Forests are of immense importance due to their socio-economic and ecosystem services (Pan et al., 2013). However, a decline in conifer forests is ongoing worldwide at an unprecedented rate due to a rise in temperature, drought stress, windthrows, and pest infestation (Safranyik et al., 2010; Hlásny et al., 2019). Most of the insect pest population outbreaks are temperature-dependent, and climate-driven intensification in the frequency, severity and cyclicity of forest pest outbreaks is already well documented (Bentz et al., 2010, 2019; Cudmore et al., 2010; de la Giroday et al., 2012; Haynes et al., 2014; Bentz and Jönsson, 2015). Besides, forest insects have expanded their geographic range by exploiting native hosts previously unexplored due to low temperature (Williams and Liebhold, 2002; Cipollini and Rigsby, 2015; Ramsfield et al., 2016). Such range expansion also causes widespread tree mortality, decreasing forest productivity and carbon storage and substantially enhancing discharge from the decay of dead tree woods (Kurz et al., 2008). Thus, severe depletion of trees due to pest outbreaks or range expansion may cause trajectories outside the resilience limits of forest ecosystems resulting in irreversible ecosystem regime shifts (Dhar et al., 2016).

Coleopteran forest pests such as Emerald ash borer (Agrilus planipennis, EAB), Asian longhorn beetles (Anoplophora glabripennis, ALB) and bark beetles took advantage of ongoing climate change and cause severe damage to the forests worldwide (Aukema et al., 2011; Meng et al., 2015; Hlásny et al., 2019). For instance, EAB is a devastating, tree-killing phloem-feeding beetle from northeastern Asia recently invaded North America through solid wood packaging material (Poland and McCullough, 2006). EAB has already killed millions of North American Ash (Fraxinus sp.) and became one of the costliest insect pest invaders in American history (Aukema et al., 2011). EAB larvae disrupt the translocation of essential nutrients and water in the infested plants while feeding on phloem tissues, leading to the death of the Ash trees within 3–4 years of infestation (Haack et al., 2002). Management of these notorious tree killers is a daunting task, and superior methods can bring hope. ALB, similar to EAB, is native to China and Korea and is a globally recognized invader with a history of attacking more than 100 different species of trees (Haack et al., 2010; Meng et al., 2015). Estimation of loss due to ALB infestation would be a staggering $889 billion if the ALB population left uncontrolled (McKenna et al., 2016). Interestingly, trunk injection of systemic insecticides such as imidacloprid was documented effective against ALB infestation. However, the cost and environmental impact of deploying chemical pesticides jeopardize such strategies and call for better alternatives for ALB management.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are the most severe and destructive pests of conifer forests worldwide (Fairweather, 2006; Keeling et al., 2016; Hlásny et al., 2019). Since abiotic factors are primary drivers for bark beetle population growth (Biedermann et al., 2019), outbreaks of these aggressive forest pests are expected to increase frequency and severity due to ongoing climate change (Kurz et al., 2008). Warming temperatures promote bark beetle population growth due to reduced winter mortality and development time by allowing additional generations per year. A recent study featuring a tree-ring iso-demographic approach further supports the notion that temperature is more critical than drought for amplifying the eruptive bark beetle outbreaks (Pettit et al., 2020). Most bark beetle species breed on weak and dead trees during an endemic stage serving a crucial function in the forest ecosystem by recycling the nutrients from the dead plant tissues. However, once the bark beetle population increases to an epidemic level, they start attacking the healthy trees leading to an outbreak (Fairweather, 2006; Boone et al., 2011; Hlásny et al., 2019). Currently, frequent outbreaks have been a major disturbing factor for conifer forests in Europe and North America (Bentz et al., 2010; Meddens et al., 2012; Hicke et al., 2016; Hlásny et al., 2019; Lubojacký, 2019) that affects forest ecosystem functioning (Grégoire et al., 2015; Thom and Seidl, 2016), climate and carbon loss mitigation, water retention (Grégoire et al., 2015) and country economy via losses in timber and tourism revenue (Holmes, 1991; SFA, 2010; Pye et al., 2011; Arnberger et al., 2018; Cahyanto et al., 2018). Some aggressive bark beetles, such as the southern pine beetle (Dendroctonus frontalis) and the mountain pine beetle (D. ponderosae), undergo a substantial range expansion in the US due to favorable warmer climate and cause frequent outbreaks leading to catastrophic tree loss (Regniere, 2003; Chen and Goodwin, 2011; Lesk et al., 2017).

