A collaboration of six German research groups has recently published their joint effort of a large-scale RNAi screen in the red flour beetle Tribolium castaneum. Within the iBeetle project, scientists from the Universities of Göttingen, Erlangen and Cologne describe the effects of the knock-down of 3400 genes, discovering new factors that play roles in embryonic and adult patterning, metamorphosis and wing development. This opens the way to hypothesis independent gene identification in an insect apart from Drosophila – going beyond the candidate gene approach, opening new fields of research and using the beetle as alternative screening platform.
This post was authored by Dr. Johannes Schinko and Anna Gilles, Institut de Génomique Fonctionnelle de Lyon (IGFL), France and contributed by IGTRCN Participant Dr. Gregor Bucher
The iBeetle screen comprises the results of two parallel screening procedures: a pupal screen, where dsRNA (double stranded RNA) was injected into female pupae. This screen recovered genes that have an effect on late metamorphosis and oogenesis (in the injected animals), as well as embryonic development (in the offspring). In the larval screen, late larvae (stage L6) were injected so that effects on metamorphosis could be investigated. Moreover, the adult odiferous glands were analyzed for developmental abnormalities. These organs were of special interest since many insects use similar glands for intraspecific communication or defense against predators.
While the pupal screen is still ongoing and has until today covered 5300 genes, the larval screen has been concluded with a total of 4480 genes investigated. The paper presents the joint results of both screens for 3400 overlapping candidates. Around half of these genes (56.3%) yielded a phenotype in either screen. Interestingly, 13% of all the genes that gave a phenotype were specific for coleopterans, thus producing an interesting set of candidates of around 250 genes that evolved within this order.
The project has overcome current limitations in insect science on three levels:
First, testing candidate genes identified in Drosophila screens in other species has been a very successful approach but has its limitations. Namely, it will not reveal genes acting in other species but not the fly. One exciting example is the knock-down phenotype of the gene Tc-homeobrain, which results in a mirror-image duplication of the abdomen. Tribolium researchers interested in early embryonic patterning had unsuccessfully tried to identify a gene producing this phenotype relying on the candidate gene approach. However, since Drosophila homeobrain does not play a role in embryonic patterning, it was never included on their lists. This gene as well as other phenotypes recovered in the screen clearly illustrate the limitations of the candidate gene approach and the need for unbiased approaches like iBeetle.
Second, some aspects of insect biology cannot be studied in Drosophila simply because of the enormous diversity insects and insect biology. Insect scientists need an alternative model with comprehensive screening possibility. As an example, the authors detect a number of genes required for development and physiology of stink glands. Such glands play important roles in several insect taxa (think of the bombardier beetles) but Drosophila does not have them.
Third, the use of Drosophila as the only screening platform may prohibit the identification of important genes relevant in insects generally. As an example, a set of 49 genes showed wing blister phenotypes. This was characterized before in Drosophila and is a sign of defective adhesion between epithelial sheets. Several of these candidates were subsequently tested in Drosophila, and four of these produced wing phenotypes that were previously unknown in this species. Using Tribolium as a screening platform complementary to Drosophila thus helps to uncover new genes that play a role in cell biology.
While funding for research projects in fundamental research is getting more and more sparse, iBeetle is a refreshing example of a project opening up many exciting lines of research beyond conventional model organisms like Drosophila. Importantly, as the RNAi templates are generated and are available to the community, additional large scale screens are within reach.
Schmitt-Engel, C., Schultheis, D., Schwirz, J., Strohlein, N., Troelenberg, N. et al., 2015 The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun 6: doi: 10.1038/ncomms8822.