The red flour beetle T. castaneum: elaborate genetic toolkit and unbiased large scale RNAi screening to study insect biology and evolution

Abstract

The red flour beetle Tribolium castaneum has emerged as an important insect model system for a variety of topics. With respect to studying gene function, it is second only to the vinegar fly D. melanogaster. The RNAi response in T. castaneum is exceptionally strong and systemic, and it appears to target all cell types and processes. Uniquely for emerging model organisms, T. castaneum offers the opportunity of performing time- and cost-efficient large-scale RNAi screening, based on commercially available dsRNAs targeting all genes, which are simply injected into the body cavity. Well established transgenic and genome editing approaches are met by ease of husbandry and a relatively short generation time. Consequently, a number of transgenic tools like UAS/Gal4, Cre/Lox, imaging lines and enhancer trap lines are already available. T. castaneum has been a genetic experimental system for decades and now has become a workhorse for molecular and reverse genetics as well as in vivo imaging. Many aspects of development and general biology are more insect-typical in this beetle compared to D. melanogaster. Thus, studying beetle orthologs of well-described fly genes has allowed macro-evolutionary comparisons in developmental processes such as axis formation, body segmentation, and appendage, head and brain development. Transgenic approaches have opened new ways for in vivo imaging. Moreover, this emerging model system is the first choice for research on processes that are not represented in the fly, or are difficult to study there, e.g. extraembryonic tissues, cryptonephridial organs, stink gland function, or dsRNA-based pesticides.

Keywords: Arthropod model system; Behavior; CRISPR genome editing; Coleoptera; Evolution of development; Genome-wide screen; Insect model system; Pest control; Physiology; Systemic RNAi.

Fig. 1

The life cycle of T. castaneum. A Face-to-face with a wild type Tribolium castaneum. B Ventral view of a male under dark field illumination. White eyes (vermilion mutant) allow for sensitive detection of transgene eye markers. The size of an adult beetle is 3.5 mm (length). C Life cycle of Tribolium: eggs are laid into the substrate (flour) and embryonic development takes 3 days at 32 °C. Only two out of a variable number of larval stages (ca. 7) are depicted. The pupa libera allows visual inspection of external structures facilitating phenotypic studies of metamorphosis. Female beetles need a few days after hatching until they start laying eggs, which they continue for 3–4 months (drawings not to scale). Life cycle sketch by Benjamin Schwarz

Fig. 2

Cuticle phenotypes and in situ stainings. A, B Cuticle autofluorescence images of a wildtype 1^st instar larva (A) and two larvae homozygous for a transposon insertion into the first exon of the Tc-cycD gene. Reduced cell proliferation results in smaller embryos with shortened thoracic appendages (B). (C, D) Hybridization chain reaction staining of the pair rule genes Tc-runt and Tc-odd-skipped together with Tc-wingless as segmental marker in an elongating germ band (C) and of Tc-odd-skipped in the embryonic legs (D). Anterior is up in all panels. The larva in (A) is 1.4 mm in length, the embryo in (C) is 0.87 mm long. Image (C) by Felix Kaufholz, (D) by Christine Zellner