RNA interference (RNAi) is a eukaryotic phenomenon where short double-stranded RNA molecules (dsRNAs) repress homologous sequences. In insects RNAi has been widely observed and has proved extremely useful as a reverse genetics tool to elucidate the function of newly identified genes, as well as showing potential as a novel insecticide. Unfortunately, however, not all insect species are equally susceptible to RNAi. This thesis explores whether persistence of dsRNA in insect hemolymph, uptake of dsRNA into insect tissue, or activation of RNAi genes could be limiting factors in RNAi experiments. Trials were conducted with the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. In M. sexta larvae dsRNA disappeared rapidly from the hemolymph in vivo. By comparison, exogenous dsRNA persisted longer in the hemolymph of B. germanica adults. These findings lead me to propose that the rate of persistence of dsRNA in insect hemolymph may be a key factor in determining the susceptibility of insect species to RNAi. Despite such rapid breakdown of dsRNA in M. sexta larvae uptake of exogenous dsRNA into hemocytes, fat body and midgut could be detected by quantitative RT-PCR in vivo and was experimentally investigated in hemocytes in vivo and in vitro using fluorescently labelled dsRNA. Furthermore, quantitative-RT-PCR revealed that the expression of two M. sexta RNAi genes dicer-2 and argonaute-2 (partial sequences of which were isolated during this study) was specifically upregulated in response to injection with dsRNA.
|Date of Award||1 Sep 2011|
|Supervisor||Stuart Reynolds (Supervisor)|