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

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3 0 UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR-mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.

Drosophila rhabdomeric terminal photoreceptor differentiation is an extended process taking several days to complete. Following ommatidial patterning by the morphogenetic furrow, photoreceptors are sequentially recruited and specified, and terminal differentiation begins. Key events of terminal differentiation include establishment of apical and basolateral domains, rhabdomere and stalk formation, inter-rhabdomeral space formation, and expression of phototransduction machinery. While many key regulators of these processes have been identified, the complete network of transcription factors to downstream effector molecules necessary for regulating each of these major events remains incomplete. Here we report an RNAi screen to identify additional molecules and cellular pathways required for photoreceptor terminal differentiation. First, we tested several eye-specific GAL4 drivers for correct spatial and temporal specificity and identified Pph13-GAL4 as the most appropriate GAL4 line for our screen. We screened lines available through the Transgenic RNAi Project (TRiP) and isolated lines that when combined with Pph13-GAL4 resulted in the loss of the deep psuedopupil, as a readout for abnormal differentiation. In the end, we screened 6,189 lines, representing 3,971 genes, and have identified 64 genes, illuminating potential new regulatory molecules and cellular pathways for the differentiation and organization of Drosophila rhabdomeric photoreceptors.

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3’ UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. For each construct, we were able to generate a line that is homozygous viable, though variable reductions in reproductive success with each construct were noted. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR-mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.