RNA interference (RNAi) is an endogenous mechanism of gene silencing and has been utilised in aquaculture as a powerful gene knockdown tool. RNAi allows for potent and specific gene silencing using custom double stranded RNA (dsRNA) molecules, and in a vast majority of crustacean models works extremely efficiently to study gene function, protect from disease, or generate desirable phenotypes. In the tropical rock lobster Panulirus ornatus and related spiny lobsters however, this process is difficult to induce because of the animals’ intrinsic biology, which limits the capacity to manipulate the genes of these aquaculture species. This study aims to identify the mechanisms of RNAi inhibition in P. ornatus, which will inform methodologies to induce gene silencing in spiny lobsters to support new aquaculture biotechnology.
Preliminary studies have revealed that P. ornatus is deficient in the core enzymes and machinery which processes dsRNA for silencing when compared to closely related species where silencing is highly effective (Fig.1). This lack of protein expression likely translates to reduced silencing in vivo, even if dsRNA would be highly bioavailable. In addition, whole animal fluorescent microscopy utilising the unique larvae of these lobsters revealed potent mechanisms to sequester and secrete dsRNA molecules via the antennal gland, preventing its access to cells and tissues for silencing. These data show that dsRNA localises only in the antennal gland for subsequent secretion, and not systemically in the body of the animal (Fig.2). This implies that even if the core RNAi machinery was functional, dsRNA would still be unable to reach cells for silencing. Elucidation of these mechanisms exposes the dysfunctional RNAi system of P. ornatus, and in turn informs on which mechanisms must be overcome to enable effective silencing in this species.