of field-evolved resistance into future resistance management strategies can help to minimize the

drawbacks and maximize the benefits of current and future generations of transgenic crops.”

Consequently, again we will have a situation, where opponents to the technology will not read the

publication properly and herald loudly that the reistance against the pink bollworm and other major

cotton pests is now agricultural reality – which might well develop in the future, if the measures proposed

by Tabashnik in the same paper are not translated into reality (based on more field-research).

It is enigmatic to the author of this report why then certain proponents of organic farming, who use Bt

spray formulations – often not very wisely – are scared that Bt crops could make their own microbial

pesticides worthless by triggering Bt resistance to major pest insects (Greenpeace, 2002; Wallimann,

2000) – the facts show another picture. The reference list of literature on external Bt applications

collected counts already now over 300 items.

http://www.botanischergarten.ch/Bibliography/Bibliography-Bt-Sprays-20080316.pdf

In a final chapter the whole question about developing Bt resistance of target pests will be discussed in

extenso. Although up to now no resistance has developed of agronomic importance, this will almost

certainly happen in the future. The answer against will be stacked Bt genes or also artificially enhanced Bt

genes (Christou et al., 2006) – but read about the caveats in (Tabashnik et al., 2009b) commented above.

18

According to (de Maagd, 2007) applications of Bt sprays nowadays, consisting of some form of

spore/crystal-mixture, are found in three major areas:

1. Forest pest control. Particularly in North America, aerial Bt-sprays are used extensively for control of

forest pests (spruce budworm, gypsy moth).

2. Mosquito control. Particularly in the Middle East and Africa, but also in Europe (German Rhine

valley), sprays are used for control of mosquitoes, such as the vectors for malaria.

3. Organic agriculture. As Bt sprays are considered a natural pesticide, it is one of the few pesticides

that can be used in organic agriculture (particularly on horticultural crops).

Despite its attractiveness as a natural pesticide, Bt has never conquered a large share of the global

pesticide market. Although it is the most widely used biological pesticide, it takes up only about 1% of the

total insecticide market. Several reasons for this can be identified:

Low persistence. The crystal protein is rapidly inactivated by solar UV-radiation.

Limited activity spectrum. Each Bt strain is active only against a few pest species, so one

product is never sufficient for all pests encountered in the field.

Many important pest species are insensitive to all known Bt strains.

Bt sprays, as many chemical insecticides, are not very effective against insects that bore

into the crop tissue. There may be only a limited time window in which sprays can be

effective. This is particularly true for cotton bollworm and European corn borer. This

requires extensive monitoring by farmers to time spraying properly.

According to (Dutton et al., 2003) Dipel (a commercial Bt spray) had no effect on aphids;

however, negative effects on spider mites were observed. Spider mites reared on Bt-

sprayed plants had a significantly lower intrinsic rate of natural increase compared to

those reared on control plants. Similarly, S. littoralis larvae were significantly affected by

Dipel as the developmental time required by larvae which were fed Bt-sprayed plants was

prolonged when compared to larvae on untreated plants. Negative effects on C carnea

larvae were also shown through prey-mediated exposure to Dipel.

In contrast to those effects, the results of (Romeis et al., 2004) strongly suggest that C.

carnea larvae are not sensitive to Cry1Ab and that earlier reported negative effects of Bt-

maize were prey-quality mediated rather than direct toxic effects. These results, together

with the fact that lepidopteran larvae are not regarded as an important prey for C. carnea

in the field, led the authors to conclude that transgenic maize expressing Cry1Ab poses a

negligible risk for this predator, a clear advantage for the transgenic strategy of Bt

applications.

19

1.1.3.Transgenic Bt maize

1.1.3.1. Conventional breeding of insect resistant maize not very successful

Attempts about conventional breeding of insect resistant maize without using genetic engineering have

been often made, but so far they have not been successful (Bohn et al., 2001; Bohn et al., 1996; Bohn et

al., 1997; Bohn et al., 2003). The key parameter for conventional marker assisted breeding is The key

parameter for assessing the prospects of Marker Assisted Breeding (MAS) is a high number of selection

plants in large populationsIn order to obtain reliable and high values for quantitative traits, large

population sizes and (n>500) and a high number of test environments have to be employed. However,

mandatory large-scale experiments are not an option for most breeders due to financial and logistic

restrictions

1.1.3.2. How it all started, the (re)view of two initiators

In a review, Herman Höfte from Plant Genetic Systems N.V. in Gent, Belgium and H.R. Whiteley from the

University of Washington in Seattle (Hofte & Whiteley, 1989) present an comprehensive update of the

knowledge of the late eighties of B. thuringiensis crystal proteins C and their genes. It offers an excellent

insight in the pioneer times of Bt crop development. They also propose a nomenclature and classification

scheme for crystal proteins based on their structure (deduced from the deoxyribonucleic acid DNA

sequence as well as their host range which later has been thoroughly changed). Nevertheless, their review

let us understand how the Bt insect resistance as a major strategy of pest control in modern agriculture

was developed:

“Bacillus thuringiensis is a gram-positive soil bacterium characterized by its ability to produce crystalline

inclusions during sporulation. These inclusions consist of proteins exhibiting a highly specific insecticidal

activity (reviewed in references (Aronson et al., 1986; Whiteley & Schnepf, 1986). Many B. thuringiensis

strains with different insect host spectra have been identified (Burges, 1981)) They are classified into

different serotypes or subspecies based on their flagellar antigens. Most strains are active against larvae

of certain members of the Lepidoptera, but some show toxicity against dipterian (reviewed in (Federici et

al., 1991)) or coleopteran (Krieg et al., 1983) species. For several crystal-producing strains. no toxic

activity has yet been demonstrated. B. thuringiensis crystalline inclusions dissolve in the larval midgut,

releasing one or more insecticidal crystal proteins (also called delta-endotoxins) of 27 to 140 kilodaltons

(kDa). As described in the following section, most crystal proteins are protoxins that are proteolytically

converted into smaller toxic polypeptides in the insect midgut. The activated toxin interacts with the

midgut epithelium cells of susceptible insects. Electrophysiological (Harvey et al., 1983) and biochemical

(Knowles & Ellar, 1987) evidence suggests that the toxins generate pores in the cell membrane, thus

disturbing the osmotic balance. Consequently, the cells swell and lyse. The larva stops feeding and

eventually dies. For several B.thuringiensis toxins, specific high-affinity binding sites have been

demonstrated to exist on the midgut epithelium of susceptible insects (Hofmann & Luthy, 1986; Hofmann

et al., 1988a). This could, at least in part, explain the extreme specificity of these proteins.

Formulations of B. thuringiensis have been used for more than two decades as biological insecticides to

control agricultural pests and, more recently, insect vector-s of a variety of human and animal diseases.

Recently, the cloning of insecticidal crystal protein genes (Whiteley & Schnepf, 1986) and their expression

20

in plant-associated microorganisms (Obukowicz et al., 1986) or transgenic plants (Barton et al., 1987;

Fischhoff et al., 1987; Vaeck et al., 1987) has provided potentially powerful alternative strategies for the

protection of crops against insect damage. These applied aspects are to a large extent responsible for an

increased interest in this bacterium and its crystal proteins in recent years. Extensive screening programs

are being carried out by various groups to search for B. thuringiensis strains with new insecticidal spectra.

Numerous publications report the identification of crystal proteins and the cloning and sequencing of

crystal protein genes. One problem related to this is the lack of a uniform nomenclature for these genes

and their products, which makes the literature rather confusing.”

More molecular details can be found in a previous publication of the same author team: (Hofte et al.,

1986).

Some years later, the classification has been changed and based completely on the molecular structure of

the growing number of proteins, first on the sequence of amino acids after (Crickmore et al., 1998). Today

the classification is entirely built on phylogenetic relationships of the molecular structure, as shown below

in Fig. 4-6 from (de Maagd et al., 2001). For the latest views on taxonomy and nomenclature of the Bt

proteins see the website with a regularly updated database structure of (Crickmore et al., 2008)

http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/ .

1.1.3.3. First cloning of a Bt gene

The first gene coding for an insecticidal protein was cloned by Schnepf and Whiteley (Schnepf &

Whiteley, 1981), working with the HD 1 isolate of B. thuringiensis kurstaki, the strain used in the

commercial product Dipel. Following this, Whiteley, her co-workers, and others showed that there were

actually four endotoxin genes in this strain that coded for insecticidal proteins. According to Sussex

(Sussex, 2008), the following three research groups under Jeff Schell, Rob Horsch, and Mary-Dell Chilton

(Chilton et al., 1978; Chilton et al., 1977; Chilton et al., 1980; Drummond et al., 1977) all reported at a

Miami Winter Symposium in January 1983 success in producing chimeric genes that functioned in

transformed plant cells (Downey et al., 1983). All three groups used the nopaline synthetase (NOS)

promoter spliced to the bacterial NPT II (neomycin phosphotransferase) coding sequence as a dominant

selectable marker and NOS polyadenylation signals or a variation of this strategy. NOS was discovered by

(Van Montagu et al., 1980; Willmitzer et al., 1980; Willmitzer et al., 1983).

1.1.3.4. Genetically engineered insect resistant Bt maize

1.1.3.4.1. Transformation with Agrobacterium tumefaciens

Transformation with Agrobacterium was difficult in the beginning: The graminaceous monocots, including

the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a

natural gene transfer system (De Cleene & Deley, 1976) that has been successfully exploited for

transferring foreign genes into higher plants (Caplan et al., 1983; De Block et al., 1984; Fraley et al., 1983;

Zambryski et al., 1984). Therefore, direct transfer techniques that are potentially applicable to all plant

species have been developed using a few dicot and monocot species as model systems (Fromm et al.,

1985; Fromm et al., 1986; Lorz et al., 1985; Potrykus et al., 1985). Only recently have we begun to

understand how Agrobacterium hijacks host factors and cellular processes during the transformation

process (Dafny-Yelin et al., 2008; Lacroix et al., 2006; Tzfira & Citovsky, 2006).

