Integrated Pest Management Reviews 5: 151173, 2000. British Crown Copyright 2001. Printed in the Netherlands.

Bt transgenic crops: Risks and benets

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 signicant increases in yields and prots. 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 Bt crops, 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 for Bt crops 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 Bt Cry toxins are described. Field monitoring of non-target entomofauna has not revealed signicant differences in the abundance or biodiversity of benecial insects associated with Bt maize. Indeed, laboratory studies of effects on parasitoids suggest that Bt plants 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 Bt crops with non-target invertebrates and to dene the complex relationship between IRM, target species and their natural enemy assemblages. 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 benets and hazards are reviewed. Introduction This review concerns the utilisation of insecticidal 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, Bt crops transgenically expressing cry genes derived from Bt isolates (Peferoen 1997), and mainly work published since a previous review (Cannon 1996).

152 The reader is referred to other reviews concerning Bt -endotoxins, for details on molecular biology (Schnepf et al. 1998), mode of action (Knowles 1994), phylogenetic relationships (Bravo 1997), structure/function relationships (Grochulski et al. 1995; Ellar 1997) and insect resistance (Tabashnik 1997; Gould 1998; Mellon & Rissler 1998).

R.J.C. Cannon 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; Bradsch 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 discovered. For example, a novel ICP (Cry9Ca1) from Bt var. tolworthi, is active against cutworms, such as Agrotis segetum (Denis & Schifferm uller) 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 of genes encoding 18 different Cry proteins, with multiple toxicity to a very wide range of species including coleopterans, lepidopterans, dipterans, hymenopterans and nematodes was reported by Osman et al. (1999). Finally, a novel class (Vip3A) of vegetatively produced, lepidopteran-active Bt proteins with no homology with known -endotoxins has been discovered (Estruch et al. 1996).

Biology and ecology of naturally-occurring Bt isolates Distribution and occurrence Bt is a ubiquitous soil bacterium, which is also present in 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). Many Bt isolates, particularly those from soil samples, are non-toxic to a wide range of insects (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 classied on the basis of their agellar (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 Bt isolates, in general, shows no apparent relationship with serovar, although common gene combinations 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 activity against a wide variety of new target species. These

Bt transgenic crops and trees Bt genes have been used to transform at least 26 different 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 different cry genes have been utilised to date (Bauer 1997; Schuler et al. 1998). The expression of cry genes is inuenced by a number of different factors, including both the genetic (where in the genome the gene construct is inserted) and physical environment (eld site), plant age and tissue type (Sachs et al. 1998; Greenplate 1999). Transgenic Bt crops rst appeared on the market on a large scale in 1996, in the USA. By 1997, insect-resistant cotton and maize were grown globally 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, Bt crops were grown in relatively small areas in 1999: Spain (30,000 ha), France (1000 ha), Portugal

Bt transgenic crops: risks and benets

Table 1. Bt crop products marketed in different countries1 Product name I. Maize StarLink DeKalBt YieldGard NatureGard NR Knockout II. Sweet corn Attribute III. Cotton BXN Cotton Bollgard cotton IV. Potato NewLeaf
1

153 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 signicant pests of cotton in the southern USA, are the fall armyworm Spodoptera frugiperda (Abbott and Smith) and the beet armyworm, S. exigua (H ubner). 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 signicant increases in yields in most years for some regions (ERS 1999). Despite the requirement for additional sprays for insects not susceptible to Bt (see below), the adoption of Bt cotton was associated with signicant increases in yields and prots, and decreased pesticide use (Merritt 1998; ERS 1999; Gianessi & Carpenter 1999). However, this decline in the application of broad-spectrum insecticides had the result that populations of non-Bt susceptible 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 signicant 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 (H ubner), is a major pest of eld 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 sufcient to meet the standards of vegetable processors (Bartels et al. 1995). For example, in sweet corn, a high-quality product free of insect contamination or damage could only be guaranteed by the use of chemicals (Bartels & Hutchinson 1995). However, in lower-quality fodder maize, losses due to the ECB were largely tolerated, and chemical control was not generally carried out. As a result, the introduction of Bt maize only resulted in

