Bt Research 2025, Vol.16, No.2, 79-85 http://microbescipublisher.com/index.php/bt 82 methods (Apirajkamol et al., 2025). The screening method will combine automated protein expression, activity screening and high-throughput bioassays to verify the functions of a large number of candidate toxins in a short period of time. Badran et al. (2016) found that the PACE system can rapidly obtain Bt toxin variants that bind with high affinity to specific insect receptors during continuous evolution. Genomic analysis and bioassays were also used to screen and confirm Bt strains with high activity against important pests (Ross et al., 2025). 5.2 Key findings: novel toxin identification and functional validation The Cry1Ac toxin was subjected to directed evolution using the PACE system, resulting in a new Cry1Ac variant that can bind with high affinity and kill insect receptors resistant to wild-type Cry1Ac, such as TnCAD. The insecticidal activity of these variants against resistant pests was 335 times higher than that of the original Cry1Ac (Badran et al., 2016). Apirajkamol et al. (2025) found in their genomic and bioactivity analyses of different Bt strains that some strains (such as Bt_02) could kill multiple resistant pests even when no known toxin genes were detected, suggesting the possible existence of new toxins or active molecules. High-throughput bioassays further confirmed that these new toxins have a strong insecticidal effect on a variety of important agricultural pests, including Cry1Ac resistant strains (Ross et al., 2025). 5.3 Implications for pest management and resistance mitigation High-throughput screening technology has accelerated the discovery and functional optimization of new Bt toxins, providing a new approach for pest resistance management. The new toxins obtained through directed evolution and diversity screening can effectively overcome the resistance of pests to traditional Bt toxins and extend the service life of biological pesticides (Badran et al., 2016; Apirajkamol et al., 2025). The combination of multiple toxins or their use together with other bioactive molecules can also slow down the development of resistance and enhance the sustainability of integrated pest management (Ross et al., 2025). These advancements have provided crucial technical support for global agricultural pest management and green control. 6 Challenges and Limitations of HTS in Bt Research 6.1 Technical challenges: false positives, assay miniaturization, data complexity High-throughput screening (HTS) poses many technical challenges in the research of Bt toxins. First of all, false positives are very common. Especially in cases of low-abundance samples or complex backgrounds, PCR bias and sample contamination can easily bring false signals, affecting the accuracy of screening results. Secondly, after the detection system was miniaturized, although the throughput increased, the requirements for experimental conditions, liquid handling and the precision of automated equipment also became higher. If the operation is not precise enough, systematic errors may be introduced (Figure 2) (Liu et al., 2020). In addition, HTS generates a huge amount of data, and the analysis process is also very complex, involving many software, algorithms and databases. Researchers without a background in bioinformatics will encounter great difficulties in data processing and result interpretation. 6.2 Biological considerations: expression variability, host-specific activity The expression level of Bt toxin varies greatly under different expression systems, strains or environmental conditions. The activity expression of the same toxin gene may be completely different in different contexts. In addition, the insecticidal activity of Bt toxin has strong host specificity. The sensitivity of different insect species or strains to the same toxin also varies greatly. This makes the biometric design and result interpretation of high-throughput screening more difficult (Liu et al., 2020). These biological variations increase the complexity of functional verification and may also cause some potential toxins to be overlooked during the initial screening. 6.3 Cost, scalability, and accessibility issues in developing regions Although HTS has greatly improved the screening efficiency, its equipment investment, reagent consumption and data storage and analysis costs are all very high, which limits its promotion in resource-limited areas (Liu et al., 2020). Some developing regions lack automation platforms and high-performance computing resources, making it difficult for them to independently carry out large-scale HTS projects. In addition, the complex analysis process and reliance on professional technicians also make the popularization and expansion of HTS technology rather difficult.
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