21

Attempts were successful in the Ghent school, first by systematically working out the procedures on how

to transfer plasmids of Agrobacterium tumefaciens to higher plants under the lead of Jeff Shell and Marc

van Montagu: (Deblock et al., 1985; Genetello et al., 1977; Holsters & Schell, 1975; Schell, 1977; Van

Montagu, 1997; Van Montagu et al., 1980; Van Montagu et al., 1983; Vanlarebeke et al., 1977). Later the

Gent research group also succeeded to transfer bacterial genes with the same methods into tobacco

plants. The first peer reviewed papers on a successful transformation were published by Herrera-Estrella

et al. including the senior authors Shell and van Montagu (Herrera-Estrella et al., 1983a; Herrera-Estrella

et al., 1983b). The genes, cloned from a Bacillus thuringiensis strain were expressed in transgenic

tobacco, which synthesized insecticidal proteins protecting the plants from feeding damage by larvae of

the tobacco hornworm (Fischhoff et al., 1987; Vaeck et al., 1987). Another early and successful attempt to

express Bt endotoxines Tobacco plants was published by (Barton & Umbeck, 1989; Barton et al., 1987).

1.1.3.4.2. Transformation with biolistic methods

The most successful and widely used transformation methods for maize and many other crops was

particle bombardment by the gene gun, developed by John Sanford and colleagues at Cornell University in

1984 (Klein et al., 1987; Sanford, 2000). Another success with electroporation was published by Fromm

(Fromm et al., 1986).

Also Rhodes achieved with electroporation transgenic protoplasts containing plasmid DNA with a

gene coding for neomycin phosphotransferase NPr II), from which the first transgenic maize

plants were grown (Rhodes et al., 1988). Two years later, it was the research group of Gordon-

Kamm (Gordon-Kamm et al., 1990) who created a reproducible system for the generation of

fertile, transgenic maize plants. Details from the recent historical account of Sussex (Sussex,

2008)

“These workers at DEKALB Plant Genetics (now Monsanto) bombarded cells from maize embryogenic suspension

cultures with tungsten particles coated with plasmids containing the selectable marker gene bar. This gene confers

resistance to the herbicide bialaphos, which was used to select transformed callus cells. Transformed calli were

shown to contain the integrated bar gene and to express the enzyme phosphinothricin acetyltransferase encoded by

bar. Fertile transformed plants were produced from the calli, and of 53 progeny tested, 29 had phosphinothricin

acetyltransferase activity. In other experiments, they cotransformed embryogenic suspension culture cells with a

mixture of two plasmids, one containing the bar gene and the other containing the gene encoding b-glucuronidase.

Regenerated plants expressed both genes. The authors concluded that ‘‘this system provides a new, powerful tool

for both the study of basic plant biology and the introduction of important agronomic traits into one of the world’s

major crops’’

See also Beegle, Shah and Songstad for further details and references (Beegle & Yamamoto, 1992; Shah et

al., 1987; Songstad et al., 1993).

In 1995 (Hill et al., 1995) followed with the first Bt maize, where the transgene has been inserted with

biolistic methods. Ever since then, the race to apply this technology and to make use of such genetically

improved pest resistant cultivars has been rapid and intense, it is summarized in a comprehensive way by

Moellenbeck and Sairam (Moellenbeck et al., 2001; Sairam et al., 2005).

22

A more specific view on Bt crops and their early development has been summarized by (Peferoen, 1997),

see also (Bohorova et al., 1999; Bohorova et al., 1995a; Bohorova et al., 1995b). Some early critical and

very detailed notes on the overall performance of Bt crops were given by (Hilder & Boulter, 1999; Obrycki

et al., 2001), they point to early developing difficulties and knowledge gaps of the eighties and early

nineties and offer thus indirectly the assurance, that performance and safety of the latest Bt crops is

nowadays considerably enhanced. Some more insight in the early development stories of the US is given

by (Koziel et al., 1993; Koziel et al., 1996).

New evidence shows, that the MON810 Yield Gard maize transgene situation is more complex than

anticipated:

Rosati, A., P. Bogani, A. Santarlasci and M. Buiatti (2008). "Characterisation of 3 ' transgene insertion site and

derived mRNAs in MON810 YieldGard (R) maize." Plant Molecular Biology 67(3): 271-281.<Go to

ISI>://WOS:000255414000006 AND http://www.botanischergarten.ch/Bt/Rosati-Characterisation-Mon810-publ-

2008.pdf

“The construct inserted in YieldGard (R) MON810 maize, produced by Monsanto, contains the CaMV 35S

promoter, the hsp70 intron of maize, the cryI(A)b gene for resistance to lepidopterans and the NOS terminator. In a

previous work a truncation event at the 3' end of the cryI(A)b gene leading to the complete loss of the NOS

terminator was demonstrated. The 3' maize genome junction region was isolated in the same experiment not

showing any homology with known sequences. The aim of the experiments here reported was therefore to isolate

and characterize a larger portion of the 3' integration junction from genomic DNA of two commercial MON810 maize

lines. Specific primers were designed on the 3' integration junction sequence for the amplification of a 476 bp

fragment downstream of the sequence previously detected. In silico analysis identified the whole isolated 3' genomic

region as a gene putatively coding for the HECT E3 ubiquitin ligase. RT-PCR performed in this region produced cDNA

variants of different length. In silico translation of these transcripts identified 2 and 18 putative additional

aminoacids in different variants, all derived from the adjacent host genomic sequences, added to the truncated

CRY1A protein. These putative recombinant proteins did not show homology with any known protein domains. Our

data gave new insights on the genomic organization of MON810 in the YieldGard (R) maize and confirmed the

previous suggestion that the integration in the genome of maize caused a complex recombination event without,

apparently, interfering with the activity of the partial CRY1A endotoxin and both the vigor and yield of the YieldGard

(R) maize.” (Rosati et al., 2008)

The absence of the NOS terminator in the MON810 transgene was already suggested by (Hernandez et

al., 2003)

1.1.3.4.3. The breakthrough to the widespread industrial production of Bt crops

Though with many of the earliest introduced transgenic traits the primary benefits go to growers, some of

those crops also provide secondary benefits to consumer health (Kershen, 2006). Bt corn is such an

example (there will be an extensive chapter dedicated to its proven low mycotoxin contents later in this

report). Transgenic maize is one of the most commonly grown transgenic crops in the world today. On a

commercial basis and regulated in numerous countries it contains genes from Bacillus thuringiensis,

encodes proteins toxic mainly to a very few members of the order Lepidoptera (Butterflies), Diptera (Flies)

and Coleoptera (Beetles). These include the common corn pests European corn borer Ostrinia nubilalis,

South Western corn borer Diatraea grandiosella, corn earworm Helicoverpa zea and corn rootworm

Diabrotica spp. More testing has revealed a limited toxicity to the following groups of insects: Orders of

23

Lepidoptera, Coleoptera, Hymenoptera, Homoptera, Dictyoptera, Orthoptera and Mallophaga, in addition

to nematodes (Strongylida, Tylenchida), protozoa (Diplomonadida) and Acari (mites) (Aronson & Shai,

2001; Ballester et al., 1999; Bravo et al., 2007; de Maagd et al., 1999; English & Slatin, 1992; Gahan et al.,

2005; Gomez et al., 2006; Gomez et al., 2003; Gomez et al., 2001; Gomez et al., 2007; Griffitts et al., 2005;

Griko et al., 2007; Heckel et al., 2007; Jurat-Fuentes & Adang, 2006; Munoz-Garay et al., 2006; Pardo-

Lopez et al., 2006; Potvin et al., 1998; Rang et al., 1999; Rausell et al., 2000b; Rausell et al., 2004b; Rausell

et al., 2004c).

The latest assessments come from ISAAA briefs (James, 2009) and a recent ENDS-Report (ENDS Report,

2008).

The benefits of Bt maize have been summarized several times in great detail by (Brookes, 2008b; Brookes

& Barfoot, 2008; Brooks et al., 2005b; Carpenter, 2001; Carpenter et al., 2001; Carpenter et al., 2004;

Sankula & Blumenthal, 2004; Silvers & Gianessi, 2001)

See also the chapter on the present and future development of new Bt maize traits, the research and

development within the companies and in public research is in full expansion, mainly for reasons to

overcome beginning Bt resistance to pests and also in order to enlarge the palette of target pests etc.

As a selected example on the situation of the industrial development a table is given here from the

comprehensive OECD report (OECD Consensus Documents, 2007): Consensus Document on Safety

Information on Transgenic Plants Expressing Bacillus thuringiensis - Derived Insect Control Protein. The

consensus document provides a lot of basic information about Bt maize toxins and their industrial use in

maize.

The table below demonstrates the various expression levels of bt crops across the industrially developed

traits:

24

Table 1 An example of variation in expression levels of δ -endotoxin in different maize constructsexpressing five different δ –endotoxins from (OECD Consensus Documents, 2007)

25

1.1.4. Chemistry, Biology and nomenclature of Bt toxins

If we want to understand the full advantages of the Bt insect tolerance strategy, we will have to analyse

some of the mechanisms on how the Bt toxins work:

The mode of action of the Bt toxins on a restricted set of insect species is complex and varies from case to

case of the specific protein employed. Most studies on toxicity mechanisms are only available since a few

years (Broderick et al., 2006). It is interesting to note that Bacillus thuringiensis requires enterobacteria in

the midgut of the target insect to unfold its insecticidal activity described already in detail by (Broderick

et al., 2009; Broderick et al., 2003; Broderick et al., 2000; Broderick et al., 2004; Crickmore et al., 1998;

Schnepf et al., 1998). The extremely targeted and restricted toxic impact on a few lepidopteran species is

the major reason for the success of the pest management with Bt proteins. See for more details in 1.1.5.

below and also in the chapter on food safety.

