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 mechanisms underlying the evolution of morphological novelties have remained enigmatic but co-option of existing gene regulatory networks (GRNs), recruitment of genes and the evolution of orphan genes have all been suggested to contribute. Here, we study a morphological novelty of beetle pupae called gin-trap. By combining the classical candidate gene approach with unbiased screening in the beetle , we find that 70% of the tested components of the wing network were required for gin-trap development. However, many downstream and even upstream components were not included in the co-opted network. Only one gene was recruited from another biological context, but it was essential for the anteroposterior symmetry of the gin-traps, which represents a gin-trap-unique morphological innovation. Our data highlight the importance of co-option and modification of GRNs. The recruitment of single genes may not be frequent in the evolution of morphological novelties, but may be essential for subsequent diversification of the novelties. Finally, after having screened about 28% of annotated genes in the genome to identify the genes required for gin-trap development, we found none of them are orphan genes, suggesting that orphan genes may have played only a minor, if any, role in the evolution of gin-traps.

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum , as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ∼8,500 and ∼5,000 genes, respectively, of a total of ∼16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other arthropods. To help closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-GFP tester strains.Injections into larvae were followed by the analysis of the adult thoracic muscle patterns, and injections into pupae by the analysis of the late embryonic/larval muscle patterns. Herein we focus on the results of our pupal injection screen for late embryonic/larval muscle phenotypes, which covered 8.500 of a total of ~16.500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded larval body wall and leg muscle phenotypes. We present the main candidates that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.