BACKGROUND
the ability to reconstruct the evolution of animals relies heavily on the availability of comparative data to facilitate understanding of evolutionary trends from the molecular to the organismal level.
among the non-bilaterian animals, recent utilization of phylogenomics has increased our understanding of metazoan relationships  <cit> .
comparative genomic approaches were used to reconstruct ancestral genomes like the putative genome of the last common ancestor of the metazoa   <cit> .
moreover, the same genomic data is used to evaluate hypotheses regarding the evolution of metazoan-specific pathways like those involved in development  <cit>  or cell signaling  <cit> .
for example, the genome of the demosponge amphimedon queenslandica is playing a key role in deciphering evolutionary trends in metazoan specific gene networks and pathways .
nevertheless, evolutionary biology relies on more than genomic data.
functional studies based on reverse genetics techniques are needed to test the hypotheses provided by genomic analyses.
however, the availability of genomic resources and reverse genetic techniques in the non-bilaterian animals is limited compared to the other animals .
for the cnidaria, the placozoa, and the ctenophora genomic data and reverse genetic techniques are available .
however, thus far, only some genomic data is available for the porifera, and the sister group of metazoa, the choanoflagellata.
reverse genetic techniques for these animal groups is of high priority for a comprehensive analysis of evolution and development of the non-bilaterian metazoa.
apart from permanent knockout and/or transgenic lines , rna interference  is a powerful tool to alter the expression of genes  <cit> .
rnai can be mediated in a variety of ways, for example soaking animals in double-stranded rna  or morpholinos  <cit> , injecting dsrna or morpholinos  <cit> , or feeding dsrna expressing bacteria  <cit> .
among these techniques, certain limitations have to be considered:  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.
so far, soaking techniques have only been adapted to eleutheria dichotoma, aiptasia pallida and trichoplax adhaerens  <cit> .
in some studies, the salinity has been altered  <cit> , which cannot be tolerated by most marine organisms.
the injection of dsrna or morpholinos is only efficient if early embryos can be investigated.
feeding of dsrna-expressing bacteria requires efficient uptake of bacteria either naturally or by mimicking other food sources  <cit> .
because sponges represent a key group for our understanding of metazoan evolution including the changes in gene regulation that took place leading to multicellularity and complex body plans in this lineage, our aim was to test and establish rnai protocols in one freshwater and one marine sponge species.
by addressing both living modes we intended to provide a broad basis for future adaptations of this method to other sponge species.
the marine species tethya wilhelma sarà, sarà, nickel & brümmer,  <dig>  and the freshwater species ephydatia muelleri   are emerging basal animal model organisms.
ephydatia is an abundant and common sponge found on all major land masses in the world  <cit> .
recent research has lead to a better understanding of ephydatia biology including cellular physiology and function as well as the role of conserved signaling pathways .
one of the most important aspects is that during winter ephydatia forms thousands of gemmules which are cysts containing totipotent stem cells .
these gemmules can be collected, stored at 4°c for up to one year, and easily induced to hatch into fully differentiated adults on the laboratory bench.
this cultivation routine provides a system to study stem cell differentiation and animal body plan development in these animals  <cit> .
a genome and a transcriptome are currently in the process of being sequenced for this species.
tethya wilhelma is one of the few marine sponges that can currently be permanently maintained in the laboratory based on its capability to reproduce asexually through buds  <cit> .
in addition, this species has been studied on a broad scale including functional anatomy  <cit> , contractile and movement behavior  <cit> , physiology of integration  <cit>  and bud development  <cit> .
a transcriptome has been sequenced recently and is currently being annotated, and sequencing of the genome is in the planning stage.
to enable functional studies in sponges we developed a protocol for bacterial mediated rnai-induced silencing based on gene silencing techniques developed for caenorhabditis elegans  <cit> .
the protocol has been adapted to the marine and freshwater environments and tested by knocking down cytoplasmic actin mrna levels in juvenile specimens of t. wilhelma and e. muelleri.
our results indicate that this pathway is able to function in both marine and freshwater sponge species.
RESULTS
actin sequences used for knockdown experiments
from a tethya cdna library, we identified a  <dig> bp fragment of t. wilhelma as a putative beta-actin ortholog .
the tw-actb fragment contains a putative 5'utr  and the 5' part of the putative coding sequence  .
this sequence is quite similar to the homo sapiens β-actin sequence  with  <dig> % pairwise identities overall.
a motif search in the translated putative cds region of tw-actb revealed prosite  <cit>  actin signature  <dig>  and  <dig>  at residues 54- <dig> and 105- <dig> respectively.
actin signature  <dig>  has not been identified; it may reside in the uncloned 3' terminus.
using beta-actin degenerate pcr primers, we isolated a  <dig> bp fragment of e. muelleri as a putative beta-actin ortholog .
the em-actb fragment is 100% identical to the putative tethya beta-actin ortholog at the amino acid level.
our em-actb fragment also contains prosite actin signature  <dig>  both fragments were cloned into the l <dig> plasmid  and transformed into escherichia coli ht <dig>  which was induced to produce dsrna.
in silico evaluation of the actin sequences used for knockdown experiments
in order to ensure that we are targeting cytoplasmic actin and not another actin isoform or actin-related protein, we performed an in silico analysis.
using species specific blast searches  <cit>  we translated both e. muelleri and t. wilhelma actin coding sequences and aligned them to other actins.
these actin sequences were obtained from a protein sequence alignment used in a previous study  <cit>  as well as contig translations  based on tblastn searches using h. sapiens β-actin  against the following transcriptomes housed at ncbi dbest  <cit> : monosiga ovata , amphimedon queenslandica , leucetta chagosensis , oscarella carmella , nematostella vectensis , hydra vulgaris , mnemiopsis leidyi , and trichoplax adhaerens .
