Coral reef ecosystems are well known for their high biodiversity and productivity, despite low ambient nutrient availability. Numerous studies on the nitrogen cycle in coral reefs have linked high local primary production to benthic biological activities, including the efficient recycling of nitrogen between algae and invertebrate hosts and benthic nitrogen fixing communities contributing to a “new” nitrogen source (Dugdale and Goering, 1967; Webb and Wiebe, 1978; O'Neil and Capone, 2009). Marine sponges are important habitat forming organisms in coral reef benthic communities. Their filter-feeding lifestyle can remove large amounts of organic particles in the size range between 0.2 and 10 μm, mainly bacterioplankton and phytoplankton, from the water column (Pile et al., 1996). These microorganisms are consumed as part of the sponge diet. Sponges also serve as hosts to many microbes that live inside the sponge mesohyl as their symbionts. The terms “symbiont” and “symbiosis” are used here consistent with Taylor (Taylor et al., 2007), according to the original definition by de Bary, to refer to two or more organisms found living together for a long period, and do not imply that the organisms benefit or harm each other. The density of microbial symbionts in sponges can reach a billion cells per ml volume, approximately three orders of magnitude higher than in the surrounding seawater (Taylor et al., 2007). Considering these high densities, symbionts are likely to play important roles in the hosts. Molecular tools and high throughput sequencing techniques have helped to overcome the constraints imposed by difficulty in culturing many of these symbionts, and have expanded our knowledge of the sponge microbiome, revealing their connections with host chemical defense, immunity and metabolism (Hentschel et al., 2012). Bacteria are the major driving force in the element biogeochemical cycle (Falkowski et al., 2008). In the sponge mesohyl, frequent water exchange between the sponge and outer environment can create an oxygen gradient and brings in a supply of nutrients (Hoffmann et al., 2005); these conditions may facilitate the essential redox reactions by symbiotic microorganisms (Fiore et al., 2010). The high abundance of microbial cells and suitable conditions are likely to result in significant nutrient flux mediated by the microbial community, which could be important for the local ecosystem.

A classic early study showed the transfer of a photosynthetic carbohydrate from symbiotic cyanobacteria to the sponge hosts (Wilkinson, 1983). The continuous influx of photosynthetic product that is rich in carbon but devoid of nitrogen could trigger the imbalance of C:N ratio in the symbiont-sponge system, leading to a nitrogen source deficiency. Field incubation experiment showed the uptake of ¹⁵N labeled ammonium and nitrate by both sponge cells and bacterial fractions and suggested the translocation of labeled nitrogen from bacteria to hosts (Fiore et al., 2013; Freeman et al., 2013). However, coral reef ecosystems are characterized by low dissolved nitrogen availability in the water column, conditions that might cause the sponge holobiont to seek an alternative nitrogen source to balance their budget. Nitrogen fixation, an anabolic pathway carried out only by prokaryotes, accounts for half of the reactive N supply that sustains ocean primary production (Gruber and Galloway, 2008). This pathway requires an anaerobic microenvironment and significant energy supply for N₂ reduction. To provide suitable conditions for nitrogen fixation, some diazotrophs like Anabaena develop heterocysts as a spatial compartment to create the anaerobic condition; other groups like unicellular cyanobacterium Cyanothece conduct nitrogen fixation at night, temporally separated from the oxygenic photosynthesis that occurs during the daytime (Dixon and Kahn, 2004; Welsh et al., 2008). In a field study in 2007, we found consistently lower δ¹⁵N values from tissues of the sponge Ircinia strobilina, indicating that these sponge individuals obtain their nitrogen from nitrogen fixation, whereas samples from the sponge Mycale laxissima showed higher δ¹⁵N ratios, suggesting less reliance on nitrogen fixation. Subsequent molecular studies demonstrated the presence of diverse nifH genes from cyanobacteria along with heterotrophic bacteria in both sponges. However, the only nifH gene transcripts were those belonging to cyanobacteria (Mohamed et al., 2008). In the current study, we applied a high throughput sequencing method that allowed deeper coverage of the community, and expanded the sampling strategy to monitor the nitrogen fixing activities during a diel cycle. Through our study, we would like to provide more details to major questions regarding the diazotrophic communities associated with sponge hosts. Is the symbiotic community species specific? How stable is the community in the long term? Do active members shift over the diel cycle?

