Implications of transgenic, insecticidal plants for insect and plant biodiversity

Genetically modified plants are widely grown predominantly in North America and to a lesser extent in Australia, Argentina and China but their regions of production are expected to spread soon beyond these limited areas also reaching Europe where great controversy over the application of gene technology in agriculture persists. Currently, several cultivars of eight major crop plants are commercially available including canola, corn, cotton, potato, soybean, sugar beet, tobacco and tomato, but many more plants with new and combined multiple traits are close to registration. While currently agronomic traits (herbicide resistance, insect resistance) dominate, traits conferring “quality” traits (altered oil compositions, protein and starch contents) will begin to dominate within the next years. However, economically the most promising future lies in the development and marketing of crop plants expressing pharmaceutical or “nutraceuticals” (functional foods), and plants that express a number of different genes. From this it is clear that future agricultural and, ultimately, also natural ecosystems will be challenged by the large-scale introduction of entirely novel genes and gene products in new combinations at high frequencies all of which will have unknown impacts on their associated complex of non-target organisms, i.e. all organisms that are not targeted by the insecticidal protein. In times of severe global decline of biodiversity, pro-active precaution is necessary and careful consideration of the likely expected effects of transgenic plants on biodiversity of plants and insects is mandatory.In this paper possible implications of non-target effects for insect and plant biodiversity are discussed and a case example of such non-target effects is presented. In a multiple year research project, tritrophic and bitrophic effects of transgenic corn, expressing the gene from Bacillus thuringiensis (Bt-corn) that codes for the high expression of an insecticidal toxin (Cry1Ab), on the natural enemy species, Chrysoperla carnea (the green lacewing), was investigated. In these laboratory trials, we found prey-mediated effects of transgenic Bt-corn causing significantly higher mortality of C. carnea larvae. In further laboratory trials, we confirmed that the route of exposure (fed directly or via a herbivorous prey) and the origin of the Bt (from transgenic plants or incorporated into artificial diet) strongly influenced the degree of mortality. In choice feeding trials where C. carnea could choose between Spodoptera littoralis fed transgenic Bt-corn and S. littoralis fed non-transgenic corn, larger instars showed a significant preference for S. littoralis fed non-transgenic corn while this was not the case when the choice was between Bt- and isogenic corn fed aphids. Field implications of these findings could be multifold but will be difficult to assess because they interfere in very intricate ways with complex ecosystem processes that we still know only very little about. The future challenge in pest management will be to explore how transgenic plants can be incorporated as safe and effective components of IPM systems and what gene technology can contribute to the needs of a modern sustainable agriculture that avoids or reduces adverse impacts on biodiversity? For mainly economically motivated resistance management purposes, constitutive high expression of Bt-toxins in transgenic plants is promoted seeking to kill almost 100% of all susceptible (and if possible heterozygote resistant) target pest insects. However, for pest management this is usually not necessary. Control at or below an established economic injury level is sufficient for most pests and cropping systems. It is proposed that partially or moderately resistant plants expressing quantitative rather than single gene traits and affecting the target pest sub-lethally may provide a more meaningful contribution of agricultural biotechnology to modern sustainable agriculture. Some examples of such plants produced through conventional breeding are presented. Non-target effects may be less severe allowing for better incorporation of these plants into IPM or biological control programs using multiple control strategies, thereby, also reducing selection pressure for pest resistance development.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.     

Characterization of cell lines developed from the colorado potato beetle, Leptinotarsa decemlineata say (coleoptera: Chrysomelidae)

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One, Trident, MYX-1806, Teknar-HPD, and Thuricide) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.     

