What Are the Functional Roles of Piwi Proteins and piRNAs in Insects?

Research on Piwi proteins and piRNAs in insects has focused on three experimental models: oogenesis and spermatogenesis in Drosophila melanogaster, the antiviral response in Aedes mosquitoes and the molecular analysis of primary and secondary piRNA biogenesis in Bombyx mori-derived BmN4 cells. Significant unique and complementary information has been acquired and has led to a greater appreciation of the complexity of piRNA biogenesis and Piwi protein function. Studies performed in other insect species are emerging and promise to add to the current state of the art on the roles of piRNAs and Piwi proteins. Although the primary role of the piRNA pathway is genome defense against transposons, particularly in the germline, recent findings also indicate an expansion of its functions. In this review, an extensive overview is presented of the knowledge of the piRNA pathway that so far has accumulated in insects. Following a presentation of the three major models, data from other insects were also discussed. Finally, the mechanisms for the expansion of the function of the piRNA pathway from transposon control to gene regulation were considered.

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.

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.

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.

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.

Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo.

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60-73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA.

Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges.

RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed.

Asian Citrus Psyllid RNAi Pathway - RNAi evidence.

Diaphorina citri, known as the Asian citrus psyllid, is an important pest of citrus because it transmits a phloem-limited bacteria strongly implicated in huanglongbing (citrus greening disease). Emerging biotechnologies, such as RNA interference, could provide a new sustainable and environmentally friendly strategy for the management of this pest. In this study, genome and functional analysis were performed to verify whether the RNAi core genes are present in the Asian psyllid genome and if the RNAi machinery could be exploited to develop a management strategy for this pest. Analyses of RNAi-related genes in the Asian citrus psyllid genome showed an absence of sequences encoding R2D2, a dsRNA-binding protein that functions as a cofactor of Dicer-2 in Drosophila. Nevertheless, bioassays using an in Planta System showed that the Asian citrus psyllid was very sensitive to ingested dsRNA, demonstrating a strong RNAi response. A small dose of dsRNA administered through a citrus flush was enough to trigger the RNAi mechanism, causing significant suppression of the targeted transcript, and increased psyllid mortality. This study provides evidence of a functional RNAi machinery, which could be further exploited to develop RNAi based management strategies for the control of the Asian citrus psyllid.