the raxml tree we generated from these data showed that both t. wilhelma and e. muelleri sequences clearly group within all other metazoan putative cytoplasmic actins and are clearly separated from other actins or actin-related proteins .
an alignment of the partial coding sequences of tw-actb and em-actb used for rnai with  <dig> additional holozoan orthologs produced pairwise identity of  <dig> % on the nucleotide level  and  <dig> % on the amino acid level .
raxml tree and alignment analyses confirm that cytoplasmic actin was targeted in our rnai knockdown experiments and thus we expect cytoplasmic actin specific phenotypic effects.
evaluation of a soaking method for dsrna delivery
we first attempted a soaking method for dsrna delivery in e. muelleri.
as negative controls, we used dsrna for green fluorescent protein  since sponges do not express gfp.
we also used double-stranded dna  generated by pcr amplifying the e. muelleri beta-actin gene since dsdna should not be processed through the rnai pathway.
soaking sponges in high concentrations  of any nucleic acid  leads to potentially cytotoxic effects to the sponges.
freshwater sponges are sensitive to changes in osmolarity  <cit> , which could be the cause of the observed cytotoxicity.
when exposed to high nucleic acid concentrations, the tissues regressed, choanocyte chambers seemed to collapse, and sponges ceased pumping.
thus, in titration experiments we tested for concentrations at which dsdna and gfp dsrna would not have an observable effect on sponge morphology, while actin dsrna would.
a concentration of 1- <dig> μg/ml of beta-actin dsrna led to distinct observable morphologies; including loss of contact of the growing edge of tissue with the culture plate, loss of definition in the canal structure of the sponge, and poorly defined choanocyte chambers .
longer exposures to beta-actin dsrna resulted in more pronounced phenotypes .
control experiments with exposure to gfp dsrna or beta-actin dsdna at similar concentrations, did not exhibit phenotype changes .
interestingly, removal of the dsrna beta-actin solution and replacement with normal culture media lead to full recovery of the sponge tissue after one to two days including canals, distinct connections to the culture plate and a developed osculum .
our data indicate that a soaking method is viable for induction of the rnai pathway in e. muelleri.
however, the tight range in the amount of dsrna that seemed to be effective to produce an expected phenotype will require titration experiments for each gene addressed in future rnai studies.
we did not attempt a soaking method for t. wilhelma since it is necessary to alter the salinity  of the culturing media in order to ensure rna stability.
we know that t. wilhelma and most other marine sponges do not tolerate salinity changes in this range without experiencing changes in contractile behavior and most likely phenotypic changes .
thus, a soaking method would likely not be viable for marine species.
feeding method for the freshwater sponge ephydatia muelleri
since sponges naturally feed on bacteria, a feeding method for dsrna delivery might be more universally applicable in marine and freshwater sponge systems.
in e. muelleri, we fed sponges ht115/l <dig> e. coli expressing em-actin dsrna, for  <dig> hours.
afterwards, sponges were washed several times in culture media to remove bacteria and tissue was harvested within one to two hours .
sponges were also fed ht <dig> or bacteria expressing gfp to monitor feeding behavior and to test for a response to induced bacteria.
gfp-fed sponges did not show any response that differed from sponges fed bacteria lacking expression vector .
to test the ability of actin dsrna to reduce actin expression, we performed quantitative-rt-pcr  assays.
for qrt-pcr analysis, we fed sponges either dsrna-expressing bacteria, untransformed bacteria, or gfp-expressing bacteria for  <dig> hours.
sponge tissue was harvested and we utilzed qrt-pcr to access actin expression levels.
a relative quantification procedure was used to calculate a ratio of treatment to control expression levels, either for the gene of interest  or for the non-target genes .
if the target gene was not affected by actin rnai we would expect a ratio of  <dig>  the ratio of paired treatment and control expression levels were averaged and are shown in figure 2b.
we performed eleven total qrt-pcr experiments with l4440-em-actb -fed sponges and controls.
in ten of these experiments, em-actb levels were lower in dsrna-fed sponges than controls, when normalized to the housekeeping gene ef1-alpha expression levels.
these results were significant  as the relative em-actb mrna expression levels dropped down to 75% of the control values .
longer treatments  with the same bacteria did not lead to a greater effect .
to test for nonspecific effects, we repeated the qrt-pcr experiment using sponges fed bacteria expressing dsem-actb and then assayed expression levels for em-annexin and em-six1/ <dig>  em-annexin is a structural protein expressed in the archaeocytes, and in developing and mature choanocyte chambers  <cit>  while em-six is a transcription factor expressed in multiple cell types including archaeocytes and choanocytes throughout development .
of three separate experiments totaling about  <dig> sponges per condition, em-annexin and em-six mrna levels were not significantly different in dsem-actb-treated sponges compared to ht115-fed control sponges .
any differences were small and non-directional, suggesting that dsem-actb did not affect em-annexin or em-six expression.
to further demonstrate, on the molecular level, that it is possible to knockdown genes other than actin, we present rt-pcr, qrt-pcr and western blot data for experiments conducted where sponges were either fed bacteria or soaked with dsrna directed to em-annexin and em-six genes.
we used rt-pcr and gel-based analysis to evaluate dsem-annexin-treated sponges using both the soaking  and feeding-based methods .
in both cases, reduced expression of em-annexin is apparent when compared with control-treated sponges .
we used qrt-pcr to demonstrate that sponges fed ht115/l <dig> em-six dsrna constructs showed reduced em-six expression when compared to control-treated tissues.
