DataSheet1_Simultaneous silencing of juvenile hormone metabolism genes through RNAi interrupts metamorphosis in the cotton boll weevil.PDF

The cotton boll weevil (CBW) (Anthonomus grandis) is one of the major insect pests of cotton in Brazil. Currently, CBW control is mainly achieved by insecticide application, which is costly and insufficient to ensure effective crop protection. RNA interference (RNAi) has been used in gene function analysis and the development of insect control methods. However, some insect species respond poorly to RNAi, limiting the widespread application of this approach. Therefore, nanoparticles have been explored as an option to increase RNAi efficiency in recalcitrant insects. Herein, we investigated the potential of chitosan–tripolyphosphate (CS-TPP) and polyethylenimine (PEI) nanoparticles as a dsRNA carrier system to improve RNAi efficiency in the CBW. Different formulations of the nanoparticles with dsRNAs targeting genes associated with juvenile hormone metabolism, such as juvenile hormone diol kinase (JHDK), juvenile hormone epoxide hydrolase (JHEH), and methyl farnesoate hydrolase (MFE), were tested. The formulations were delivered to CBW larvae through injection (0.05–2 µg), and the expression of the target genes was evaluated using RT-qPCR. PEI nanoparticles increased targeted gene silencing compared with naked dsRNAs (up to 80%), whereas CS-TPP-dsRNA nanoparticles decreased gene silencing (0%–20%) or led to the same level of gene silencing as the naked dsRNAs (up to 50%). We next evaluated the effects of targeting a single gene or simultaneously targeting two genes via the injection of naked dsRNAs or dsRNAs complexed with PEI (500 ng) on CBW survival and phenotypes. Overall, the gene expression analysis showed that the treatments with PEI targeting either a single gene or multiple genes induced greater gene silencing than naked dsRNA (∼60%). In addition, the injection of dsJHEH/JHDK, either naked or complexed with PEI, significantly affected CBW survival (18% for PEI nanoparticles and 47% for naked dsRNA) and metamorphosis. Phenotypic alterations, such as uncompleted pupation or malformed pupae, suggested that JHEH and JHDK are involved in developmental regulation. Moreover, CBW larvae treated with dsJHEH/JHDK + PEI (1,000 ng/g) exhibited significantly lower survival rate (55%) than those that were fed the same combination of naked dsRNAs (30%). Our findings demonstrated that PEI nanoparticles can be used as an effective tool for evaluating the biological role of target genes in the CBW as they increase the RNAi response.

棉铃象甲（cotton boll weevil, CBW，学名Anthonomus grandis）是巴西棉花的主要害虫之一。当前棉铃象甲的防治主要依赖杀虫剂施用，该方式成本高昂且无法实现有效的作物保护。RNA干扰（RNA interference, RNAi）已被用于基因功能分析及害虫防治方法的开发，但部分昆虫物种对RNAi响应较差，限制了该技术的广泛应用。因此，纳米颗粒被探索作为提升对RNAi响应不佳的昆虫RNAi效率的备选方案。本研究探讨了壳聚糖-三聚磷酸钠（chitosan–tripolyphosphate, CS-TPP）与聚乙烯亚胺（polyethylenimine, PEI）纳米颗粒作为双链RNA（double-stranded RNA, dsRNA）载体系统，以提升棉铃象甲RNAi效率的潜力。我们测试了多种靶向保幼激素代谢相关基因的纳米颗粒-dsRNA配方，这些基因包括保幼激素二醇激酶（juvenile hormone diol kinase, JHDK）、保幼激素环氧水解酶（juvenile hormone epoxide hydrolase, JHEH）以及法尼酸甲酯水解酶（methyl farnesoate hydrolase, MFE）。通过注射（剂量0.05–2 μg）将该制剂施用于棉铃象甲幼虫，并采用实时定量逆转录PCR（RT-qPCR）评估靶基因的表达水平。与裸露dsRNA相比，PEI纳米颗粒可提升靶基因沉默效率（最高可达80%）；而CS-TPP-dsRNA纳米颗粒则会降低基因沉默效果（0%–20%），或仅达到与裸露dsRNA相当的沉默水平（最高50%）。我们进一步评估了通过注射裸露dsRNA或与PEI复合的dsRNA（剂量500 ng），分别靶向单个基因或同时靶向两个基因时，对棉铃象甲存活率与表型的影响。总体而言，基因表达分析显示，无论靶向单个还是多个基因，PEI处理组的基因沉默效果均优于裸露dsRNA组（沉默效率约60%）。此外，注射dsJHEH/JHDK（无论裸露还是与PEI复合）均会显著影响棉铃象甲的存活率（PEI纳米颗粒组为18%，裸露dsRNA组为47%）与变态发育过程。表型改变（如化蛹不完全或蛹体畸形）表明，JHEH与JHDK参与了棉铃象甲的发育调控。此外，用dsJHEH/JHDK + PEI（1000 ng/g）处理的棉铃象甲幼虫存活率（55%）显著低于饲喂相同组合裸露dsRNA的幼虫（30%）。本研究结果表明，PEI纳米颗粒可作为评估棉铃象甲靶基因生物学功能的有效工具，因其能够增强RNAi应答反应。