Abstract
RNA interference (RNAi) has emerged as a promising, highly specific and environmentally sustainable strategy for pest management. Given the urgent need for a targeted and effective solution against the cabbage seedpod weevil (CSW) Ceutorhynchus obstrictus (Marsham), a major pest of oilseed rape, RNAi-based management offers a promising opportunity. This review explores the application of RNAi within the Curculionidae family, with a focus on CSW, and evaluates the key challenges in implementation. A major barrier to effective RNAi in CSW is ensuring the stability of double-stranded RNA (dsRNA) until it reaches its target site. We examine the factors influencing dsRNA stability and their implications for RNAi efficacy. In addition, we discuss strategies for selecting suitable target genes and highlight potential candidate genes for silencing in CSW. Species-specific traits of CSW, including exoskeletal structure and feeding behavior, are also considered for their impact on dsRNA uptake and RNAi efficacy. While RNAi holds great potential, overcoming biological and technical barriers will be essential for optimizing RNAi-based control. This review provides insights into these challenges and outlines future directions for advancing RNAi-based pest management. By addressing current limitations and improving methodologies, RNAi can become a promising and sustainable method for controlling CSW populations.
1 Introduction
The interplay between farming practices and protection of biodiversity poses a complicated challenge with broad consequences for both food security and ecosystem health. In particular, the widespread adoption of intensive agricultural practices is a major driver contributing to the dramatic decrease in insect species richness and abundance (Hallmann et al., 2017; Seibold et al., 2019). This rapid global loss of biodiversity is threatening essential ecosystem services, ultimately putting human populations at risk (Díaz et al., 2019). Despite advances in agricultural pest control, herbivorous insects continue to pose a significant threat to food security. These challenges point to an urgent need for sustainable farming practices that control target pests while safeguarding biodiversity and ensuring consistent crop yields (Oerke, 2006; ; ; Willow et al., 2019).
Oilseed rape (OSR), also known as canola, Brassica napus L. (Brassicales: Brassicaceae), serves as a valuable source of both oil and protein. Its cultivation has gained increasing relevance in light of the growing demand for renewable resources. Countries such as Canada, China, India, Australia, France, Germany, the Russian Federation, Ukraine, Poland, and the United States are among the major global producers (FAOSTAT, 2023). Insect pest damage during the growing season poses a major threat to rapeseed production. Among the most important pests, the cabbage seedpod weevil (CSW), Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae) is widespread across Europe and Canada and can cause substantial yield losses (; ; Veromann et al., 2010; Tansey et al., 2011; Kovács et al., 2019; Hussain et al., 2023). CSW reduces OSR seed yield through both adult and larval feeding. Overwintered adults emerge in spring once temperatures reach 13–15 °C (Free and Williams, 1978) and feed on flower buds, causing blossom blasting and pod abortion. While this can reduce yields by up to 14% in Canada, such damage typically occurs only under very heavy infestation by adults (). The greatest losses, however, are associated with the larval stages (Free and Williams, 1978). Females are reproductively immature upon emergence from diapause and require a period of maturation feeding before mating. They lay a single egg inside a small developing pod (20–40 mm long) (Figure 1). After oviposition, the pod is marked with a pheromone to prevent multiple larvae from developing in the same pod (Dosdall and Cárcamo, 2011). The eggs hatch 1–2 weeks later, and the larvae feed on the developing seeds for 2–3 weeks (depending on temperature feeding can be twice as long). Each larva consumes three to six seeds, which can cause 20 to 30% of seed loss in each pod, and in rare cases of very high weevil density, two to three larvae may infest a single pod, leading to near-total seed loss within that pod (). To exit pods, larva chew holes, which can facilitate fungal infection and subsequent seed damage under humid conditions (Dosdall et al., 2001). The mature larvae drop to the soil to pupate for 9–23 days. New generation adults emerge after 11–24 days and disperse into neighboring fields, where they feed on oilseed rape and wild brassicaceous plants for 1–2 weeks. This feeding activity can damage the pods and allows moisture to enter, potentially triggering premature germination of undamaged seeds (). Afterwards, the weevils seek overwintering sites in non-cropped habitats and woodlands.
Figure 1
In Europe, parasitoids can significantly suppress CSW populations, with larval parasitism rates reaching up to 80% (Veromann et al., 2010; Kovács et al., 2019; Langer and Jensen, 2024; Sulg et al., 2024). Generalist predators also contribute to larval mortality (Williams, 2010), and cultural practices, such as crop rotation, can further reduce pest pressure in fields (; Sulg et al., 2024). Nevertheless, OSR growers continue to rely heavily on insecticides. These are typically applied not only to control CSW but also to prevent damage caused by the cabbage pod midge, Dasineura brassicae (Winnertz) (Diptera: Cecidomyiidae), which is often associated with CSW, though such association is not always required for infestation (Hausmann, 2021). In Canada, a high number of parasitoids associated with CSW has been established in about two-thirds of the fields, but parasitism rates vary widely (Dosdall et al., 2006; Dosdall and Mason, 2010; Haye et al., 2018; Desroches et al., 2023; ). This variability, combined with pest pressure, has contributed to the continued reliance on insecticides. However, the limited availability of active ingredients is increasing the risk of resistance development, and the use of broad-spectrum pesticides poses a threat to non-target organisms (Whaley et al., 2016; Hussain et al., 2023). Addressing this issue requires a targeted scientific approach to establish integrated pest management strategies that both protect OSR yields and ensures the long-term sustainability of agricultural systems.
