THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE
ISIS Report 31/10/11
Bt Resistant Rootworm Spreads
The emergence of more Bt-resistant pests is further proof of the futility of Bt crops Dr Eva Sirinathsinghji
A fully referenced version of this report is posted on ISIS (Institute of Science in Society) members website.
Bt is a toxin from the soil bacterium Bacillus thuringiensis, which produces a large family of similar proteins that target different insect pests; and quite a few of them have been incorporated in genetically modified crops to act as ‘biopesticides’. Unfortunately, the pests soon develop resistance to it.
Bt resistance has not only been documented in the laboratory, but also in the wild, with at least 8 populations of insects have developed resistance, with 2 populations resistant to Bt sprays and at least 6 species resistant to Bt crops [1-10].
The emergence of resistance has pushed GM scientists to attempt new strategies of delaying its spread. New strategies include the genetic modification of the Bt toxin to kill pests that have already acquired resistance. Bruce Tabashnik and his colleagues at the University of Arizona, along with collaborators in Mexico, China and Germany, published a study in Nature Biotechnology this month showing that these modified toxins bypass their usual interaction with cadherin, a receptor in target insects that binds the Bt toxin in the first of a multi-step process causing bursting of cells in the insect gut . As some Bt resistant insects have been found to carry mutations in the cadherin receptor, they hypothesised that making Bt toxicity independent of cadherin binding would make pests susceptible to Bt once again. What they found however, was the opposite. Modified Bt toxin provided ‘little or no advantage’ against pests with cadherin mutations, while increasing Bt potency in pests where resistance was independent of cadherin mutations. The agrobiotech business Pioneer has significantly invested in these modified toxins despite the authors conclusions that ‘insects can probably adapt to modified Bt toxins used alone, or in combinations with other toxins’. This study exposes the lack of scientific understanding of Bt resistance as well as our inability to control it. As researchers search for ways of delaying resistance, resistance is evolving in the fields.
Resistant rootworms in Iowa fields
The first evidence of Bt-resistant western rootworm in the wild has been reported by a team of scientists in Iowa State University . Bt resistant pests have been emerging over the last few years, but as many scientists had warned, evidence now suggests that their resistance might not be recessive, i.e. need two copies of the Bt resistance gene to survive Bt crops. Instead, only one copy will do. This is hugely significant in terms of controlling the spread of Bt resistance through the pest populations. It also diminishes the efficacy of natural Bt toxin sprays used by organic farmers for pest control.
Following reports by farmers that their Bt maize fields (containing the
Cry3Bb1 toxin) were showing signs of severe rootworm injury, Aaron Gassmann and his colleagues at Iowa State University decided to investigate the possibility of resistance to Bt toxin evolving in these pests. Their research is important, particularly in Iowa where the western rootworm is abundant. Further reports of Bt resistance in neighbouring Minnesota has also been documented .
To assess whether the rootworms found in the damaged fields were resistant to Bt toxin, the researchers performed survival bioassays. This was done first by collecting samples of rootworm from damaged Bt maize fields as well as healthy Bt and non Bt-maize fields as controls. These sample populations of adult beetles were maintained in the lab, allowed to lay eggs, and the newly emerged larvae transferred to Bt maize producing Cry3Bb1 toxin and other maize varieties. The numbers surviving after 17 days were recorded, at which point they would have completed the larval developmental stage. The survival rates of larvae collected from problem fields averaged 3 times that of larvae from healthy fields. Furthermore, there was significant positive correlation between the numbers of years Cry3Bb1 Bt maize had been grown. All the problem fields had grown Cry3Bb1 Bt maize for at least 3 years. This is the first detection of Bt resistance in one of North America’s most destructive maize pests. Based on the speed with which resistance had evolved, the scientists speculate that the resistance in these fields was due to non-recessive genes, and/or the fact that 50 percent of farmers in the US are not complying with the requirement to cultivate adjacent non-Bt maize fields as refuges, which is intended to slow down the evolution of resistance.
Rootworm resistance to Cry3B1 toxin only
Bt crops have been created to express one or more Cry toxins. There are 54 known Cry toxins produced by various strains of Bacillus thuringiensis
(Bt) bacteria, each differing in their DNA sequence as well as the type of insect they target. Cry1A and Cry2A toxins are effective in targeting Lepidoptera (moths and butterflies) including the cotton bollworm and the European cornborer, while Cry3Bb toxins, grown in the Iowa fields that were analysed, target coleopteran (beetles) such as the corn rootworm. The toxins work through binding to cadherin proteins, on the cell surface of the insect midgut, leading to lysis of the cells and death of the insect. The effectiveness of these toxins depends on the susceptibility of targeted insects.
Insect adaptation to Bt toxins is expected, even by Monsanto
With Bt crops, high selection pressures are being placed on target insects to adapt, especially considering their widespread cultivation. Resistance is not a controversial issue, but an acknowledged evolutionary process. Even Monsanto stated that  “resistance is natural and expected, so measures to delay resistance are important.”
