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Integrated Pest Management Reviews 5: 151173, 2000. British Crown Copyright2001. Printed in the Netherlands.

Bt transgenic crops: Risks and benefits

Raymond J.C. Cannon

Central Science Laboratory, MAFF, Sand Hutton, York, YO41 1LZ, UK (Tel.: 44-1904-462-000;

Fax: 44-1904-462-111)

Received 26 May 2000; Accepted 9 September 2000

Key words: Bacillus thuringiensis, Bt, crops, GM, risks

Abstract

Bt crops, predominantly maize and cotton hybrids, transgenically expressing cry genes derived from Bacillus

thuringiensis, were planted on approximately 14 million hectares (worldwide) in 1999. Preliminary reports suggest

that in most cases pesticide use was reduced, and in some situations there were significant increases in yields and

profits. However, assemblages of secondary pests such as aphids, plant bugs and thrips also exist in Bt crops,

and although the overall need for scouting and chemical control is reduced in Btcrops, there may be a requirement

for additional, conventionally applied chemicals to control such non-target pests.

Naturally-occurring Bt toxins with activity against a wide variety of pest species have been discovered and are

thus potentially available for engineering into Bt crops to control a broader spectrum of pests than are currently

targeted. New Bt crops and second-generation products incorporating an expanding range of Cry toxins and other

arthropod targeted genes are in development and could become available for introduction to the market within the

next few years.Insecticide resistance management (IRM) strategies forBtcrops are reviewed in the context of studies on selection

pressures and the potential for resistance development in target populations. The so-called, high dose strategy,

combined with the use of refuges, is widely agreed to be the best technical approach for managing resistance, and

evidence is accumulating that separate refuges are more effective at conserving pest susceptibility than mixed

refuges. A widespread consensus on the necessity for such measures, and an appreciation of the importance of

multi-tactical approaches, has developed. Monitoring programmes, protocols and studies relevant to detecting the

early development of resistance to BtCry toxins are described.

Field monitoring of non-target entomofauna has not revealed significant differences in the abundance or biodi-

versity of beneficial insects associated with Bt maize. Indeed, laboratory studies of effects on parasitoids suggest

that Btplants may even have an environmental advantage over broad spectrum pesticides. However, more complex,

multi-trophic, long-term experiments are needed to thoroughly assess the compatibility of Btcrops with non-target

invertebrates and to define the complex relationship between IRM, target species and their natural enemy assem-blages. Studies on the effects of transgenically-expressed Cry toxins on non-target insects, and their persistence in

soil and on leaves, is reviewed. It is suggested that there is currently no generally agreed framework, or methodology,

within which ad hoc experimental results can be accommodated, and each crop-transgene combination has to be

assessed on a case-by-case basis. Studies proposing a conceptual approach to evaluating risks associated with Bt

crops are highlighted and potential benefits and hazards are reviewed.

Introduction

This review concerns the utilisation of insectici-

dal crystal proteins (ICPs) (Kumar et al. 1996)

or -endotoxins derived from the Gram-positive,

spore-forming bacterium, Bacillus thuringiensis (Bt),

in transgenic crops. The emphasis is on so-called,

Btcrops transgenically expressing cry genes derived

from Bt isolates (Peferoen 1997), and mainly work

published since a previous review (Cannon 1996).

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152 R.J.C. Cannon

The reader is referred to other reviews concern-

ing Bt -endotoxins, for details on molecular biol-

ogy (Schnepf et al. 1998), mode of action (Knowles

1994), phylogenetic relationships (Bravo 1997), struc-

ture/function relationships (Grochulski et al. 1995;

Ellar 1997) and insect resistance (Tabashnik 1997;

Gould 1998; Mellon & Rissler 1998).

Biology and ecology of naturally-occurring

Bt isolates

Distribution and occurrence

Btis a ubiquitous soil bacterium, which is also presentin considerable diversity in a variety of above-ground

niches, including the phylloplane of a range of species

(Ohba 1996; Bel et al. 1997; Damgaard et al. 1997;

1998; Hansen et al. 1998; Mizuki et al. 1999a). Bt

is sparsely distributed, but occurs frequently and is

widespread, both locally and worldwide (Bernhard

et al. 1997). However, dust from stored product mills

and grain silos, as well as from insects collected from

the wild, are more potent sources of Bt isolates than

soil samples (Chaufaux et al. 1997; Iriarte et al. 1999;

Morris et al. 1999). ManyBtisolates, particularlythose

from soil samples, are non-toxic to a wide range ofinsects (Roh et al. 1996; Park et al. 1998), although

some have a cytocidal effect on human cancer cells

(Mizuki et al. 1999b).

Bt strains have been classified on the basis of their

flagellar (H) antigens into at least 58 serotypes (Laurent

et al. 1996), and the list continues to grow as more are

isolated from different habitats throughout the world

(e.g. Ferrandis et al. 1999a). The distribution of cry

genes within Btisolates, in general, shows no apparent

relationship with serovar, although common gene com-

binations are associated with toxicity to certain species

(Ferrandis et al. 1999b).

Novel pesticidal strains

A new nomenclature for Bt -endotoxins, and cry

genes, was proposed by Crickmore et al. (1995a,b).

At least 180 cry and cyt genes, and approximately 88

holotype Cry and Cyt toxins, have been discovered and

new ones are regularly added to the list (Crickmore

et al. 2000).

Extensive screening programmes and a worldwide

search for novel isolates has revealed toxins with activ-

ity against a wide variety of new target species. These

include, certain protozoa, platyhelminths, nematodes,

lice, aphids, mites, hemipterans, cockroaches and ants

(Feitelson et al. 1992; Payne & Cannon 1993; Schnepf

et al. 1996; Stockhoff & Conlan 1996; Payne et al.

1993; 1997; Bradfisch et al. 1998).

In addition, novel proteins with activities against

recalcitrant or refractory species, i.e. those relatively

insensitive to most Cry proteins, have also been dis-

covered. For example, a novel ICP (Cry9Ca1) from

Bt var. tolworthi, is active against cutworms, such as

Agrotis segetum (Denis & Schiffermuller) and Agrotis

ipsilon (Hufnagel) (Lambert et al. 1996), and binds

to different receptors to other ICPs currently used

in transgenic crops (van Frankenhuyzen et al. 1997).

Another, highly unusual strain, with a large complex ofgenes encoding 18 different Cry proteins, with multi-

ple toxicity to a very wide range of species including

coleopterans, lepidopterans, dipterans, hymenopter-

ans and nematodes was reported by Osman et al.

(1999).

Finally, a novel class (Vip3A) of vegetatively pro-

duced, lepidopteran-activeBtproteinswithnohomol-

ogy with known -endotoxins has been discovered

(Estruch et al. 1996).

Bt transgenic crops and trees

Bt genes have been used to transform at least 26 dif-

ferent crop and tree species, although codon-optimised

(i.e. for higher plant expression) genes have only been

used in a smaller sub-set of these; at least ten differ-

ent cry genes have been utilised to date (Bauer 1997;

Schuler et al. 1998). The expression of cry genes is

influenced by a number of different factors, including

both the genetic (where in the genome the gene con-

struct is inserted) and physical environment (field site),

plant age and tissue type (Sachs et al. 1998; Greenplate

1999).

Transgenic Bt crops first appeared on the marketon a large scale in 1996, in the USA. By 1997,

insect-resistant cotton and maize were grown glob-

ally on 1.1 million ha and 3 million ha, respectively

(Merritt 1998). In 1999, GM crops were planted on an

area of ca. 40 million ha (world-wide), predominantly

in the USA, China, Argentina and Canada (Anon.

1999b). Bt crops including some which were also

herbicide-tolerant accounted for approximately 34%

of these plantings (ca. 14 million ha) (Anon. 1999b). In

Europe, Btcrops were grown in relatively small areas

in 1999: Spain (30,000 ha), France (1000 ha), Portugal

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Bt transgenic crops: risks and benefits 153

Table 1. Btcrop products marketed in different countries1

Product name Company Country2

I. Maize

StarLink AgrEvo, Inc. US, CA

DeKalBt DeKalb Genetics Corp US

YieldGard Monsanto US, CN, AR

NatureGard Mycogen Corp US

NR Knockout Novartis Seeds3 US, FR, ES,

AR, CN

II. Swe et corn

Attribute Novartis Seeds US

III. Cotton

BXN Cotton Calgene US

Bollgard cotton Monsanto US, AU, CN,

MX, ZA, AR

IV. Potato

NewLeaf Monsanto US, CN

1Only Bt products with insecticide-resistance traits alone are

shown; other products, such as Bollgard with BXN cotton are

also herbicide resistant.2Isocodes for countries of the world.3Two products: Bt-176 and Bt-11 (due to merger of Sandoz and

Ciba-Geigy). Both express CryIAb. (Grady 1998; Gianessi &

Carpenter 1999; Anon. 1999c,d.)

(1000 ha) and Germany (500 ha) (Anon. 1999b; 2000).

Products introduced to date have predominantly been

maize and cotton hybrids, and to a lesser extent Bt

potatoes (Table 1).

Cotton

Bt cotton was planted on approximately 17% of

the USA cotton growing belt in 1998 (Gianessi &

Carpenter 1999). Bollgard/Ingard cotton was also

planted in Australia (200,000 acres), China (130,000

acres), Mexico (100,000 acres), South Africa (30,000

acres) and Argentina (20,000 acres) in 1998 (Anon.

1999f).

Transgenic Bt cotton lines expressing Bollgard

genes were reportedly taller, produced better yields andthe value of the fibre was higher, compared to parent

varieties sprayed according to scouting recommenda-

tions (Kerby 1995; Benedict et al. 1996; Jones et al.

1996).

