Larval RNA Interference in the Red Flour Beetle, <em>Tribolium castaneum</em>

Abstract

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle's body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum

“…The RNAi response in T. castaneum is one of the strongest and sustained in insects tested to date 42 . RNAi in both adult and larva beetles is systemic and can be triggered by injection of dsRNA in any tissue or stage 43 . Despite this already substantial reaction, we demonstrated that the effect of RNAi can be increased further by employing a nanostructured delivery agent.…”

“…T. castaneum larvae were size and age sorted and held in flour/bran media at 28 °C/80% RH prior to trials (adapted from Linz et al 43 ). Larvae were restrained using strips of adhesive tape at the anterior and posterior ends and microinjected in the dorsal side of the abdominal segments using heat-pulled micro-capillary borate silicon needles with a bore size of ~ 25 µm in conjunction with a Nanoject II system (Harvard Apparatus, USA).…”

Key factors determining variations in RNA interference efficacy mediated by different double‐stranded RNA lengths in Tribolium castaneum

“…The dsRNAs were diluted to a concentration of 1 μg/μl with T. castaneum injection solution (Linz et al, 2014). Then, 100 nl of the solution (100 ng/larva) was injected into the haemocoel at the dorsal side of the thorax of sixth instar T. castaneum larvae using a Nanoliter 2010 injector with a MICRO4 Controller (World Precision Instruments, Sarasota, FL, USA), and the injected larvae were reared under the conditions indicated.…”

Hox-logic of body plan innovations for social symbiosis in rove beetles

“…Using this transcriptome, we synthesized dsRNAs from template cDNAs of the Dalotia coriaria white (Dcw) and vermillion (Dcver) loci, which have been shown to control eye pigmentation in beetles (46,47). We developed an RNAi protocol based on the systemic larval RNAi method used in Tribolium (48)(49)(50)(51).…”