Numerous papers have been published on the nomenclature and chemistry of Bt toxins, and still today

there is some confusion about classification. Some basic papers have been summarized by de Maagd 2001

(de Maagd et al., 2001), including some very helpful illustrations on the Bt toxins.

Fig. 3 Above: Primary and tertiary structure of Cry toxins. (a) Relative lengths of Cry protoxins and position of the five conserved blocks, if present. More details on these conserved blocks, as well as the identification of three more blocks in the C-terminal ends of the longer protoxins, can be found in . The positions of the three domains of the activated toxin are

26

indicated for Cry1 and vary with the positions of blocks 2 and 3 for the other toxins. The remainder of the protoxin, consisting of short N-terminal part (20–40 amino acids) preceding the first domain and the C-terminal part following the third domain in the longer protoxins, is digested away by gut proteases during the activation process. (b) Three-dimensional structure of an activated toxin, Cry1Aa (Ref. 7). The toxin has three structural domains. Domain I (blue) is involved in membrane insertion and pore formation. Domain II (green) and domain III (yellow-red) are involved in receptor recognition and binding. Conserved block 1 is in the central helix of domain I, block 2 is at the domain I–II interface, block 3 is at the boundary between domains II and III, block 4 is in the central β-strand of domain III and block 5 is at the end of domain III. From (de Maagd et al., 2001)

Fig. 4 Transmission electron micrograph of a sporulating Bacillus thuringiensis (Bt) cell. δ-Endotoxins are produced as regularly shaped crystals (PB; protein body) – hence the name crystal (Cry) proteins – next to a spore (SP). The vegetative cell wall will eventually break to release the spore and crystal. The cell shown is approximately 2 μm long. From (de Maagd et al., 2001)

Fig. 5 Spores and crystals of Bacillus thuringiensis serovar morrisoni strain T08025 Microscopy by Jim Buckman from

http://commons.wikimedia.org/wiki/File:Bacillus_thuringiensis.JPG

27

De Maagd et al. (de Maagd et al., 2001) point rightly so to the fact that there also exist other toxic

substances in the arsenal of Bacillus thuringiensis, they might also be used in the development of new

insect resistant crops:

“Besides the Cry proteins (PB in Fig. 2), which are often the only focus, cytolysins (Cyt toxins), which act by a different

mechanism, are also found within the crystal. Bt produces various virulence factors other than the crystal proteins,

including secreted insecticidal protein toxins, α-exotoxins, β-exotoxins, hemolysins, enterotoxins, chitinases and

phospholipasesa (Hansen & Hendriksen, 2001). The spore itself contributes to pathogenicity, often synergizing the

activity of the crystal proteins (Bernstein et al., 1999; Bernstein et al., 2003; Johnson et al., 1998). All of these factors

might have a role in insect pathogenesis under natural conditions, helping the bacterium to develop in the dead or

diseased insect larvae, but the exact contribution of each factor is often unknown. Although the Cry proteins are

commonly referred to as ‘Bt toxins’, a few Cry proteins were found in Bacillus popilliae and in Clostridium

bifermentans” (Schnepf et al., 1998)

Some years later, the classification has been changed, it is now based completely on the molecular

structure of the growing number of proteins, first on the sequence of amino acids (Crickmore et al., 1998).

Today the classification is entirely built on phyogenetic relationships of the molecular structure, as shown

below in Fig. 4-6 from de Maagd (de Maagd et al., 2001). For the latest views on taxonomy and

nomenclature of the Bt proteins see the website with an updated database structure of Crickmore

(Crickmore et al., 2008) http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/.

Fig. 6 Scanning electron microscopy of spore (s) and crystals (c) of strains S285 (1); S447 (2); S479 3); S550 (4); S1255 (5); and Bti (6). From (Monnerat et al., 2005) Most of the Bt proteins toxic to Mosquitos have a round shape, in contrast to the Bt proteins usually inserted in Bt maize.

28

Separate phylogenetic analysis of the three domains of the active toxin is more likely to yield interesting

insights into how the extensive variety in structure and specificity came into existence (Bravo, 1997;

Crickmore et al., 1998). Such an analysis is shown in the following 3 figures below, domains I – III, with the

insect-order specificity shown as color-coded branches, redrawn from (Bravo, 1997). The role of the

protoxin-specific part in the evolutionary development of insect specificity should not be discarded

however, as the presence of a C-terminal extension might affect activity as mentioned above for Cry1Ba

and Cry7Aa. A classic example is the co-evolution of Spodoptera frugiperda Smith (Lepidoptera :

Noctuidae) populations from Latin America, parallel to its susceptibility with various Bt Cry toxins

(Monnerat et al., 2006)

A brief comparison of the trees for domains I and II shows that their overall structure is very similar,

having identical main branches that correspond to the current classification of the protoxins; that is, the

nearest neighbors of Cry1Aa are the other Cry1A toxins, the nearest neighbor of Cry1Ca is Cry1Cb, and so

on. Domains I and II of Cry9Aa behave differently; they don’t cluster with the other Cry9 toxins. The

classification of Cry9Aa is therefore primarily based on its homology with Cry9Ea in domain III and with all

Cry9 proteins in the protoxinspecific part31,32, suggesting that Cry9Aa evolved independently from the

other Cry9 proteins but obtained a Cry9-specific C-terminal extension more recently, perhaps by a

recombination event. See also the section of Cry9Ab in the food safety chapter.

29

Unrooted trees show the phylogenetic relationships of the separated domains I, II, and III:

Domain I

Fig. 7 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees were constructed basically as described earlier31, except that toxin alignments were made using DbClustal45, and updated with Cry protein sequences that were released since 1997. Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show similarities with the rest of the Cry protein family, see also Fig. 2(a). Shown are consensus trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program. Branches are color-coded according to the insect order specificity of the toxins, as far as is known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001)

30

Domain II

Fig. 8 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees were constructed basically as described earlier31, except that toxin alignments were made using DbClustal45, and updated with Cry protein sequences that were released since 1997. Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show similarities with the rest of the Cry protein family, see also Fig. 2(a). Shown are consensus trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program. Branches are color-coded according to the insect order specificity of the toxins, as far as is known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001)

31

Domain III

Fig. 9 Phylogenetic relationships of the separate domains. Unrooted phylogenetic trees of domains I, II and III of 79 known subgroups of Cry proteins obtained by the parsimony method. Trees were constructed basically as described earlier31, except that toxin alignments were made using DbClustal45, and updated with Cry protein sequences that were released since 1997. Cry6, Cry15, Cry22 and Cry23 sequences were not included because they do not show similarities with the rest of the Cry protein family see also Fig. 2(a). Shown are consensus trees resulting from 100 analyses using the bootstrapping tool and the CONSENSE program. Branches are color-coded according to the insect order specificity of the toxins, as far as is known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. From de Maagd (de Maagd et al., 2001)

32

As a summary the phylogenetic tree excluding the C-terminal parts can be found in fig. 2 of de Maagd (de

Maagd et al., 2003)

Fig. 10 Phylogenetic relationships between the entomocidal toxins. Four distinct homology groups have been identified within the family of toxins (Crickmore, 2003), three of which are shown here. The Cyt group is not shown. The branches are color coded according to insect order specificity of the toxin where known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. Solid and dashed double-headed arrows indicate associations of proteins in binary toxins or suspected binary toxins, respectively. Panel A: 3-domain Cry proteins. The C-terminal extension (if present) was not included for the phylogenetic analysis. Hence, proteins that have the same primary rank based mainly on sequence similarity between their C-terminal extensions may sometimes end up on different branches. Panel B: Binary and Bin-like toxins; Panel C: Mtx2, Mtx3 and Mtx2/3-related proteins. From de Maagd et al. (de Maagd et al., 2003)

Much more information can be found on a specialized website, maintained and updated by Neil

Crickmore on the following table, downloaded from his website, you can see the development of a VIP

nomenclature (Vegetatively expressed and secreted proteins), first reported in the mid 1990s, (Estruch et

al., 1996; Lee et al., 2003b; Yu et al., 1997) as separated from the CRY proteins (Crickmore et al., 2008),

there is a committee taking care of the new nomenclature, given on the website.

33

Crickmore (Crickmore et al., 2008) comments on the Bt nomenclature:

http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

“The current nomenclature, based solely on amino acid identity, allows closely related toxins to be ranked

together and removes the necessity for researchers to bioassay each new toxin against a growing series of

organisms. Biological specificity being a component of the orginal nomenclature. Roman numerals have

also been exchanged for Arabic numerals in the primary rank (eg CryIIIA became Cry3A). Each new toxin is

assigned a unique name incorporating four ranks. A completely new toxin might therefore be assigned the

name Cry50Aa1. For the sake of convenience we propose that the use of the quaternary rank (which

distinguishes between toxins that are more than 95% identical) is optional, only being used for the sake of

clarity. Note that quaternary ranks are assigned to each independently sequenced toxin gene, thus despite

the fact that some toxins have different quaternary ranks - they may in fact be identical.”

