The Mechanism Of Double-strand RNA Uptake And Function Of Related Genes In Red Flour Beetle(Tribolium Castaneum)

RNA interference (RNAi) is a highly conserved mechanism among eukaryote organism and is used as a research tool to investigate gene function in numerous organisms. The red flour beetle (Tribolium castaneum) was considered as an emerging model organism that exhibits robust systemic RNAi response, whereas, the mechanism of dsRNA uptake in T. castaneum has not been described in detail. In this study, we proposed the possible mechanisms of dsRNA uptake in T. castaneum. With the combination of pharmacological endocytosis inhibitors and RNAi technique, we found clathrin-dependent endocytosis mediated dsRNA uptake in T.castaneum. In this study, two pharmacological endocytosis inhibitors, chlorpromazine (CPZ) and bafilomycin-Al (BafA) can disrupt exogenous dsRNA entry and decrease RNAi efficiency in T. castaneum. Midgut cell of T. castaneum was investigated to localize Cy3-labelled dsRNA and found the amount of dsRNA uptaking in cells was reduced after inhibitor application. In addition, we found that medium chain of adaptor protein2(TcAP50), clathrin heavy chain (TcChc), subunit H of vacuolar (H+)-ATPase (TcVhaSFD) and ras-related protein (TcRab7) were also involved in dsRNA uptake process in T. castaneum. This study for the first time demonstrated that dsRNA uptake is an active process involving clathrin-dependent endocytosis in T. castaneumn.Lethal giant larvae (Lgl) protein encoded by the Lgl gene plays a critical role in epithelia cell polarization. However, current knowledge on the characteristics and functions of Lgl in other insects are very limited. In this study, we sequenced and characterized TcLgl from the red flour beetle (Tribolium castaneum). Analyses of stage-and tissue-specific expression patterns revealed that TcLgl was constitutively expressed throughout all different developmental stages and was predominately expressed in the gut. In addition, the function of TcLgl was investigated using RNA interference (RNAi) in early larvae (8-d larva), late larvae (20-d larva) and early pupae (1-d pupae). The transcript level of TcLgl was dramatically suppressed after the injection of double-starnded RNA (dsRNA) of TcLgl (dsTcLgl) at all these stages. The early larvae injected with dsTcLgl failed to pupate, and led to100%cumulative mortality in two weeks after the injection. When the late larvae were injected with100,200and400ng of dsTcLgl, the pupation rates were only50.3,36.0and18.2%, respectively. However, these pupae failed to eclose and completely died at two days after eclose in normal condition. The remaining injected late larvae continue keeping larvae stage more than one week and gradually died. Similarly, when early pupae were injected with100,200,400ng of dsTcLgl, the eclosion rates were only22.46,18.03and11.20%, respectively, compared with the control pupae injected with dsGFP7d after the injection. All emerged adults which were injected with dsTcLgl during early pupal stage died within12d after eclosion. Our results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.Endocytosis has long been thought of as simply a way for cells to internalize nutrients and membrane associated molecules. However, current knowledge on endocytosis process in insect is very limited. In this study, we have identified four major genes (TcChc,TcAP50, TcVhaSFD and TcRab7) involved in clathrin-dependent endocytosis in red flour beetle(Tribolium castaneum). Genome structure analysis show that they located on different chromosomes. TcVhaSFD have two alternative spliced transcript named TcVhaSFDa and TcVhaSFDβ. Analyses of stage-specific gene expression revealed that these four genes are constitutively expressed throughout all developmental stages and have predominately expression in pupal stage. In addition, these genes were mainly expressed in the gut and fat body tissues, except TcAP50was predominately expression in carcass. RNAi of these genes revealed that absence of TcChc, TcAP50, TcVhaSFD and TcRab7lead to100%cumulative mortality in the late larval stages. For dsRNAs injections in the early pupal stage, abnormal eclosion were observed when TcChc, TcVhaSFD and TcRab7knockdown. The obvious phenotype show that newly eclosion insects have adult’s head and thorax combined with pupal abdomen, unfortunately, these insects died within2-day post-eclosion. Early pupae of TcAP50silenced eclosion normally, however, these adults died at6-day post-eclosion and lead to100%cumulative mortality. Strikingly, we also found that TcChc and TcVhaSFD have correlation with the egg production through microscopic examine of ovaries between control and treatment. These results demonstrate for the first time that major genes involved in clathirn-dependent endocytosis, including TcChc,TcAP50, TcVhaSFD and TcRab7have crucial role in development of red flour beetle.