In the nematode Caenorhabditis elegans (Maupas) (Nematoda: Rhabditidae), exposure to exogenous double-stranded RNA (dsRNA) was found to trigger a highly specific and efficient post-transcriptional gene-silencing response, leading to the groundbreaking discovery of RNA interference (RNAi) as a powerful molecular technique (Fire et al., 1998). In 2007, studies on the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and the western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae) were among the first to experimentally demonstrate the potential of RNAi as a promising strategy for insect pest control (; Mao et al., 2007). More recently, the RNAi-based insecticide Calantha has shown considerable effectiveness in managing the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) (Pallis et al., 2023), offering efficient pest controls while minimizing non-target effects and supporting biodiversity (Wenninger et al., 2025). This advancement marks a significant milestone in the development of RNAi-based pesticides. The high specificity and effectiveness of these technologies stem from the conserved molecular mechanism of RNAi. Endogenous small RNAs (sRNAs), including MicroRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs), regulate gene expression through post-transcriptional mechanisms, such as translational repression and mRNA degradation (Ghildiyal and Zamore, 2009; Jonas and Izaurralde, 2015). RNAi-based pest control primarily exploits the siRNA pathway. Following uptake of exogenous dsRNA, the RNAi machinery mediates sequence-specific degradation of complementary target mRNAs (Siomi and Siomi, 2009). RNAi is initiated when dsRNA enters the cell and is recognized and cleaved by the RNase III endonuclease Dicer into 21–24 nucleotide siRNAs. These siRNAs are subsequently incorporated into the RNA-induced silencing complex (RISC), where the passenger strand is discarded, whereas the guide strand directs the catalytic Argonaute protein to complementary messenger RNA (mRNA) transcripts. Argonaute-mediated cleavage of the target mRNA leads to transcript degradation, which prevents protein synthesis and reduces expression of the target gene (Siomi and Siomi, 2009). RNAi relies on nucleotide sequence complementarity for target recognition, which enables highly specific gene silencing with minimal effects on non-target organisms lacking homologous target sequences. Furthermore, dsRNA is a biodegradable molecule with relatively low environmental persistence compared with conventional synthetic insecticides, which reduces the risk of long-term environmental accumulation (Dalakouras et al., 2024). The combination of high target specificity and relatively low environmental persistence makes RNAi an attractive and environmentally sustainable approach for agricultural pest management.
2 Target gene selection in CSW
2.1 Potential target genes in weevils
Beetles are generally considered highly responsive to RNAi, with gene silencing resulting in a wide range of phenotypic effects, including reduced reproduction, decreased survival, and disruption of key metabolic processes (Joga et al., 2021; Willow and Veromann, 2021; ; Hanamasagar et al., 2024). The physiological similarities between CSW and other RNAi-responsive species in the weevil family (Curculionidae), together with the recent functional validation of RNAi in CSW (Sabnis et al., 2026), provide valuable insight into the selection of potential target genes for RNAi-based control. Previous RNAi studies in Curculionidae have shown that silencing genes involved in essential physiological functions can cause high mortality or severe developmental abnormalities, with the level of response largely depending on dsRNA dose and delivery approach, supporting the potential of RNAi as a tool for sustainable CSW management (Table 1). The first study investigating RNAi in CSW provided a transcriptomic resource and experimentally validated the functionality of the RNAi pathway in this species. Candidate genes involved in essential cellular processes, including vesicle trafficking (coatomer subunit alpha, αCOP; Ras opposite, Rop), transcription (RNA polymerase II 140 kDa subunit, RpII140; Spt5; Spt6), chromatin regulation (dre4), and RNA processing (nucampholin, ncm), exhibited substantial transcript knockdown accompanied by increased adult mortality, with αCOP and Rop producing the strongest effects. Together, these findings demonstrate that CSW is amenable to RNAi and provide a foundation for the identification and optimization of target genes for future RNAi-based management strategies (Sabnis et al., 2026).
Table 1
| Species | Target | Function | Application strategy | Dose of dsrna (ng) | Reduction in gene expression | Phenotypic outcome | Reference |
|---|---|---|---|---|---|---|---|
| A. grandis | α-amylase | digestion | oral administration in larvae | 20,000 | 80% after 7 days | 60% mortality in 14 days | (Salvador et al., 2021) |
| A. grandis | cytochrome P450 | defense and detoxification | oral administration in larvae | 20,000 | – | ~40% mortality in 14 days | (Salvador et al., 2021) |
| A. grandis | AgraRelish | regulation of innate immune signaling pathway | microinjection in adults | 500 | 73% at 2 days | 94.74% mortality in 12 days after infecting with Metarhizium anisopliae (LT50 is 4.20 days) | (Moreira-Pinto et al., 2021) |
| A. grandis | juvenile hormone epoxide hydrolase (JHEH) | developmental regulation | microinjection in larvae | 500 | after combining with PEI nanoparticles, ~70–80% in 24 h | 58% mortality in 14 days after combining with PEI nanoparticles | (Vasquez et al., 2023) |
| A. grandis | juvenile hormone diol kinase (JHDK) | developmental regulation | microinjection in larvae | 500 | after combining with PEI nanoparticles, ~70–80% in 24 h | 82% mortality in 13 days after combining with dsJHEH and PEI nanoparticles | (Vasquez et al., 2023) |
| C. buqueti | Pheromone-binding proteins (CbuqPBP2) | binding and transporting sex pheromones | microinjection in adult males | 7500 | ~41.7% after 48 h | reduced the electroantennography responses of male adults to dibutyl phthalate and styrene | (Liu et al., 2023) |
| C. obstrictus | coatomer subunit alpha (αCOP) | vesicular transport | microinjection in adults | 200 | 89.5% at 5 days | 86.0% mortality in 15 days | (Sabnis et al., 2026) |
| C. obstrictus | Ras opposite (Rop) | vesicle trafficking and membrane fusion | microinjection in adults | 200 | 80.0% at 5 days | 76.7% mortality in 15 days | (Sabnis et al., 2026) |
| D. armandi | antifreeze protein (DaAFP) | cold tolerance | microinjection in larvae | 50 | ~70–% after 72 h | ~75% mortality after 72 h + 1 h cold exposure at −6 to −10 °C | (Fu et al., 2022) |
| D. armandi | 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) | hormone-guided signaling | microinjection in adult males | 200 | ~25% after 72 h | reduces the synthesis of frontalin (pheromone) to 1.92% | (Sun et al., 2021) |
| D. armandi | 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) | hormone-guided signaling | microinjection in adult males | 200 | ~18.2% after 72 h | reduces the synthesis of frontalin (pheromone) to 2.60% | (Sun et al., 2021) |
| H. hampei | chitin synthase (HhCHS1) | exoskeleton formation | oral administration in adult females | 2000 | 84% after 7 days | no significant mortality | (Ossa-Ossa et al., 2022) |
| L. oryzophilus | Trehalose-6-phosphate synthase (LoTPS) | cold tolerance | oral administration in adults | 100 | ~58% after 24 h | ~60–65% mortality after 24 h + 4 h cold exposure at 0 °C followed by −8 °C for 2 h | (Zhang et al., 2023) |
| R. ferrugineus | vitellogenin (RfVg) | egg formation | microinjection in adult female pupae | 2000 | 95.3% after 15 d | atrophied ovaries or no oogenesis and ultimately eggs were not hatched | (Rasool et al., 2021) |
| S. zeamais | COX-I | metabolic control, cell signaling and survival | microinjection in larvae | 100 | 43.42% after 24 h | 75.55% mortality after 72 h | (Zhang et al., 2021) |
| S. zeamais | COX-II | metabolic control, cell signaling and survival | microinjection in larvae | 100 | 95.17% after 24 h | 71.88% mortality after 72 h | (Zhang et al., 2021) |
| S. zeamais | COX- III | metabolic control, cell signaling and survival | microinjection in larvae | 100 | 99.92% after 24 h | 82.22% mortality after 72 h | (Zhang et al., 2021) |
Summary of RNAi studies in Curculionidae relevant for potential target gene selection in the cabbage seedpod weevil (CSW), Ceutorhynchus obstrictus.