Company AgrEvo, Inc. DeKalb Genetics Corp Monsanto Mycogen Corp Novartis Seeds3

Country

US, CA US US, CN, AR US US, FR, ES, AR, CN US US US, AU, CN, MX, ZA, AR US, CN

Novartis Seeds Calgene Monsanto

Monsanto

Only Bt products with insecticide-resistance traits alone are shown; other products, such as Bollgard with BXN cotton are also herbicide resistant. 2 Isocodes for countries of the world. 3 Two 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 and the value of the bre was higher, compared to parent varieties sprayed according to scouting recommendations (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 gossypiella (Saunders). Bt cotton varieties do not control the cotton bollworm (also called the corn earworm), Helicoverpa zea (Boddie), as well as they control

154 modest savings from reduced insecticide applications, although its use was associated with signicantly 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 rst introduced in 1996 in the USA, and by 1998, 18% of the national acerage was planted with Bt maize (14.4 million acres). In Europe, Novartis Bt maize 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 introduced in the US (Table 1) signicantly 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 number, refers to the unique genetic transformation event when the modied 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 kernels, whereas Events BT11 and MON810, result in full season expression in leaf, pollen, tassel, silk and kernel tissue (Fearing et al. 1997; Andow & Hutchinson, 1998). CryIAb protein concentration levels in transgenic 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 transgenic 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). However, Fusarium infections of the ears and kernels, and symptomless infection of kernels, were consistently reduced in Bt maize (Munkvold et al. 1997). Transgenic maize lines and hybrids developed primarily for ECB resistance have also demonstrated good control of other stemborers, such as Diatraea grandiosella Dyar (Bergvinson et al. 1997), and Sesamia nonagrioides (Lefebvre) (Na bo 1999).

R.J.C. Cannon 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 toxins expressed Bt maize (Anon. 1998b; Lynch et al. 1999). Potato Bt potatoes (New Leaf ) were planted on 50,000 acres in the USA in 1998, exclusively for the control of the Colorado potato beetle (CPB), Leptinotarsa decemlineata 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 technology reects, 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 insecticides such as imidacloprid, a systemic compound highly effective against both the CPB and sucking pests such as aphids (Elbert et al. 1990), also contributed to the slow adoption of the Bt potato technology. Novel crops and new developments A number of other Bt crops, including alfalfa, tomatoes, sunower, soybeans, oil seed rape (canola) and wheat are being developed and could be introduced to the market within the next ve years (SeongLyul 1995; Anon. 1998a; 1999c). Additionally, second generation 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 incorporating new and more powerful promoters, new and tighter tissue-specic promoters as well as a better understanding of the principles governing plant gene expression (Estruch et al. 1997). Horticultural and ornamental plants have also been modied to express cry genes, including petunias (Omer et al. 1997) and chrysanthemums (Dolgov et al. 1995). Transgenic expression also opens up the possibility of delivering a toxic dose of a selected Bt toxin to pests which would not have encountered conventionalapplied 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).

Bt transgenic crops: risks and benets The development and use of agricultural biotechnology 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 Guen ee, 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 Fr ohner and Coffea arabica L., to confer resistance to coffee leaf miner, Perileucptera coffeella (Gu erin-M eneville) (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 Scirpophaga incertulas (Walker), resistance was enhanced in transformed rice (cv. Tarom Molaii) via the expression of a cryIAb gene in the rice leaf blades; the toxin was not detectable in the dehulled, mature grain (Ghareyazie et al. 1997).

