RNA interference (RNAi) is a conserved post-transcriptional gene silencing mechanism mediated by small RNA molecules that regulate gene expression in a sequence-dependent manner. First described in Caenorhabditis elegans (Fire et al., 1998), RNAi has since been identified across eukaryotes, including insects, where it contributes to antiviral defense, suppression of transposable elements, developmental regulation, and physiological adaptation (Hannon, 2002, Huvenne and Smagghe, 2010, Zhu and Palli, 2020).

In insects, three RNAi pathways are recognized: the small interfering RNA (siRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA) branches. The siRNA pathway is of particular relevance for pest management, as it can be activated by exogenous double-stranded RNA (dsRNA) molecules (Christiaens and Smagghe, 2014, Svoboda, 2020). Following cellular uptake, dsRNA is processed by Dicer-2 into ∼21 nucleotide duplexes (Liu et al., 2003). These duplexes are incorporated into the RNA-induced silencing complex (RISC), which contains Argonaute-2 (Ago2) and double-stranded RNA-binding proteins (dsRBPs) such as R2D2 (Liu et al., 2003, Marques et al., 2010) and Loquacious (Förstemann et al., 2005, Yoon et al., 2018). Within RISC, one strand of the duplex is removed, and the guide strand directs Ago2 to complementary mRNAs, resulting in target cleavage and degradation.

The miRNA and piRNA pathways primarily regulate endogenous processes, including developmental gene expression and transposon silencing. In contrast, the siRNA pathway is uniquely responsive to external dsRNA and is therefore the primary route for experimental gene silencing and RNAi-based pest management. Efficient silencing requires uptake of dsRNA, protection from nuclease activity, and effective intracellular trafficking; these steps may be hindered by degradation in extracellular fluids or by endosomal entrapment (Dowling et al., 2016, Shukla et al., 2016, Swevers et al., 2021).

The first practical demonstration of this approach in crop protection was the development of SmartStax PRO maize, which expresses dsRNA targeting Diabrotica virgifera virgifera (western corn rootworm) (Head et al., 2017). This example demonstrated the feasibility of RNAi-based control but also indicated that consistent silencing is difficult to achieve across insect taxa. RNAi efficiency is influenced by several factors, including dsRNA uptake, nuclease activity, and the functionality of core proteins such as Dicer, Argonaute, and dsRBPs (Christiaens et al., 2020a, Christiaens et al., 2020b, Pacheco et al., 2022). Hemipteran insects frequently show variable RNAi responsiveness, which has been associated with differences in dsRBP expression and domain organization (Hunter and Wintermantel, 2021).

In addition to its applied value, RNAi has become a standard tool in insect functional genomics. Gene knockdown by RNAi has facilitated studies of development, endocrinology, reproduction, neurobiology, and immunity, particularly in species lacking efficient transgenesis or genome-editing systems (Tomoyasu et al., 2008, Huvenne and Smagghe, 2010, Swevers and Smagghe, 2012, Wynant et al., 2014). Delivery strategies currently under development include host-induced gene silencing (HIGS) in transgenic plants, spray-induced gene silencing (SIGS), and nanocarrier-mediated formulations (Zotti et al., 2018, Christiaens et al., 2022). These methods offer specificity and environmental safety but are limited by heterogeneous outcomes, particularly in Lepidoptera and Hemiptera (Christiaens et al., 2020a, Christiaens et al., 2020b, Jain et al., 2021, Joga et al., 2016).

The molecular basis of RNAi refractoriness is multifactorial. Degradation of dsRNA in the gut and hemolymph reduces signal stability (Christiaens and Smagghe, 2014, Singh et al., 2017). Uptake across the gut epithelium is often inefficient, partly due to low expression of SID-1-like transporters (Dowling et al., 2016, Shukla et al., 2016, Pacheco et al., 2022). Recent structural analyses have revealed that SID-1 family proteins directly bind and transport double-stranded RNA via a transmembrane channel architecture, providing molecular evidence for their role in systemic RNAi (Hirano and Shimizu, 2024, Wang et al., 2024). Although these proteins lack canonical dsRBDs, their dsRNA-recognition capacity positions them among functional dsRNA-interacting cofactors. Furthermore, differences in the expression and domain organization of Dicer-2, Ago2, and dsRBPs such as R2D2 and Loquacious contribute to interspecific variation in silencing responses (Yoon et al., 2018, Kim et al., 2021). In Hemiptera, several core components of the RNAi pathway show low or tissue-restricted expression and sequence divergence compared with dipteran and coleopteran homologs, suggesting lineage-specific adaptations that may underlie reduced RNAi efficiency (Hunter and Wintermantel, 2021).

Taken together, these findings indicate that double-stranded RNA-binding proteins are integral to RNAi efficiency, as they coordinate substrate recognition, processing, stabilization, and RISC assembly. A comparative analysis of these proteins is therefore required to explain taxonomic variation in RNAi and to inform the design of pest management strategies adapted to RNAi-refractory species.

This review focuses on insect dsRBPs, with emphasis on their structural organization, mechanistic roles in RNAi pathways, and evolutionary diversification. In addition to canonical dsRBD-containing cofactors such as R2D2, Loquacious, and Staufen, this review also considers functionally relevant dsRNA-binding or dsRNA-transporting proteins, including SID-1-like channels, whose recently resolved structures have clarified their role in dsRNA uptake and systemic signaling (Wang et al., 2024). Particular attention is given to R2D2, Loquacious, and Staufen, which represent the best-studied examples with distinct but complementary contributions. Hemiptera are highlighted due to their limited RNAi responsiveness and the possibility that lineage-specific differences in dsRBP biology underlie this phenotype. By combining structural, functional, and applied perspectives, this review examines the contribution of dsRBPs to insect RNAi and their potential relevance for crop protection.

To ensure a representative comparative framework, this review focuses on four insect orders – Diptera, Coleoptera, Lepidoptera, and Hemiptera – which collectively encompass the major experimental models of RNA interference in insects. These groups differ markedly in their RNAi responsiveness and systemic silencing capacity: coleopterans exhibit robust and systemic RNAi effects, dipterans display moderate and well-characterized responses, whereas lepidopterans and hemipterans are generally refractory to dsRNA-induced silencing. Focusing on these lineages therefore allows the structural and functional diversity of double-stranded RNA-binding proteins to be analyzed in relation to distinct RNAi phenotypes across insects.

This review aims to provide a synthesis that integrates recent structural discoveries – including high-resolution cryo-electron microscopy of Dicer-2-R2D2 and Dicer-2-Loquacious complexes (Jouravleva et al., 2022, Deng et al., 2023) – with comparative and evolutionary perspectives on double-stranded RNA-binding proteins across insects. While previous reviews have primarily focused on either the molecular mechanisms of RNA interference or its applied aspects in pest management, this article bridges these viewpoints by examining how structural diversity, domain organization, and expression patterns of dsRBPs contribute to inter-order variation in RNAi efficiency. By connecting structural insights with evolutionary and applied contexts, the present synthesis advances understanding of RNAi variability and highlights molecular determinants that may guide the optimization of RNAi-based pest control strategies.

Literature was retrieved from PubMed, Scopus, and Web of Science using combinations of the following keywords: “RNA interference”, “dsRNA-binding protein”, “R2D2”, “Loquacious”, “Staufen”, “SID-1-like”, “Hemiptera”, and “insects”. Publications from 2000 to 2025 were considered, with priority given to peer-reviewed original research and recent structural or functional studies. Earlier landmark works were included where relevant. Only English-language articles were considered, and reference lists of selected papers were screened to identify additional sources.