[font face=Serif][font size=5]Insect resistance to Bt crops: lessons from the first billion acres[/font]

Bruce E Tabashnik¹, Thierry Brévault² & Yves Carrière¹

[font size=4]Evolution of resistance in pests can reduce the effectiveness of insecticidal proteins from Bacillus thuringiensis (Bt) produced by transgenic crops. We analyzed results of 77 studies from five continents reporting field monitoring data for resistance to Bt crops, empirical evaluation of factors affecting resistance or both. Although most pest populations remained susceptible, reduced efficacy of Bt crops caused by field-evolved resistance has been reported now for some populations of 5 of 13 major pest species examined, compared with resistant populations of only one pest species in 2005. Field outcomes support theoretical predictions that factors delaying resistance include recessive inheritance of resistance, low initial frequency of resistance alleles, abundant refuges of non-Bt host plants and two-toxin Bt crops deployed separately from one-toxin Bt crops. The results imply that proactive evaluation of the inheritance and initial frequency of resistance are useful for predicting the risk of resistance and improving strategies to sustain the effectiveness of Bt crops.[/font]

[font size=3]Transgenic crops are one of the most widespread and controversial applications of biotechnology^1–4. To reduce reliance on insecticide sprays, scientists have genetically engineered corn and cotton plants to make insecticidal proteins encoded by genes from the common bacterium Bacillus thuringiensis (Bt)⁵. These Bt proteins kill some devastating insect pests, but cause little or no harm to most other organisms, including people^4,5. Benefits of Bt crops include reduced insecticide use, pest suppression, conservation of beneficial natural enemies, increased yield and higher farmer profits^6-12. The area planted with Bt crops worldwide increased from 1.1 million hectares in 1996 to 66 million hectares in 2011, with a cumulative total of more than 420 mil- lion hectares (>1 billion acres) (Fig. 1). Bt corn accounted for 67% of corn planted in the United States during 2012 (http://www.ers.usda.gov/Data/BiotechCrops/) and Bt cotton accounted for 79–95% of cotton planted in Australia, China, India and the United States during 2010 to 2012 (Fig. 2).

The remarkable ability of insects to adapt to insecticides and other control tactics supports the conclusion that evolution of resistance by pests is the main threat to the continued success of Bt crops^13-23. Many previous reviews have addressed pest resistance to Bt crops^13-23, including a 2011 mini-review emphasizing four successful cases of the high-dose?refuge resistance management strategy in North America²² and our 2009 review of 17 cases involving 11 species of lepidopteran pests and four Bt toxins (B.E.T., Van Renburg, J.B.J. & Y.C.)²¹. Several papers have compared field outcomes for resistance to Bt crops with predictions from theory, but the rigor of these previous comparisons has been limited by small sample sizes for both the field outcomes and the factors predicted to affect resistance^19-22.

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[font face=Serif][center][font size=5]Current Opinion in Insect Science[/font]

Volume 15, June 2016, Pages 111–115

Pests and resistance * Behavioural ecology[/center]

[font size=5]Resistance to Bt maize by western corn rootworm: insights from the laboratory and the field[/font]

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[font size=4]Abstract[/font]

[font size=3]Western corn rootworm is a serious pest of maize. Beginning in 2003, management of western corn rootworm included transgenic maize that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). The first Bt maize hybrids produced Cry3Bb1, but additional Bt toxins have since been introduced, including eCry3.1Ab, mCry3A and Cry34/35Ab1. Laboratory selection experiments found that western corn rootworm could develop resistance to all types of Bt maize following three to seven generations of selection. By 2009 cases of field-evolved resistance to Cry3Bb1 maize had been identified, with populations also showing cross-resistance to mCry3A maize. Factors likely contributing to resistance were the lack of a high dose of Bt toxin for maize targeting rootworm and minimal fitness costs of resistance.[/font]

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[font size=4]Field-evolved resistance[/font]

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Initial cases of field-evolved resistance displayed a common pattern of association with continuous maize cultivation and continuous planting of Cry3Bb1 maize |21 and 25•|. However, over time, pest dispersal also appeared to become important because some fields with Cry3Bb1 resistance sampled in Iowa during 2012 had only been planted to maize for 2 years |5•|. Additionally, while Cry3Bb1 maize was the primary type of maize grown in fields where resistance was detected, resistance also was found to mCry3A maize, implying cross-resistance between mCry3A and Cry3Bb1 |5• and 25•|. The high degree of structural similarity between these Bt toxins suggests a similar mode of action for mCry3A and Cry3Bb1, which are both three-domain Bt toxins, and this likely facilitated the development of cross-resistance |5•, 26 and 27|.