The main lepidopterous cotton pests in the US

cotton belt and the main targets for Bt cotton

are the tobacco budworm, Heliothis virescens (F.),

and the pink bollworm (PBW), Pectinophora gossyp-

iella (Saunders). Bt cotton varieties do not control

the cotton bollworm (also called the corn earworm),

Helicoverpa zea (Boddie), as well as they control

H. virescens and P. gossypiella, and conventionally

applied sprays may be necessary when infestations

of this pest exceed economic thresholds (as in 1996)

(Kaiser 1996; Macilwain 1996; Lambert 1997; Luttrell

et al. 1999).

Two other sporadic, but significant pests of cotton in

the southern USA, are the fall armyworm Spodoptera

frugiperda (Abbott and Smith) and the beet army-

worm, S. exigua (Hubner). Transgenic cotton had 75%

as many S. exigua as other, non-Bt varieties, but

S. frugiperda numbers were unaffected (Hardee &

Bryan 1997).

The widespread adoption of Bt cotton in the USA

was associated with significant increases in yields in

most years for some regions (ERS 1999). Despite therequirement for additional sprays for insects not sus-

ceptible to Bt (see below), the adoption of Bt cot-

ton was associated with significant increases in yields

and profits, and decreased pesticide use (Merritt 1998;

ERS 1999; Gianessi & Carpenter 1999). However, this

decline in the application of broad-spectrum insecti-

cides had the result that populations of non-Bt sus-

ceptible phytophagous species, such as plant bugs,

aphids and thrips, increased (Turnipseed et al. 1995).

The southern green stink bug, Nezara viridula (L.),

and tarnished plant bug, Lygus lineolaris (Palisot de

Beauvois), cause significant damage to cotton bolls,and the development of treatment thresholds for such

secondary pests is suggested (Greene et al. 1999).

Farmers are also advised to monitor transgenic cotton

crops for other, non-target pest species, such as the

boll weevil, Anthonomus grandis grandis (Boheman),

which can be very damaging in certain regions of

the USA.

Maize

The European corn borer (ECB), Ostrinia nubilalis

(Hubner), is a major pest of field corn (maize),

causing yield losses in the region of $1 billion(1994) in the USA (Carozzi & Koziel 1997). In

some years, Bt bioinsecticides did not provide a level

of control sufficient to meet the standards of veg-

etable processors (Bartels et al. 1995). For exam-

ple, in sweet corn, a high-quality product free of

insect contamination or damage could only be guar-

anteed by the use of chemicals (Bartels & Hutchinson

1995). However, in lower-quality fodder maize, losses

due to the ECB were largely tolerated, and chem-

ical control was not generally carried out. As a

result, the introduction of Bt maize only resulted in

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154 R.J.C. Cannon

modest savings from reduced insecticide applications,

although its use was associated with significantly

higher yields in most years for some regions (ERS

1999). Nevertheless, a reduction in insecticde use as

a result of Bt maize planting occurred on 2.5% of

the total acreage, resulting in 2 million fewer acre

treatments with insecticides (Gianessi & Carpenter

1999).

Bt maize was first introduced in 1996 in the USA,

and by 1998, 18% of the national acerage was planted

withBtmaize (14.4 million acres). In Europe, Novartis

Btmaize was planted on a very small scale in 1998: in

Spain (ca. 20,000 ha) and France (ca. 2000 ha) (Anon.

1999d).

Whilst all transgenic Bt maize products introducedin the US (Table 1) significantly reduced injury

from 1st generation ECB, differences were evident

in terms of performance against the 2nd generation

(Ostlie et al. 1998a,b). YieldGard hybrids (Events

BT11 and MON810) provided 98% control of both

1st and 2nd generation ECB, whereas Event 176

hybrids (Knockout and NatureGard46), controlled

only 5075% (Rice & Pilcher 1998). The event num-

ber, refers to the unique genetic transformation event

when the modified Bt gene is inserted into the maize

genome. Event 176, expresses the toxin in green plant

tissue, pollen and the stalk, but not in the silk and ker-nels, whereas Events BT11 andMON810, resultin full

season expression in leaf, pollen, tassel, silk and ker-

nel tissue (Fearing et al. 1997; Andow & Hutchinson,

1998). CryIAb protein concentration levels in trans-

genic corn silks the typical food source for newly

hatched ECB larvae varied from 0.0 to 1.28 g/g,

but exhibited only a weak negative correlation with

damage, possibly as a result of the concentration of

naturally-occurring plant resistance compounds, such

as maysin (Sims et al. 1996). Variation between trans-

genic maize genotypes was less than 10-fold, except

for whole plants at anthesis, where the range was about

15-fold (Fearing et al. 1997).Plants with stalk damage caused by ECB have a

higher incidence of stalk rot caused by fungi such as

Fusarium moniliforme (Carozzi & Koziel 1997). How-

ever, Fusarium infections of the ears and kernels, and

symptomless infection of kernels, were consistently

reduced in Btmaize (Munkvold et al. 1997).

Transgenic maize lines and hybrids developed pri-

marily for ECB resistance have also demonstrated

good control of other stemborers, such as Dia-

traea grandiosella Dyar (Bergvinson et al. 1997),

and Sesamia nonagrioides (Lefebvre) (Nabo 1999).

However, a number of other, non-target pests such

as white grub, seed corn maggots, nitidulid beetles

and wireworms are not susceptible to the Cry tox-

ins expressed Bt maize (Anon. 1998b; Lynch et al.

1999).

Potato

Btpotatoes (New Leaf) were planted on 50,000 acres

in the USA in 1998, exclusively for the control of the

Colorado potato beetle (CPB), Leptinotarsa decem-

lineata Say (Gianessi & Carpenter 1999). Although

New Leaf potatoes were extremely effective against

(susceptible) CPB no larvae were found to survive

the small percentage of growers utilising this tech-

nology reflects, among other things, the requirement

to control other pests, such as aphids, unaffected by

the Cry toxins in Bt potatoes (Gianessi & Carpenter

1999). Additionally, the introduction of novel insec-

ticides such as imidacloprid, a systemic compound

highly effective against both the CPBand sucking pests

such as aphids (Elbert et al. 1990), also contributed to

the slow adoption of the Btpotato technology.

Novel crops and new developments

A number of other Bt crops, including alfalfa, toma-

toes, sunflower, soybeans, oil seed rape (canola) and

wheat are being developed and could be introduced to

the market within the next five years (SeongLyul 1995;

Anon. 1998a; 1999c). Additionally, second genera-

tion products, such as corn rootworm-resistant maize,

incorporating different Cry toxins such as Cry9C

targeting different toxin receptors, are also close to

production (Anon. 1999c,e; Ferber 2000). Companies

expect to expand our technology base by incorporat-

ing new and more powerful promoters, new and tighter

tissue-specific promoters as wellas a better understand-

ing of the principles governing plant gene expression(Estruch et al. 1997). Horticultural and ornamental

plants have also been modified to express cry genes,

including petunias (Omer et al. 1997) and chrysanthe-

mums (Dolgov et al. 1995).

Transgenic expression also opens up the possibil-

ity of delivering a toxic dose of a selected Bt toxin to

pests which would not have encountered conventional-

applied Bt biopesticides, either as a result of their

cryptic habitats (e.g. stem borers and leaf miners), or

particular feeding habits (e.g. pests with sucking or

piercing mouthparts) (Cannon 1993).

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Bt transgenic crops: risks and benefits 155

The development and use of agricultural biotech-

nology in developing countries has recently been the

topic of much debate (e.g. Wambugu 1999; Simms

1999). A number of tropical plant species have been

transformed using Bt genes. For example, brinjal

plants (Solanum melongena cv. Pusa Purple Long)

expressing a synthetic cryIAb gene for control

of the larvae of Leucinodes orbonalis Guenee, a

pyralid fruit borer (Kumar et al. 1998); groundnut

plants (Arachis hypogaea L.) against lesser cornstalk

borer, Elasmopalpus lignosellus (Zeller) (Singsit et al.

1997); coffee, Coffea canephora Pierre ex Frohner

and Coffea arabica L., to confer resistance to coffee

leaf miner, Perileucptera coffeella (Guerin-Meneville)

(Leroy et al. 1999); and both japonica and indica rice,Oryza sativa L. (Fujimoto et al. 1993; Nayak et al.

1997).

Stem borer, Chilo supressalis (Walker) and Scir-

pophaga incertulas (Walker), resistance was enhanced

in transformed rice (cv. Tarom Molaii) via the expres-

sion of a cryIAb gene in the rice leaf blades; the

toxin was not detectable in the dehulled, mature grain

(Ghareyazie et al. 1997).

The threat of resistance

Cross-resistance and transgene design

In diamondback moth (DBM), Plutella xylostella (L.),

a single autosomal gene can confer resistance to four

Bt toxins, including some to which the resistant strain

had not been exposed (Tabashnik et al. 1997a). This

suggests, the possibility at least, that a pest such as

H. zea, attacking Bt cotton expressing Cry1Ac, might

become cross-resistant to Btmaize producing Cry1Ab.

In other words, pests may evolve resistance to some

groups of toxins much faster than expected. However,

other studies have suggested that Btresistance mecha-

nisms might be specific to individual toxin subclasses,and may not extend broadly to other toxin types (Tang

et al. 1996). For example, resistance to Cry1Ab was

not linked with resistance to Cry1C in DMB (Liu &

Tabashnik 1997b)

Cross-resistance is most likely when ICPs share key

structural features (Tabashnik et al. 1996). Thus, the

choice of cry gene(s), as well as transgene design

including synthetic genes and modes of expression,

all contribute to the success of post-expression tactics,

such as theuse of refuges or seed mixtures (Bauer1995;

Strizhov et al. 1996; Tabahsniket al. 1997b).