Genes essential for development represent attractive RNAi targets because their silencing can prevent successful development and completion of the life cycle. Several studies have demonstrated that silencing genes involved in metabolism and digestion effectively impairs insect survival and development. Silencing of α-amylase and cytochrome P450 genes increases the larval mortality in cotton boll weevils, Anthonomus grandis (Boheman) (Coleoptera: Curculionidae) (Salvador et al., 2021), whereas suppression of HhCHS1 gene disrupts exoskeleton formation and impairs growth in the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) (Ossa-Ossa et al., 2022). In CSW, silencing genes involved in carbohydrate digestion, such as α-amylase, could impair larval development by reducing the ability of larvae to utilize carbohydrate reserves in developing seeds within the pod (Schiebold et al., 2011). Likewise, silencing genes involved in chitin biosynthesis could disrupt molting and exoskeleton formation. This could reduce larval survival and prevent successful adult emergence. Despite their potential, targeting these genes in CSW remains challenging because larvae develop concealed within seed pods, where they are largely protected from direct exposure to foliar-applied dsRNA. Consequently, adults remain the most accessible life stage for effective dsRNA delivery. Nevertheless, advances in plant-mediated RNAi, systemic root uptake of dsRNA, and nanoparticle-based formulations that improve dsRNA stability, plant uptake, and systemic movement could provide promising strategies to enhance delivery to concealed larval stages. Nevertheless, advances in plant-mediated RNAi, systemic root uptake of dsRNA, and nanoparticle-based formulations that improve dsRNA stability, plant uptake, and systemic movement may enable more effective delivery to concealed larval stages in the future (a; Wytinck et al., 2020).
Genes involved in pheromone-mediated communication are also promising RNAi targets for pest control. As these genes act later in the life cycle, their silencing can substantially reduce population growth by interfering with key adult behaviors. Chemical communication is essential for behaviors such as mating and aggregation, and can be disrupted by silencing genes encoding pheromone-binding proteins of mevalonate pathway, as demonstrated in studies on the bamboo-snout beetle, Cyrtotrachelus buqueti (Guérin-Méneville) (Liu et al., 2023) and the Chinese white pine beetle, Dendroctonus armandi (Tsai & Li) (Coleoptera: Curculionidae) (Sun et al., 2021). Furthermore, RNAi can also be used to interfere with insect reproduction. Reproduction-related genes represent targets whose effects occur later but can strongly suppress subsequent generations. For example, silencing the vitellogenin gene, which is necessary for egg formation, resulted in reduced egg production and weakened ovaries in red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) (Rasool et al., 2021). Similarly, in the A. grandis, larval metamorphosis was disrupted by knocking down genes associated with juvenile hormone metabolism, including those encoding juvenile hormone diol kinase (JHDK) and juvenile hormone epoxide hydrolase (JHEH) (Vasquez et al., 2023). Since juvenile hormone regulates vital biological processes in insects, including metamorphosis, growth, fertility, and diapause, these results highlight its significance as potential RNAi target and offer valuable insights for selecting reproduction-related target genes to control CSW using the RNAi.
Immune-related and defense mechanisms-related genes are other putative targets for RNAi-based pest control. Such targets may be particularly useful when RNAi is combined with biological control agents. For example, the knockdown of AgraRelish, an essential regulator of innate immune signaling pathways in insects, increased the susceptibility of A. grandis to infection by the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) (Hypocreales: Clavicipitaceae) (Moreira-Pinto et al., 2021). Silencing immune-related genes in CSW could increase susceptibility to pathogens, providing a complementary tool for integrated pest management. To survive harsh winter conditions, insects have evolved a range of behavioral and physiological adaptations, including the synthesis of polyols and antifreeze proteins responsible for cold adaptation (Zachariassen, 1985; ). Genes associated with overwintering survival represent additional RNAi targets that can reduce population persistence. Silencing the trehalose-6-phosphate synthase (TPS) and antifreeze proteins (daAFP) genes can disrupt these cold-adaptation mechanisms. In the D. armandi and the rice water weevil, Lissorhoptrus oryzophilus (Kuschel) (Coleoptera: Curculionidae), silencing DaAFP and LoTPS, respectively, significantly reduces cold tolerance in these species, offering a strategy to lower overwintering survival (Fu et al., 2022; Zhang et al., 2023). Applying a similar approach to CSW could decrease its viability during diapause, thereby reducing adult emergence in spring. To counter plant defense mechanisms, some insect pests, such as the greater grain weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae), and the wood boring weevil Pagiophloeus tsushimanus (Morimoto) (Coleoptera: Curculionidae), possess genes that encode proteins that detoxify plant secondary metabolites (Zhang et al., 2021; Li et al., 2023). CSW feeds on cruciferous crops that are rich in glucosinolates, compounds with a key role in plant defense against herbivores (Hopkins et al., 2009). Silencing detoxification genes could impair its ability of CSW to neutralize these toxic plant metabolites, thereby strengthening the efficacy of the plant natural defenses.