155 Certain, naturally-occurring combinations of toxins (e.g. Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa) in Bt var. israelensis, provide an advantage in suppressing resistance, compared with single toxins (Wirth & Georghiou 1997). This CytA/CryIV model (old terminology) could provide a molecular genetic strategy for engineering resistance management for Cry proteins directly into transgenic plants (Wirth et al. 1997). Bt strains with unique combinations of cry genes can also be designed and engineered using molecular recombinant systems (Baum et al. 1996). Fitness costs A much lower tness and intrinsic rate of increase in a resistant, laboratory population of DBM, suggested a trade-off between Bt-resistance and tness (Shirai et al. 1998). However, Tang et al. (1997) found that Bt resistance in DBM did not confer detectable levels of reduced tness in the absence of exposure to Bt. Liu et al. (1995) found that stronger expression of resistance occurred in 3rd instar DBM, than in neonates, and suggested that it might be disadvantageous for resistance to increase uniformly in all instars. Fitness costs have also been reported for Cry3Aresistant CPB, including reduced larval weight, reduced fecundity, shortened oviposition period, reduced egg-mass size, increased overwintering mortality and reduced population growth rate (Trisyono & Whalon 1997; Alyokhin & Ferro 1999c). Baseline susceptibility and eld monitoring Surveillance of transgenic crops in the form of scouting for insect survivors is the most straightforward 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 Bt crops, is essential for detecting the early development of resistance. Monitoring protocols, e.g. for the ECB in Europe (SCP 1999) and the USA (Anderson 2000), emphasise 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 susceptibility have been detected (Huang et al. 1997). However, variability in tolerance to CryIAb among ECB populations was unrelated to prior exposures to (Bt) pesticides (Siegfried et al. 1995).

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 Bt maize producing Cry1Ab. In other words, pests may evolve resistance to some groups of toxins much faster than expected. However, other studies have suggested that Bt resistance mechanisms might be specic 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 the use of refuges or seed mixtures (Bauer 1995; Strizhov et al. 1996; Tabahsnik et al. 1997b).

156 Preliminary monitoring of different H. 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 eld doses of MVPII the closest in toxicological properties of all Bt insecticides to the CryIAc protein expressed in transgenic cotton in spray chamber bioassays. However, transgenically expressed protein in maize is only 65% homologous to the original protein, and results obtained using puried, naturally-occurring toxins, or biopesticides, may not translate to the transgenic version (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 puried proteins and commercial formulations were observed amongst populations of H. 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 concentrations be taken, in addition to maintaining a viable integrated pest management (IPM) programme. Relatively simply methods for detecting signicant 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 rst commercial plantings of transgenic cotton varieties, was estimated as 1.5 103 (Gould et al. 1997). Roush & Shelton (1997) questioned whether this initial resistance frequency is actually a single major gene, or instead a resistance phenotype that may be under control of more than one locus. However, genetic linkage analysis 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

R.J.C. Cannon 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 the frequency of resistance alleles in a ECB population (< 0.013), and concluded that alleles for partial resistance may be reasonably common in natural ECB populations, but alleles for complete resistance may be rare. Resistance was dened as when neonate ECB larvae could (both) survive and develop into the 2nd instar on Bt corn. The sensitivity of the F2 screen is such that the frequency of resistance alleles can be detected down to p = 2 104 , from sampling 1200 females. However, Schneider (1999) has since shown that the number of individuals that must be screened to achieve a 95% level of condence using the F2 screen is only ca. 750 mated females, and Andow and Alstad (1999) agree that they originally overestimated the effort needed for the F2 screen. The USEPA require biotechnology companies marketing Bt corn products in the USA to investigate the feasibility and utility of the F2 screen as a condition of their continued registration (Anderson 2000). Resistance management for transgenic crops and trees Potential insecticide resistance management (IRM) strategies for Bt crops, include: mixtures of toxins; the use of synergists; temporal and spatial rotations of toxins; refuges; low doses to produce sub-lethal effects; ultrahigh doses to kill resistant heterozygotes and homozygotes; and gene regulation of toxin expression (Bauer 1995; Roush 1996; Gould 1998). However, the high dose strategy, combined with the use of refuges and avoiding mosaics of different pesticides and toxins, is widely agreed to be the best technical approach for managing resistance (Roush 1997a; Gould 1998). A high dose is dened as a titre of toxin capable of causing the mortality of all heterozygous individuals which feed on the transgenic tissue (Alstad & Andow 1999). In practice, the EPA has endorsed a concentration of toxin which is at least 25-fold higher than the dose which kills 99% of the susceptible pest species (op. cit. Renner 1999). The basic goals of the high dose/refuge strategy are to reduce the tness between susceptible and resistant insects, and the degree to which a resistant insect can pass on its phenotypic trait to its offspring (Gould 1998). The inclusion of non-transformed host plants (in refuges) close to the crop is the best way to ensure the