Certain, naturally-occurring combinations of tox-

ins (e.g. Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa) in

Bt var. israelensis, provide an advantage in suppress-

ing resistance, compared with single toxins (Wirth &

Georghiou 1997). This CytA/CryIV model (old ter-

minology) could provide a molecular genetic strat-

egy for engineering resistance management for Cry

proteins directly into transgenic plants (Wirth et al.

1997).Btstrains with unique combinations ofcry genes

can also be designed and engineered using molecular

recombinant systems (Baum et al. 1996).

Fitness costs

A much lower fitness and intrinsic rate of increase ina resistant, laboratory population of DBM, suggested

a trade-off between Bt-resistance and fitness (Shirai

et al. 1998). However, Tang et al. (1997) found that

Bt resistance in DBM did not confer detectable lev-

els of reduced fitness in the absence of exposure to Bt.

Liu etal. (1995) found that stronger expression of resis-

tance occurred in 3rd instar DBM, than in neonates,and

suggested that it might be disadvantageous for resis-

tance to increase uniformly in all instars.

Fitness costs have also been reported for Cry3A-

resistant CPB, including reduced larval weight,

reduced fecundity, shortened oviposition period,reduced egg-mass size, increased overwintering mor-

tality and reduced population growth rate (Trisyono &

Whalon 1997; Alyokhin & Ferro 1999c).

Baseline susceptibility and field monitoring

Surveillance of transgenic crops in the form of

scouting for insect survivors is the most straightfor-

ward and simple method of detecting resistance (Riebe

1999). However, establishing a monitoring programme

for determining baseline susceptibilities to the cry gene

products, prior to the widespread planting of Btcrops,

is essential for detecting the early development of resis-tance.Monitoring protocols, e.g. for the ECB in Europe

(SCP 1999) and the USA (Anderson 2000), empha-

sise the requirement to target geographically distinct

populations.

Baseline susceptibilities have been determined for

ECB populations in some maize-growing regions of

the USA, and regional differences in susceptibilityhave

been detected (Huang et al. 1997). However, variabil-

ity in tolerance to CryIAb among ECB populations

was unrelated to prior exposures to (Bt) pesticides

(Siegfried et al. 1995).

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156 R.J.C. Cannon

Preliminary monitoring of differentH. virescens and

H. zea populations in the southern USA showed no

shifts in baseline susceptibility to Bt one year after

the introduction of Bt cotton (Hardee et al. 1997).

In this case, the insects were exposed to field doses

of MVPII the closest in toxicological properties of

all Bt insecticides to the CryIAc protein expressed in

transgenic cotton in spray chamber bioassays. How-

ever, transgenically expressed protein in maize is only

65% homologous to the original protein, and results

obtained using purified, naturally-occurring toxins, or

biopesticides, may not translate to the transgenic ver-

sion (Pilcher et al. 1997a). [NB. synthetic cry genes

may be truncated and codon optimised to achieve

higher levels of protein expression (Cannon, 1996;Duck & Evola 1997).] Wider ranges of variation in

susceptibilities to Cry toxins purified proteins and

commercial formulations were observed amongst

populations ofH. virescens by Luttrell et al. (1999).

Mascarenhas et al. (1998) considered that for pests

such as soybean loopers, Pseudoplusia includens

(Walker), which move between soybean and cotton

in areas were they are grown in close proximity, it is

imperative that a proactive approach of establishing

baseline mortality data and discriminating concentra-

tions be taken, in addition to maintaining a viable inte-

grated pest management (IPM) programme.Relatively simply methods for detecting signifi-

cant levels of resistance to Bt have been developed,

e.g. using discriminating concentrations or diagnostic

doses (Huang et al. 1997; Bailey et al. 1998).

Detecting levels of resistance alleles

Gould et al. (1995) suggested it might not be possible

to detect low frequency, major genes that code for

high levels of resistance by carrying out short-term

selection studies, i.e. of less than 10 generations. The

frequency of alleles that confer resistance to CryIAc

in H. virescens, collected before the first commercialplantings of transgenic cotton varieties, was estimated

as 1.5 103 (Gould et al. 1997). Roush & Shelton

(1997) questioned whether this initial resistance fre-

quency is actually a single major gene, or instead a

resistance phenotype that may be under control of

more than one locus. However, genetic linkage analy-

sis revealed the existence of a major locus responsible

for ca. 80% of the total Cry1Ac resistance levels in

H. virescens (Heckel et al. 1997).

Andow and Alstad (1998) proposed an F2 screening

procedure to estimate the frequency of rare resistance

alleles in natural populations. Compared to a

discriminating-dose assay, the F2 screen extends the

sensitivity of allele-frequency estimation for recessive

traits by more than an order of magnitude. Andow et al.

(1998) determined thefrequency of resistance alleles in

a ECB population (

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Bt transgenic crops: risks and benefits 157

availability of susceptible insects, and is likely to be an

effective way of slowing the evolution of resistance to

these crops (Roush 1996).

The optionavailable to cotton growers underthe EPA

(1997) mandate, was, i) for every 100 ha ofBt cotton,

4 ha (i.e. slightly less than 4%) of non-Bt cotton must

be planted and these cannot be sprayed with insecti-

cides which kill the major lepidopteran pests of cotton,

and ii) every 100 ha of cotton must contain 25 ha of

non-Bt cotton which can be treated with an insecti-

cide, except Btbioinsecticides. Tabashnik (1997) con-

sidered that the first option might work well under ideal

conditions, but if the optimistic assumptions regard-

ing inheritance and mating are violated, the number

of homozygous susceptible individuals generated bya 4% refuge may not be enough to stem the tide of

resistance. Similarly, in the case of the second option,

the suppression of homozygous susceptible individu-

als by conventional insecticide treatments in the non-Bt

cotton, could essentially eliminate the refuge.

Since older larvae are generally less susceptible to

transgenicBtplants than neonate larvae (e.g. Wierenga

et al. 1996), IRM strategies should allow for the least

susceptible stage (Huang et al. 1999a) because lar-

vae may have opportunities to grow and develop on

non-Bt crops, or alternate hosts, before they attack Bt

crops.Pyramiding, combining two or more resistance traits

(genes) in the same plant particularly those with

completely different modes of actions/target recep-

tors could be a useful strategy for increasing the

durability of the Cry toxin (Sachs et al. 1996), and

could also greatly reduce the requirement for refuges

(Roush 1998).

Refuges and other strategies

There has been a lively debate concerning the size and

placement of refuges in relation toBtcrops. However, a

widespread consensus on the importance of such mea-sures has developed, and recommendations have been

made by a numberof authorities andalliances (e.g. EPA

1997; Feldman & Stone 1997; Andow & Hutchinson

1998; Gould et al. 1998; Gould & Tabashnik 1998;

McGaugheyetal. 1998; EPA 1999; SCP1999; Vlachos

et al. 1999; Anderson 2000). For example, recom-

mendations for transgenic crops specify that between

20 and 50% of any given area should include non-

transgenic crops (EPA 1999). The EPA stipulates that

Btcorn registrants in the USA must ensure that grow-

ers plant a minimum structured refuge of at least 20%

non-Bt corn, and for Bt corn grown in cotton grow-

ing areas, at least 50% non-Bt corn must be planted

(Anderson 2000).

Computer models and theoretical

considerations

Computer simulations suggest that 100% mortality of

heterozygotes could delay resistance for more than 200

generations, but it is probably unrealistic to expect that

mortality of heterozygotes will exceed 95.5% for most

transgenic crops (Roush 1997b). Other simulations

have shown that late season survival of ECB larvae

on maize, as a result of plant senescence, could result

in resistance developing after 542 years (Onstad &Gould 1998a). Where the resistance alleles are at least

partially resistant, it should take at least 10 years

for Bt resistance become a problem (Gould et al.

1997).

Simulation models have also shown that both spatial

structuring, e.g.patchworks ofBtandnon-Btfields, and

the temporal pattern of refuges influence the develop-

ment of resistance (Alstad & Andow 1995; Peck et al.

1999). However, Ives (1996) concluded that changing

the distribution of toxic plants among fields has lit-

tle potential for controlling resistance evolution. Sepa-

rate refuges are superior to seed mixtures for delayingresistance in the ECB population to transgenic maize

(Onstad & Gould 1998b). However, strips of 612 rows

of non-transgenic maize (making up 20% of the field)

were equally effective to separate blocks of the same

percentage, in terms of delaying resistance (Onstad &

Guse 1999). Recent field experiments using a model

system incorporatingBtbroccoli plants and DBM have

confirmed that separate refuges will be more effective

at conserving susceptible larvae than mixed refuges

(Shelton et al. 2000).

In theory, the required size of a refuge is dependent

upon the amount of time one wishes to delay resis-

tance, and the amount of safety error one desires tobuild into the system (Caprio 1998). Theory also pre-

dicts that when there is only a single resistance locus in

the genome, andthe mortality of the heterozygousindi-

viduals exposed to transgeniccrops exceeds95%, resis-

tance canbe delayed for more than 40 generations,even

when resistance is initially as commonas 103 and only

10% of the pest population develops on refuge hosts

(Roush 1998). However, if the mortality of the het-

erozygotes falls below 90%, then a refuge size of 20%

is needed to delayresistance > 20 generations. In addi-

tion, in species which are not particularly susceptible

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158 R.J.C. Cannon

to Bt toxins (including the ECB), and in circum-

stances where fewer than 90% of naturally-occurring,

unselected larvae of these species would be killed,

very large refuges would be required to delay resis-

tance to single-toxin plants (Gould et al. 1997; Roush

1998).

Most simulation models are of the deterministic type

and are based on a number of critical assumptions, par-

ticularly that mating occurs randomly between adults

of different genotypes (Tabashnik 1994). However, as

Gould (1996) describes, there are exceptions to these

assumptions. A notable recent example, is the finding

by Bourguet et al. (2000) that ECB populations found

on non-maize plants (hop and sagebrush) in the Nord-

Pas-de-Calais region of northern France may constitutea separate subpopulation from those on maize in the

same region.