2.2 Considerations for dsRNA design
2.2.1 Safety considerations
Designing effective dsRNA molecules for pest management in species with limited RNAi resources requires careful consideration of multiple factors, particularly balancing efficacy against the target pest with safety for non-target organisms (). To achieve this, the selected dsRNA sequence should be highly conserved within the target species while remaining sufficiently divergent from homologous sequences in non-target organisms (Luo et al., 2024; ; Mendoza-Alatorre et al., 2025). This consideration is particularly important for the management of CSW using dsRNA products, as non-target organisms, including parasitoids, pollinators and predators, that co-occur in the field during periods when CSW is present may differ among regions and may be inadvertently exposed during treatments. Genomic resources for these non-target species are therefore essential to support accurate sequence comparisons and to minimize the risk of off-target gene silencing.
2.2.2 Efficacy considerations
dsRNA length is one of several factors that influence RNAi efficacy. For example, in the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), 520 bp dsRNAs induced stronger gene silencing than 69 bp fragments, whereas short (30 bp) dsRNAs were largely ineffective (Miller et al., 2012). Another study in the same species reported that RNAi efficacy was highest for dsRNAs of 240 and 480 bp, moderate for 60 and 120 bp, and minimal for 21 bp fragments, suggesting a necessary length range of 240–480 bp (Wang et al., 2019). Additionally, studies in D. v. virgifera demonstrated that dsRNAs within the size range of 200–500 base pairs are needed for efficient uptake and subsequent RNAi efficacy (Flynt, 2021). However, the influence of dsRNA length on RNAi efficacy is species-dependent and cannot be generalized across insect species. These interspecific differences are likely influenced by variation in dsRNA uptake, nuclease activity, RNAi machinery, and systemic RNAi capacity among insect species (Darrington et al., 2017). Although dsRNAs of approximately 200–500 bp frequently induce strong silencing in several coleopteran species, this range should not be considered universally optimal because the minimum effective dsRNA length differs among insect species (Darrington et al., 2017). In species where longer dsRNAs are more effective, this response has been attributed to greater cellular uptake efficiency and enhanced structural stability, although the underlying mechanisms vary among species (Mao et al., 2007; Darrington et al., 2017). Furthermore, studies in humans have shown that Dicer plays a role in selecting and loading siRNAs into the RISC. In this system, short RNA duplexes (<24 bp) bypass Dicer and are incorporated less efficiently into RISC (Sakurai et al., 2011). Consistent with these findings, identify several parameters critical for RNAi efficiency in insects, including the absence of siRNA secondary structures and thermodynamic asymmetry that promotes selection of the antisense strand as the guide strand (). Taken together, these findings indicate that optimal dsRNA length should be determined for each target species rather than applying a universal design criterion (He et al., 2020; Mehlhorn et al., 2021; Mendoza-Alatorre et al., 2025). Additionally, candidate sequences should be designed to avoid internal palindromes as they can form secondary structures (Reynolds et al., 2004) that can interfere with Dicer processing, reduce dsRNA stability, and limit cellular uptake. Sequence redundancy should also be avoided, as it may produce multiple siRNAs that act on the same or similar transcripts and thus increase the likelihood of off-target effects (Jackson et al., 2003). Sequences with excessively high GC content can form stable secondary structures that impede processing, whereas those with very low GC content may exhibit insufficient duplex stability with the target mRNA, while a moderate GC level (about 50%) provides sufficient stability to prolong the longevity of dsRNA (Sakurai et al., 2011).
2.3 Screening methods
The success of RNAi-based plant protection largely depends on the selection of target genes, whose knock-down results in mortality or significant fitness reduction () ultimately leading to population suppression. Although recent advances have established transcriptomic resources and demonstrated RNAi functionality in CSW, identifying the most effective target genes remains a critical step in the development of RNAi-based control strategies. Three main approaches can be employed to screen potential RNAi target genes for pest control.
A first approach is small scale screening, a focused method that tests a limited number of genes, usually between two and ten, for preliminary functional validation. Selection of targets is guided by key criteria, including core survival genes essential for basic cellular function, conserved genes identified through homology with related species, and genes involved in crucial developmental processes such as molting, digestion, or neural activity. For instance, a recent study using small-scale screening in the pollen beetle, Brassicogethes viridescens (Fabricius) (Coleoptera: Nitidulidae) (Willow et al., 2025) identified SNF7, αCOP, and RPS13 as effective targets with gene-specific knock-down effects. Likewise, a study in the pollen beetle, Brassicogethes aeneus (Fabricius) (Coleoptera: Nitidulidae) demonstrated αCOP as a validated RNAi target, exhibiting significant mortality upon its silencing (Willow et al., 2021). Similar approaches have identified potential target genes in the model organism T. castaneum, with successful gene silencing replicated in the D. virgifera virgifera and the, B. aeneus (Knorr et al., 2018, 2021). More recently, small-scale screening in CSW evaluated seven candidate target genes. Although all induced substantial transcript knockdown and increased adult mortality, αCOP and Rop produced the strongest phenotypic effects, making them the most promising candidates among the genes tested (Sabnis et al., 2026). These findings demonstrate the applicability of small-scale screening for functional assessment of candidate RNAi target genes in CSW.
The second approach involves mid-scale screening, which evaluates dozens to a few hundred genes based on functional predictions, literature reviews, or homology with known essential genes in related species. For example, Baum et al. screened 290 potential target genes in D. virgifera virgifera and identified Snf7 as the most promising target gene (). Similarly, a study in the cabbage stem flea beetle, Psylliodes chrysocephala (Linnaeus) (Coleoptera: Chrysomelidae), tested twenty-seven genes, identifying four, primarily proteasome subunit genes, that caused 100% mortality and significantly reduced feeding and mobility of beetles (). Mid-scale screening is particularly advantageous because it enables the identification of highly effective RNAi targets that are often missed in small-scale screens, while avoiding the need for genome-wide functional analysis, which is not feasible for most pest species. Although a reference genome for CSW is available through NCBI (King et al., 2023), recent transcriptomic analyses have expanded the molecular resources available for RNAi target discovery (Sabnis et al., 2026). Together, these genomic resources make CSW particularly well suited to a mid-scale screening approach (; Mendoza-Alatorre et al., 2025). Coding sequences (CDSs) derived from transcriptomic resources can be integrated into dsRNA selection and RNAi prediction pipelines, such as the dsRIP web platform, an online tool for predicting and optimizing dsRNA target regions (https://dsrip.uni-goettingen.de/) (), thereby facilitating the rapid identification and optimization of additional candidate RNAi targets for CSW.