these results were significant and the relative em-six mrna expression levels dropped down to ~69% of the control values  .
it is clear that several parameters will need to be optimized to consistently achieve > 50% knockdown.
these include time and length of exposure to dsrna, age of sponges and ability to consistently induce bacteria and control for amount of bacteria fed to sponges.
to demonstrate that knockdown is also evident at the protein level, we performed western blot analysis for both actin and six proteins after treatment with either dsem-actb or dsem-six rnai .
the variation in knockdown levels for actin protein found in western analysis was consistent with the variation in gene expression levels found by qrt-pcr after dsem-actb treatment.
two different representative western blots showing dsem-actb-treated tissue compared to control-treated tissue are shown and illustrate that actin protein levels are lower after dsem-actb treatment .
in response to dsem-six treatment, protein levels for six were also reduced while the same blot showed that actin protein levels remain relatively constant .
feeding method for the marine sponge tethya wilhelma
using the same bacteria and a similar expression vector construct as described above, but a different feeding profile  we fed t. wilhelma specimens tw-actb dsrna expressing ht <dig>  larger specimens of t. wilhelma were cultured over a period of  <dig> and  <dig> hours, respectively, and pulse-fed with dstw-actb expressing bacteria or a control strain for  <dig>  hours each day.
this resulted in actin knockdown in rnai-treated sponges .
after  <dig> hours of cultivation, including two pulse-feeding cycles, relative tw-actb expression levels dropped down to  <dig> % in relation to control animals, representing an average knockdown by  <dig> %.
after an additional dsrna feeding pulse within a total cultivation time of  <dig> hours, the relative actin mrna levels remained within a reduced range of  <dig> % in relation to control animals.
thus, all dstw-actb-treated specimens  showed significant reductions compared to endogenous mrna levels .
in contrast, pulse feeding of dstw-actb expressing bacteria for a reduced time of  <dig> minutes did not reveal any knockdown effect within  <dig> or  <dig> hours  for this species.
to determine if greater knockdown is observed in t. wilhelma after longer-term exposure to dstw-actb, we repeated the experiment over a period of eight days.
between  <dig> and  <dig> hours after initial feeding, single specimens were tested for relative tw-actb expression levels, which were between  <dig> %  to  <dig> %  to the level of tw-actb in control sponges.
while this is a wide range, the average , is close to the total average of the short-term experiment , which represents significant reductions in expression levels .
to determine viability of sponges affected by long-term actin knockdown, all specimens that were used for quantification in the long-term experiment were time-lapse imaged during the 24-hour period preceding sampling and preparation for rna quantification.
all specimens displayed typical contractile behavior  demonstrating the general viability of the rnai-treated specimens.
thus, our results demonstrate that daily pulse feeding for  <dig>  hours does not result in lethal effects as it has been observed by direct dsrna incubation in a dose-dependent manner  <cit> .
evaluation of morphological changes in response to actin 'knockdown' in ephydatia muelleri
to examine morphological changes due to actin knockdown in e. muelleri, we stained dsem-actb-treated sponges and control sponges with bodipy-558/ <dig> conjugated phalloidin.
we examined three major regions of the sponge tissue by confocal laser scanning microscopy : 1) the outer edge of sponge growth, 2) the regions containing newly developed choanocyte chambers, and 3) the endopinacocyte-lined canals surrounded by choanocyte chambers.
an extensive actin network was present at the edge of sponge growth.
the outer surface of the sponge that forms attachments to the substrate is composed of exopinacoderm and basopinacoderm and is made of continuous epithelium where some connective and migratory  cells are observed.
the region also contains filopodia of multipolar cells leading from the mesohyl to the exopinacoderm.
small foci represent the edge of two abutting cells when viewed from above, and larger foci likely represent focal adhesion points where actin bundles highlight filopodia of mesohyl cells.
sponges fed dsem-actb have a less extensive actin network at the growing edge of the sponge.
many fibers do not run continuously through the sponge tissue and the continuous fibers often lack the network organization seen in control sponges.
these data likely indicate that there is a reduced cellular mobility in the mesohyl .
the sponge region containing newly developed choanocyte chambers  was chosen as it is a relatively thin region but allows us to see the overall pattern of developed sponge tissue.
in this region the actin-rich choanocytes themselves look very similar in treated and control sponges.
to further investigate, we performed transmission electron microscopy on a control and a dsem-actb-treated sponge to see whether ultrastructural aspects of choanocytes were affected after rnai treatment.
while a more thorough sampling strategy is required, we found one trait in the microvillar collar that may differ between treatment and control sponges.
actin rnai-treated choanocytes had a greater percentage of cells with misshapen/folded collars .
the control sponges, on the other hand, contained numerous perfectly round collars, which were not observed in the actin rnai-treated sponges .
however, the pattern of endopinacocytes connecting the choanocytes differed markedly.
control sponges have canals that are clearly lined by actin-rich endopinacocytes.
dsem-actb-fed sponge canals are far less clearly lined .
in dsem-actb-treated sponges, the endopinacocytes that span the spaces between choanocytes are thin and display a weaker actin signal.
the actin cytoskeletal network is thus reduced in dsem-actb-fed pinacocytes, but the choanocytes appear to be unaffected at the level observed with light microscopy.
in the canals lined by actin-containing pinacocytes, the actin fibers run continuously around the canals, over the span of many cells.
control sponges have clearly outlined canals in most focal planes.
dsem-actb-fed sponges, however, have canals that are not as well-defined and are lacking clear phalloidin-stained pinacocytes in many planes .