Laboratory tests of IRM strategies

Liu and Tabashnik (1997a) tested the so-called refuge

tactic in laboratory experiments using DBM, where

refuges were created by using untreated leaf-discs.

A 10% refuge helped to maintain susceptibility of

DBM larvae, but the correspondence of these exper-

iments with field outcomes is uncertain.

Therefuge/high dose strategy would not be expectedto work when resistance is not recessive (Tabashnik

et al. 1997b). Laboratory studies suggested that resis-

tance in the ECB to the conventional Bt insecticide,

Dipel ES, is inherited as an incompletely dominant

autosomal gene (i.e. it is not sex-linked) (Huang et al.

1999b). If field resistance in this species turns out to be

similar, the usefulness of the high-dose/refuge strategy

to resistance management in Bt maize may be dimin-

ished. However, Dipel ES differs substantially from the

toxin expressed inBtmaize; it also contains spores and

at least three other toxins and in addition, damage by

neonates is not a reliable indicator of survival on trans-

genic plants. Finally, there is no evidence that eitherlarvae from the Dipel ES-R strain, or heterozygous lar-

vae could survive to maturity on Bt maize (Tabashnik

et al. 2000).

Liu et al. (1999), found that a resistant strain of PBW

took longer, 5.7 days on average, to develop on Bt

cotton than susceptible larvae on non-Bt cotton. This

suggested that the resulting developmental asynchrony

between resistant and susceptible adults would favour

assortative mating among resistant strains and gener-

ate a disproportionately high number of homozygous

resistant insects, thereby accelerating the development

of resistance andreducingthe benefits of refuges. How-

ever, it is not clear to what extent this effect would

be affected by other factors, such as variation in toxin

expression, weather and overlap between generations.

Movement and dispersal

Movementof insects,bothbetweenBtandnon-Btcrops

and within Btcrops, can affect the rate at which resis-

tance develops (Pecket al. 1999).

Bt cotton plants were not toxic to 5th instar H. zea

and H. virescens, although movement of larvae from

plant to plant occurred more rapidly on transgenic than

non-transgenic cotton (Parker & Luttrell 1998; 1999).

Movement of 5th instars from non-transgenic plantsonto transgenic cotton plants could result in feeding

damage (Halcomb et al. 1996) andmay allow more het-

erozygous individuals to survive, thus increasing the

rate of resistance development. Ramachandran et al.

(1998a), also reported that when Bt transgenic, and

non-transgenic canola, B. napus, plants were grown in

contact with each other in a seed mixture, there was a

possibility that at least a few older larvae would feed

on transgenic plants and move to non-transgenic ones

before acquiring a lethal dose. Thus, the use of mixed

stands as a refugium for susceptible individuals may

not be as effective at maintaining susceptibility as purestands. Ramachandran et al. (1998a), suggested that

transgenic and non-transgenic plants grown in sepa-

rate rows with a wider row spacing (i.e. strip plant-

ing) would minimise larval movement, and reduce

damage to non-transgenic plants. Onstad and Gould

(1998b) also thought that planting two-row strips may

be as good as separate refuges in delaying resistance,

but considered that their adoption carries greater risk

because of the uncertainty surrounding movement and

survival of neonates.

Wierenga et al. (1996) suggestedthat non-transgenic

(potato) plants in a refugia would facilitate the survival

of CPB stages beyond the 2nd instar (less susceptibleto Bt potato than neonates), and they could then grow

large enough to move ontotransgenicplants andreceive

a sub-lethal dose.

Laboratory-selected, resistant CPB continuously fed

on transgenicBtpotato foliage (NewLeaf) were capa-

ble of flight and reproduction, although it took them

longer to initiate flight behaviour and their fecundity

was lower (Alyokhin & Ferro 1999a). Suppression of

flight as a result of ingestion of Bt potato could keep

the beetles within transgenic fields, thus increasing

selection pressure for the development of resistance

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Bt transgenic crops: risks and benefits 159

(Alyokhin et al. 1999). However, Alyokhin and Ferro

(1999b) showed that susceptible CPB males arriving in

transgenic potato fields from refugia can mate with res-

ident resistant females, and the resulting heterozygote

offspring are not able to survive on transgenic crops

(Alyokhin & Ferro 1999a).

Selective feeding, i.e. theability of thelarvaeto move

and switch their feeding to other tissues containing less

Cry protein, could potentially influence dose acquisi-

tion since toxin concentrations can be lower in certain

parts of the plant ( e.g. in kernel, stalk, silks and pollen

of maize, and the fruiting structure of cotton) in some

transgenic varieties. In a field situation, insects may

be able to feed predominantly on Bt-free plant tissue

as a result of behavioural avoidance of Bt-expressingtissues (Stapel et al. 1998).

No significant differences were found in oviposition

preferences for H. virescens on transgenic cotton, nor

for DMB on Bt canola (Ramachandran et al. 1998a)

or Btbroccoli (Tang et al. 1999). Similarly, no signifi-

cant differences were found in ECB egg mass densities

between transgenic and isogenic corn (Orr & Landis

1997).

Environmental and ecosystem effects

Effects on natural enemies

Short term risks to natural enemies will be a function of

the intrinsic susceptibility of the organism and the level

of exposure to the toxin (Jepson et al. 1994). Sublethal

effects of exposure also have important consequences

for natural enemies, and intergenerational effects could

be one of the most sensitive indicators in risk assess-

ment studies.

No acute detrimental effects were observed in terms

of the abundance, or predatory ability, of generalist

predators of ECB in Bt maize, compared with non-

Bt maize (Orr & Landis 1997; Pilcher et al. 1997b).Similarly, field monitoring of non-target entomofauna

showed no differences in the abundance or biodiver-

sity of beneficial insects associated with transgenic Bt

maize, (Goy et al. 1995; Jarchow 1999; Lozzia 1999)

Bt cotton (Sims 1995) or Bt sweet corn (Wold et al.

1999) compared to non-transgenic controls.

Effects of conventional Bt sprays on parasitoids

Laboratory studies have shown that conventional Bt

formulations may be harmful to immature parasitoids

(Blumberg et al. 1997), but applications at recom-

mended field rates had negligible impact on emergence

of adults (Atwood et al. 1999). Studies using the bra-

conidwasp, Cotesia plutellae Kurdyumov, showed that

highly resistant DBM hosts, which were not suscepti-

ble to infection by the pathogen, provided a refugium

from competition (with Bt) for the parasitoid, whereas

in susceptible hosts the pathogen effectively outcom-

peted the parasitoid (Chilcutt & Tabashnik 1997a). In

other words, adverse mortality effects on C. plutel-

lae occurred when developing parasitoid larvae were

exposed to Bt within susceptible hosts (Chilcutt &

Tabashnik 1999). Therefore, in susceptible DBM pop-

ulations, Bt applications combined with C. plutellae

would be highly effective, whereas in resistant pop-ulations, applications of Bt would exert little control

(Chilcutt & Tabashnik 1997b).

Tritrophic studies

Laboratory studies of the effects of Bt crops on para-

sitoids of target pests, are rather few in number. Schuler

etal. (1999b) investigatedthe behaviourofC. plutellae,

parasitising DBM larvae feeding on Btoilseed rape in

a model ecosystem. No effects on the survival, or host

seeking ability, of the parasitoid were detected, indicat-

ingthatBtplants may have an environmental advantageover broad spectrum pesticides.

In a series of laboratory feeding experiments using

Bt maize plants and artificial diets, Hilbeck et al.

(1998ac) studied the effects of Bt-fed herbivorous

prey on the predator, Chrysopa carnea Stephens.

Although the development time of chrosopid larvae

was prolonged when feeding onBtmaize-fed ECB, the

result was probably a combined effect of exposure toBt

and nutritional deficiency caused by sick prey, and no

conclusions were drawn as to how these results might

translate in the field (Hilbeck et al. 1998a). Exper-

iments using an artificial liquid diet (encapsulated

Cry1Ab toxin) showed higher mortalities in larvae con-sistently exposed to the toxin, but no, or only small, dif-

ferences in development times were observed (Hilbeck

et al. 1998b). However, the results were interpreted

as demonstrating that Cry1Ab is toxic to C. carnea

by using an appropriate bioassay system. Further

work using Bt diet-fed prey (Spodoptera littoralis

[Boisduval] larvae) confirmed increased mortalities of

immature chrysopid larvae, relative to controls, and

also revealed the existence of prey/herbivore-by-plant

interactions (Hilbeck et al. 1998c). In other words, in

addition to prey/herbivore-by-Bt interactions which

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160 R.J.C. Cannon

enhanced the impact of the Bttoxins (predator mortal-

ity was higherthan prey mortalityat equivalent doses)

mortalities on transgenic plants were 10% higher than

equivalent doses in diet.

The distribution of the CPB predator, Coleomegilla

maculata (De Geer), at a plant-to-plant level in potato

fields is driven by CPB egg mass density (Arpaia

et al. 1997). However, egg consumption was inversely

related to egg mass density, and as such is expected

to slow the rate of adaptation by the pest to the toxin.

Thus, C. maculata predatory behaviour could decrease

the rate at which CPB adapted to Bt-toxins, if plot-

to-plot mixed plantings were used. In other words, in

such mixed plots, a much lower density of CBP egg

masses is expected on transgenic plants, and if preda-tors prey in an inversely-density-dependent way, then

the likelihood of a Bt-resistant CPB individual beetle

reaching adult stage (and reproducing) on transgenic

plants will be lowered, thus slowing the rate at which

resistance allelles increase in frequency. Riddick and

Barbosa (1998) did not detect any significant impact of

Cry3A-intoxicated CPB on the consumption, develop-

ment and fecundity ofC. maculata, and suggested that

this species will not be deterred from feeding on CPB

in fields of transgenic potatoes.