An updated list can bee downloaded at:

http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html

VIP proteins (Vegetative insecticidal proteins)

Since the vegetatively expressed, and secreted, VIP proteins were first reported in the mid 1990s (Estruch

et al., 1996) the rate of discovery of related toxins has increased in a similar fashion to that seen with the

Cry toxins a decade previously. Given that each discovering lab are adopting their own naming systems for

these toxins the idea was recently put forward that a nomenclature system, along the same lines used for

the Cry and Cyt toxins, be adopted. On this web page the results of applying the Bt Cry toxin

nomenclature analyses to the VIP and VIP-related toxins are presented. The following table shows the

proposed names for these toxins that result from such an analyses. A dendrogram showing the

relationship between each toxin can be found under

http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/viptree.pdf

The mode of action of Vip3A differs from that of Cry1Ab endotoxin

Vegetative insecticidal proteins (VIPs), produced during the vegetative stage of their growth in Bacillus

thuringiensis, are a group of insecticidal proteins and represent the second generation of insecticidal

trans-genes that will complement the novel δ-endotoxins in future (Wu et al., 2007).

34

1.1.5. Mode of action of Bt proteins and selectivity of the toxins

If we want to understand the full advantages of the Bt insect tolerance strategy, we will have to analyze

some of the mechanisms on how the Bt toxins work:

The mode of action of the Bt toxins on a restricted set of insect species is complex and varies from case to

case. Most studies on toxicity mechanisms are only available since a few years (Gomez et al., 2007; Heckel

et al., 2007; Soberon et al., 2007)

(Broderick et al., 2006). It is interesting to note that Bacillus thuringiensis requires enterobacteria in the

midgut of the target insect to unfold its insecticidal activity described already in detail by (Crickmore et

al., 1998; Schnepf et al., 1998). The extremely targetet and restricted toxic impact on a few lepidopteran

species is the major reason for the success of the pest management with Bt proteins.

In a summary, (de Maagd, 2007) writes: the large variety of natural Cry toxins, which each are active

against only a small number of species constitute an extensive arsenal of tools for insect control in

agriculture. An example of differences in specificity is shown in a table 2 below. It has been shown for

several insect species that the midgut epithelial cells contain receptors for Bt toxins. The presence of

specific receptors on the epithelial cells of different insect larvae, together with the proteolytic processing

under alkaline conditions, determines the specificity of Bt. Differences in sensitivity of insects for a

particular Bt toxin have been explained by differences in the concentrations of receptor molecules on the

epithelial cell membranes and by differences in affinity for particular Bt toxins.

Whereas previous binding studies have been done with fluorometric analysis which is not specific enough

(Hofmann & Luthy, 1986), using labelling with iodine-125 provides more precision (Hofmann et al., 1988a)

and specifity studies made progress (Hofmann et al., 1988b)

Some B. thuringiensis δ-endotoxins active against Manduca sexta (Tobacco Hornworm) compete for

binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or

coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on Plasmodiophora

brassicae vesicles. Together with other studies such as (Rie et al., 1989), these data provide evidence that

binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with

respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.

Earlier papers on the basics of Bt toxins have been published by (Crickmore et al., 1998; Schnepf et al.,

1998). According to (Federici & Siegel, 2007): As far as is known, Cyt proteins do not require a protein

receptor but, instead, bind directly to the nonglycosylated lipid portion of the microvillar membrane.

Once within the membrane, they appear to aggregate, forming lipind faults that cause an osmotic

imbalance that results in cell lysis (Butko, 2003; Nevels & Butko, 2007).

35

Following again (de Maagd, 2007): When ingested by a susceptible insect larva, Bt crystals are solubilized

by the high pH in the larval midgut and release proteins mostly varying in size between 70 and 130

kiloDaltons (kDa). These so-called protoxins (which themselves are non-toxic) are subsequently processed

by insect midgut proteases via stepwise degradation into true toxins of approximately 65 kDa (Figures 1

and 2 from (de Maagd, 2007)). The efficiency by which the crystals are solubilized and processed into the

corresponding toxins depends on the pH and the proteases present in the insect midgut. These protoxin

process control factors contribute to the specificity of Bt crystal proteins in addition to the receptor-toxin

interaction. The toxin proteins bind to the brush border membrane of midgut epithelial cells. After

binding the toxins presumably form pores in the cell membranes which disrupt the semi-permeability of

the membranes, thus leading to free ion transport. As a consequence the epithelial cells will swell and

eventually lyse. This part of the mode of action is still subject of debate, as there are also reports claiming

that binding by itself sets in motion a chain of intracellular events leading to cell death, here just a few

examples of the literature on protoxin binding from the last 3 years: (Abdullah et al., 2006; Chen et al.,

2007; de Barros Moreira Beltrao & Silva-Filha, 2007; Fabrick & Tabashnik, 2007; Fiorito et al., 2007; Griko

et al., 2007; Higuchi et al., 2007; Hossain et al., 2007; Ibiza-Palacios et al., 2008; Ikanovic et al., 2007;

Jurat-Fuentes & Adang, 2007; Krishnamoorthy et al., 2007; Luo et al., 2006; Oestergaard et al., 2007;

Shimada et al., 2006; Siqueira et al., 2006; Tang et al., 2007; Xu & Wu, 2008)

Fig. 11 Mode of action of Bacillus thuringiensis crystal proteins. Fig 1 from (de Maagd, 2007)

36

Finally, disintegration of the intestinal tract and death of the larva follows. Afterwards, germination of

spores and bacterial multiplication in the moribund or dead insect larvae can occur.

For the 130 kDa crystal proteins the toxic fragment roughly comprises the N-terminal half of the protoxin

molecule, whereas the C-terminal half is involved in crystal formation (Figure 2 A).

The N terminal half of the protoxin, when encoded by a truncated gene in heterologous systems such as

Escherichia coli or plants (see below), is as active as the toxin generated after cleavage of the crystal

protein by insect midgut juices in vivo.

Some of the 70 kDa crystal protein genes (e.g. cry3A), which occur naturally, resemble these engineered

truncated genes. This observation has been important for the engineering of other organisms, both

bacteria and plants.

The three-dimensional structure of the toxic fragment of several crystal proteins have been resolved, and

explained many of the biological features of this toxin. The N-terminal fragment consists of three domains

with specific roles in the toxin action (Figure 2 B).

The first domain consists of seven α-helices and is involved in pore formation. The second and third

domains contain ß-sheets and are involved in receptor binding.

37

Fig. 12. Primary structure of Cry proteins indicating the variety in length of the protoxin and the extent of the activated toxin after digestion of the protoxin by gut proteases, as well as the position of the three structural domains. Bar indicates number of amino acids. B. Tertiary structure of Cry1Aa toxin. Clearly recognizable are the three structural domains (Roman numerals). Fig 2 from (de Maagd, 2007)

A helpful illustration on how Bt toxins are binding is given by (de Maagd et al., 2001)

Fig. 13 Mode of action of Cry toxins. (a) After ingestion by the insect, crystals dissolve in the gut juice. (b) Gut proteases subsequently clip off the C-terminal extension in the longer Cry proteins (purple) as well as a small N-terminal fragment (yellow). (c) The resulting ‘activated’ toxin (i.e. the structure depicted in Fig. 2b) binds to receptors on the epithelial cell membrane, a process in which both domain II and domain III are involved. (d) Structural rearrangement of domain I might follow allowing a two-helix hairpin to insert into the membrane. (e) Inserted toxins form pores probably as oligomers, but the architecture of the pore is still unknown. From (de Maagd et al., 2001)

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this

bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high

degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces

during sporulation. (de Maagd et al., 2001) describe the properties of these toxin proteins and the current

knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the

38

active toxin and experimental results indicate how sequence divergence in combination with domain swapping by

homologous recombination might have caused this extensive range of specificities.

Another illustration of the mode of action of the insecticidal Bt protein is given by (Whalon & Wingerd,

2003)

Fig. 14 Mechanism of Cry protein toxicity. A: Ingestion of spores or recombinant protein by phytophagous larva. B: In the midgut, endotoxins are solubilized from Bt spores (s) and inclusions of crystallized protein. (cp). C: Cry toxins are proteolytically processed to active toxins in the midgut. Active toxin binds receptors on the surface of columnar epithelial cells. Bound toxin inserts into the cellular membrane. D: Cry toxins aggregate to form pores in the membrane. E: Pore formation leads to osmotic lysis. F: Heavy damage to midgut membranes leads to starvation or septicemia. From (Whalon & Wingerd, 2003)

39

There is a lot more literature about the specificity of mode of action of Bt toxins existing, but this is not

the main topic of this review, and therefore a small selection of citations may be enough:

(Abdul-Rauf & Ellar, 1999; Aronson & Shai, 2001; Aronson et al., 1995; Bravo et al., 2007; de Maagd et al.,

1999; de Maagd et al., 2000; Garcia-Robles et al., 2001; Gomez et al., 2003; Gomez et al., 2007; Gonzalez-

Cabrera et al., 2006; Heckel et al., 2007; Herrero et al., 2004; Hofmann et al., 1988b; Ibiza-Palacios et al.,

2008; Jenkins & Dean, 2001; Jurat-Fuentes & Adang, 2000, 2001, 2006; Jurat-Fuentes et al., 2004;

Karumbaiah et al., 2007; Keeton & Bulla, 1997; Nakanishi et al., 2002; Oestergaard et al., 2007; Rausell et

al., 2000a; Rausell et al., 2004a; Rie et al., 1989; Soberon et al., 2007; Vanrie et al., 1989; Whalon &

Wingerd, 2003; Wu & Aronson, 1992).

1.1.6. Bt crops: present and future developments, synthetic Bt genes

1.1.6.1. Present situation in Europe

According to the latest report of Brookes (Brookes, 2008b) Only Bt 176 and MON 810 – resistant to the

Lepidopteran pests Ostrinia nubilalis European Corn Borer (ECB) and Sesamia nonagroides Mediteranean

Stem Borer (MSB) have been planted in Europe to date. Currently, only varieties of the event MON 810

are available for cultivation: 41 varieties in Spain, 6 in France, 5 in Germany and 36 have been registered

on the EU Common Variety Catalogue (status of December 2006). In total, the area planted to Bt maize in

the EU was just below a still rather modest 65,000 ha in 2006, equivalent to approximately 0.6% of total

‘EU 25’ maize plantings (including forage maize area) (James, 2007).

