ABSTRACT The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given

the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of

sponge-associated bacteria are active _in situ_. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain

insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in _Ancorina alata_, and a much lower diversity in _Polymastia_

example, _Actinobacteria_, _Chloroflexi_ and _Gemmatimonadetes_. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of

sponge-associated microorganisms. SIMILAR CONTENT BEING VIEWED BY OTHERS MICROBIOME OF THE FRESHWATER SPONGE _EPHYDATIA MUELLERI_ SHARES COMPOSITIONAL AND FUNCTIONAL SIMILARITIES WITH THOSE

OF MARINE SPONGES Article 29 July 2022 UNVEILING MICROBIAL GUILDS AND SYMBIOTIC RELATIONSHIPS IN ANTARCTIC SPONGE MICROBIOMES Article Open access 16 March 2024 PHYLOGENETIC DIVERSITY AND

FUNCTIONAL POTENTIAL OF THE MICROBIAL COMMUNITIES ALONG THE BAY OF BENGAL COAST Article Open access 25 September 2023 INTRODUCTION Many marine sponges harbour dense and diverse microbial

communities of considerable ecological and biotechnological importance (Hentschel et al., 2006; Taylor et al., 2007b). These communities, which can include bacteria, archaea and eukaryotic

microorganisms, are often quite specific to sponges, with many microbial phylotypes appearing to live exclusively within sponge hosts and not in the surrounding seawater (Hentschel et al.,

2002; Taylor et al., 2007b; Schmitt et al., 2008). Although our understanding of the microbial diversity in sponges is rapidly improving, much remains unknown about the activity of these

microbes (Taylor et al., 2007a). Specific microbially mediated processes within sponges, such as photosynthesis, sulphate reduction, nitrogen fixation and nitrification, have been quantified

and in many cases the relevant microbes have been identified (Wilkinson and Fay, 1979; Wilkinson, 1983; Diaz and Ward, 1997; Wilkinson et al., 1999; Hoffmann et al., 2005, 2009; Hallam et

al., 2006; Bayer et al., 2008; Mohamed et al., 2008a, 2009; Steger et al., 2008). These studies, which have utilized methods such as isotope enrichments, metagenomics and functional gene

analyses, have extended our knowledge of symbiont function in sponges, yet they remain focused on specific processes or particular functional groups of organisms. What is lacking to date is

a community-wide assessment of microbial activity in marine sponges. This would be useful, as identification of those sponge-associated microbes that are active _in situ_ is an important

step towards elucidating their ecological role and contribution to the host. All living organisms contain ribosomal RNA, and these molecules have become the gold standard for microbial

ecology and taxonomy (Ludwig and Schleifer, 1999; Tringe and Hugenholtz, 2008). The analysis of 16S rRNA genes through clone libraries and fingerprinting approaches such as denaturing

gradient gel electrophoresis (DGGE) has greatly extended our knowledge about the phylogenetic richness of sponge-associated bacteria and archaea (Webster et al., 2001, 2004; Hentschel et

al., 2002; Taylor et al., 2004, 2007b; Holmes and Blanch, 2006; Longford et al., 2007; Schmitt et al., 2007, 2008; Thiel et al., 2007; Mohamed et al., 2008b; Zhu et al., 2008). Researchers

in other systems have taken this approach one step further, yielding insights into both richness and activity by comparing 16S rRNA gene- and 16S rRNA-derived sequences, respectively

(Moeseneder et al., 2001, 2005; Winter et al., 2001; Troussellier et al., 2002; Mills et al., 2005; Gentile et al., 2006; Martinez et al., 2006; Brinkmann et al., 2008; McIlroy et al., 2008;

West et al., 2008; Rodriguez-Blanco et al., 2009). In general, cellular concentrations of rRNA are correlated with growth rate and activity (DeLong et al., 1989; Poulsen et al., 1993),

hence—with acknowledgement of certain caveats (e.g., for ammonia-oxidizing bacteria; Morgenroth et al., 2000)—the rRNA itself can yield useful information about which community members are

active. In this study we investigated bacterial community composition (16S rRNA gene) and activity (16S rRNA) in two marine sponges from northeastern New Zealand. Clone libraries, generating

a total of 313 sequences, were constructed from the high-microbial-abundance sponge _Ancorina alata_ (Demospongiae: Astrophorida: Ancorinidae) and the low-microbial-abundance sponge

_Polymastia_ sp. (Demospongiae: Hadromerida: Polymastiidae). The existence of both high- and low-microbial-abundance sponges is well documented (Vacelet and Donadey, 1977; Reiswig, 1981;

Hentschel et al., 2006; Weisz et al., 2008), although the exact reasons for these differences in microbial loads are uncertain. In addition to the well-characterized taxa, such as the

_Alpha_- and _Gammaproteobacteria_, activity was inferred for uncultivated, sponge-specific lineages within phyla, including the _Gemmatimonadetes_, _Chloroflexi_ and a taxon of uncertain

affiliation related to the sponge-specific ‘_Poribacteria_’ (Fieseler et al., 2004). Moreover, our results show the potential of rRNA gene vs rRNA comparisons to provide insights into the

activity of sponge-associated microorganisms. MATERIALS AND METHODS SPONGE SAMPLING Small samples were taken from three individuals of each of the sponges _A. alata_ and _Polymastia_ sp.