Riddicket al. (1998) investigated the relative abun-

dance of two natural enemies of theCPB in seed-mixed,and 100% pure, fields of Cry3A-transgenic and non-

transgenic potato. The results demonstrated that Lebia

grandis Hentz larvae, specialist carabid ectoparasitoids

of CPB, will not persist in seed-mixed and 100% trans-

genicpotato fields, and willgradually disperse, because

of the low densities of CPB in these fields. However,

C. maculata adults and larvae, which are generalist coc-

cinelid predators of early CPB larvae and other prey,

are likely to thrive and flourish in fields containing

transgenic potato. Therefore, predation by C. macu-

lata could decrease the rate at which CPB adapt to the

transgenic crop (Arpaia et al. 1997).

Non-target Lepidoptera

Losey et al. (1999) found that larvae of the Monarch

butterfly, Danaus plexippus L., reared on milkweed,

Asclepias currasavica L., leaves dusted with pollen

from Bt corn, ate less, grew more slowly and suffered

higher mortality than larvae reared on leaves dusted

with untransformed corn. However, criticism of this

work pointed to the lack of choice in the experimental

design, poor quantification and the use of inappropri-

ate controls (Hodgson 1999). More recently, Wraight

et al. (2000) concluded that Bt pollen from event 810

maize would be unlikely to affect wild populations of

black swallowtails, Papilio polyxenes Fabricius.

Experiments investigating the effects of conven-

tional Bt sprays on other, non-target lepidopterans

have shown that it is difficult to generalise about

susceptibility to Bt, particularly for mid- to late-instar

larvae, and that susceptibility must be dealt with on a

species-to-species basis (Peacock et al. 1998). Leong

et al. (1992) demonstrated the low sensitivity of over-

wintering D. plexippus to conventional Btpesticides.

Persistence in soil and on leaves

A soil microcosm experiment, usingBtcotton, demon-

strated an initially rapid decline of Cry toxin overthe first 14 days possibly due to biotic degrada-

tion but low amounts persisted for several weeks or

months (Palm et al. 1996). Cry toxins become resis-

tant to microbial utilisation, and remained insectici-

dal for at least 40 days, when bound on clay minerals

(Koskella & Stotzky 1997). However, Yu et al. (1997)

found no detrimental side effects in two non-target soil

arthropods after exposure to the Cry toxins for ca. two

months.

Insecticidally-active, CryIA(b) protein is released

into the rhizosphere ofBtcorn seedlings duringgrowth,

and could add to the amount of toxin introducedinto the soil from other transgenic plant sources,

including pollen and plant residues after harvest-

ing the crop (Saxena et al. 1999). Donegan et al.

(1996) reported few significant differences in phyllo-

plane bacteria between transgenic Bt-producing potato

plants and potato plants treated with microbial Bt var.

tenebrionis.

Discussion

Initial performance

Initial reports on the performance ofBtinsect-resistant

crops in the USA were generally highly favourable.

For example, the overall performance of the Bt crops

introduced into the USA in 1996 was described by

Gelernter (1997) as superlative, and Merritt (1998),

of Monsanto Life Sciences, reported that the perfor-

mance of Bt maize (YieldGard hybrids) was con-

sistently excellent. Similarly, the performance of Bt

sweet corn against H. virescens and S. frugiperda

was described by Lynch et al. (1999) as exceptional.

Control of the PBW in field plots of Bt cotton in

Arizona, USA, in 1993 and 1994 was so successful

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Bt transgenic crops: risks and benefits 161

that Flint et al. (1995) considered that the possibil-

ity exists that the PBW can be eradicated from the

SW USA. These reports echo the optimistic predic-

tions of earlier researchers that wide-area deployment

of insect-resistant crops could depress overall popula-

tions of target pests to the point that refuges incur little

damage (op. cit. Gould 1998).

The preliminary results of an on-going study by the

Economic Research Service of the USDA (ERS 1999),

showed that in most cases, the adoption of insect-

resistant crops reduced pesticide use, although in some

cases such as Bt corn the effect was small and

insignificant. In certain situations, such as the ofBtcot-

ton in southeast USA, increased adoption of this new

technology was associated with significant increases inyields and profits.

A step change or an extension

of traditional methods?

The use of transgenic plants in pest control is seen

by some as a natural extension of plant domestication

(e.g. Duck & Evola 1997). More specifically, Gould

(1998) considered that transgenic insecticidal culti-

vars represent an extension of one form of classical

host-plant resistance, namely antibiosis (or alleleopa-

thy). However, alleleopathic substances, such as alka-loids and phenolics, are usually sublethal in their

effects.

Regal (1994) considered that rDNA genetic engi-

neering is fundamentally different from traditional

plant breeding, for three main reasons: 1) because of

the movement of fully functional genetic traits between

completely different sorts of organisms (phyloge-

netic leap-frogging); 2) the absence of the debilitating

trade-offs associated with radical improvements pro-

duced via conventional breeding; and 3) the potential

to access, or reprogramme, non-Mendelian (hidden)

portions of the genome. According to Regal (1994),

these features imply that some types of GMO are morerisky than those which could be produced via selective

breeding, but this does not mean that every GMO is

ecologically dangerous.

Donegan etal. (1999)believethat there will be unin-

tentional changes in plant characteristics as a result of

genetic manipulation which may impact on soil and

plant biota and processes.

Potential benefits and hazards

Potential benefits claimed for Bt crops include:

more effective and full-season insect control; reduced

scouting and monitoring costs; a reduction in

conventional foliar insecticide use (with concomitant

improvements in safety); and a reduced impact on non-

target organisms (Fischhoff 1996; Hutchinson 1998;

Hails 2000).

Potential hazards identified for GM, insect-

resistant crops, include: adverse effects on non-target

invertebrates; the transfer of insect resistance (e.g. to

genes to closely related, non-crop species); the devel-

opment of resistance in target pests; and the need for

additional types of pest control (Anon. 1999a).

The magnitude of a risk associated with a particu-

lar hazard is dependent on the scale (size) of its intro-

duction and the time of its realisation, the so-called

wedge-effect (Harding & Harris 1997). The key toresolve questions concerning the safety of agricultural

biotechnology is to devise a rigorous set of relevant

questions, and according to van Dommelen (op. cit.

Duvick 1999)these questionsshould be concernedwith

hazard identification (identifying a potentially bad out-

come) rather than with risk analysis (calculating the

odds of a bad outcome).

Evaluating potential risks

Oneof the main problems associated with assessing the

risks posed byBtcrops is that there is no agreed frame-work, or methodology, into which ad hoc experimental

results can be accommodated. In other words there is a

paucity of general principles (Hails 2000), and each

crop-transgene combination has to be assessed on a

case-by-case basis prior to commercialisation (Anon.

1996; Rissler & Mello 1996).

However, there have been a number of studies and

discussions which have attempted to formulate prin-

ciples and recommendations for the safe deployment

of Bt crops. Jepson et al. (1994) proposed a concep-

tual framework to evaluate the risks of Bt plants: a

combination of laboratory tests, field experiments and

longer-term monitoring. Determining the pattern andfrequency of exposure of sensitive indicator species

(Jepson 1993) to Cry toxins expressed by Bt crops,

was proposed. Edwards (1994) also emphasised the

requirement for evaluating the effects of GMOs on

dynamic (soil) ecosystem processes, such as organic

matter breakdown, nutrient cycling and respiration.

Hokkanen and Wearing (1994) summarised the con-

clusions and recommendations of an OECD workshop

on the ecological implications ofBtcrops, and Schuler

et al. (1999a) emphasised the importance of vigorous

and standardised methodologies for ecotoxocological

evaluations.

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162 R.J.C. Cannon

Decisions to deploy Bt crops should also be made

on a country-by-country basis, with relevant data on

features of the crop that could impact on selection,

and the host range of the target pest and its propen-

sity to develop resistance (Whalon & Norris 1996).

There is also a need for coordination in the production

and release of differentBtcrops (Wearing & Hokkanen

1995).

Human health and safety issues

Conventional Btpesticides are generally considered to

be very safe (Nielson-LeRoux et al. 1998), although

there is a small risk to human health, largely asso-

ciated with the presence of diarrhoeal enterotoxins

(Damgaard 1996). However, they have been widely

used for many years, and have not caused an over-

whelming number of cases of gastro-enteritis to

occur (Hendriksen & Hansen 1998). Some naturally-

occurringBtstrains do not contain the enterotoxin gene,

and in strains where they do not occur it is possible

to remove enterotoxin genes by genetic engineering

(Asano et al. 1997).

Daily food intake of non-transgenic plant material

including organically-grown cabbage (Damgaard et al.

1997; Hansen et al. 1998) could contain naturally-

occurringBtat substantial levels (Mizuki et al. 1999a).Other studies (Hernandez et al. 1998) have shown

that the strain ofBtH34-konkukian can be pathogenic

for immunocompromised mice, but further studies are

needed to evaluate the potential pathogenicity for mam-

mals of this bacterium. This was amongst the most

commonly collected serovar in a survey of Spain car-

ried out by Iriarte et al. (1999). However, such risk

factors could be removed by transgenic expression.

Assessment criteria: the tiered approach

The approach to risk assessment recommended by theUK DETR (Anon. 1993), which is also being intro-

duced into EC Directive 09/220/EEC, with the objec-

tive of harmonising the approach to risk assessment

for GMOs across the EC (Anon. 1999a), involves the

following procedural steps: 1) identify the hazardous

characteristics of the GMO; 2) assess the likelihood of

those hazards being realised under the conditions of the

proposed release; 3) assess the magnitude of the conse-

quences for human health and the environment, should

those hazards be realised; 4) assess the risk; 5) con-

sider implementation of risk management procedures;

and 6) take into account and risk management proce-

dures and come to an estimation of the overall risk.