The third option is large-scale screening, which involves testing thousands of genes across the genome using high-throughput techniques to identify those involved in essential biological processes such as development, reproduction, and survival, without relying on prior gene selection. This approach is best suited to model organisms with well-annotated genomes and extensive functional genomics resources, such as T. castaneum (Ulrich et al., 2015; Schultheis et al., 2019). Due to the early stage of RNAi research in CSW, large-scale screening is currently not feasible.
3 Challenges and opportunities for RNAi-based pest control in CSW
3.1 Barriers for RNAi
The effectiveness of RNAi for insect control can be impeded by numerous factors. Before reaching the target insect, the dsRNA can be degraded due to environmental factors such as ultraviolet radiations, high temperatures, microbial activities, or be diluted and dispersed by precipitation. These processes can reduce the amount of dsRNA available for exposure, potentially leading to low RNAi efficacy. Upon delivery of dsRNA, physical structures of the insect such as exoskeleton, gut lining, and cellular membranes can hinder the uptake and distribution inside the insect body (; Mendoza-Alatorre et al., 2025). Furthermore, variations in gut pH, nucleases in the saliva and gut, and presence of functional RNAi machinery can affect the integrity, uptake and processing of dsRNA (; Mendoza-Alatorre et al., 2025). Additionally, immune-driven degradation of dsRNA or compensatory backup gene activation can mitigate the consequences of RNAi in insects (Ortolá and Daròs, 2024; Mendoza-Alatorre et al., 2025). Assessing all potential barriers is essential for applying RNAi as a pest management strategy in CSW.
3.1.1 Environmental barriers
Even though coleopterans are, overall, highly sensitive to RNAi (), the instability of dsRNA in environmental conditions can considerably reduce its potential use in the field conditions (He et al., 2022). Besides the known environmental challenges, including UV rays, high temperatures, rainfall and microbial activity (Niu et al., 2024; Mendoza-Alatorre et al., 2025), the physicochemical properties of plant surfaces can further influence the persistence of topically applied dsRNA by affecting its stability, retention, and availability for insect uptake (; ; Hoang et al., 2022). CSW adults emerging in spring, feed on the flower buds of OSR plants, the surface characteristics and chemical composition of these tissues may also influence dsRNA persistence and bioavailability. Although glucosinolates are abundant secondary metabolites in OSR (Daun, 1986), and are particularly concentrated in floral tissues (Drozdowska and Rogozińska, 2013), whether they directly affect dsRNA stability remains unknown. Experimental studies are therefore needed to determine how the properties of OSR tissues influence dsRNA persistence and, ultimately, RNAi efficacy against CSW.
3.1.2 Uptake and translocation barriers
The efficacy of RNAi in coleopterans is significantly impacted by several intrinsic structures that affects the efficient transport, uptake, and systemic distribution of dsRNA. dsRNA has limited ability to penetrate insect cuticles due to their structural complexity and low permeability, posing a challenge for RNAi via topical application (Lu et al., 2023). In CSW, the chitinous exoskeleton and protective elytra may further reduce the efficiency of dsRNA delivery through this method. Upon ingestion, dsRNA encounters a first physical barrier in the gut environment: the peritrophic matrix, a semi-permeable sieve-like structure compartmenting the gut lumen and providing a protective barrier against abrasion and pathogens (Terra, 2001). The peritrophic matrix represents a significant barrier to dsRNA delivery, as its pore size restricts molecular passage and its negatively charged proteoglycans contribute to electrostatic repulsion, thereby limiting dsRNA penetration (Kunte et al., 2020). Several studies in coleopteran species have reported the presence of a peritrophic matrix in the gut (; Liu et al., 2014). Accordingly, CSW may also possess this structure, however, the fate of ingested dsRNA in the presence of the peritrophic matrix in this species remains unexplored. After passing through the peritrophic matrix, a second physical barrier is encountered: the epithelial cell membrane. This barrier is selective and can be crossed by specialized pathways, such as receptor mediated internalization, clathrin-dependent endocytosis or passive diffusion (Kunte et al., 2020; Pugsley et al., 2021; Darlington et al., 2022; He et al., 2022; Mendoza-Alatorre et al., 2025). However, due to its large size and negative charge, dsRNA molecules cannot easily enter the cell. In insects, dsRNA uptake typically occurs through clathrin-dependent endocytosis, facilitated by scavenger receptors or other membrane-associated proteins (Wytinck et al., 2020; He et al., 2022). While systemic RNAi defective (SID)-like proteins have been identified in several insects and are known to participate in dsRNA uptake and transport in other organisms, such as C. elegans, their direct role in insect cellular uptake and systemic RNAi remains uncertain (Vélez and Fishilevich, 2018; Darlington et al., 2022). Transcriptomic analysis of CSW identified putative systemic RNAi-associated genes, including silA, silB, silC, and RSD3, supporting the presence of molecular components potentially involved in dsRNA uptake and systemic RNAi in this species (Sabnis et al., 2026). After uptake through clathrin-dependent endocytosis, the dsRNA is located in endosomes. To be able to induce a RNAi response, the dsRNAs need to escape the endosomes before they are degraded due to fusion of the endosomes with lysosomes (Yoon et al., 2017; Kunte et al., 2020). This poses yet another barrier of RNAi. Variation in the efficiency of endosomal escape is observed between different insect species, with coleopteran insects generally exhibiting efficient endosomal release of dsRNA, whereas lepidopteran species tend to show limited or inefficient escape from endosomes (Shukla et al., 2016; Pugsley et al., 2021).