Bt transgenic crops: risks and benets availability of susceptible insects, and is likely to be an effective way of slowing the evolution of resistance to these crops (Roush 1996). The option available to cotton growers under the EPA (1997) mandate, was, i) for every 100 ha of Bt cotton, 4 ha (i.e. slightly less than 4%) of non-Bt cotton must be planted and these cannot be sprayed with insecticides 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 insecticide, except Bt bioinsecticides. Tabashnik (1997) considered that the rst option might work well under ideal conditions, but if the optimistic assumptions regarding inheritance and mating are violated, the number of homozygous susceptible individuals generated by a 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 individuals by conventional insecticide treatments in the non-Bt cotton, could essentially eliminate the refuge. Since older larvae are generally less susceptible to transgenic Bt plants than neonate larvae (e.g. Wierenga et al. 1996), IRM strategies should allow for the least susceptible stage (Huang et al. 1999a) because larvae 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 receptors 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 to Bt crops. However, a widespread consensus on the importance of such measures has developed, and recommendations have been made by a number of authorities and alliances (e.g. EPA 1997; Feldman & Stone 1997; Andow & Hutchinson 1998; Gould et al. 1998; Gould & Tabashnik 1998; McGaughey et al. 1998; EPA 1999; SCP 1999; Vlachos et al. 1999; Anderson 2000). For example, recommendations for transgenic crops specify that between 20 and 50% of any given area should include nontransgenic crops (EPA 1999). The EPA stipulates that Bt corn registrants in the USA must ensure that growers plant a minimum structured refuge of at least 20%

157 non-Bt corn, and for Bt corn grown in cotton growing 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 of Bt and non-Bt elds, and the temporal pattern of refuges inuence the development of resistance (Alstad & Andow 1995; Peck et al. 1999). However, Ives (1996) concluded that changing the distribution of toxic plants among elds has little potential for controlling resistance evolution. Separate refuges are superior to seed mixtures for delaying resistance in the ECB population to transgenic maize (Onstad & Gould 1998b). However, strips of 612 rows of non-transgenic maize (making up 20% of the eld) were equally effective to separate blocks of the same percentage, in terms of delaying resistance (Onstad & Guse 1999). Recent eld experiments using a model system incorporating Bt broccoli plants and DBM have conrmed 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 resistance, and the amount of safety error one desires to build into the system (Caprio 1998). Theory also predicts that when there is only a single resistance locus in the genome, and the mortality of the heterozygous individuals exposed to transgenic crops exceeds 95%, resistance can be delayed for more than 40 generations, even when resistance is initially as common as 103 and only 10% of the pest population develops on refuge hosts (Roush 1998). However, if the mortality of the heterozygotes falls below 90%, then a refuge size of 20% is needed to delay resistance > 20 generations. In addition, in species which are not particularly susceptible

158 to Bt toxins (including the ECB), and in circumstances where fewer than 90% of naturally-occurring, unselected larvae of these species would be killed, very large refuges would be required to delay resistance 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, particularly 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 nding by Bourguet et al. (2000) that ECB populations found on non-maize plants (hop and sagebrush) in the NordPas-de-Calais region of northern France may constitute a 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 experiments with eld outcomes is uncertain. The refuge/high dose strategy would not be expected to work when resistance is not recessive (Tabashnik et al. 1997b). Laboratory studies suggested that resistance 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 eld 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 diminished. However, Dipel ES differs substantially from the toxin expressed in Bt maize; it also contains spores and at least three other toxins and in addition, damage by neonates is not a reliable indicator of survival on transgenic plants. Finally, there is no evidence that either larvae from the Dipel ES-R strain, or heterozygous larvae 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 generate a disproportionately high number of homozygous resistant insects, thereby accelerating the development