Survival and fecundity of Bt-susceptible Colorado potato beetle adults after consumption of transgenic potato containing Bacillus thuringiensis subsp. tenebrionis Cry3A toxin

Survival and fecundity of Colorado potato beetle adults, Leptinotarsa decemlineata (Say), that had or had not fed previously on non-transgenic potato before exposure to transgenic potato containing the Bacillus thuringiensis subsp. tenebrionis Cry3A toxin (Bt) was investigated. In the laboratory, < 5% of first-generation adults survived after two weeks when restricted to Bt foliage since eclosion, but over 85% of adults that had fed initially on non-Bt potato survived exposure to Bt potato for two weeks. In field experiments, less than 0.5% of adults that were exclusively provided Bt potato plants survived overwinter, whereas 44% to 57% survived overwinter when fed non-Bt potato plants for two weeks before being provided Bt potato as a final pre-overwintering host. Survival through the winter increased as the duration of initial feeding on non-Bt potato increased and was similar for beetles provided either tubers or Bt potato plants as a final pre-overwintering host. Only overwintered beetles that fed initially on non-Bt potato before encountering either tubers or Bt potato as a final pre-overwintering host laid eggs the following spring. Survival and reproduction of potato beetle adults after colonizing Bt potato fields should not be adversely affected as long as they have had sufficient time to feed initially on non-Bt potato. Implications for how potato production practices in the Mid-Atlantic US may affect the utility of general resistance management plans for Bt potato are discussed.     

Managing Insect Resistance to Plants Producing Bacillus thuringiensis Toxins

ABSTRACT:?

Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance to B. thuringiensis insecticidal proteins and related aspects, including the development of insect-resistant transgenic plants, B. thuringiensis toxins, their mode of action, mechanisms, stability, and genetics of resistance and management strategies for delaying resistance.     

Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae

Commercial biopesticides based on the fungal pathogen Beauveria bassiana strain GHA and the bacterial pathogen Bacillus thuringiensis tenebrionis were applied alone and in combination (tank mixed) against larval populations of the Colorado potato beetle, Leptinotarsa decemlineata, in small plots of potatoes over three field seasons. Interactions between the two products were evaluated in terms of pest-control efficacy. B. bassiana (formulated as Mycotrol) was applied at low and medium label rates of 1.25 and 2.5 x 10(13) conidia/ha, and B. thuringienis (formulated as Novodor) was applied at low and high label rates of 40.3 and 120.8 x 10(6) Leptinotarsa units/ha. Two weekly applications of the bacterial pesticide alone provided 50-85% control of beetle larvae within 14 days after the initial application, while applications of the mycopesticide alone produced no greater than 25% control. Maximum control, in nearly all tests, was produced by the combination of the two products. The combined treatments produced a statistically significant 6-35% greater reduction in larval populations than would have been predicted had the two biopesticides acted independently. This low-level synergistic interaction was observed during all field seasons and resulted from combinations at all rates, including, in one of two tests, the low rates of each product. These results indicate that B. thuringiensis and B. bassiana have strong potential for integrated biologically based management of Colorado potato beetle.     

Predation on Colorado Potato Beetle Eggs by Generalist Predators in Research and Commercial Potato Plantings

Field studies quantified predation on Colorado potato beetle [Leptinotarsa decemlineata(Say)] eggs and determined the relationship between predation and egg mass abundance in research and commercial potato plantings in eastern North Carolina. Predator exclusion experiments were conducted weekly in research plantings. In addition, egg mass density and predation on egg masses were monitored throughout the season in research plots and commercial potato fields. Predation was an important source of mortality for Colorado potato beetle eggs. Survivorship of eggs exposed to predators was consistently, significantly lower than survivorship of eggs protected from predation. Averaged over 2 years, the mean survivorship of eggs protected from predation was 69%, compared with 26% survivorship of eggs exposed to predation. Regression analysis failed to detect any relationship between egg mortality due to predation and egg abundance. These results imply that efforts to reduce Colorado potato beetle populations selectively will not be offset by an according decline in abundance of natural enemies and therefore should be fully compatible with naturally occurring biological control.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin-receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.     

An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor

Bacillus thuringiensis insecticidal proteins toxic action relies on the interaction with receptor molecules on insect midgut target cells. Here, we describe an ADAM metalloprotease as a novel type of B. thuringiensis toxin receptor on the basis of the following data: (i) by ligand blot and N-terminal analysis, we detected a Colorado potato beetle Cry3Aa toxin binding molecule that shares homology with an ADAM10 metalloprotease; (ii) Colorado potato beetle brush border membrane vesicles display ADAM activity since it cleaves an ADAM fluorogenic substrate; (iii) Cry3Aa acts as a competitor of the cleavage of the ADAM fluorogenic substrate; (iv) Cry3Aa sequence contains the recognition motif R(345)FQPGYYGND(354) present in ADAM10 substrates. Accordingly, a peptide representative of the recognition motif localized within loop 1 of Cry3Aa domain II (Ac-F(341)HTRFQPGYYGNDSFN(358)-NH(2)) effectively prevented Cry3Aa proteolytic processing and nearly abolished pore formation, evidencing the functional significance of the Cry3Aa-ADAM interaction in relation to this toxin mode of action.     