An industry approach to safety evaluation of the Bt

maize involves three different types of tests to assess

the specificity of the truncated form of the Cry pro-

tein expressed in the crops: in vitro dietary tests using

selected lepidopteran targets; field monitoring of the

entomofauna associated with the crop; and toxicity

studies against selected non-target organisms, such

as earthworms and bees (Jarchow 1999). However,

tritrophic level studies are necessary to assess the long-

term compatibility of insecticidal plants with natu-

ral enemies, and to define the complex relationship

between resistance management, target and non-target

herbivores, and their natural enemies (Arpaia et al.1997; Hilbecket al. 1998b).

Levels of toxin and exposure models

On a per acre basis, the highest level of Cry protein

in Event 176 Btmaize plants was 24 g/acre (at anthe-

sis) (Fearing et al. 1997). CryIAb levels were markedly

lower in late-season, senescing plants, for which total

Cry protein/acre was estimated to be less than 0.2 g.

Conventional applications ofBt-based insecticides cor-

respond to ca. 45 g/acre. Therefore, 1020-fold less Bt

protein is present per acre in these transgenic maizeplants derived from Event 176, compared to conven-

tional applications of Bt biopesticides (immediately

after application). However,BtCry protein expressed in

plants is considerably more long-lived, in the sense that

it is not rapidly degraded like conventional Bt(Cannon

1996), and thus potentially available as a hazard to sus-

ceptible organisms via direct or indirect exposure.

Constitutive expression in transgenicBtplants is not

100% controllable; trace amounts of CryIAb protein

below the limit of detection (ca. 8 ng/g fresh weight)

were detected in the pith and roots of Event 176 Bt

maize plants (Fearing et al. 1997).

Levels of Cry1Ac toxin expressed in Bollgard

cotton declined steadily as the growing season pro-

gressed, e.g. from 57.1 g/g dry wt. (53 days after

planting) to 6.7 g/g dry wt. (116 days after planting)

(Greenplate, 1999).

Ecosystem effects

In general, conventional applications Bt biopesticides

at recommended rates are compatible with major nat-

ural enemies (Melin & Cozzi 1990). Indeed, judicious

timing ofBtapplications can enhance the performance

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Bt transgenic crops: risks and benefits 163

of parasitoids, at least in certain cases (Chenot & Raffa

1998). Synergistic interactions between natural ene-

mies, selectively preying on stunted larvae feeding

on transgenic hosts, have been hypothesised, but not

proven (Mascarenhas & Luttrel 1997).

The discussions and controversy surrounding reports

of toxicity studies such as Losey et al. (1999) have

not been placed in the context of conventional foliar

insecticides (Sanborn 1999), including biopesticides.

In addition to possible non-target effects, ecolog-

ical risk assessment of GMOs should also consider

likely effects on the agroecosystem, including whether

the transgenic crop plant will promote or impede

sustainable development (Burn 1999; Pascher &

Gollmann 1999).

IRM

Altieri (1998) argued that insect resistant transgenic

crops reinforce the pesticide treadmill in agroecosys-

tems. However, the assumption that transgenic plants

per se willcauseresistance faster thansprays is, accord-

ing to Roush (1996), not necessarily true; transgenic

plants may actually delay resistance more effectively

than sprays in some cases. However, the increased use

ofBttoxins via transgenic crops could result in the

more rapid evolution of resistance. As a consequence,the rules for IRM have changed, and there is a greater

requirement for co-operation and the development of

more proactive plans in the case of transgenic crops

(Hutchinson 1998).

According to Whalon and Norris (1996), resis-

tance management programmes rely on four key

management strategies: 1) diversification of mortal-

ity sources; 2) reduction of selection pressure and use

of refugia; 3) prediction and monitoring of resistance;

and 4) policy implementation. However, none of the

possible operational tactics present clear advantages in

all environments, with all pests, except perhaps mea-

sures to encourage survival or immigration of sus-ceptible genotypes (Whalon & McGaughey 1998). In

some circumstances, the use of refuges could enhance,

rather than reduce, the development of resistance

(Wierenga et al. 1996), and assumptions concerning

gene flow between non-crop refuges and Bt crops

must not be taken for granted (e.g. Bourguet et al.

2000).

Bt plants are not stand-alone products and should

be integrated with other pest management strategies

(Peferoen 1997). Hoy (1998) considered that the term

resistance management is inappropriate, since at

best resistance can only be delayed, hence a more

realistic goal is to mitigate resistance. However,resis-

tance mitigation programmes will not be sustainable

if based on single-tactic strategies, and Hoy (1995)

recommended a multi-tactic strategy, including: mon-

itoring pests densities; evaluating economic injury

levels; deploying and conserving biological control

agents; and using host-plant resistance, cultural, bio-

rational and genetic controls.

The requirement for integration and interaction

between all parties concerned with transgenic crops,

e.g. regulators, manufacturers of the technology, seed

companies, agri-chemical distributors and dealers,

agricultural educators and end-users of the technology,

was emphasised by Riebe (1999). In addition, prac-tical considerations such as whether IRM strategies

can be easily incorporated into existing farm manage-

ment systems, or be universal enough to apply to very

diverse production environments, are crucial. One of

the most critical ideas for growers to accept, is the

simple premise that susceptible individuals need to

be preserved (Hoy 1999). It is clearly essential that

this concept which is somewhat counterintuitive in

terms of conventional chemical control approach is

widely accepted, in order to successfully implement

IRM strategies.

Testing alternative strategies suchas bigger refugesversus alternating transgenic and non-transgenic cotton

between years (Tabashnik 1997) in large areas under

commercial conditions is difficult, and the opportunity

to take effective action to delay the evolution of resis-

tance in PBW is passing (Flint & Parks 1999).

Resistance management practices for long rotation,

transgenic tree crops with high economic damage

thresholds present particularly challenging tasks, pri-

marily because of their longevity (Bauer 1997).

Development of resistance

The historical lack of Bt resistance in an insect pop-ulation could be simply the result of limited expo-

sure (Gould 1998). For example, selection pressures

on ECB populations in maize crops prior to the intro-

duction of Bt maize were minimal, due to a combi-

nation of inadequate coverage by conventional foliar

Btbiopesticides, their limited field persistence and the

narrow window of opportunity for application (Bolin

et al. 1999). However, there appears to be a high poten-

tial for resistance development to Bt in ECB (Huang

et al. 1999c), although resistance in the field has not

been reported, and Lang et al. (1996) found that after

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164 R.J.C. Cannon

13 generations of selection pressure, no ECB colony

survived on transgenic Btmaize hybrids.

In the case of the tobacco budworm, H. virescens, a

major pest of cotton in the USA, the magnitude of the

resistance conferred by a major locus, together with the

high initial frequency of the resistance allele, strongly

suggests that field populations of this species have the

potential to rapidly attainresistance toBtcotton (Gould

et al. 1997; Heckel et al. 1997).

Strains of DBM from Hawaii, which were resis-

tant to conventional Bt insecticides were reported

by Tabashnik et al. (1997b), and a Cry1Ac-resistant

strain from Hawaii selected for extremely high

levels of resistance in the laboratory was able to

develop on transgenic canola, Brassica napus L., with-out any adverse effect (Ramachandran et al. 1998b).

Tang et al. (1999) also found that resistant DBM lar-

vae evolved via exposure to foliar sprays of Bt in

commercial crucifer fields in Florida were able to

complete development from egg to adult, and cycle

for multiple generations, on broccoli (Brassica oler-

acea L. subsp. italica) expressing the Cry1Ac toxin.

CPB larvae resistant to formulated Bt var. tenebrionis

insecticides, remained susceptible to Bt potato plants

that express high concentrations of toxin (Altre et al.

1996).

Strategies for mitigating resistance

The use of individual toxins (or protoxins) in trans-

genic plants may be less durable, and might induce

resistance more readily, than formulated materials

containing multiple Cry proteins and spores (Liu

et al. 1996). Additive, or synergistic, effects have

been suggested between some Cry toxins, but the

extent of synergism will depend on factors such as

the strain of insect, the type of spore and the set

of toxins (Liu et al. 1998). Certain toxins, such as

Cry1Fa, appear to show no cross-resistance (Muller-

Cohn et al. 1996) and could be promising candidatesfor use in resistance management strategies. Expres-

sion of Cry toxins in chloroplasts may offer some

potential for overcoming Bt-resistance (Kota et al.

1999).

Koskella and Stotzky (1997) suggested that the

potential persistence and retention of activity of Cry

toxins in the soil environment could circumvent all

other resistance management strategies. For example,

certain species could be simultaneously exposed to two

different toxins (one in the plant and the other in the

soil) as a result of persistence from previous plantings

of transgenic crops.

Deployment of Bt crops in IPM

Duck and Evola (1997) considered that transgenic

plants fit well with integrated strategies for pest

management, with the advantages that they do not

require scouting (=monitoring) at least not for pri-

mary pest targets or the application of chemicals.

Similarly Fischhoff (1996) considered that Bt cotton

would be a useful tool in IPM schemes, in part because

it only affected a few, targeted species.

Although mixtures of conventional Bt formulations

with low doses of chemical products, were particu-larly effective at reducing the overall usage of syn-

thetic chemicals in sweet corn (Bartels & Hutchinson

1995), this integrated approach required more accu-

rate application timings i.e. in relation to pest pop-

ulation levels by the farmer, and hence a need

for monitoring, which the transgenic crop does not.