Several conventional approaches such as sanitation felling (Wermelinger, 2004; Seidl et al., 2016), removal of wind felled trees (Leverkus et al., 2018), and deployment of pheromone-baited and poisoned log tripod traps (Wermelinger, 2004) is used for the last few decades to manage the bark beetle population levels in endemic phase. However, the success of all these approaches is questionable in managing the recent bark beetle outbreaks (Billings, 2011; Hlásny et al., 2019). Furthermore, similar to other wood-boring forest insects, several synthetic pesticides have also been used to suppress bark beetles over the past years (Williamson and Vité, 1971). However, many of these compounds caused other problems such as environmental pollution, detrimental effects on non-target organisms, and widespread pesticide resistance (Feder, 1979; Baum et al., 2007; Billings, 2011). Therefore, questions have been raised about the feasibility, effectiveness, and purpose of conventional phytosanitary measures. Hence, novel and aggressive management of these devastating coleopteran wood-boring forest pests is the highest priority in the Anthropocene.

RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism, which is triggered by exogenous double-stranded RNA (dsRNA) (Fire et al., 1998; Zhu and Palli, 2020). Thus, RNAi becomes a promising tool for forest pest management in this era of genomics (Baum et al., 2007). Recent advancements in sequencing technology and platforms lead to higher availability of coleopteran forest pest genomes and transcriptomes that can serve as valuable resources for species-specific dsRNA design (Keeling et al., 2012, 2013; Scully et al., 2013; McKenna et al., 2016; Powell et al., 2020). It was pretty well known that coleopteran insects are usually susceptible to RNAi (Figure 1; Baum et al., 2007; Zhu et al., 2011; Palli, 2014; Prentice et al., 2015; Ulrich et al., 2015; Fishilevich et al., 2016; Li et al., 2018a; Bramlett et al., 2020; Mehlhorn et al., 2021; Willow et al., 2021). Recently, Yoon et al. (2018) reported the underlying cause of coleopteran insect susceptibility toward RNAi. With intriguing evidence of RNAi susceptibility in coleopterans, researchers started exploring the potential of RNAi in managing coleopteran forest pests (Table 1). Recent high-quality publications demonstrated the entomotoxicity of RNAi against wood-boring coleopteran forest pests such as southern pine beetle, mountain pine beetles, emerald ash borer, Asian longhorn beetles, and Chinese White pine beetle (Rodrigues et al., 2017a, b; Rodrigues et al., 2018; Kyre et al., 2019; Dhandapani et al., 2020a, b; Kyre et al., 2020). However, the potential of RNAi in coleopteran forest pest management is not yet comprehensively summarized elsewhere. Hence, it is essential to capture all aspects of such studies together and critically evaluate the future potential of RNAi against wood-boring coleopteran pest management. The current review focuses on synthesizing key challenges for RNAi-mediated forest pest management (Table 2). It can also serve as a valuable source of information for general foresters, private forest owners, forest managers, and researchers worldwide who are currently using RNAi or planning to use RNAi as a tool against coleopteran pests inside the forests.

RNAi refers to a post-transcriptional gene silencing mechanism prohibiting protein formation by introducing environmental RNA (Fire et al., 1998). Three RNAi pathways have been characterized so far. These are common in insects but not in plants or other animals and include the small interfering RNA (siRNA) pathways, microRNA (miRNA) pathway, and piwiRNA (piRNA) pathway. However, siRNAs are highly sequence-specific to target transcripts, and miRNAs are partial complementarity to target transcripts (Lam et al., 2015; Zhu and Palli, 2020). In contrast to siRNAs and miRNAs, the piRNAs pathway is likely less understood (Farazi et al., 2008).

The RNAi technology application depends on introducing dsRNA into the insect pest body to silence a target gene, subsequently activating the siRNA pathway. Briefly, upon entry of exogenous dsRNA into the cell, the dsRNA is processed into siRNAs by an enzyme ribonuclease III, called Dicer-2. These siRNAs (21–24 nucleotide duplexes) are incorporated in the silencing complex, called the RNA-induced silencing complex (RISC), where the siRNA duplex is unwound. Subsequently, a protein called Argonaute2 (AGO2) cleaves the sense (passenger) strand, and the antisense (guide) strand remains connected with the RISC. Afterward, the antisense strand of the siRNA guides the RISC and allows base pairing to the complementary target mRNA. Subsequently, AGO2 protein degrades or cleaves the target mRNA, and specific post-transcriptional gene silencing occurs (Agrawal et al., 2003; Pecot et al., 2011).

