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Keywords:

  • Bt crops;
  • non-target organisms;
  • risk assessment;
  • IPM

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Acknowledgements
  5. References

inline imageKong-Ming Wu (Corresponding author)

The application of recombinant DNA technology has resulted in many insect-resistant varieties by genetic engineering (GE). Crops expressing Cry toxins derived from Bacillus thuringiensis (Bt) have been planted worldwide, and are an effective tool for pest control. However, one ecological concern regarding the potential effects of insect-resistant GE plants on non-target organisms (NTOs) has been continually debated. In the present study, we briefly summarize the data regarding the development and commercial use of transgenic Bt varieties, elaborate on the procedure and methods for assessing the non-target effects of insect-resistant GE plants, and synthetically analyze the related research results, mostly those published between 2005 and 2010. A mass of laboratory and field studies have shown that the currently available Bt crops have no direct detrimental effects on NTOs due to their narrow spectrum of activity, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and clear benefits on the environment and farmer health. Consequently, Bt crops can be a useful component of integrated pest management systems to protect the crop from targeted pests.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Acknowledgements
  5. References

Within the context of integrated pest management (IPM), insect pest-resistant cultivars, developed through conventional plant breeding methods, have been used with great effectiveness against important pests in numerous cropping systems. It was estimated that the economic value of host-plant resistance to major pests of wheat in the USA was US $192 million per year (Smith 2005). In addition to high efficiency, using insect-resistant cultivars for pest control is user friendly and safe to the environment. However, the widespread use of host-plant resistance had been constrained by the limited availability of elite cultivars possessing high levels of resistance to key pest species before the appearance of recombinant DNA technology (Kennedy 2008). The application of recombinant DNA technology has eliminated the constraint and provided a more efficient tool to develop insect-resistant varieties by genetic engineering (GE). Compared with conventional plant breeding procedures, the new technology has a number of advantages. First, because the techniques of GE allow genes to be inserted directly into advanced crop breeding lines or cultivars, the time required to develop commercially-acceptable, resistant cultivars is greatly reduced (Smith 2005). Second, the potential array of available resistance traits that can be used to obtain insect-resistant crops is greatly increased. Further, it is possible to identify and use insect-resistant genes from any organism. Finally, since the gene products that confer resistance can be well defined, it is possible to test them directly to address questions regarding health and environmental effects (Kennedy 2008).

The first insect-resistant GE plants were produced in 1987, when genes coding for a Cry toxin derived from a soil bacterium Bacillus thuringiensis (Bt) Berliner were expressed in tobacco (Vaeck et al. 1987). Since then, many novel Cry proteins and more new insecticide proteins, such as protease inhibitors, lectins, and α-amylase inhibitors, have been exploited (Malone et al. 2008). In 1995, the transgenic crop cultivars of Bt maize and Bt cotton were first approved for commercial release in the USA, and first planted in 1996 (Hellmich et al. 2008; Naranjo et al. 2008; James 2009). Since then, the number of countries electing to grow biotech crops has increased steadily. In 2009, more than 14 million farmers in 25 countries were cultivating biotech crops, with a global production area of 134 million hectares (James 2009; Marshall 2010).

Bt crops not only provide an effective alternative tool for controlling target insects (Wu et al. 2008), but also provide many social, environmental, and economic benefits, such as reducing the use of chemical insecticides, benefiting the environment and human health, and increasing farm income (Wang 2007, Brookes and Barfoot 2010; Choudhary and Gaur 2010; Huang et al. 2010; Hutchison et al. 2010; Tabashnik 2010). For example, the direct global farm income benefit from Bt cotton was $ 2.9 billion in 2008. Within this, 65% of the farm income gain has derived from yield gains (less pest damage) and the balance (35%) from reduced expenditure on crop protection (spraying of insecticides) (Brookes and Barfoot 2010). Nevertheless, as with any technology, there have been questions about the potential risks transgenic plants might have on the environment. One of the major ecological concerns regarding the environmental risks of insect-resistant GE plants is their potential effects on non-target organisms (NTOs) (Romeis et al. 2006, 2008, 2009).

In order to safely and sustainably utilize transgenic biotechnology in pest control, the potential impact of Bt crops on NTOs, including pest natural enemies, pollinators, microbes, and mammalians, have been extensively studied worldwide in the last 20 years. A significant amount of research data is available on this. To make the data more accessible and easily understood, in the present study, we summarized and analyzed the data regarding the development and commercial use of transgenic Bt insect-resistant varieties, the assessment methods and procedure for non-target effects of insect-resistant GE plants, and the related assessment results mostly published between 2005 and 2010. The present study provides a general insight into the development and use, as well as the risk assessment, of Bt crops worldwide.

Crop Varieties Transformed with Bt Genes

Many crops, such as cotton, maize, potato, tomato, rice, eggplant, and crucifer vegetables, have been genetically transformed with genes derived from soil bacteria Bt coding for proteins that are highly active against many important pests (Table 1). In addition to the δ-exotoxins, such as vegetative insecticidal protein-3A (VIP3A), the well-known insecticidal endotoxins are highly selective and represent a class of numerous proteins with insecticidal action on larvae from various orders: Cry1 and Cry2 are toxic for lepidopteran pests, Cry2A for lepidopteran and dipteran pests, and Cry3 for coleopteran pests (Malone et al. 2008) (Table 1). The first generation of Bt crops normally expressed single Cry proteins (Cry1A) with specific activity against lepidopteran pests (Bollgard I expressing Cry1Ac). To broaden the spectrum of protection and to delay the evolution of pest resistance to Bt, other insecticidal-active Bt toxins, such as Cry1F, Cry2A and VIP3A, have been added to the list of commercialized traits, and often they are presented as pyramided genes in a single variety (e.g. Dow Agrosciences’ Wide Strike cotton (Cry1F+Cry1Ac) and Monsanto's Bollgard II (Cry1Ac+Cry2Ab2)) (James 2009) (Table 1). The use of non-Bt insect-resistance traits with different modes of action, such as protease inhibitors or lectins, solely or in combination with Bt, has long been advocated as a means of delaying selection for resistant pest (Malone et al. 2008), although, to date, there have been no transgenic plants commercialized (James 2010a). In the long term, new transgenic plants expressing novel Cry or other insecticidal proteins, stacked genes, or fusion proteins will increase in importance (Ferré et al. 2008).