overall, our observations suggest a general knockdown of the actin cytoskeleton with a less prominent actin network.
evaluation of morphological changes in response to actin 'knockdown' in tethya wilhelma
to examine morphological changes due to actin knockdown in t. wilhelma, we fixed sponges that had been pulse-fed induced ht115/tw-actb bacteria expressing dsrna and control ht <dig> bacteria daily  after  <dig> and  <dig> hours.
using whole-mount preparations we examined two major regions of the sponge by clsm that were stained using alexa-488-conjugated phalloidin: 1) the exopinacocyte, and 2) the cortical mesohyl.
phalloidin staining revealed an extensive actin network in the exopinacocytes with actin-rich ripple-like structures on the surface of the exopinacocytes , structures typical in t. wilhelma and resembling lamellipodia extensions of fibroblasts .
in the dstw-actb-treated specimens, after  <dig> hours the exopinacocytes showed fewer actin-rich ripples  than in the control, while after  <dig> hours, ripples were almost absent .
in addition, the actin network was overall less prominent in the treated sponges than in the control specimens.
the largest effect was observed in the cortical mesohyl.
the control sponges showed a large number of extremely long spindle-shaped bipolar cells , which are typical morphotypes of highly mobile cells  <cit>  'frozen' in movement by fixation.
in contrast, the dstw-actb-treated sponges were characterized by atypical, extremely large atrophic multipolar cells  and only few bipolar cells.
in comparison, multipolar cells in the control sponges were much smaller .
the changes in the pinacocytes with the reduced actin-rich lammellae and atypical mesohyle with the atrophic multipolar cells and the lack of bipolar cells can be explained by decreased actin expression.
while the actin turnover rate is not high enough to produce lethal effects or cessastion of contractility, processes based on actin dynamics like cell mobility or lamellipodia formation seem to be strongly affected.
since the atrophic multipolar cells correlate with a lack of mobile bipolar cells, it seemed that both forms actually represent extreme morphotypes of a single archeocyte-like cell type.
CONCLUSIONS
for the first time, we report successful rnai techniques for gene knockdown in the sponges e. muelleri  and t. wilhelma .
we used dsrna soaking methodology for e. muelleri and a bacterial feeding method adapted to freshwater and marine culture media for both species.
our experiments demonstrate a significant reduction of endogenous mrna levels in both species after feeding and represent the successful proof-of-principle for bacteria-mediated feeding induced rna interference in marine and freshwater demosponges.
we tested dsrna methodology using cytoplasmic actin orthologs, a ubiquitously expressed gene involved in cell shape and motility.
in e. muelleri, qrt-pcr analysis showed a modest  but significant  knockdown of em-actb after feeding sponges bacteria expressing dsrna against em-actb for  <dig> hours compared to controls, with a maximum knockdown of about 55%.
t. wilhelma sponges treated for  <dig> or  <dig> hours showed similarly modest but significant  reductions.
t. wilhema treated for up to  <dig> days showed actin mrna reductions throughout the entire period of the experiment.
the maximum knockdown caused by dstw-actb-expressing bacteria was almost 50% compared to the control-group mrna level.
we also demonstrated in e. muelleri, at both the mrna and protein level, that it is possible to knock down additional genes , thus illustrating this method should be applicable for other target genes.
the reduction in expression for actin demonstrates an efficient silencing for a highly expressed gene that may account for about 5% of the total protein content of a cell  <cit> .
furthermore, in order to increase the level of reduction, longer periods of treatment with dsrna might be required for highly expressed genes or stable proteins like actin.
hints of this were seen in the long-term t. wilhelma feeding and e. muelleri soaking experiments .
it is possible that the rnai effect may be greater than measured, because the knockdown effect may only be measured in surviving cells.
sponges are known to have high proliferation activity, a short cell cycle duration and cell shedding that is predominantly confined to the choanocytes .
this high cell turnover rate may be part of the reason that actin  will not be knocked down at great levels.
due to the biology of these organisms and the method used, feeding sponges dsrna will likely be a technique that can be utilized to knockdown, but not knockout gene expression.
however, the overall significantly reduced mrna levels during the entire period of the experiment clearly demonstrated the potential of bacterial-mediated rnai-induced silencing for functional gene knock down studies in poriferans.
after feeding em-actin dsrna, e. muelleri did not display a macroscopic phenotype that could be clearly described at the cellular level.
however, the primary actin knockdown phenotype shown by phalloidin staining was a less extensive actin network that was manifested in a "crumpled" edge, where tissues should be adhering to the substrate and migrating outwards.
additionally, a reduction of cellular connections in the choanocyte-rich regions and canals due to loss of actin in endopinacocytes was observed.
t. wilhelma showed neither a reduced survival rate nor alterations of rhythm or kinetics of body contractions.
like in e. muelleri, rnai treated specimens of t. wilhelma displayed no distinct macroscopic phenotype.
however, we found strong effects on mesohyle and pinacoderm cell anatomy of t. wilhelma.
apart from reduced actin content and altered actin network appearance in pinacocytes, the most prominent effects were hypertrophic mesohyle cells and the lack of mobile bipolar cells, indicating a reduced mobility and cell shape dynamics in dsrna-treated specimens.
rnai methods allow us to begin to study sponge gene function in a directed manner, including questions regarding evolution of gene regulatory networks, nervous system and muscle evolution, and also functional studies concerning biomineralization and other metabolic processes.
our results also suggest that rnai might be a valuable tool to investigate cell-type functionality in sponges, as has been shown for other organisms, like hydra .
this would facilitate studies on the evolution of cell differentiation in sponges.
additionally, it might help us to differentiate variable morphotypes of one single cell type versus other cell types.
thus, rnai would help solve a long-standing problem for sponge biologists and evolutionary biologists interested in cell type evolution  by allowing unique studies to clearly define and distinguish sponge cell types, most likely in conjunction with in situ hybridization.
we assume that our protocol will be readily applicable to other marine sponge species like the homoscleromorph oscarella lobularis  <cit>  or the calcareous sycon ciliatum and s. raphanus  <cit> .
it will also be adaptable to the freshwater species e. fluviatilis and spongilla lacustris, which are also commonly used to study sponge biology  <cit> .
in conclusion, our present study closed an existing gap for a functional genomics protocol for the porifera and will thus stimulate a number of comparative functional studies, which will deepen our understanding of metazoan evolution.

