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Generation of alpaca-derived nanobody fragments as potential insecticidal agents against Plutella xylostella via cadherin toxin binding region-based screening

Cheng Shen,Guochen Li,Tianyi Han,Naila Ilyas,Meng He,Ziyan Huang,Xiao Zhang,Chongxin Xu,Chengyu Chen,Wei Chen,Xianjin Liu,Qing Zhu,Yuan Liu

BACKGROUND Bacillus thuringiensis (Bt) toxins are widely used in pest control owing to their effectiveness as bioinsecticides. However, the extensive use of these toxins has resulted in the development of resistance in various pest populations, including Plutella xylostella , which is a significant pest of cruciferous crops. This increasing resistance highlights the need for alternative insecticidal strategies that can complement or replace traditional methods. RESULTS A novel approach involves using insect cadherin receptors to screen for nanobodies with potential insecticidal activity that selectively target the toxin binding region of the Plutella xylostella cadherin toxin via phage display technology. Through three rounds of panning with a natural alpaca nanobody phage display library, we identified nanobody B1, which specifically binds to the CR9-CR11 region of Plutella xylostella cadherin. This nanobody significantly inhibited the binding of Cry1Ac toxin to cadherin, a key factor in insect susceptibility to the toxin. In bioassays, the application of soluble B1 protein (80 μg/cm 2 ) resulted in 25 ± 4.5% mortality in Plutella xylostella larvae. Histological examination of the midgut tissue revealed substantial damage to the epithelial layer, supporting the bioassay findings. Enzymatic hydrolysis assays demonstrated that, while B1 exhibited some stability, it showed limited resistance to enzymatic degradation in vitro . This could be a significant contributing factor to the low insecticidal activity of B1. CONCLUSION This study proposes a novel strategy for insecticide development based on the application of nanobodies to target cadherins within the midgut of Plutella xylostella . The identification of nanobody B1 presents a novel approach to circumvent resistance mechanisms in pest populations, overcoming resistance mechanisms in pest populations, and thus advancing sustainable pest management practices. Furthermore, these findings establish a foundation for designing alternative insecticidal proteins that could diminish the reliance on traditional formulations. © 2025 Society of Chemical Industry.