The Sex-Specific Splicing of Doublesex in Brine Shrimp Artemia franciscana.

The understanding of sex determination and differentiation in animals has recently made remarkable strides through the use of advanced research tools. At the gene level, the Mab-3-related transcription factor (Dmrt) gene family, which encodes for the typical DNA-binding doublesex/Mab-3 (DM) domain in their protein, is known for its contribution to sex determination and differentiation in insects. In this study, DNA-binding DM domain screening has identified eight transcripts from Artemia franciscana transcriptomic that encode proteins containing one conserved DNA-binding DM domain. The genome mapping confirmed that these eight transcripts are transcribed from six different loci on the A. franciscana genome assembly. One of those loci, the Af.dsx-4 locus, is closely related to Doublesex, a gene belonging to the Dmrt gene family. This locus could be transcribed into three alternative transcripts, namely Af.dsx4, Af.dsxF and Af.dsxM. While Af.dsx4 and Af.dsxF could putatively be translated to form an identical Af.dsxF protein of 186 aa long, Af.dsxM translates for an Af.dsxM protein of 289 aa long but shares a DNA-binding DM domain. Interestingly, Af.dsxF and Af.dsxM are confirmed as sex-specific transcripts, Af.dsxF is only present in females, and Af.dsxM is only present in male individuals. The results suggest that the sex-specific splicing mechanism of the doublesex described in insects is also present in A. franciscana. Af.dxs-4 locus can be used in further studies to clarify the sex determination pathways in A. fracnciscana.

Risk assessment of RNAi-based pesticides to non-target organisms: Evaluating the effects of sequence similarity in the parasitoid wasp Telenomus podisi.

RNA interference (RNAi)-based pesticides are promising novel pest management products that might reduce environmental impacts compared to other pesticides. Their sequence-guided mode of action facilitates a high species-selectivity, preventing harm on non-target organisms. However, there is currently no consensus on the minimum needed sequence similarity for efficient RNAi in insects and studies have shown that adverse effects in non-targets cannot always be ruled out a priori. This study investigates the effects of exposing the parasitoid wasp Telenomus podisi to double-stranded RNA (dsRNA) which is lethal to its host, the Neotropical brown stink bug Euschistus heros. Feeding T. podisi with wasp-specific dsRNA targeting the vATPase A and actin-2 genes led to 76.4 ± 9.9% and 76.7 ± 8.8% mortality respectively, demonstrating that dietary RNAi is functional in T. podisi. When feeding T. podisi with E. heros-specific dsRNA targeting the same genes, no lethal or sublethal effects were observed. To link sequence similarity to potential gene silencing effects in the parasitoids, the expression of genes showing the highest degree of similarity (17-21 nucleotide matches) with these two target genes was monitored and was found unaffected by the E. heros-specific dsRNA. Our study confirms that RNAi was in this case highly specific and that for E. heros, RNAi-based pesticides can be used complementary to biological control in an integrated pest management context.

Identification and Full Characterisation of Two Novel Crustacean Infecting Members of the Family Nudiviridae Provides Support for Two Subfamilies.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein.

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

RNAi efficacy is enhanced by chronic dsRNA feeding in pollen beetle.

Double-stranded RNAs (dsRNAs) represent a promising class of biosafe insecticidal compounds. We examined the ability to induce RNA interference (RNAi) in the pollen beetle Brassicogethes aeneus via anther feeding, and compared short-term (3 d) to chronic (17 d) feeding of various concentrations of dsRNA targeting αCOP (dsαCOP). In short-term dsαCOP feeding, only the highest concentration resulted in significant reductions in B. aeneus survival; whereas in chronic dsαCOP feeding, all three concentrations resulted in significant mortality. Chronic dsαCOP feeding also resulted in significantly greater mortality compared to short-term feeding of equivalent dsαCOP concentrations. Our results have implications for the economics and development of dsRNA spray approaches for managing crop pests, in that multiple lower-concentration dsRNA spray treatments across crop growth stages may result in greater pest management efficacy, compared to single treatments using higher dsRNA concentrations. Furthermore, our results highlight the need for research into the development of RNAi cultivars for oilseed rape protection, given the enhanced RNAi efficacy resulting from chronic, compared to short-term, dsRNA feeding in B. aeneus.

Involvement of clathrin-dependent endocytosis in cellular dsRNA uptake in aphids.

