Insects, lacking adaptive immunity, rely solely on their innate immune system for defense against pathogens, a trait that has enabled their survival for over 480 million years (Liu et al., 2025a). Among the key pathways governing this system, the Toll signaling pathway plays a central role in recognizing pathogen associated molecular patterns (PAMPs) and coordinating immune responses. Toll-like receptors (TLRs), acting as pattern recognition receptors (PRRs) in vertebrates and some invertebrates, detect microbial components such as peptidoglycan (PGN) and lipopolysaccharide (LPS) through their extracellular structural domains (Fitzgerald and Kagan, 2020). In the classical pathway that was elucidated in Drosophila, Spätzle cytokines bind the Toll receptor to activate an intracellular cascade of reactions, culminating in the production of antimicrobial peptides (AMPs) (Lemaitre and Hoffmann, 2007). While the role of Toll receptors in antibacterial and antifungal immunity is well documented, their involvement in antiviral defense remains poorly understood, particularly in response to double-stranded RNA (dsRNA), a hallmark of viral replication. This knowledge gap is particularly evident in the silkworm (Bombyx mori), an economically and scientifically important model organism representative for Lepidoptera, for which the understanding of the immune mechanisms is crucial for pest control and disease management (Kawaoka et al., 2008, Tanaka and Yamakawa, 2011).

Toll receptors and the Toll signaling pathway were first identified as highly evolutionarily conserved (Brennan and Anderson, 2004), sparking significant interest in elucidating how Toll receptors activate immune signaling cascades in insects (Lindsay and Wasserman, 2014). Insect Toll receptors exhibit a striking structural and functional similarity to mammalian TLRs, and both are recognized as critical regulators of the innate immune system across taxa (Lemaitre and Hoffmann, 2007). B. mori, a lepidopteran model organism, exhibits remarkable functional diversity within its Toll gene family, which comprises 14 members in the genome (Tanaka et al., 2008). For instance, the Toll family member 18 Wheeler (18 W) is implicated in the bacterial infection response, selectively inducing AMP genes such as cecropin-A and gloverin 2 (Wang et al., 2019). BmToll and BmToll-2 are highly expressed in the ovary, while BmToll-10 and BmToll-11 exhibit testis-specific expression, suggesting their potential involvement in sex-specific biological functions (Cheng et al., 2008). BmToll-6 and BmToll-7 may play roles in embryogenesis (Eldon et al., 1994). Lepidoptera may differ from other arthropods in that BmToll9-1 functions as a PRR by forming complexes with myeloid differentiation-2-like proteins (MD-2)-related lipid-recognition (ML) protein family members, a mechanism similar to mammalian TLR4 members (Zhang et al., 2021). Our research group's latest study has revealed that BmToll9 homologous genes play unique and interrelated roles in silkworm immunity and development. BmToll9-1, previously believed to be involved in silkworm innate immunity (Wu et al., 2010), has been found to be a key mediator in maintaining intestinal homeostasis. Specifically, LPS inhibits the transcriptional expression of BmToll9-1 (Liu et al., 2013) and reduces the expression of genes in the Toll, immune deficiency (IMD), and Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways as well as the downstream AMP effector genes (Liu et al., 2014, Liu et al., 2024b). Through this mechanism, the balance of the intestinal microbiota is disturbed that results in the impairment of larval growth and the reduction of body weight, indicating its dual role in pathogen immunity and intestinal microbiota symbiosis (Liu et al., 2024b, Liu et al., 2025a). In contrast, BmToll9-2 plays a specialized role in systemic humoral immunity. Recent studies indicate that BmToll9-2 acts as a sensor for bacterial infections, exhibiting high sensitivity to Gram-negative bacterial components such as LPS from Escherichia coli and PGN from Staphylococcus aureus (Liu et al., 2024c). Notably, BmToll9-2 deficiency leads to impaired AMP gene expression and increased susceptibility to bacterial challenges, highlighting its critical role as a positive regulator of Toll-mediated immune responses (Liu et al., 2024a).

The potential of dsRNAs to induce gene silencing for pest control is well established, yet their immunostimulatory role in lepidopteran insects remains controversial (Kolliopoulou and Swevers, 2014). In mammals, extracellular dsRNAs are recognized by TLR3/MDA5 as conserved PAMPs, triggering interferon production (Chen and Hur, 2022). In contrast, insect studies have mainly attributed dsRNA-induced immunity to indirect mechanisms, such as gut microbiota perturbation or RNAi pathway activation (Velez and Fishilevich, 2018). In D. melanogaster, the Toll pathway contributes to defense against RNA viruses, including Drosophila C virus (DCV), suggesting a conserved antiviral function (Chauhan et al., 2025). Similarly, the midgut epithelial cells of B. mori serve as natural targets for B. mori cytoplasmic polyhedrosis virus (BmCPV), a dsRNA virus, providing an ideal model to investigate antiviral Toll signaling (Liu et al., 2015). BmCPV infection activates host immune responses, including the production of circular RNAs that modulate NF-κB-like pathways (Zhao et al., 2022).

Previous studies have demonstrated that dsRNA injection can suppress the expression of BmToll9-1 in silkworm larvae (Liu et al., 2013). It is known that BmToll9-1 and BmToll9-2, as two closely related genes, exhibit similar functions in the innate immunity of silkworm, as demonstrated by their transcriptional response to exogenous pathogens and their positive regulation of components and effectors in the Toll pathway (Liu et al., 2024a, Liu et al., 2024b, Liu et al., 2024c, Liu et al., 2025a). However, whether dsRNA or dsRNA virus challenge directly engages BmToll9-2 remains unresolved. This ambiguity underscores a critical question: Does exogenous dsRNA function as a PAMP to activate Toll-mediated immunity? Addressing this question would provide insights into the mechanism by which dsRNA viruses could activate the immune response and offering insights into the evolutionary conservation of Toll-mediated antiviral defense.

By integrating molecular and immunological approaches, this work advances our understanding of Toll receptor plasticity in pathogen recognition. Our findings also have practical implications for exogenous dsRNA delivery methods used in RNAi-based pest control and emphasize the need to circumvent enzymatic degradation strategies. This study not only elucidates the immune signaling of BmToll9-2 in response to exogenous dsRNA but also reveals an antiviral defense mechanism that has evolved in Lepidoptera.