VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS

Transcription

1 II MODIFICATION RELATED CONCERNS 1 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS G. SCHÜTTE FSP BIOGUM University of Hamburg Research Center for Biotechnology, Society and the Environment Ohnhorststr Hamburg [email protected] February 2000 Updated: 2001

2 2 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS About FSP BIOGUM Our research center for Biotechnology, Society and the Environment (head of FSP BIOGUM: Prof. Dr. Beusmann) was established at the University of Hamburg in 1993 parallel to the research centers for applied molecular biology at the Institute for Botany and molecular neurobiology at the Medical University Clinic. The task of FSP BIOGUM is research, teaching and communication in technology assessment of modern biotechnologies. Risks to society and the environment in comparison to technical and institutional alternatives shall be discussed.

3 II MODIFICATION RELATED CONCERNS 3 GENERAL AND MODIFICATION RELATED ECOLOGICAL RISKS OF TRANSGENIC PLANTS AND REGULATION CRITERIA 4 VARIETIES RESISTANT AGAINST ANIMAL PESTS Years of field experience with transgenic plants have shown that they are able to deliver ecological G. SCHÜTTE and economic benefits. To keep risks of transgenic cultivars below acceptable limits is the challenge for science FSP and BIOGUM regulation. A comprehensive and thorough assessment is a prerequisite for the responsible Technology development Assessment of the potentials on Biotechnology of green biotechnology. University of Hamburg Ohnhorststr. 18 The reports are based Hamburg on a broad review by SCHÜTTE et al. (1998) for the German Federal Environmental Agency (Umweltbundesamt) and on experiences gained by conducting biosafety [email protected] workshops for African countries in South-Africa and Zimbabwe (1997 & 1998). Dr. K.-H. Wolpers, GTZ (Gesellschaft für Technische Zusammenarbeit) encouraged us to publish summaries of the main risk themes in English for use in developing countries. It turned out that risk discussion and regulation developed so fast that a substantial update was necessary. In order to make the material available as soon as possible, it was decided to publish the chapters step by step as contributions to a series. Meanwhile, at GTZ Dr. W. Kasten took over the work of Dr. Wolpers, who retired. We thank both of them for their cooperation and the GTZ for financial support. The different reports will focus on several general and modification-related scopes of the risk discussion. A coherent description and interpretation of the risk research and discussion, tables with summarized facts, recommendations and a list of selected literature on special subtopics are and will be presented. I General concerns Unexpected effects Gene transfer and invasiveness of transgenic plants or their hybrid progeny The translation of this issue was financed by Deutsche II Modification related concerns Gesellschaft für Technische Zusammenarbeit (GTZ) GmbH Herbicide resistant plants (published) Varieties resistant against invertebrate pests (this issue) Virus resistant plants Disease resistant plants Plants tolerant against abiotic stress (published) Plants with changed compounds Plant varieties producing pharmaceuticals Antibiotic resistance and horizontal gene transfer (published)

4 4 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS CONTENT INTRODUCTION... 6 SYNOPSIS... 7 TOXICITY AND SIDE EFFECTS OF RESISTANT PLANTS... 7 Effects of B.t. toxins in sprays on non-target insects... 7 Effects of B.t. toxins in sprays on other taxa... 8 Effects of B.t. toxins in plants on non-target insects... 8 Comparison of B.t. toxins in sprays and in plants... 9 Summary...10 Specificity and side effects of new insecticidal toxins in resistant plants...10 Conclusion...12 RESISTANCE...12 B.t. plants...12 Resistance to new insecticidal toxins...13 RESISTANCE MANAGEMENT...14 RESISTANCE MANAGEMENT BY THE HIGH-DOSE/REFUGE CONCEPT...14 Premises of the current management concept for B.t. plants...15 Can the premises be met?...16 ALTERNATIVE and future MANAGEMENT CONCEPTS...17 High dose in combination with biopesticides and refuges...17 Moderate dose in combination with refuges...18 Moderate and low doses in combination with natural enemies...18 Premises of a management concept for a moderate or low dose in combination with natural enemies...20 Resistance management by sequential or mixed releases and pyramiding...20 Alternative management recommendations for three different B.t. crop species...21 MONITORING ANd Resistance assessment...22 Monitoring resistances by rearing field samples...23 Monitoring resistances when species cannot be reared or rearing is too expensive due to the number of crucial species...23

5 II MODIFICATION RELATED CONCERNS 5 MANAGING POPULATIONS WHEN RESISTANCE OCCURRED...24 CONCLUSIONS...24 STATE OF KNOWLEDGE...26 Criteria for resistance assessment...27 Recommendations for risk assessment...28 Recommendations for the approval and use of high-dose varieties...29 Recommendations for the approval and use of lower dose varieties...30 LITERATURE...31

6 6 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS INTRODUCTION Decreasing the use of pesticides is one of the major aims of plant breeding. Insecticides make up 40% of the pesticides used worldwide. Insecticides and nematicides have many side effects on insect predators and parasitoids as well as on vertebrates and cause human health problems. From 1996 to 1998 insect resistance was thus the most often field tested transgenic trait. Most of the insecticides (about 44%) are used against Lepidoptera (BOULTER 1993). Known Bacillus thuringiensis (B.t.) strains and their toxin-groups are toxic mainly to Lepidoptera, Coleoptera or Diptera. The necessary dose to control a pest differs depending on species and toxin. Hence the expression of B.t. toxins has become the first approach to breed for insect resistance by genetic engineering. Other insecticidal substances such as vegetative insecticidal proteins ( VIPs from B.t.; ESTRUCH et al. 1997, WARREN 1997), lectins (CZAPLA 1997, STOGER et al. 1999, PEUMANS et al. 1997) and proteinase inhibitors from plants (ALTPETER et al. 1999, COWGILL et al. 1999), terpenoids, cholesterol oxidases (from Streptomyces spec.), insect chitinases and fungal chitinolytic enzymes (KRAMER et al. 1997, DING et al. 1998), bacterial insecticidal proteins (BOWEN et al. 1998, BOWEN & ENSIGN 1998, STRAUSS 1998) and early recognition resistance genes (ROSSI et al. 1998, COOK 1998) are also currently being tested. The use of proteinase inhibitors as shown by WU et al. (1997), might cause a growth inhibition but not always an increased larval mortality. VIPs will enlarge the available range of toxins against coleopteran pests (see WARREN 1997). Early recognition resistance genes (ROSSI et al. 1998, COOK 1998) and cysteine proteinase inhibitors (URWIN et al. 1995) also confer resistance to nematodes. B.t. cotton, B.t. corn and B.t. potato have been commercialized in the USA and planted on millions of acres since In B.t. cotton insecticide use was declined and cotton yield has increased in the USA (MONSANTO 1997). More than three different B.t. toxins are currently used and some others are being tested. The level of control and selection pressure of B.t. plants and of some other transgenic insecticidal plants is expected to be higher than with conventional insecticides, especially because of the mode of expression (McGAUGHEY et al. 1998). Hence the duration of resistance is doubted and the resistance management preferences are still an issue of much disagreement and of research (FISCHHOFF 1996, TRAYNOR 1995, MELLON & RISSLER 1998, FERRO in press). Side effects of insecticidal or nematicidal plants are also addressed by different studies (HILBECK in press., STEWART JR. 1999, DEML & DETTNER 1998 and others, see below).

7 II MODIFICATION RELATED CONCERNS 7 A new aspect of risk assessment of insecticidal plants was discussed by STEWART JR. (1999). For example he argued that insect resistance could be transferred to related weeds from oilseed rape and lead to an increased fitness in such weeds. This aspect is currently investigated by Stewart et al. in oilseed rape and Snow et al. in sunflower (USDA 1998, USDA 1999). SYNOPSIS TOXICITY AND SIDE EFFECTS OF RESISTANT PLANTS Persistence of B.t. toxins Spores and toxins persist in soil and fresh water and are degraded with a half-life between one week and a few months. Toxins decay under UV-light within a few hours or a few days, and spores need nutritious soils for germination (reviewed in SCHÜTTE & RIEDE 1998). B.t. toxins can persist in soil when adsorbed to humic acids. Their insecticidal activity is retained and they are more resistant to biodegradation. The toxins acumulate increasing with an increase in the amount of humic acids until a plateau is reached (CRECCHIO & STOTZKY 1998, SAXENA et al. 1999). Effects of B.t. toxins in sprays on non-target insects Side effects of sprays against lepidoptera Side effects of B.t. kurstaki toxins have very often been tested. They kill a large portion of lepidopteran species and only a few insects of other orders (field tests reviewed in REARDON & WAGNER 1995 and WAGNER et al. 1996). Some parasitoids and some predators are directly harmed by B.t. toxins (laboratory tests reviewed in DEML & DETTNER 1998, MELIN & COZZI 1990 and FLEXNER et al. 1986, BIGLER et al. 1997, DOGAN et al. 1996, VINSON 1990). B.t. kurstaki toxins from sprays had a direct negative effect on predators (in brackets on parasitoids) in 24 (24) cases and no negative effect in 78 (87) cases (review of DEML & DETTNER 1998). Indirect negative effects through nutritional quality of preys on predators (parasitoids) have been found in 12 (8) cases and positive effects in 6 (2) cases. After FLEXNER et al. (1986) who reviewed studies of side effects of the same toxins on 42 beneficial arthropods, the spray exhibited toxicity to 2 species, medium toxicity to 3 and low toxicity to 29 species (80-100%, 40-80%, 10-40% mortality). Side effects of sprays against coleoptera