Table 1. Crop varieties transformed with Bacillus thuringiensis genes for resistance to target pests
CropInsect toxin genesTarget pest orderMajor target pests
  1. VIP3A, vegetative insecticidal protein. Source: James 2009, US EPA, genetically-modified crop database, ISAAA.org.

CottonCry1AcLepidopteraTobacco budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), cotton bollworm (Helicoverpa zea, Helicoverpa armigera)
Cry1Ab/cLepidopteraHeliothis virescens, Pectinophora gossypiella, Helicoverpa armigera, Helicoverpa zea
Cry1Ac+CpTILepidopteraPectinophora gossypiella, Helicoverpa armigera
Cry1Ac+Cry2AbLepidopteraHeliothis virescens, Pectinophora gossypiella, Helicoverpa armigera, Helicoverpa zea
Cry1A+Cry1FLepidopteraSpodoptera spp.
Cry1FLepidopteraHeliothis virescens, Helicoverpa zea, beet armyworm (Spodoptera exigua), and soybean looper (Pseudoplusia includens)
Vip3ALepidopteraHelicoverpa zea, Heliothis virescens, Pectinophora gossypiella, Spodoptera exigua, Pseudoplusia includens, cabbage looper (Trichoplusia ni), fall armyworm (Spodoptera frugiperda), and cotton leaf perforator (Bucculatrix thurberiella).
Vip3A+Cry1AbLepidopteraHeliothis virescens, Pectinophora gossypiella, Helicoverpa armigera, Helicoverpa zea
MaizeCry1AbLepidopteraEuropean corn borer (Ostrinia nubilalis)
Cry1Ab+mCry3ALepidoptera, coleopteraOstrinia nubilalis, corn rootworm (Diabrotica spp.)
Cry1FLepidopteraOstrinia nubilalis, Spodoptera frugiperda, southwestern corn borer (Diatraea grandiosella), western bean cutworm (Striacosta albicosta), black cutworm (Agrotis ipsilon), Helicoverpa zea
Cry34Ab1+Cry35Ab1ColeopteraDiabrotica spp.
Cry34Ab1+Cry35Ab1Lepidoptera, Coleoptera,Ostrinia nubilalis, Diabrotica spp.
 +Cry1Fa2+Cry1Fa2  
mCry3AColeopteraWestern corn rootworm (Diabrotica vigifera vigifera), northern corn rootworm (Diabrotica berberi), and Mexican corn rootworm (Diabrotica vigifera zeae)
Cry3Bb1ColeopteraDiabrotica spp.
Cry1A.105+Cry2Ab2LepidopteraOstrinia spp., Spodoptera frugiperda,
Cry1A.105+Cry2Ab +Cry3Bb1+Cry34Ab1 +Cry35Ab1+Cry1Fa2Lepidoptera, ColeopteraAbove-ground insects: Helicoverpa zea, Ostrinia nubilalis, Spodoptera frugiperda, Diatraea grandiosella, sugarcane borer (Diatraea saccharalis), Striacosta albicosta and Agrotis ipsilon; below-ground insects: Diabrotica virgifera virgifera, Diabrotica barberi and Diabrotica virgifera zeae
Vip3Aa20+mCry3A +Cry1AbLepidoptera, ColeopteraAbove-ground insects: Ostrinia nubilalis, Diatraea grandiosella, Helicoverpa zea, Spodoptera frugiperda, Spodoptera exigua, Agrotis ipsilon, Striacosta albicosta, Diatraea saccharalis, armyworm (Pseudaletia unipunctata), southern cornstalk borer (Diatraea crambidoides), common stalk borer (Papaipema nebris); below-ground insects: Diabrotica virgifera virgifera, Diabrotica barberi, and Diabrotica virgifera zeae
PotatoCry3AColeopteraColorado potato beetle (Leptinotarsa decemlineata)
TomatoCry1AcLepidopteraHeliothis virescens, Pectinophora gossypiella, Helicoverpa armigera, Helicoverpa zea
RiceCry1AbLepidopteraRice stem borers(Scirpophaga incertulas, Chilo suppressalis)
Cry1Ab/cLepidopteraScirpophaga incertulas, Chilo suppressalis
EggplantCry1AcLepidopteraFruit and shoot borer (Leucinodes orbonalis)
Crucifer vegetablesCry1LepidopteraDiamondback moth (Plutella xylostella)

