In recent years, the research on the potential of using RNA interference (RNAi) to suppress crop pests has made an outstanding growth. However, given the variability of RNAi efficiency that is observed in many insects, the development of novel approaches toward insect pest management using RNAi requires first to unravel factors behind the efficiency of dsRNA-mediated gene silencing. In this review, we explore essential implications and possibilities to increase RNAi efficiency by delivery of dsRNA through non-transformative methods. We discuss factors influencing the RNAi mechanism in insects and systemic properties of dsRNA. Finally, novel strategies to deliver dsRNA are discussed, including delivery by symbionts, plant viruses, trunk injections, root soaking, and transplastomic plants.

This report is the outcome of an EFSA procurement (OC/EFSA/GMO/2015/02) which aims at reviewing relevant scientific information on RNA interference (RNAi) that could serve as baseline information for the environmental risk assessment of RNAi‐based genetically modified (GM) plants. The report is based on a systematic literature search on the use of RNAi molecules in arthropods, nematodes, annelids and molluscs with dsRNA, siRNA and miRNA applied primarily through feeding and soaking (oral ingestion). The numbers of retrieved publications covering these areas are reported, along with the species name, life stages tested, the target gene and its function, details of the test substances and their concentrations used, methods of delivery and effects. Separate sections discuss the available information on: (1) the uptake and systematic spread of RNAi activity, including a description of the various components involved in this process; (2) the mechanisms of dsRNA‐, siRNA‐ and miRNA‐elicited gene silencing and the different factors involved in RNAi efficiency; (3) routes of exposure of the biotic and abiotic environment to dsRNA, siRNA and miRNA from GM plants; (4) the environmental fate of dsRNA, siRNA and miRNA; and (5) the various factors that may limit non‐target effects including exposure, factors influencing the silencing efficiency of dsRNA, siRNA and miRNA, possible unintended and off‐target effects, and their mechanisms. Finally, an overview of the species of arthropods, nematodes, annelids and molluscs for which genomic data are available is also presented. The report identifies some of the challenges involved in developing plants with RNAi systems which affect invertebrate gene expression. The report also concludes that, currently, knowledge on issues such as exposure, specificity, offtarget effects, sequence similarities and bioinformatics is very limited, as only a few RNAi expressing plants which specifically target invertebrate species have been developed and comprehensively studied.

Graphical AbstractThree-tier impact of forest protection products (FPPs).

Eurasian spruce bark beetle (Ips typographus [L.]) causes substantial damage to spruce forests worldwide. Undoubtedly, more aggressive measures are necessary to restrict the enduring loss. Finishing genome sequencing is a landmark achievement for deploying molecular techniques (i.e., RNA interference) to manage this pest. Gene expression studies assist in understanding insect physiology and deployment of molecular approaches for pest management. RT-qPCR is a valuable technique for such studies. However, accuracy and reliability depend on suitable reference genes. With the genome sequence available and the growing requirement of molecular tools for aggressive forest pest management, it is crucial to find suitable reference genes in Ips typographus under different experimental conditions. Hence, we evaluated the stability of twelve candidate reference genes under diverse experimental conditions such as biotic (developmental, sex and tissues) and abiotic factors (i.e., temperature and juvenile hormone treatment) to identify the reference genes. Our results revealed that ribosomal protein 3a (RPS3-a) was the best reference gene across all the experimental conditions, with minor exceptions. However, the stability of the reference gene can differ based on experiments. Nevertheless, present study provides a comprehensive list of reference genes under different experimental conditions for Ips typographus and contributes to “future genomic and functional genomic research”.