R.J.C. Cannon of resistance and reducing the benets of refuges. However, 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 Movement of insects, both between Bt and non-Bt crops and within Bt crops, can affect the rate at which resistance develops (Peck et 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 plants onto transgenic cotton plants could result in feeding damage (Halcomb et al. 1996) and may allow more heterozygous 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 pure stands. Ramachandran et al. (1998a), suggested that transgenic and non-transgenic plants grown in separate rows with a wider row spacing (i.e. strip planting) 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) suggested that non-transgenic (potato) plants in a refugia would facilitate the survival of CPB stages beyond the 2nd instar (less susceptible to Bt potato than neonates), and they could then grow large enough to move onto transgenic plants and receive a sub-lethal dose. Laboratory-selected, resistant CPB continuously fed on transgenic Bt potato foliage (NewLeaf ) were capable of ight and reproduction, although it took them longer to initiate ight behaviour and their fecundity was lower (Alyokhin & Ferro 1999a). Suppression of ight as a result of ingestion of Bt potato could keep the beetles within transgenic elds, thus increasing selection pressure for the development of resistance

Bt transgenic crops: risks and benets (Alyokhin et al. 1999). However, Alyokhin and Ferro (1999b) showed that susceptible CPB males arriving in transgenic potato elds from refugia can mate with resident resistant females, and the resulting heterozygote offspring are not able to survive on transgenic crops (Alyokhin & Ferro 1999a). Selective feeding, i.e. the ability of the larvae to move and switch their feeding to other tissues containing less Cry protein, could potentially inuence dose acquisition 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 eld situation, insects may be able to feed predominantly on Bt-free plant tissue as a result of behavioural avoidance of Bt-expressing tissues (Stapel et al. 1998). No signicant differences were found in oviposition preferences for H. virescens on transgenic cotton, nor for DMB on Bt canola (Ramachandran et al. 1998a) or Bt broccoli (Tang et al. 1999). Similarly, no signicant differences were found in ECB egg mass densities between transgenic and isogenic corn (Orr & Landis 1997).

159 (Blumberg et al. 1997), but applications at recommended eld rates had negligible impact on emergence of adults (Atwood et al. 1999). Studies using the braconid wasp, Cotesia plutellae Kurdyumov, showed that highly resistant DBM hosts, which were not susceptible to infection by the pathogen, provided a refugium from competition (with Bt) for the parasitoid, whereas in susceptible hosts the pathogen effectively outcompeted the parasitoid (Chilcutt & Tabashnik 1997a). In other words, adverse mortality effects on C. plutellae occurred when developing parasitoid larvae were exposed to Bt within susceptible hosts (Chilcutt & Tabashnik 1999). Therefore, in susceptible DBM populations, Bt applications combined with C. plutellae would be highly effective, whereas in resistant populations, applications of Bt would exert little control (Chilcutt & Tabashnik 1997b). Tritrophic studies Laboratory studies of the effects of Bt crops on parasitoids of target pests, are rather few in number. Schuler et al. (1999b) investigated the behaviour of C. plutellae, parasitising DBM larvae feeding on Bt oilseed rape in a model ecosystem. No effects on the survival, or host seeking ability, of the parasitoid were detected, indicating that Bt plants may have an environmental advantage over broad spectrum pesticides. In a series of laboratory feeding experiments using Bt maize plants and articial 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 on Bt maize-fed ECB, the result was probably a combined effect of exposure to Bt and nutritional deciency caused by sick prey, and no conclusions were drawn as to how these results might translate in the eld (Hilbeck et al. 1998a). Experiments using an articial liquid diet (encapsulated Cry1Ab toxin) showed higher mortalities in larvae consistently exposed to the toxin, but no, or only small, differences 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) conrmed 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