Commercial Planting of Insect-Resistant Bt Crops

So far, Bt maize and Bt cotton are the only insect-resistant GE crops for commercial planting (James 2010a). Bt genes (Cry1Ac, Cry1Ab, Cry2Ab, and Cry1F) of cotton were commercialized in 11 countries in 2009 (Table 2), and the total planting area reached 15 million hectares, which comprised approximately half of all the cotton grown in the world in 2009 (Figure 1) (Naranjo 2010). The total area where Bt cotton was planted globally in 2010 was 19.6 million hectares, up by 4.6 million hectares in 2009 (Figure 1) (James 2010b). China and India are the two major cotton-growing countries. In 2009, 8.4 million hectares of hybrid Bt cotton were planted in India, which made India displace China as the largest Bt cotton-growing country. To delay the development of pest resistance, Bt cotton varieties containing two different Cry proteins (Bollgard II and Wide Strike) have been gradually adopted by some countries in recent years. Since 2004, growers in Australia have been exclusively using Bollgard II (expressing Cry1Ac and Cry2Ab) instead of Bollgard I (expressing Cry1Ac) (Naranjo et al. 2008). Bt cotton varieties with two Cry proteins is becoming common, and most Bt cotton is also genetically engineered to be herbicide tolerant (Naranjo 2010). Maize transformed with Bt genes (Cry1Ab, Cry1F, Cry3Bb1, VIP3A, Cry34Ab1/Cry35Ab, Cry2Ab) was commercially planted in 16 countries worldwide in 2009 (Table 2), and the total planting area reached 35.3 million hectares (Figure 1). In 2010, Bt maize was grown on 39 million hectares, an increase of 3.0 million hectares, or a year-over-year growth rate of 10% (Figure 1) (James 2010b). After the USA, Brazil is the second largest Bt maize-growing country, with 5 million hectares in 2009 (Marshall 2010). There were seven countries (the USA, Argentina, Canada, the Philippines, South Africa, Honduras, and Chile) planting maize with double-stacked traits for herbicide tolerance and insect resistance. The USA and Canada are the only two countries to grow triple-stack maize with one gene for the European corn borer, a second for root worm, and a third for herbicide tolerance (James 2009). It seems that the growth of biotech maize stacked with double and triple genes versus single genes is typical of the shift in all countries that deploy stacked genes in maize.

Table 2. Global status of commercialized Bacillus thuringiensis (Bt) cotton and Bt maize
CropCountryYear of commercializationInsect toxin genesTotal hectares in 2009
  1. aNo planting from 2000 to 2004; bCry1A represents a fusion gene of Cry1Ac and Cry1Ab.

  2. Sources: Clive James 2009 and genetically-modified crop database.

CottonArgentina1998Cry1Ac245 000
Australia1996, 2002Cry1Ac,Cry1Ac+Cry2Ab180 000
Brazil2005Cry1Ac 
 2009Cry1Ac+Cry2Ab116 000
 2009Cry1Ac+Cry1F 
Burkina Faso2008Cry1Ac+Cry2Ab15 000
China1997Cry1Ac 
 1999Cry1Ab+CpT I3 400 000
Colombia2002Cry1Ac23 000
Costa Rica2009Cry1Ac 
 2009Cry1Ac+Cry2Ab<1 500
 2009Cry1Ac+Cry1F 
India2002Cry1Ac 
 2006Cry1Ac+Cry2Ab8 400 000
 2006Cry1Ac+Cry1Ab 
Mexico1997Cry1Ac46 000
South Africa1997Cry1Ac 
 2005Cry1Ac+Cry2Ab7 300
USA1996Cry1Ac 
 2002Cry1Ac+Cry2Ab1 491 000
 2004Cry1Ac+Cry1F 
 2004Cry1F 
MaizeArgentina1998Cry1Ab 
 2005Cry1F1 960 000
Brazil2008Cry1Ab 
 2009Cry1F5 000 000
 2009VIP3Aa20 
 2009Cry1A.105+Cry2Ab 
Canada1997Cry1Ab 
 2002Cry1F>1 300 000
 2003Cry3Bb1 
 2005Cry34Ab1+Cry35Ab1 
 2007mCry3A 
 2008Cry1A.105+Cry2Ab2 
Chile2007Cry1Ab28 000
Czech Republic2005Cry1Ab6 480
Egypt2008Cry1Ab1 000
Honduras2002Cry1Ab12 000
 2009Cry1F 
The Philippines2003Cry1Ab392 000
Portugala1999Cry1Ab5 094
Poland2007Cry1Ab3 000
Romania2007Cry1Ab3 244
Slovakia2006Cry1Ab875
Spain1998Cry1Ab76 057
South Africa1997Cry1Ab1 600 000
Uruguay2003Cry1Ab 
 2006Cry1F90 000
USA1996Cry1Ab 
 1996Cry3Bb1 
 2001Cry1F 
 2005Cry34Ab1+Cry35Ab1 
 2007mCry3A 
 2008Cry1A.105+Cry2Ab2>17 000 000
image

Figure 1. Global planting area of Bacillus thuringiensis (Bt) cotton, Bt maize, and both, 1996–2010 (million hectare). Source: James 2002–2010.

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Assessment of Insect-Resistant, Genetically-Engineered Plant Effects on NTOs

The assessment of the potential effects of insect-resistant, genetically-engineered (IRGE) plants on NTOs includes two phases, namely premarket risk assessment (PMRA), which is carried out prior to the commercialization of a GE plant, and post-market monitoring (PMM), which is conducted after commercialization of a GE plant (Sanvido et al. 2005, 2009). Approval for the commercial cultivation of a specific transformation event is based on the PMRA, where potential adverse effects of the genetically-modified (GM) plant on the environment are assessed on a case-by-case basis. However, PMRA cannot completely eliminate any uncertainty (Hill and Sendashonga 2003; Levidow 2003; Sanvido et al. 2005). Therefore, a GE plant is, in principle, only approved for limited release after PMRA. Subsequently, PMM should be carried out to cope with the scientific uncertainties inherent to the risk analysis in PMRA before the large-scale release of the GE plant to the market.

Premarket Risk Assessment

Assessment procedure and methods

To assess the effects of IRGE plants on NTOs, problem formulation should first be established by analyzing available information on GE plants (introduced traits, expression pattern, and mode of action of insecticidal proteins), and identifying the potential exposure of any NTO to insecticidal proteins. Problem formulation is used to define the scope of the risk assessment, and to generate testable scientific hypotheses and measured end-points that are relevant for decision-making (Wolt et al. 2010).

