Background: RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

BackgroundInsect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening.ResultsWe use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.ConclusionsOur results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1880-y) contains supplementary material, which is available to authorized users.

The iBeetle-Base (http://ibeetle-base.uni-goettingen.de) makes available annotations of RNAi phenotypes, which were gathered in a large scale RNAi screen in the red flour beetle Tribolium castaneum (iBeetle screen). In addition, it provides access to sequence information and links for all Tribolium castaneum genes. The iBeetle-Base contains the annotations of phenotypes of several thousands of genes knocked down during embryonic and metamorphic epidermis and muscle development in addition to phenotypes linked to oogenesis and stink gland biology. The phenotypes are described according to the EQM (entity, quality, modifier) system using controlled vocabularies and the Tribolium morphological ontology (TrOn). Furthermore, images linked to the respective annotations are provided. The data are searchable either for specific phenotypes using a complex ‘search for morphological defects’ or a ‘quick search’ for gene names and IDs. The red flour beetle Tribolium castaneum has become an important model system for insect functional genetics and is a representative of the most species rich taxon, the Coleoptera, which comprise several devastating pests. It is used for studying insect typical development, the evolution of development and for research on metabolism and pest control. Besides Drosophila, Tribolium is the first insect model organism where large scale unbiased screens have been performed.

INTRODUCTIONTribolium castaneum is exceptionally amenable to gene knockdown by RNA interference (RNAi) which, in this insect, is systemic (spreading throughout the organism and to the next generation), highly penetrant, and able to phenocopy genetic null phenotypes. Hence, any gene function can be knocked down at any stage in (apparently) all tissues upon injection of double-stranded RNA (dsRNA). The RNAi effect is elicited both in the injected animal and, if female pupae or adults have been injected, transferred to the offspring. Embryonic RNAi (eRNAi) usually generates the strongest phenotypes in the injected individual, but suffers from elevated lethality caused by injection injury. Pupal RNAi (pRNAi), in which female pupae are injected and phenotypes scored in the offspring, is the easiest to perform. However, in some cases, the knockdown of a gene leads to sterility of the injected female. This problem can be circumvented in many cases by injecting adult females (aRNAi) or using eRNAi. In order to interfere with processes during metamorphosis, injection into last-stage larvae is used (lRNAi). Up to two genes in a single experiment have been successfully knocked down via RNAi. The inclusion of more than two genes usually leads to a dilution effect, which lowers phenotypic strength. This protocol describes the production of dsRNA from a polymerase chain reaction (PCR) template, injection procedures for each Tribolium life stage, and important controls for effective analysis.