Porifera Research: Biodiversity, Innovation and Sustainability - 2007 165

Microbial nitrification in Mediterranean sponges: possible involvement of ammonia-oxidizing Kristina Bayer, Susanne Schmitt, Ute Hentschel(*)

Research Center for Infectious Diseases, University of Wuerzburg. Roentgenring 11, D-97070 Wuerzburg, Germany. Tel.: +49-931-312581. Fax: +49-931-312578. [email protected], [email protected], [email protected]

Abstract: The aim of this study was to assess the potential for nitrification in Aplysina aerophoba Schmidt 1862 using a combined physiological and molecular approach. Whole animals were incubated in experimental aquaria and the concentrations of ammonia, nitrate and nitrite were determined in the incubation water using colorimetric assays. Nitrate excretion rates reached values of up to 3.6 µmol g-1 fresh weight day-1 (equivalent to 830 nmol g-1 dry weight h-1) and were matched by ammonia excretion rates of up to 0.56 ± 0.09 µmol g-1 fresh weight day-1. An accumulation of nitrite was not detected in any of the experiments. Control experiments without sponges showed no variation in nitrogen species in the incubation water. A slight increase in ammonia excretion was observed over 11 days of maintenance in holding tanks that were constantly supplied with fresh, untreated Mediterranean seawater. Other sponges from the same habitat (Dysidea avara Schmidt 1862, Tethya sp., Chondrosia reniformis Nardo 1847) showed high rates of ammonia excretion but nitrate excretion was significantly reduced or absent. Using specific PCR primers, 16S rRNA genes of the betaproteobacterial clade of the Nitrosospira cluster 1 were recovered from A. aerophoba, D. avara and Tethya sp. tissues. In conclusion, this study provides physiological and molecular evidence for the presence of nitrifying in A. aerophoba while the potential for nitrification in the other sponges remains to be investigated.

Keywords: Ammonia-oxidizing Betaproteobacteria, microbial consortia, nitrification, 16S rRNA gene, sponge

Introduction 40 - 60 % of the total biomass exceeding concentrations of seawater by two to four orders of magnitude. Molecular Sponges (Porifera) are evolutionarily ancient metazoans diversity analyses showed that the sponge microbiota is with a fossil record dating back nearly 600 million years in phylogenetically complex, yet highly sponge-specific. time (Li et al. 1998). Today, an estimated 13,000 species, Members of eight eubacterial phyla [ classified in three classes (Demospongiae, Calcarea, (Alpha-, Gamma-, Deltaproteobacteria), Acidobacteria, Hexactinellida) populate virtually all benthic marine and Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, freshwater habitats (Hooper and van Soest 2002). Sponges Gemmatimonadetes and Nitrospira], members of the recently have a primitive morphology lacking true organs or tissues. discovered candidate phylum ‘Poribacteria’ (Fieseler et Most metabolic functions are carried out by totipotent, al. 2004), and of one archaeal phylum (Crenarchaeota) are amoeboid cells, termed archaeocytes that move freely numerically abundant and metabolically active in sponges. through the mesohyl matrix. Inhalant and exhalant canals As none of these sponge-specific microorganisms have been build an aquiferous system through which water is actively obtained in pure culture, their function, metabolism, and pumped by flagellated choanocytes (Brusca and Brusca possibly nutritional interactions with the host sponge are 1990). As filter-feeders, sponges efficiently take up nutrients virtually unknown. like organic particles and microorganisms from the seawater, In this study, we aimed to investigate the process of leaving the expelled water essentially sterile (Reiswig 1974, microbial nitrification in Mediterranean demosponges.