For practical reasons, only a small fraction of all possible terrestrial organisms can be considered for regulatory testing. Therefore, to assess the effect of IRGE plants on NTOs, appropriate species should be selected (Dutton et al. 2003; Garcia-Alonso et al. 2006; Romeis et al. 2008). First, the species should represent different ecological functions, such as herbivory, pollination of cultivated and wild plants, predation and parasitism of pest organisms, and decomposition in the soil. In addition, species with special aesthetic or cultural value, or species classified as threatened or endangered, should also be considered for risk assessment (Romeis et al. 2008). Since risk is a function of hazard, and the likelihood that this hazard will be realized, the NTOs will not be affected if they have not had the opportunity to be in contact with the insecticidal protein by the plant (exposure). Thus, the species that is highly exposed to the insecticidal protein and is most likely affected by the protein should be chosen. Finally, practical considerations, including the ease of working with a species, the potential for unambiguous taxonomic recognition, the ability to rear the species in captivity, the availability of permanent source colonies, and validated and accepted test methods, should be considered for species selection (USEPA 2007; Romeis et al. 2008).

Once the surrogate test species are selected, they are evaluated through the tiered testing procedure that has been recommended and well accepted by regulators and risk assessors (EPA 2002; Dutton et al. 2003; Garcia-Alonso et al. 2006; Romeis et al. 2006; USEPA 2007; Romeis et al. 2008). The procedure starts with laboratory tests (lower tier), followed by semifield (glasshouse) and field (higher tier) tests (Figure 2). Lower-tier tests serve to identify potential hazards, and are typically conducted in controlled conditions. Lower-tier tests are designed to measure a specific end-point (or set of end-points) under worst conditions using protein concentrations that are normally 10–100 times higher than those present in plant tissues. In general experiments, typical measurement end-points are mortality, fecundity, development duration, body mass, or the percentage of individuals that reach a certain life stage (Dutton et al. 2003; Romeis et al. 2011). Under these conditions, a lack of adverse effects might provide confidence that there is no risk, and no further data would be needed (Romeis et al. 2011). However, if potential hazards were detected, or if unacceptable uncertainties about possible hazards remain, the higher-tier tests should be conducted that include more complex semifield (i.e. under containment using live GE plant material) or open field tests. These tests can serve to confirm whether an effect can still be detected under more realistic rates and routes of exposure to the protein. Field tier tests should provide more ecological information and answer questions related to the effects observed in laboratory and semifield tests (Dutton et al. 2003). The structure and species diversity of organisms’ communities in general were investigated as measurement end-points (Romeis et al. 2008). In cases where uncertainty about the risk remains after higher-tier studies, one can always return to lower tiers to conduct additional studies. In exceptional cases, higher-tier studies, or studies using alternative designs, might be conducted at the initial stage of the risk assessment process when lower-tier tests are not possible. Movement between tiers takes place either because the available information is insufficient to accept the risk hypothesis of “no effect” or because the results of risk hypothesis have the adverse effects. Where no hazard or risk is detected, effective tiered processes prevent costly and unnecessary testing.

image

Figure 2. Sequential test procedure for assessing the effects of genetically-modified plants on non-target organisms using a tiered scheme.

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Effects on NTOs

Pest predators and parasitoids

The effects of Bt cotton and Bt maize on predators and parasitoids have been extensively assessed, with most studies conducted using tritrophic systems, including plants, herbivores, and natural enemies. In the present study, we summarized the data that were mostly published from 2005 to 2010 (Table 3). From the previous data, it can be found that adverse effects on predators (larval survival, consumption rate, and body mass) were only reported in the studies where Bt-susceptible insects were used as prey (Zhang et al. 2006; Chen et al. 2009; Lawo et al. 2010); no effects were detected when Bt-unsusceptible, or even sublethally-damaged herbivores, were used as prey (Davidson et al. 2006; Obrist et al. 2006; Zhang et al. 2006; Álvarez-Alfageme et al. 2008; Lewandowski and Górecka 2008; Álvarez-Alfageme et al. 2009, 2011; Meissle and Romeis 2009; García et al. 2010; Li and Romeis 2010). Likewise, no negative effects were found when predators were directly fed Bt plant tissues (e.g. maize pollen) (Ferry et al. 2007; Li et al. 2008; Duan et al. 2008b; Meissle and Romeis 2009) (Table 3). In addition, studies feeding predators with high concentrations of purified Cry proteins revealed no direct toxicity to Chrysoperla carnea (Li et al. 2008), Adalia bipunctata (Álvarez-Alfageme et al. 2011), or Orius insidiosus (Duan et al. 2008b). In their study, Schmidt et al. (2009) reported toxicity of Escherichia coli-produced recombinant Cry1Ab and Cry3Bb to first-instar Adalia bipunctata. However, the results of the study have been questioned due to the methodological shortcomings that undermine the study's conclusion, which also prevent the reconstruction of the study (Álvarez-Alfageme et al. 2011). The recent study by Álvarez-Alfageme et al. (2011), using tritrophic and bitrophic experimental systems, clarified that Adalia bipunctata is not sensitive to Cry1Ab and Cry3Bb1, and the detected harmful effects reported by Schmidt et al. (2009) were artifacts of poor study design and procedures. These results, together with earlier data, demonstrate that the negative effects observed were a consequence of sublethally-intoxicated prey due to Bt ingestion being of apparently lower nutritional quality, but was not caused by the direct toxicity of Bt toxins (Romeis et al. 2006).

Table 3. Studies (2005–2010) under confined conditions assessing effects of Bacillus thuringiensis (Bt) plants on insect pest predators and parasitoids
CropToxinPredator/parasitoid speciesPrey/host order/plant tissueIs the host/prey susceptible to the toxin?Reported effectsReference
  1. aIn the presence of the corn leaf aphid. (–), negative effect; (+), positive effect.