Previous findings of Bt resistance across the globe
This is not the first report of resistance to Bt toxins, although it took a few years for reports to emerge. It can be expected that resistance takes a few years to develop, and now we are beginning to see evidence of that. In 2009, cotton bollworm in four states of India devastated cotton crops, which was acknowledged by Monsanto . Field studies in Northern China and Australia have also documented resistance to Bt cotton crops in 2010 [14, 15].
Bad science has led to non-recessive resistance
As shown in the study in Iowa , current insect management systems are not successful in controlling resistance. Such adopted strategies include the ‘high-dose/refuge strategy’ where doses of Bt toxin are expressed at
25 times the level required to kill 99 percent of susceptible pests. This high dose is designed to kill any heterozygote insects (with one copy of resistance gene) that have partial resistance, thereby making the resistant trait functionally recessive. By concomitantly cultivating a high-dose Bt crop next to a non-Bt crop refuge, resistant pests from the Bt fields can breed with susceptible pests living on the refuge, resulting in susceptible heterozygote offspring.
The success of the high-dose/refuge strategy depends on the size of the refuge and most critically, the resistant gene being recessive. If dominant resistance develops, then a refuge is ineffective in delaying it from spreading, as heterozygotes will be resistant and therefore the trait will spread more rapidly through the population. One may even argue that a refuge is counter-effective with dominant resistance, as the refuge may provide more potential breeding mates when initial numbers of resistant insects is low in the population. It is hard to determine the soundness of this strategy, as little long-term field studies have been performed to test the hypothesis.
Experiments performed by Monsanto and independent scientists showed that the dose of the Cry3Bb1 maize, which was released in 2003, is not high enough to make resistance functionally recessive . In fact, around half of susceptible larvae are able to survive on this plant. This was known before the release of the crop, and scientists recommended that the EPA impose a 50 percent refuge strategy to try and reduce selection pressure for resistance to develop. But the EPA followed Monsanto’s recommendation and implemented a 20 percent refuge as compulsory with Bt Cry3Bb maize lines, making the crop more economically viable for Monsanto , but not for farmers. Further, resistance to Bt crops may well be exacerbated by the documented variability in expression of Bt toxins throughout their lifetime, as well as in different parts of the plant. Low levels of expression allow partially resistant insects to survive. Bt crops also have prolonged expression of the Bt transgene, which increases selection pressure on pests to adapt. This is in contrast to Bt sprays that degrade in the sunlight and can be applied only when necessary.
Other evidence for dominant resistance in rootworm
Potential for dominant resistance to Bt toxin was shown back in 1998 in laboratory experiments with the European corn borer. Corn borers showed partial dominant resistance to the Bt toxin spray Dipel ES . More recently, a lab study analysed rootworm resistance to Cry3Bb1 Bt maize, the same Bt crop studied by Gassmann’s group. Increased survival of rootworm developed over just three generations, and resistance was not recessive. Survival for resistant rootworms was 11.7 times that of larvae that had not been exposed to Bt maize after 6 generations . Genetic experiments on field-evolved resistant pests will need to be done to confirm the mode of inheritance in the wild.
Delays in the emergence of resistance is expected
Although industry and GM crop proponents are claiming that the lack of documented resistance to date is proof that their strategies were working, the delay in resistance seen until now can be explained by the fact that there are a number of Bt toxins expressed in different crops, and cross resistance appears to be low, even though it is possible that pests could develop a devastating resistance to all Bt toxins. Broad-spectrum resistance to Bt toxins has indeed been documented in lab studies of the cotton bollworm, but such cases are rare . Another factor is the spraying of insecticides on refuge sites as well as Bt crops. This practice has been encouraged by regulators and actually diminishes the whole rationale for using Bt crops in the first place, but may have killed off any resistant pests that were developing in the fields (see  No Bt resistance? SiS 20).
New industry strategies to combat resistance are futile
As it appears that even Monsanto expects resistance to develop at some point, newer GM crops have now been commercialised that express more than one toxin, taking advantage of the low level of cross-resistance observed in pests. Second generation GM cotton Bollgard II express both Cry1Ac and Cry2Ab, whereas the first generation Bollgard expressed only Cry1Ac. In Australia, Bollgard II was released in 2004/2005 season. However, resistance to Bollgard II has already been reported in Australian fields . The latest Smartstax varieties have 8 GM traits ‘stacked’ together, 6 for insect resistance and 2 for herbicide tolerance (see  SmartStax Maize a Medley of Transgenes with Problems, SiS 46). It is a matter of time before resistance to multiple toxins will emerge.
Bt crops are fast becoming futile. They do not reduce pesticide use, as they are not always toxic enough to kill pests, and now resistant populations are emerging in numerous continents. Alternative organic, sustainable methods of farming provide a realistic alternative, independent of reliance on agrobiotech corporations (see  Food Futures Now: *Organic *Sustainable *Fossil Fuel Free, ISIS publication).
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