The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata , we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.

INTRODUCTION: Tribolium castaneum is a small, low-maintenance beetle that has emerged as a sophisticated model system for studying the evolution of development and that complements (in some cases, even rivals) Drosophila for functional genetic analysis of basic biological questions. Although Tribolium and Drosophila are both holometabolous insects, they differ fundamentally in larval and adult morphology. Even generally conserved developmental features, such as body segmentation, are achieved by quite different means. Thus, comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Genetic tools available for Tribolium include genetic maps for visible and molecular markers, chromosomal rearrangements that enable lethal mutations to be balanced in true-breeding stocks, transposon-based transformation systems, a completed and annotated genome sequence, and systemic RNA interference (RNAi), which makes it possible to knock down any given gene and even particular splice variants in the offspring or in any tissue of the injected animal. Inactivating gene functions at various developmental stages provides new opportunities to investigate post-embryonic development, as well as larval and adult physiology, including hormonal control, host-parasite interactions, and pesticide resistance.

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.