Predators feeding on prey reared on Bt plants
 CottonCry1AcPropylaea japonica (Coleoptera: Coccinellidae)LepidopteraYesYes (–)Zhang et al. 2006
 Chrysoperla carnea (Neuroptera: Chrysopidae)LepidopteraYesYes (–)Lawo et al. 2010
   NoNo 
Cry1Ab/cPropylaea japonica (Coleoptera: Coccinellidae)LepidopteraNoNoZhang et al. 2006
 MaizeCry1AbNeoseiulus cucumeris (Acari: Phytoseiidae)AcariNoNoObrist et al. 2006
 Stethorus punctillum (Coleoptera: Coccinellidae)AcariNoNoÁlvarez-Alfageme et al. 2008
 Chrysoperla carnea (Neuroptera: Chrysopidae)HomopteraNoNoLewandowski and Górecka 2008
 Poecilus cupreus (Coleoptera: Carabidae)LepidopteraYesNoÁlvarez-Alfageme et al. 2009
 Adalia bipunctata (Coleoptera: Coccinellidae)AcariNoNoÁlvarez-Alfageme et al. 2011
 Atheta coriaria (Coleoptera: Staphylinidae)AcariNoNoGarcía et al. 2010
Cry3Bb1Theridion impressum (Araneae: Theridiidae)LepidopteraYesNoMeissle and Romeis 2009
  AcariNoNo 
 Adalia bipunctata (Coleoptera: Coccinellidae)AcariNoNoÁlvarez-Alfageme et al. 2011
 Stethorus punctillum (Coleoptera: Coccinellidae)AcariNoNoLi and Romeis 2010
 PotatoCry1Ac9, Cry9Aa2Micromus tasmaniae (Neuroptera: Hemerobiidae)HemipteraNoNoDavidson et al. 2006
 RiceCry1AbPirata subpiraticus (Araneae: Lycosidae)LepidopteraYesYes (–)Chen et al. 2009
Predators directly feeding on Bt plant tissue
 MaizeCry1AbChrysoperla carnea (Neuroptera: Chrysopidae)Pollen NoLi et al. 2008
Cry3Bb1Orius insidiosus (Heteroptera: Anthocoridae)Pollen NoDuan et al. 2008b
 Theridion impressum (Araneae: Theridiidae)pollen NoMeissle and Romeis 2009
 PotatoCry3AHarmonia axyridis (Coleoptera: Coccinellidae),    
 Nebria brevicollis (Coleoptera: Carabidae)Pollen and flower NoFerry et al. 2007
Parasitoids developing in hosts reared on Bt plants
 cottonCry1A+CpTI, Cry1AcCampoketis chlorideae (Hymenoptera: Ichneumonidae)LepidopteraYesYes (–)Liu et al. 2005a
Cry1A+CpTI, Cry1AcMicroplitis mediator (Hymenoptera: Braconidae)LepidopteraYesYes (–)Liu et al. 2005b
 MaizeCry1AbCotesia marginiventris (Hymenoptera: Braconidae)LepidopteraYesYes (–)Vojtech et al. 2005Ramirez-Romero et al. 2007
 Cotesia marginiventris (Hymenoptera: Braconidae)LepidopteraYesYes (+)aFaria et al. 2007
 Campoletis sonorensis (Hymenoptera: Ichneumonidae)LepidopteraYesYes (–)Sanders et al. 2007
 PotatoCry1Ac9, Cry9Aa2Apanteles subandinus (Hymenoptera: Braconidae)LepidopteraYesNoDavidson et al. 2006
 BroccoliCry1Ac, Cry1C, Cry1Ac+Cry1CPteromalus puparum (Hymenoptera: Pteromalidae)LepidopteraYesYes (–)Chen et al. 2008
Cry1AcDiadegma insulare (Hymenoptera: Ichneumonidae)LepidopteraNoNoLiu et al. 2010
 Chinese cabbageCry1AcMicroplitis mediator (Hymenoptera: Braconidae)LepidopteraNoNoKim et al. 2008
Parastoids directly feeding on Bt plant tissue
 Cry1AbTrichogramma ostriniae (Hymenoptera: Trichogrammatidae)Maize pollen NoWang et al. 2007

Adverse effects of Bt crops on survival, the development, and reproduction of some parasitoid species were also observed when Bt-susceptible herbivores were used as hosts in several studies (Liu et al. 2005a,b; Vojtech et al. 2005; reviewed in Romeis et al. 2006; Ramirez-Romero et al. 2007; Sanders et al. 2007; Chen et al. 2008). Likewise, it was subsequently confirmed that the deleterious effects observed on parasitoids were due to the lower quality of hosts caused by Bt toxin ingestion, but not the direct toxicity of Bt toxins (Davidson et al. 2006; Faria et al. 2007; Wang et al. 2007; Chen et al. 2008; Kim et al. 2008; Liu et al. 2010). Since parasitoids have particularly close relationships with their hosts, and often possess a relatively narrow host range, they are more likely than predators to suffer significant negative impacts from GE crops if their Bt-susceptible hosts are treated with Bt toxins and are weakened or killed (Romeis et al. 2006).

Similar to laboratory or glasshouse studies, field surveys (higher-tier tests) did not find convincing and meaningful negative effects on the population density, abundance, species richness, and diversity of natural enemies when transgenic Bt cotton or maize was cultivated, which were assessed using different methods (Lopez et al. 2005; reviewed in Romeis et al. 2006; Chen et al. 2009; Balog et al. 2010). Naranjo et al. (2005) detected minor changes in the abundance of a few non-target taxa occurring with the cultivation of Bt corn and cotton, while almost all these effects were explained by expected changes in target pest populations. A recent meta-analysis suggested that there were no uniform effects of Bt cotton, maize, and potato on the functional guilds of non-target arthropods, but insecticide effects were much greater than those of Bt crops (Wolfenbarger et al. 2008).