δ¹⁵N values from I. strobilina samples were 1.01 ± 0.97 (SD, n = 24), consistently lower than the values 3.86 ± 0.92 (SD, n = 24) from M. laxissima (ANOVA between sponge species, p < 0.01) and no significant difference (Multiple factorial ANOVA, p > 0.05) between the collection years (Figure 1). We also measured the δ¹⁵N data during the diel cycle. The outcome confirmed the difference in species level, but did not reveal any patterns over a 24 h time span (data not shown).

The diversity of the bacterial diazotrophic communities revealed by nifH gene sequences amplified from total DNA was quite similar between the two sponge species. In the current study, deep sequencing of nifH amplicons revealed a greater diversity of nitrogen fixing groups in the communities. Based on 90% amino acid sequence similarity, we recovered 22 OTUs from the sponge I. strobilina, and a slightly higher diversity (24 OTUs) from M. laxissima (Table 1). The communities were dominated by cyanobacteria, alpha-proteobacteria and, gamma-proteobacteria. Strict anaerobes belonging to delta-proteobacteria were also found. The diversity of nifH genes in the surrounding seawater was lower than in the sponges with 17 OTUs, all from heterotrophic bacteria (Figure 2) and, OTU composition was different from those detected in sponge samples (ANOVA, between sponges and seawater P < 0.001). Community compositions based on sequence reads from individuals were consistent in gDNA source, with no significant difference in both individuals (ANOVA between individuals, p > 0.05) and, species level (ANOVA between sponge species, p > 0.05). Detailed individual community compositions are shown in supplemental Figure S1.

The dominant cyanobacterial OTUs were found in all sponges, regardless of location and time of collection. In fact, the representative sequence from OTU1, belonging to the cyanobacterial genus Leptolyngbya, shared 100% identity with the DNA sequences found in the same sponge species collected in 2004 and 2005 at Conch Reef, Key Largo.

In the cDNA dataset, transcripts from cyanobacteria, alpha-proteobacteria and gamma-proteobacteria accounted for at least 80% of sequence reads in each sample. The diversity of nifH genes from the cDNA libraries was lower than from the gDNA libraries. 13 OTUs were shared between sponge species, although two gamma-proteobacterial OTUs were found exclusively in M. laxissima cDNA samples, and one cyanobacterial and one gamma-proteobacterial OTU were found only in I. strobilina samples. Cyanobacterial transcripts were found from filamentous cyanobacteria, including heterocyst-forming genera like Anabaena, and non-heterocyst forming genera like Leptolyngbya and, from unicellular cyanobacteria, closely related to Cyanothece. However, no group showed a consistent expression pattern corresponding to particular times in the light/dark cycle. Most transcripts from heterotrophic bacteria were classified either as alpha-proteobacteria closely related to aerobic genus Xanthobacter or as gamma-proteobacteria closely related to facultative anaerobe Klebsiella (Table 2). A complete list of OTUs found in this study is provided (Table S1) and their phylogenetic relationships with cultured nitrogen fixer and closest environmental clones are listed (Figure S2).

We selected sequences from three major groups: cyanobacteria, gamma-proteobacteria and, alpha-proteobacteria. For these groups, we normalized sequence reads by minimum sample reads across all samples and compared the composition of the actively transcribed components of the community during day and night. When we combined transcript reads from six individuals of the two sponge species we found that cyanobacterial transcripts were dominant in the daytime, accounting for 94.1 ± 10.7% (SD, n = 6) of the total sequence reads from both sponge species. The percentage pattern changed significantly at night (One-Way ANOVA, p < 0.01), with 72.8 ± 37.4% (SD, n = 6) of transcripts deriving from heterotrophic bacteria (Figures 3A,B). In the species level, day/night difference was more significant for I. strobilina (ANOVA between symbiont species during day/night, p = 0.04) and less significant for M. laxissma (ANOVA between symbiont species during day/night, p = 0.11), largely due to a relative high proportion of cyanobacterial transcripts found in the nighttime sample of the third M. laxissma individual.

Within sponge samples, cyanobacteria transcripts were consistently dominant at dusk and heterotrophic bacteria were more abundant at dawn. In contrast, community structure at noon and, at night showed variation among different individuals at each time point. For example, two I. strobilina individuals showed dominance of heterotrophic bacterial transcripts at 10:00 PM, whereas individual 2 at the same time point had transcripts exclusively from cyanobacteria (Figure 3A).

In the nMDS plot of community structure, nifH gene sequences derived from sponge samples clustered together with no obvious distinction in sequences between the two sponge species, whereas nifH gene sequences derived from seawater samples were clearly separated from the sponge-derived sequences (Figure 4).

The δ¹⁵N values in the two sponges M. laxissima and I. strobilina were consistent over two sampling periods 8 years apart, suggesting that the sources of fixed nitrogen for each of these sponge species remain the same over long periods. The fact that I. strobilina is considered to be a high microbial abundance (HMA) sponge and M. laxissima to be a low microbial abundance (LMA) sponge (Reiswig, 1973, 1974) might translate to a relative higher prokaryote activity like nitrogen fixation in the I. strobilina. Based on field observation, adult I. strobilina individuals are generally denser than M. laxissima and show lower pumping rate, and this could create a less efficient oxygen-penetrated mesohyl for I. strobilina thus provide more anaerobic niches for nitrogen fixation by non-heterocyst cyanobacteria and heterotrophic bacteria. The δ¹⁵N values likely reflected the combined effect of biologically available fixed nitrogen input and loss in the holobiont. Studies on nutrient flux through sponges showed that sponges serve as a net source of nitrate (Jiménez and Ribes, 2007; Hoffmann et al., 2009; Fiore et al., 2013), although not much nitrate and ammonium release were reported for M. laxissima and I. strobilina (Southwell et al., 2008). This observation is potentially contradictory to the hypothesis that there is nitrogen deficiency in the sponge microbiome community that requires the activity of nitrogen fixing bacteria to provide additional fixed nitrogen. However, the net export of nitrate does not reflect the spatial heterogeneity in the sponge mesohyl (Webster et al., 2001; Radax et al., 2012). Whether the total sponge mesohyl is a nitrogen limited environment or not, carbohydrate input from photosynthesis and inorganic nitrogen species removal by ammonium oxidation and denitrification (Mohamed et al., 2009) likely requires localized replenishment of fixed nitrogen to the bacterial community. Previous research found similar low δ¹⁵N values in spongin fractions derived from sponge tissue, indicated the transfer of nitrogen to the hosts (Weisz, 2006). Measurement of δ¹⁵N values of bacteria and sponge cells separated from the same sample could provide more direct evidences on whether the hosts benefit more from fixed nitrogen produced by microbial symbionts.

In agreement with our previous study (Mohamed et al., 2009), we found similar nitrogen fixing communities in the two distantly related sponge species, suggesting that the difference in δ¹⁵N values observed in these two species might reflect differences in the rates of nitrogen-fixation rather than being the consequence of different nitrogen-fixing communities. In order to confirm the relatively lower δ¹⁵N data observed in I. strobilina, quantitation of nifH transcripts using real-time PCR could provide insights into the relative nitrogen fixation activities of the two similar nitrogen-fixing communities found in the two sponge species.

Studies of coral reef ecosystems have shown that nitrogen-fixing bacteria are widely distributed in the water column and corals (Hewson et al., 2007; Lema et al., 2014). Our results show a consistent presence of nitrogen-fixing cyanobacterial groups in marine sponges from two geographic locations of the Caribbean coral reef (Sweetings Cay, Bahamas and Key Largo, Florida). The fact that cyanobacterial-derived nifH transcripts are dominant in sponge samples collected during the daytime, suggests that cyanobacteria are key nitrogen-fixing symbionts in the two sponges. The coexistence of heterocyst-forming filamentous cyanobacteria and unicellular diazotrophic cyanobacteria in both sponges also suggests that, in order to protect nitrogenase from an oxidative environment, both spatial and, temporal separation strategies might be adopted by the community. However, when comparing the presence of heterocyst-forming filamentous and, unicellular cyanobacterial transcripts under light/dark conditions, neither group showed a consistent diel pattern in this study.

A previous study on nifH gene diversity associated with corals showed a high proportion of heterotrophic bacteria in those communities (Lema et al., 2014). In our study, high throughput sequencing revealed rare phyla like delta-proteobacteria that were not detected by our previous study in which we used a cloning and sequencing method. Unlike the cyanobacteria, nitrogen fixing heterotrophic bacteria seems to be more active in nitrogen fixation at night. The communities were not well conserved between the two sponge species or between sponges of the same species collected at each location, suggesting that the heterotrophic diazotrophic bacteria may not be as closely associated with their host as the cyanobacterial groups. The fact that, based on phylogeny (Figure S2), the closest environmental clones of many heterotrophic bacterial OTUs found in this study are connected with benthic community (i.e., associated either with coral, sea-grass or from the marine sediment) suggests that the heterotrophic nitrogen-fixing bacterial selection may be controlled by a “first come first served” process (Fan et al., 2012), which proposes that local geographic factors matter the most in shaping some bacterial communities.

A recent study on natural community in the Hawaii Ocean Time-Series station found a diel expression pattern in which photosynthesis related transcripts from Prochlorococcus peaked at dawn or dusk (Ottesen et al., 2014). Although not directly influenced by sunlight, nitrogen fixation is regulated by local environmental factors include pH, O₂, NH⁺₄ and, organic carbon availability which is strongly impacted by a photosynthetic process (Stacey et al., 1992). A study of diazotrophs in the open ocean found temporal patterns of nifH transcript abundance in different cyanobacteria phylotypes but no obvious pattern for heterotrophic gamma-proteobacteria (Church et al., 2005). Though the existence of horizontal transfer in nifH gene in proteobacteria (Cantera et al., 2004) could potentially complicate the phylogenetic assignment, so far no HGT of nifH gene between cyanobacteria and heterotrophs has been reported, thus the multiple taxon nifH gene expression in our study likely reflected bacterial responses to sporadic and transient environmental cues inside the hosts. Sponge mesohyl can undergo spatial gradients that fluctuate through active pumping (Hoffmann et al., 2005). The associated diazotrophic community must control N₂ fixation under these oscillating environmental conditions. We detected diurnal patterns in those members of the bacterial community that are actively expressing nitrogen fixation genes. We speculate that this pattern may reflect a combined effect of energy supply from photosynthesis and oxic states in localized regions of the holobiont during the light/dark cycle. The low light intensity at dusk, at the end of the light cycle, may be a time at which energy is still available from the day-time photosynthesis to power nitrogen fixation in cyanobacteria while accumulated oxygen could limit nitrogen fixation in heterotrophic bacteria. Conversely, at dawn, at the end of the dark cycle, the energy gained from photosynthesis by autotrophs may be exhausted, and, bacterial respiration may create an anoxic state by oxygen consumption, favoring nitrogen fixation from heterotrophic bacteria. The other two time points in our dataset may reflect the intermediate state between the two scenarios described above, thus resulting in individual variation in active nitrogen fixers. Meanwhile, some sampling constraints might limit the interpretation of the current results. First, the stress effects incurred by each time of tissue collection on the sponge hosts and associated microbial communities are worth considering. Though the actual stress impacts are difficult to assess, an additional sampling point during the daytime showing the dominance of cyanobacterial nifH transcripts immediately after the dark cycle, would strengthen our hypotheses. Alternatively, the detection of the expression of stress gene marker like hsp70 gene for the host (Lopez-Legentil et al., 2008) or dnaK gene (Glatz et al., 1999) for symbiotic cyanobacteria over the time course in future studies might provide insights into stress effects in the sponge during the course of sampling.

The imbalance of the global nitrogen budget indicates a potential underestimation of biological nitrogen fixation (Karl et al., 2002). Nitrogen fixation by non-cyanobacterial groups has been overlooked and could play an important role in marine environments (Moisander et al., 2014). Nitrogen fixation by non-cyanobacterial groups in sponges may also contribute to nitrogen input from the benthic community to nutrient limited coral reef ecosystems.

Our study showed that cyanobacteria are a dominant and consistent group in the diazotrophic community within sponges, and, various heterotrophic bacteria groups can be important components in the community. Composed of “core” cyanobacteria and flexible heterotrophic bacteria, nitrogen fixers in sponges represent an optimal combination to replenish the nitrogen pool.