8 8 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Toxins from B.t. tenebrionis sprays used against coleoptera exhibited no toxicity in nine of eleven species from different insect families. Two species were harmed (higher adult mortality, reduced fecundity) (DEML & DETTNER 1998). Honey bees were not affected by the CryIIIB toxin from transgenic plants (ARPAIA 1996). Effects of B.t. toxins in sprays on other taxa Neither B.t.k nor B.t.t. strains exhibit toxicity to microorganisms, water organisms (KREUZWEISER et al. 1996, and review of MELIN & COZZI 1990) or vertebrates with the exception of a soluted toxin of B.t. israeliensis (used against Diptera), which was toxic to mice but seemingly due to a protein other than the δ-endotoxins used in plants (reviews of DOBRNIEWSKI 1994, SAIK et al. 1990, STIRN 1998, RIEDE & SCHÜTTE 1998 and BRAKE & VLACHOS 1998). GOLDBURG and TJADEN (1990) did not find binding sites for B.t. toxins in rats. Therefore it appears that B.t. toxins are not as harmful as the majority of insecticides. Effects of B.t. toxins in plants on non-target insects DOGAN et al. (1996) did not find negative effects when aphids from B.t. potato were fed to coccinellidae. Experimental results of HILBECK et al. (1998) and LOSELY et al. (1999) have raised a public discussion because a mortality of more than 50% was found for a Chrysopa species fed with B.t.-contaminated preys from transgenic corn and 45% mortality of monarch larvae (Danaus plexiplus) fed on leaves contaminated with pollen from B.t. corn. The first finding is not really new. FLEXNER (1986) already found a low to medium toxicity of B.t. kurstaki toxins. A reduced growth rate for Chrysopa sp. due to B.t. toxins was proved as well (SALAMBA cited in VINSON 1990). Regarding the monarch larvae it should be taken into account, that this species is predominantly endangered by the control of its primary host plant, milkweed (Asclepias sp.), in the corn belt. Furthermore the preliminary study of LOSELEY et al. (1999) has been completed by SEARS et al. (2000). They found, that the density of pollen grains on plates (after pollen shed in corn on leaves of the associated flora) within one meter distance from the plant reaches only half of the density corresponding to the LD 50 dose at 96 hours for first instar larvae on average. The abundance of different predator groups including Chrysopa carnea did not decrease in field tests with transgenic B.t. corn (ORR & LANDIS 1997, PILCHER et al. 1997). Monitoring of predators in different B.t. crops (FLINT et al. 1995, LUTTRELL et al. 1995) also showed no significant decrease of abundance. But even large field tests and monitoring projects of three or more years duration may be too small and too short to detect a decrease in abundance (due to a toxin), because the insect

9 II MODIFICATION RELATED CONCERNS 9 population sizes naturally fluctuate in repeating cycles of several generations up to twenty years. Furthermore, test plots have to be very large (up to about one ha depending on the species) in order to be suitable to measure the effects on mobile insects such as Chrysopa sp., Cocinellidae or wasps. The insects move from field to field. The field tests of FLINT et al. (1995) and PILCHER et al. (1997) ended after two years on test plots of 36 and 23m 2 respectively. The work of ORR & LANDIS (1997) lasted only one year on 0,4ha test plots. The longest study on bigger plots was done by LUTTRELL et al. (1995). They sampled different arthropods on 2ha plots of cotton (sprayed with insecticides, unsprayed B.t. cotton and unsprayed control) for three years: Results of LUTTRELL et al. (1995) based on sweep net samples and visual whole-plant searches: other insects (not beneficials or pests which make up 29% of all sampled insects) highest numbers on unsprayed plots, 6% less on B.t. plots and 28% less on sprayed plots total arthropods minus aphids highest numbers on unsprayed plots, 13% less on B.t. plots and 32% less on sprayed plots all lepidoptera beneficial insects highest numbers on unsprayed plots, 52% less on sprayed plots and 84% less on B.t. plots highest numbers on unsprayed plots, 2-3% less on B.t.- plots and 31% less on sprayed plots Abundance activity total lepidoptera (including the target pests) was less on B.t. plots compared to sprayed plots (written on grey background). Abundance activity of other insects (not beneficial or pests), total arthropods minus aphids and beneficial insects were higher on B.t. plots compared to sprayed plots. These results show, how positive B.t. plants could influence the insect fauna, when insecticides are replaced. Cotton is still Unfortunately insecticides are used 3-5 times each season on B.t. cotton on average. LUTTRELL et al. (1995) still conceded that the plot size was not big enough to estimate the effects on all arthropods. Of the beneficials polyphagous predators have a better chance of surviving because not all of their prey is contaminated. It is also one reason why field test results often are in contradiction to laboratory tests. And it shows the necessity to differentiate between them and specialized prasitoids (they often are important antagonists) which was not done in this study. Comparison of B.t. toxins in sprays and in plants It is not clearly evaluated whether B.t. sprays or B.t. plants have minor side effects on the insect fauna (PILCHER et al. 1997, DOGAN et al. 1996, DALY 1994, further references within SCHÜTTE & RIEDE 1998). The mostly truncated and activated toxins in B.t. plants

10 10 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS are constitutively expressed in high doses and thereby kill faster whereas B.t. toxins from traditional sprays have to be processed and activated by insect enzymes and persist only shortly under light. Arthropod species which have binding sites for a toxin but cannot activate it could be harmed by toxins from transgenic plants. Changes in toxicity due to the exchange of 120 amino acids have been shown (GOLDBURG & TJADEN 1990). Also protoxins can be activated in different ways leading to changes in specificity (HAIDER et al. 1986). On the other hand, the side effects of toxins from transgenic plants are limited to insects feeding on the crop or on its pollen and indirectly to some of their antagonists. Therefore toxicity test results from sprays cannot be substituted by tests with plants (DEML & DETTNER 1998). It is known from studies with beneficial insects which are fed on prey from B.t. plants, that some parasitoids are directly or indirectly affected and others not, which is also dependant on the dose the receive (HILBECK et al. 1998, SCHULER et al. 1999a+b, JOHNSON & GOULD 1992). Summary The B.t. kurstaki toxins are more specific than most insecticides (SCHÜTTE & RIEDE 1998, THEILING & CROFT 1988) although they are harmful to different non-target insects (see above). B.t.k and B.t.t toxins from sprays do not exhibit toxicity to microorganisms, aquatic organisms and vertebrates/mammalians. Lepidoptera-specific B.t. toxins (CryIA[b]) expressed in transgenic tomato plants were not toxic to rats and mice (NOTEBORN et al. 1995, KUIPER & NOTEBORN 1996). It is not clearly evaluated whether B.t. sprays or B.t. plants have minor side effects on arthopods. B.t. plants have an overall positive effect on the arthropod fauna compared to conventional insecticides when additional insecticide spraying is avoided. Specificity and side effects of new insecticidal toxins in resistant plants Cholesterol oxidases (from Streptomyces) and VIPs (vegetative insecticidal proteins) are toxic to lepidoptera and coleoptera respectively through lysis of their ephithelial midgut cells. It is known for VIPs to be toxic to a broader range of insects (ESTRUCH et al. 1996, WARREN 1997). Lectins and chitinases also have a broader host range compared to B.t. toxins (STEWART JR. 1999). Experiments have shown that ladybirds (Adiala bipunctata) feeding on aphids from transgenic lectin-potato (providing a low level of insect resistance) were harmed (decrease of fecundity, lifetime and viability of eggs) (BIRCH et al. in press). BELL et al. (1999) tested

11 II MODIFICATION RELATED CONCERNS 11 the effects of hosts fed with diets containing the snowdrop lectin (GNA) on a parasitic wasp and did not find negative effects, Lectins from fresh food are also of concern because they cause human health effects because they serve as mitogens to human T-cells (PEUMANS et al. 1997, STRIN 1998, BIRCH et al. in press). CZAPLA (1997) discussed the variable effects of different lectins. GNA (from Galanthus nivalis) and a Vicia faba-lectin have less antinutritional effects than others (PUSZTAI et al. 1990, RUBIO et al both cited in CZAPLA 1997). But the insecticidal doses needed is quite high and the dose for the consumer must be low in order to prevent the unwanted effects. After GATEHOUSE (2000) the GNA-lectin enhanced parasitism by the parasitoid (Eulophus pennicornis) of a lepidopteren pest (tomato moth) in greenhouse. Thus the necessary dosis may be smaller when the parasitoid is abundant. CZAPLA (1997) demanded long-term exposure studies for lifestock and humans and proposed to decrease the dose by mixing/pyramiding lectin genes with other insecticidal genes. Proteinase inhibitors are of a narrow specificity and resistance is often unsatisfactory according to GATEHOUSE et al. (1993). ASHOURI et al. (1998) and WALKER et al. (1998) showed that pentatomids and ladybirds were harmed when they fed on proteinase-inhibitorcontaminated prey. Side effects on epigaeic arthropods could be reduced by tissue-specific expression of a proteinase resistance gene against nematodes (COWGILL et al. 1999). Some insects can produce proteinases insensitve to inhibition (WU et al. 1997, JONGSMA et al. 1995). New approaches to test the level of resistance of different inhibitory domains (of proteinase inhibitors) displayed in phages or by computer modeling could lead to a more effective protection (ALTPETER et al.1999, ROBERTS et al. 1992). But, some proteinase inhibitors are anti-nutritional for humans and other vertebrates (ALTPETER et al. 1999, LIENER 1986, FRIEDMAN 1986), which also has to be tested. Photorhabdus luminescens toxins have shown to be toxic to several orders of insects such as Coleoptera, Hymenoptera, Dictyoptera (BOWEN & ENSYGN 1998). The toxins damage midgut epithelial cells. According to BOWEN et al. (1998) side effects could possibly be reduced by selection and use of parts of the toxic protein complexes. Further studies on side effects are under way (SRAUSS 1998). Early recognition resistance genes (ROSSI et al. 1998, COOK 1998) are toxic to different animal phyla. The MI gene causes resistance to nematodes and aphids but is on the other hand specifically toxic to only some isolates of the aphid species. Other genes of this nucleotid-binding and leucine rich family confer resistance to bacterial and fungal pathogens and to viruses (COOK 1998).