(both class Demospongiae). Sampling was carried out by SCUBA diving at depths of 3–10 m at Mathesons Bay (36°18′S, 174°47′E) and Jones Bay (36°23′S, 174°49′E), northeastern New Zealand, in

November/December 2008. Tissue samples were transferred into RNAlater (Applied Biosystems/Ambion, Foster City, CA, USA), then transported to the laboratory on ice before freezing at −80 °C.

Samples were subsequently freeze-dried and stored again at −80 °C. Tissue samples for electron microscopy were cut into small pieces of about 1 mm3, fixed in 2.5% glutaraldehyde

double-distilled water, and kept overnight at 4 °C before processing. TRANSMISSION ELECTRON MICROSCOPY (TEM) Fixed sponge samples (three individuals per sponge) were washed five times in

cacodylate buffer (50 mM, pH 7.2), fixed in 2% osmium tetroxide for 90 min, washed five times in double-distilled water, and incubated overnight in 0.5% uranyl acetate. After dehydration in

an ethanol series (30%, 50%, 70%, 90%, 96%, and three times at 100% for 30 min each), samples were incubated three times for 30 min in 1 × propylene oxide, maintained overnight in 1:1

(vol/vol) propylene oxide-Epon 812 (EM bed-812, Electron Microscopy Science, Hatfield, PA, USA), incubated twice for 2 h in Epon 812, and finally embedded in Epon 812 for 48 h at 60 °C.

Samples were then sectioned with an ultramicrotome (Leica, Wetzlar, Germany; EM UC6) and examined by TEM (Philips CM12, Fei Company, Hillsboro, OR, USA). NUCLEIC ACID EXTRACTION AND

CONSTRUCTION OF 16S RRNA GENE/RRNA CLONE LIBRARIES Total DNA and RNA were co-extracted from 5–6 mg of freeze-dried sponge tissue using the AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany).

Extractions were performed separately for all three individuals of both species (_A. alata_ and _Polymastia_ sp.). The RNA extract was subsequently purified by DNA digestion for 60 min

using RQ1 RNase-free DNase (Promega, Madison, WI, USA) and RNA was reverse-transcribed into cDNA using random hexamers in the SuperScript III First Strand Synthesis System (Invitrogen,

Carlsbad, CA, USA). Afterwards, DNA, cDNA and RNA were PCR-amplified with the universal bacterial primers 616V (5′-AGAGTTTGATYMTGGCTC-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′) (Kane et al.,

1993; Juretschko et al., 1998), spanning a ∼1500 bp region of the 16S rRNA gene. The RNA template served as a control in the PCR and did not give any products. Cycling conditions on a T1

Thermocycler (Biometra, Goettingen, Germany) were as follows: initial denaturing step at 95 °C for 5 min, 30 cycles of denaturing at 95 °C for 1 min, primer annealing at 54 °C for 1 min and

elongation at 72 °C for 90 s, followed by a final extension step at 72 °C for 10 min. The PCR products from each sponge species representing the respective DNA and cDNA fractions were pooled

and ligated into the pGEM-T-easy vector (Promega) and clone libraries were constructed according to the manufacturer's instructions (four libraries in total). Pooling of PCR products

was justified given that corresponding denaturing gradient gel electrophoresis profiles based on DNA and RNA revealed high inter-individual similarities (data not shown). Clones that

contained an insert (as evaluated by blue/white colony screening) were grown overnight at 37 °C. After a lysis step for 30 min at 94 °C, a PCR was performed with the vector-specific primers

PGEM-F and PGEM-R (Aislabie et al., 2009), to determine which clones contained a correct-sized insert. Cycling conditions were the same as described above. In total, 375 (192 for

_Polymastia_ sp. and 183 for _A. alata_) PCR products were sequenced by Macrogen (Seoul, Korea), and, after the removal of poor quality sequences and chimeric sequences (_n_=62) detected

with Pintail (Ashelford et al., 2005), the final sequence data (81 near-full length (>1200 bp) and 232 partial sequences) were submitted to the DDBJ/EMBL/GenBank databases under accession

numbers FJ900272–FJ900584. STATISTICAL AND PHYLOGENETIC ANALYSES The nonparametric richness estimator Chao1, used to evaluate how much of the richness in each library was sequenced, was

calculated at different operational taxonomic unit (OTU) thresholds using DOTUR implented in Mothur (Schloss and Handelsman, 2005). Preliminary phylogenetic affiliations were obtained for

all DNA- and cDNA (RNA)-derived sequences using the NCBI's BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences from _A. alata_ and _Polymastia_ sp., as well as their

closest relatives identified by BLAST, were aligned using the web-based SINA aligner, then imported into a 16S rRNA ARB-SILVA database (Ludwig et al., 2004; Pruesse et al., 2007) (version

96, containing 271 543 bacterial rRNA sequences) for subsequent manual refinement of the alignment (inspection of the automatic alignment by eye and manual corrections where appropriate

using the editor tool in the ARB software package). Maximum likelihood, maximum parsimony and neighbor-joining trees were calculated in ARB using long (⩾1200 bp) sequences only. Shorter

sequences were added using the parsimony interactive tool in ARB without altering the tree topology. Phylogenetic consensus trees, using the maximum likelihood tree as a backbone, were

manually constructed (Ludwig et al., 1998). Maximum parsimony bootstraps (1000 resamplings) were performed to further assess the stability of observed branching patterns. RESULTS Examination

of sponge mesohyl (three individuals per species) by TEM revealed the presence of large numbers of extracellular, morphologically diverse microorganisms within the sponge _A. alata_,

whereas very few microbial cells were seen in _Polymastia_ sp. (Figure 1). We thus deem _A. alata_ to be a ‘high-microbial-abundance’ sponge (sensu; Hentschel et al., 2006), whereas

_Polymastia_ sp. is a ‘low-microbial-abundance’ sponge. A total of 313 sequences was obtained from the sponges _A. alata_ and _Polymastia_ sp., all of which were of bacterial origin. Of the

157 sequences from _A. alata_, 78 were derived from the 16S rRNA gene and 79 from 16S rRNA. For _Polymastia_ sp. we obtained 85 16S rRNA gene sequences and 71 sequences from 16S rRNA.