Wood-boring insects thrive inside the bark, trunk on a nutritionally limiting diet. They tunnel in the inner layer where water and nutrients are available. They attack either healthy or weakened and dead trees based on their statuses like primary invader or secondary invader. It is worth mentioning that primary invaders (i.e., Ips typographus, Eurasian spruce bark beetle) mostly kill the infested tree. However, most often, the damage caused by an infestation of wood borers remains unnoticed until the tree showed visible symptoms or external signs of damage, such as the entry hole of a wood borer or sawdust (Hlásny et al., 2019). This hidden lifestyle of wood borers, contrary to most other agricultural pests, causes considerable impediments for control measures, even for RNAi-based FPPs. Perhaps choosing a suitable strategy to deliver dsRNA is a big challenge in RNAi-based forest protection methods (Table 2). Several possible dsRNA application strategies are available that can be deployed against forest pests. They are transiently transformative (recombinant symbiont or virus) and non-transformative (nanoparticles, trunk injections and spraying, root soaking, and soil drench) methods (Figure 2). Due to the transient feature of the molecules used in non-transformative delivery methods, target pests have limited exposure to the dsRNA molecules, delaying the resistance development. Deploying the RNAi based Plant-incorporated protectants (RNAi-PIPs) via transgenic trees (transformative approach) seems to be a less viable solution against wood-boring forest pests due to public and scientific concern (i.e., gene flow), lack of suitable tree transformation protocols, high development time and cost, and extensive regulatory processes (Cagliari et al., 2019). Hence, in the present review, we omitted the discussion on the transformative approaches. However, it is worth mentioning here that researchers already developed efficient and stable plastid transformation protocols for poplar, which can be considered for developing RNAi-PIPs against pests infesting the green tissues of poplar (Wu et al., 2019).

RNAi has proved the potential to suppress pests and save beneficial insects from diseases and parasites. For example, a non-target organism screening with the dsRNA-based biocontrol product targeting L. decemlineata revealed the selectivity and safety of the dsRNA sequence even for closely related species and beneficial insects (Bramlett et al., 2020). Similarly, a genome-wide off-target screen in important bumblebee pollinators of Bombus terrestris with dsRNA targeting pollen beetle αCOP revealed no reduction in the transcript level for all putative off-targets, including an off-target with a 20-continuous-nucleotide match (Taning et al., 2021). Also, for a set of potential targets in the EAB, off-target effects were screened. After confirming the dsRNA’s specificity, they are qualified as potential targets to suppress EAB populations (Rodrigues et al., 2018). However, deploying RNAi-based FPPs presents unique challenges for ecological, environmental, and human risk assessments.

In our opinion, for forestry application, safety assessments should include evaluating environmental safety for the NTOs (Figure 3B). Due to its rapid environmental degradation, the trees treated with the exogenous applications of dsRNAs to control forest pests will not be considered genetically modified organisms (GMOs) (Shew et al., 2017; Arpaia et al., 2021). However, it is significant to follow biosafety assessments for FPPs before deployment. The noticeable effects on non-target organisms exposed to RNAi will provide significant evidence for ensuring safety. Bioinformatics tools perform an essential role in the development of species-specific targets (Figure 3A). However, the availability of a limited number of genome sequences from forest pests limits risk estimates. Furthermore, the effects of dsRNA on soil and other plant-associated beneficial microorganisms need to be evaluated to measure the effect of dsRNA in forest ecosystems accurately (Figure 3B).

The deployment of genetically engineered bacteria, fungi, or viruses capable of delivering the RNAi-based FPPs in the forest needs to be under some regulatory framework as they will also be considered GM products. Unfortunately, there is no risk assessment protocol for genetically engineered microorganisms delivering dsRNA (in short, RNAi-microbes) so far as RNAi-based GM crops (Papadopoulou et al., 2020). One putative reason is that the deployment of RNAi-microbes is an intriguing idea (i.e., SMR) that has just begun to blossom (Zhang et al., 2019). Regulatory agencies worldwide need to devise the environmental risk assessment protocol dedicated to deploying RNAi-microbes in the forest. In our opinion, RNAi-microbes can be evaluated for their mechanism of action, specificity (including the designing the dsRNA), active ingredient, environmental fate, ecotoxicology [impact on humans, other NTOs (including soil organisms), other microbes in the microhabitat, soil and water], Toxicity-Exposure-Ratio (TER), off-site movement, the requirement of the periodic application, immune response and resistance management in target pest while formulating the legal framework relating to the risk of dsRNA-based FPPs. Bioinformatics tools can assist in the selection of surrogate species for tiered toxicology testing. Nevertheless, RNAi pesticides occur naturally inside target organisms and thus a potentially safer alternative to synthetic pesticides.

