Chitosan is a polycationic polysaccharide that occurs naturally and is produced when chitin is partially deacetylated (Figure 1). Chitosan has several physicochemical characteristics, including molecular weight, viscosity, degree of deacetylation, and crystallinity (Kas, 1997; Riseh et al., 2022). A primary amine group with a pKa value of around 6.5 is present in every deacetylated subunit of chitosan; as a result, chitosan is soluble in acidic pH, like acetic acid but insoluble in neutral and alkaline pH. The amount of chitosan© deacetylation, molecular weight, ionic strength of the solution, and pH significantly impact its solubility (Mao et al., 2010). Chitosan dissolved in acetic acid and spontaneous mechanical churning at room temperature leads to caused nanoparticles. In addition, adjusting the chitosan-to-stabilizer ratio altered the particle size and surface charge (Hosseini et al., 2015). Several methods have been described for synthesizing RCNPs, such as electrostatic interaction, encapsulation, and adsorption (Figure 1). When chitosan is dissolved in acidic circumstances, the degree of chitosan deacetylation (DCDA) value influences the positive charge density; more DCDA results in an enhanced positive charge, allowing a better dsRNA/siRNA binding capacity (Liu et al., 2007; Mao et al., 2010). The ionotropic gelation method uses the electrostatic contact between a negatively charged group of nucleotides (e.g., in dsRNA) and the amine group of chitosan and self-assembled to form the polyelectrolyte complex (PEC) as a result of a decrease in hydrophilicity caused by charge neutralization between the cationic polymer and dsRNA. When dsRNA is added to chitosan (in acetic acid) solution and with continuous stirring at room temperature, the RCNPs can be produced spontaneously (Figure 1). Also, chitosan© molecular weight affects the physicochemical characteristics of RCNPs, including their size, zeta potential, shape, and complex stability (Mao et al., 2010). The surface charge of the RCNPs is dependent on the molar ratio of chitosan nitrogen (N) to dsRNA phosphate (P) (N/P ratio), which affects the particle capacity to efficiently condense dsRNA and interact with negatively charged cells, which in turn affects the transfection efficiency (Köping-Höggård et al., 2001; Huang et al., 2005; Jeong et al., 2007; Nafee et al., 2007). The chitosan salt form also impacted the RCNPs, such as chitosan glutamate, which had a larger molecular weight, created smaller complexes with dsRNA/siRNA, and had a higher siRNA loading efficiency than chitosan hydrochloride (Katas and Alpar, 2006). The amount of dsRNA at a certain point within the RCNPs plays a fundamental role in host cell transfection efficiency, whereas more concentration of dsRNA will increase the diameter of the particles and form an aggregation, and will decline the transfection (MacLaughlin et al., 1998; Romøren et al., 2003; Zhao et al., 2006; Mao et al., 2010). Chitosan can be employed as a dsRNA-chitosan complex as well as a coating material for symbiotic microbes that express dsRNA to provide a flexible technology platform for the management of forest insect pests (Figure 1; Mao et al., 2010; Joga et al., 2021; Riseh et al., 2022).

RNAi-chitosan nanopesticides extend to precision use due to their minuscule dimensions, high surface area, enhanced permeability, thermal stability, dispersion, and biodegradability to improve forest yield and to control target action based on insect pests or microbes infection (Figure 1; Adisa et al., 2019; Kumar et al., 2019). For applying RCNPs in forestry, several methods like foliar application, trunk injection, and soil drenching can be considered (Figure 1; Joga et al., 2021; Mogilicherla et al., 2022). Chitosan nanoparticle-mediated RNAi has been developed over the last 10 years as an alternative to traditional pest control methods (Table 1).