RNAi is an essential technology for studying gene function in eukaryotes, and is also considered to be a potential strategy for pest control. However, the mechanism behind the cellular uptake of dsRNA in aphids, a group of important agricultural sucking pests, remains unknown. Here, using the pea aphid Acyrthosiphon pisum as model for aphids, we identified two core genes of clathrin-dependent endocytosis (CDE), Apchc and Apvha16. We confirmed that expression of Apchc, Apvha16 and RNAi core component genes (ApAgo2, ApDcr2 and ApR2d2) were simultaneously induced at 12 h after feeding dsRNA. By using an RNAi-of-RNAi approach, we demonstrated that suppression of Apchc and Apvha16 transcripts by RNAi significantly impaired RNAi efficiency of selected reporter genes (RGs), including ApGNBP1, Apmts and Aphb, suggesting the involvement of CDE in cellular dsRNA uptake in aphids. Further confirmation was also provided using two inhibitors, chlorpromazine (CPZ) and bafilomycin A1 (BafA1). Administration of CPZ and of BafA1 both led to an impaired silencing efficiency of the RGs in the pea aphid. Finally, these RNAi-of-RNAi results were reconfirmed in the peach aphid Myzus persicae. Taking these findings together, we conclude that CDE is involved in cellular dsRNA uptake in aphids.

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezaraviridula.

Variability in RNA-interference (RNAi) efficacy among different insect orders poses a big hurdle in the development of RNAi-based pest control strategies. The activity of double-stranded ribonucleases (dsRNases) in the digestive canal of insects can be one of the critical factors affecting oral RNAi efficacy. Here, the involvement of these dsRNases in the southern green stinkbug Nezaraviridula was investigated. First, the full sequence of the only dsRNase (NvdsRNase) in the transcriptome of N. viridula was obtained, followed by an oral feeding bioassay to evaluate the effect of NvdsRNase-silencing on oral RNAi efficacy. The NvdsRNase was first silenced in nymphs by NvdsRNase-dsRNA injections, followed by exposure to an artificial diet containing a lethal αCop-specific dsRNA. A significantly higher mortality was observed in the NvdsRNase-silenced nymphs when placed on the dsαCop-containing diet (65%) than in the dsGFP injected and dsαCop fed control (46.67%). Additionally, an ex vivo dsRNA degradation assay showed a higher stability of dsRNA in the saliva and midgut juice of NvdsRNase-silenced adults. These results provide evidence for the involvement of NvdsRNase in the reduction of oral RNAi efficacy in N. viridula. This information will be useful in further improving potential RNAi-based strategies to control this pest.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9-16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3-4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

RNAi-mediated mortality in southern green stinkbug Nezara viridula by oral delivery of dsRNA.

BACKGROUND: The southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae), is an important emerging polyphagous pest infesting soybean in the United States, Brazil and Argentina. The indiscriminate use of synthetic insecticides to control stinkbugs has limited the effectiveness of current management strategies. Alternatively, RNA interference (RNAi) has emerged as a novel mode of action to control pests in an eco-friendly manner. RESULTS: Here, we assessed the potential of RNAi technology by oral delivery of double-stranded RNA (dsRNA) for the control of N. viridula. Initially, ten candidate genes were tested by microinjection assay to select the best target genes for oral delivery. Seven genes resulted in more than 90% mortality after microinjection. To evaluate RNAi efficacy by oral delivery of dsRNA, five genes were tested by feeding the insects on gene-specific dsRNA mixed with an artificial diet. Significant mortality of 43% and 45% was observed after 14 days of treatment with dsαCop and dsvATPase A, respectively. To elucidate the lower RNAi efficacy via oral delivery of dsRNA, ex vivo dsRNA degradation in the saliva and the midgut juice was performed, which indicated that the reduced RNAi efficacy is accompanied by a rapid degradation of dsRNA by digestive secretions. CONCLUSION: This study proves that RNAi can be triggered by orally delivered dsRNA in N. viridula and can be exploited to control this economically important pest. The reduced stability of dsRNA in saliva and midgut that was observed indicates a need to further improve RNAi efficacy, for example by use of specific formulations.

Parental RNA interference as a tool to study genes involved in rostrum development in the Neotropical brown stink bug, Euschistus heros.