 the ability to reconstruct the evolution of animals relies heavily on the availability of comparative data to facilitate understanding of evolutionary trends from the molecular to the organismal level. among the non-bilaterian animals, recent utilization of phylogenomics has increased our understanding of metazoan relationships. comparative genomic approaches were used to reconstruct ancestral genomes like the putative genome of the last common ancestor of the metazoa. moreover, the same genomic data is used to evaluate hypotheses regarding the evolution of metazoan-specific pathways like those involved in development or cell signaling. for example, the genome of the demosponge amphimedon queenslandica is playing a key role in deciphering evolutionary trends in metazoan specific gene networks and pathways. nevertheless, evolutionary biology relies on more than genomic data. functional studies based on reverse genetics techniques are needed to test the hypotheses provided by genomic analyses. however, the availability of genomic resources and reverse genetic techniques in the non-bilaterian animals is limited compared to the other animals. for the cnidaria, the placozoa, and the ctenophora genomic data and reverse genetic techniques are available. however, thus far, only some genomic data is available for the porifera, and the sister group of metazoa, the choanoflagellata. reverse genetic techniques for these animal groups is of high priority for a comprehensive analysis of evolution and development of the non-bilaterian metazoa. apart from permanent knockout and/or transgenic lines, rna interference is a powerful tool to alter the expression of genes. rnai can be mediated in a variety of ways, for example soaking animals in double-stranded rna or morpholinos, injecting dsrna or morpholinos, or feeding dsrna expressing bacteria. among these techniques, certain limitations have to be considered: 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. so far, soaking techniques have only been adapted to eleutheria dichotoma, aiptasia pallida and trichoplax adhaerens. in some studies, the salinity has been altered, which cannot be tolerated by most marine organisms. the injection of dsrna or morpholinos is only efficient if early embryos can be investigated. feeding of dsrna-expressing bacteria requires efficient uptake of bacteria either naturally or by mimicking other food sources. because sponges represent a key group for our understanding of metazoan evolution including the changes in gene regulation that took place leading to multicellularity and complex body plans in this lineage, our aim was to test and establish rnai protocols in one freshwater and one marine sponge species. by addressing both living modes we intended to provide a broad basis for future adaptations of this method to other sponge species. the marine species tethya wilhelma sarà, sarà, nickel & brümmer, and the freshwater species ephydatia muelleri are emerging basal animal model organisms. ephydatia is an abundant and common sponge found on all major land masses in the world. recent research has lead to a better understanding of ephydatia biology including cellular physiology and function as well as the role of conserved signaling pathways. one of the most important aspects is that during winter ephydatia forms thousands of gemmules which are cysts containing totipotent stem cells. these gemmules can be collected, stored at 4°c for up to one year, and easily induced to hatch into fully differentiated adults on the laboratory bench. this cultivation routine provides a system to study stem cell differentiation and animal body plan development in these animals. a genome and a transcriptome are currently in the process of being sequenced for this species. tethya wilhelma is one of the few marine sponges that can currently be permanently maintained in the laboratory based on its capability to reproduce asexually through buds. in addition, this species has been studied on a broad scale including functional anatomy, contractile and movement behavior, physiology of integration and bud development. a transcriptome has been sequenced recently and is currently being annotated, and sequencing of the genome is in the planning stage. to enable functional studies in sponges we developed a protocol for bacterial mediated rnai-induced silencing based on gene silencing techniques developed for caenorhabditis elegans. the protocol has been adapted to the marine and freshwater environments and tested by knocking down cytoplasmic actin mrna levels in juvenile specimens of t. wilhelma and e. muelleri. our results indicate that this pathway is able to function in both marine and freshwater sponge species.  actin sequences used for knockdown experiments from a tethya cdna library, we identified a bp fragment of t. wilhelma as a putative beta-actin ortholog. the tw-actb fragment contains a putative 5'utr and the 5' part of the putative coding sequence. this sequence is quite similar to the homo sapiens β-actin sequence with % pairwise identities overall. a motif search in the translated putative cds region of tw-actb revealed prosite actin signature and at residues 54- and 105- respectively. actin signature has not been identified; it may reside in the uncloned 3' terminus. using beta-actin degenerate pcr primers, we isolated a bp fragment of e. muelleri as a putative beta-actin ortholog. the em-actb fragment is 100% identical to the putative tethya beta-actin ortholog at the amino acid level. our em-actb fragment also contains prosite actin signature both fragments were cloned into the l plasmid and transformed into escherichia coli ht which was induced to produce dsrna. in silico evaluation of the actin sequences used for knockdown experiments in order to ensure that we are targeting cytoplasmic actin and not another actin isoform or actin-related protein, we performed an in silico analysis. using species specific blast searches we translated both e. muelleri and t. wilhelma actin coding sequences and aligned them to other actins. these actin sequences were obtained from a protein sequence alignment used in a previous study as well as contig translations based on tblastn searches using h. sapiens β-actin against the following transcriptomes housed at ncbi dbest: monosiga ovata, amphimedon queenslandica, leucetta chagosensis, oscarella carmella, nematostella vectensis, hydra vulgaris, mnemiopsis leidyi, and trichoplax adhaerens. the raxml tree we generated from these data showed that both t. wilhelma and e. muelleri sequences clearly group within all other metazoan putative cytoplasmic actins and are clearly separated from other actins or