Evidence of nitrification and denitrification in h

Authors: MarieLise Schläppy Sandra I Schöttner Gaute Lavik Marcel M M Kuypers Dirk de Beer Friederike Hoffmann

Publish Date: 2009/11/24

Volume: 157, Issue: 3, Pages: 593-602

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Abstract

Aerobic and anaerobic microbial key processes were quantified and compared to microbial numbers and morphological structure in Mediterranean sponges Direct counts on histological sections stained with DAPI showed that sponges with high microbial abundances HMA sponges have a denser morphological structure with a reduced aquiferous system compared to low microbial abundance LMA sponges In Dysidea avara the LMA sponge rates of nitrification and denitrification were higher than in the HMA sponge Chondrosia reniformis while anaerobic ammonium oxidation and sulfate reduction were below detection in both species This study shows that LMA sponges may host physiologically similar microbes with comparable or even higher metabolic rates than HMA sponges and that anaerobic processes such as denitrification can be found both in HMA and LMA sponges A higher concentration of microorganisms in the mesohyl of HMA compared to LMA sponges may indicate a stronger retention of and hence a possible benefit from associated microbesSponges are evolutionary ancient Metazoa the first multicellular organisms on the tree of life They can harbor great amounts and a large variety of microbes see Taylor et al 2007 for review Microscope studies showed that in some species 33 of the space in the sponge tissue is occupied by microbes Vacelet and Donadey 1977 These species have been termed ‘bacteriosponge’ Reiswig 1981 or ‘high microbial abundance’ HMA sponges Hentschel et al 2003 and can have as many as 108–1010 cells g−1 of sponge wet weight which is 2–4 orders of magnitude higher than the microbial concentration in seawater The microorganisms in HMA species are spongespecific and differ from those in the water column in number and nature Hentschel et al 2003 In contrast ‘low microbial abundance’ LMA sponges have ≤106 microbes g−1 of sponge wet weight Hentschel et al 2003 Microorganisms can be present in the sponge matrix mesohyl between sponge cells within sponge cells Vacelet and Donadey 1977 and even within the nucleus Friedrich et al 1999 Vacelet 1975Molecular studies revealed the presence of a large variety of microbes associated to sponges which belong to 14 different phyla Taylor et al 2007 Spongeassociated microorganisms are very similar across sponge species and within species across latitudinal gradients Hentschel et al 2002 Unfortunately the everincreasing knowledge of the taxonomic nature of spongeassociated microbes infers little about their metabolic function activity and benefit to the sponge Thus microbially mediated biochemical processes must be uncovered if the nature of the spongemicrobe association is to be understood To date several microbial processes have been found in sponges nitrification Bayer et al 2007 Bayer et al 2008 Corredor et al 1988 Diaz and Ward 1997 Jiménez and Ribes 2007 Southwell et al 2008 nitrogen fixation Wilkinson and Fay 1979 Mohamed et al 2008 photosynthesis Arillo et al 1993 methane oxidation Vacelet et al 1996 and sulfate reduction Hoffmann et al 2005aUntil recently sponges were assumed to have an exclusively aerobic metabolism similarly to most invertebrates Their enormous capacity to filtrate water eg Reiswig 1971 was thought to provide adequate oxygenation across the whole sponge at all times The discovery of anaerobic microbes in Porifera led to the proposition of anoxic niches within sponges Webster et al 2001 When oxygen concentrations were measured with microelectrodes inside sponges it became evident that sponges that do not ventilate become quickly anoxic This is the case for Aplysina aerophoba Hoffmann et al 2008 Geodia barretti explants Hoffmann et al 2005a Dysidea avara Schläppy et al 2007 Chondrosia reniformis Schläppy et al in press and Cliona orientalis Schönberg et al 2004 The spatial and/or temporal anoxic microniches within the tissues of those species could result in the activation of the anaerobic microbes present in the sponge The link between anoxia in sponges and anaerobic microbial processes was made by Hoffmann et al 2005b who reported the simultaneous occurrence of anoxic zones in sponge tissues and of sulfate reduction an anaerobic microbial process in Geodia barretti a coldwater spongeVacelet and Donadey 1977 observed that sponges with a dense mesohyl and a reduced aquiferous system host large amount of microorganisms while species with a looser mesohyl and well developed aquiferous system host comparatively less mircroorganisms Recently Weisz et al 2008 provided supporting evidence for this original observation and established that HMA sponges are denser heavier and have a lower pumping rate as the LMA species In this study we investigate whether dense tissue and a high number of associated microbes lead to more diverse microbial processes including anaerobic ones We present a new method of quantification counting microbes directly on tissue sections to determine both microbial numbers and tissue density We chose a LMA sponge Dysidea avara and a HMA sponge Chondrosia reniformis Additionally we included Aplysina aerophoba for which microbial cell counts using the classical sponge slurry approach were already made Friedrich et al 1999 and for which microbial processes have already been described Bayer et al 2008 Jiménez and Ribes 2007The Dysidea avara specimens came from two different locations and dates 1 Adriatic Sea Limski Canal Croatia close to Rovinj Croatia 44°6750′N 13°370′ Sec April 2005 and 2 Northern Adriatic Sea Muntanya de Montgó Punta del Romani at Cala Illa Mateua in the township of L’Escala Girona Spain 42°06863′N 03°10116′E Northern Mediterranean in March 2006 Aplysina aerophoba and Chondrosia reniformis specimens only came from the Croatian site Sponges were immediately fixed in 2 formalin after collection dehydrated in a 30 50 70 ethanol series and stored in 70 ethanol Three tissue blocks were cut from each individual at different zones of the sponge apex middle and basis Each block contained the continuum from sponge surface to sponge core After saturation with liquid cryomedium Jung Tissue Freeze Medium® Leica Microsystems Nussloch for 12 h at 4°C the blocks were trimmed into 1 × 05 cm pieces They were embedded in base molds with fresh cryomedium and left to harden for 12 h at −80°C For histological analysis 5 μmlongitudinal sections were made using a cryostat microtome HM 505E Microm Walldorf Germany at −35°C All sponge sections were mounted on gelatinized glass slides and prepared for microscopic analysis by using 02 4 6diamino2phenyindole DAPI Sigma Since autofluorescence was expected to occur on the sections stained with DAPI and thus give a spurious DAPI signal we also used a second staining method fluorescence in situ hybridization FISH to confirm that the DAPI signals really represented microbial DNA FISH was performed with sections of each species using the Cy3labeled oligonucleotide probe mix EUB IIII Daims et al 1999 Formamide concentrations in the hybridization solution and washing buffer were 35 vv After rinsing and airdrying the sections were mounted in CitiflourMicroscopical analysis was first carried out at 100× magnification with a Zeiss Axiophot microscope equipped with Zeiss filters for DAPI and Cy3 on the entire tissue section to obtain an overview and train visual perception Color micrographs were taken using a Zeiss Axiolmager M1 microscope with an AxiCam MRc camera system Digital image processing was performed using AxioVision 44 software An ocular with a 122 μm × 122 μmcounting grid and scale bar was used for determining the proportion of different tissue types eg cortex mesohyl and for counting of DAPI or FISHstained microbial cells at 1000× magnification To determine microbial abundance thirty visual fields were inspected per specimen on a transect from the sponge’s surface to its core by counting 150 gridcells per tissue block With three tissue blocks per specimen this reveals n = 450 gridcell counts for determination of microorganism numbers per specimen A percentage estimation of the aquiferous system proportion of choanocyte chamber and canals to mesohyl in was carried out for each grid and averaged for each species

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