Jisheng Liu

Insects lack adaptive immunity and rely exclusively on innate immune system for pathogen defense. However, the specific role of BmToll9-2, a key Toll receptor in the silkworm (Bombyx mori), in mediating immune responses, particularly against bacterial challenges in the larval fat body, remains incompletely understood. To address this gap, this study employed a combination of molecular approaches, including RNA interference (RNAi) targeting BmToll9-2, bacterial challenges with heat-inactivated Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), and quantitative real-time PCR (qPCR) to assess the transcriptional changes of BmToll9-2 and immune-related genes in the fat body, a key immune tissue. Quantitative analysis showed that after BmToll9-2 RNAi, bacterial challenges significantly upregulated BmToll9-2 expression in the fat body at 12 h post-challenge, whereas BmToll9-2 silencing alone notably downregulated most downstream signaling genes of the Toll pathway by 53.07–83.14%, as well as 11 immune effector genes, including antimicrobial peptide genes, by 64.48–93.40%. Importantly, feeding bacteria post-RNAi reversed these downregulations: E. coli induced a stronger upregulation of both signaling and effector genes compared to S. aureus. This study provides transcriptional evidence that BmToll9-2 may act as a positive regulator of the Toll pathway signaling and antimicrobial peptides in silkworm larval fat body, facilitating robust and rapid immune signaling. This study deepens our understanding of Lepidopteran innate immunity by elucidating key molecular mechanisms, thereby providing a solid foundation for refining RNAi-based pest control strategies.

The advent of RNA interference (RNAi) in 1998, marked by the discovery of gene silencing triggered by double-stranded RNA (dsRNA) in nematodes, opened a transformative chapter in molecular biology

Conventional chemical pesticides have long protected global crop yields, but their environmental and health impacts, including contamination, biodiversity loss, residue accumulation, and pest resistance, demand sustainable alternatives. RNA interference (RNAi) represents a genetic breakthrough in crop protection, enabling precise, sequence‐specific silencing of essential pest genes via double‐stranded RNA (dsRNA). Innovations such as nanoparticle‐based delivery, host‐induced gene silencing (HIGS), and spray‐induced gene silencing (SIGS) have transformed RNAi into a practical, field‐ready technology. Integration with multi‐omics and computational biology accelerates target discovery and enhances specificity and scalability. Beyond pest management, RNAi applications extend to vector control and pollinator protection, exemplifying One Health principles. RNAi‐based pesticides therefore, redefine sustainable agriculture through molecular precision and cross‐sectoral genetic innovation.

In contrast to mammals, insects possess a separate machinery for processing of long dsRNAs into siRNAs for the cleavage of viral RNAs. The process of RNAi is considered very efficient in all insects once the delivery in the cytoplasm occurs such as during RNA virus replication. For the application of RNAi as insecticide to succeed, efficient uptake of intact dsRNA into the cytoplasm therefore is necessary, which seems to occur by natural mechanisms in the leaf beetles for which RNAi-based insecticides already have been marketed. In most insects, relatively high amounts of dsRNA are required to trigger gene silencing which raises questions regarding potential side effects. Besides, RNAi is considered as the major antiviral defense mechanism, at least in Drosophila , but not necessarily in all other insects. Following increasing evidence from the recent literature, it has become prudent to include the sensing of dsRNA as an immune trigger to evaluate the extent of the RNAi mechanism that is triggered by dsRNA. In this review, an overview of mechanisms is presented regarding how the recognition of dsRNA as a “pathogen-associated molecular pattern”, the multiple additional functions of the canonical siRNA factors and the modulation of the function of Dicer-2 and Ago-2 by dsRNA-binding proteins may complicate the efficiency of the exo-RNAi process and aggravate its application for pest control.