RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleriby ingested dsRNA expressing bacteria

Abstract

BackgroundThe marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available.ResultsWe show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene.ConclusionThis technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Blue-light-receptive cryptochrome is expressed in a sponge eye lacking neurons and opsin

“…Although a gene knockdown approach (Rivera et al, 2011) could confirm that Aq-Cry2 plays some role in larval phototaxis, it may not be able to distinguish between a specific role as the photoreceptor regulating the ciliary rudder, an ongoing role in settlement timing, or both. Indeed, both duplicate sponge cryptochromes are expressed throughout development and it would not be surprising if they had multiple functions at different developmental time points.…”

“…Indeed, both duplicate sponge cryptochromes are expressed throughout development and it would not be surprising if they had multiple functions at different developmental time points. Another hurdle is that techniques for gene knockdown in sponges are limited to feeding adults and have not yet been developed for embryos or larvae, the stage when the animals are phototactic (Rivera et al, 2011). In light of this, experiments manipulating magnetic fields (Henbest et al, 2008) or examining a potential role for cryptochromes in the regulation of ciliary movements via calcium signaling (Liscum et al, 2003;Tamm and Terasaki, 1994) might be used to provide mechanistic insight into Aq-Cry2 function.…”

Elements of a ‘nervous system’ in sponges

“…The attractiveness of this model, which was highlighted by Yoko Watanabe through the film 'Life of the freshwater sponge' (Tokyo Film Corporation http://tokyocinema.net/EnglVieo.htm), has led to more recent studies on signalling and coordination of sponge behaviour (Elliott and Leys, 2007;Elliott and Leys, 2010), epithelia Adams, 2010), patterning and most recently, sensory cells (Ludeman et al, 2014). And since freshwater sponges are easily obtained and cultured in Europe, Japan and North America, there is a body of knowledge on the genetics of development (Richelle-Maurer et al, 1998;RichelleMaurer and Van de Vyver, 1999;Nikko et al, 2001;Funayama et al, 2005a;Funayama et al, 2005b;Mohri et al, 2008;Funayama et al, 2010;Holstien et al, 2010;Funayama, 2013) and even the possibility of using RNA interference methods (Rivera et al, 2011). Typically, gemmules are collected during winter months and kept refrigerated to hatch as needed in the lab, but it is also possible to keep a population over the long term by returning hatched batches to lakes.…”

RNA interference in early branching metazoans

“…dsRNA delivery has been attempted in the freshwater sponge Ephydatia muelleri, through soaking and through the feeding of dsRNA-expressing bacteria (Rivera et al 2011). The authors also fed dsRNA-expressing bacteria to the marine sponge Tethya wilhelma but did not attempt soaking in this species as they concluded that RNA instability in seawater would render this form of delivery unworkable.…”

“…Two papers have been published on experimental RNAi in two other sponge species however their success is questionable due to a lack of evidence confirming both RNA delivery and successful knockdown of the target (Rivera et al 2011(Rivera et al , 2013. The lack of functional tools for interrogating gene function is a significant limiting factor in the further development of A. queenslandica as a model sponge species.…”