Indeed, the costs associated with scouting and spray-

ing for a sporadic pest such as ECB can exceed the

costs of losses caused by damage (Carozzi & Koziel

1997).

van Emden (1999) emphasised the disadvantages of

single toxin transgenic plant resistance, compared toplant resistance obtained by traditional plant breeding,

and suggested that both biological control agents and

chemical insecticides often act synergistically with the

latter. However, Meade and Hare (1995) found that the

combined effects of host-plant resistance in celery and

Bt insecticides was additive under field conditions.

Conclusions

In purely commercial terms, Bt crops have overcome

many of the disadvantages associated with conven-

tional microbial biopesticides, effectively breaking outof a niche which represented less than 1% of the

global crop protection market (Lisansky 1997). This

has created the potential for the utilisation of Cry tox-

ins on a vastly increased scale. Although Bt crops

contain an order of magnitude less toxin, on a gram

per hectare basis, than conventional applications of Bt

biopesticides, the protein remains viable, and unde-

graded, within the plant for a considerably longer

period, effectively throughout the growing season. As

such, it has the advantage of being in the right place

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Bt transgenic crops: risks and benefits 165

at the right time, for example when a neonate larvae

takes its first bite of a transgenic crop, but conversely

presents a potential hazard to susceptible non-target

organisms should they be exposed. Potential routes

of exposure are many and varied, including seep-

age from the roots of the Bt plant, the incorpora-

tion of plant residues within soil, and the passage

through trophic systems to higher levels exposing

predators, parasites and scavengers. Such mechanisms

and processes need to be explored thoroughly to estab-

lish the extent of the ecotoxicological risk profile.

There are also the consequences for insecticide resis-

tance as a result of the increased scale and extent of

usage.

Bt crops have, to some extent, removed the neces-sity for scouting and monitoring target pests and the

requirement for accurate timing of conventional insec-

ticide treatments. However, there is still a requirement

to monitor and treatnon-target, non-susceptiblespecies

using conventional control techniques. Whilst emerg-

ing products are aimed at removing, or diminishing this

need, there is a wide consensus that to avoid the devel-

opment of resistance, multi-tactical approaches to pest

control are needed. In addition, somewhat counterintu-

itive concepts, such as the preservation of a sufficiently

large pool of susceptible individuals of the pest pop-

ulation, in refuges, are essential to the continued andlong-term effectiveness of these products. This, and the

requirement for the placement and planting of accept-

ably large non-Bt refuges, will place other demands

on the farmer. The requirement for regional cooper-

ation and a collaborative approach is increased. This

is a factor which will have to be addressed in devel-

oping countries, where extension services and gov-

ernment support may not be as well funded as in the

developed world.

Interactions between both target and non-target

species, their natural enemies, and the Btplant need to

be evaluated in the context of risk to beneficial species

andIRM strategies. Risk assessments will haveto focuson the wider ecological environment (and conserva-

tion issues), as well as the immediate agroecological

situation in which the crop is located. However, evalu-

ating risks in terms of current practices or at least

the most harmless options rather than against a

utopian zero risk scenario, is to be encouraged. Eco-

logical risk assessments must also take account of

the geographical dimensions of target and non-target

species, and include appropriate hypotheses for poten-

tial hazards, which can then be empirically tested. Such

experiments need to take account of the different scales

within which such processes act, and ensure that all

extrapolations, i.e. from small-scale to large-scale and

short-term to long-term, are valid and well-tried and

tested.

References cited

Alstad, D.N. and Andow, D.A. (1995) Managing the evolu-

tion of insect resistance to transgenic plants. Science 268,

18941896.

Alstad, D.N. and Andow, D.A. (1999) Implementing manage-

ment of insect resistance to transgenic crops. http://www.

agbiotechnet.com/review/misc/alstad.html.

Altieri, M.A. (1998)The environmental risks of transgenic crops:an agroecological assessment. AgBiotech News Inform. 10,

405N410N.

Altre, J.A., Grafius, E.J. and Whalon, M.E. (1996) Feed-

ing behaviour of CryIIIA-resistant and susceptible Col-

orado potato beetle (Coleoptera: Chrysomelidae) larvae

on Bacillus thuringiensis tenebrionis-transgenic CryIIIA-

treated and untreated potato foliage. J. Econ. Entomol. 89,

311317.

Alyokhin, A.V. and Ferro, D.N. (1999a) Modifications in disper-

sal and oviposition ofBt-resistant and Bt-susceptible Colorado

potato beetles as a result of exposure to Bacillus thuringien-

sis subsp. tenebrionis Cry3A toxin. Entomol. Exp. Appl. 90,

93101.

Alyokhin, A.V. and Ferro, D.N. (1999b) Mating ability of Col-

orado potato beetle (Coleoptera: Chrysomelidae) males fedtransgenic potato (Solanaceae) foliage. Canad. Entomol. 131,

539540.

Alyokhin, A.V. and Ferro, D.N. (1999c) Relative fitness of Col-

orado potato beetle (Coleptera: Chrysomelidae) resistant and

susceptible to the Bacillus thuringiensis Cry3A toxin. J. Econ.

Entomol. 92, 510515.

Alyokhin, A.V., Ferro, D.N., Hoy, C.W. and Head, G. (1999)

Laboratory assessment of flight activity displayed by Colorado

potato beetle (Coleoptera: Chrysomelidae) fed on transgenic

and Cry3a toxin-treated potato foliage. J. Econ. Entomol. 92,

115120.

Anderson, J.L. (2000) Letter to Bt corn registrants 12/20/99.

United States Environmental Protection Agency. http://www.

epa.gov/pesticides/biopesticides/otherdocs/bt corn ltr.html.

Andow, D.A. andAlstad,D.N. (1998)F2 screenfor rare resistancealleles. J. Econ. Entomol. 91, 572578.

Andow,D.A. and Alstad, D.N. (1999) Credebilityintervalfor rare

resistance allele frequencies. J. Econ. Entomol. 92, 755758.

Andow,D.A. andHutchison, W.D.(1998)Bt-cornresistance man-

agement. In M. Mellon and J. Rissler (eds) Now or Never:

Serious New Plans to Save a Natural Pest Control , pp. 1966.

Cambridge, MA, USA: Union of Concerned Scientists.

Andow,D.A., Alstad, D.N., Pang,Y.-H., Bolin, P.C. and Hutchin-

son, W.D. (1998) Using an F2 screen to search for resis-

tance alleles to Bacillus thuringiensis toxin in European

corn borer (Lepidoptera: Crambidae). J. Econ. Entomol. 91,

579584.

8/22/2019 a:1011347122894.pdf

16/23

166 R.J.C. Cannon

Anon. (1993) The Regulation and Control of the Deliber-

ate Release of Genetically Modified Organisms. DOE/ACRE

Guidance Note No. 1. DETR publication.Anon. (1996) Position statement on transgenic insect-resistant

crops: potential benefits and hazards. Amer. Entomol. 42,

181182.

Anon. (1998a) News Release: Agribiotech, Inc. and Myco-

gen to develop insect resistant alfalfa using Bt technology.

http://www.mycogen.com.

Anon. (1998b) News Release: Know this years pests to develop

next years pest control. http://www.mycogen.com.

Anon. (1999a) Advisory Committee on Releases to the Environ-

ment Annual Report No. 5: 1998. DETR publication: Crown

copyright.

Anon. (1999b) Global GM crop planting up 44% in 1999. Agrow

340, 22.

Anon. (1999c) Biotech Food Products. Bio Member Survey. No.368. http://www.biotechknowledge.com

Anon. (1999d) Novartis website. http://www.seeds.novartis.com.

Anon. (1999e) News release: in-plant protection out-performs

rootworm insecticides. http://www.mycogen.com.

Anon. (1999f) Continued growth in 1998: acreage totals of Mon-

santosbiotechnology crops.Monsanto Today Newsletter1142.

http://www.biotechknowledge.com.

Anon. (2000) Germany bans Btmaize planting. Agrow 347, 9.

Arpaia, S., Gould, F. and Kennedy, G. (1997) Potential impact

ofColeomegilla maculata predation on adaptation ofLeptino-

tarsa decemlineata to Bt-transgenic potatoes. Entomol. Exp.

Appl. 82, 91100.

Asano, S.-I., Nukumizu, Y., Bando, H., Iizuka, T. and

Yamamoto, T. (1997) Cloning of novel enterotoxin genes

from Bacillus cereus and Bacillus thuringiensis. Appl. Envi-ron. Microbiol. 63, 10541057.

Atwood, D.W., Kring, T.J. and Young, S.Y. III (1999) Micropli-

tis croceipes (Hymenoptera: Braconidae) development in in

tobacco budworm (Lepidoptera: Noctuidae) larvae treated with

Bacillus thuringiensis and thiodocarb. J. Entomol. Sci. 34,

249259.

Bailey, W.D., Zhao, L.M., Carter, L.M., Gould, F., Kennedy, G.G.

and Roe, R.M. (1998) Feeding disruption bioassay for species

and Bacillus thuringiensis resistance diagnosis for Heliothis

virescens and Helicoverpa zea in cotton (Lepidoptera: Noctu-

idae). Crop Protect. 17, 591598.

Bartels, D.W. and Hutchinson, W.D. (1995) On-farm efficacy of

aerially applied Bacillus thuringiensis for European corn borer

(Lepidoptera: Pyralidae) and corn earworm (Lepidoptera: Noc-

tuidae) control in sweet corn. J. Econ. Entomol. 88, 380386.Bartels, D.W., Hutchinson, W.D., Fritz, V.A. and Klacan, G.R.

(1995) Effect of Bacillus thuringiensis application interval on

European corn borer (Lepidoptera: Pyralidae) control in sweet

corn. J. Entomol. Sci. 30, 374389.

Bauer, L.S. (1995) Resistance: a threat to the insecticidal crys-

tal proteins of Bacillus thuringiensis. Florida Entomol. 78,

414443.