The pipeline is much more important in numbers and innovation, see the table 2 below.

1.1.6.2. Recent and future developments of Bt maize breeding

Some of the later developments are summarized in the following table2 below, collated by (de Maagd,

2007).

Table 2 below: Commercial Bt-crops registered by the EPA in the United States up to the year 2001, including pending registrations. An event name refers to the particular individual transformed plant from which all others originate by crossing and multiplication. Additional stacked events are not included. For a complete and updated overview, visit the Agbios GM database at http://www.agbios.com/dbase.php ECB= European corn borer (Ostrinia nubilalis), SWCB= Southwestern corn borer (Diatraea grandiosella); MCB= Mediterranean corn borer (Sesamia nonagroides); BCW=Black cutworm (Agrotis ipsilon); FAW=Fall Armyworm (Spodoptera frugiperda); CRW=Corn rootworm (Diabrotica virgifera virgifera); TBW= Tobacco budworm (Heliothis virescens), CBW=Cotton bollworm (Helicoverpa zea or H. armigera), PBW=Pink Bollworm (Pectinophora gossypiella); CPB= Colorado potato beetle (Leptinotarsa decimlineata). *These contain Bt genes for other proteins than the 3-domain toxins.

40

An example of a most recent cloning of a new Cry1Ab toxin for Asian pest insects demonstrates a

constant flow of scientific innovation, the table above needs updating (Xue et al., 2008).

Xue et al. cloned a novel cry1A from Bacillus thuringiensis and expressed it in the B. thuringiensis

acrystalliferous mutant HD73_. The gene, designated cry1Ah1, encoded a protein with a molecular weight

of 134 kDa. The toxin expressed in the mutant revealed high toxicity against Ostrinia furnacalis,

Helicoverpa armigera, Chilo suppressalis, and Plutella xylostella, which makes it a potential candidate for

insect biocontrol, for more details see fig.3.

41

Table 3 All the strains and plasmids used or constructed (Xue et al., 2008)are listed in Table fig. 15. Escherichia coli JM110 was used for plasmid propagation, and SCS110 was used to produce nonmethylated plasmid DNA for the transformation of B. thuringiensis. Escherichia coli strains were grown in Luria–Bertani medium (LB) at 37 1C, while B. thuringiensis strains were grown in peptone-beef extract (PB) medium (0.5% peptone, 0.3% beef extract) at 30 1C. Liquid cultures were grown in a rotary shaker at 230 r.p.m. Antibiotics were added to autoclaved media as follows: ampicillin, 100 mgmL

-1 (for E. coli); erythromycin

10 mgmL-1

(for B. thuringiensis), Table 1 from (Xue et al., 2008)

Recent developments within the Bt insect resistance strategies have also been summarized by Singh et al.

(Singh et al., 2004): Enhanced efficacy of Bt Cry proteins was achieved by creating fusions between

domain III of Cry1Ac and domains I and II of various other Cry1 proteins (Naimov et al., 2003) Similarly, a

hybrid toxin was developed against Spodoptera litura, a polyphagous pest that is tolerant to most Bt

toxins (Baute et al., 2002). A poorly active domain in the naturally occurring Cry1Ea toxin was replaced

with a highly homologous 70 amino acid region of Cry1Ca in domain III. The synthetic gene was further

42

optimized for high-level expression in plants and was introduced into tobacco and cotton plants.

Resulting plants were found to be extremely toxic to Spodoptera litura at all stages of larval development.

A hybrid Bacillus thuringiensis gene was constructed using a synthetic and truncated cry1Ba gene as the

scaffold for inserting part of cryIIa gene encoding domain II (Baute et al., 2002). Transgenic potato plants

expressing this hybrid toxin were resistant to several insect pests, including both Coleoptera (Colorado

potato beetle) and Lepidoptera (potato tuber moth and European corn borer). As the target receptor

recognition of this hybrid protein is expected to be different from Cry proteins currently in use to control

these pests, this strategy provides new opportunities for resistance management studies involving

multiple transgenes in crops.

According to Cécile Rang et al. (Rang et al., 2004), who studied the binding and competition of five Bacillus

thuringiensis toxins - Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, and Cry1E – it is possible to develop a tropical Bt

maize resistant to several stem borers. On Spodoptera frugiperda, Cry1Ab and Cry1Ac compete for the

same binding site; Cry1Ba and Cry1Ca compete for a second binding site. Cry1Ea recognizes a third

specific binding site in S. frugiperda and does not compete with any of the other toxins. On Diatraea

grandiosella and D. saccharalis, Cry1Ac competes with Cry1Ab and not with Cry1Ba and Cry1Ca.

Cry1Ba and Cry1Ca recognize each a specific binding site and do not compete with any of the other four

toxins. Cry1Ea does not recognize any binding site on Diatraea species. Combinations of toxins studied by

the authors are proposed to develop transgenic maize resistant to the three stemborers while allowing

resistance management. For an evolutionary view about those relationships see (Monnerat et al., 2006)

and comments in 1.1.4 below fig. 4.

A comprehensive study on future developments in insect resistant transgenic crops has been published by

Christou et al. 2006 (Christou et al., 2006). The authors concentrate on agronomically feasable traits for

the near future and distinguish (besides giving suggestions how to prevent resistance) on:

Second generation transgenic plants: transgenic plants containing, in addition to the selectable

marker, one or two transgenes encoding simple agronomic traits (such as pest and herbicide

resistance).

Third generation transgenic plants: transgenic plants that contain multiple transgenes targeting

multiple pests and diseases, often in a temporal or spatial manner. These might also express

additional value-added or agronomic traits.

The word is on targeting phloem feeding insects using the root phloem-specific promoter AAP3 (Okumoto

et al., 2004) and many other highly interesting examples, supported by an extensive list of references.

Future developments point to gene pyramiding, the combination of useful transgenes and management

systems, this is why Onishi et al (Onishi et al., 2005) propose novel multiplex polymerase chain reaction

(PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within

a single reaction. Another possibility has been suggested by (Ammann, 2009 in press ): An appropriate mix

of seeds, each trait with a specific resistance against pest insects, in a combination adapted to local needs.

43

Another trend is emerging: The search for novel toxic proteins with selective characters which could serve

in future as novel endotoxins, the search comprises toxins of organisms with known toxins which might

show potential: Sea anemones, spiders, scorpions etc. Although there are certain communication

problems and a lot of safety research to be done first, the work has started, as manifested in the following

publications: (Bosmans & Tytgat, 2007; Gordona et al., 2007; Gruber et al., 2007; Gurevitza et al., 2007;

King, 2007; Nicholson, 2007a; Nicholson, 2007b; Rohou et al., 2007; Whetstone & Hammock, 2007).

Karlova et al. (Karlova et al., 2005) investigated the role of domain III of Bacillus thuringiensis- endotoxin

Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac

with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this

way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic, promising

prospects for future commercialization and resistance management of Bt crops.

An outlook into the future risks related to new technologies has been given by (Karlova et al., 2005): in

the survey, involving more than 450 researchers, which has then been discussed properly, GM crops of

the first generation play only a minor role.

In a recent paper, Gatehouse et al. (Gatehouse, 2008) outline the future of Bt crop development: Since

not all pests are adequately targeted by the Bt toxins used at present, and there is still a need to develop

solutions to specific problems, such as resistance to sap-sucking pests and pests of stored products, some

developments to the basic Bt strategy and selected alternative methods for engineering insect resistance

are outlined: Basically, it is about:

Improving expression levels of Bt toxins

Gene stacking (multiple Bt toxins)

New Bt toxins

Here only the last possibility about new Bt toxins is given as an illustrated example: the aim is to change

the nature of the Bt toxin itself, in order to change its range of toxicity:

44

Mutagenesis of the toxin-receptor, example 1:

Residues mutated in Domain II

Fig. 15 Engineering specificity in a three-domain Cry toxin; mutagenesis of the toxin-receptor interaction loop in domain II. Threedimensional structure of Cry3A (1dlc; RCSB) is shown in ribbon format. Domain I (helices) is at top right, and domain II (sheet structure) is at bottom left. Domain III (carbohydrate-binding domain; sheet structure) is behind the other domains, central in this view. Residues mutated (Wu et al., 2000) to increase toxicity toward yellow mealworm (Tenebrio molitor), Colorado potato beetle, and cottonwood leaf beetle (Chrysomela scripta) are shown in ball-and stick representation. From (Gatehouse, 2008)

One of the current commercial transgenic maize variety with resistance to corn rootworm, MON863,

expresses a modified version of the Bt Cry3Bb1 toxin (Vaughn et al., 2005). Unmodified Cry3Bb1 is active

against a number of coleopteran species, but toxicity toward Western corn rootworm was not sufficient

to give adequate protection at levels of expression achievable in maize. A large number of variants of the

native Cry3Bb, incorporating a series of specific mutations that aimed to improve the channel-forming

ability of the toxin, were produced and screened for activity (English et al., 2003). Mutations (see Fig. 11)

were carried out to: (1) increase hydrophobicity of the protein in regions in domain I containing sheets of

bound water molecules and in loop regions; (2) increase the mobility of the channel-forming helices in

domain I by disrupting hydrogen-bond formation; (3) increase the mobility and flexibility of loop regions

45

in domain I; (4) alter potential ion-pair interactions and metal-binding sites; and (5) reduce or eliminate

binding to carbohydrates in the insect gut by mutation of a loop region between domains I and II. The

toxicity of the protein towards the corn rootworm was increased approximately 8-fold, giving a product

that could be expressed at levels sufficient for adequate protection against rootworm.

