Beyond Bt crops

C&I Issue 5, 2012

Insect-protected transgenic crops, also known as Bt crops, have been grown commercially for more than 15 years and, in 2010, global production exceeded 58m ha. This is the fastest adoption of any crop technology in recent years and uptake has been driven by the economic and environmental benefits experienced by farmers growing transgenic crops in both industrial and developing countries.

However, widespread cultivation of Bt crops has been hailed as one of the world’s largest pressures for resistance selection by pests to overcome the insecticidal effect in the field. While Bt technology has succeeded beyond expectation, and has surpassed the typical predicted lifespan in the field before resistance to most conventional neurotoxic pesticides emerges, increasing numbers of reports of Bt resistant target pests have nevertheless begun to appear. Examples of resistance have been documented, for example, in pests including cereal stem borer, fall armyworm, various bollworm species and, most recently, in western corn rootworm.

Bt technology

Bt crops express insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) that produces a diverse group of insecticidal protein toxins with narrow specificity towards different insects. These toxins, called Crystal (Cry) and Cytolitic (Cyt) proteins, accumulate in crystalline inclusion bodies in the bacteria. Cry proteins kill insects by binding to specific target sites in the pest’s gut, where they disrupt membrane integrity.

More than 200 have been identified and are classified based on primary sequence similarity into 54 types (Cry1– Cry54) and many sub-types, for example, Cry1Aa and Cry1Ba. They are active against a limited number of insects, including butterflies and moths; coleopterans, such as beetles and weevils; dipterans, including flies and mosquitoes; and against nematodes or roundworms. Despite the large number of known Cry toxins, only a dozen – Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D, Cry1E, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B and Cry34/Cry35 – are used commercially as sprays or in Bt crops.

Cry proteins are pro-toxins that need to be activated in the insect gut, and critical to this step is an alkaline environment and specific protease enzymes, which cleave the innocuous pro-toxin into its active form. Bt toxins have been extensively studied and consist of three domains of which domains II and III determine the insect specificity and interact with specific receptors located on the insect mid-gut surface.

This binding catalyses clustering of the toxin molecules into a pre-pore structure that can insert into the host membrane. Domain I then facilitates insertion into the target membrane to form a pore through the membrane, whereupon leakage destroys the cells and kills the insect. Generally the toxin needs to be expressed at concentrations of more than 0.2% of total soluble protein in the appropriate tissue in a transgenic plant to be effective.

Insect resistance

Pest species have evolved various ways to resist Bt technology to overcome this toxicity. To date, the low incidence of observed resistance to Bt crops has been ascribed to the fitness cost to resistant individuals, especially those obtained in laboratory studies, which are not able to survive in the field; the low frequency of resistance mutations; dilution of resistant mutations through mating with insects that have fed on non-Bt crops or that have not yet developed resistance; and the high toxin dose that is delivered by commercialised Bt crops.

The risk of insect resistance is further mitigated by the use of a so-called refuge strategy on large commercial farms, where a small part of the crop planted is non-Bt in order to provide a source of food for the target pest that does not promote resistance development in the pest. Such refuges are either planted under contract to ensure that farmers adhere to this strategy or alternatively, non-Bt seed is mixed with the Bt seed prior to sale. 

In developing country agricultural systems, refuges are supplied by the abundance of non-Bt host plants present on smallholder farms, where intercropping with other hosts to the target pest is common. Refuges provide ample susceptible individuals to mate with the scarce resistant ones surviving on Bt crops, yielding hybrid progenies, and are most effective when resistance is inherited recessively from resistant insects so that hybrid offspring are killed on Bt crops.

Resistance is further affected by the environment, including the presence of alternative host plants, competition, overwintering and natural enemies. Therefore, refuges that increase fitness costs or result in more recessive resistance genes enhance resistance management.

Genetic approaches

Expression of multiple ‘Cry’ genes is a more recent strategy to combat resistance. These genes each target different receptors in the pests and therefore require multiple mutations for resistance to develop.

Field evolved resistance to various Cry genes have been reported since 2005 in several species of major insect pests, including fall armyworm in Puerto Rico; corn earworm in the US; cereal stem borer in South Africa; pink bollworm (Pectinophora gossypiella) in India; and bollworm (Helicoverpa punctigera) in Australia; and, most recently, western corn rootworm (Diabrotica virgifera virgifera) in the US.

The first generation of commercialised insect resistant crops expressed single Bt Cry genes, which posed a high risk that insects would evolve resistance if they were not managed properly. Second and third generation products have reduced this risk by stacking or pyramiding different genes, such as multiple but different Cry genes as well as Cry genes combined with other insecticidal proteins, which target different receptors in insect pests but also provide resistance to a wider range of pests.

Alternatively, synthetic variants of Cry genes have been employed as in MON863 maize, which expresses a variant of the Bt kumamotoensis Cry3Bb1 gene against corn rootworm, and which is eight times more effective than the native, non-modified version. This robust new generation of Bt crops requires multiple mutations/adaptations within the same insect for target pests to develop resistance.

Constitutive expression, where the Bt protein is constantly produced throughout every cell in the plant, has been very successful, but in some cases tissue specific expression is a better option, for example, in epidermal cells, which first come under attack from insects or in the phloem for sap sucking insects. It has been shown that Bt expression can be regulated by protein transcription factors or chemical induction and, with this technique, it is possible to create refuges within single plants where parts of the plants do not express the genes and act as non-GM refuges.