actin-related proteins. an alignment of the partial coding sequences of tw-actb and em-actb used for rnai with additional holozoan orthologs produced pairwise identity of % on the nucleotide level and % on the amino acid level. raxml tree and alignment analyses confirm that cytoplasmic actin was targeted in our rnai knockdown experiments and thus we expect cytoplasmic actin specific phenotypic effects. evaluation of a soaking method for dsrna delivery we first attempted a soaking method for dsrna delivery in e. muelleri. as negative controls, we used dsrna for green fluorescent protein since sponges do not express gfp. we also used double-stranded dna generated by pcr amplifying the e. muelleri beta-actin gene since dsdna should not be processed through the rnai pathway. soaking sponges in high concentrations of any nucleic acid leads to potentially cytotoxic effects to the sponges. freshwater sponges are sensitive to changes in osmolarity, which could be the cause of the observed cytotoxicity. when exposed to high nucleic acid concentrations, the tissues regressed, choanocyte chambers seemed to collapse, and sponges ceased pumping. thus, in titration experiments we tested for concentrations at which dsdna and gfp dsrna would not have an observable effect on sponge morphology, while actin dsrna would. a concentration of 1- μg/ml of beta-actin dsrna led to distinct observable morphologies; including loss of contact of the growing edge of tissue with the culture plate, loss of definition in the canal structure of the sponge, and poorly defined choanocyte chambers. longer exposures to beta-actin dsrna resulted in more pronounced phenotypes. control experiments with exposure to gfp dsrna or beta-actin dsdna at similar concentrations, did not exhibit phenotype changes. interestingly, removal of the dsrna beta-actin solution and replacement with normal culture media lead to full recovery of the sponge tissue after one to two days including canals, distinct connections to the culture plate and a developed osculum. our data indicate that a soaking method is viable for induction of the rnai pathway in e. muelleri. however, the tight range in the amount of dsrna that seemed to be effective to produce an expected phenotype will require titration experiments for each gene addressed in future rnai studies. we did not attempt a soaking method for t. wilhelma since it is necessary to alter the salinity of the culturing media in order to ensure rna stability. we know that t. wilhelma and most other marine sponges do not tolerate salinity changes in this range without experiencing changes in contractile behavior and most likely phenotypic changes. thus, a soaking method would likely not be viable for marine species. feeding method for the freshwater sponge ephydatia muelleri since sponges naturally feed on bacteria, a feeding method for dsrna delivery might be more universally applicable in marine and freshwater sponge systems. in e. muelleri, we fed sponges ht115/l e. coli expressing em-actin dsrna, for hours. afterwards, sponges were washed several times in culture media to remove bacteria and tissue was harvested within one to two hours. sponges were also fed ht or bacteria expressing gfp to monitor feeding behavior and to test for a response to induced bacteria. gfp-fed sponges did not show any response that differed from sponges fed bacteria lacking expression vector. to test the ability of actin dsrna to reduce actin expression, we performed quantitative-rt-pcr assays. for qrt-pcr analysis, we fed sponges either dsrna-expressing bacteria, untransformed bacteria, or gfp-expressing bacteria for hours. sponge tissue was harvested and we utilzed qrt-pcr to access actin expression levels. a relative quantification procedure was used to calculate a ratio of treatment to control expression levels, either for the gene of interest or for the non-target genes. if the target gene was not affected by actin rnai we would expect a ratio of the ratio of paired treatment and control expression levels were averaged and are shown in figure 2b. we performed eleven total qrt-pcr experiments with l4440-em-actb -fed sponges and controls. in ten of these experiments, em-actb levels were lower in dsrna-fed sponges than controls, when normalized to the housekeeping gene ef1-alpha expression levels. these results were significant as the relative em-actb mrna expression levels dropped down to 75% of the control values. longer treatments with the same bacteria did not lead to a greater effect. to test for nonspecific effects, we repeated the qrt-pcr experiment using sponges fed bacteria expressing dsem-actb and then assayed expression levels for em-annexin and em-six1/ em-annexin is a structural protein expressed in the archaeocytes, and in developing and mature choanocyte chambers while em-six is a transcription factor expressed in multiple cell types including archaeocytes and choanocytes throughout development. of three separate experiments totaling about sponges per condition, em-annexin and em-six mrna levels were not significantly different in dsem-actb-treated sponges compared to ht115-fed control sponges. any differences were small and non-directional, suggesting that dsem-actb did not affect em-annexin or em-six expression. to further demonstrate, on the molecular level, that it is possible to knockdown genes other than actin, we present rt-pcr, qrt-pcr and western blot data for experiments conducted where sponges were either fed bacteria or soaked with dsrna directed to em-annexin and em-six genes. we used rt-pcr and gel-based analysis to evaluate dsem-annexin-treated sponges using both the soaking and feeding-based methods. in both cases, reduced expression of em-annexin is apparent when compared with control-treated sponges. we used qrt-pcr to demonstrate that sponges fed ht115/l em-six dsrna constructs showed reduced em-six expression when compared to control-treated tissues. these results were significant and the relative em-six mrna expression levels dropped down to ~69% of the control values. it is clear that several parameters will need to be optimized to consistently achieve > 50% knockdown. these include time and length of exposure to dsrna, age of sponges and ability to consistently induce bacteria and control for amount of bacteria fed to sponges. to demonstrate that knockdown is also evident at the protein level, we performed western blot analysis for both actin and six proteins after treatment with either dsem-actb or dsem-six rnai. the variation in knockdown levels for actin protein found in western analysis was consistent with the variation in gene expression levels found by qrt-pcr after dsem-actb