12 12 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Conclusion Since most alternative toxins to the B.t. toxins have a broader host range they will have a higher impact on non-target insects (STEWART Jr. 1999) but this will have to be explored case by case. The can be useful to prolong resistance when spatially or sequentially mixed with B.t. toxins. Investigations of tritrophic systems including predators and parasites are of relevance not only for the assessment of side effects but also for resistance management (see below). RESISTANCE B.t. plants On average resistance to insecticides occurs after 10 years (GEORGHIOU 1990). In contrast, the first field resistance against B.t. toxins was reported years after introduction. It only occurred because of excessive use (45-72 applications per year, BAUER 1995, MARRONE & MACINTHOSH 1993). Compared with other insecticides B.t. sprays have been unbeaten in preventing resistance because of their short life time and composition of multiple toxins. Both conditions are not fulfilled in transgenic plants. Knowledge from laboratory strains Different resistance mechanisms have been detected in laboratory tests, even within different populations of one species against the same toxin or toxin group. Degradation of toxins, mutation of toxin binding sites, prevented penetration through membranes and changes of insect behavior are the known mechanisms. Also the level of resistance greatly varies between species and populations. One can find factors of 1 to 5000 (lethal dose for 50% of selected population/lethal dose for 50% of control population, reviewed in SCHÜTTE & RIEDE 1998, TABASHNIK et al. 1993). Mostly the factors layed under 100 and often under twenty. Very broad resistances against a wide range of B.t. toxins as well as restricted resistances have been detected, and they can be inherited in a recessive, dominant or partial fashion. The majority of documented resistances to B.t. toxins is inherited in a recessive or partially recessive way (GOULD et al. 1994). An incompletely dominant autosomal resistance gene to a foliar B.t. product was detected in an important target species of B.t. corn (Ostrinia nubilalis) (HUANG et al. 1999), but resistance to B.t. corn could not yet be detected (TABASHNIK et al. 1999). Multiple resistances have evolved against a range of more than 20 B.t. strains (GOULD et al. 1992, WHALON unpublished data in BAUER 1995). VAN RIE et al. (1990) and TABASHNIK et al. (1993) found resistances due to altered binding sites to

13 II MODIFICATION RELATED CONCERNS 13 be quite restricted to a few toxins. There may be a chance to predict cross resistances based on this mechanism (TABASHNIK et al. 1996). Also insect could become resistant by producing new binding molecules which prevent the binding of B.t. proteins to midgut binding sites (HECKEL et al. 1999). Other mechanisms (such as behavioral changes or proteolytic degradation) seem to be connected with smaller resistance levels and higher fitness costs, but the latter will be optimized by the pests depending on the duration of the coevolution process. Of the current target pests of B.t. plants, the Leptinotarsa decemlineata (Colorado potato beetle), the cotton pests Heliothis virescens (tobacco budworm) and species of the genus Spodoptera can develop broad (multiple) resistances against B.t. toxins (WHALON unpublished data in BAUER 1995, GOULD et al. 1992, MOAR et al. 1995, STONE et al. 1998a in MARRONE & MACINTOSH 1993). Three of the pests presently controlled with transgenic B.t. plants without much management experience belong to the 12 worldwide leading insects with respect to their ability to develop and break resistances: Leptinotarsa decemlineata, Heliothis virescens, Helicoverpa armigera (GEORGHIOU 1990). The resistance can remain for 8 to 30 generations without selection pressure in different Lepidoptera species or populations, sometimes quite slowly diminishing, but reselection works quickly (WHALON & WIERENGA 1994, TABASHNIK 1994, BAUER 1995, TRISYONO & WHALON 1997, MARRONE & MACINTOSH 1993). Resistances which evolved in field are more stable due to lesser fitness costs according to ROUSH (1997) who analyzed different laboratory and field studies (LIU & TABASHNIK 1996, TANG et al ROUSH & CROFT 1998: all cited in RUOSH 1997). Resistance mechanisms with low fitness costs and resistances inherited by dominant genes cause long remaining resistances and fast reselection. Resistance to new insecticidal toxins Resistance mechanisms to other toxins which are presently tested might be assessed in future. It is known that different insect species (JONGSMA et al. 1995, WU et al. 1997) that they can adapt to proteinase inhibitors by elevating the levels of insensitive classes of proteinases (chymotrypsin elastase in Helicoverpa armigera, tryptic activity in Spodoptera). The use of combinations of inhibitors and the elaborated screening of suitable inhibitors (see above) are discussed to prevent the adaptation. For lectins several possible modes of action in the midgut of insects are listed up by SZAPLA (1997). A toxin which has several modes of action could lead to a more stable resistance, but they are still not clearly known for lectins.

14 14 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS RESISTANCE MANAGEMENT The initial frequency of resistance genes, their mode of inheritance and the selection pressure, influence the lifetime of resistance. In theory, the selection pressure can be reduced by lower toxin doses, induced expression, tissue specific expression or mixing resistant plants and non-resistant plants or plants with different resistance genes. Seed mixtures of resistant and susceptible plants are only helpful when the feeding instars do not move between the different plants (MALLET & PORTER 1992, McGAUGEY & WHALON 1992). When the larvae move between plants, the planting of refuges should be preferred. Simulation models indicate, that the durability of resistance can effectively be prolonged when 25% of the pests population is unexposed to selection (ANDOW & HUTCHINSON 1998). Spatial structures of refuges should enable random mixing of unexposed and exposed biotypes. RESISTANCE MANAGEMENT BY THE HIGH-DOSE/REFUGE CONCEPT The EPA and most industrial companies clearly pursue the high-dose/refuge approach (FISCHHOFF 1996). The following management regulations have been established for resistance management of (more or less - discussion see below) high dose expressing transgenic insecticidal plants: A 4-5% refuge without insecticide use or about 20-25% with insecticide spraying were demanded by the EPA for the first corn and cotton varieties and are still demanded for cotton. Some additional regulations were given for counties with high cotton acreage (over 75%) according to the structure of the refuges. The B.t. corn acreage was limited to 5% in counties with more than 400 ha cotton acreage because some pests feed on both plants. Nevertheless for three B.t. corn varieties there are no management requirements to date. Institutions such as the Union of Concerned Scientists (Cambridge, MA), the USDA and a research group sponsored by industry all recommended to plant larger refuges. The recommendations for corn are very similar ranging from 20-25% refuge without to 40-50% with application of insecticides. 50% refuges were recommended in areas, were B.t. cotton is grown beside B.t. corn. A new management plan for corn, submitted by the big seed producers in conjunction with the National Corn Growers Association (St. Louis, MO), requires the planting of 20% refuges and neither to distinguish between sprayed or unsprayed refuges nor between high-dose varieties and varieties with lower doses. This is at least regarded to be a step in the right direction by entomologists (DOVE 1999). The US cotton market is dominated by Monsanto and thus this company requires compliance with their management plans. The company requires specific management plans

15 II MODIFICATION RELATED CONCERNS 15 for the southern states (DOVE 1999) where the tobacco budworm (Heliothis virescens) and cotton bollworm (Helicoverpa zea), which overwinters in the southern states, co-occur. Monsanto instituted a fine of more than $ 3000 for planting B.t. cotton illegally (GOULD & TABASHNIK 1998). Whereas the B.t. cotton in Australia was first planted with a 5% refuge, regulation changed to a general limitation of B.t. cotton to 20% of the total cotton crop acreage. In China a B.t. cotton expressing two toxins has been approved, and management is based on big areas of uncultivated ( natural ) refuges (SHI-RONG in press). The high-dose concept combined with refuge provisions in the USA is not accepted as the best practice by some entomologists (FERRO 1993, McGAUGHEY & WHALON 1992, SHELTON et al. 1993, MELLON & RISSLER 1998). GOULD et al. (1997) predicted a 10- year period without resistance problems concerning Heliothis virescens and only a threeyear period for the bollworm complex (Pectinophora gossypiella - pink bollworm, Helicoverpa zea - cotton bollworm or corn earworm, other Helicoverpa sp.) (taking into account a proportion of 4% refuge without insecticide use in cotton). These predictions have been based on monitoring the frequency of resistance alleles in the USA. Although computer simulations and population genetics theory, laboratory and greenhouse tests support this high-dose/refuge concept (GOULD 1994, GOULD & TABASHNIK 1998) there are serious reasons not to rely on it too much. The models, simulations or greenhouse tests do not take into account a wide range of environmental irregularities. Temporary, very high pest pressures due to regional climate differences, the occurrence of dominant resistance alleles in some populations, long range flights of resistant genotypes or varying expression levels due to different promotors, silencing or plant age can, for example, undermine the concept in nature. All these factors are critical because of the special premises connected with the high-dose/refuge concept. Premises of the current management concept for B.t. plants The concept is valid when 99% of the individuals (by all resistant varieties) and 100% of the resistant heterozygotes are killed (which means that all resistance alleles must be recessive). Therefore, the toxin expression must be uniform in time and space as well (GOULD & TABASHNIK 1998). The frequency of resistant alleles should also not be higher than according to ANDOW and HUTCHINSON (1998). This could also be true for Helicoverpa armigera in Australia (AHMAD & ROUSH 1999). The more target insects that are included in the control by the resistant variety the more difficult are the assumptions for the refuges to meet. The refuges should be as large as they need to be in order to prevent feeding instars from moving to non-resistant plants and small enough to make many

16 16 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS individuals mate in the refuge (MALLET & PORTER 1992, TABASHNIK 1994b, GOULD & TABASHNIK 1998). Can the premises be met? 99% mortality of pest populations of each target species When there is more than one target pest, and the susceptibility is low for at least one of them, problems will occur. In the southern parts of the USA more than nine Lepidopteran (thus more or less susceptible) corn pests, and more than seven Lepidopteran cotton pests occur (ANDOW & HUTCHINSON 1998, GOULD & TABASHNIK 1998). In cotton, Helicoverpa zea and Spodoptera exigua (beet armyworm) are not susceptible enough to the CryIA(c) toxin in transgenic plants to meet the 99% kill (or high dose) premise (GOULD & TABASHNIK 1998). The pollen expression is lowered in cotton (MONSANTO 1997). Helicoverpa species in Australia are also not susceptible enough. Problems might occur in corn because the high dose cannot be expressed by all varieties. Varieties other than YieldGard and BiteGard exhibit spatially or temporary lowered expression (B.t. Xtra in kernels, stalk, silk and pollen; NatureGard and KnockOut in kernels and silk and after pollen shed even in other tissues, ANDOW & HUTCHINSON 1998). Furthermore, not all of the plants of a B.t. variety express the toxin (2% of cotton and 1-5% of corn plants do not express the toxin according to ANDOW & HUTCHINSON 1998 and GOULD & TABASHNIK 1998) and pests feeding on those plants could survive. Some pests may avoid resistant plants and thereby reduce the toxin dose to a moderate one and survive. This was observed for Ostrinia nubilalis (references within ANDOW, HUTCHINSON 1998) and for Leptinotarsa decemlineata (WHALON & FERRO 1998) and it could happen at field margins near refuges. Also, when the refuge is not adequate, mating of resistant biotypes could result in higher survival rates in the following generation (see below). Killing all heterozygous resistant individuals In 1996, when a high pest pressure occurred in cotton crops in Texas, B.t. plants did not kill all heterozygous cotton bollworms. Theoretically the premise cannot be met whenever the gene pool of a population provides a dominant resistance allele. This could be true for Pectinophora gossypiella in cotton in the USA (GOULD & TABASHNIK 1998) and for Ostrinia nubilalis in corn. HUANG et al. (1999) detected incompletely dominant resistance genes in laboratory studies with the European corn borer. Dominant resistance alleles are