Systemic RNAi occurs when gene silencing initiated locally and spreads from cell to cell throughout the organism (Timmons and Fire, 1998). By extending gene silencing beyond the initial site of dsRNA uptake, effective systemic RNAi greatly increases the probability of mortality in the insects (Joga et al., 2016). Numerous studies in coleopteran insects have demonstrated that RNAi in this group is both efficient and systemic (Li et al., 2018; Yoon et al., 2020; Klingler and Bucher, 2022; Shimomura et al., 2025), providing a solid foundation for applying RNAi-based approaches in CSW. Consistent with these observations, transcriptomic analyses identified the core components of the RNAi machinery in CSW, and functional assays confirmed that dsRNA-mediated gene silencing induces substantial transcript knockdown and mortality in this species (Sabnis et al., 2026). Fragments of dsRNA or siRNA can cross epithelial barriers and enter the hemolymph, gaining access to distant cells for cytoplasmic entry and subsequent knock down of target genes (Maori et al., 2019; Mingels et al., 2020; Mendoza-Alatorre et al., 2025). The systemic distribution of dsRNA in insects is limited by multiple barriers, including the absence or malfunction of dsRNA transporters (Huvenne and Smagghe, 2010), structural constraints from extracellular matrices like the basal lamina (Shukla et al., 2016; Cooper et al., 2019), degradation by nuclease-rich hemolymph (Garbutt et al., 2013), and inefficient endosomal escape (Cooper et al., 2019), which together restrict dsRNA diffusion and intercellular transport, confining RNAi effects to targeted cells and limiting systemic spread (Mendoza-Alatorre et al., 2025).
3.1.3 Enzymatic barriers
In addition to the structural obstacles, RNAi in insects is frequently impeded by a number of biochemical barriers in the gut and hemolymph. The dsRNases in gut fluid, saliva and hemolymph can quickly break down dsRNA, reducing the amount of molecules for cellular uptake and gene downregulation (; Peng et al., 2020). Although dsRNase activity has been documented to diminish RNAi efficiency in certain coleopteran species, it is not consistently the primary limitation, as RNAi often remains effective despite the presence of these nucleases (Willow and Veromann, 2021). Apart from the enzymatic breakdown, pH of the gut and hemolymph can also contribute to the instability of dsRNA. Studies conducted on multiple insect species have shown that, hemolymph pH influences dsRNA stability and the effectiveness of RNAi machinery and high pH speeds up dsRNA degradation by increasing dsRNase activity in the gut (Peng et al., 2018; Kunte et al., 2020; He et al., 2022; Mendoza-Alatorre et al., 2025). Therefore, understanding the physiological pH conditions of CSW is crucial for designing effective RNAi-based pest management strategies. Multiple studies have confirmed the presence of core RNAi machinery in coleopteran insects (Tomoyasu et al., 2008; Petek et al., 2020; List et al., 2022); however, differences in how these components are expressed in different species can greatly affect the strength and reliability of gene silencing (Koo and Palli, 2025). Recent transcriptomic analyses have identified the core RNAi machinery in CSW, including genes involved in small RNA biogenesis, RISC assembly, and putative systemic RNAi (Sabnis et al., 2026). Nevertheless, further molecular characterization of the RNAi response, including analysis of RISC-associated small RNAs, is required to improve our understanding of dsRNA processing and the factors governing RNAi efficiency in this species.
3.1.4 Immunological barriers
Several immunological factors can also affect the efficiency of RNAi in insects. In addition to triggering RNAi-mediated gene silencing, dsRNA also stimulates immunological responses. For instance, in certain Diptera, Dicer-2 recognizes dsRNA, and triggers the janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway and immunological effectors such the Vago peptide (). In Lepidoptera, dsRNA can upregulate Dicer2 and modify Toll pathway genes which demonstrates the interaction between RNAi and immunological signaling (Liu et al., 2013). Furthermore, exposure to dsRNA can modulate the expression of antimicrobial peptides and activate distinct antiviral genes. These responses vary depending on cell type, growth stage, and species, all of which may influence the overall efficacy of gene silencing (Flenniken and Andino, 2013). CSW RNAi studies should account for potential immune responses, as dsRNA can trigger both gene silencing and activate immune pathways, which may influence RNAi efficacy.
Another important immunological factor is the availability of RNAi machinery for externally introduced dsRNA. The RNAi efficacy may be limited when the host is already burdened by the presence of viruses or transposable elements, which compete for the same silencing pathways (Swevers et al., 2013; Kontogiannatos et al., 2021). Specifically, either Dicer/RISC activity can be disrupted or dsRNA can be bound by viral suppressors of RNAi (Li et al., 2002; Qi et al., 2012; ). A recent study on T. castaneum has demonstrated that viral proteins are capable of suppressing the RNAi pathway (Kaufholz et al., 2024), providing a basis for investigating the complexities of RNAi pathways and their modulation by viral infections in CSW. Whether viral infections interfere with RNAi efficiency in CSW remains unknown and warrants further investigation.
The insect gut microbiota may also influence the outcome of RNAi treatment. Studies have shown that gut bacteria can contribute to RNAi-associated mortality by interacting with physiological changes following dsRNA ingestion rather than by directly enhancing gene silencing (Xu et al., 2021). In addition, changes in gut microbial composition may influence immune responses and other physiological processes that affect insect survival (Huang et al., 2025). Although the mechanisms underlying these interactions remain incompletely understood, investigating the gut microbiota of CSW and its response to dsRNA exposure could provide valuable insights into microbiome-mediated factors that influence RNAi efficacy in this species.
3.2 DsRNA delivery
As RNAi efficacy can be influenced by various barriers, selecting an appropriate dsRNA application strategy is crucial, especially in non-model insect species. dsRNA delivery methods including microinjection, oral ingestion, and topical application have been employed at different stages of RNAi research, offers valuable insights for developing effective RNAi-based strategies in CSW (Lu et al., 2023; ; Mendoza-Alatorre et al., 2025; Siddique et al., 2025). Microinjection is one of the most direct and reliable routes for exposing insects to dsRNA. Multiple studies on coleopterans have proven that microinjection is an efficient method in achieving effective gene knock-down (Pinheiro et al., 2020; Rasool et al., 2021; Willow et al., 2021; ). Through microinjection, dsRNA can be directly delivered into the hemolymph or certain target tissues, bypassing the barriers associated with oral uptake of dsRNA. In CSW, microinjection has recently been successfully used to validate RNAi functionality and evaluate candidate target genes (Sabnis et al., 2026), demonstrating its value as a laboratory tool for functional gene studies. However, because it is impractical for large-scale application, microinjection is primarily suited to laboratory-based functional studies rather than field-scale pest management.