Pile 1997, Wehrl et al. 2007). Nitrification describes the oxidation of ammonia3 (NH ) - Despite the fact that sponges feed on microorganisms, to nitrite (NO2 ) by ammonia-oxidizing bacteria (AOB) - large amounts of extracellular microorganisms populate the and archaea (AOA) and subsequently to nitrate (NO3 ) mesohyl matrix of many demosponges (for recent reviews, by nitrite-oxidizing bacteria (NOB) for energy purposes see Hentschel et al. 2003, 2006, Imhoff and Stöhr 2003, (Kowalchuk and Stephen 2001). Several lines of evidence Hill 2004). Bacterial numbers may constitute as much as suggest that marine sponges are indeed a reservoir for 166 nitrifying microorganisms. Firstly, sponges and many other DNA extraction and PCR marine invertebrates release ammonia as a metabolic waste For the amplification of 16S rRNA genes from ammonia- product (Wang and Douglas 1998, Davy et al. 2002) and oxidizing Betaproteobacteria, the primers AOB189f (5’- nitrate excretion has already been documented in Caribbean GGA GAA AAG CAG GGG ATC G-3’) and AOB1224r (Corredor et al. 1988, Pile 1996, Diaz and Ward 1997) and (5’-CGC CAT TGT ATT ACG TGT GA-3’) were used, that Mediterranean (Jimenez and Ribes 2007) sponges. Secondly, originally had been designed as the FISH probes NSO190 16S rRNA gene sequences of several clades of ammonia- and NSO1224, respectively (Loy et al. 2003). The PCR oxidizing Beta- and Gammaproteobacteria and nitrite- reaction mix contained 1 x PCR reaction buffer (Qiagen), 2 oxidizing Nitrospina were recovered from sponge tissues, mM of each primer, 0.2 mM dNTPs (Sigma) and 1.25 U Taq making microbial nitrification a likely scenario (Hentschel et Polymerase (Qiagen) in a final volume of 50 µl. The PCR protocol was as follows: 1 min initial denaturation at 94°C al. 2002, Diaz et al. 2004). In the present study, a combination followed by 30 cycles of denaturation at 94°C for 30 sec, of physiological and phylogenetic approaches was employed primer annealing at 56°C for 30 sec and elongation at 72°C to explore the potential of microbial nitrification in Aplysina for 50 sec. The PCR was terminated with a final elongation aerophoba Schmidt 1862 and several other Mediterranean step at 72°C for 5 min. demosponges. Cloning, RFLP-Analysis and Sequencing Materials and methods Purified PCR products (PCR purification kit, Qiagen) Animal collection were ligated into the pGEMT-easy vector (Promega) and transformed by electroporation into competent E. coli XL 1- Whole, intact colonies of the sponges A. aerophoba, Blue cells. The enzymes Msp I and Hae III were used for Dysidea avara Schmidt 1862, Tethya sp. and Chondrosia restriction fragment length polymorphism (RFLP) analysis. reniformis Nardo 1847 were collected by SCUBA diving Plasmid DNA was isolated from selected clones by standard offshore Banyuls-sur-Mer (France) (42°43’N, 10°08’E) and miniprep procedures (Sambrook et al. 1989) and sequencing from Rovinj (Croatia) (45°05’N, 13°38’E) at depths from 2- was performed on an ABI 377XL automated sequencer 20 m. The animals were in the range of 30-50 g wet weight (Applied Biosystems). (about 15 g for D. avara). Small tissue pieces were removed from freshly collected animals, immediately frozen in liquid Phylogenetic analysis nitrogen and stored at -80°C until use. Whole, intact animals Sequences obtained in this study were checked for were maintained in > 1000 L volume, flow-through holding chimeras with the program Pintail. Sponge sequences tanks that were constantly supplied with fresh, untreated together with reference sequences [received from GenBank Mediterranean seawater prior to the experiments. using BLAST (http://www.ncbi.nlm.nih.gov/BLAST)] were aligned automatically with ClustalX and the alignment was Sponge incubations subsequently corrected manually in Align. Neighbor Joining Individual specimens were placed into aquaria containing (with Jukes-Cantor correction) and Maximum Parsimony three liters of fresh, untreated Mediterranean seawater. A trees were constructed using the ARB software package constant water current was generated by small aquarium (Ludwig et al. 2004). pumps (Vita Tech 300, Vitakraft, Germany). Only sponges that were in good physiological condition as judged by their Results and discussion regular filtration activity were chosen for the experiments. The experiments were performed in triplicate while a fourth In vivo sponge incubations aquarium without a sponge served as a control. In time For A. aerophoba, nitrate excretion rates of 3.6 ± 0.27 µmol intervals, 10 ml aliquots were removed from each aquarium, g-1 fresh weight day-1 (equivalent to 830 nmol g-1 dry weight placed on ice and frozen at -20°C until use. h-1) were determined which corresponded to an ammonia excretion of 0.56 ± 0.09 µmol g-1 fresh weight day-1 (n = 4 ± Determination of ammonia, nitrate and nitrite S.E.) (Fig. 1A). The nitrate excretion rate was about six fold concentrations higher than the ammonia excretion rate. Ammonia and nitrate The ammonia concentration was determined with the did not appear in the incubation water in sponge-free control Indol-phenol-blue reaction (Parsons et al. 1984). The aquaria. Nitrite was not detected in any of the incubations. - In order to measure ammonia uptake rates, 100 or 200 µM concentration of nitrite (NO2 ) was determined by the Griess - NH Cl final concentrations were added to the incubation reaction (Parsons et al. 1984). Nitrate (NO3 ) concentrations 4 were measured indirectly after conversion to nitrite by the nir- water. Aplysina aerophoba was capable of ammonia uptake mutant E. coli strain JBC 606 as described in Pospesel et al. which corresponded to a nitrate excretion rate of 9.2 and 5.0 (1998). Ammonia and nitrate standard curves ranging from µmol g-1 fresh weight day-1 (Fig. 1B). Aplysina aerophoba 0-100 µM were performed for each measurement series and was not capable of taking up nitrate which was tested at a fresh standards were prepared on a weekly basis. concentration of 100 µM (data not shown). 167

Fig. 1: A. Ammonia and nitrate excretion by A. aerophoba (n = 4 ± S.E.). B. Ammonia uptake and nitrate excretion (n = 2). Symbols represent ammonia (♦) and nitrate (▲) concentrations.