In insects, the identity of body segments is controlled by homeotic genes and the knockdown of these genes during embryogenesis can lead to an abnormal development and/or atypical phenotypes. The main goal of this study was to investigate the involvement of labial (lab), deformed (dfd), sex comb reduced (scr), extradenticle (exd) and proboscipedia (pb) in rostrum development in the Neotropical brown stink bug Euschistus heros, using parental RNAi (pRNAi). To achieve this objective, 10-days-old adult females were first microinjected with double-stranded RNAs (dsRNA) targeting these five genes. Then, the number of eggs laid per female, the percentage of hatched nymphs with normal or abnormal phenotype and target gene silencing were evaluated. Except for the dsDfd-treatment, the number of eggs laid per female per day was not affected by the different dsRNA-treatments compared to the control (dsGFP). However, treatment with either dsLab, dsDfd, dsScr or dsExd caused a strong reduction in egg hatching. The dsExd-treatment caused no apparent change in phenotype in the nymphs while hatched nymphs from the dsDfd, dsScr and dsPb-treatment showed abnormalities in the rostrum. Particularly for the dsPb-treatment, 91% of the offspring displayed a bifurcated rostrum with a leg-like structure. Overall, these results indicate that these five genes are involved in E. heros embryonic development and that the knockdown of dfd, scr and pb leads to an abnormal development of the rostrum. Additionally, this study demonstrates the efficiency of pRNAi in studying genes involved in embryogenesis in E. heros, with clear phenotypes and a strong target gene silencing in the next generation, after treatment of the parent female adult with gene-specific dsRNA.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

First Evidence of Bud Feeding-Induced RNAi in a Crop Pest via Exogenous Application of dsRNA.

Spray-induced gene silencing (SIGS) is a potential strategy for agricultural pest management, whereby nucleotide sequence-specific double-stranded RNA (dsRNA) can be sprayed onto a crop; the desired effect being a consumption of dsRNA by the target pest, and subsequent gene silencing-induced mortality. Nucleotide sequence-specificity is the basis for dsRNA's perceived biosafety. A biosafe approach to pollen beetle (Brassicogethes aeneus) management in oilseed rape (Brassica napus) agroecosystems is needed. We examined the potential for SIGS in B. aeneus, via bud feeding, a field-relevant dsRNA exposure route. Oilseed rape buds were uniformly treated with dsRNA designed to target αCOP in B. aeneus. Our model control dsRNA (dsGFP) remained detectable on buds throughout the entire 3 d exposure period. When applied at 5 µg/µL, dsαCOP induced significant αCOP silencing 3 d after dietary exposure to buds treated with this dsαCOP concentration. We also observed a trend of increased αCOP silencing with increasing concentrations of dsαCOP at both 3 and 6 d. Furthermore, we observed a marginally significant and significant reduction in B. aeneus survival at 10 and 15 d, respectively. Our results suggest potential for developing a SIGS approach to B. aeneus management-though further experiments are needed to more fully understand this potential.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

RNAi in Insects: A Revolution in Fundamental Research and Pest Control Applications.

In this editorial for the Special Issue on 'RNAi in insect pest control', three important applications of RNA interference (RNAi) in insects are briefly discussed and linked to the different studies published in this Special Issue. The discovery of the RNAi mechanism revolutionized entomological research, as it presented researchers with a tool to knock down genes, which is easily applicable in a wide range of insect species. Furthermore, RNAi also provides crop protection with a novel and promising pest control mode-of-action. The sequence-dependent nature allows RNAi-based control strategies to be highly species selective and the active molecule, a natural biological molecule known as double-stranded RNA (dsRNA), has a short environmental persistence. However, more research is needed to investigate different cellular and physiological barriers, such as cellular uptake and dsRNA degradation in the digestive system in insects, in order to provide efficient control methods against a wide range of insect pest species. Finally, the RNAi pathway is an important part of the innate antiviral immune defence of insects, and could even lead to applications targeting viruses in beneficial insects such as honeybees in the future.

Biosafety of GM Crop Plants Expressing dsRNA: Data Requirements and EU Regulatory Considerations.