Feeding-based approaches, such as Virus Induced Gene Silencing (VIGS), Spray-Induced Gene Silencing (SIGS), and Host-Induced Gene Silencing (HIGS) represent promising dsRNA delivery strategies for herbivorous insects (; Koch and Wassenegger, 2021; Pampolini and Rieske, 2023). Among these approaches, SIGS has emerged as a promising non-transgenic alternative to HIGS that may alleviate regulatory constraints associated with genetically modified crops while retaining the advantages of RNAi-based pest management (Koch and Wassenegger, 2021). However, the successful implementation of SIGS under field conditions depends on improving dsRNA stability and delivery, making formulation strategies such as nanoparticle-based carriers particularly important, as discussed in Section 3.3 (; Qiao et al., 2024). Despite the barriers associated with oral delivery, coleopteran species are highly sensitive to ingested dsRNA (Willow and Veromann, 2021; ). For example, the L. decemlineata was successfully managed by foliar spray application of Calantha (ledprona), an RNAi-based insecticide, in which dsRNA is ingested by feeding beetles, inducing gene silencing that disrupts protein processing and causes mortality (Pallis et al., 2023). Multiple studies in other coleopteran species have also reported significant results using dsRNA ingestion (; Willow et al., 2021). Adult CSW, which feed on buds, leaves and seedpods of cruciferous plants have well-developed chewing mouthparts, making them good candidates for oral dsRNA delivery (Dosdall and McFarlane, 2004; Gariepy et al., 2024). Since the eggs and subsequent larval stages of CSW develop within the seedpods, they are not directly accessible to environmentally applied dsRNAs. Delivery strategies that enhance the uptake and systemic movement of dsRNA within plant tissues (; Koch and Wassenegger, 2021) may improve the targeting of these concealed life stages.
Topical exposure is another potential dsRNA acquisition route, in which dsRNA is applied to the insect cuticle. This approach has been successfully demonstrated in soft-bodied insects, such as the yellow fever mosquito, Aedes aegypti (Hasselquist) (Diptera: Culicidae) (Pridgeon et al., 2008), the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) (Niu et al., 2019), and the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) (Finetti et al., 2023). While this indicates that topical application of dsRNA could potentially be used to suppress pest population, cuticular permeability poses a significant challenge in coleopteran species. In adult CSW, the presence of a chitinous exoskeleton and protective elytra can hinder dsRNA penetration and reduce topical RNAi efficiency. Although CSW larvae have a soft body structure, topical application is not feasible due to their location inside the seedpods, limiting direct exposure. Subsequently, the mature larvae burrow into the soil for pupation, which further complicates direct dsRNA application. In addition, dsRNA typically degrades in soil within two days, rendering soil-based delivery of naked dsRNA ineffective (Dubelman et al., 2014). Considering all developmental stages of CSW, adults remain the most accessible life stage for topical dsRNA application. Advanced dsRNA formulations that improve cuticular penetration represent a promising strategy for enhancing topical RNAi in several insect species (Yan et al., 2021; Lu et al., 2023). However, whether this approach is effective in CSW remains to be determined.
3.3 Use of formulations for improved dsRNA delivery
Recent advances have focused on addressing key barriers to effective RNAi, such as poor delivery of dsRNA, using nanoparticles to enhance its stability and uptake in cells (Qiao et al., 2024; Quilez-Molina et al., 2024). Nanoparticles employed for RNAi are classified according to their material composition, structural characteristics, and functional modifications as these parameters critically influence the stability, delivery efficiency, cellular targeting, and controlled release of dsRNA molecules (Pugsley et al., 2021; Yan et al., 2021; Mat Jalaluddin et al., 2023; ; Kallavus et al., 2026). Although nanoparticle-based formulations have shown considerable promise for improving dsRNA stability and delivery, their application in CSW may present unique challenges. Their effectiveness is dependent on not only the physicochemical properties of the nanoparticles but also on the physiological parameters of CSW, which can significantly influence RNAi outcomes (; Qiao et al., 2024). Even after surviving the gut environment, nanoparticles with specific sizes, surface charges, and chemical characteristics compatible with CSW’s endocytic pathways are necessary for effective cellular uptake (Ma et al., 2022; McGraw et al., 2022). Furthermore, certain nanoparticles may cause toxicity or other unexpected physiological effects (Martínez-Cisterna et al., 2024), or may not provide sufficient protection against nucleolytic degradation in the digestive tract and hemolymph (Rana et al., 2023). Studies on the T. castaneum may provide insights into nanoparticle-mediated dsRNA delivery in CSW, given their comparable RNAi responsiveness (; Edwards et al., 2020). In order to achieve target gene knock-down, nanoparticle formulations must be carefully developed, optimized, and tested on CSW because RNAi sensitivity varies greatly amongst insect species and their growth phases.