Pollinators and butterflies

Pollinators play an important functional role in most terrestrial ecosystems. As the world's most abundant and widespread pollinators, honey bees have drawn much attention, and they were used as indicators for the PMRA of Bt crops (EPA 2001). Feeding tests with Bt plant pollen have been extensively preformed on honey bees, and no effect was observed on their longevity, feeding and learning behavior, development of hypopharyngeal glands, superoxide dimutase activity, and intestinal bacterial communities in most of the studies (Bailey et al. 2005; Babendreier et al. 2005; Liu et al. 2005; Babendreier et al. 2007; Rose et al. 2007; Hofs et al. 2008; Liu et al. 2009a; Han et al. 2010b). Contrarily, Han et al. (2010a) showed that honey bee feeding behavior was disturbed during a 7-d oral exposure to cotton pollen-expressed Cry1Ac+CpTI toxins. However, a lack of long-term exposure did not provide enough evidence to support the results. A recent meta-analysis of 25 independent studies suggested that the Bt proteins used in GE crops to control lepidopteran and coleopteran pests do not negatively impact the survival of larvae or honey bee adults (Duan et al. 2008a). Likewise, no effects were detected on the abundance, diversity, colony activity, and development of honey bees in field surveys (Rose et al. 2007; Hofs et al. 2008).

Butterflies are more than just useful indicator species; they represent some of the most spectacular and visually-appealing organisms in the world, and play many vital roles in ecosystems (Bonrbrake et al. 2010). The observations that pollen from a Bt corn line dusted onto milkweed leaves caused mortality of monarch larvae (Losy et al. 1999) prompted much public interest. Later, more thorough research indicated that the likely effect of Bt maize on the monarch butterfly was negligible because of limited exposure and low toxicity of Bt maize pollen to monarch larvae (Sears et al. 2001; Stanley-Horn et al. 2001; Hellmich et al. 2008), although some adverse effects were observed on mortality, the development, body weight, and larval behavior of butterflies in laboratory or glasshouse experiments where the test insects were artificially exposed to high levels of insecticidal Bt proteins (Mattila et al. 2005; Lang and Vojtech 2006; Prasifka et al. 2007; Perry et al. 2010).

Microorganisms and macroorganisms in soil

Bt toxins from transgenic plants can enter soil in three different ways: (i) plant pollen deposited in and around Bt crop fields during anthesis; (ii) root exudate; and (iii) plant residues after harvest (Heckmann et al. 2006; Li et al. 2007; Vaufleury et al. 2007; Zwahlen et al. 2007). The potential impacts of Bt plants on soil organisms depend, at least in part, on the persistence of the transgenic-derived protein and its biological activity in soil. Research has shown that Bt toxins can bind to clay particles and humic substances from soils, which renders the proteins resistant to biodegradation, but with retention of larvicidal activity (Zwahlen et al. 2003; Clark et al. 2005; Viktorov 2008; Saxena et al. 2010). Most studies have suggested that Bt proteins from transgenic plants break down relatively rapidly in the early stage after entering soil, and that only a small amount of them can remain for long, so that Bt proteins do not bio-accumulate in soil (Ahmad et al. 2005; Li et al. 2007; Icoz and Stotzky 2008a, 2008b; Margarit et al.2008; Rauschen et al. 2008; Shan et al. 2008; Daudu et al. 2009; Zurbrügg et al. 2010).

The effects of Bt crops on soil macroorganisms, including mites, collembola, earthworm, and snails, have been studied. In general, no toxic effects of Cry proteins on macroorganisms have been reported in laboratory and field experiments (Ahmad et al. 2005; Heckmann et al. 2006; Vercesi et al. 2006; Vaufleury et al. 2007; Zwahlen et al. 2007; Hönemann et al. 2008; Hönemann and Nentwig 2009; Liu et al. 2009b; Bai et al. 2010). To our knowledge, only one laboratory study reported that Bt maize has a negative impact on growth and egg hatchability of snails (Kramarz et al. 2009). However, the risk was not well established, due to the lack of certain important information, and additional tests should be conducted. Field investigations suggested that crop management practices and/or environmental conditions (e.g. heavy rainfall during the growing season) and pesticide use had the greatest impact on these species’ diversity and evenness, rather than the crop itself (Bt or isoline) (Birch et al. 2007; Cortet et al. 2007; Griffiths et al. 2007a).

The effects of Bt crops on microbes have been a concern in recent years. A number of studies on the effects of Bt crops on soil microorganisms have failed to find any significant effects in laboratory experiments, in microcosm, and under field conditions (Baumgarte and Tebbe 2005; Griffiths et al. 2005; Shen et al. 2006; Devare et al. 2007; Griffiths et al. 2007b, Knox et al. 2008; Oliveira et al. 2008; Miethling-Graff et al. 2010; Tan et al. 2010). In contrast, a study by Castaldini et al. (2005) reported differences between Bt176 maize and non-Bt maize in rhizospheric eubacterial communities, mycorrhizal colonization, soil respiration, bacterial communities, and mycorrhizal establishment, while the risk was not well established. In general, the conclusion has been drawn that Bt toxins do not directly cause negative effects on microbes, but other factors, such as plant growth stage, duration of plant straw decomposition, plant hybrid, and variety, might have stronger effects on the microorganisms than the presence of the Cry protein.