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 assessment 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 nonBt maize (Orr & Landis 1997; Pilcher et al. 1997b). Similarly, eld monitoring of non-target entomofauna showed no differences in the abundance or biodiversity of benecial 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

160 enhanced the impact of the Bt toxins (predator mortality was higher than prey mortality at 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 elds 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 plotto-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 predators 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 signicant impact of Cry3A-intoxicated CPB on the consumption, development and fecundity of C. maculata, and suggested that this species will not be deterred from feeding on CPB in elds of transgenic potatoes. Riddick et al. (1998) investigated the relative abundance of two natural enemies of the CPB in seed-mixed, and 100% pure, elds of Cry3A-transgenic and nontransgenic potato. The results demonstrated that Lebia grandis Hentz larvae, specialist carabid ectoparasitoids of CPB, will not persist in seed-mixed and 100% transgenic potato elds, and will gradually disperse, because of the low densities of CPB in these elds. However, C. maculata adults and larvae, which are generalist coccinelid predators of early CPB larvae and other prey, are likely to thrive and ourish in elds containing transgenic potato. Therefore, predation by C. maculata 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 buttery, 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 quantication and the use of inappropriate controls (Hodgson 1999). More recently, Wraight et al. (2000) concluded that Bt pollen from event 810

R.J.C. Cannon maize would be unlikely to affect wild populations of black swallowtails, Papilio polyxenes Fabricius. Experiments investigating the effects of conventional Bt sprays on other, non-target lepidopterans have shown that it is difcult 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 overwintering D. plexippus to conventional Bt pesticides. Persistence in soil and on leaves A soil microcosm experiment, using Bt cotton, demonstrated an initially rapid decline of Cry toxin over the rst 14 days possibly due to biotic degradation but low amounts persisted for several weeks or months (Palm et al. 1996). Cry toxins become resistant to microbial utilisation, and remained insecticidal 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 of Bt corn seedlings during growth, and could add to the amount of toxin introduced into the soil from other transgenic plant sources, including pollen and plant residues after harvesting the crop (Saxena et al. 1999). Donegan et al. (1996) reported few signicant differences in phylloplane bacteria between transgenic Bt-producing potato plants and potato plants treated with microbial Bt var. tenebrionis.

Discussion Initial performance Initial reports on the performance of Bt insect-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 performance of Bt maize (YieldGard hybrids) was consistently 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 eld plots of Bt cotton in Arizona, USA, in 1993 and 1994 was so successful

Bt transgenic crops: risks and benets that Flint et al. (1995) considered that the possibility exists that the PBW can be eradicated from the SW USA. These reports echo the optimistic predictions of earlier researchers that wide-area deployment of insect-resistant crops could depress overall populations 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 insectresistant crops reduced pesticide use, although in some cases such as Bt corn the effect was small and insignicant. In certain situations, such as the of Bt cotton in southeast USA, increased adoption of this new technology was associated with signicant increases in yields and prots. 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 specically, Gould (1998) considered that transgenic insecticidal cultivars represent an extension of one form of classical host-plant resistance, namely antibiosis (or alleleopathy). However, alleleopathic substances, such as alkaloids and phenolics, are usually sublethal in their effects. Regal (1994) considered that rDNA genetic engineering 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 (phylogenetic leap-frogging); 2) the absence of the debilitating trade-offs associated with radical improvements produced 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 more risky than those which could be produced via selective breeding, but this does not mean that every GMO is ecologically dangerous. Donegan et al. (1999) believe that there will be unintentional changes in plant characteristics as a result of genetic manipulation which may impact on soil and plant biota and processes. Potential benets and hazards Potential benets claimed for Bt crops include: more effective and full-season insect control; reduced