The formulations of RCNPs have significant potential to control the attack of several common pests like aphids, moths, and beetles (Sahab et al., 2015; Gurusamy et al., 2020a). Silencing of the CHS1, CHS2, semaphorin-1a, and vestigial (vg) genes by feeding chitosan-dsRNA nanoparticles to mosquitoes (Anopheles gambiae and Aedes aegypti) showed more pesticide-susceptible (Zhang et al., 2010, 2015; Mysore et al., 2013; Kumar et al., 2016). Our group and colleagues successfully knocked down the target genes (CAD, AMN, CHS, JHAMT, AMY, V-ATPase, IAP1, V-ATPase B, Sec23, SNF7, and SRC) using chitosan-dsRNA nanoparticles and observed decent mortality in A. aegypti and Spodoptera frugiperda (Das et al., 2015; Gurusamy et al., 2020a). Also, the complexes of chitosan-sodium tripolyphosphate-dsRNA (CS-TPP-dsRNA) showed improved mortality in A. aegypti (Dhandapani et al., 2019). In another study, the chitosan-dsRNA nanopesticides showed good stability, cellular uptake, and mortality in Chilo suppressalis (Wang et al., 2020). Helicoverpa armigera was significantly controlled when RCNPs were applied topically to chickpea plants (Kolge et al., 2021). Additionally, RCNPs were stable for 5 days on leaf surfaces, effectively protected from nuclease degradation and insect gut pH, and efficiently knocked down the targeted genes (JHAMT and AChE), resulting in 100% insect mortality, whereas the non-targeted insects like Spodoptera litura and Drosophila melanogaster were unaffected and showed no signs of toxicity (Kolge et al., 2021, 2023). A recent study demonstrated that topically applying dsRNA-coated with rosin-modified PEG and chitosan (dsRNA/ROPE@C) to Nilaparvata lugens (Brown plant hopper) causes excellent gene knockdown and mortality (Lyu et al., 2023). Recently, our team created chitosan-dsRNA nanopesticides, fed them to bollworms (Earias vittella), and observed considerable target gene knockdown and mortality (Sandal et al., 2023). Additionally, the price drop from $12500 to $2 for 1 g of dsRNA has increased the likelihood that RNAi technology will be applied in the field (Zotti et al., 2018). Our colleagues successfully applied bacterially expressed dsRNA in a tropical setting and observed a significant reduction in Colorado potato beetle (CPB) infection (Máximo et al., 2020; Petek et al., 2020). Most recently, researchers developed an RNAi-based biopesticide known as “ledprona” against the CPB, which inhibits enzyme expression, facilitates protein breakdown, and ultimately causes mortality (Pallis et al., 2023). These investigations could pave the path for creating and using RCNPs as a safe, effective, and novel way to protect crops and forest trees.

Furthermore, researchers used the chitosan domain to encapsulate metal-based nanoparticles (Ag, Au, Fe, Co, Cu, TiO2, ZnO, SiO2, and CaCO3) to increase plant resilience to salt, drought, and heavy metal environments (Souri et al., 2017; Behboudi et al., 2019; Sen et al., 2020; Ali et al., 2021; Sheikhalipour et al., 2021) and improve their health for protecting themselves from other biotic stresses (Naidu et al., 2023). The previous studies successfully used double-layered hydroxide (LDH), carbon quantum dots (CQD), branched amphiphilic peptide capsules (BAPCs), and lipid nanoparticle-based dsRNA formulations to address biotic stress caused by insects (Mitter et al., 2017; Christiaens et al., 2020; Gurusamy et al., 2020b; Kaur et al., 2020). Such findings encourage researchers to adopt similar approaches to improve forest health. However, the above-mentioned nanomaterials have some limitations, i.e., manufacturing synthetic nanomaterials is expensive, and excessive nanoparticle concentrations may negatively impact forest soil health and microfauna. Dedicated studies can evaluate the feasibility of these nanoparticles in forest protection.

Chitosan is frequently utilized as a carrier for encasing microbial agents because of its ability to take the form of particles, films, capsules, gels, fibres, and porous forms and its unquestionable success in field applications (Lakkis, 2016; Saberi Riseh et al., 2021). Three potential methods (diffusion, osmotic burst, and erosion or breakdown) will work separately or together and release the microbial substances from chitosan encapsulations. Encapsulating chitosan-microbes (chitosan-ATCC393 and chitosan-139S1) can protect against several environmental challenges (Li et al., 2011; Vejan et al., 2019). Moreover, the Harpinpss-chitosan, BMI-chitosan, B. thuringiensis-chitosan, B. cereus-chitosan, E. fergusonii-chitosan, B. thuringiensis-chitosan, and Pseudomonas-chitosan encapsulations tested on tomato, soybean, cotton, tobacco, bean, corn, and Hyaloptera peroni plants showed a reduction in egg-laying in female insects, thereby reducing the population and insect damage (Badawy and El-Aswad, 2012; Zeng et al., 2012; Chandrashekharaiah et al., 2015; Sahab et al., 2015; Badawy and Rabea, 2016; Kitherian, 2017; Ureña Saborío et al., 2017; Nadendla et al., 2018; De Oliveira et al., 2021). Based on the aforementioned findings, RNAi molecules expressed in microbes that can be encapsulated with chitosan are a viable technology and can be used as RNAi-biopesticides in forest pest management (Figure 1). However, such potential demands further dedicated studies and pilot field experiments.