Mutagenesis of the toxin-receptor, example 1:

Residues mutated in Domain I and in region linking Domain I and II.

Fig. 16 Engineering specificity in a three-domain Cry toxin; mutagenesis to improve channel-forming ability. Three-dimensional structure of Cry3Bb (1ji6; RCSB) is shown in ribbon format in the same view as Figure 1. Residues mutated (English et al., 2003) to increase toxicity toward corn rootworm are shown in ball-and-stick representation. Mutations are made in helices of domain I and in the region linking domains I and II. The mutation sites shown are taken from the most active toxin produced; a range of other sites for mutation were explored. From (Gatehouse, 2008)

46

1.2. Fundamental statements about risk assessment,

particularly related to Bt maize.

1.2.1.The situation in risk and benefit assessment of Bt maize worldwide and in Europe

The Bt maize approvals of EPA have been contested by Greenpeace, but successfully rebutted by EPA, and

factually Greenpeace has withdrawn the petition subsequently, since chances were minimal to be

successful: Scientific arguments against the petition were overwhelming, for the full documentation see

Fox (Fox, 2000), with additional comments (Fox, 1995, 1997, 2003a, b), further comments under USEPA

(US Environmental Protection Agency (USEPA), 2001). In a reaction to two papers on possible hazards for

non-target arthropods (US Environmental Protection Agency (USEPA), 1995) subsequent field studies (all

cited in the chapter on the monarch butterfly), the EPA has released an extensive documentation by

stating that Bt crops are safe for commercialization (Wilson et al., 2005), thus confirming that the earlier

risk assessment (Burkness et al., 2002) of the same agency was correct.

The subsequent reality in politics and regulation looks less positive, the transatlantic rift is becoming over

the years more and more conspicuous. It is mainly based on a fundamental difference between the US

and Europe in regulation. Whereas the US insists on an assessment of the risks of GM crops in reality,

compared to all other crops, Europe concentrates – without giving proper scientific reasoning, on the

view that GM crops need separate and special attention, this will be discussed in the chapters on

environmental risk assessment, here just a few publications explaining this rift: (Bonny, 2003; Hodgson,

2008; Miller, 1994a, b; Miller, 2007; Nature-Editorial, 1992, 2007; Ramjoue, 2007a, b)

here just the controversy in some short impressions about the latest decision of Ilse Aigner, minister of

agriculture of Germany: A purely political negative decision related to the approval of Bt maize is

camouflaged with so called factual arguments. The author just got the official list of scientific publications

which are the fundament of the negative decision. ALL paper which were presented are questionable, of

low quality in expermimental planning and statistical interpretation. Since the political debate is still

ongoing, the remarks here may suffice. It should be made clear, that the Web of Science offers with a

short search related to keywords as MON810 and MON863 reveals more than two hundred publications

in scientific journals, not a single one with clear cut negative results which could provoke a negative

regulatory decision.

There are a lot of publications available, a short search in the Web of Science has revealed over 200

publications related to the two keywords MON81 and MON863.

http://www.botanischergarten.ch/Bt-Bibliography/Bibliography-MON810-MON863-WOS-20090416.pdf

1.2.2 The agricultural reality:

A recent survey by (Gatehouse et al., 2002) in one of the main cultivation areas of the American Midwest

produces a very positive picture of many years of agronomic practice. Pilcher et al. (Pilcher et al., 2002)

also demonstrated in the same region, that besides clear cut and persistent benefits in economy and

47

environmental impact, there is also a certain consistency in performance, regardless of the pest complex,

pest density, or geographic location in the Midwestern US. Further positive performance is also

summarized in numerous surveys and comments (Ammann, 2005; Betz et al., 2000; Chapman & Burke,

2006; Conner et al., 2003; Dale, 2002; de Maagd, 2002; Gray, 2004; Nap et al., 2003; Navon, 2000;

Noteborn et al., 1995; Obrycki et al., 2001; Romeis et al., 2007; Sanvido & Romeis, 2007; Sanvido et al.,

2006; Widmer, 2007) and in (Christou et al., 2006; Manyangarirwa et al., 2006; Mehlo et al., 2005) . It will

be important to consider seriously all those positive experiences from abroad also in Europe.

Overall, there are also many earlier literature reviews confirming that the use of Bt crops can be

considered safe (Bajaj, 1994; Freeling, 1994; Gay, 1999; Peterson & Bianchi, 1999).

In a reference manual, now published by the Springer Verlag, a whole range of experienced authors give

broad insight in the history of maize breeding: (Romeis et al., 2008b).

One review from Indian ICRISAT researchers deserves special mention, insofar as Sharma and his co-

authors (Sharma et al., 2004) strive to give more critical views about the downside of GM crops resistant

to pest insects. It is a plea to develop regional breeding strategies in order to avoid future problems with

Bt resistant pest insects. Also they call for more research to adapt Bt maize to tropical climates. It should

also be mentioned that the authors cannot come up with any concrete negative effect or event, and they

show nicely the great future potential of the whole pest resistance strategy with transgenic crops.

In dozens of papers researchers have dealt with future resistance problems, and solutions are available in

the case that Bt-resistant pests will develop. A separate report will deal with the issue. Detection methods

are summarized in a recent paper (Ferre et al., 2008), who itself offers the simultaneous detection of

eight events of Bt maize. In the recently published book, (Romeis et al., 2008c) their chapter presents a

broad and comprehensive survey on the relevant questions and management of upcoming Bt resistant

pests, see the chapter 1.1.2.2. on resistant management.

1.2.3. And what about Europe ?

There are considerable differences in the culture of risk assessment between the United States and

Europe, summarized by (Drobnik, 2007). A minority of European ecologists tries to re-invent the wheel in

risk assessment of crops and asks questions related to the safety of Bt crops which have long been

answered in science with dozens of papers published in peer reviewed journals and also in the agricultural

reality of the American Midwest. In addition, some ecologists apply the wrong principles to the ecological

risk assessment of GM crops: Used to work in pristine or slightly disturbed natural habitats to assess the

ecological science of ecosystems and food webs, they hardly realize that dealing with agriculture means to

cope up to a totally different dynamics of agricultural ecology with all its rapid changes and crop rotation

and succession from year to year (Ammann et al., 2004).

Delayed start of GM crop cultivation in Europe

In Europe the start of GM crop cultivation has been delayed also for other reasons, mainly political ones, a

recent account about the basically still negative regulatory situation in Europe has been given by

(Eurobarometer, 2006): Its time to relax on GM crop regulation, particularly on the first generation of GM

48

insect resistant crops: There is enough experience gained during 10 years of GM crop applications to

seriously evaluate the ratio of risk to benefit and reduce the existing regulation in Europe – but it is still

difficult to evaluate benefit and risk when GM crops are not used. The precautionary approach (= legal

name, not precautionary principle) is applied exclusively to GM crops, never or rarely to alternative

solutions of, e.g., pest biocontrol.

Europe risks in a dramatic way to lag behind, with serious consequences in agricultural production, trade

and welfare overall. The question is, whether it is still possible to avoid serious negative effects stemming

from this overall restrictive agricultural policy related to technological progress.

As early as 1992 many US authorities asked seriously whether there is enough reason to single out the

transformed crops: (Eurobarometer Special, 2008). The time will soon come when we will have to rethink

the process oriented regulation of the first generation GM crops which are rapidly spreading all over the

world.

As we will see below (2.2.), the biosafety research situation regarding to peer reviewed publications has

dramatically improved in the last few years – in particular with Bt crops – its time for a re-assessment. It is

also time to delve into future risk posed by biotechnology and other new technologies (see 1.1.4.)

The Eurobarometer 2005 shows how propaganda inseminates public opinion with shameful nonsense (Li

et al., 2004). However the situation in 2007 looks slightly better (Demont & Tollens, 2004; Farinos et al.,

2004; Lorch & Then, 2007), the risks coming from GM crops rank considerably below some of the other

environmental risks. Voices asking for change of the “old” politics of rejecting, based on claims of negative

consumer attitude, not on science, are getting stronger, they come from the parties within the European

Parliament, British ACRE, EuropaBio, the European Federation of Biotechnology, and from Commission,

scientists and other European and non-European sources, see also (Afolabi et al., 2007; Bohorova et al.,

1999; de Groote et al., 2004; Gouse et al., 2005; Gressel et al., 2004; McGeoch & Pringle, 2005; Thomson,

2002).

In Europe, particularly in Spain, the trends are recently more promising and this will continue in the same

manner (Sutherland et al., 2008). In Africa the prospects look positive as well, and as soon as regulatory

problems are solved outside South Africa, proliferation of Bt maize will find only a few obstacles (Christou

et al., 2006).

It is also time to throw a more detailed look at the difference of the risk assessment and risk perception

cultures between Europe and the Americas: A comparative case study of genetically modified corn in the

United States of America and European Union done by (Guehlstorf & Hallstrom, 2005) reveals some hard

facts: The way governments regulate modern biotechnology is not necessarily a reflection of how their

political culture perceives the new scientific technology, it is rather how their existing regulatory structure

can create a political culture of acceptance or rejection for contested technological advancements. This

comparative study casts doubt on interpreting agricultural biotechnology decisions solely on equations of

risk analysis, and offers a detailed cultural analysis of the regulatory differentiation of modern agricultural

policy between America and Europe. It is in this context laudable that the European Commission wants to

49

put more weight on the scientific risk assessment process, trying to clearly separate science and politics –

indeed a difficult process.

Apart from such subtle and difficult transatlantic thought it is important to realize, that on a global level,

the ongoing debate in science on GM crops suffers from a lack of awareness about the basic strategic

approaches in risk assessment as we will see below (1.2.)