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program. This revised version was published online in November 2006 with corrections to the Cover Date.     

Behavioral Response of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) to Cotton with and Without Expression of the CrylAc δ-Endotoxin Protein of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Berliner

Behavioral responses of larvae and adults of cotton bollworm, Helicoverpa armigera to cotton with (GK-12) and without (SI-3) expression of the CrylAc -endotoxin protein of Bacillus thuringiensis (Bt) Berliner were observed during 2001 and 2002. Our results showed that 8.3% individuals fed with flowers and bolls of GK-12 could develop from neonate to pupa; however, pupal weight decreased by 48.6% and duration of development was delayed by 7.6 days compared with those fed with flowers–bolls of SI-3. Deterrence index (DI) of larvae decreased in later instars, which indicated that the Bt toxin decreased with age. Feeding frequency of 4th-instar larvae on GK-12 leaves decreased by 38.8%, but movement frequency increased by 37.1%. Larvae moved at least one plant away by the age of 10 days in both pure and mixed plantings of SI-3 and GK-12 in the field. Adults preferred to lay eggs on SI-3. The total number of eggs deposited on SI-3 plants in 3 days were about 232 and 95% greater than that on GK-12 plants at bud–flower stage and flower–boll stage, respectively. Based on the behavior of larva and adults in response to the transgenic cotton, the potential effect of refuge strategy in resistance management of H. armigera is discussed.     

Responses of production and storage walnut pests to Bacillus thuringiensis insecticidal crystal protein fragments

Recent advances in genetic engineering have provided the opportunity to induce walnut plants to produce Bacillus thuringiensis Berliner insecticidal crystal protein fragments (ICPFs) for insect control. We studied the effects of two ICPFs CryIA(b) and CrylA(c) previously shown to be encoded by the cryIA(b) and cryIA(c) genes in the B. thuringiensis strains HD-1 and HD-73, respectively. The lethal effects on larvae of codling moth, Cydia pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest pest Indianmeal moth, Plodia interpunctella (Hübner), were investigated. Both proteins were toxic to the three species tested. Indianmeal moth larvae were the most susceptible and navel orangeworm the least; CryIA(b) was generally more toxic to navel orangeworm. Similar relationships resulted when ICPFs were incorporated into the diet. Both ICPFs caused decreased rate of development of navel orangeworm. Effects on pupal weight occurred only at the highest concentration (100 ng/cm²). Neither ICPF affected frequency of mating or fecundity. In addition to the lethal effects, the extended development times observed could have considerable effects on the population dynamics of the navel orangeworm and possibly other species.     

Generation and Analysis of Soybean Plastid Transformants Expressing <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1Ab Protoxin

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis).     

麦套夏播转Bt基因棉R93-6对昆虫群落的影响

以转Bt(Bacillusthuringiensis)基因棉品系R93-6为试验材料,以中棉所16号为对照,研究了在麦套夏播条件下转基因棉对昆虫群落的影响。结果表明,转基因棉田昆虫群落、害虫和天敌亚群落的多样性指数和均匀度指数均低于常规棉田,而优势集中性则高于常规棉田,所以转基因棉田昆虫群落、害虫和天敌亚群落的稳定性不如常规棉田,某种害虫大发生的可能性较大。对季节性变化格局的研究表明,转基因棉田昆虫群落可以划分为前期(6月初至7月下旬)、中期(7月底至8月底)和后期(9月份以后)三个发展阶段,根据不同阶段害虫和天敌发生的特点, 提出了害虫综合治理的策略。即前期害虫的防治应以生物生态调控为主;中期以化学防治为主,以生物生态调控为辅,协调好生物防治和化学防治的矛盾;后期应以生物生态调控为主,并加强农业防治。