A large scale systemic RNAi screen in the red flour beetleTribolium castaneumidentifies novel genes involved in insect muscle development

Abstract

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other arthropods. To help closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-GFP tester strains.Injections into larvae were followed by the analysis of the adult thoracic muscle patterns, and injections into pupae by the analysis of the late embryonic/larval muscle patterns. Herein we focus on the results of our pupal injection screen for late embryonic/larval muscle phenotypes, which covered 8.500 of a total of ~16.500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded larval body wall and leg muscle phenotypes. We present the main candidates that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

RNAi screen inTriboliumreveals involvement of F-BAR proteins in myoblast fusion and visceral muscle morphogenesis in insects

“…In Drosophila mutants for genes with less prominent functions, with weak alleles, or with partial functional rescue by maternal products, the muscles are thinner as well due to the reduced uptake of fusion competent cells (e.g., Hamp et al 2016). We propose that the 'thin muscle' phenotypes in Tribolium knockdowns of most myoblast fusion genes (including some weak phenotypes with Tcas kirre/rst; Schultheis et al 2019) result from similar effects of incomplete functional knock-down and rapid disappearance of the unfused myoblasts. The absence of the GFP marker at earlier stages unfortunately prevented the detection of unfused myoblasts in control and RNAi treated embryos to confirm this explanation.…”

“…As described in the accompanying paper (Schultheis et al 2019), we participated in large-scale screens with systemic RNAi in the flour beetle Tribolium castaneum aiming to identify new genes that regulate the development of the somatic musculature. One screen was for knock-down phenotypes in muscles of late stage embryos and first instar larvae, which involved injecting double stranded RNAs into pupae of a tester strain that expressed EGFP in all somatic (and visceral) muscles.…”

Metamorphosis in Insect Muscle: Insights for Engineering Muscle-Based Actuators

“…This process is conserved in beetles, where knocking down duf and sns homologs results in thinner muscles, likely due to blocked myoblast fusion. 59 Such myotube filopodia and Duf/Sns interactions are likely to be general to all AMP fusion, as similar features are required in vertebrate myoblast fusion. 90,91 Much remains to be learned about the process of myoblast fusion, but what is known points to the importance of functional cytoskeletal proteins, adhesion proteins, and Notch signaling.…”

“…However, Duf has a conserved role in muscle fusion, and its function as a marker is likely to be useful in identifying these cells. 59 The other population of Duf-expressing cells, which we term ''surrogate IPs,'' is morphologically similar to FCMs and appears not to seed myotube formation in healthy animals. However, when the IPs are ablated, many more muscle fibers form in a manner that is dependent on Duf expression.…”

Progress in the use of genetic methods to study insect behavior outside Drosophila

“…Injections may be given at any of an insect's life stages (e.g. embryos [23,108], larvae [109][110][111], pupae [91,[112][113][114], adults [115,116]). While labor-intensive, this method normally provides the highest efficiency of gene silencing, with the caveat that giving the injection may impair an animal's survival.…”