1.2.3. There are three major reasons, why we should develop a critical view, strictly based on

scientific data, when we judge risk assessment research and the resulting scientific

publications related to Bt crops.

1.2.3.1..Assessment of the impact of GM crops should also take into account the benefits and

balance it against the risks.

Article 19 of the CBD http://www.cbd.int/convention/articles.shtml?a=cbd-19 which is the basis of all

biosafety assessment activity and which is legally also the root article in the CBD on which the Cartagena

Protocol is building upon, makes it crystal clear that benefits of biotechnology should be taken into

account – this is already obvious from the title of Article 19: Handling of Biotechnology and Distribution of

its Benefits. Unfortunately, the negotiations of the Cartagena Protocol and the drafted and later agreed

articles concentrate nearly exclusively on the risk side, thus clearly not following the original task given by

Article 19 of the CBD. Many opponents of the GM crop technology now call upon this fact and argue, that

in the negotiated part of the Cartagena Protocol there is no explicit mention of benefits. But this is a truly

tautological argument, since it is obvious that the negotiations did not follow the legal task given by the

CBD in Article 19.

Conclusion: there is no legal basis to exclude the benefits in the risk assessment prescribed by the

Cartagena Protocol and to balance it out with the risks of the new technology.

A typical example on how economic benefits can be linked directly to the damage of the European corn

borer has been given by:

Baute, T. S., M. K. Sears and A. W. Schaafsma (2002). "Use of transgenic Bacillus thuringiensis Berliner corn

hybrids to determine the direct economic impact of the, European corn borer (Lepidoptera : Crambidae) on field

corn in eastern Canada." Journal of Economic Entomology 95(1): 57-64.<Go to ISI>://WOS:000178371100008 AND

http://www.botanischergarten.ch/Bt/Baute-Direct-Economic-2002.pdf

“Transgenic corn expressing Bacillus thuringiensis Berliner (Bt corn) (Maximizer and Yieldgard hybrids, Novartis

Seeds), non-Bt isolines and high-performance.(check) hybrids were evaluated for European corn borer, Ostrinia

nubilalis. (Hubner), damage and grain yield in commercial strip plots across Ontario in 1996 and 1997. Bt corn

hybrids reduced stalk tunneling damage by 88-100%. In 1996, minimal damage was found in locations where only

one generation of European corn borer occurred per year. Bt corn proved its greatest potential for reducing the

50

number and length of, cavities below the primary ear in locations where two generations of European corn borer

were present. A yield response to using Bt hybrids only occurred when levels of tunneling damage exceeded 6 cm in

length. European corn borer infestations resulted in a 6 and 2.4% reduction in yield for 1996 and 1997, respectively,

when Bt hybrids were compared with their non-Bt isolines. A linear relationship was found between tunnel length per

plant in centimeters (x) and yield protection (%) obtained from using Bt corn (y) (y = 1.02 + 0.005x, r(2) = 0.7217). At a

premium of $34.58 Canadian (CDN) per hectare for Bt corn seed, an infestation of at least 6 ern of corn borer

tunneling per plant was required to break even at a market price for corn of $2.50 per bushel CDN. During the period

of study, low infestations (0-2 cm) of European corn borer occurred at 25% of the locations assessed, moderate

infestations (4-6 cm) occurred at 42% of the locations, and high infestations (>6 cm) occurred at 33% of the locations.

At a corn price of $3.00 per bushel CDN and seed premiums of $34.58 per hectare CDN, 5 ern of tunneling was

required for a return on investment in Bt seed, comprising only 55% of the growers in the study. With infestations of

more than 6 cm of tunneling occurring only 33% of the time, a return on seed investment would be realized in only

one of three growing seasons. At a seed premium of $24.70 per hectare CDN per year, at least $74 per hectare CDN

in the year of infestation would be required to make up for the two years of no. return. In this study, a $74 per

hectare CDN return at a corn price of $9.26 per hectare CDN with >16 cm of tunneling damage would have occurred

only 7.3% of the time”. (Baute et al., 2002)

It is beyond the scope of this report to add an extensive chapter on the benefits of Bt crops, there is only

room for one of the latest and most comprehensive studies which has been recently updated by Brookes:

(Brookes, 2007, 2008a; Brookes, 2008b).

1.2.3.2.Assessment of the impact of GM crops should also compare to non GM crops

An early and insightful comment has been given by Pimentel and Raven 2000: (Pimentel & Raven, 2000),

here the conclusions:

Pimentel, D. S. and P. H. Raven (2000). "Bt corn pollen impacts on nontarget Lepidoptera: Assessment of

effects in nature." Proceedings of the National Academy of Sciences of the United States of America 97(15): 8198-

8199.<Go to ISI>://WOS:000088273900003 AND http://www.botanischergarten.ch/Bt/Pimentel-Impacts-nontarget-

Lepidoptera-2000.pdf

In conclusion, the introduction of resistance factors in corn through the application of genetic engineering

technology is an effective strategy to reduce the extensive crop damage caused by corn rootworm and European

corn borer populations. Yet, the diverse reactions of the different Lepidoptera to corn genotypes with varying levels

of Bt endotoxin in their pollen signal that additional testing and development is required to limit or entirely prevent

damage to nontarget butterfly and other beneficial insect species.

Although Bt corn pollen under certain circumstances has the potential of adversely affecting the population levels of

Monarch butterflies and other nontarget Lepidoptera, we consider these impacts to be minimal when compared

with habitat loss and the widespread use of pesticides throughout the ecosystem. Broad agricultural investigations

should focus on improving pest-management strategies in the context of sustainability and productivity to reduce

the use of pesticides and make agriculture more environmentally and economically sound. In addition, broadly

based research should continue to be conducted on the reasons for the decline of populations of Monarch

butterflies and other species, and remedial steps should be directed to the more important of these factors as they

are identified (Pimentel & Raven, 2000)

51

It is a scientific imperative that risk assessment studies with GM crops should compare to non-GM crops,

including the agronomic practice linked to the traits. This is what it is all about in agriculture: Innovation in

crop breeding and agricultural management strives to improve production step by step, as it has always

been done, it would be ahistoric to overlook this aspect. But this includes also the view that improvement

steps cannot be judged along the scale of absolute certainty and absolute safety. This is seemingly a

modest and unquestionable principle, but strangely enough only a very few studies are meeting those

criteria in comparison and judging improvement.

An often neglected principle should be to compare properly GM crops with non-GM crops, in order to

evaluate natural variation in both cases. Only by doing this you can get a proper balance and judgement

about any kind of risk related to GM crops. In a well planned study this has been done by (Turlings et al.,

2005)

Turlings, T. C. J., P. M. Jeanbourquin, M. Held and T. Degen (2005). "Evaluating the induced-odour emission of a Bt

maize and its attractiveness to parasitic wasps." Transgenic Research 14(6): 807-816.<Go to ISI>://000233628500004

AND http://www.botanischergarten.ch/Bt/Turlings-Evaluating-Induced-Odor-2005.pdf

“The current discussion on the safety of transgenic crops includes their effects on beneficial insects, such as

parasitoids and predators of pest insects. One important plant trait to consider in this context is the emission of

volatiles in response to herbivory. Natural enemies use the odours that result from these emissions as cues to locate

their herbivorous prey and any significant change in these plant-provided signals may disrupt their search efficiency.

There is a need for practical and reliable methods to evaluate transgenic crops for this and other important plant

traits. Moreover, it is imperative that such evaluations are done in the context of variability for these traits among

conventional genotypes of a crop. For maize and the induction of volatile emissions by caterpillar feeding this

variability is known and realistic comparisons can therefore be made. Here we used a six-arm olfactometer that

permits the simultaneous collection of volatiles emitted by multiple plants and testing of their attractiveness to

insects. With this apparatus we measured the induced odour emissions of Bt maize (Bt11, N4640Bt) and its near-

isogenic line (N4640) and the attractiveness of these odours to Cotesia marginiventris and Microplitis rufiventris, two

important larval parasitoids of common lepidopteran pests. Both parasitoid species were strongly attracted to

induced maize odour and neither wasp distinguished between the odours of the transgenic and the isogenic line. Also

wasps that had previously experienced one of the odors during a successful oviposition divided their choices equally

between the two odours. However, chemical analyses of collected odours revealed significant quantitative

differences. The same 11 compounds dominated the blends of both genotypes, but the isogenic line released a

larger amount of most of these. These differences may be due to altered resource allocation in the transgenic line,

but it had no measurable effect on the wasps' behaviour. All compounds identified here had been previously

reported for maize and the differential quantities in which they were released fall well within the range of

variability observed for other maize genotype”s. (Turlings et al., 2005)

“Some GEOs are expected to provide environmental benefits, as outlined in Box 1. However, in this report, we focus

more on potential environmental risks of GEOs than on their benefits for two reasons: risks are a more immediate

concern for ecologists, regulatory agencies, and the public, and many environmental benefits have yet to be

developed or rigorously documented.”

52

The role of ecologists is not only to point (rightly so) to potential and real risks of using GMOs, it is also

likewise important to balance the risks out against the benefits – since otherwise we are just barring

progress from agriculture – and in times of growing famine and environmental pressure we have no

reason to stop such century old procedures from creating stepwise agricultural innovation. Logically we

should also evaluate the risk of delaying the cultivation of GM crops in Europe. But there is more to say

about the complexity of the task in the next paragraph:

1.2.3.3.Dealing with complex structures of parameters in risk assessment calls for scientific

scrutiny done by experts

The way risk assessment is handled in the United States is less controversial than in Europe, because it is

done by strong agencies with good funding and an old scientific tradition (EPA, FDA etc.). Risk assessment

gets ample support from independent governmental scientific bodies, and it is also good to see that US

politicians keep to the rule that scientific judgment should first be done by the experts, later politicians

have the task to implement risk/benefit assessment decisions in society. In Europe, this rule has been

broken several times on a national and European level.