SMR is a potent tool, and researchers have developed endogenous symbionts to express target dsRNAs for insect control (Chen et al., 2015; Hu and Wu, 2016; Whitten et al., 2016; Hu and Xia, 2019). Recent research identified the bacterial symbionts, used them to express dsRNA effectively, and controlled the two evolutionarily divergent insect species (R. prolixus and F. occidentalis) (Whitten et al., 2016). Additionally, entomopathogenic fungi were identified and used to induce fungal-induced gene silencing (FIGS) in the insects B. tabaci and L. migratoria (Chen et al., 2015; Hu and Xia, 2019). Our colleagues from the United States are deploying fungal-induced gene silencing (FIGS) technology to manage bark beetles, i.e., genetically modifying the bark beetle-associated yeast Ogataea pini, to generate specific dsRNA molecules that target Ips calligraphus (information based on personal communication). Our team has also successfully identified and isolated insect-symbiotic bacteria and fungi (Chakraborty et al., 2020a,b, 2023b) and may use them as a CSMR for tropical application to control the bark beetles and termites (Gupta et al., 2023). Recently, our group identified 69 core bacterial genera and 19 fungal genera among six bark beetles (Ips typographus, Ips duplicatus, Ips cembrae; Ips sexdentatus, Ips acuminatus, and Polygraphus poligraphus). Notably, the most abundant bacterial genera were Erwinia, Sodalis, Serratia, Tyzzerella, Raoultella, Rahnella, Wolbachia, Spiroplasma, Vibrio, and Pseudoxanthomonas whereas the most abundant fungal genera belong to the phylum Ascomycota (Chakraborty et al., 2020a,b, 2023a). Further, our group focused on exploring how varying ages of Norway spruce wood and different terpene concentrations affect the microbial compositions associated with two termite species, Reticulitermes flavipes and Microcerotermes biroi (Chakraborty et al., 2023b). In termite-infested wood samples, the relative abundance of bacterial genera like Pseudomonas, Massilia, and Rhizobium was high, and Spirochaeta and Treponema revealed notable changes in relative abundance between these two species. Moreover, within termite-infested wood, fungal communities affiliated with the Eurotiales, Sordariales, Hypocreales, Trichospornales, and Ophiostomatales orders were identified, notably, the fungal genera Apiotrichum, Fusarium, Hawksworthiomyces, Lasiodiplodia, Sporothrix, Trichosporon, and Trichoderma displayed substantial prevalence in the termite-infested wood. As described thoroughly in our recent review, some identified microbial associates of bark beetle or termites can be good candidates for Symbiont-mediated RNAi or SMR (Gupta et al., 2023). Nevertheless, SMR technology can be considered for its potential in forest conservation; additional refinements are necessary before applications.

In order to increase forest production and health, RCNPs will be utilized more frequently in forestry and dispersed into the environment. Due to their biodegradable nature, these substances may not harm non-target organisms; they may not bioaccumulate and not interact with other environmental contaminants and dissolved organic matter, which means they will not harm the environment as well as humans and animals (Chandy and Sharma, 1990; Aspden et al., 1997; Rao and Sharma, 1997). RNAi-biopesticides made of chitosan are easily dissolved in nature and unable to accumulate in the food chain, stunt plant growth, or potentially harm people and animals. Although studies have shown that nanoparticles pose a risk to the environment, they have also sparked much interest in environmental cleanup (Roy et al., 2021). Therefore, more research is necessary to comprehend RCNPs and their relationship to the environment thoroughly. Understanding the characteristics of various RNAi-chitosan bioformulation is crucial, as is making comparisons between pure active ingredients and both nanoformulations and traditional formulations to see how the behaviour of the active components changes (Kah et al., 2018).

Applications for RCNP in the forest have numerous obstacles, including developing different delivery strategies for various microbes and insect pests, facilitating plant uptake and in planta systemic movement of RCNPs, looking for synergistic effects, such as dsRNAs targeting multiple genes and combining RNAi with other pest control methods, and establishing a congruent confluence, building a regulatory framework that is widely accepted for the commercialization, therefore lowering the price for their widespread use. Recently, RCNPs were used in the field, and the result demonstrated their compact size, cationic charge, effective loading, resistance to degradation, effective cellular uptake, stability, and adhesion to leaf surfaces (Petek et al., 2020; Kolge et al., 2021, 2023). Few RNAi-based insecticides have so far been licensed and will soon be available on the market (Li et al., 2023; Pallis et al., 2023).

The advancement of RNAi-nanotechnology has been beneficial to forestry. However, strict controls are in place for forests to ensure the security of feed and food sources, possible risks to human and animal health, non-target organisms and beneficial microbes, and the long-term environmental effects of the deliberate release of RNAi-nanomaterials (Kumar et al., 2019; Gilbertson et al., 2020; Hofmann et al., 2020; Mogilicherla et al., 2022). The European Union is developing regulatory rules for engineered RNAi-nanomaterials in forestry food safety (Lowry et al., 2019). Preparing regulatory guidelines for RNAi-nanomaterials is more difficult due to several factors, including the difficulty in defining nanomaterials, tracing their sources and transport pathways, quantifying them in environmental samples, assessing their bioavailability, and interpreting their toxicity (Lai et al., 2018; Hofmann et al., 2020; Gottardo et al., 2021). Under such circumstances, creating cutting-edge analytical methods for regulatory purposes is necessary.

The capacity of researchers and scientists to develop forest pest-specific dsRNAs will increase as more omics data for forest insects, helpful microorganisms, and non-target organisms become accessible and help to reduce possible risks. Fortunately, our group and CZU colleagues (CZU, Prague) have recently involved forest insects (bark beetles and termites) genome and transcriptome sequencing and their symbiotic microbes transcriptome sequencing, which along with other excellent efforts from colleagues worldwide, will significantly enhance sequence information on forest insect pests and facilitate future species-specific RNAi-based biopesticides development.