Bacterial phylum-level richness was much higher in the _A. alata_ libraries, with members of eight phyla (including three _Proteobacteria_ classes) recovered from the DNA fraction, and seven

of these also occurring in the RNA fraction (Figures 2, 3, 4 and 5, Supplementary Figures 1–4). When a 99% sequence similarity threshold was used to define an OTU, 43 OTUs were found in the

_A. alata_ DNA-derived library, whereas 31 OTUs were found when a 95% OTU definition was applied. The Chao1 estimates of total OTU richness were 73 and 35 for the 99% and 95% OTUs,

respectively. In the _A. alata_ RNA library, thirty-eight 99% OTUs were identified (Chao1 estimate=74), whereas thirty 95% OTUs were found (Chao1=45). The _Polymastia_ sp. DNA library

comprised only two bacterial phyla, with three represented in the RNA library (Figures 2, 3, 4 and 5, Supplementary Figures 1–4). Fourty-five 99% OTUs were found in the DNA library

(Chao1=279), with twenty-two 95% OTUs recovered (Chao1=37). In the RNA library from _Polymastia_ sp., sixteen 99% OTUs were found (Chao1=21), compared with thirteen 95% OTUs (Chao1=15). At

the phylum level, the compositions of the DNA- and RNA-derived libraries from _A. alata_ were very similar (Figure 2). The most abundant taxa in both libraries were the class

_Gammaproteobacteria_ (21% of DNA clones and 34% of RNA clones) and the _Chloroflexi_-affiliated organisms (32% of DNA clones and 20% of RNA clones). Other taxa represented in both libraries

were the _Acidobacteria_, _Actinobacteria_, _Bacteroidetes_, _Gemmatimonadetes_, _Alpha_- and _Deltaproteobacteria_, and a lineage of uncertain affiliation that seems to fall within the

_Planctomycetes_–_Verrucomicrobia_–_Chlamydiae_ (PVC) superphylum (Wagner and Horn, 2006). The only major taxon to be recovered from only one library was the _Nitrospirae_, for which a

single sequence was found in the DNA-derived library. Phylogenetic consensus trees were constructed for all obtained sequences, and examination of these reveals the extent of overlap between

DNA and RNA libraries at the phylotype level as indicated by clusters A1–A16 in Figures 3, 4 and 5 and Supplementary Figures 1–4. For example, concordance between the libraries was high

within the _Actinobacteria_ (Figure 3), in which both DNA and RNA clones (cluster A1) occurred within a sponge-specific cluster. In addition, there was an RNA phylotype without a

corresponding DNA sequence, and DNA phylotypes with no matching RNA sequences. Similar results were evident for the other bacterial phyla (Figures 4 and 5, Supplementary Figures 1–4), in

which there were typically some overlapping DNA and RNA sequences but also examples of DNA or RNA sequences on their own. The _Polymastia_ sp. clone libraries were much less diverse than

those of _A. alata_ (Figure 2). The DNA library was dominated (84% of clones) by a single _Alphaproteobacteria_ lineage (Supplementary Figure 2), with the remaining sequences falling

elsewhere within the _Alphaproteobacteria_ and within one _Actinobacteria_ lineage (Figure 3). Both the _Alphaproteobacteria_ (cluster P2; Supplementary Figure 2) and _Actinobacteria_

(cluster P1; Figure 3) were also represented in the RNA-derived library, although the _Alphaproteobacteria_ comprised only 24% of the sequenced clones. Although absent from the DNA library,

the _Gammaproteobacteria_ were well represented in the RNA library, with several phylotypes together making up 45% of the recovered clones (Supplementary Figure 1). _Spirochaetes_ were also

abundant in the RNA library, comprising 18% of clones (Supplementary Figure 4), but were absent from the DNA library. Consistent with previous studies, many of the sequences obtained in this

study fell into monophyletic, sponge-specific sequence clusters (Hentschel et al., 2002). These occurred to varying extents in all recovered bacterial phyla, although some clusters within

the _Chloroflexi_ (Figure 4), _Gemmatimonadetes_ (Figure 5), _Nitrospirae_ and _Deltaproteobacteria_ (both Supplementary Figure 4) also contained at least one coral-derived sequence.