RNAi is a robust technology that can bring a new paradigm in forest pest management, but some obstacles yet limit its implementation. Researchers are developing a variety of methods for boosting RNAi in wood-boring coleopterans, i.e., selection of appropriate target genes by using sequencing data, genetic modification of microbes and plants, identifying the components of extracellular vesicles, dsRNA complexation/encapsulation with nanomaterials (Sinisterra-Hunter and Hunter, 2018; Cooper et al., 2019; Christiaens et al., 2020a). Furthermore, forest protection with RNAi-based pesticides would be a novel integrated pest management strategy (IPM) due to its high sequence-dependent specificity and better safety than conventional pesticides (Wang et al., 2016; Mitter et al., 2017; Christiaens et al., 2020a; Taning C.N. et al., 2020). Hence, the researchers are focusing on controlling forest pests by using this technology, and its potential to control several coleopteran wood-boring forest pests such as bark beetles, EAB, ALB is already experimentally proven (Rodrigues et al., 2017a, b, 2018; Kyre et al., 2019, 2020; Dhandapani et al., 2020a).

However, the dsRNA production cost is still remarkably higher, although dsRNA production costs were lowered to 2 USD per gram in 2017 from 12,500 USD per gram back in 2008 (Zotti et al., 2018). The current dsRNA production capacity may be increased dramatically soon to produce vaccines against the COVID-19 (SARS-CoV2) pandemic to the global community. Furthermore, the same vaccine production platforms could be converted to produce large-scale RNAi-based pesticides, enabling dsRNA-based pesticides to be much cheaper (Taning C.N.T. et al., 2020).

It is a well-known phenomenon that the symbiont, blue-stain fungi pave the way for the successful colonization of coniferous bark beetles by acting as a source of bark beetles semiochemicals (Kandasamy et al., 2016), depletes spruce defense chemicals (Lahr and Krokene, 2013), and provide nutrient supplements (Bentz and Six, 2006; Six and Wingfield, 2011; Davis et al., 2019; Six, 2020; Six and Elser, 2020). Likewise, fungi also benefit from bark beetles getting inoculated into the phloem as they cannot penetrate bark alone (Franceschi et al., 2000). Thus, it concludes that the conifer bark beetles and their symbiont collectively causing to extensive Norway spruce forest mortality. For that reason, the researchers should work simultaneously on controlling symbionts and bark beetles by using the RNAi tool. Most interestingly, in our current research, we have seen that the RNAi is functional and highly efficient against the European spruce bark beetle, Ips typographus, in laboratory conditions (unpublished results).

Foliar application of RNAi-based pesticides may not be applicable for controlling the wood-boring insect pests due to the size of the trees and the presence of thick outer bark. In comparison, the delivery of dsRNA pesticides through the host such as trunk injection, soil drench, symbiotic microorganisms of plant and target pest, and viruses may promise the long-lasting protection of trees from insect pests and pathogens.

Restricting the off-target and non-target effects would be challenging with RNAi-based FPPs. However, species-specific and target-specific RNAi targets have to be identified for the effectiveness of this technology (Christiaens et al., 2018). A bioinformatics pipeline can be helpful here in finding HDRs in the target pest. However, the genome sequences of forest pests will be prerequisites for such strategies. Hence, research endeavors toward more forest insect genomes and tissue-specific transcriptomes are necessary for the future to obtain superior species-specific targets for dsRNA applications.

Lastly, the resistance against dsRNA-based FPPs would be a critical barrier for the deployment of this strategy. For example, Khajuria et al. (2018) reported that WCR got resistant upon exposure of several generations to DvSnf7 dsRNA. Insect pests could evolve resistance in different possible ways. For instance, mutations of target and RNAi core machinery genes decreased dsRNA uptake and increased dsRNA degradation (Zhu and Palli, 2020). However, changes in the target gene selection could help delay the resistance, and delivery of dsRNA through nanoparticles and liposomes could improve the efficacy, stability, and dsRNA uptake mechanism by gut epithelial cells (Wytinck et al., 2020). The careful optimization of target gene selection, dsRNA design, synthesis and delivery, nanocarriers or symbiotic microbes, or virus-induced gene silencing can be highly effective for suppressing and causing mortality in forest insect pest species RNAi. However, field tests, environmental safety, and non-target effects are lacking for these efforts, and optimization is necessary for producing various RNAi-based FPPs. In addition, researchers must take the initiative to create a perception of RNAi-based FPPs to state forest agencies, forest owners, and general foresters to facilitate its deployment for long-term forest protection. Nevertheless, RNAi-based FPPs in conjugation with existing forest pest management practices (i.e., silvicultural, biological) can aid a multi-faceted management approach that keeps the tree-killing forest pest populations in the endemic stage while conserving the beneficial species.

AR conceptualized the manuscript structure. MJ, KM, and AR wrote the first draft and did the revision. GS commented on the initial draft and revised the manuscript. All the authors approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.