161 scouting and monitoring costs; a reduction in conventional foliar insecticide use (with concomitant improvements in safety); and a reduced impact on nontarget organisms (Fischhoff 1996; Hutchinson 1998; Hails 2000). Potential hazards identied for GM, insectresistant crops, include: adverse effects on non-target invertebrates; the transfer of insect resistance (e.g. to genes to closely related, non-crop species); the development of resistance in target pests; and the need for additional types of pest control (Anon. 1999a). The magnitude of a risk associated with a particular hazard is dependent on the scale (size) of its introduction and the time of its realisation, the so-called wedge-effect (Harding & Harris 1997). The key to resolve 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 questions should be concerned with hazard identication (identifying a potentially bad outcome) rather than with risk analysis (calculating the odds of a bad outcome). Evaluating potential risks One of the main problems associated with assessing the risks posed by Bt crops is that there is no agreed framework, 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 principles and recommendations for the safe deployment of Bt crops. Jepson et al. (1994) proposed a conceptual framework to evaluate the risks of Bt plants: a combination of laboratory tests, eld experiments and longer-term monitoring. Determining the pattern and frequency 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 conclusions and recommendations of an OECD workshop on the ecological implications of Bt crops, and Schuler et al. (1999a) emphasised the importance of vigorous and standardised methodologies for ecotoxocological evaluations.

162 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 propensity to develop resistance (Whalon & Norris 1996). There is also a need for coordination in the production and release of different Bt crops (Wearing & Hokkanen 1995). Human health and safety issues Conventional Bt pesticides are generally considered to be very safe (Nielson-LeRoux et al. 1998), although there is a small risk to human health, largely associated with the presence of diarrhoeal enterotoxins (Damgaard 1996). However, they have been widely used for many years, and have not caused an overwhelming number of cases of gastro-enteritis to occur (Hendriksen & Hansen 1998). Some naturallyoccurring Bt strains 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 naturallyoccurring Bt at substantial levels (Mizuki et al. 1999a). Other studies (Hernandez et al. 1998) have shown that the strain of Bt H34-konkukian can be pathogenic for immunocompromised mice, but further studies are needed to evaluate the potential pathogenicity for mammals of this bacterium. This was amongst the most commonly collected serovar in a survey of Spain carried 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 the UK DETR (Anon. 1993), which is also being introduced into EC Directive 09/220/EEC, with the objective 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 consequences for human health and the environment, should those hazards be realised; 4) assess the risk; 5) consider implementation of risk management procedures;

R.J.C. Cannon and 6) take into account and risk management procedures 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 specicity of the truncated form of the Cry protein expressed in the crops: in vitro dietary tests using selected lepidopteran targets; eld 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 longterm compatibility of insecticidal plants with natural enemies, and to dene the complex relationship between resistance management, target and non-target herbivores, and their natural enemies (Arpaia et al. 1997; Hilbeck et al. 1998b). Levels of toxin and exposure models On a per acre basis, the highest level of Cry protein in Event 176 Bt maize plants was 24 g/acre (at anthesis) (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 of Bt-based insecticides correspond to ca. 45 g/acre. Therefore, 1020-fold less Bt protein is present per acre in these transgenic maize plants derived from Event 176, compared to conventional applications of Bt biopesticides (immediately after application). However, Bt Cry 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 susceptible organisms via direct or indirect exposure. Constitutive expression in transgenic Bt plants 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 progressed, 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 natural enemies (Melin & Cozzi 1990). Indeed, judicious timing of Bt applications can enhance the performance

Bt transgenic crops: risks and benets of parasitoids, at least in certain cases (Chenot & Raffa 1998). Synergistic interactions between natural enemies, 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, ecological 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 agroecosystems. However, the assumption that transgenic plants per se will cause resistance faster than sprays is, according to Roush (1996), not necessarily true; transgenic plants may actually delay resistance more effectively than sprays in some cases. However, the increased use of Bt toxins 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), resistance management programmes rely on four key management strategies: 1) diversication of mortality 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 measures to encourage survival or immigration of susceptible 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 ow 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