DISCUSSION This study represents the first community-wide approach to investigating bacterial activity in marine sponges. Previous studies have provided valuable information on specific

metabolic processes and/or the organisms involved, but to gain a broader picture of microbial diversity and activity within any system it is essential to consider the whole community. By

comparing 16S rRNA gene and 16S rRNA profiles of bacterial identity and activity, respectively, we were able to provide insights into the _in situ_ activity of uncultivated, sponge-specific

bacterial lineages. Taxa such as the _Actinobacteria_, _Chloroflexi_, _Gemmatimonadetes_ and _Acidobacteria_ contain large sponge-specific clusters from diverse host sponges; yet, a failure

to obtain most of these organisms in pure culture has led to a paucity of information about their activities and likely function within the host. BACTERIAL 16S RRNA GENE VS 16S RRNA

COMPARISONS IN SPONGES Earlier studies have successfully used the 16S rRNA gene vs rRNA approach to investigate the activity of, for example, marine plankton, sediment and gas hydrate

communities (Moeseneder et al., 2001, 2005; Mills et al., 2005; Gentile et al., 2006; Rodriguez-Blanco et al., 2009). Similar to earlier studies, we found substantial overlap between the

DNA- and RNA-derived libraries (clusters A1–16 and P1 and 2 in Figures 3, 4 and 5, and Supplementary Figures 1–4), but also many cases in which a particular DNA or RNA sequence occurred

alone (e.g., both phenomena can be seen for the _Actinobacteria_ in Figure 3). Each of these cases can be readily explained—or at least speculated upon (Moeseneder et al., 2005). First,

phylotypes from the same sponge species represented in both DNA and RNA libraries are clearly present and—as indicated by the detection of their rRNA—are presumably active as well. This is

the case for, for example, cluster A1 in Figure 3. On this same tree, the _A. alata_ DNA clones AncD11, AncA12, AncD8 and AncE1 may represent abundant bacteria and/or those that contain

multiple 16S rRNA gene operons. The absence of matching clones in the RNA library implies that the bacteria represented by these sequences may have only low activity. RNA clones without

corresponding DNA clones (e.g., AncK22, AncL22 and AncL39 in Figure 3) may represent bacteria that are uncommon but metabolically highly active. It is worth noting here that a conservative

approach was taken, with any inferences about bacterial activity remaining strictly qualitative. Discrepancies between DNA- and RNA-derived libraries can often be explained within a

biological context, as discussed above. However, it is also worthwhile to consider methodological factors that could potentially contribute to such variation among libraries. Most

critically, the entire approach is based on the use of rRNA as a proxy for activity. Although it is now known that ammonia-oxidizing bacteria retain appreciable cellular concentrations of

rRNA even during idle periods when activity is expected to be minimal (Morgenroth et al., 2000), it is widely accepted that in most bacteria rRNA levels are typically correlated with

cellular growth rate and activity (DeLong et al., 1989; Poulsen et al., 1993). The approach therefore seems valid for most bacteria in most situations, although further investigation of

host-associated microbes is required to establish definitively the relationship between rRNA level and activity for symbionts. In addition, small cells are likely to have a lower ribosomal

RNA content when compared with larger cells, even though they could be metabolically more active. Another point to consider is that a specific sequence could be detected in one (i.e., either

DNA or RNA) library but missed in the corresponding library because of insufficient numbers of clones being sequenced. Thus, a difference between DNA and RNA libraries would seem to be

present, when in fact there is none. Given the considerable overlap between our DNA- and RNA-derived libraries, we do not believe this to be a major factor in our case, although this does

depend on the phylotype definition used. If an exclusively monophyletic grouping of DNA- or RNA-derived sequences is used to define a phylotype, then concordance between the libraries is

high. However, one can also consider the case in which phylotypes are defined based on a—somewhat arbitrary—sequence similarity threshold (e.g., 99% may represent ‘species’, whereas 95% may

approximate ‘genus’ level). Using a 95% threshold, observed OTU richness was almost as high in the _A. alata_ DNA library as the total richness predicted by the Chao1 estimator, suggesting

that further sequencing of this library would have revealed very few additional genera. Predictably, the observed and predicted OTU richness values diverge more when a more stringent (99%)

OTU threshold is used. Similar results can be seen for other libraries, indicating that further sequencing could potentially eliminate some of the observed differences between DNA- and

RNA-derived libraries. Irrespective of this, the overall conclusion, that a large fraction of sponge-associated bacteria are active _in situ_, is not affected. What is worth noting is the

seemingly low coverage of the DNA-derived library from _Polymastia_ sp. The observed number of 99% OTUs in this library was 45, whereas an OTU richness of 279 was estimated by the Chao1

analysis. Interestingly, only twenty-two 95%-OTUs were found, whereas the corresponding Chao1 estimate fell to only 37. The vast majority of sequences in this library fall into one

monophyletic cluster within the _Alphaproteobacteria_, with a minimum pairwise similarity of 93.7%; thus, there is evidently a high level of microdiversity (‘species-level’) in this cluster,

but almost all sequences belong to the same ‘genus’ (95%). We used clone libraries to provide the highest degree of phylogenetic information (through the generation of full-length 16S

sequences) while demonstrating the effectiveness of the combined DNA vs RNA approach for sponges. However, other techniques are more appropriate in situations requiring high sample numbers,

for example when examining biological and/or environmental variability. Fortunately, the DNA vs RNA approach is equally applicable to high-throughput community fingerprinting techniques such

as denaturing gradient gel electrophoresis (Winter et al., 2001; Troussellier et al., 2002), terminal restriction fragment length polymorphism (T-RFLP) (Moeseneder et al., 2001) and

single-stranded conformational polymorphism (SSCP) (West et al., 2008; Rodriguez-Blanco et al., 2009). It could also be used in conjunction with next-generation sequencing methods such as

