Mating system and genetic variation impede the spread of gene drives, which target natural populations of disease-vectoring insects.

The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. However, many standard genetic tools are lacking or underdeveloped in this system. Here, we present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that labels cell membranes and nuclei with different fluorophores for the analysis of cellular morphology. This approach also demonstrates the functionality of the viral T2A peptide for bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve as a blueprint for the further development of this powerful model’s genetic toolkit.

22The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. 23However, many standard genetic tools are lacking or underdeveloped in this system. Here, we 24 present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a 25 new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression 26 of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that 27 labels cell membranes and nuclei with different fluorophores for the analysis of cellular 28 morphology. This approach also demonstrates the functionality of the viral T2A peptide for 29 bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines 30 with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color 31 loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, 32 the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve 33 as a blueprint for the further development of this powerful model's genetic toolkit. 34 35

Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum, we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of CRISPR/Cas9-based synthetic gene. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many diseasevectoring arthropods. We conclude that designing such a drive will require characterization of genetic variability and the mating system within and among targeted populations.