Ammonia and nitrate excretion rates of A. aerophoba were determined in correlation to the maintenance time in holding tanks (Fig. 2). After one day of maintenance, the ammonia excretion rate was 0.54 ± 0.07 µmol g-1 fresh weight day-1. After six and 11 days of maintenance, the ammonia excretion rates were slightly increased (1.04 ± 0.05 and 1.11 ± 0.05 µmol g-1 fresh weight day-1, respectively, Krustal-Wallis Test p=0.051). Nitrate excretion rates were similar over time of maintenance, ranging from 0.74 ± 0.15 (Fig. 2A), to 0.94 ± 0.36 (Fig. 2B) and 0.87 ± 0.43 µmol g-1 fresh weight day-1 (Fig. 2C). Interestingly, a correlation between ammonia and nitrate excretion and sponge pumping activity was evident. Fig. 2: Ammonia and nitrate excretion by A. aerophoba over time While ammonia was excreted at almost double rates in non of maintenance in flow-through holding tanks (n = 3 ± S.E.). The pumping sponges, nitrate was not excreted in sponges whose rates were measured after one day (A), six days (B) and 11 days (C) after collection. Symbols represent ammonia (♦) and nitrate (▲) osculi were closed as judged by visual inspection (data not concentrations. shown). The observation that the mesohyl of non-pumping sponges becomes anaerobic within 15 min (Hoffmann et al. unpublished) would be consistent with an inhibition With respect to microbial loads in the mesohyl tissue, of the aerobic process of nitrification. However, possible sponges are characterized as high and low microbial abundance differences in the diffusion process of ammonia and nitrate in sponges (Hentschel et al. 2006). Accordingly, ammonia and non pumping sponges cannot be excluded. nitrate excretion rates were determined in the Mediterranean 168 weight day-1 (Fig. 3C) respectively, nitrate excretion was reduced or not detectable in any of the three sponge species investigated. Furthermore, Tethya sp. and C. reniformis were not able to take up ammonia (data not shown).

Phylogenetic analysis In total, 200 clones were compared by restriction fragment length polymorphism analysis and four major restriction patterns were detected. After removal of five chimeras, nine, one, and two sequences from A. aerophoba, D. avara and Tethya sp. libraries, respectively, were used for phylogenetic tree construction (Fig. 4). All twelve sequences fell into the marine Nitrosospira cluster 1 of the Betaproteobacteria together with marine seawater and sediment sequences. Except Aplysina aerophoba (F) clone 5, the sequences obtained in this study build a subcluster with a high in-cluster similarity (98.5-99.9%). It is noteworthy that sequences were also recovered from the bacteria-free sponge D. avara as well as Tethya sp. whose mesohyl shows moderate amounts of microorganisms (Thiel et al. 2007). While it cannot be excluded that the cloned AOB sequences represent seawater bacteria rather than true sponge symbionts, the high nitrate excretion rates, at least for A. aerophoba, would argue for a specific and probably symbiotic association. Although primers used in this study also match species, no such bacteria could be detected in the clone libraries. Our findings expand those by Diaz (1997) and Diaz et al. (2004) who had reported on the identification of members of the Nitrosomonas eutropha/europae lineage (Betaproteobacteria) in five tropical sponges.

Modelling nitrogen fluxes in the sponge-microbe association The following scenario, depicted in Fig. 5, is proposed based on this and other studies. The sponge host excretes ammonia as a metabolic waste product, which in turn, is oxidized to nitrite by ammonia-oxidizing bacteria (AOB), such as Nitrosospira (this study), Nitrosococcus (Hentschel et al. 2002) or members of the Nitrosomonas eutropha/ europaea l lineage (Diaz et al. 2004). Nitrite is further oxidized to nitrate by nitrite-oxidizing bacteria (NOB), such as Nitrospina or members of the phylum Nitrospira (Hentschel et al. 2002). The coordinated action of members of these two groups might then be responsible for the conversion of ammonia to nitrate in A. aerophoba and possibly also in other sponges. In addition to eubacteria, the involvement of archaea should Fig. 3: Ammonia and nitrate excretion by D. avara (A), Tethya sp. be considered in future studies. Recent literature illustrates (B), and C. reniformis (C), (each species, n = 3 ± S.E.). Symbols represent ammonia (♦) and nitrate (▲) concentrations. Fig. 4: Distance 16S rRNA (1053 bp) gene phylogeny showing the relationships between ammonia-oxidizing Betaproteobacteria low microbial abundance sponges D. avara and Tethya sp. using the ARB program package. Sponge derived sequences and the high microbial abundance sponge C. reniformis (Fig. are shown in bold. Neighbor-joining and maximum parsimony (100 pseudoreplicates) bootstrap values are indicated. Arrow to 3). While ammonia was excreted at similar rates of 3.0 ± 0.39 outgroup (Nitrosococcus oceani Nc1 (AJ298727)). Designation -1 -1 - µmol g fresh weight day (Fig. 3A), 6.95 ± 0.08 µmol g of clusters is adapted from Freitag and Prosser (2003). Scale bar 1 fresh weight day-1 (Fig. 3B) and 5.8 ± 0.9 µmol g-1 fresh indicates 1% sequence divergence. 169 170

Fig. 5: Schematic diagram showing the inferred nitrogen-fluxes resulting from nitrification in the sponge-microbe-association.

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