tag pyrosequencing (Sogin et al., 2006; Huse et al., 2008). Our laboratory is currently investigating this for sponges, with the aim of detecting bacterial activity in both rare and abundant

community members. NEW INSIGHTS INTO THE _IN SITU_ ACTIVITY OF SPONGE-ASSOCIATED BACTERIA Marine sponges fall into one of two main categories with respect to their associated

microorganisms. High-microbial-abundance sponges contain very dense communities of diverse microorganisms. In these sponges, microbes occur at densities of up to 1010 cells per gram wet

weight of sponge and their collective biomass may rival that of the host sponge cells (Vacelet, 1975; Friedrich et al., 2001). Our TEM data indicate that the New Zealand sponge _A. alata_

belongs to this category (Figure 1A). Our second sponge, _Polymastia_ sp., exhibited much lower densities of microbes in the mesohyl (Figure 1B) and therefore seems typical of a

low-microbial-abundance sponge, which tend to have microbial densities of 105–106 cells per gram, similar to that of seawater (Hentschel et al., 2006). In addition, _A. alata_ and

_Polymastia_ sp. also differ in the fact that the latter sponge has a much lower bacterial sequence richness, with only three phyla detected compared with eight in _A. alata_ (Figure 2).

Within each sponge, the DNA and RNA libraries showed considerable overlap, indicating that a substantial fraction of the bacterial community within both sponges was physiologically active.

This is evident at the levels of both phylum and specific sequence types, with overlapping phylotypes marked by boxes in Figures 3, 4 and 5 and Supplementary Figures 1–4. In _Polymastia_

sp., corresponding DNA and RNA phylotypes were found in two major clusters, one within the _Actinobacteria_ (P1, Figure 3) and the other within the _Alphaproteobacteria_ (P2, Supplementary

Figure 2). Interestingly, about one-third of all RNA clones, and almost all DNA clones, fell within these two clusters. Phylotypes P1 and P2 might therefore represent true symbionts of

_Polymastia_ sp., although this needs to be confirmed by future studies. In contrast, many remaining RNA clones, mainly within _Gammaproteobacteria_ and _Spirochaetes_, could represent

highly active bacteria serving as food or being contaminants from seawater. Many of these clones cluster with sequences from non-sponge sources (Figures 3, 4 and 5, Supplementary Figures

1–4). In _A. alata_, corresponding DNA and RNA phylotypes were distributed among seven different phyla, thus encompassing almost the entire bacterial diversity found in this sponge. Activity

was therefore not restricted to a specific phylogenetic group of bacteria, but rather to a phylogenetically complex bacterial community. Many of the detected phylotypes in _A. alata_

(including those found at both DNA and RNA levels) were similar to sequences derived from other sponges, or even fell within monophyletic sponge-specific clusters. Much recent attention has

been focused on the phenomenon that even distantly related sponges from different oceans share a subset of their microbial communities that is not found outside sponge hosts (Hentschel et

al., 2002; Taylor et al., 2007b). This is particularly the case for the high-microbial-abundance sponges, which contain numerous lineages that are apparently absent from other environments.

However, despite this interest, there is still little known about the nature of these symbioses. The function of sponge-specific microbes has only been determined for certain microbial

groups that are responsible for, for example, photosynthesis, nitrification or sulphate reduction in sponges (Wilkinson, 1983; Hoffmann et al., 2005; Bayer et al., 2008). This study suggests

that in fact many of the sponge-specific symbionts are active within their respective host sponges. The activity patterns described here for microbial consortia in _A. alata_ and

_Polymastia_ sp. represent only a snapshot at a single time point. Microbial activities may be heavily influenced by host biology and other environmental factors. For example, the pumping

activity of a sponge influences the oxygenation of its mesohyl matrix (Hoffmann et al., 2008). The mesohyl of the Mediterranean sponge _Aplysina aerophoba_ was well oxygenated while the

sponge was pumping water through its body, but became anoxic minutes after pumping ceased (Hoffmann et al., 2008). It is easily conceivable that in the first circumstance aerobic microbes

are active, whereas under anoxic conditions anaerobic microbes would be more active. Additional, chemical and physical factors that might influence the activity of sponge-associated microbes

include changes in temperature, salinity, light or turbulence. The combined 16S rRNA and 16S rRNA gene approach offers a way to evaluate activity changes within the overall sponge microbial

community, whereas analyses of mRNA could give more insights into which specific pathways are being affected. CONCLUDING REMARKS In this study we have successfully shown the application of

the 16S rRNA gene vs rRNA approach to marine sponge-associated bacteria. In the process, we were able to provide the first insights into the _in situ_ activity of uncultivated,

sponge-specific bacterial lineages. There is a compelling argument for including both rRNA gene and rRNA analyses in future investigations of sponge-associated microorganisms, as the

combined approach allows identification of phylotypes that would remain hidden when DNA or RNA clones are examined in isolation. Furthermore, it helps us to tackle one of the key focal

points for sponge microbiology research (Taylor et al., 2007a), the challenge of elucidating symbiont activity and function. ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * FJ900272–FJ900584

REFERENCES * Aislabie J, Jordan S, Ayton J, Klassen JL, Barker GM, Turner S . (2009). Bacterial diversity associated with ornithogenic soil of the Ross Sea region, Antarctica. _Can J

Microbiol_ 55: 21–36. Article  CAS  PubMed  Google Scholar  * Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ . (2005). At least 1 in 20 16S rRNA sequence records currently held

in public repositories is estimated to contain substantial anomalies. _Appl Environ Microbiol_ 71: 7724–7736. Article  CAS  PubMed  PubMed Central  Google Scholar  * Bayer K, Schmitt S,

Hentschel U . (2008). Physiology, phylogeny and _in situ_ evidence for bacterial and archaeal nitrifiers in the marine sponge _Aplysina aerophoba_. _Environ Microbiol_ 10: 2942–2955. Article

  CAS  PubMed  Google Scholar  * Brinkmann N, Martens R, Tebbe CC . (2008). Origin and diversity of metabolically active gut bacteria from laboratory-bred larvae of _Manduca sexta_

(Sphingidae, Lepidoptera, Insecta). _Appl Environ Microbiol_ 74: 7189–7196. Article  CAS  PubMed  PubMed Central  Google Scholar  * DeLong EF, Wickham GS, Pace NR . (1989). Phylogenetic

stains: ribosomal RNA-based probes for the identification of single cells. _Science_ 243: 1360–1363. Article  CAS  PubMed  Google Scholar  * Diaz MC, Ward BB . (1997). Sponge-mediated

nitrification in tropical benthic communities. _Mar Ecol Prog Ser_ 156: 97–107. Article  CAS  Google Scholar  * Fieseler L, Horn M, Wagner M, Hentschel U . (2004). Discovery of the novel

candidate phylum ‘_Poribacteria’_ in marine sponges. _Appl Environ Microbiol_ 70: 3724–3732. Article  CAS  PubMed  PubMed Central  Google Scholar  * Friedrich AB, Fischer I, Proksch P,

Hacker J, Hentschel U . (2001). Temporal variation of the microbial community associated with the Mediterranean sponge _Aplysina aerophoba_. _FEMS Microbiol Ecol_ 38: 105–113. Article  CAS 

Google Scholar  * Gentile G, Giuliano L, D′Auria G, Smedile F, Azzaro M, De Domenico M _et al_. (2006). Study of bacterial communities in Antarctic coastal waters by a combination of 16S

rRNA and 16S rDNA sequencing. _Environ Microbiol_ 8: 2150–2161. Article  CAS  PubMed  Google Scholar  * Hallam SJ, Mincer TJ, Schleper C, Preston CM, Roberts K, Richardson PM _et al_.

(2006). Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine _Crenarchaeota_. _PLoS Biol_ 4: e95. Article  PubMed  PubMed Central 

Google Scholar  * Hentschel U, Usher KM, Taylor MW . (2006). Marine sponges as microbial fermenters. _FEMS Microbiol Ecol_ 55: 167–177. Article  CAS  PubMed  Google Scholar  * Hentschel U,

Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J _et al_. (2002). Molecular evidence for a uniform microbial community in sponges from different oceans. _Appl Environ Microbiol_ 68:

4431–4440. Article  CAS  PubMed  PubMed Central  Google Scholar  * Hoffmann F, Larsen O, Thiel V, Rapp HT, Pape T, Michaelis W _et al_. (2005). An anaerobic world in sponges. _Geomicrobiol

J_ 22: 1–10. Article  Google Scholar  * Hoffmann F, Radax R, Woebken D, Holtappels M, Lavik G, Rapp HT _et al_. (2009). Complex nitrogen cycling in the sponge _Geodia barretti_. _Environ

Microbiol_ 11: 2228–2243. Article  CAS  PubMed  Google Scholar  * Hoffmann F, Roy H, Bayer K, Hentschel U, Pfannkuchen M, Brummer F _et al_. (2008). Oxygen dynamics and transport in the

Mediterranean sponge _Aplysina aerophoba_. _Mar Biol_ 153: 1257–1264. Article  CAS  PubMed  PubMed Central  Google Scholar  * Holmes B, Blanch H . (2006). Genus-specific associations of

marine sponges with group I crenarchaeotes. _Mar Biol_ 150: 759–772. Article  Google Scholar  * Huse SM, Dethlefsen L, Huber JA, Welch DM, Relman DA, Sogin ML . (2008). Exploring microbial

diversity and taxonomy using SSU rRNA hypervariable tag sequencing. _PLoS Genet_ 4: e1000255. Article  PubMed  PubMed Central  Google Scholar  * Juretschko S, Timmermann G, Schmid M,

Schleifer KH, Pommerening-Roser A, Koops HP _et al_. (1998). Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: _Nitrosococcus mobilis_ and

_Nitrospira_ like bacteria as dominant populations. _Appl Environ Microbiol_ 64: 3042–3051. CAS  PubMed  PubMed Central  Google Scholar  * Kane MD, Poulsen LK, Stahl DA . (1993). Monitoring

the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. _Appl Environ Microbiol_ 59:

682–686. CAS  PubMed  PubMed Central  Google Scholar  * Longford SR, Tujula NA, Crocetti GR, Holmes AJ, Holmström C, Kjelleberg S _et al_. (2007). Comparisons of diversity of bacterial

communities associated with three sessile marine eukaryotes. _Aquat Microb Ecol_ 48: 217–229. Article  Google Scholar  * Ludwig W, Schleifer K-H . (1999). Phylogeny of _Bacteria_ beyond the

16S rRNA standard. _ASM News_ 65: 752–757. Google Scholar  * Ludwig W, Strunk O, Klugbauer S, Klugbauer N, Weizenegger M, Neumaier J _et al_. (1998). Bacterial phylogeny based on comparative

sequence analysis. _Electrophoresis_ 19: 554–568. Article  CAS  PubMed  Google Scholar  * Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar _et al_. (2004). ARB: a software

environment for sequence data. _Nucleic Acids Res_ 32: 1363–1371. Article  CAS  PubMed  PubMed Central  Google Scholar  * Martinez RJ, Mills HJ, Story S, Sobecky PA . (2006). Prokaryotic

diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of Mexico. _Environ Microbiol_ 8: 1783–1796. Article  CAS  PubMed  Google Scholar

  * McIlroy S, Porter K, Seviour RJ, Tillett D . (2008). Simple and safe method for simultaneous isolation of microbial RNA and DNA from problematic populations. _Appl Environ Microbiol_ 74:

6806–6807. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mills HJ, Martinez RJ, Story S, Sobecky PA . (2005). Characterization of microbial community structure in Gulf of Mexico

gas hydrates: comparative analysis of DNA- and RNA-derived clone libraries. _Appl Environ Microbiol_ 71: 3235–3247. Article  CAS  PubMed  PubMed Central  Google Scholar  * Moeseneder MM,

Arrieta JM, Herndl GJ . (2005). A comparison of DNA- and RNA-based clone libraries from the same marine bacterioplankton community. _FEMS Microbiol Ecol_ 51: 341–352. Article  CAS  PubMed 

Google Scholar  * Moeseneder MM, Winter C, Herndl GJ . (2001). Horizontal and vertical complexity of attached and free-living bacteria of the eastern Mediterranean Sea, determined by 16S

rDNA and 16S rRNA fingerprints. _Limnol Oceanogr_ 46: 95–107. Article  CAS  Google Scholar  * Mohamed NM, Colman AS, Tal Y, Hill RT . (2008a). Diversity and expression of nitrogen fixation

genes in bacterial symbionts of marine sponges. _Environ Microbiol_ 10: 2910–2921. Article  CAS  PubMed  Google Scholar  * Mohamed NM, Rao V, Hamann MT, Kelly M, Hill RT . (2008b).

Monitoring bacterial diversity of the marine sponge _Ircinia strobilina_ upon transfer into aquaculture. _Appl Environ Microbiol_ 74: 4133–4143. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Mohamed NM, Saito K, Tal Y, Hill RT . (2009). Diversity of aerobic and anaerobic ammonia-oxidizing bacteria in marine sponges. _ISME J_, doi:10.1038/ismej.2009.84. Article  PubMed

  Google Scholar  * Morgenroth E, Obermayer A, Arnold E, Brühl A, Wagner M, Wilderer PA . (2000). Effect of long-term idle periods on the performance of sequencing batch reactors. _Water Sci

Technol_ 41: 105–113. Article  CAS  Google Scholar  * Poulsen LK, Ballard G, Stahl DA . (1993). Use of rRNA fluorescence _in situ_ hybridization for measuring the activity of single cells

in young and established biofilms. _Appl Environ Microbiol_ 59: 1354–1360. CAS  PubMed  PubMed Central  Google Scholar  * Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J _et

al_. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. _Nucleic Acids Res_ 35: 7188–7196. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Reiswig HM . (1981). Partial carbon and energy budgets of the bacteriosponge _Verongia fistularis_ (Porifera: Demospongiae) in Barbados. _PSZNI Mar Ecol_ 2:

273–293. Article  CAS  Google Scholar  * Rodriguez-Blanco A, Ghiglione J-F, Catala P, Casamayor EO, Lebaron P . (2009). Spatial comparison of total vs active bacterial populations by

coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea. _FEMS Microbiol Ecol_ 67: 30–42. Article  CAS  PubMed  Google Scholar  * Schloss PD, Handelsman J .

(2005). Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. _Appl Environ Microbiol_ 71: 1501–1506. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Schmitt S, Angermeier H, Schiller R, Lindquist N, Hentschel U . (2008). Molecular microbial diversity survey of sponge reproductive stages and mechanistic insights

into vertical transmission of microbial symbionts. _Appl Environ Microbiol_ 74: 7694–7708. Article  CAS  PubMed  PubMed Central  Google Scholar  * Schmitt S, Weisz JB, Lindquist N,

Hentschel U . (2007). Vertical transmission of a phylogenetically complex microbial consortium in the viviparous sponge _Ircinia felix_. _Appl Environ Microbiol_ 73: 2067–2078. Article  CAS

  PubMed  PubMed Central  Google Scholar  * Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR _et al_. (2006). Microbial diversity in the deep sea and the underexplored ‘rare

biosphere’. _Proc Natl Acad Sci USA_ 103: 12115–12120. Article  CAS  PubMed  PubMed Central  Google Scholar  * Steger D, Ettinger-Epstein P, Whalan S, Hentschel U, de Nys R, Wagner M _et

al_. (2008). Diversity and mode of transmission of ammonia-oxidizing archaea in marine sponges. _Environ Microbiol_ 10: 1087–1094. Article  CAS  PubMed  Google Scholar  * Taylor MW, Hill RT,

Piel J, Thacker RW, Hentschel U . (2007a). Soaking it up: the complex lives of marine sponges and their microbial associates. _ISME J_ 1: 187–190. Article  PubMed  Google Scholar  * Taylor

MW, Radax R, Steger D, Wagner M . (2007b). Sponge-associated microorganisms: evolution, ecology and biotechnological potential. _Microbiol Mol Biol Rev_ 71: 295–347. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Taylor MW, Schupp PJ, Dahllof I, Kjelleberg S, Steinberg PD . (2004). Host specificity in marine sponge-associated bacteria, and potential implications for

marine microbial diversity. _Environ Microbiol_ 6: 121–130. Article  PubMed  Google Scholar  * Thiel V, Neulinger SC, Staufenberger T, Schmaljohann R, Imhoff JF . (2007). Spatial

distribution of sponge-associated bacteria in the Mediterranean sponge _Tethya aurantium_. _FEMS Microbiol Ecol_ 59: 47–63. Article  CAS  PubMed  Google Scholar  * Tringe SG, Hugenholtz P .

