Bt Research 2025, Vol.16, No.4, 168-181 http://microbescipublisher.com/index.php/bt 176 of the resistant Cry1F toxin was found to be mutations in the receptor protein on the microvilli of the midgut brush membrane, and the toxins no longer bind. Changes in brush edge membrane enzymes or transport proteins can also confer resistance. Studies have found that the upregulation of the expression of some vesicle transport proteins in resistant bollworms may promote the clearance of Bt toxin in cells. Protease-mediated blockage of toxin activation is also an important mechanism. For example, in some larvae with Cry1Ac resistance, the activity of midgut proteases (trypsin) is significantly reduced, resulting in the intake of Cry1Ac prototoxin that cannot be effectively activated, and thus greatly reduced toxicity. In addition, insect intestinal symbionts and pathogenic viruses can also affect the effects of Bt toxins. An interesting case is that the cotton bollworm skewers (HaDV2) are often detected in the bodies of cotton bollworms that survive in China's cotton area. This symbiotic virus can improve the resistance of bollworms to Cry1Ac toxin in Bt cotton by interfering with the host immune signaling pathway. This phenomenon of symbiotic virus-assisted resistance suggests the complexity of our pest resistance evolution. 6.2 The role of multigene stacking and gene editing technology in resistance management Delaying and controlling pest resistance is a long-term and complex task. Fortunately, biotech itself also provides a powerful tool for coping. One of the most effective methods is multigene stacking (gene aggregation). By transferring two or more insect-resistant genes with different modes of action into the same crop, it is extremely difficult for pests to evolve resistance to multiple toxins at the same time. Based on simple model calculations, the probability that the pests develop resistance to two Bt proteins simultaneously is a reduction in square order of a single protein (Salim et al., 2020). In addition to the traditional Bt gene combination, scientists have also explored the combination of Bt genes with other insect-resistant mechanism genes. For example, superposition of Bt toxic protein genes with phytoprotein inhibitor genes, insect-proof secondary metabolic pathway genes, etc. makes pests face "double obstacles" at the same time. These innovative strategies have shown good results in experimental research (Figure 3) (Nagaraj and Rajasekaran, 2024). Another type of emerging technology is gene editing, which has potential applications in resistance governance. Using tools such as CRISPR, key resistance genes of pests can be modified or target plants can be accurately improved. Figure 3 Multiple Bt gene pyramiding and silencing (PyramidingBt gene + RNAi) (Adopted from Nagaraj and Rajasekaran, 2024) 6.3 Case analysis: the resistance of American bollworm to Bt cotton and its countermeasures The United States was one of the earliest countries to plant Bt cotton. It established a relatively complete resistance management system in the early stages of its promotion, and its experience is representative. In the mid-2000s, researchers began to find early signs of resistance to Cry1Ac toxin in cotton bollworm populations such as Arizona (Tabashnik and Carrière, 2010). To prevent the spread of resistance, the U.S. Environmental Protection Agency (EPA) stipulated as early as 1996 that cotton farmers must plant a certain proportion of non-Bt cotton near Bt cotton fields as a "refuge" to provide wild-type insect sources with possible resistant insects to dilute resistance genes. The EPA requires that if a unit-price Bt cotton is used, the proportion of sheltered fields must reach 20%; while the sheltered fields can be reduced to 5% when planting double-price Bt cotton. Thanks to the strict implementation of the asylum strategy, the United States did not experience any field failure cases
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