163 best resistance can only be delayed, hence a more realistic goal is to mitigate resistance. However, resistance mitigation programmes will not be sustainable if based on single-tactic strategies, and Hoy (1995) recommended a multi-tactic strategy, including: monitoring pests densities; evaluating economic injury levels; deploying and conserving biological control agents; and using host-plant resistance, cultural, biorational 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, practical considerations such as whether IRM strategies can be easily incorporated into existing farm management 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 such as bigger refuges versus alternating transgenic and non-transgenic cotton between years (Tabashnik 1997) in large areas under commercial conditions is difcult, and the opportunity to take effective action to delay the evolution of resistance 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, primarily because of their longevity (Bauer 1997). Development of resistance The historical lack of Bt resistance in an insect population could be simply the result of limited exposure (Gould 1998). For example, selection pressures on ECB populations in maize crops prior to the introduction of Bt maize were minimal, due to a combination of inadequate coverage by conventional foliar Bt biopesticides, their limited eld persistence and the narrow window of opportunity for application (Bolin et al. 1999). However, there appears to be a high potential for resistance development to Bt in ECB (Huang et al. 1999c), although resistance in the eld has not been reported, and Lang et al. (1996) found that after

164 13 generations of selection pressure, no ECB colony survived on transgenic Bt maize 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 eld populations of this species have the potential to rapidly attain resistance to Bt cotton (Gould et al. 1997; Heckel et al. 1997). Strains of DBM from Hawaii, which were resistant 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., without any adverse effect (Ramachandran et al. 1998b). Tang et al. (1999) also found that resistant DBM larvae evolved via exposure to foliar sprays of Bt in commercial crucifer elds in Florida were able to complete development from egg to adult, and cycle for multiple generations, on broccoli (Brassica oleracea 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 transgenic 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 (M ullerCohn et al. 1996) and could be promising candidates for use in resistance management strategies. Expression 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

R.J.C. Cannon 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 t well with integrated strategies for pest management, with the advantages that they do not require scouting (= monitoring) at least not for primary 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 particularly effective at reducing the overall usage of synthetic chemicals in sweet corn (Bartels & Hutchinson 1995), this integrated approach required more accurate application timings i.e. in relation to pest population levels by the farmer, and hence a need for monitoring, which the transgenic crop does not. Indeed, the costs associated with scouting and spraying 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 to plant 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 eld conditions.

Conclusions In purely commercial terms, Bt crops have overcome many of the disadvantages associated with conventional microbial biopesticides, effectively breaking out of 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 toxins 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 undegraded, 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

Bt transgenic crops: risks and benets at the right time, for example when a neonate larvae takes its rst 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 seepage from the roots of the Bt plant, the incorporation 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 establish the extent of the ecotoxicological risk prole. There are also the consequences for insecticide resistance as a result of the increased scale and extent of usage. Bt crops have, to some extent, removed the necessity for scouting and monitoring target pests and the requirement for accurate timing of conventional insecticide treatments. However, there is still a requirement to monitor and treat non-target, non-susceptible species using conventional control techniques. Whilst emerging products are aimed at removing, or diminishing this need, there is a wide consensus that to avoid the development of resistance, multi-tactical approaches to pest control are needed. In addition, somewhat counterintuitive concepts, such as the preservation of a sufciently large pool of susceptible individuals of the pest population, in refuges, are essential to the continued and long-term effectiveness of these products. This, and the requirement for the placement and planting of acceptably large non-Bt refuges, will place other demands on the farmer. The requirement for regional cooperation and a collaborative approach is increased. This is a factor which will have to be addressed in developing countries, where extension services and government 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 Bt plant need to be evaluated in the context of risk to benecial species and IRM strategies. Risk assessments will have to focus on the wider ecological environment (and conservation issues), as well as the immediate agroecological situation in which the crop is located. However, evaluating 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. Ecological risk assessments must also take account of the geographical dimensions of target and non-target species, and include appropriate hypotheses for potential hazards, which can then be empirically tested. Such

165 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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