Bauer, L.S. (1997) Fiber farming with insecticidal trees.

J. Forestry 95, 2023.

Baum, J.A., Kafefuda, M. and Gawron-Burke, C. (1996)

Engineering Bacillus thuringiensis bioinsecticides with an

indigenous site-specific recombination system. Appl. Environ.

Microbiol. 62, 43674373.

Bel, Y., Granero, F., Alberola, T.M., Martnez-Sebastian,M.J. and

Ferre, J. (1997) Distribution, frequency and diversity ofBacil-

lus thuringiensis in olive tree environments in Spain. System.Appl. Microbiol. 20, 652658.

Benedict, J.H., Sachs, E.S., Altman, D.W., Deaton, W.R.,

Kohel, R.J., Ring, D.R. and Berberich, S.A. (1996) Field per-

formance of cottons expressing transgenic CryIA insecticidal

proteins for resistance to Heliothis virescens and Helicoverpa

zea (Lepidoptera: Noctuidae). J. Econ. Entomol. 89, 230238.

Bergvinson, D., Willcox, M. and Hoisington, D. (1997) Efficacy

and deployment of transgenic plants for stemborer manage-

ment. Insect Sci. Applic. 17, 157167.

Bernhard, K., Jarrett, P., Meadows, M., Butt, J., Ellis, D.J.,

Roberts, G.M., Pauli, S., Rodgers, P. and Burgess, H.D. (1997)

Natural isolates ofBacillus thuringiensis: worldwide distribu-

tion, characterisation,and activityagainst insectpests.J. Invert.

Pathol. 70, 5968.

Blumberg, D., Navon, A., Keren, S., Goldenberg, S. andFerkovich, S.M. (1997) Interactions among Helicoverpa

armigera (Lepidoptera: Noctuidae), its larval endoparasitoid

Microplitis croceipes (Hymenoptera: Braconidae), and Bacil-

lus thuringiensis. J. Econ. Entomol. 90, 11811186.

Bolin, P.C., Hutchinson, W.D. and Andow, D.A. (1999) Long-

term selection for resistance to Bacillus thuringiensis Cry1Ac

endotoxin in a Minnesota population of European corn borer

(Lepidoptera: Crambidae). J. Econ. Entomol. 92, 10211030.

Bourguet, D., Bethenod, M.T., Trouve, C. and Viard, F. (2000)

Host-plant diversity of the European corn borer Ostrinia nubi-

lalis: what value for sustainable transgenic insecticidal Bt

maize? Proc. R. Soc. Lond. B 267, 11771184.

Bradfisch, G.A.,Stockhoff, B. andMuller-Cohn, J. (1998) Pestici-

dal Bacillus thuringiensis strains. Patent No. WO9840491 A2.

Bravo, A. (1997) Phylogenetic relationships of Bacillus

thuringiensis -endotoxin family proteins and their functional

domains. J. Bacteriol. 179, 27932801.

Burn, A.J.C. (1999) Genetically modified crops: the research

requirements of the conservation agencies. In F. Amijee,

C.J. Gliddon and A.J. Gray (eds) Environmental Impact

of Genetically Modified Crops Research Report No. 10

pp. 239247. London: DETR Publication.

Cannon, R.J.C. (1993) Prospects and progress for Bacillus

thuringiensis-based pesticides. Pesticide Sci. 37, 331335.

Cannon, R.J.C. (1996) Bacillus thuringiensis use in agriculture:

a molecular perspective. Biol. Rev. 71, 561636.

Caprio, M.A.(1998) Evaluating resistancemanagementstrategies

for multiple toxins in the presence of external refuges. J. Econ.

Entomol. 91, 10211031.

Carozzi, N.B. and Koziel,M.G. (1997) Transgenic maizeexpress-

ing a Bacillus thuringiensis insecticidal protein for control of

European corn borer. In N.B. Carozzi, and M.G. Koziel (eds)

Advances in Insect Control: The Role of Transgenic Plants,

pp. 6374. London: Taylor & Francis Ltd.

Chaufaux, J.,Marchal, M., Gilois, N., Jehanno, I. andBuisson, C.

(1997) Recherche de souches naturellesduBacillus thuringien-

sis dans differents biotopes, a travers le monde. Canad. J.

Microbiol. 43, 337343.

Chenot, A.B. and Raffa, K.F. (1998) Effects of parasitoid strain

and host instar on the interaction of Bacillus thuringiensis

subsp. kurstaki with the gypsy moth (Lepidoptera: Lymantri-

idae) larval parasitoid Cotesia melanoscela (Hymenoptera:

Braconidae). Environ. Entomol. 27, 137147.

8/22/2019 a:1011347122894.pdf

17/23

Bt transgenic crops: risks and benefits 167

Chilcutt, C.F. and Tabashnik, B.E. (1997a) Host-mediated com-

petition between the pathogen Bacillus thuringiensis and the

parasitoid Cotesia plutellae of the diamondback moth (Lepi-doptera: Plutellidae). Environ. Entomol. 26, 3845.

Chilcutt, C.F. and Tabashnik, B.E. (1997b) Independent and

combined effects of Bacillus thuringiensis and the parasitoid

Cotesia plutellae (Hymenoptera: Braconidae) on susceptible

and resistant diamondback moth (Lepidoptera: Plutellidae).

J. Econ. Entomol. 90, 397403.Chilcutt, C.F. and Tabashnik, B.E. (1999) Effects of Bacillus

thuringiensis on adults of Cotesia plutellae (Hymenoptera:

Braconidae), a parasitoid of the diamondback moth (Lepi-

doptera: Plutellidae). Biocont. Sci. Technol. 9, 435440.Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van

Rie, J.,Lereclus, D.,Baum,J. andDean,D.H. (1995a) Revision

of the nomenclature for the Bacillus thuringiensis pesticidal

crystal proteins. Microbiol. Molec. Biol. Rev. 62, 807813.

Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E.,Lambert, B., Lereclus, D., Baum, J. and Dean, D.H. (1995b)

Revision of the nomenclature for the Bacillus thuringiensis

pesticidal cry genes. In Programme and Abstracts of the 28th

Annual Meeting of the Society for InvertebratePathology,p.14.

Society for Invertebrate Pathology, Bethesda, MD, USA.Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J.,

Lereclus, D., Baum, J, Bravo, A. and Dean, D.H. (2000) Bacil-

lus thuringiensistoxin nomenclature. http://www.biols.susx.ac.

uk/Home/Neil Crickmore/Bt/ index. html.Damgaard, P.H. (1996) Diarrhoel enterotoxin production by

strains of Bacillus thuringiensis isolated from commercial

Bacillus thuringiensis-based insecticides. FEMS Immunol.

Medical Microbiol. 12, 245250.Damgaard, P.H., Hansen, B.M., Pedersen, J.C. and Eilenberg, J.

(1997) Natural occurrence ofBacillus thuringiensis on cabbagefoliage and in insects associated with cabbage crops. J. Appl.

Microbiol. 82, 253258.Damgaard, P.H., Abdel-Hameed, A., Eilenberg, J. and Smits, P.H.

(1998) Natural occurrence of Bacillus thuringiensis on grass

foliage. World J. Microbiol. Biotech. 14, 239242.Dolgov, S.V., Mityshkina, T.U., Rukavtsova, E.B. and

Buryanov, Y.I. (1995) Production of transgenic plants of

Chrysanthemum morifolium Ramat with the gene of Bac.

thuringiensis -endotoxin. Acta Hort. 420, 4647.Donegan, K.K., Schaller, D.L., Stone, J.K., Ganio, L.M.,

Reed, G., Hamm, P.B. and Seidler, R.J. (1996) Microbial pop-

ulations, fungal species diversity and plant pathogen levels in

field plots of potatoplants expressing theBacillus thuringiensis

var. tenebrionis endotoxin. Transgen. Res. 5, 2535.

Donegan, K.K., Seidler, R.J., Doyle, J.D., Porteous, L.A.,Digiovanni, G., Widmer, F. and Watrud, L.S. (1999) A

field study with genetically engineered alfalfa inoculated

with recombinant Sinorhizobium meliloti. J. Appl. Ecol. 36,

920936.Duck, N. and Evola, S. (1997) Use of transgenes to increase

host plant resistance to insects: opportunities and challenges.

In N.B. Carozzi andM.G. Koziel (eds)Advances in Insect Con-

trol: The Roleof TransgenicPlants, pp.120. London: Taylor&

Francis Ltd.Duvick, D.N. (1999) How much caution in the fields? Science

286 (15 Oct 1999), 418419. [A review of van Dommelen, A.

(1999) Hazard Identification of Agricultural Biotechnology:

Finding Relevant Questions. Utrecht, The Netherlands: Inter-

national Books.]

Edwards, C.A. (1994) The risks of releasing genetically engi-

neered organisms into the environment with particular refer-

ence to plants and soil communities. Agricul. Zool. Rev. 6,277290.

Elbert, A., Overbeck, H., Iwaya, K. and Tsuboi, S. (1990) Imida-

cloprid, a novel systemic nitromethylene analogue insecticide

for crop protection. In Brighton Crop Protection Conference

Pests and Diseases, Vol. I, pp. 2128. Farnham, Surrey, UK:

The British Crop Protection Council.

Ellar, D.J. (1997) The structure and function of Bacillus

thuringiensis -endotoxins and prospects for bioinsecticide

improvement. Microbial Insecticides: Novelty or Necessity?

BCPC Symposium Proceedings No. 68. pp. 83100.

EPA (1997) Plant pesticide resistance management. Fed. Regist.

62, 82428244.

EPA (1999) EPA and USDA position paper on insect resistance

management in Bt crops. (27 April 1999.) http://www.epa.

gov/oppbppd1/biopesticidesotherdocs /bt position paper 6