Aquatic organisms

Although aquatic organisms are not likely to be exposed to the insecticidal Bt proteins through their expressions in crop plants, with the exception of the aerial deposition of pollen or run-off transport of soil-bound Bt residue and crop material, several studies have been conducted to evaluate the potential effects of Bt plants on aquatic organisms (Pokelsek et al. 2007; Swan et al. 2009; Jensen et al. 2010). Bøhn et al. (2008, 2010) showed that the Cry1Ab toxin expressed in maize reduced the fitness performance of Daphnia magna, which is a crustacean (phylum: Arthropoda) invertebrate that inhabits ponds and lakes in most regions of the world. However, given the poor experimental design, the physiological relevance of the findings is questionable; conclusions of adverse effects cannot be drawn from these studies (Monsanto 2010). Rosi-Marshall et al. (2007) reported that the consumption of Cry1Ab expressed in plant parts, such as corn pollen, stalks, and cobs, increased mortality and reduced growth in caddisflies, which are related to the targeted insects, lepidopteran pests of the Cry1Ab protein expressed in Bt corn. However, the study missed important background information on methodology and plant materials used in the study by Beachy et al. (2008), so that the conclusions drawn in the paper seemed premature for speculative ecosystem effects. Chambers et al. (2010) assessed the influence of Bt maize detritus on benthic macro-invertebrate abundance, diversity, biomass, and functional structure in situ in 12 streams adjacent to Bt maize or non-Bt maize fields using combined laboratory and field approaches. There were no significant differences in the total abundance or biomass of benthic macro-invertebrate and trichopterans between Bt and non-Bt streams. These studies demonstrate that further studies with better experimental design are needed for the assessment of the potential effects of Bt crops on aquatic organisms.

Birds and mammals

There have been several articles published since 2005 that describe the impacts of Bt maize on mammalians and birds. Studies were carried out to compare the performances, such as growth rate, weight gain, food intake, feed efficiency, fecundity, and broilers, of the animals feeding on transgenic plant tissues with those feeding on control plant tissues (Aeschbacher et al. 2005; Flachowsky et al. 2005; Rossi et al. 2005; Hammond et al. 2006; MacKenzie et al. 2007; Wiedemann et al. 2007; Finamore et al. 2008; Trabalza-Marinucci et al. 2008). The possible health effects of Bt crops through multigeneration in mammals and poultry were also measured to clarify and shed light on the safety of long-term Bt crop consumption (Flachowsky et al. 2005; Halle et al. 2005; Kilic and Akay 2008). All the current datasets show that Bt plants have no toxicity effects on mammalian development and health, due to the fact that the normal mode of toxic action for Bt proteins is unlikely to occur in the vertebrate digestive systems (Siegel et al. 1987; McClintock et al. 1995; Broderick et al. 2006).

PMM

Assessment procedure and methods

PMM ensures the detection and prevention of adverse effects on the environment, possibly deriving from the commercial cultivation of GM crops, which can be divided into case-specific monitoring (CSM) and general surveillance (GS) (European Community 2001; European Council 2002; European Union 2003). CSM aims to assess the anticipated adverse effects that cannot be identified with certainty in PMRA, but might occur during commercial cultivation. In contrast, GS aims at detecting adverse effects on the environment that were not anticipated during PMRA. Therefore, GS has to be performed in all cases, while CSM might not be required when the conclusions of PMRA identify an absence of risk or negligible risk (European Council 2002). Although there have not been widely-accepted PMM strategies established for GM plant cultivation so far, some articles have described the conceptual frameworks for the design of environmental PMM programs for GM plant cultivation based on current governmental legislation and common risk analysis procedures (Sanvido et al. 2005; Sanvido et al. 2007; Sanvido et al. 2008, 2009). Because GS has to be done in PMM programs, we introduced a general procedure for GS. The procedures of GS have included defining safeguard subjects, collecting reports on adverse incidents via existing surveillance programs and reporting systems on adverse environmental effects, analyzing reports on adverse incidents, evaluating reports on adverse incidents from GS and determining the likelihood with GM plant cultivation, determining possible causalities with GM plants, and making a final decision (Sanvido et al. 2005). For instance, to monitor the non-target effects of GE plants, the general protection goal in monitoring of Bt crops is the biodiversity by using GS (Sanvido et al. 2005). The GS plan might comprise the following elements: (i) farm questionnaires and/or other surveillance approaches; (ii) literature review; (iii) information for operators and farmers; (iv) alert “hotline”; and (v) integration of information from surveillance programs by third parties (Wilhelm 2010).

Effects of Bt Crop on Arthropod Population Dynamics

Bt cotton

A relatively large number of pest species that are not susceptible to the Bt toxins expressed in transgenic cottons affect cotton production worldwide. The sucking pests, including the cotton aphid, thrip, whitefly, leafhopper, and spider mites, are the major non-target pests in Bt cotton fields, which are not susceptible to the Bt proteins currently used (Wu and Guo 2005; Arshad and Suhail 2010; Manna et al. 2010). In general, most of these species exhibit the same pest status and continue to be managed identically in Bt and conventional cotton systems. However, due to the reduced use of insecticides for bollworms and the change of pest management regimes in Bt cotton fields, these secondary pest populations have increased and gradually evolved into key pests in the USA, India, China, Australia, and other countries (Gouse et al. 2004; Sharma et al. 2005; Williams 2006; Wilson et al. 2006; Ho and Xue 2008; Lu et al. 2008; Li et al. 2010; Zhao et al. 2011). For example, in Australia, the green mirid (Creontiades dilutus), green vegetable bug (Nezara viridula), leaf hopper (Austroasca viridigrisea and Amrasca terraereginae), and thrip (Thrips tabaci, Frankliniella schultzei, and Frankliniella occidentalis) have become more prominent (Lei et al. 2003; Wilson et al. 2006). In India, the reduction in insecticide use increased the incidence of sucking and other pests, such as the mired bug, mealy bug, thrip, and leaf-eating caterpillar (Karihaloo and Kumar 2009; Nagrare et al. 2009). Field surveys conducted over 10 years in six major cotton-growing provinces (i.e., Henan, Hebei, Jiangsu, Anhui, Shangdong, and Shanxi) in northern China showed that mirid bugs (Heteroptera: Miridae) have progressively increased and acquired pest status in Bt cotton fields (Lu et al. 2010). In addition, it was also found that spider mites have been observed to occur at higher levels in Bt cotton during the drought season (Wu and Guo 2005). These emergent pests have forced Chinese farmers to continue using chemical pesticides; however, the increase in insecticide use for the control of these secondary insects is lower than the reduction in total insecticide use due to Bt cotton adoption (Wang et al. 2009).