The use of RNA interference (RNAi) enables the silencing of target genes in plants or plant-dwelling organisms, through the production of double stranded RNA (dsRNA) resulting in altered plant characteristics. Expression of properly synthesized dsRNAs in plants can lead to improved crop quality characteristics or exploit new mechanisms with activity against plant pests and pathogens. Genetically modified (GM) crops exhibiting resistance to viruses or insects via expression of dsRNA have received authorization for cultivation outside Europe. Some products derived from RNAi plants have received a favourable opinion from the European Food Safety Authority (EFSA) for import and processing in the European Union (EU). The authorization process in the EU requires applicants to produce a risk assessment considering food/feed and environmental safety aspects of living organisms or their derived food and feed products. The present paper discusses the main aspects of the safety assessment (comparative assessment, molecular characterization, toxicological assessment, nutritional assessment, gene transfer, interaction with target and non-target organisms) for GM plants expressing dsRNA, according to the guidelines of EFSA. Food/feed safety assessment of products from RNAi plants is expected to be simplified, in the light of the consideration that no novel proteins are produced. Therefore, some of the data requirements for risk assessment do not apply to these cases, and the comparative compositional analysis becomes the main source of evidence for food/feed safety of RNAi plants. During environmental risk assessment, the analysis of dsRNA expression levels of the GM trait, and the data concerning the observable effects on non-target organisms (NTO) will provide the necessary evidence for ensuring safety of species exposed to RNAi plants. Bioinformatics may provide support to risk assessment by selecting target gene sequences with low similarity to the genome of NTOs possibly exposed to dsRNA. The analysis of these topics in risk assessment indicates that the science-based regulatory process in Europe is considered to be applicable to GM RNAi plants, therefore the evaluation of their safety can be effectively conducted without further modifications. Outcomes from the present paper offer suggestions for consideration in future updates of the EFSA Guidance documents on risk assessment of GM organisms.

Exploration of the virome of the European brown shrimp (Crangon crangon).

Crangon crangon is economically a very important species. Recently, promising culture attempts have been made, but a major problem is the uncontrollable mortality during the grow-out phase. As of yet, the life cycle of C. crangon is not closed in captivity so wild-caught individuals are used for further rearing. Therefore, it is important to investigate the virome of C. crangon both in wild-caught animals as in cultured animals. In recent years, next-generation-sequencing (NGS) technologies have been very important in the unravelling of the virome of a wide range of environments and matrices, such as soil, sea, potable water, but also of a wide range of animal species. This will be the first report of a virome study in C. crangon using NGS in combination with the NetoVIR protocol. The near complete genomes of 16 novel viruses were described, most of which were rather distantly related to unclassified viruses or viruses belonging to the Picornavirales, Bunyavirales Nudiviridae, Parvoviridae, Flaviviridae, Hepeviridae, Tombusviridae, Narnaviridae, Nodaviridae, Sobemovirus. A difference in virome composition was observed between muscle and hepatopancreatic tissue, suggesting a distinct tissue tropism of several of these viruses. Some differences in the viral composition were noted between the cultured and wild shrimp, which could indicate that in sub-optimal aquaculture conditions some viruses become more abundant. This research showed that a plethora of unknown viruses is present in C. crangon and that more research is needed to determine which virus is potentially dangerous for the culture of C. crangon.

Double-Stranded RNA Technology to Control Insect Pests: Current Status and Challenges.

Exploiting the RNA interference (RNAi) gene mechanism to silence essential genes in pest insects, leading to toxic effects, has surfaced as a promising new control strategy in the past decade. While the first commercial RNAi-based products are currently coming to market, the application against a wide range of insect species is still hindered by a number of challenges. In this review, we discuss the current status of these RNAi-based products and the different delivery strategies by which insects can be targeted by the RNAi-triggering double-stranded RNA (dsRNA) molecules. Furthermore, this review also addresses a number of physiological and cellular barriers, which can lead to decreased RNAi efficacy in insects. Finally, novel non-transgenic delivery technologies, such as polymer or liposomic nanoparticles, peptide-based delivery vehicles and viral-like particles, are also discussed, as these could overcome these barriers and lead to effective RNAi-based pest control.

RNA-based biocontrol compounds: current status and perspectives to reach the market.

Facing current climate challenges and drastically reduced chemical options for plant protection, the exploitation of RNA interference (RNAi) as an agricultural biotechnology tool has unveiled possible new solutions to the global problems of agricultural losses caused by pests and other biotic and abiotic stresses. While the use of RNAi as a tool in agriculture is still limited to a few transgenic crops, and only adopted in restricted parts of the world, scientists and industry are already seeking innovations in leveraging and exploiting the potential of RNAi in the form of RNA-based biocontrol compounds for external applications. Here, we highlight the expanding research and development pipeline, commercial landscape and regulatory environment surrounding the pursuit of RNA-based biocontrol compounds with improved environmental profiles. The commitments of well-established agrochemical companies to invest in research endeavours and the role of start-up companies are crucial for the successful development of practical applications for these compounds. Additionally, the availability of standardized guidelines to tackle regulatory ambiguities surrounding RNA-based biocontrol compounds will help to facilitate the entire commercialization process. Finally, communication to create awareness and public acceptance will be key to the deployment of these compounds. © 2019 Society of Chemical Industry.

Generation of Virus- and dsRNA-Derived siRNAs with Species-Dependent Length in Insects.

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals.