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The HMA-LMA dichotomy revisited: an electron microscopical survey of 56 sponge species.

Introduction

Sponges (Porifera) represent an evolutionarily ancient phylum with a fossil record dating back to Precambrian times (Li et al., 1998). Today, sponges are important components of the marine benthos and play an important role in the coupling of benthic and pelagic environments owing to their immense filter-feeding capacities (Bell, 2008; de Goeij et al, 2013). Within their mesohyl tissues, many sponges harbor a great diversity of symbiotic microorganisms from the three domains of life: Archaea, Bacteria, and Eukaryota. To date, representatives from more than 28 bacterial phyla (including candidate phyla such as Poribacteria and Tectomicrobia) and two archaeal lineages were identified from marine sponges (Hentschel et al., 2012; Schmitt et al., 2012; Simister et al., 2012). The vast majority of sponge-associated microbes remain uncultivated and are thus functionally largely uncharacterized (Taylor et al., 2007).

The presence of microorganisms in marine sponge tissues has been known for almost a century. Dosse (1939) and Levi and Porte (1962) were among the first to describe microorganisms in the sponge mesohyl matrix using transmission electron microscopy. It was soon discovered that while some sponge species harbored dense microbial consortia within their mesohyl tissues, the mesohyl of other species from the same habitat were notably devoid of microorganisms (Reiswig, 1974; Vacelet and Donadey, 1977, Wilkinson, 1978). Accordingly, two general categories were identified that were termed "bacterial sponges" and "non-symbiont harboring, normal sponges" (Reiswig, 1981). Later on, the terms "low microbial abundance" (LMA) and "high microbial abundance" (HMA) sponges were coined to acknowledge the additional presence of archaea in sponge tissues (Hentschel et al, 2003). A typical HMA sponge contains 108 to 1010 microorganisms/g sponge tissue, which can make up to 20%-35% of the sponge biomass (Reiswig, 1981; Webster et al, 2001; Hentschel et al., 2012); in contrast, only [10.sup.5] to [10.sup.6] bacteria/g sponge tissue are found in LMA sponges, which is roughly equivalent to the microbial abundances in seawater (Hentschel et al., 2006). This pattern extends to reproductive propagules in that the larvae of HMA sponges contain dense bacterial assemblages at the larval center, while the interior of LMA sponge larvae is largely free of microbes (Ereskovsky and Tokina, 2004; Maldonado, 2007; Schmitt et al., 2007; Gloeckner et al, 2013a, b). Vertical microbial transmission from the parent to the larva, a hallmark of symbiotic host-microbe associations (Bright and Bulgheresi, 2010), is now considered an important and presumably evolutionarily ancient component of HMA sponge symbioses. The microbial community is likely complemented by horizontal acquisition of microbes from seawater, although this process has never been demonstrated (Schmitt et al., 2008a; Webster et al., 2010).

Besides bacterial abundances, there are also noticeable differences in microbial diversity between HMA and LMA sponges. Several studies, employing a variety of 16S rRNA gene-based methods, consistently demonstrated a lower microbial diversity in LMA than in HMA sponges (Weisz et al., 2007; Kamke et al.,2010; Erwin et al., 2011; Schmitt et al., 2012; Giles et al., 2013; Gloeckner et al, 2013a; Poppell et al., 2013; Moitinho-Silva et al., 2014). Each LMA species was dominated by a large clade of Proteobacteria (Alpha-, Beta-, or Gamma-) or Cyanobacteria (genus Synechococcus), and there was little overlap between the LMA sponge microbiomes under investigation. The HMA sponge communities, however, showed more phylum-level diversity, with Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, candidate phylum Poribacteria, and other phyla as dominant community members. Differences extend also to the microbial physiology of the respective microbial consortia, particularly with respect to nitrogen metabolism (Bayer et al., 2008; Schlappy et al., 2010; Ribes et al., 2012). Distinct differences were further noted regarding the distribution of certain polyketide synthase genes (sup-APKS) that were found in all HMA but not in the LMA sponges under investigation (Hochmuth et al., 2010). Clearly, much remains to be learned about the metabolism, physiology, and function of sponge-associated microbial consortia, particularly in the context of the HMA or LMA dichotomy.

With respect to animal morphology, Vacelet and Donadey (1977) observed early on that the HMA sponges generally display a denser mesohyl, narrower aquiferous canals, and smaller choanoycte chambers than their LMA counterparts. In other words, the HMA sponges appear to be less well "irrigated." The narrower canals and smaller choanocyte chambers may result in a reduced water flow when compared to LMA sponges (Weisz et al., 2008; Schlappy et al., 2010). In the present study, we revisited the original observations on microbial abundances in sponges; we used electron microscopy complemented by DAPI cell-counting. Altogether, 56 demosponge species from four different geographic locations were investigated. This survey significantly expands previously published datasets on HMA and LMA sponges (Hentschel et al., 2003; Schmitt, 2007; Schmitt et al., 2008a; Weisz et al., 2008), and the findings are interpreted in a taxonomic framework of the animal hosts.

Materials and Methods

Sponge collection

Scuba divers from the research vessels R/V Seward Johnson and F. G. Walton Smith, Harbor Branch Oceanographic Institution, Florida, collected sponges from various locations around the Bahamas (termed "BAH") over the years 2003-2013 (Table 1). Sponges were further obtained by scuba diving in 2004 in Key Largo, Florida, USA (25[degrees]01 'N, 80[degrees]23'W) ("FL"); offshore Rovinj, Croatia (45[degrees]08'N; 13[degrees]64'E) ("MED") in 2012; at Souda Bay, Crete, Greece (35[degrees]31'N; 24[degrees]09'E) ("MED") in 2013; and at other Mediterranean locations. Sponge samples were further collected at Fsar reef, Thuwal, Red Sea, Saudi Arabia (22[degrees]23'N; 39[degrees]03'E) ("RS") in 2010 (Table 1). Three to five sponge individuals were collected per species and brought to the surface in separate resealable plastic bags. The samples were processed as described below within a few hours after sampling.

Electron microscopy

Freshly collected sponge material of a few cubic millimeters in size was fixed in 2.5% glutaraldehyde/phosphate-buffered saline for 12 h, rinsed three times for 20 min each in PBS, and postfixed in 2% osmium tetroxide/PBS for 12 h. Several pieces per individual (hereafter termed "technical replicates") were dehydrated in an ethanol series (30%, 50%, 70%, 90%, 3 X 100%), incubated three times for 20 min each in propylene oxide and polymerized in Epon 812 (Serva, Germany) for 4 days at 60[degrees]C. The embedded sponge pieces were sectioned with an ultramicrotom (OM U3, C. Reichert, Austria). For contrasting, 70-80-nm thick sections were post-stained with 0.5% uranyl acetate in methanol for 10 min and Reynolds lead citrate for 5 min. The resulting sections were investigated by electron microscopy (Zeiss EM 10, Zeiss, Germany). Several different images of three biological specimens were inspected for each species.

Bacterial quantification protocol

A piece of sponge tissue of about 1 g was removed with an ethanol-sterilized scalpel blade and rinsed three times with 0.2-[micro]m filter-sterilized seawater. The tissue cube was cut so that one side always represented the surface tissue. A 10 X dilution was obtained by adding 1 ml of sponge tissue to 9 ml of 0.2-[micro]m filter-sterilized seawater. The tissue was homogenized with a mortar and pestle and poured through Nitex (100-[micro]m pore size) to remove unground tissue pieces. The suspension was fixed in paraformaldehyde to a final concentration of 3.7% and stored at 4[degrees]C until use. The tissue remainders were rinsed off the Nitex sheet with 0.5-ml filter-sterilized seawater and homogenized, using a mortar and pestle, in 4.5 ml of filter-sterilized seawater. The suspension was poured through Nitex, fixed in 3.7% paraformaldehyde final concentration, and stored at 4[degrees]C until use. Dilutions ranging from [10.sup.-1] to [10.sup.-3] were prepared from each homogenate. One milliliter of each dilution was stained with 0.7 [micro]g/ml DAPI (4,6-Diamidino-2-phenylindole) final concentration in the dark for 30 min. The DAPI stock solution (100 [micro]g [ml.sup.-1]) was prepared weekly and stored at 4[degrees]C. A volume of 1 ml of stained homogenate was added to 9 ml of filter-sterilized seawater and filtered onto a black, 0.2-[micro]m polycarbonate membrane 25 mm in diameter (Millipore, Germany) that was supported by an 0.45-[micro]m cellulose nitrate filter (Schleicher und Schuell, Germany). Vacuum (<10 cm Hg) was applied carefully with a hand pump. The filters were washed once with filter-sterilized seawater and subsequently rinsed with 3 ml of 70% ethanol. The 0.2-[micro]m polycarbonate filter was then mounted with Citifluor (Citifluor Ltd., UK) onto a microscope slide. Bacterial numbers were determined after epifluorescence microscopy using a 100 X magnification oil lense (Axiolab microscope, Zeiss, Germany). Three independent specimens were processed for each species. Each sample represents an average bacterial number from 10 different counting fields. For each sample, the numbers of bacteria and nuclei were counted, and the results from the first and second homogenate were summed. The number of cyanobacteria was counted using the red and green fluorescent filter set.

18S rRNA sponge phytogeny

Nearly full-length 18S rRNA gene sequences published in NCBI GenBank (http://www.ncbi.nlm.nih.gov) were analyzed. In a few cases, the 18S rRNA gene sequences were unavailable and the sequence of a closely related congeneric (and in the case of Batzella rubra, of a confamiliar sequence) were therefore substituted, taking the current changes in demosponge classification into consideration (Redmond et al., 2013). Altogether 45 sponge species were included in the phylogenetic tree, and the GenBank accession numbers are provided in Figure 3. The sequences were aligned using the Sponge Genetree Server with 18S rRNA gene secondary structure information included in the analysis (Erpenbeck et al, 2008). Positions that could not be aligned were excluded from further analyses. Maximum-likelihood reconstruction was inferred with RAxML 7.2.5 (Stamatakis, 2006) using the GTRGAMMI model of nucleotide substitution as suggested by jModeltest ver. 0.1.1. (Darriba et al., 2012) under the Akaike Information Criterion (Akaike, 1974) and 100 fast bootstrap replicates. Analyses were performed on the 64-node Linux cluster of the Molecular Geo- and Palaeobiology Labs, Ludwig Maximilian University, Munich.

Results and Discussion

In the present study, 56 sponge species were inspected by transmission electron microscopy (TEM) for the presence of microorganisms in the

mesohyl matrix. Of the 41 sponge species examined from the greater Caribbean (Bahamas and Florida locations, Table 1), 24 were identified as high microbial abundance (HMA) and 17 as low microbial abundance (LMA) (Tables 2, 3). Of the 12 species sampled from the various Mediterranean locations, four were identified as HMA and eight as LMA. From the Red Sea collection site, one sponge was identified as HMA and three as LMA. For previous TEM-based surveys of HMA and LMA sponge patterns, the reader is referred to the literature: Hentschel et al. (2003) and references cited therein; Schmitt et al. (2008b); Weisz et al. (2008); Popell et al. (2013). The question arises whether the sponges fall into strict HMA or LMA categories or whether they rather represent a continuum. Judging from our data, the HMA-LMA dichotomy may be best described as a continuum with a highly biomodal distribution, in the sense that most investigated species are found at the extreme ends (either HMA or LMA) of the continuum.

The HMA sponge tissues contained dense and morphologically diverse microorganisms that are located largely extracellularly in the mesohyl matrix (Fig. 1A-I). In extreme cases, the microbial cells are much more abundant than sponge cells, as shown here for Aplysina aerophoba (Fig. 1A) and Spheciospongia vesparium (Fig. 1B) as well as elsewhere, for example, for the order Verongida (Vacelet, 1975; Friedrich et al., 1999). Our data support the results of Weisz et al. (2008) but disagree with those of Popell et al. (2013) in that S. vesparium is classified here as an HMA sponge. While the potential for phenotypic plasticity in microbial amount cannot be ruled out, we have found microbial abundance to be a highly conserved trait on the level of sponge species.

In general, there is a remarkably stable presence of certain bacterial morphotypes, of which some appear to contain intracellular compartments and unusual membrane structures (Fig. 1C). Some of these originally described morphotypes (i.e., types A and C according to Vacelet, 1975, and Friedrich et al., 1999) can readily be identified in the present pictures of HMA sponge tissue. The amount of bacteria in the HMA sponge tissues can, however, be variable, ranging from densely packed mesohyl tissues for the order Verongida (Fig. 1A) to moderately dense microbial consortia such as those of the taxa Ircinia or Agelas (Fig. 1G, H). However, additional literature reports based on 16S rRNA gene sequencing and inspections of microbial abundances in larval tissues clearly identified these species as HMA sponges (Schmitt et al., 2007, 2008a,b; Schmitt, 2007). Experimental artefacts may have arisen for sponges that were difficult to cut with a diving knife (i.e., Ircinia), or for sponges having tissues that are less cohesive (i.e., Xestospongia). However, the consistency between technical replicates (representing samples from the same individual) and between biological replicates (representing samples from different individuals) is remarkably high in our experience.

Relative to HMA sponge species, the mesohyl of LMA sponges was noticeably devoid of microorganisms (Fig. 2). A few intracellular bacteria in various stages of digestion were present that likely represent food bacteria (Fig. 2F). Occasionally, bacterial morphotypes were observed in TEM pictures of LMA sponges (Gloeckner et al., 2013a); how ever, the morphotype diversity appeared unlike that of HMA sponges in that the compartmentalized cells and those with unusual membranes were missing (Vacelet, 1975; Friedrich et al., 1999). Therefore, on the basis of electron microscopical observations, the combination of bacterial abundance and morphotype diversity determines whether a sponge species belongs to the HMA or LMA category. However, in some sponges with intermediate bacterial abundances, TEM may not be sufficient to determine whether a given species is HMA or LMA, and in these exceptional cases, additional methods are needed.

When the status of a sponge species as either HMA or LMA was equivocal on the basis of microscopical observations, an independent line of evidence was sought in microbial enumeration by staining with DAPI (Table 4). Sponge tissue homogenates were used for bacterial and cyanobacterial quantification, and numbers were either expressed in total or as ratios relative to the number of sponge nuclei. Further, sponge homogenates were screened for the presence or absence of bacteria, using defined HMA and LMA sponge homogenates as controls. The sponge species subjected to this analysis formed two distinct groups of HMA and LMA sponges, which mirrored the TEM observations. Siphonodictyon coralliphagum was the only exception, in which the low bacterial numbers obtained by DAPI staining indicated it was an LMA sponge, but the TEM observations identified it as an HMA sponge (Schmitt, 2007). The DAPI method can be prone to errors; for example, if the bacterial cells are disrupted upon tissue homogenization, particularly as a result of the presence of cytotoxic secondary metabolites. However, this problem happened only once in 56 species investigated and appeared to be a rare scenario. The ratio of bacteria per sponge cell nuclei was variable, ranging from 17 to 95 bacterial cells per sponge cell nucleus for Xestospongia muta and Agelas insularis, respectively, and from 0 to 5 bacterial cells per sponge cell nucleus for the LMA sponges (Table 4). These data support the TEM observations that HMA sponges contain variable amounts of microorganisms in the mesohyl tissues. Major disadvantages of the DAPI-staining method were the large amount of background staining and the limited applicability to sponges with high intrinsic autofluorescence. Also, some sponge tissues were difficult to macerate and yielded clumpy homogenates, making it difficult to determine precise numbers. In these cases, confocal microscopy on sponge tissue sections, as recently employed by Ribes and coworkers (Ribes et al., 2012), is a suitable alternative for microbial visualization and quantification in sponge tissues.

Several recent publications addressed the HMA-LMA dichotomy by 16S rRNA gene sequencing. Whether obtained by clone libraries (Kamke et al., 2010; Giles et al., 2013), DGGE (Weisz et al., 2007; Gloeckner et al., 2013a; Poppell et al., 2013,), terminal restriction fragment length polymorphism (T-RFLP) (Erwin et al., 2011), or amplicon sequencing (Schmitt et al., 2012; Moitinho-Silva et al., 2014), the data consistently revealed a different bacterial composition in LMA sponges than in HMA sponges. Similarly, in comparative DGGEs, the LMA sponges consistently displayed less complex banding patterns than their HMA counterparts (Weisz et al., 2007; Gerce et al., 2011; Popped et al., 2013). Even small 16S rRNA gene clone libraries were sufficient to detect the major phylogenetic lineages (Moitinho-Silva and Hentschel, unpubl. data). 16S rRNA gene data have therefore proven to be very useful to infer the HMA or LMA status of the host sponge.

The host phylogeny based on nearly full-length 18S rRNA gene sequences corroborates the current molecular phylogenetic hypotheses of demosponges (e.g., Morrow et al., 2012; see also Redmond et al. (2013) for the most comprehensive 18S rRNA gene phylogeny). However, as the new nomenclature is yet to be finalized, we refer to taxon names as currently used in the World Porifera Database (van Soest et al., 2014). Several clades were recovered that in the present sample set consisted exclusively of HMA taxa (Verongida, Agelasida (Agelas)), while the Poecilosclerida (sensu stricto) clade with representatives from six families consisted exclusively of LMA species (Fig. 3). For the remaining clades, of which many orders and families are currently under redefinition (see, e.g., Redmond et al., 2013), distinct HMA/LMA distribution patterns were not recognized. Additional and distinct patterns might be revealed on lower taxonomic levels given a more representative taxonomic sampling. It is noteworthy, however, that the HMA/LMA characteristics are conserved in closely related species over time and space when collected from different geographic regions (Wilkinson, 1978; Montalvo and Hill, 2011).

The question remains open as to what causes the HMALMA dichotomy. A survey of the literature revealed no apparent correlation with host defense status (Chanas and Pawlik, 1995; Pawlik et al., 1995), with reproductive mode (oviparous vs. viviparous), or with ecological parameters, as both types of sponges coexist in the same habitat (Schiller, 2006). A phylogenetic signature is present only to a moderate extent. At this point it appears most likely that sponge morphology is an important determining factor. The HMA sponges are frequently large and massive and generally have a firm touch and fleshy consistency, while LMA sponges are generally smaller and feel fragile, soft, brittle, or tough (U.H., pers. obs.). Similarly, the architecture of the sponge interior plays a determining role (Vacelet and Donadey, 1977; Weisz et al., 2008). A higher choanocyte chamber density in LMA sponges was reported by Poppell (2013) and Schlappy (2010). A current hypothesis states that LMA sponges invest more energy into feeding structures, whereas the nutrition of HMA sponges is supplemented by their microbial symbionts (Poppell et al., 2013). With respect to the postulated role of symbionts in the "sponge loop" (de Goeij et al., 2013), an improved understanding of the different feeding strategies of the sponge "holobiont" is clearly a worthwhile undertaking. It is further safe to speculate that HMA sponges are morphologically adapted to house microbial consortia within their tissues. In evolutionary terms, the question arises whether the sponges are preconditioned to host microbes or whether the animal tissue morphology is a consequence of it containing the microbes. If preconditioned, it is conceivable that the extracellular matrix (ECM) of HMA sponges may be structurally altered to accommodate the presence of sponge symbionts; and vice versa, that the sponge symbionts may have mechanisms to survive within ECM, modifying it with their activities. Indeed, a recent study provides compelling evidence obtained by single-cell genomics that poribacterial sponge symbionts can degrade ECM for nutritional purposes (Kamke et al., 2013).

Although the present study is the most comprehensive survey for HMA and LMA patterns to date, more investigations are needed with greater taxonomic depth, including analysis of specimens from other sponge classes besides Demospongiae; and including more locations, such as the Great Barrier Reef, the deep-sea, and the polar seas. With regard to methodologies, we propose a combination of transmission electron microscopy and 16S rRNA gene sequence data to reliably determine the HMA or LMA status of the host sponge. The latter is particularly recommended when TEM data yield ambiguous results. The present sponge survey will help pave the way for a functional understanding of the HMA-LMA dichotomy in sponges.

Abbreviations: HMA, high microbial abundance; LMA, low microbial abundance.

Acknowledgments

We gratefully acknowledge the marine operations personnel at the National Undersea Research Center (Key Largo, Florida), the Ruder Boskovic Institute (Rovinj, Croatia), the Hellenic Center for Marine Research (HCMR, Crete, Greece), and the Coastal and Marine Resources Core Lab at KAUST (Thuwal, Saudi Arabia), in particular T. Ravasi and M. Berumen, for expert help during sponge collection. The crews of the R/V Seward Johnson II and F. G. Walton Smith (Harbor Branch Oceanographic Institute) are acknowledged for excellent support during field work. Financial support was provided, by the DFG, SFB567TPC3 to U.H. LM-S was supported by a grant from the German Excellence Initiative to the Graduate School of Life Sciences, University of Wuerzburg.

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VOLKER GLOECKNER [1], MARKUS WEHRL [1], LUCAS MOITINHO-SILVA [1], CHRISTINE GERNERT [1], PETER SCHUPP [2], JOSEPH R. PAWLIK [3], NIELS L. LINDQUIST [4], DIRK ERPENBECK [5], GERT WORHEIDE [5,6], AND UTE HENTSCHEL [1] *

[1] Julius-von-Sachs Institute for Biological Sciences, Department of Botany II, University of Wuerzburg, Julius-von-Sachs Platz 3, 97082 Wuerzburg, Germany; [2] Carl-von-Ossietzky University Oldenburg, Institute for Chemistry and Biology of the Marine Environment (ICBM), Schleusenstr. 1, 26382 Wilhelmshaven, Germany; [3] Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, North Carolina, USA; [4] Institute of Marine Sciences, University of North Carolina at Chapel Hill, North Carolina, USA; [5] Department of Earth and Environmental Sciences, Palaeontology & Geobiology, & GeoBio-Center, Ludwig-Maximilians-Universitat Munchen, Richard-Wagner-Str. 10, 80333 Munchen, Germany; and [6] SNSB-Bayerische Staatssammlung fur Palaontologie und Geologie, Richard-Wagner-Str. 10, 80333 Munchen, Germany

Received 24 February 2014; accepted 28 May 2014.

The first two authors contributed equally to this work.

* To whom correspondence should be addressed. E-mail: ute. hentschel@uni-wuerzburg.de

Table 1
Sponge collection sites

Location        Collection Site             Sponge Species

BAH,            Little San Salvador         Agelas cilrina, Agelas
Bahamas         24[degrees]34.39'N;         dilatata, Aiolochroia
Islands         75[degrees]58.00'W          crassa, Siphonodictyon
                                            coralliphagum, lotrochota
                                            birotulata, Plakortis sp.

                San Salvador                Agelas dispar, Aplysina
                24[degrees]01.14'N;         cauliformis var. thick,
                74[degrees]32.68'W          Aplysina insularis,
                                            Verongula gigantea,
                                            Ptilocaulis sp., Cliona
                                            varians

                Sweetings Cay               Cribrochalina vasculum,
                26[degrees]36.0'N;          Dysidea etheria
                77[degrees]52.60'W

                Chub Cay                    Aplysina cauliformis var.
                25[degrees]23.365'N;        thin, Monanchora
                77[degrees]52.127'W         arbuscula

                Grand Bahama Island         Aplysina archeri,
                25[degrees]1.282'N;         Aplysina fistularis,
                77[degrees]34.56'W          Chalinula molitba

                Exuma Cay 24[degrees]       Myrmekioderma gyroderma,
                25.642'N;76[degrees]        Spheciospongia vesparium,
                40.464'W                    Erylus formosus

                Bimini                      Batzella rubra,
                25[degrees]45.316'N;        Cinachyrella alloclada
                79[degrees]18.061'W

                Great Inagua                Plakortis lita, Svenzea
                21[degrees]05.945'N;        zeai
                73[degrees]40.216'W

                Cat Cay                     Callyspongia plicifera
                25[degrees]31.480'N;
                79[degrees]17.938'W

MED,            Rovinj, Croatia             Aplysina aerophoba,
Mediterranean   45[degrees]05'N,            Chondrosia reniformis,
                13[degrees]38'E             Axinella polypoides,
                                            Tethya aurantium,
                                            Suberites domuncula,
                                            Dysidea avara

                Marseille, France           Aplysina cavernicola,
                43[degrees]11 '48.92'N;     Oscarella lobularis
                5[degrees]21'48.62'E

                Banyuls-sur-Mer, France     Crambe crambe
                42[degrees]29'N;
                03[degrees]08'E

                Souda, Crete, Greece        Petrosia sp., Acanthella
                36[degrees]76.759'N;        acuta, Axinella cannabina
                24[degrees]51.422'E

FL, Florida     Key Largo, USA              Agelas wiedenmayeri,
                24[degrees]56.863'N;        Xestospongia muta,
                80[degrees]27.230'W         Ircinia felix, Ircinia
                                            strobilina, Smenospongia
                                            aurea, Ecytoplasia ferox,
                                            Scopalina ruetzleri,
                                            Tedania ignis, Mycale
                                            Icixissima, Niphates
                                            erecta, Niphates
                                            digitalis, Amphimedon
                                            compressa, Callyspongia
                                            vaginalis, Aplysina
                                            lacunosa

Red Sea         Fsar Reef, Jeddah, Saudi    Amphimedon ochracea,
                Arabia                      Crella cyathophora,
                22[degrees]23.096'N;        Stylissa carteri,
                39[degrees]02.856'E         Xestospongia testudinaria

Table 2
A compilation of high microbial abundance (HMA) sponge species

Species               Collection     Transmission       Higher Taxon
                        Site *    Electron Microscopy    ([section])
                                  Reference ([theta])

Agelas citrina           BAH      Wehrl 2006           Agelasida
Agelas dilatata          BAH      Wehrl 2006           Agelasida
Agelas dispar            BAH      This study           Agelasida
Agelas wiedenmayeri       FL      Wehrl 2006;          Agelasida
                                  Schmitt et al.
                                  2008b
Aiolochroia crassa       BAH      This study           Verongida
Aplysina aerophoba       MED      Hentschel et al.     Verongida
                                  2003; Siegl et
                                  al. 2008

Aplysina archeri         BAH      Wehrl 2006           Verongida
Aplysina                 BAH      Wehrl 2006           Verongida
  cauliformis,
  thick morphotype
Aplysina                 BAH      Wehrl 2006           Verongida
  cauliformis,
  thin morphotype
Aplysina                 MED      Wehrl 2006;          Verongida
  cavernicola                     Friedrich et al.
                                  1999, 2001
Aplysina fistularis      BAH      Wehrl 2006;          Verongida
                                  Gloeckner 2013
Aplysina insularis       BAH      Wehrl 2006           Verongida
Aplysina lacunosa         FL      Wehrl 2006           Verongida
Chondrosia               MED      Wehrl 2006           Chondrosida
  reniformis
Cribrochalina            BAH      Schiller 2006        (marine)
  vasculum                                             Haplosclerida
Ectyoplasia ferox         FL      Schmitt et al.       Raspailiidae
                                  2008a,b; Gloeckner
                                  et al. 2013
Ircinia felix             FL      Schmitt et al. 2007  Dictyoceratida
Ircinia strobilinci       FL      Schmitt 2007         Dictyoceratida
Myrmekioderma            BAH      Gloeckner 2013       "Halichondrida"
  gyroderma
Petrosia sp.             MED      This study           (marine)
                                                       Haplosclerida
Plakortis lita           BAH      This study           Homoscleromorpha
Plakortis sp.            BAH      Laroche et al. 2007  Homoscleromorpha
Siphonodictyon           BAH      Schiller 2006;       (marine)
  coralliphagum                   Schmitt et al.       Haplosclerida
                                  2008b
Smenospongia aurea        FL      Schmitt et al.,      Dicytoceratida
                                  2008b; Gloeckner
                                  2013
Spheciospongia           BAH      This study           "Hadromerida"
  vesparium
Svenzea zeai             BAH      This study           "Halichondrida"
Verongula gigantea       BAH      Wehrl 2006           Verongida
Xestospongia muta         FL      Wehrl 2006;          (marine)
                                  Hentschel et al.     Haplosclerida
                                  2006
Xestospongici             RS      This study           (marine)
  testudinaria                                         Haplosclerida

* BAH, Bahama Islands; FL, Florida; MED, Mediterranean; RS, Red Sea.

([dagger]) Some transmission electron microscopy data were reported in
Master's (Schiller, 2006) and Ph.D. theses (Wehrl, 2006; Schmitt, 2007;
Gloeckner, 2013).

([section]) Higher taxon names in quotation marks indicate orders
recognized as non-monophyletic which may be subject to emendings in
the future.

Table 3
A compilation of low microbial abundance (LMA) sponge species

Species              Collection  Transmission        Higher Taxon
                       Site *       Electron          ([section])

                                  Microscopy
                                   Reference
                                   ([dagger])

Acanthella acuta        MED      This study      "Halichondrida"
Amphimedon               FL      Angermeier et   (marine) Haplosclerida
  compressa                      al. 2012
Amphimedon               RS      This study      (marine) Haplosclerida
  ochracea
Axinella cannabina      MED      This study      "Halichondrida"
Axinella                MED      Wehrl 2006      "Halichondrida"
  polypoides
Batzella rubra          BAH      Gloeckner 2013  Poecilosclerida (s.s.)
Callyspongia            BAH      Gloeckner 2013  (marine) Haplosclerida
  plicifera
Callyspongia             FL      Schiller 2006;  (marine) Haplosclerida
  vaginalis                      Wehrl 2006
Chalinula molitba       BAH      Schiller 2006;  (marine) Haplosclerida
                                 Wehrl 2006
Cinachyrella            BAH      Gloeckner 2013  Spirophorida
  alloclada
Cliona varians          BAH      Schiller 2006   "Hadromerida"
Crambe crambe           MED      Wehrl 2006      Poecilosclerida (s.s.)
Crella cyathophora       RS      Giles et al.    Poecilosclerida (s.s.)
                                 2012
Dysidea avara           MED      Wehrl 2006      Dictyoceratida
Dysidea etheria         BAH      Schiller 2006   Dictyoceratida
Erylus formosus         BAH      This study      Astrophorida
Iotrochota              BAH      Wehrl 2006      Poecilosclerida (s.s.)
  birotulata
Monanchora              BAH      This study      Poecilosclerida (s.s.)
  arbuscula
Mycale Icixissima        FL      Wehrl 2006      Poecilosclerida (s.s.)
Niphates digitalis       FL      Schiller 2006;  (marine) Haplosclerida
                                 Wehrl 2006
Niphates erecta          FL      Wehrl 2006      (marine) Haplosclerida
Oscarella               MED      Gloeckner et    Homoscleromorpha
  lobularis                      al. 2013
Ptilocaulis sp.         BAH      Wehrl 2006      "Halichondrida"
Scopalina                FL      Wehrl 2006;     "Halichondrida"
  ruetzleri                      Gloeckner 2013
Stylissa carteri         RS      Giles et        "Halichondrida"
                                 al. 2013
Suberites               MED      Wehrl 2006      "Hadromerida"
  domuncula
Tedania ignis            FL      Schiller 2006;  Poecilosclerida (s.s.)
                                 Wehrl 2006
Tethya aurantium        MED      Wehrl 2006      "Hadromerida"

* BAH, Bahama Islands; FL, Florida; MED, Mediterranean; RS, Red Sea.

([dagger]) Some transmission electron micrography data were
reported in Master's (Schiller 2006) and PhD theses (Wehrl 2006;
Schmitt 2007; Gloeckner 2013).

([section]) Higher taxon names in quotation marks indicate orders
recognized as non-monophyletic and may be subject to emendings in
the future.

Table 4
Quantification of bacteria, cyanobacteria, and sponge
nuclei by DAPI staining (per ml sponge homogenate)

                           Bacteria Mean        Cyanobacteria
Species                     [+ or -] SE        Mean [+ or -] SE

High microbial
    abundance spongest
  Agelas citrina          7.0 X [10.sup.8]     2.6 X [10.sup.6]
                          [+ or -]  2.1 X       [+ or -] 2.0 X
                             [10.sup.8]           [10.sup.6]
  A. dilatata             1.9 X [10.sup.9]     1.5 X [10.sup.6]
                          [+ or -]  8.7 X       [+ or -] 9.6 X
                             [10.sup.8]           [10.sup.5]
  Aplysina archeri        1.4 X [10.sup.9]     1.1 X [10.sup.7]
                          [+ or -]  1.9 X       [+ or -] 5.8 X
                             [10.sup.7]           [10.sup.6]
  A. insularis            6.2 X [10.sup.9]     2.9 X [10.sup.8]
                           [+ or -] 4.2 X       [+ or -] 7.3 X
                             [10.sup.8]           [10.sup.7]
  Cribochalina            5.5 X [10.sup.9]     1.1 X [10.sup.9]
    vasculum               [+ or -] 2.3 X       [+ or -] 1.7 X
                             [10.sup.9]           [10.sup.7]
  Xestospongia muta       7.0 X [10.sup.9]     1.2 X [10.sup.9]
                           [+ or -] 5.5 X       [+ or -] 2.8 X
                             [10.sup.8]           [10.sup.8]
  Ircinia felix           1.0 x [10.sup.9]     5.7 X [10.sup.8]
                           [+ or -] 7.3 X       [+ or -] 7.1 X
                             [10.sup.7]           [10.sup.6]
  Plakortis sp.           4.3 X [10.sup.9]     3.6 X [10.sup.7]
                           [+ or -] 8.0 x       [+ or -] 1.6 x
                             [10.sup.8]           [10.sup.7]
  Ecytoplasia ferox       8.7 X [10.sup.9]           n.d.
                           [+ or -] 1.7 X
                             [10.sup.8]

Low microbial
    abundance sponges
    ([paragraph])
  Iotrochota              2.4 X [10.sup.9]           n.d.
    birotulata             [+ or -] 2.8 X
                             [10.sup.8]
  Siphonodictyon         2.0 x [10.sup.8] +          n.d.
    coralliphagum         1.5 X [10.sup.7]
    ([section])
  Dictyonella                   n.d.                 n.d.
      funiculciris
  Tedania ignis                 n.d.                 n.d.
  Chalinula molitba       3.5 X [10.sup.6]           n.d.
                           [+ or -] 3.5 X
                             [10.sup.6]
  Niphates digitalis            n.d.                 n.d.
  Amphimedon compressa          n.d.                 n.d.
  Callyspongia            2.2 x [10.sup.6]     1.8 X [10.sup.6]
    vaginalis              [+ or -] 1.6 X       [+ or -] 1.8 x
                             [10.sup.6]           [10.sup.6]
  Ptilocaulis sp.               n.d.                 n.d.
  Cliona varians                n.d.                 n.d.
  Dysidea etheria         3.1 X [10.sup.6]     1.3 X [10.sup.6]
                           [+ or -] 1.2 X       [+ or -] 7.6 X
                             [10.sup.6]           [10.sup.5]
  Caribbean seawater

                           Bacteria Total       Nuclei Mean      Ratio
Species                   Mean [+ or -] SE      [+ or -] SE        *

High microbial
    abundance spongest
  Agelas citrina          7.0 X [10.sup.8]    3.2 X [10.sup.7]   22.16
                           [+ or -] 2.1 X      [+ or -] 5.7 X
                             [10.sup.8]          [10.sup.6]
  A. dilatata             1.9 X [10.sup.9]    5.7 X [10.sup.7]   33.40
                           [+ or -] 8.7 X      [+ or -] 1.7 X
                             [10.sup.8]          [10.sup.7]
  Aplysina archeri        1.4 X [10.sup.9]    3.5 X [10.sup.7]   39.16
                           [+ or -] 2.4 X      [+ or -] 1.8 X
                             [10.sup.7]          [10.sup.7]
  A. insularis            6.5 X [10.sup.9]    6.8 X [10.sup.7]   95.71
                           [+ or -] 4.9 X      [+ or -] 2.2 X
                             [10.sup.8]          [10.sup.6]
  Cribochalina            6.6 X [10.sup.9]    2.1 X [10.sup.8]   31.71
    vasculum               [+ or -] 2.4 X      [+ or -] 3.3 X
                             [10.sup.7]          [10.sup.7]
  Xestospongia muta       8.2 X [10.sup.9]    4.8 X [10.sup.8]   16.96
                           [+ or -] 7.7 X      [+ or -] 4.1 X
                             [10.sup.8]          [10.sup.7]
  Ircinia felix           1.6 X [10.sup.9]    6.1 X [10.sup.7]   25.67
                           [+ or -] 7.4 X      [+ or -] 1.2 X
                             [10.sup.7]          [10.sup.7]
  Plakortis sp.           4.3 X [10.sup.9]    4.8 X [10.sup.7]   89.23
                           [+ or -] 7.9 X      [+ or -] 1.6 X
                             [10.sup.8]          [10.sup.7]
  Ecytoplasia ferox       8.7 X [10.sup.9]    3.8 X [10.sup.8]   22.94
                           [+ or -] 1.7 X      [+ or -] 2.6 X
                             [10.sup.8]          [10.sup.7]

Low microbial
    abundance sponges
    ([paragraph])
  Iotrochota              2.4 X [10.sup.9]    5.2 X [10.sup.8]    4.67
    birotulata             [+ or -] 2.8 X      [+ or -] 4.3 X
                             [10.sup.8]          [10.sup.7]
  Siphonodictyon          2.0 X [10.sup.8]    2.2 X [10.sup.8]    0.93
    coralliphagum          [+ or -] 1.5 X      [+ or -] 6.2 X
    ([section])              [10.sup.7]          [10.sup.7]
  Dictyonella                   n.d.          3.2 X [10.sup.8]    0.00
      funiculciris                             [+ or -] 1.4 X
                                                 [10.sup.7]
  Tedania ignis                 n.d.          8.9 X [10.sup.8]    0.00
                                               [+ or -] 5.2 X
                                                 [10.sup.7]
  Chalinula molitba       3.5 X [10.sup.6]    6.1 X [10.sup.8]    0.01
                           [+ or -] 3.5 X      [+ or -] 7.4 X
                             [10.sup.6]          [10.sup.7]
  Niphates digitalis            n.d.          4.6 X [10.sup.8]    0.00
                                               [+ or -] 1.3 X
                                                 [10.sup.8]
  Amphimedon compressa          n.d.          1.4 X [10.sup.8]    0.00
                                               [+ or -] 2.3 X
                                                 [10.sup.7]
  Callyspongia            4.0 X [10.sup.6]    5.5 X [10.sup.8]    0.01
    vaginalis              [+ or -] 2.0 X      [+ or -] 1.1 X
                             [10.sup.6]          [10.sup.7]
  Ptilocaulis sp.               n.d.          3.7 X [10.sup.8]    0.00
                                               [+ or -] 3.2 X
                                                 [10.sup.7]
  Cliona varians                n.d.          2.0 X [10.sup.8]    0.00
                                               [+ or -] 2.1 X
                                                 [10.sup.7]
  Dysidea etheria         4.4 X [10.sup.6]    1.9 X [10.sup.8]    0.02
                           [+ or -] 1.9 X      [+ or -] 8.1 X
                             [10.sup.6]          [10.sup.6]
  Caribbean seawater      3.5 X [10.sup.5]
                           [+ or -] 5.7 X
                             [10.sup.4]

* Ratio of total bacteria/nuclei.

([dagger]) The following additional sponges were identified as HMA
by qualitative DAPI screening: Ectyoplasia ferox, Myrmekioderma
gyroderma, Agelas dispar, Ircinia felix, Ircinia strobilina,
Smenospongia aurea, Aplysina cauliformis, Aplysina fistularis,
Verongula gigantea, Aiolochroia crassa, Geodia neptuni, Chondrosia
collectrix. Calyx podatypa.

([paragraph]) The following additional sponges were identified as
LMA by qualitative DAPI screening: Erylus formosus, Mycale
(Arenochalina) laxissima, Batzella rubra, Monanchora arbuscula,
Scopalina ruetzleri, Callyspongia plicifera, Callyspongia
vaginalis, Cinachyrella alloclada.

([section]) For Siphonodictyon coralliphagum, the low bacterial
bacterial numbers determined by DAPI staining contradict its status
as an HMA sponge, as determined by transmission electron microscopy
(Schmitt et at, 2008b).

n.d. = not detected.
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Publication:The Biological Bulletin
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Date:Aug 1, 2014
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