treatment. two different representative western blots showing dsem-actb-treated tissue compared to control-treated tissue are shown and illustrate that actin protein levels are lower after dsem-actb treatment. in response to dsem-six treatment, protein levels for six were also reduced while the same blot showed that actin protein levels remain relatively constant. feeding method for the marine sponge tethya wilhelma using the same bacteria and a similar expression vector construct as described above, but a different feeding profile we fed t. wilhelma specimens tw-actb dsrna expressing ht larger specimens of t. wilhelma were cultured over a period of and hours, respectively, and pulse-fed with dstw-actb expressing bacteria or a control strain for hours each day. this resulted in actin knockdown in rnai-treated sponges. after hours of cultivation, including two pulse-feeding cycles, relative tw-actb expression levels dropped down to % in relation to control animals, representing an average knockdown by %. after an additional dsrna feeding pulse within a total cultivation time of hours, the relative actin mrna levels remained within a reduced range of % in relation to control animals. thus, all dstw-actb-treated specimens showed significant reductions compared to endogenous mrna levels. in contrast, pulse feeding of dstw-actb expressing bacteria for a reduced time of minutes did not reveal any knockdown effect within or hours for this species. to determine if greater knockdown is observed in t. wilhelma after longer-term exposure to dstw-actb, we repeated the experiment over a period of eight days. between and hours after initial feeding, single specimens were tested for relative tw-actb expression levels, which were between % to % to the level of tw-actb in control sponges. while this is a wide range, the average, is close to the total average of the short-term experiment, which represents significant reductions in expression levels. to determine viability of sponges affected by long-term actin knockdown, all specimens that were used for quantification in the long-term experiment were time-lapse imaged during the 24-hour period preceding sampling and preparation for rna quantification. all specimens displayed typical contractile behavior demonstrating the general viability of the rnai-treated specimens. thus, our results demonstrate that daily pulse feeding for hours does not result in lethal effects as it has been observed by direct dsrna incubation in a dose-dependent manner. evaluation of morphological changes in response to actin'knockdown' in ephydatia muelleri to examine morphological changes due to actin knockdown in e. muelleri, we stained dsem-actb-treated sponges and control sponges with bodipy-558/ conjugated phalloidin. we examined three major regions of the sponge tissue by confocal laser scanning microscopy: 1) the outer edge of sponge growth, 2) the regions containing newly developed choanocyte chambers, and 3) the endopinacocyte-lined canals surrounded by choanocyte chambers. an extensive actin network was present at the edge of sponge growth. the outer surface of the sponge that forms attachments to the substrate is composed of exopinacoderm and basopinacoderm and is made of continuous epithelium where some connective and migratory cells are observed. the region also contains filopodia of multipolar cells leading from the mesohyl to the exopinacoderm. small foci represent the edge of two abutting cells when viewed from above, and larger foci likely represent focal adhesion points where actin bundles highlight filopodia of mesohyl cells. sponges fed dsem-actb have a less extensive actin network at the growing edge of the sponge. many fibers do not run continuously through the sponge tissue and the continuous fibers often lack the network organization seen in control sponges. these data likely indicate that there is a reduced cellular mobility in the mesohyl. the sponge region containing newly developed choanocyte chambers was chosen as it is a relatively thin region but allows us to see the overall pattern of developed sponge tissue. in this region the actin-rich choanocytes themselves look very similar in treated and control sponges. to further investigate, we performed transmission electron microscopy on a control and a dsem-actb-treated sponge to see whether ultrastructural aspects of choanocytes were affected after rnai treatment. while a more thorough sampling strategy is required, we found one trait in the microvillar collar that may differ between treatment and control sponges. actin rnai-treated choanocytes had a greater percentage of cells with misshapen/folded collars. the control sponges, on the other hand, contained numerous perfectly round collars, which were not observed in the actin rnai-treated sponges. however, the pattern of endopinacocytes connecting the choanocytes differed markedly. control sponges have canals that are clearly lined by actin-rich endopinacocytes. dsem-actb-fed sponge canals are far less clearly lined. in dsem-actb-treated sponges, the endopinacocytes that span the spaces between choanocytes are thin and display a weaker actin signal. the actin cytoskeletal network is thus reduced in dsem-actb-fed pinacocytes, but the choanocytes appear to be unaffected at the level observed with light microscopy. in the canals lined by actin-containing pinacocytes, the actin fibers run continuously around the canals, over the span of many cells. control sponges have clearly outlined canals in most focal planes. dsem-actb-fed sponges, however, have canals that are not as well-defined and are lacking clear phalloidin-stained pinacocytes in many planes. overall, our observations suggest a general knockdown of the actin cytoskeleton with a less prominent actin network. evaluation of morphological changes in response to actin'knockdown' in tethya wilhelma to examine morphological changes due to actin knockdown in t. wilhelma, we fixed sponges that had been pulse-fed induced ht115/tw-actb bacteria expressing dsrna and control ht bacteria daily after and hours. using whole-mount preparations we examined two major regions of the sponge by clsm that were stained using alexa-488-conjugated phalloidin: 1) the exopinacocyte, and 2) the cortical mesohyl. phalloidin staining revealed an extensive actin network in the exopinacocytes with actin-rich ripple-like structures on the surface of the exopinacocytes, structures typical in t. wilhelma and resembling lamellipodia extensions of fibroblasts. in the dstw-actb-treated specimens, after hours the exopinacocytes showed fewer actin-rich ripples than in the control, while after hours, ripples were almost absent. in addition, the