The HMA-LMA dichotomy revisited: an electron microscopical survey of 56 sponge species.
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
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.
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.
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 , MARKUS WEHRL , LUCAS MOITINHO-SILVA , CHRISTINE GERNERT , PETER SCHUPP , JOSEPH R. PAWLIK , NIELS L. LINDQUIST , DIRK ERPENBECK , GERT WORHEIDE [5,6], AND UTE HENTSCHEL  *
 Julius-von-Sachs Institute for Biological Sciences, Department of Botany II, University of Wuerzburg, Julius-von-Sachs Platz 3, 97082 Wuerzburg, Germany;  Carl-von-Ossietzky University Oldenburg, Institute for Chemistry and Biology of the Marine Environment (ICBM), Schleusenstr. 1, 26382 Wilhelmshaven, Germany;  Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, North Carolina, USA;  Institute of Marine Sciences, University of North Carolina at Chapel Hill, North Carolina, USA;  Department of Earth and Environmental Sciences, Palaeontology & Geobiology, & GeoBio-Center, Ludwig-Maximilians-Universitat Munchen, Richard-Wagner-Str. 10, 80333 Munchen, Germany; and  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. email@example.com
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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|Author:||Gloeckner, Volker; Wehrl, Markus; Moitinho-Silva, Lucas; Gernert, Christine; Schupp, Peter; Pawlik,|
|Publication:||The Biological Bulletin|
|Date:||Aug 1, 2014|
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