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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.
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