actin network was overall less prominent in the treated sponges than in the control specimens. the largest effect was observed in the cortical mesohyl. the control sponges showed a large number of extremely long spindle-shaped bipolar cells, which are typical morphotypes of highly mobile cells'frozen' in movement by fixation. in contrast, the dstw-actb-treated sponges were characterized by atypical, extremely large atrophic multipolar cells and only few bipolar cells. in comparison, multipolar cells in the control sponges were much smaller. the changes in the pinacocytes with the reduced actin-rich lammellae and atypical mesohyle with the atrophic multipolar cells and the lack of bipolar cells can be explained by decreased actin expression. while the actin turnover rate is not high enough to produce lethal effects or cessastion of contractility, processes based on actin dynamics like cell mobility or lamellipodia formation seem to be strongly affected. since the atrophic multipolar cells correlate with a lack of mobile bipolar cells, it seemed that both forms actually represent extreme morphotypes of a single archeocyte-like cell type.  for the first time, we report successful rnai techniques for gene knockdown in the sponges e. muelleri and t. wilhelma. we used dsrna soaking methodology for e. muelleri and a bacterial feeding method adapted to freshwater and marine culture media for both species. our experiments demonstrate a significant reduction of endogenous mrna levels in both species after feeding and represent the successful proof-of-principle for bacteria-mediated feeding induced rna interference in marine and freshwater demosponges. we tested dsrna methodology using cytoplasmic actin orthologs, a ubiquitously expressed gene involved in cell shape and motility. in e. muelleri, qrt-pcr analysis showed a modest but significant knockdown of em-actb after feeding sponges bacteria expressing dsrna against em-actb for hours compared to controls, with a maximum knockdown of about 55%. t. wilhelma sponges treated for or hours showed similarly modest but significant reductions. t. wilhema treated for up to days showed actin mrna reductions throughout the entire period of the experiment. the maximum knockdown caused by dstw-actb-expressing bacteria was almost 50% compared to the control-group mrna level. we also demonstrated in e. muelleri, at both the mrna and protein level, that it is possible to knock down additional genes, thus illustrating this method should be applicable for other target genes. the reduction in expression for actin demonstrates an efficient silencing for a highly expressed gene that may account for about 5% of the total protein content of a cell. furthermore, in order to increase the level of reduction, longer periods of treatment with dsrna might be required for highly expressed genes or stable proteins like actin. hints of this were seen in the long-term t. wilhelma feeding and e. muelleri soaking experiments. it is possible that the rnai effect may be greater than measured, because the knockdown effect may only be measured in surviving cells. sponges are known to have high proliferation activity, a short cell cycle duration and cell shedding that is predominantly confined to the choanocytes. this high cell turnover rate may be part of the reason that actin will not be knocked down at great levels. due to the biology of these organisms and the method used, feeding sponges dsrna will likely be a technique that can be utilized to knockdown, but not knockout gene expression. however, the overall significantly reduced mrna levels during the entire period of the experiment clearly demonstrated the potential of bacterial-mediated rnai-induced silencing for functional gene knock down studies in poriferans. after feeding em-actin dsrna, e. muelleri did not display a macroscopic phenotype that could be clearly described at the cellular level. however, the primary actin knockdown phenotype shown by phalloidin staining was a less extensive actin network that was manifested in a "crumpled" edge, where tissues should be adhering to the substrate and migrating outwards. additionally, a reduction of cellular connections in the choanocyte-rich regions and canals due to loss of actin in endopinacocytes was observed. t. wilhelma showed neither a reduced survival rate nor alterations of rhythm or kinetics of body contractions. like in e. muelleri, rnai treated specimens of t. wilhelma displayed no distinct macroscopic phenotype. however, we found strong effects on mesohyle and pinacoderm cell anatomy of t. wilhelma. apart from reduced actin content and altered actin network appearance in pinacocytes, the most prominent effects were hypertrophic mesohyle cells and the lack of mobile bipolar cells, indicating a reduced mobility and cell shape dynamics in dsrna-treated specimens. rnai methods allow us to begin to study sponge gene function in a directed manner, including questions regarding evolution of gene regulatory networks, nervous system and muscle evolution, and also functional studies concerning biomineralization and other metabolic processes. our results also suggest that rnai might be a valuable tool to investigate cell-type functionality in sponges, as has been shown for other organisms, like hydra. this would facilitate studies on the evolution of cell differentiation in sponges. additionally, it might help us to differentiate variable morphotypes of one single cell type versus other cell types. thus, rnai would help solve a long-standing problem for sponge biologists and evolutionary biologists interested in cell type evolution by allowing unique studies to clearly define and distinguish sponge cell types, most likely in conjunction with in situ hybridization. we assume that our protocol will be readily applicable to other marine sponge species like the homoscleromorph oscarella lobularis or the calcareous sycon ciliatum and s. raphanus. it will also be adaptable to the freshwater species e. fluviatilis and spongilla lacustris, which are also commonly used to study sponge biology. in conclusion, our present study closed an existing gap for a functional genomics protocol for the porifera and will thus stimulate a number of comparative functional studies, which will deepen our understanding of metazoan evolution. 
we 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. 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.
 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. thus far, functional methods for these organisms have not been available.  we 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.  this technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals. 