Complex structures in risk assessment parameters should be carefully scrutinized by experts who know

their science, and there is no reason, why the complex debate should deteriorate into something like a

self-service supermarket of arguments, where everybody is allowed to shop along the lines of his own

world view.

We address in a separate chapter 1.2.4. specifically the false dynamics generated by the DG environment

of the EU, who in crucial questions ignores the scientific and independent judgment of the EFSA, a

process, which should be criticized and corrected.

Even members of the German government call lately for more respect for the science on GM crops,

before major political decisions are provoked. It has to be admitted, that it is often difficult to still keep

oversight of the complexity of the debate. Some papers deal with the complex structure of agricultural

biodiversity and the resulting food webs, without coming to workable conclusions (Brooks et al., 2005a;

Perry et al., 2004).

A reasonable way forward would be to come to a consensus of pre-selected nontarget organisms for

monitoring. Recently, those thoughts have been taken up and reinforced by the now published ECOGEN

studies: (Andersen et al., 2007; Birch et al., 2007; Bohanec et al., 2007; Cortet et al., 2007; de Vaufleury et

al., 2007; Debeljak et al., 2007; Griffiths et al., 2007; Krogh & Griffiths, 2007; Krogh et al., 2007; Wesseler

et al., 2007)..These studies demonstrate how it is possible to deal in a scientific way with agricultural

complexity, without loosing a pragmatic, solution oriented vision.

It is ironic to see that ecological complexity leaves a free room for argumentation in all directions, a

complex ecosystem can thus be compared with a supermarket of arguments of all prices.

53

1.2.4.Example of a complex structure of risk assessment, an analysis related to the scientific

questions alone: The British Farm Scale Experiments

To give an well known example, even the British Farm Scale Experiments (FSE) have become subject to an

ardent debate on the outcome, after having been much praised in the beginning, and although they have

been planned over years according to highest scientific field research standards and agreed upon by all

participating parties (Perry et al., 2003). Indeed, the committee decided on a layout which seemingly

compares systematically GM-crops with non-GM-crops (herbicide tolerance). But another important rule

is neglected: The rule to adjust to the agricultural management practice of the used transgenic and non-

transgenic varieties for the field experiments. Herbicide tolerant GM crops need to be treated differently

from the non-transgenic varieties, more details on future perspectives in agricultural management in

(Hails, 2002) . So, actually, the FSE are asking the wrong questions, i.e. comparison of non-GM-crops with

GM-crops, rather they give precise answers to the impact of different kinds of herbicides (Ammann,

2005; Chassy et al., 2003). Another obvious lacune is that there are no yield data available. And on top of

this, the results are often communicated incorrectly by the scientific community: The question was,

whether biodiversity would be harmed with GM crops, and in a first publication round it was summarized,

that only GM maize demonstrated better results related to biodiversity, Beet and Canola would do worse.

And maize would do only better, because broadband pesticides were used like Atrazine in the non-GM

crop controls, which is likely to be banned soon. But it was demonstrated through additional data

analysis within the FSE that this is the wrong conclusion and that maize will still do better with other

herbicides as well (Perry et al., 2004). Later, in a more thorough data analysis, it was demonstrated, that

also GM beet does better, related to biodiversity: (May et al., 2005). The case gives evidence on how

complex environmental questions can be related to the performance of GM crops. See also a similar

study published later: (Sanvido et al., 2006). Comparable conclusions in a recent and thorough analysis of

the FSE are drawn by Bohan (Bohan et al., 2005). The question remains as to how best to manage

biodiversity at a landscape scale, but there may well be opportunities to trade-off the convenience of

some GM systems with the more intensive habitat management required to deliver specific biodiversity

targets. Ecological research has provided much information on the relationship between habitat

management and biodiversity, and it is likely that specific biodiversity targets will be reached more

efficiently by the implementation of specific measures rather than the general promotion of a less

intensive agriculture (Dollaker & Rhodes, 2007)

It also should be mentioned that there is no doubt that it is imperative to involve knowledge and

conclusions from the social sciences and philosophy to fulfil the task of modern risk assessment, but this is

not the task of this review, on the contrary, the reviewer is concerned that science arguments are more

and more pushed into the background, when they should be intricately interwoven into decision making

processes taking up the notion of the ‘Symmetry of Ignorance’ (Fischer, 2000) and making sure, that a

professional discourse is initiated in the spirit of the ‘Systems Approach’ (Ammann & Papazova Ammann,

2004).

54

1.2.6.Conclusion: Proposed procedure for future risk assessments

In this report, those basic thoughts (about scientific data and risk assessment) will be kept in mind when

we review the papers on Bt crops. The study here builds on extensive literature searches, including more

than 5800 scientific papers and major reports and books, and through intensive search work and

networking, the author of the study has downloaded ca. 1300 full text documents, all linked to the

bibliographical references, in the widespread format of Endnote Version 10. The database Endnote 10 is

appended to the report in .enl format and can be used for specific searches for keywords in various

combinations, since most references are collected over the Web of Science, which provides with

professional data structure and well chosen keywords for most publications.

There are lots of papers written about the best way of assessing the risk for non-target arthropods related

to the cultivation of Bt-crops. A proposal by (Scholte & Dicke, 2005) seems at first sight logical, namely to

start with the “most important” ecological evaluation. But knowing about the nearly infinite complexity of

most ecosystems, this would be to start a risk assessment the wrong way around.

Time will come in the Cartagena Biosafety Protocol MOP5 negotiations in Japan 2010

http://www.cop10.jp/aichi-nagoya/english/committee/index.html, when the enhancement and streamlining of

the risk assessment procedures must be discussed without prejudice. This could include ideas and

reconsiderations of various origins (Anonymous, 1992; Borch & Rasmussen, 2000; Gruere & Rosegrant,

2008).

The probably best way of handling the complex matter of risk assessment and management is suggested

by a worldwide consortium of specialists on risk assessment related to non-target arthropods , starting

with a comprehensive study in the laboratory which builds on realistic feeding and toxicity levels: (Romeis

et al., 2008a).

The consortium proposes an assessment continuum within a tiered scheme of ecological risk assessment.

The decision to reject the risk hypothesis includes consideration of remaining uncertainties. With

increasing tiers, the assessment becomes more complex and realistic, but with conclusions that are more

specific. The assessment can stop at any stage during the process as soon as sufficient information has

been compiled to address the risk hypothesis. Thus collection of data irrelevant to the risk assessment is

minimized. It is crucial to focus first, before the risk assessment starts, on a clear-cut risk hypothesis.

55

Fig. 17, Assessment continuum within a tiered scheme of ecological risk assessment. The decision to reject the risk hypothesis includes consideration of residual uncertainties. With increasing tiers, the assessment becomes more complex and realistic, with conclusions that are more specific. The assessment can stop at any stage during the process as soon as sufficient information has been compiled to address the risk hypothesis. Thus collection of data irrelevant to the risk assessment is minimized. N, level of risk assessment tier; NTA, nontarget arthropod. From (Romeis et al., 2008a)

From Fig. 6 it is also clear, that any risk assessment for non-target arthropods needs an adoption of risk

hypothesis according to the organisms in question, similar to thoughts published by Wolfenbarger et al.

2008 (Wolfenbarger et al., 2008), who divide their organisms into functional/ecological units which they

call ‘functional guilds’.

56

Fig. 18, Reconstruction of NTA risk assessment for Bt maize expressing Cry1Ab showing that different risk hypotheses require different types of data and synthesis at different tiers. From (US Environmental Protection Agency (USEPA), 1995) taken from (Romeis et al., 2008a) and see also the fully cited accompanying text below:

(Romeis et al., 2008a):

“In the evaluation of Bt maize expressing Cry1Ab, entities of concern included biological control organisms belonging

to, for example, the orders of Coleoptera (lady beetles), Neuroptera (lacewings) and Hymenoptera (parasitoid

wasps), as well as pollinators such as bees (also Hymenoptera), decomposers such as soil arthropods (for example,

springtails) and nontarget Lepidoptera. The problem formulation identified several risk hypotheses that were

subsequently addressed in the analytical phase of the risk assessment (Fig. 6).

Because analysis of the available precursor information revealed with sufficient certainty that the only meaningful

difference between Bt maize and its nontransformed comparators was the expression of the Cry1Ab protein, early

tier (worst-case) studies were conducted using elevated doses of purified protein or plant tissue. These studies

confirmed existing knowledge (precursor information) that these proteins are not likely to affect nonlepidopteran

insects (risk hypotheses 1–3) , . Testing could thus be terminated at this early tier.

The potential hazard to nontarget Lepidoptera (risk hypothesis 4) was recognized initially but it was concluded that

the risk is negligible . Additional studies under more realistic exposure conditions were triggered once a note and a

more comprehensive study had revealed a hazard of Cry1Ab to larvae of the monarch butterfly. Consecutive studies

57

were conducted under semi-field conditions. These studies concluded that the risk of Cry1Ab maize to monarch

populations is negligible because larval exposure to Cry1Ab toxin under field conditions is low confirming the initial

risk assessment .”

It is time to delve into future risks and benefits posed by biotechnology and leave the issues of the first

Bt crop generation behind, it would be simply a waste of time and money to continue with extensive

biosafety research related to the widespread and vastly commercialized crops, which will in most cases

deal with very interesting agro-ecological questions, but they are not anymore relevant for the risks of

agricultural production compared to conventional methods. This are questions which are situated in the

realms of the “nice-to-knows”.

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60

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