(2008). A renaissance for the pioneering 16S rRNA gene. _Curr Opin Microbiol_ 11: 442–446. Article  CAS  PubMed  Google Scholar  * Troussellier M, Schafer H, Batailler N, Bernard L, Courties

C, Lebaron P _et al_. (2002). Bacterial activity and genetic richness along an estuarine gradient (Rhone River plume, France). _Aquat Microb Ecol_ 28: 13–24. Article  Google Scholar  *

Vacelet J . (1975). Etude en microscopie electronique de l’association entre bacteries et spongiaires du genre _Verongia_ (Dictyoceratida). _J Microsc Biol Cell_ 23: 271–288. Google Scholar

  * Vacelet J, Donadey C . (1977). Electron microscope study of the association between some sponges and bacteria. _J Exp Mar Biol Ecol_ 30: 301–314. Article  Google Scholar  * Wagner M,

Horn M . (2006). The _Planctomycetes_, _Verrucomicrobia_, _Chlamydiae_ and sister phyla comprise a superphylum with biotechnological and medical relevance. _Curr Opin Biotechnol_ 17:

241–249. Article  CAS  PubMed  Google Scholar  * Webster NS, Negri AP, Munro MM, Battershill CN . (2004). Diverse microbial communities inhabit Antarctic sponges. _Environ Microbiol_ 6:

288–300. Article  PubMed  Google Scholar  * Webster NS, Wilson KJ, Blackall LL, Hill RT . (2001). Phylogenetic diversity of bacteria associated with the marine sponge _Rhopaloeides

odorabile_. _Appl Environ Microbiol_ 67: 434–444. Article  CAS  PubMed  PubMed Central  Google Scholar  * Weisz JB, Lindquist N, Martens CS . (2008). Do associated microbial abundances

impact marine demosponge pumping rates and tissue densities? _Oecologia_ 155: 367–376. Article  PubMed  Google Scholar  * West NJ, Obernosterer I, Zemb O, Lebaron P . (2008). Major

differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean. _Environ Microbiol_ 10: 738–756. Article  CAS 

PubMed  Google Scholar  * Wilkinson CR . (1983). Net primary productivity in coral reef sponges. _Science_ 219: 410–412. Article  CAS  PubMed  Google Scholar  * Wilkinson CR, Fay P . (1979).

Nitrogen fixation in coral reef sponges with symbiotic cyanobacteria. _Nature_ 279: 527–529. Article  CAS  Google Scholar  * Wilkinson CR, Summons RE, Evans E . (1999). Nitrogen fixation in

symbiotic marine sponges: ecological significance and difficulties in detection. _Mem Queensland Mus_ 44: 667–673. Google Scholar  * Winter C, Moeseneder MM, Herndl GJ . (2001). Impact of

UV radiation on bacterioplankton community composition. _Appl Environ Microbiol_ 67: 665–672. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhu P, Li Q, Wang G . (2008). Unique

microbial signatures of the alien Hawaiian marine sponge _Suberites zeteki_. _Microb Ecol_ 55: 406–414. Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We gratefully

acknowledge the help of K Lau, G Lear and S Boycheva with RNA analyses, A Turner with TEM, M Mawdsley for assistance with sample collection, and P Deines (all University of Auckland) for

helpful discussions. This research was supported by a University of Auckland New Staff Research Fund grant (Project: 9341 3609286) to MWT and a German Research Foundation (DFG) grant (SCHM

2559/1-1) to SS. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Biological Sciences, The University of Auckland, Auckland, New Zealand Janine Kamke, Michael W Taylor & Susanne

Schmitt Authors * Janine Kamke View author publications You can also search for this author inPubMed Google Scholar * Michael W Taylor View author publications You can also search for this

author inPubMed Google Scholar * Susanne Schmitt View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Michael W Taylor.

ADDITIONAL INFORMATION Supplementary Information accompanies the paper on The ISME Journal website (http://www.nature.com/ismej) SUPPLEMENTARY INFORMATION SUPPLEMENTARY MATERIAL (PDF 116

KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kamke, J., Taylor, M. & Schmitt, S. Activity profiles for marine sponge-associated bacteria

obtained by 16S rRNA vs 16S rRNA gene comparisons. _ISME J_ 4, 498–508 (2010). https://doi.org/10.1038/ismej.2009.143 Download citation * Received: 05 October 2009 * Revised: 25 November

2009 * Accepted: 28 November 2009 * Published: 07 January 2010 * Issue Date: April 2010 * DOI: https://doi.org/10.1038/ismej.2009.143 SHARE THIS ARTICLE Anyone you share the following link

with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt

content-sharing initiative KEYWORDS * activity * bacteria * marine sponges * microbial symbionts * 16S rRNA