To date, there have been no confirmed monitoring results of the negative effects of Bt cotton on insect pest predators. Based on field investigations, population densities of major predator species, such as predatory spiders, coccinellids, chrysopids, and small flower bugs, in transgenic Bt cotton fields were not different from those in conventional Bt cotton fields, but were significantly greater than those in conventional cotton fields applied with pesticides (Sisterson et al. 2007; Sharma et al. 2007; Dhillon and Sharma 2009). In India, it was found that predator populations (Chrysoperla spp., Orius spp., Coccinella spp., Brumus spp., Vespa spp., Lycosa spp., and Aranews spp.) were similar on Bollgard I, Bollgard II, and conventional cotton (Manna et al. 2010). As expected, the population densities of parasitic wasps (Trichogramma confusum, Microplitis spp., Campoletis chlorideae, and Meteorus pulchricornis) decreased significantly due to poor quality and lower density of Helicoverpa armigera in Bt cotton fields because of the close relationship between parasitoids and their hosts (Wu and Guo 2005; Yang et al. 2005; Xia et al. 2007). A 3-year field survey showed that the planting of Bt cotton increased the diversity of arthropod communities (Men et al. 2003), and the results confirmed Cui et al.'s (2005) study with natural enemy subcommunities. Thus, the biological control function of natural enemies in Bt cotton fields did not change compared with conventional Bt cotton (Naranjo 2005; Wolfenbarger et al. 2008; viewed in Naranjo 2009).

Bt maize

As with Bt cotton, Bt maize did not affect the non-target arthropods at population levels in Bt maize in long-term or short-term monitoring by the sampling methods used, including visual surveillance, sticky cards, pitfall traps, and litterbags (Pons et al. 2005; Eizaguirre et al. 2006; Higgins et al. 2009; Virla et al. 2010). Serious problems with secondary pests stemming from experiences with Bt cotton were not found in Bt maize due to declining insecticide use against target lepidopteran pests, although minor pests species increased in Bt maize in some countries with commercial planting (Hellmich et al. 2008). For example, in Germany, the 6-year monitoring of non-target arthropods in Bt maize (expressing the Cry1Ab toxin) did not find differences in the population densities of aphids, thrips, heteropterans, aphid specific predators, spiders, and carabids (Schorling and Freier 2006). Abundance and species richness of foliage-dwelling spiders (Araneae) were equal or higher in Bt maize fields and adjacent field margins than non-transgenic maize fields (Ludy and Lang 2006). In the USA, transgenic Bt maize also did not affect the community abundance of non-target arthropods, based on a 3-year field investigation (Higgins et al. 2009). Predaceous arthropods were equal or more abundant on Bt than non-Bt maize (Daly and Butin 2005; Eizaguirre et al. 2006). Overall, the studies indicated no major effects against natural enemies in Bt maize fields compared with non-Bt maize fields, with the occasional exception of taxa that were dependent on Bt-susceptible pests as hosts (Eizaguirre et al. 2006; Rose and Dively 2007). Likewise, no effects were found in Cry1Ab and Cry3Bb1 maize on the diversities of macroorganisms and microorganisms in long-term and short-term field studies in the USA (Icoz et al. 2008; Priestley and Brownbridge 2009; Zeilinger et al. 2010). A recent report by Monsanto (2010) confirmed that there is a negligible impact from the cultivation of MON810 expressing Cry1Ab on biodiversity, abundance, or survival of non-target species via the analysis of 240 questionnaires from a survey of farmers cultivating MON810 in six European countries in 2009, and through a detailed analysis of more than 30 publications. It can be concluded that the biodiversity of non-target arthropods was seemingly more easily affected by the agro-ecological system than the Cry toxin (De la Poza et al. 2005; Farinós et al. 2008).

Transgenic Bt Crops and IPM

In summary, recombinant DNA technology provides an efficient tool to develop insect-resistance breeding for certain crops. The technology has enabled the direct insertion of foreign genes that can be derived from any kind of living organism into crop plants, allowing plants to completely express new pest-resistance properties. The use of this approach has resulted in many insect-resistant varieties, and the crops expressing Cry toxins derived from Bt have been planted worldwide. Thus far, the laboratory and field studies conducted have shown that the currently-used Bt crops generally do not cause apparent unexpected detrimental effects on NTOs or their ecological functions, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests due to the reduction of pesticide use. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application, and clear benefits on environment and farmer health have been reported. Consequently, Bt crops can be a useful component of IPM systems to protect the crop from targeted pests. In fact, Bt cotton and Bt maize have revolutionized pest control strategies in a number of countries and have changed conventional IPM practices. Certainly, the sole use of Bt crops cannot solve all of the problems related to pest regulation; it needs to be used with other IPM tactics, including chemical pesticides, for controlling pests. For example, to control secondary pests, such as mirids and spider mites, in Bt cotton, chemical control, especially the use of more specific, less disruptive compounds, remains important, together with the use of other IPM tactics, such as crop rotation and intercropping. As an important component of IMP, the ideal use of Bt crops would include reduction of insecticide use with maintenance of other traditional IPM practices.

(Co-Editor: Weicai Yang)

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Acknowledgements
  5. References

We would like to thank Dr Jorg Romeis for providing valuable literature for use in this study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Acknowledgements
  5. References
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