17 II MODIFICATION RELATED CONCERNS 17 very rare and therefore difficult to detect (see below). Some corn varieties might also not kill all heterozygotes because of the spatial or temporal lowered toxin expression. Implementing a suited refuge system ALYOKHIN et al. (1999) measured that feeding on transgenic B.t. potato had a strong negative effect on the proportion of beetles that flew, and on the average number of flights per beetle. The suppression of flight activity is discussed to make less resistant beetles fly into the refuge which may undermine the refuge strategy. Furthermore, LIU et al. (1999) found out, that the development time of resistant pink bollworm larvae differed from non resistant ones. The authors concluded that this difference may lead to an enhanced probability of resistant bollworms mating, resulting in homozyous resistant progeny. Pectinophora gossypiella does not move far before mating which makes it necessary to plant the refuge very close to the field. The maximum distance for random mating must be known for every target pest. (GOULD & TABASHNIK 1998, ANDOW & HUTCHINSON 1998). When pests such as the cotton bollworm move between regions, refuge systems have to be implemented even on a regional and international scale (e.g. populations on B.t. corn in Mexico and in the USA). In the future, polyphagous pests will feed on different B.t. host plants (which will be commercialized) and must be taken into account when a refuge structure is established. Cotton bollworm feeds on cotton, corn, sorghum, peanuts, alfalfa, soybean and many vegetables. Tobacco budworm feeds on cotton, tobacco, tomato and sometimes soybean. The Pseudoplusia includens (soybean looper) also feeds on cotton and soybean (GOULD & TABASHNIK 1998). Most cotton Lepidoptera are polyphagous (FITT 1994). ALTERNATIVE AND FUTURE MANAGEMENT CONCEPTS High dose in combination with biopesticides and refuges Biological control measures, e.g. the planned introduction of Trichogramma, baculoviruses (RIEDE 1998), releases of sterile moths (HENNEBERY et al. 1985) or entomophagous fungi (HASSANI et al. 1998) are useful as long as they do not prefer susceptible to resistant prey, thus accelerating resistance development. The best way to use these measures in combination with the high dose is to apply them only in B.t. acreage and not in the refuge (ANDOW & HUTCHINSON 1998) or, to apply them in the refuge after mating because susceptible insects should not be harmed before mating with resistant ones. Nevertheless, it might be difficult to find parasitoids which are able to develop in infected hosts from high-

18 18 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS dose plants. As SCHULER et al. (1999) measured in experiments with Plutella xylostella and its parasitoid Cotesia plutellidae the wasps need seven days to develop but larvae died after five days. A moderate dose (high dose defined as causing 99% mortality) should not kill more than 80% of the population in order not to accelerate resistance (ROUSH 1997). Moderate dose in combination with refuges The use of B.t. plants with a moderate or low dose was also discussed as a possible resistance management concept. ANDOW and HUTCHINSON (1998) recommended to use a moderate dose approach in combination with refuges at least for those corn varieties which do not express a high dose in all tissues and not to all target pests. Yet, it has not been assessed how large a refuge should be in combination with a moderate or low dose and therefore the authors recommended a 50% unsprayed refuge area. Moderate and low doses in combination with natural enemies It is known from partial and highly resistant plants, which are comparable to low- and highdose resistances, that natural enemies and especially monophagous (density dependent) antagonists are indirectly affected. This effect can lead to pest gradations in the following years because of the lack of antagonists (MORRILL et al. 1994). It is also known from toxicological studies that some antagonists cannot develop in B.t. infested/susceptible larvae or are directly harmed by B.t. toxins in their preys (see above). Thus, a combination of resistance with natural enemies seems to be more adequate to partial and lower doses resistances. VAN EMDEN (1966) showed that a low level of plant resistance can lead to a sufficient control of aphids in combination with predators, when pest multiplication does not exceed 1,15 (per day). Other greenhouse and field experiments (BOTTRELL & BARBOSA 1997, HARE 1992, JOHNSON & GOULD 1992, STARKS et al. 1972) also showed that the use of partial resistances or low dose B.t. plants in combination with antagonists can control pests. JOHNSON & GOULD (1992) reported a 4- to 5 fold increase in parasitism on toxic (low dose B.t.) plants in two of their field test plots. But they detected an increased mortality of susceptible larvae of Heliothis virescens on toxic plants. It was discussed to be due to the prolonged larval development, the increased probing and moving of larvae which both could make them vulnerable for parasitoids and predators (BOTTRELL & BARBOSA 1997, JOHNSON & GOULD 1992). In this case the antagonistic species would enhance resistance as they prefer susceptible preys (JOHNSON & GOULD 1992, JOHNSON et al. 1997, VAN EMDEN 1986, STARKS et al. 1972). This possibility has to be considered and depends on the tritrophic system (JOHNSON et al. 1997, GOULD et al. 1991).

19 II MODIFICATION RELATED CONCERNS 19 Another tritrophic system studied by SCHULER et al. (1999a) (Plutella xylostella and its parasitoid Cotesia plutellidae on B.t. oilseed rape) would probably be suitable to enlarge the lifetime of resistance when combined with moderate dose plants as the level of parasitism of the parasitoid wasp on B.t. plants and on susceptible plants did not differ. In this case, the hurdle was the high dose from which the host died too early to let the parasitoid develop and sustain its population. As SCHULER et al. (1999) concluded, a sublethal toxin level could also increase the level of parasitism by impairing the host s immune response. JOHNSON (1997) concluded from a study on a parasitic wasp (Campoletis sonorensis) of Heliothis virescens feeding on low dose B.t. plants that a low dose was of limited practicability. JOHNSON (1997) found differences in the attraction of parasitoids which may be due to missing volatile and tactile clues on toxic plants as a consequence of less feeding activity. But the larger development time of hosts (pests) on toxic plants seemingly provided higher rates of parasitism compared to that of hosts (pests) on non-toxic plants. The breeding options reviewed and discussed by BOTTRELL & BARBOSA (1997) may become of special relevance on this background. BOTTRELL & BARBOSA (1997) pleaded for an incorporation of natural enemy-enhancing traits/genes by breeding. The use of plant volatiles attracting specialized parasitoids induced by herbivore attack, morphological traits (e.g. leaf surfaces) and of nutritional traits (amount of pollen and nectar production) supporting antagonists was discussed. Theoretically, it would be possible to influence parasitoid and predator abundance in mixtures by incorporating the enemy enhancing traits into the toxic plants (and repellents into susceptible plants). The fitness differential of resistant pest and susceptible biotypes caused by antagonists might then be increased. The main problem might be mortality due to entomopathogens because susceptible larvae which exhibit increased movement and probing might be more likely to encounter a lethal infection (JOHNSON et al. 1997). Obviously the latter management approach (the moderate or low resistance plus antagonists) also depends on some premises, which would have to be assessed:

20 20 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Premises of a management concept for a moderate or low dose in combination with natural enemies The dose must be low enough to sustain populations of natural enemies depending on a certain pest density. It should be lower than 80% for the toxin used (or for each toxin when pyramided) in order to prevent a fast breakdown resistance. Important beneficials which are not harmed by the toxins in their preys and hosts (and can completely develop in infested hosts in time) should be effective enough to control the pest population on resistant plants. The cumulated pest mortality caused by the guild of antagonists to susceptible pests (or pests on susceptible/non-toxic plants in mixtures) should be lower (or at least not be higher) than on resistant pests (or pests on resistant/toxic plants). The application of natural plant elicitors (THALER 1999) could be added when pests are not sufficiently controlled due to the lower dose. Mixing moderate or low resistance genes sequentially, by seed mixtures or by pyramiding genes can further prolong lifetime of resistance. Also refuges could be planted. Resistance management by sequential or mixed releases and pyramiding Resistance to insecticidal plants can be delayed by mixing different B.t.- and other insecticidal genes. It can be done by mixing, sequential release (rotation) or by pyramiding. The toxins must exhibit negative cross resistance. Models showed, that mixtures of two different toxins can delay resistance in the pests (ROUSH 1994, GOULD et al.1997). Experiments by GEORGHIOU and WIRTH (1997) with Culex quinquefasciatus showed that three or four toxins delay resistance for another six to nine or 28 generations of the target pest respectively. GOULD (1986) predicted from simulation models, that pyramiding of toxins is the best method when resistance is inherited in a recessive way (plus strong epistasis), whereas a mixture or sequential release provides more durable resistance for the additive mode of inheritance of resistance in the pest. A partially recessive mode of inheritance would be best managed by pyramiding and refuge planting. A 30% refuge could prolong the durability from 10 to 120 generations when resistance in pests is inherited in a partially recessive mode (GOULD et al. 1994). When a recessive mode of inheritance was simulated, resistance development needed 150 (10% refuge) or 500 (30% refuge) instead of 30 generations. Furthermore, simulation models (GOULD et al.1994) showed that refuges and mixtures are not that effective when the resistance traits of the pest are additively inherited. Plutella xylostella larvae resistant to CryIA toxins and moderate levels of CryIC toxins could be controlled by high CryIC levels (CAO et al. 1999). Mosquito species resistant to CryIV toxins were susceptible to CytA toxin from B.t. (WIRTH et al. 1997). Another interesting

21 II MODIFICATION RELATED CONCERNS 21 result was published by DING et al. (1998) who controlled the tobacco hornworm (Manduca sexta) and tobacco budworm feeding on transgenic chitinase expressing tobacco in combination with a low dose of foliar B.t.. Spodoptera frugiperda (fall armyworm), which is not very susceptible to CryIA(b) toxins (WILLIAMS et al. 1998), but is susceptible to a proteinase inhibitor tested by ALPPETER et al. (1999). ANDOW and HUTCHINSON (1998) recommended to mix CryIA and Cry9C toxins in plants, but the expression of the latter toxin in plants is still a hindrance. KOTA et al. (1999) succeeded in expressing B.t. cry2aa2 genes in chloroplasts leading to a 20 to 30 fold (pro)toxin level compared to current cultivars. This level resulted in 100% mortality of highly resistant ( fold to Cry2Aa2 and 20,000-40,000fold to CryIA) beet armyworm, tobacco budworm and cotton bollworm. Alternative management recommendations for three different B.t. crop species Cotton A recommendation for the USA is to plant 50% refuge on cotton acreage and use insecticides other than foliar B.t. only when the economic injury level is reached. In areas, where the (not very mobile species) Pectinophora gossypiella occurs, the B.t. crop should be planted in not more than 40 adjacent rows followed by the same block of non-b.t. rows. In other areas 1x1 mile squares are recommended as a scale for planting refuge cotton and B.t. cotton on 50% of the acreage (GOULD & TABASHNIK 1998). The alternative recommendation is to plant about 17% refuge without insecticide use in the refuge unless they are used in the whole field (to avoid additional selection pressure on the part of the population in the refuge). This version includes the recommendation to plant two or more refuge rows in immediate adjacency to the B.t. field and at least eight non-b.t. rows every 48 rows (GOULD & TABASHNIK 1998). GOULD (1988) proposed a moderate dose approach by expressing the toxin only in the capsules. Corn Ostrinia nubilalis (European corn borer) can move over 30 km with the wind and has more than 200 host plants, but many of them cannot sustain large populations (ANDOW & HUTCHINSON 1998). It can have 1-5 generations depending on the geographic latitude (TOLLEFSON & CALVIN 1994). The extremely mobile species Helicoverpa zea could suffer high selection pressure and become resistant on B.t. cotton or B.t. corn (and probably soon on B.t. soybean, B.t. peanut, B.t. alfalfa). When B.t. soybean and others are approved, a new assessment of refuge needs will be necessary.

22 22 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS It was recommended to plant not more than 50% of the corn acreage with B.t.-corn and to provide 25% unsprayed or 75% sprayed corn-refuge planted at least every 230 acres (ANDOW & HUTCHINSON 1998). An integrated and low dose approach is to use B.t. corn predominantly as trap crops. Sown prior to the crop their major pests will prefer them according to studies by ALSTAD and ANDOW (1995) on the European corn borer. Only the first generation of pest species would be exposed to the toxin and selection pressure would then be decreased thereby. Potato Leptinotarsa decemlineata is known to move over long ranges (WHALON & FERRO 1998). Resistant biotypes can survive in diapause over long periods which could lead to a short reselection time after sequential mixing of toxins. An incompletely dominant resistant gene was detected by RAHADJA and WHALON (1995) in laboratory. The 99%-kill premise is valid for presently approved varieties (WHALON & FERRO 1998), but the species is monophagous and therefore needs large areas of non-b.t. potato as a refuge. WHALON and FERRO (1998) proposed to plant 20% refuges in strips, centers or blocks not more than 500 m from B.t. fields. Refuges can be sprayed when the economic threshold is reached but not with in-furrow and foliar imidacloprid. They also proposed crop rotation at least every third year and a reward for the detection of resistant biotypes. Rye borders and open field margins could help to make beetles fly into the refuge and maximize outcrossing of resistant alleles. In the same way as corn, B.t. potato could predominantly be used as a trap crop. Sown prior to the crop the beetle will prefer them in spring and the second pest generation will be very small and only exposed to a minimum selection pressure (WHALON & WIERENGA 1994). MONITORING AND RESISTANCE ASSESSMENT Monitoring the frequency of resistance genes, the mechanism, fitness cost, the level and inheritance with uniform test standards will be necessary (see also JUTSUM et al. 1998, ANDOW & HUTCHINSON 1998, GOULD & TABASHNIK 1998). A careful monitoring and management could help not to waste the limited number of valuable toxins. On a regional scale, preventive predictions of allele frequency shifts could be helpful. Geographic gradients in allele frequency could be analyzed and simulated by migration-selection models, when data on migration, fitness costs and movement patterns are available (LENORMAND et al. 1999, LENORMAND & RAYMOND 1998).

23 II MODIFICATION RELATED CONCERNS 23 Estimations of the frequency of resistance alleles have in some cases been unrealistic. GOULD et al. (1997) were the first to base a prediction of the durability of resistance on monitoring results, which revealed a 0,15-0,3% frequency of the resistance gene for the cotton pest Heliothis virescens (TABASHNIK 1997, GOULD et al. 1997). ROUSH (1994) had estimated the frequency of resistance to be about Cost effective monitoring methods which work as a warning tool are needed. Scouting by farmers will reveal resistances only when the frequencies of resistance are already too high. The biggest and most cost intensive problem may be to detect dominant resistance alleles because big sample sizes are necessary to find such rare alleles. Dominant alleles cause a very rapid increase of resistant phenotypes. According to GOULD and TABASHNIK (1998) it will be necessary to change the management concept when a frequency of 10-6 of dominant alleles or a frequency of 10-3 of recessive or partially recessive alleles is found (GOULD et al. 1997). Thus WHALON and FERRO (1998) proposed to offer a reward for anyone who finds resistant biotypes. Also, different approaches seem to be needed depending on the fact whether the target species can be reared in laboratory or not. Monitoring resistances by rearing field samples LC50 or LC90 assays will not work sensibly enough to detect recessive resistances or little changes in susceptibility caused by multiple minor genes (TABASHNIK 1995). The most sensitive test is to feed a non-lethal dose and then to measure the inhibition of growth (SIMS et al. 1996). For monitoring Leptinotarsa decemlineata, it was recommended to take samples of the first instar larvae (WHALON & FERRO 1998) but small changes of susceptibility may earlier be detectable in older larvae which are generally less susceptible. An in-field-screen on attractive (sentinal) plots is the most cort effektive method to detect dominant resistance (ANDOW pers. communication). The method of rearing and sib-mating isofemale lines and afterwards testing the F2 larvae (F2-screen) was described by ALSTAD and ANDOW (1996) family lines are needed for a detection level of This method is the most cost effective one to detect a recessive resistance (ANDOW pers. communication). When the resistance alleles are identified and an assay to discriminate between heterozygotes and susceptible homozygotes is available, insects can also be screened against test stocks (GOULD et al. 1997). Monitoring resistances when species cannot be reared or rearing is too expensive due to the number of crucial species

24 24 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS A probably cost effective monitoring method was developed for B.t. corn (ANDOW & HUTCHINSON 1998). Larval densities on corn ears on sentinal B.t. sweet corn plots, which do attract large numbers of second or third generation Lepidoptera and thereby guarantee high sample sizes, are compared to densities on non B.t. sweet corn. Ear sampling can quickly be done and multiple pests are monitored by one assay. Another appraoch will be the genetic mapping of resistance which can in addition be used to predict multiple resistances (HECKEL et al. 2000) MANAGING POPULATIONS WHEN RESISTANCE OCCURRED When resistance is detected, the mechanism of resistance, their mode of inheritance and possible cross resistances should be assessed. It might be necessary to discontinue sales or to use new toxin genes with negative cross resistance when resistance occurs. Dominant resistances will cause the most rapid increase of phenotypic resistance and should be managed (and monitored) with special caution. The species with the highest reproductivity, shortest life-cycle (most generations per year) and smallest range of non-b.t. host plants might more rapidly develop resistances and the resistant fraction of the population will increase more rapidly compared to other species. When the mobility of the resistant species is high, mandatory remedial management must be required for large regions. Unfortunately movement and mating patterns of many pests are not well investigated. ANDOW and HUTCHINSON (1998) proposed to discontinue variety sales when resistance is detected at a frequency of one in ten or more. For the Colorado potato beetle an eradication plan using insecticides and sampling larvae of the following year for control was recommended after detection of resistance (WHALON & FERRO 1998). CONCLUSIONS If the insecticidal plants were grown without additional insecticides much more arthropods would survive compared to conventional farming. The ecological effects of reduced insecticide use in combination with insecticidal plants are presently monitored. A reduction of insecticides could be shown in practise which has an beneficial impact on public health. Farmers reduced the application of insecticides from 5 to 3 on average in cotton (SHELTON et al. 2000). In B.t. corn, about 26% of the farmers reduced whereas 2,4% increased insecticide use (MONSANTO 2000). The high-dose/refuge approach will lead to clearly visible results and economic benefits. Thus, industry chose this concept. Smaller short-run, but longer lasting economic and more ecological benefits can be expected in combination with lower-dose- and multiple-toxin-

25 II MODIFICATION RELATED CONCERNS 25 concepts. As the compliance for the latter is low, it might be necessary to honor them at least by the money spared due to possibly superfluous management and monitoring efforts in connection with lower doses. Society should not rely on one concept and should not put all its eggs into one basket. Tissue specific expression, or signal depending expression approaches may contribute to further management concepts in future. Furthermore gene technology can make plant breeders able to use natural enemy-enhancing traits/genes from off-types and wild species as BOTTRELL et al. (1998) discussed.

26 26 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS STATE OF KNOWLEDGE The use of insecticides in cotton and corn was reduced through the planting of B.t. crops in the USA in Farmers reduced the application of insecticides from 5 to 3 on average in cotton. In B.t. corn, about 26% of the farmers reduced whereas 2,4% increased insecticide use. In conventional agriculture B.t. plants would influence the insect fauna in a very positive way, if insecticides were replaced. The number of existing B.t. toxins (which cause less side effects than most insecticides) for plant breeding is restricted, and multiple resistances against different B.t. toxins do occur. Newly tested other toxins are often less specific and less effective. Some plant pathologists and entomologists plead not to waste the beneficial toxins and to develop/implement more careful management concepts. Current B.t. varieties will induce resistance sooner than conventional B.t. sprays. If the resistance management concept was retained some species would be expected to become resistant within 3-10 years, but the US and Australian regulations have been renewed. The presently favoured high-dose/refuge resistance management concept is only valid when certain premises are met and that these premises are met is doubted for some crops, varieties and regions. Lower dose strategies and alternative strategies are also only valid when special premises are fulfilled. Alternative concepts such as the use of a moderate or low dose in combination with antagonists and biological control measures or the planting of trap crops have rarely been assessed in field. Pyramiding genes with negative cross resistance to which adaptation is inherited recessively, could be a usefull tool to prolong durability of resistance, especially in combination with refuges. This strategy combined with moderate or low doses and natural enemies (which are not susceptible to the toxins and better or equally develop in resistant compared to susceptible biotypes) could result in a sustainable and thus desirable diversification of mortality sources. It is theoretically possible to forecast the spread of resistance by the use of migrationselection models on the basis of data on fitness costs and movement patterns.

27 II MODIFICATION RELATED CONCERNS 27 CRITERIA FOR RESISTANCE ASSESSMENT Resistance problems are expected to occur sooner and to be severe if: many and highly adaptable (many generations per year, high reproductivity) target pests will be controlled by the insect resistant plant especially for high-dose/refuge systems at least one of the target pests is less susceptible than the others or mating between resistant and susceptible biotypes is hindered dominant resistance alleles exist in a population implementation and control of management and monitoring activities are unrealistic in a region or country (larger scale when long distance movement of pests occur) alternative measures of biological control or alternative resistant varieties (with different modes of toxin action) are missing or a mixture of two or more genes with cross resistance is used or adaptation to these genes is inherited in an additive mode.

28 28 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS RECOMMENDATIONS FOR RISK ASSESSMENT Toxicity and ecotoxicity of new insecticidal plants should be studied. The toxicity of B.t. toxins to Collembola and Foramicidae and particulary specialized parasitoids should be measured. Generally toxins from plants or preys from insecticidal plants should be used in these studies. One should estimate or measure the average amount of toxin (of any insecticidal plant) taken up by susceptible non-target insects via pollen. The durability of low dose and moderate dose concepts in combination with pyramiding toxin genes, IPM and biological control measures should be tested in field. Movement patterns of the main target pests before and after mating should be investigated in order to improve refuge structures. A resistance assessment including studies on the frequency of resistance genes in target species, the resistance mechanism (and possible cross resistances), fitness cost, the level and the inheritance should be done for new resistance genes. Also migration-selection studies would be helpful to assess the amount of gene flow between regions when target pests are known to migrate. It should be assessed whether lower doses do more often lead to metabolic and multiple resistances than high doses. When management strategies are implemented it should be assessed whether their premises are met. In general: Susceptability, host range and population biology of target pests, side-effects of the toxin and the infested pest on natural enemies (tritrophic systems). High-dose strategy: Level of expression in different tissues at different plant ages Refuges: Development time of resistant and susceptible target pests Lower dose strategy: Effects of natural enemies on the fitness differential of resistant and susceptible target pests

29 II MODIFICATION RELATED CONCERNS 29 RECOMMENDATIONS FOR THE APPROVAL AND USE OF HIGH-DOSE VARIETIES The range of target insects should be known as well as their relevant attributes such as their host range, generations per year, reproductivity, susceptibility, movement and mating patterns. Cost effective mandatory monitoring assays by which recessive (and dominant?) alleles can be detected at densities of should be available, implemented, publicly operated, financed and controlled for the most adaptable target species. Uniform test standards have to be laid down. Mandatory refuge structures should be laid down by experts (entomology, plant pathology, population dynamics, farming, industry) in consensus conferences and implemented (depending on the crop, variety and range of target species). Very careful management should be required when alternative biological control measures or alternative resistant varieties are missing. An upper limit of the frequency of resistant biotypes/resistance alleles should be established for at least the pest species which is expected to adapt first. Remedial action concepts for the case of exceeding this limit should also be established and the responsibility for implementation must be laid down. Patterns of refuges should be recorded/documented for each main target species on a regional, national or international scale depending on its range of migration. Isolation distances between high-dose insecticidal crops and habitats of crosscompatible wild plants with a typical and susceptible insect fauna should be mandatory in centers of diversity. It should be taken into account, whether or not organic farming solely depends on B.t. sprays in a certain crop.

30 30 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS RECOMMENDATIONS FOR THE APPROVAL AND USE OF LOWER DOSE VARIETIES The range of target insects should be known as well as their relevant attributes such as their host range, generations per year, reproductivity, susceptibility, movement and mating patterns. The pest population should suffer less than 80% mortality from one toxin (from each toxin when pyramided) in order to prevent a fast break down of resistance. A dose must be expressed in plants which enables some effective natural enemies to survive and develop in infested pests. The possible cumulative effect of natural enemies on the target pest should be estimated. The surviving natural enemies should not decrease the fitness differential between resistant and susceptible target pests Cost effective mandatory monitoring assays by which recessive (and dominant?) alleles can be detected at densities of should be available, implemented, publicly operated, financed and controlled for the most adaptable target species. Uniform test standards have to be laid down. An upper limit of the frequency of resistant biotypes/resistance alleles should be established for at least the pest species which is expected to adapt first. Remedial action concepts for the case of exceeding this limit should also be established and the responsibility for implementation must be laid down. It should be taken into account, whether or not organic farming solely depends on B.t. sprays in a certain crop.

31 II MODIFICATION RELATED CONCERNS 31 LITERATURE TECHNOLOGY AND GENERAL ASPECTS Altpeter F, Diaz I, McAuslane H, Gaddour K, Carbonero P, Vasil IK Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor Cme. Molecular Breeding 5:53-63 Bottrell DG, Barbosa P, Gould F Manipulating natural enemies by plant variety selection and modification: A realistic strategy? Annu.Rev.Entomol. 43: Boulter D Insect pest control by copying nature using genetically engineered crops. Phytochemistry 34(6): Bowen D, Rocheleau TA, Blackburn M, Andreev O, Golubeva E, Bhartia R, Ffrench- Constant RH Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280: Bowen DJ, Ensign JC Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Applied and Environmental Microbiology 64(8): Cai D, Kleine M, Kifle S, Harloff H-J, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentjin EMJ, Lange W, Stiekema WJ, Wyss U, Grundler FMW, Jung C Positional cloning of a gene for nematode resistance in sugar beet. Science 275: Cook RJ The molecular mechanisms responsible for resistance in plant-pathogen interactions of the gene-for-gene type function more broadly than previously imaged. Proc.Natl.Acad.Sci.USA 95: Ding X, Gopalakrishnan B, Johnson LB, White FF, Wang X, Morgan TD, Kramer KJ, Muthukrishnan S Insect resistance of transgenic tobacco expressing an insect chitinase gene. Transgenic Reseach 7:77-84 Estruch JJ, Carozzi, NB, Desai N, Duck NB, Warren GW Transgenic plants: An emerging approach to pest control. Nature Biotechnology 15: Gatehouse AMR, Shi Y, Powell KS, Brough C, Hilder VA, Hamilton WDO, Newell CA, Merryweather A, Boulter D, Gatehouse JA Approaches to insect resistance using transgenic plants. Phil. Trans.R.Lond.B 342, Hassani M, Zimmermann G, Langenbruch G-A, Vidal S Biologische Bekämpfung von Baumwollschädlingen: Wirkung verschiedener Bacillus thuringiensis-präparete und entomopathogener Pilze auf Spodoptera littoralis und Helicoverpa armigera. Mitt. A. d. Biol. Bundesanst. H.357:343 Hoffmann MP, Zalom FG, Wilson LT, Smilanick JM, Malyj LD, Kiser J, Hilder VA, Barnes WM Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis delta-endotoxin or cowpea trypsin inhibitor: efficacy against Helicoverpa zea (Lepidoptera: Noctuidae). Journal of Economic Entomology 85: Kramer KJ, Muthukrishnan S, Johnson L, White F Chitinases for insect control. in: Carozzi N, Koziel M. (eds.) Advances in insect control: The Role of transgenic plants: Bristol Moar WJ, Pustzi-Carey M, Van Faassen H, Bosch D, Frutos R, Rang C, Luo K, Adang MJ Development of Bacillus thuringiensis CryIC resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) Appl. Environ. Microbiol. 61: Moffat AS First nematode-resistance gene found. Science 275:757

32 32 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Peumans WJ, Winter HC, Bemer V, Van Leuven F, Goldstein IJ, Truffa-Bachi P, Van Damme EJM Isolation of a novel plant lectin with an unusual specifity from Calystegia sepium. Glycoconjugate Journal 14: Roberts BL, Markland W, Ley AC, Kent RB, White DW, Guterman SK, Ladner RC Directed evolution of a protein: Selection of potent neutrophil elestase inhibitors displayed on M13 fusion phage. Proc.Natl.Acad.Sci.USA. 89: Rossi M, Goggin FL, Milligan SB, Kaloshian I, Ullman DE, Williamson VM The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proc.Natl.Acad.Sci.USA 95: Shelton AM, Tang JD, Ruosh RT, Metz TD, Earle ED. et al Field tests on managing resistence to Bt-engineered plants. Nature Biotechnology 18(3): Stephenson P. et al. Internet Communication. Director, Absorbent Hygiene Products Manufacturers Association (UK) 46 Bridge Street, Goldaming, Surrey GU7 1HL Stoger E, Williams S, Christou P, Down RE, Gatehouse JA Expression of the insecticidal lectin from snowdrop (gananthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae. Molecular Breeding 5:63-73 Strauss E Possible new weapon for insect control. Science 280:2050 Urwin PE, Atkinson HJ, Waller DA, McPherson MJ Engineered oryzacystatin-l expressed in transgenic hairy roots confers resistance to Globodera pallida. The Plant Journal 8(1): Warren GW Vegetative insecticidal proteins: Novel proteins fot control of corn pests. in: Carozzi N, Koziel M. (eds.) Advances in insect control: The Role of transgenic plants: Bristol USDA Biotechnology Risk Assessment Research Grants Program Home Page. USDA Biotechnology Risk Assessment Research Grants Program Home Page. RESISTANCE AND ADAPTATION Ahmad M, Ruosh R Estimation of allele frequencies for Bacillus thuringiensis resistance in diamondmoth, Plutella xylostella and cotton bollworm, Helicoverpa armigera. In: Gene flow and agriculture: Relevance for transgenic Crops. British Crop Protection Council Symposium Proceedings No. 72: Bauer LS Resistance: A threat to the insecticidal crystal proteins of Bacillus thuringiensis. Florida Entomologist 78: Georghiou GP Overview of insecticide resistance. In: Green MB (ed.) Managing Resistance to Agrochemicals. From Fundamental research to Practical Strategies.ACS Series 421: Washington DC Gould F, Anderson A, Jones A, Summerford D, Heckel GG, Lopez J, Micinski S, Leanard R., Laster M Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens. Proc. Natl. Acad. Sci. USA 94: Gould F, Martinez-Ramirez A, Anderson A, Ferre J, Silva FJ, Moar WJ Broad spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens. Proc. Natl. Acad. Sci. USA 89:

33 II MODIFICATION RELATED CONCERNS 33 Huang F, Buschmann LL, Higgins RA, McGaugey WH Inheritance of resistance to Bacillus thuringiensis toxin (Dipel ES) in the European corn borer. Science 284: Jongsma MA, Bakker PL, Peters J, Bosch D, Stiekma WJ Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc.Natl.Acad.Sci.USA: Marrone PG, Macintosh SC Resistance to Bacillus thuringiensis and resistance management. In: Entwistle JS et al. (eds.) Bacillus thuringiensis, An Environmental biopesticide: Theory and practice: Monsanto BollGard cotton Uudate. Internet: 6pp. Rahardja U, Whalon, WE Inheritance of resistance to Bacillus thuringiensis subsp. Tenebrionis CryIIIA-endotoxin in Colorado potato beetle (Coleoptera: Chroysomelidae). J. Econ. Entomol. 88:21-26 Shelton A.M, Robertson JL, Tang JD, Perez C, Eigenbrode SD Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis Subspecies in the Field. Journal of Economic Entomology 86: Tabashnik BE Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39:47-79 Tabashnik BE Insecticide resistance. Trends Ecol. Evol. 10: Tabashnik BE Seeking the root of insect resistance to transgenic plants. Proc. Natl. Acad. Sci. USA 94: Van Rie J, McGaughey WH, Johnson DE, Barnett BD, Van Mallaert H Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science 247:72-74 Williams WP, Buckley PM, Sagers JB, Hanten JA Evaluation of transgenic corn for resistance to corn earworm (Lepidoptera: Noctuidae); fall armyworm (Lepidoptera: Noctuidae), and southwestern corn borer (Lepidoptera: Crambidae) in a laboratory bioassey. J.Agric.Entomol. 15(2): Wu Y, Llewellyn D, Mathews A, Dennis ES Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molecular Breeding 3: RESISTANCE MANAGEMENT AND MONITORING Alstad DN, Andow DA Managing the evolution of insect resistance to transgenic plants. Science 268: Alstad DN, Andow DA Implementing management of insect resistance to transgenic crops. AgBiotech. News Inf. 18: Alyokhin A, Ferro DN, Hoy CW, Head G Laboratory assessment of flight activity displayed by Colorado potato beetles (Coleoptera:Chrysomelidae) fed on transgenic and Cry3a toxin-treated potato foliage. Journal of Economic Entomology 92: Andow D, Hutchinson W Bt-Corn Resistance Management. In: Mellon M, Rissler J. (eds.) Now or Never: Serious New Plans to Save a Natural Pest Control: Cao J, Tang JD, Strizhov N, Shelton AM, Earle ED Transgenic brocoli with high levels of Bacillus thuringiensis CryIC protein control diamondback moth larvae resistant to CryIA or CryIC. Molecular Breeding 5: Dove A, Bt resistance plan appraised. Nature Biotechnology 17:

34 34 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Ferro DN. (in press) Transgenic potatoes: Resistance management of the Colorado potato beetle. In: Proceedings of the 5rd International Symposium on "The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms" , Braunschweig. Ferro DN Potential for resistance to Bacillus thuringiensis: Colorado potato beetle (Coleoptera: Chrysomelidae) - A model system. American Entomologist 39:38-44 Fischhoff DA Insect-resistant crop plants. In: Perseley GJ (ed.) Biotechnology and integrated pest management: Oxoxn, UK: CAB Int. 475pp Fox J Bt cotton infestations renew resistance concerns. Nature Biotechnology 14:1070 Georghiou GP, Wirth, MC Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology 63(3): Gould F, Follett P, Nault B, Kennedy GG Resistance management strategies for transgenic potato plants. In. Zehnder GW, et al. (eds.) Advances in potato pest: Biology and Management. APS Press. St. Paul, Minn.: Gould F, Kennedy GG, Johnson MT Efets of natural enemies on the rate of adaptation to resistant host plants. Entomol. Exp.Appl. 58:1-14 Gould F, Tabashnik B Bt-Cotton Resistance Management. In: Mellon M, Rissler J. (Eds.) Now or Never: Serious New Plans to Save a Natural Pest Control: : Gould F, Tabashnik B.E Bt-cotton resistance management. See Ref. 36, pp Gould F Simulation models for predicting durability of insect-resistant germ plasm: A deterministic diploid, two-locus model. Env. Entomol. 15:1-10 Gould F Genetic engineering, integrated pest management and the evolution of pests. Tree/Tibtech 3/6(4): Gould F Potentials and problems with high-dose strategies for pesticidal engineered crops. Biocontrol Sci. Technol. 4: Gould F Comments of Fred Gould on Plant Pesticide Resistance Management (EPA- Hearing ) Manuskript Gould F Sustainability of transgenic insecticidal cultivars. Ann. Rev. Entomol. 43: Gould F Evolutionary biology and genetically engineered crops. BioScience 38:26-33 Hare JG Effects of plant variation on herbivore-natural enemy interactions. In: Fritz RS, Simms EL: (eds.) Plant resistance to herbivores and pathogens: Ecology, Evolution and Genetics: Chicago Heckel DG, Gahan LJ, Liu Y-B, Tabashnik BE Genetic mapping of resistance to Bacillus thuringiensis toxins in diamondback moth using biphasic linkage analysis. PNAS USA 96: Henneberry TJ, Keaveny DF Suppression of pink bollworm by sterile moth relases. USDA Agricultural Research Service, ARS-32 Johnson MT Interaction of resistant plants and wasp parasitoids of tobacco budworm (Lepidoptera: Noctuidae). Environ. Entomol. 26(2): Johnson MT Gould F, Kennedy GG Effect of entomopathogen on adaptation of Heliothis virescens populations to transgenic host plants. Entomol. Experimentalis et Appl. 83:

35 II MODIFICATION RELATED CONCERNS 35 Johnson MT, Gould F Interaction of genetically engineered host plant resistance and natural enemies of Heliothis virescens (Lepidoptera: Noctuidae) in tobacco. Envirnm. Entomol. 21: Jutsum AR, Heaney SP, Perrin BM, Wege PJ Pesticide Resistance: Assessment of risk and the development and implementation of effective management strategies. Pesticide Science 54: Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar W Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc.Natl.Acad.Sci.USA 96: Langenbruch GA B.t.-Mais und B.t.-Kartoffeln gefährden sie den Einsatz von Bacillus thuringiensis-päparaten in Deutschland? Mitt.a.d.Biol. Bundesanst. 357: 342 Lenormand T, Bourguet D, Giullemaud T, Raymond M Tracking the evolution of insecticide resistance in the mosquito Culex pipiens. Nature 400: Lenormand T, Raymond M Resistance management: the stable zone strategy. Proc.R.Soc.Lond.B. 265: Mallet J, Porter P Preventing insect adaptation to insect-resistant crops: Are seedmixtures or refugia the best strategy? Proc. Roy. Soc. London Ser. B: McGaughey WH, Whalon ME Managing insect resistance to Bacillus thuringiensis toxins. Science 258: McGaughey, W.H., Gould, F., Gelernter, W Bt resistance management. A plan for reconciling the needs of the many stakeholders in Bt-based products. Nat. Biotechnol. 16: Mellon M, Rissler J (eds.) Now or never: Serious new plans to save a natural pest control. 147pp. Cambridge, Masschusetts Morrill WL, Kushnak GD, Bruckner PL, Gabor JW Wheat stem sawfly (Hymenoptera: Cephidae) damage, rates of parasitism, and overwinter survival in resistant wheat lines. J. Econ. Entomol. 87: Nurminsky DI, Hartl DL Development time and resistance to Bt crops. Nature 400: Riede M Folgen des Einsatzes transgener Baculoviren. In: Nutzung der Gentechnik im Agrarsektor der USA Die Diskussion von Versuchsergebnissen und Szenarien zur Biosicherheit,. Schütte G et al. Umweltbundesamt Texte Nr. 47/98: Berlin. Roush RT Managing pests and their resistance to Bacillus thuringiensis: Can transgenic crops be better than sprays? Biocontrol Science and Technology 4: Roush RT Managing resistance to transgenic crops. in: Carozzi N, Koziel M (eds.) Advances in Insect Control: The Role of Transgenic Plants: Bristol Schuler T, Poppy GM, Potting PJ, Denholm I, Kerry BR. 1999a. Interactions between insect tolerant genetically modified plants and natural enemies. In: Gene flow and agriculture: Relevance for transgenic Crops. Brithish Crop Protection Council Symposium Proceedings No. 72: Schuler T, Potting PJ, Denholm I, Poppy GM. 1999b Parasitoid behaviour and Bt plants. Nature 400: Shi-Rong J. (in Press) Development of insect resistance management strategies of Bt cotton in China. In: Proceedings of the 5rd International Symposium on "The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms , Braunschweig

36 36 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Sims SB, Greenplate JT, Stone TB, Caprio MA, Gould FL Monitoring strategies for early detection of Lepidoptera resistance to Bacillus thuringiensis insecticidal proteins. In: Brown TM (ed.) Molecular genetics and evolution of pesticide resistance. ACS Symposium Series 645: Starks KJ, Muniappan R, Eikenbary RD Interaction between plant resistance and parasitism against the greenbug or barley and sorghum. Ann.Entomol.Soc.Am. 65: Tabashnik BE, Groeters FR, Finson N, Liu Y-B, Johnson MW, Heckel DG, Luo K, Adang M Resistance to Bacillus thuringiensis in Plutella xylostella The moth heard round the world. Paper 4142 of the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series. University of Hawaii, Honolulu Tabashnik BE, Malvar T. Liu Y-B, Finson N, Borthakur D, Shin B-S, Park S-H, Masson L, de Maagd RA, Bosch D Cross-resistance of the diamond moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Applied and Environmental Microbiology: Tabashnik BE, Roush RT, Earle ED, Shelton AM Science paper on Bt resistance in corn borers challenged. ISB News Report 9/99 Tabashnik BE Delaying insect adaptation to transgenic plants: Seed mixtures and refugia reconsidered. Proc. Roy. Soc. Lond. Ser. B 255:7-12 Thaler JS Induced resistance in agricultural crops: Effects of jasmonic acid on herbivory and yield in tomato plants. Environmental Entomology 28(1):30-37 Tollefson JJ, Calvin DD Sampling arthropod pests in field corn. in: Pedigo LP, Buntin GD (eds.) Handbook of sampling methods for arthropods in agriculture: CRC Press, Boca Raton Traynor P National biological impact program. NBIAP News Rep. Dec. 16pp. Trisyono A, Whalon MA Fitness costs of resistance to Bacilllus thuringiensis in Colorado potato beetle (Coleoptera: Chrysomelidae). Journal of Economic Entomology 90(2): Van Emden HF Plant insect relationships and pest control. In: World review of pest control 5: Whalon M, Ferro D Bt-Potato Resistance Management. in: Mellon M, Rissler J. (Eds.) Now or Never: Serious New Plans to Save a Natural Pest Control: Whalon ME, Wierenga JM Bacillus thuringiensis resistant Colorado potato beetle and transgenic plants: Some operational and ecological implications for deployment. Biocontrol Science and Technology 4: Wirth MC, Georghiou GP, Federici BA CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc.Natl.Acad.Sci.USA:

37 II MODIFICATION RELATED CONCERNS 37 TOXICITY AND ECOTOXICITY Arpaia S Ecological impact of Bt-transgenic plants: 1. Assessing possible effects of CryIIIB toxin on honey bee (Apis mellifera L.) colonies. Journal of Genetics and Breeding 50: Ashouri AS, Overney S, Michaud D, Cloutier C Fitness and feeding are affected in the two spotted stinkbug, by the cysteine proteinse inhibitor, Oryzacystatin I. Archives of Insect Biochemistry & Physiology 38:74-83 Bell HA, Fitches EC, Down RE, Marris GC, Edwards JP Gatehouse JA The effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on Eulophus pennicornis (Hymenoptera: Eulopidae), a parasitoid of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae). Journal of Insect Physiology 45(11): Bigler F, Keller B, Keller M Risikoforschung an gentechnisch verändertem Bt-Mais. in: Eidgenössische Forschungsgemeinschaft für Agrarökologie und Landbau (ed.) Medieninformation Birch ANE, Geogham IE, Majerus M, McNicol JW, Hackett C, Gatehouse AMR, Gatehouse JA. in press. Ecological impact on predatory 2-spot ladybirds of transgenic potaoes expressing snowdrop lectin for aphid resistance. Journal of Molecular Breeding Brake J, Vlachos D Evaluation of transgenic event 176 Bt-corn in broiler chickens. Poultry science 77(5): Cowgill SE, Coates D, Atkinson HJ Non-target effects of proteinase inhibitors expressed in potato as an anti-nematode defence. In: Gene flow and agriculture: Relevance for transgenic Crops. British Crop Protection Council Symposium Proceedings No. 72: Crecchio C, Stotzky G Insecticidal activity and biodegradation of the toxin from Bacillus thuringiensis Subs. kurstaki bound to humic acids from soil. Soil. Biol. Biochem. 30(4): Crecchio C, Stotzky G Insecticidal activity and biodegradation of the toxin from Bacillus thuringiensis Subs. Kurstaki bound to humic acids from soil. Soil. Biol. Biochem 30(4): Czapla TH Plant lectins as insect control proteins in transgenic plants. in: Carozzi N, Koziel M. (eds.) Advances in insect control: The Role of transgenic plants: Bristol Daly JC Ecology and resistance management for Bacillus thuringiensis transgenic plants. Biocontrol Science and Technology 4: Deml R, Dettner K Wirkungen Bacillus thuringiensis-toxin produzierender Plfanzen auf Ziel- und Nichtzielorganismen eine Standortbestimmung. Umweltbundesamt Texte 36/ S. Dogan EB, Berry RE, Reed GL, Rossignol PA Biological parameters of convergent lady beetle (Coleoptera: Coccinellidae) feeding an aphids (Homoptera: Aphididae) on transgenic potato. Journal of Economic Entomology 89: Drobniewski FA The Safety of Bacillus Species as Insect Vector Control Agents. Journal of Applied Bacteriology 76: Fitt GP Field evaluation of transgenic cotton in Australia: Environmental consideratons and consequences of expanding trial size. In: Jones DD (ed.) Proceedings of the 3rd International Symposium on "The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms : Monterey, California, Oakland

38 38 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Flexner JL, Lighthart B, Croft BA The effects of microbial pesticides on non-target, beneficial arthropods. Agriculture, Ecosystems and Environment 16: Flint HM, Henneberry TJ, Wilson FD, Holguin E, Parks N, Buehler RE The effects of of transgenic cotton, Gossypium hirsutum L.; containing Bacillus thuringiensis toxin genes for the control of the pink bollworm, Pectinophora gossipiella (Saunders) Lepidoptera, Gelechiidae and other arthropods. Southwestern Entomologist 20: Friedman M (ed.) Nutritional and toxicolical significance of enzyme inhibitors in foods. New York Gatehouse A Effects of transgenic insect resistant crops on parasitoids and predators of insect pests. In: The environmentel implications od GM plants with insect resistance genes. ESF/AIGM Workshop Bern Gianessi LP, Carpenter JE Agricultural Biotechnology: Insect Control Benefits. Biotechnology Industry Organization (BIO) Goldburg RJ, Tjaden G Are B.T.K. Plants Really Safe to Eat? Bio/Technology 8: Goldburg RJ, Tjaden G Are B.t.k. plants really safe to eat? Bio/Technology: Haider MZ, Knowles BH, Ellar DJ Specifity of Bacillus thuringiensis var. colmeri insecticidal δ-endotoxin is determined by differential protolytic processing of the protoxin by larval gut proteases. Eur. J. Biochem. 156: Hilbeck A, Baumgartner M, Fried PM, Bigler F Effects of transgenic Bacillus thuringiensis corn-fed prey on mortality and development time of Chrysoperla carnea (Neuroptera: Chrysopidae). Environmental Entomology 27: Hilbeck, A. in Press. Effect of Bt on insect natural enemies. In: Proceedings of the 5rd International Symposium on "The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms" , Braunschweig Kreutzweiser DP, Gringorten JL, Thomas DR, Butcher JT Functional effects of the bacterial insecticide Bacillus thuringiensis var. kurstaki on aquatic communities. Ecotoxicology and enviroment safety 33: Kuiper HA, Noteborn HPJ Food Safety Assessment of Transgenic Insect-Resistant B.t. Tomatoes. OECD Food Safety Evaluation: Liener IE Trypsin inhibitors: Concern for human nutrition or not? In: Friedman M. (ed.) Nutritional and toxicolical significance of enzyme inhibitors in foods: New York Loseley JE, Raynor LS, Carter ME Transgenic pollen harms monarch larvae. Nature 399:214 Luttrell RG, Mascarenhas VJ, Schneider JC, Parker CD, Bullock PD Effect of transgenic cotton expressing endotoxin protein on arthropod populatione in Mississippi cotton. Proc. of the Beltwide cotton production research conference. National cotton council of America: Memphis TN Melin BE, Cozzi EM Safety to nontarget invertebrates of Lepidopteran strains of Bacillus thuringiensis and their β- exotoxins. In: Laird M, Lacey LA, Davidson EW (eds.) Safety of Microbial Insecticides: CRC Press, Inc, Boca Raton, Florida Monsanto Noteborn HPJ, Bienenmann-Plo, van den Berg JH, Alink GM, Zolla L Safety Assessment of the Bacillus thuringiensis Insecticidal Crystal Protein CRYIA(b) Expressed in Transgenic Tomatoes. Genetically Modified Foods - Safety Issues. ACS Symposium Series 605:

39 II MODIFICATION RELATED CONCERNS 39 Orr DB, Landis DA Oviposition of European corn borer (Lepidoptera: Pyralidae) and impact of natural enemy populations in transgenic versus isogenic corn. Journal of Economic Entomology 90: Pilcher CD, Obrycki JJ, Rice ME, Lewis LC Preimaginal development, survival, and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environ. Entomol. 26(2): Reardon RC, Wagner DL Impact of Bacillus thuringiensis on nontarget Lepidopteran species in broad-leaved forests. Biorational Pest Control Agents: Formulation and Delivery 595: Saik JE, Lacey LA, Lacey, CM Safety of microbial insecticides to vertebrates - Domestic Animals And Wildlife. In: Laird M, Lacey LA, Davidson EW. (eds.) Safety of microbial insecticides: , CRC Press, Inc., Boca Raton, Florida Saxena D, Flores S, Stotzky G Nature 402:480 Saxena D, Flores S, Stotzky G Nature 402:480 Schütte G, Riede M Bacillus thuringiensis-toxine in Kulturpflanzen. In: Nutzung der Gentechnik im Agrarsektor der USA Die Diskussion von Versuchsergebnissen und Szenarien zur Biosicherheit, Schütte G. et al.: Umweltbundesamt Texte Nr. 47/98. Berlin. Sears MK, Stanley-Horn DE, Mattila HR Preliminary report on the ecological impact of BT corn pollen on th Monarch butterfly in Ontario. Department of Environmental Biology. Univeresity of Guelph, Ontario N1G 2W1 Shelton AM, Tang JD, Ruosh RT, Metz TD, Earle ED Field tests on managing resistance to Bt-engineered plants. Nature Biotechnology 18(3): Sims SR Bacillus thuringiensis var. kurstaki (CryIA(C)) protein expressed in transgenic cotton: effects on beneficial and other non-target insects. Southwestern Entomologist 20: Stewart CN Jr Insecticidal transgenes into nature: gene flow, ecological effects, relevancy, and monitoring. In: Gene flow and agriculture: Relevance for transgenic Crops. British Crop Protection Council Symposium Proceedings No. 72: Stirn S Toxizität transgener Pflanzen. In: Schütte G. Heidenreich B; Beusmann V. Nutzung der Gentechnik im Agrarsektor der USA - Die Diskussion von Versuchsergebnissen und Szenarien zur Biosicherheit, Umweltbundesamt Texte Nr.47/89: Tapp H, Stotzky G Persistance of the insecticidal toxin from Bacillus thuringiensis Subs. kurstaki in soil. Soil. Biol. Biochem. 30(4): Theiling KM, Croft BA Pesticide side-effects on arthropod natural enemies: A database summary. Agriculture, Ecosystems and Environment 21: Vinson SB Potential impact of microbial insecticides on beneficial arthropods in the terrestrial environment. In: Laird M, Lacey LA, Davidson EW (eds.) Safety of Microbial Insecticides: CRC Press, Inc, Boca Raton, Florida

40 40 VARIETIES RESISTANT AGAINST INVERTEBRATE PESTS Wagner DL, Peacock JW, Carter JL, Talley SE Field assessment of Bacillus thuringiensis on nontarget Lepidoptera. Environ. Entomol. 25(6): Walker AJ, Ford L, Majerus MEN, Geoghegan IE, Birch N, Gatehouse JA, Gatehouse AMR Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird, Adalia bipunctata, and ist sensitivity to proteinase inhibitors. Insect Biochemistry & Molecular Biology 28:173-80