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[font size=4]Additional considerations[/font]

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Structural similarities between Bt toxins may have contributed to cross-resistance between Cry3Bb1 maize and mCry3A maize |5• and 27|. Thus, cross-resistance may extend to additional three domain Bt toxins that possess structural homologies to Cry3Bb1 and mCry3A. Additionally, pyramiding of Cry34/35Ab1 with either mCry3A or Cry3Bb1 to manage Cry3-resistant populations of western corn rootworm will place intense selection on pest populations to develop resistance to the structurally dissimilar Cry34/35Ab1 toxin, for which cross-resistance has not been detected [5•]. The use of RNA interference (RNAi) likely represents the next transgenic approach for management of western corn rootworm |38|. To the extent that RNAi will be pyramided with Bt traits for management of corn rootworm, it remains unclear to what degree past selection for resistance to Bt toxins may complicate future management of western corn rootworm.

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[font face=Serif][font size=5]Bt in organic farming and GM crops - the difference[/font]

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The bt bacteria, commercially available for organic farming is a preparation of weakened or most often dead bacteria, which is sprayed only in the case of high insect infestation and only onto the affected area.

The bacterium inside the spray contains the pro-form of the so called bt toxin. This is not an active component, it needs to be tailored (cut to size) to produce the active bt toxin, which is effective as a pesticide.

When the insect eats the dead bacterium, the toxin is partially digested in the insect gut by proteolytic (cutting) enzymes and converted to active bt toxin. This is actually a lectin which binds to the gut wall of the insects and this interferes with the digestion/absorption of food, thereby preventing growth, maturation, reproduction.

The actual bacterium, which is not eaten by any insects, degrades in the light/sun/rain pretty fast (less than a day). The chances of pests developing resistance to it are very low indeed, since all the pests which are exposed to the toxin are affected by it.

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[font face=Serif][center][font size=5]The International Journal of Biochemistry & Cell Biology[/font]

Volume 78, September 2016, Pages 106–115[/center]

[font size=5]Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases[/font]

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[font size=4]Abstract[/font]

[font size=3]The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-? and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-?B) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-? abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-?B pathways.

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[font size=4]4. Discussion[/font]

[font size=3]Cry1Ac protein has been used extensively as a biopesticide (Sansinenea, 2012), and it is expressed as a truncated toxin in transgenic plants for human and animal consumption. However, its effects in mammals have not been fully evaluated, and efforts have been centred around evaluating safety and potential toxicity (Rubio-Infante and Moreno-Fierros, 2015). Our research group has previously reported that Cry1Ac is immunogenic (Guerrero et al., 2004). In this work, we show for the first time that Cry1Ac toxin is able to induce macrophage activation by promoting the upregulation of the costimulatory molecules CD80 and CD86 and inducing overproduction of the proinflammatory cytokines TNF-? and IL-6, the MCP-1 chemokine and nitric oxide. Moreover, it was demonstrated that stimulation with Cry1Ac in RAW264.7 cells induced the phosphorylation of ERK1/2, p38 and JNK kinases and promoted the nuclear translocation of the p50 and p65 NF-?B subunits. Furthermore, our data indicate the participation of the MAPK pathways in Cry1Ac-induced macrophage activation, as the upregulation of CD80 and the production of cytokines were significantly abrogated by the p38 inhibitor SB203580, while the MEK1/2 MAPK inhibitor PD98059 increased the production of MCP-1.

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Although in the present work the Cry1Ac-induced activation of MAPK induced in macrophages was related to proinflammatory responses, ERK1/2, JNK and p38 kinases are enzymes that also modulate various cellular events and responses to physical and chemical stress and are involved in growth processes, stress, differentiation, inflammation and apoptosis (Arthur and Ley, 2013 and Plotnikov et al., 2011).

Altogether, the present findings indicate the capacity of Cry1Ac toxin to activate macrophages via MAPK pathways, contributing to our understanding of the molecular bases underlying its immunostimulatory mechanisms. However, our results also urge the need for a deeper evaluation in further studies, to determine whether MAPKs activation might be also provoked in other cellular types particularly at the intestinal mucosa. We consider it important because GM crops producing these toxins are currently considered safe (McClintock and Schaffer, 1995 and Rubio-Infante and Moreno-Fierros, 2016), and in particular, Cry1Ac is currently expressed in GM plants such as maize and eggplant, which are used for human consumption. Furthermore, exposure to Bt toxins is also expected to occur through food contamination. We assume that the food consumed by the mice used in the present study did not contain significant traces of Cry1Ac derived from transgenic plants because the sera from non-immunized mice did not exhibit significant levels of anti-Cry1Ac antibodies (data not shown), and we know that these proteins are highly immunogenic when administered via mucosal routes (Guerrero et al., 2004).

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