Expression of the Bt proteins in plant structures such as in chloroplasts is also an important target for future Bt crops. Higher levels of toxin, up to 3−5% of total leaf protein, are accumulated in chloroplasts since the plastid genome is bacterial in origin as are Bt genes. Since cytoplasmic plastids are maternally inherited, pollen does not contain these organelles so this will reduce the chances for Bt gene flow and slow the development of resistance.

Improved insect resistance has also been achieved by expression of multiple resistance genes in a single plant, known as gene stacking or pyramiding. Some are already under commercial cultivation and a whole new generation of such crops is under development or being explored as alternatives aimed at preventing or slowing down development of resistance. These include the expression of multiple Cry genes targeting one pest and hybrid Cry proteins – through domain swapping as described below – that enhance both toxicity and host range. Cry genes are also combined with plant lectins to target several pests, for example, the snowdrop (Galanthus nivalis) lectin fused to the Cry gene, delivers Bt proteins to the haemolymph, the equivalent of blood, of target insect larvae.

Other examples are of the SFII spider neurotoxin fusion protein that is lethal to moth larvae and garlic leaf lectin against peach potato aphid. Fusion proteins of Cry1A with the galactose-binding domain of the ricin B-chain increase the number of binding domains for Cry1A. Many of these strategies show promise and have to be carefully evaluated to ensure there will be no toxic and/or allergic effects to humans.

Recently, transgenic maize was obtained with six insect resistance genes against corn rootworm and moth pests and two herbicide tolerance genes, which can provide a ‘one stop solution’ to pest and weed problems through gene stacking.

Novel Bt insecticidal proteins other than the three-domain Cry proteins are also being developed, such as binary toxins of Cry34/35 and Vip1/2 toxins against corn rootworm (Diabrotica virgifera), as an alternative to the synthetic Cry3Bb1. In addition, single chain vegetative insecticidal proteins, such as Vip3, have been found to have a broader range of toxicity and can be further improved with protein engineering.

Protein engineering

The structural similarity of all three-domain Cry proteins has also led to the idea of protein engineering to exchange domains amongst these proteins. Combining domains from different proteins will create new Cry proteins with novel specificity and new opportunities to manage insect resistance. Site-directed mutagenesis in Cry toxins instead of ‘domain swapping’ is also being attempted since it was found that such mutations in the loop regions of domain II increase the toxicity of Cry3A to gypsy moth 40-fold and that of Cry3Bb against rootworm eight-fold. Another approach, to remove the 𝜶-1 helix of domain I and hence bypass the resistance mechanism developed by insects, resulted in a protein that regained toxicity to resistant insects.

Transgenic prospects

Transgenic plant technologies have been, and still are, a major scientific success, even 15 years after the deployment of the first GM crops. However, accessibility of these products is relatively restricted, especially in developing countries and Europe, due both to opposition to GM technology and a lack of regulatory mechanisms to deploy them. Often, the potential economic returns of a new product are insufficient for commercialisation and the high cost makes it difficult even for public institutions to develop products for farmers in the developing world.

Out in the field, meanwhile, farmers have already begun to feel the effects of encroaching pest resistance and novel approaches are constantly sought to ensure that this increasingly valuable technology remains effective.

Benefits of Bt crops

Unlike synthetic pesticides, Bt proteins are non-toxic to vertebrates; are very specific to particular insect pests; and have been exploited since 1938 as safe biopesticides. Similarly, Bt transgenic crops possess advantages over conventional synthetic pesticides, which often kill non-target pests and their predators, in addition to the target pest. Bt crops are also particularly suitable for small farmers since no equipment and pesticide knowledge are needed for cultivation and these crops reduce human exposure to insecticides, especially for those using hand sprayers.

In this context, cultivation of Bt maize has reduced yield losses due to rootworms and stem borers substantially without resorting to toxic organophosphate insecticides. For maize, Bt varieties substitute up to 50% of insecticides used for conventional varieties whereas for cotton, cultivation of Bt varieties has drastically reduced the number of sprays required – previously up to 30 sprays/season – which benefits both the environment and labourers’ health.

A further benefit of Bt maize is that fewer harmful mycotoxins accumulate from opportunistic fungi that infect damaged kernels since cobs without insect damage are seldom infected by fungi.

Bt crops also increase incomes through higher yields of healthier grain, as evidenced by their increasing adoption. In addition, there has been no documented proof of any negative impact on non-target insects in Bt fields.

Non-Bt approaches

Natural plant defence mechanisms, such as proteinase inhibitors, can also be harnessed to fight pests, especially those that may spoil the crop on storage. However, there are concerns about the effect of mammals and humans ingesting relatively large amounts of these proteins such as α-amylase.

Other mechanisms for pest control involve secondary metabolism compounds, such as the cyanogenic glycoside dhurrin from sorghum, the second most important cereal crop in Africa after maize, which is induced by tissue damage; or volatile communication compounds that deter insect colonisation or attract natural enemies of insect pests as deployed in the ‘push-pull’ crop protection strategy.

Another novel approach is exploiting the large number of potential insecticidal proteins produced by the bacteria species Photorhabdus luminescens. One of these proteins, Toxin A, is effective against tobacco hornworm and corn rootworm when expressed at only 0.07% of total protein.

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