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.
Highlights
The 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
Actin sequences used for knockdown experiments From a Tethya cDNA library, we identified a 707 bp fragment of T. wilhelma as a putative beta-actin ortholog (Tw-actb; Additional File 1, Figure S1)
Our experiments demonstrate a significant reduction of endogenous mRNA levels in both species after feeding and represent the successful proof-ofprinciple for bacteria-mediated feeding induced RNA interference in marine and freshwater demosponges
Summary
The 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. RNAi can be mediated in a variety of ways, for example soaking animals in double-stranded RNA (dsRNA) or morpholinos [18,19,27], injecting dsRNA or morpholinos [28,29], or feeding dsRNA expressing bacteria [24,25,30] Among these techniques, certain limitations have to be considered: (1) Soaking animals in dsRNA appears to be most problematic in seawater due to issues of RNA stability. Certain limitations have to be considered: (1) Soaking animals in dsRNA appears to be most problematic in seawater due to issues of RNA stability This might be one of the reasons why functional genomic studies in marine non-Bilateria are scarce. The salinity has been altered [18,19], which cannot be tolerated by most marine organisms. (2) The injection of dsRNA or morpholinos is only efficient if early embryos can be investigated. (3) Feeding of dsRNA-expressing bacteria requires efficient uptake of bacteria either naturally or by mimicking other food sources [30]
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