4 Safety considerations of RNAi in CSW
As with all new technologies, the use of RNAi-based products may pose certain risks for non-target organisms. Thorough knowledge of the RNAi mechanism and assessment of the associated biosafety risks is required to limit or mitigate potential adverse effects (; Gathman et al., 2025). Due to gaps in knowledge regarding RNAi in insects, in-depth discussion and analysis of important ecological and molecular questions influencing biosafety is needed. Ecological risk assessment requires a suite of reliable evaluation tools to assess potential unintended effects and ecotoxicological hazards (; ). These include sequence homology analyses for off-target prediction, laboratory toxicity and feeding bioassays using pollinators, parasitoids, and predatory insects, and environmental fate studies assessing dsRNA persistence in soil, water, and on plant surfaces, which together support tiered ecological risk assessment (; ). Recent studies on the southern pine beetle, Dendroctonus frontalis (Zimmermann) (Coleoptera: Curculionidae), demonstrated RNAi-based pest management can achieve a high degree of species specificity while minimizing adverse effects on non-target insects (Pampolini and Rieske, 2020; Hollowell and Rieske, 2022; Hollowell et al., 2023). Similar results were reported for B. viridescens, where dsRNA treatments caused insecticidal effects in the target species but showed no adverse effects on representative non-target organisms, including the parasitoid wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae), the bumblebee Bombus terrestris (Linnaeus) (Hymenoptera: Apidae), and the predator two-spotted lady beetle, Adalia bipunctata (Linnaeus) (Coleoptera: Coccinellidae) (Willow et al., 2025). Collectively, these studies demonstrate that RNAi-based pest management can achieve a high degree of target specificity while emphasizing the importance of comprehensive ecological risk assessment before field deployment. Despite these encouraging findings, the primary biosafety concern for RNAi-based management of CSW remains the possibility of unintentional gene silencing caused by incomplete sequence homology, which could result in off-target effects in non-target insects. Nanoformulations designed to stabilize dsRNA add further concerns, as they may prolong environmental exposure and introduce carrier-associated toxicities, thereby increasing uncertainty in safety assessments for non-target organisms (Dong et al., 2022; Yang et al., 2022; Quilez-Molina et al., 2024). Accordingly, the development and evaluation of nanoformulations for dsRNA delivery in CSW will require careful assessment to ensure their efficacy and environmental safety. While multiple studies indicate that unformulated dsRNA degrades rapidly in the environment, its persistence varies considerably depending on the composition and surrounding matrix (; Zarrabian and Sherif, 2024). A major threat that cannot be ignored is that certain species may become resistant to dsRNA, sometimes with no evident fitness impacts, which could reduce its long-term utility (Khajuria et al., 2018; Mishra et al., 2021; Liao et al., 2024). This risk is particularly relevant for CSW because limited knowledge of RNAi biology in this species makes it difficult to predict the mechanisms and ecological consequences of resistance evolution.
5 Logistical challenges in RNAi research of CSW
Laboratory-based research on CSW presents notable logistical challenges due to its complex life cycle, the seasonal availability of its damaging life stages, and the absence of standardized rearing protocols. The larval and pupal stages are particularly difficult to collect and maintain under laboratory conditions because larvae develop within seed pods, while pupation occurs several centimeters below the soil surface. Both stages require specific temperature, moisture, and humidity conditions, yet the precise environmental range necessary for their successful development remains undetermined. RNAi studies typically target larval and synchronized adult stages; however, due to the univoltine nature of CSW, these life stages are available only during limited periods in the field (Hussain et al., 2023). This seasonality constrains the number of individuals available for experimentation, limits replication, and impedes the continuous optimization of RNAi protocols. Effective RNAi research generally depends on the availability of a stable laboratory colony to ensure a consistent supply of insects at defined developmental stages. T. castaneum, (Gerken and Campbell, 2020) L. decemlineata (Gelman et al., 2001), and D. virgifera virgifera () have established rearing protocols, which have facilitated extensive and reproducible RNAi research. In contrast, no standardized rearing method currently exists for CSW, making researchers reliant on field-collected specimens. This reliance may introduce variability in age, physiological condition, and prior environmental exposure, potentially influencing RNAi efficacy and reproducibility. In the absence of a standardized rearing system, early RNAi studies on CSW are therefore likely to remain limited in experimental scope and developmental consistency, hindering progress toward protocol optimization.
6 Conclusion
Recent advances have established a foundation for the development of RNAi-based pest management in CSW through the generation of transcriptomic resources, the identification of promising candidate target genes, and functional validation of the RNAi pathway. However, continued efforts to identify and optimize highly effective target genes, together with improvements in dsRNA delivery and stability, will be essential for translating these advances into practical pest management strategies. For RNAi to effectively complement existing biological control agents, it is crucial to ensure that dsRNA treatments are safe for non-target organisms. Despite promising advances in target gene identification and dsRNA delivery technologies, RNAi research in CSW continues to face significant practical challenges that complicate experimental design and implementation. One of the major limitations is the availability of the insects. Currently, there is no established laboratory rearing protocol for CSW, making it difficult to maintain insect populations year-round. The lack of stable laboratory colonies, the seasonal nature of the species, and the short adult lifespan further hinder experimental design and replication. Establishing a reliable laboratory rearing protocol for CSW is needed for advancing RNAi research and optimization. Overcoming these logistical and biological constraints will facilitate the development of effective, practical, and environmentally sustainable RNAi-based approaches for managing CSW, an economically important pest of oilseed rape.
Statements
Author contributions
AS: Writing – original draft, Writing – review & editing. ÉA: Writing – review & editing. TK: Writing – review & editing. RK: Writing – review & editing. SV: Writing – review & editing. KD: Writing – original draft, Writing – review & editing. EV: Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Estonian Research Council grants PRG1056 and PRG3102, the Special Research Fund (BOF GOA, 01G01022) of Ghent University, and the Research Foundation Flanders (FWO-Vlaanderen).
Acknowledgments
This work was supported by the Estonian Research Council grants PRG1056 and PRG3102, the special Research Fund (BOF GOA, 01G01022) of Ghent University and Research Foundation Flanders (FWO-Vlaanderen). The authors acknowledge the assistance of ChatGPT (GPT-5, OpenAI, 2025) in generating the conceptual diagram in Figure 1.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was used in the creation of this manuscript. AI was used in generating the conceptual diagram in Figure 1 and for language editing (grammar and clarity).
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Summary
Keywords
cabbage seedpod weevil, Curculionidae, dsRNA, oilseed rape, pest control biotechnology
Citation
Sabnis AV, Avila dos Santos É, Kallavus T, Kaasik R, Vilumets S, De Schutter K and Veromann E (2026) RNAi-based management of Ceutorhynchus obstrictus: opportunities, challenges, and research priorities. Front. Agron. 8:1819085. doi: 10.3389/fagro.2026.1819085
Received
27 February 2026
Revised
13 July 2026
Accepted
20 July 2026
Published
31 July 2026
Volume
8 - 2026
Edited by
Jaime Cesar Pinero, University of Massachusetts Amherst, United States
Reviewed by
Jian-Hua Zhao, Chinese Academy of Sciences (CAS), China
Shakil Ahmad, Guangdong Academy of Science (CAS), China
Updates
Copyright
© 2026 Sabnis, Avila dos Santos, Kallavus, Kaasik, Vilumets, De Schutter and Veromann.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Apurva Vikas Sabnis, apurva_vikas.sabnis@emu.ee; ApurvaVikas.Sabnis@UGent.be
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