Biogeochemical interactions among benthic macrofauna, microbial communities and macrophytes in eutrophic coastal lagoons

Surface sediments are interesting spots to analyze the paradigm of biodiversity and ecosystem functioning due to the multiple physic and chemical gradients that shape the interactions among microbial communities, macrofauna and primary producers. Sediments receive large inputs of organic matter and are sites of intense biogeochemical processes, mediated by microbial communities and facilitated by macrofauna, ultimately resulting in nutrients uptake by benthic primary producers or their recycling to the water column. The relationship between diversity and ecosystem functioning was analyzed at different spatial scales in the benthic compartment of two shallow eutrophic lagoons, the Curonian Lagoon (Lithuania) and the Sacca di Goro (Italy). Special attention was given to the benthic nitrogen (N) cycle, due to the critical role of this element in aquatic ecosystem functioning and to the complex regulation of its various oxic and anoxic reactions, carried out by diverse microbes and strongly influenced by macrofauna and primary producers. Investigations were carried out at different spatial scales included whole lagoon (macro-scale) as well as single macrofauna individuals and holobionts’ microbiomes (micro-scale). At the two lagoons the benthic functioning was evaluated by quantifying rates of whole system respiration and production via gas exchange, nutrient cycling and exchange at the sediment-water interface. Multivariate statistical analyses were used to reveal the interactions between the dominant macrofauna species and net solute fluxes and speculate about underlying processes. Such approach allowed to reconstruct how different macrofauna functional groups shape benthic N cycling in different macro areas of the Sacca di Goro, and determine net loss, net recycling or different level of coupling between processes (e.g., ammonification and nitrification, or nitrification and denitrification). In the Curonian Lagoon the whole scale approach was used to verify whether macrofauna act as a natural buffer against redox-dependent phosphorus recycling during short-term events of oxygen shortage. Manipulative experimental approaches addressed specific processes at the microscale, in sediments colonized by different macrofauna functional groups, and along gradients of density. Such approaches included intact or reconstructed sediment incubation, metabolic measurements of single macrofauna individuals, and the use of 15N-labeled inorganic N forms to measure specific microbial transformations (denitrification, anammox, nitrate ammonification, N-fixation) in sediments or in macrofauna microbiota. Moreover, molecular tools were used to analyze microbial diversity (16S rRNA metabarcoding) and activity (marker genes and transcripts) in holobionts. Three organisms that are abundant in the Curonian lagoon were considered: the burrowing larvae of Chironomus plumosus, the filter feeder bivalve Dreissena polymorpha and the phytophagous gammarid Pontogammarus robustoides. Results suggest that in the Sacca di Goro lagoon macrofauna play an important role, in regulating N transformations. However, its importance also depends on the prevailing environmental factors (i.e., salinity, hydrodynamics and background nutrient concentrations). Whereas in the Curonian Lagoon bioturbation did not significantly affect the nutrient metabolism and the stability of reductive-oxidative reactions during anoxia events. Molecular studies revealed that Chironomid larvae burrows are hot-spots of microbial communities involved in N cycling and that these organisms, via bioirrigation, significantly enhance both the recycling of ammonium and N removal via denitrification. Mussels primarily enhance the recycling of N to the water column, both via direct excretion and by stimulating dissimilatory nitrate reduction to ammonium. The latter is likely an effect of mussel’s biodeposits. For these two organisms the quantification of functional genes showed a significantly higher potential for microbial denitrification, nitrate ammonification and N2-fixation in macrofauna as compared to the surrounding environment. As chironomid and dreissenid densities in eutrophic lagoons are large, animals-associated microbes may account for a substantial (and so far, overlooked) N import and recycling. P. robustoides was finally demonstrated to have an important role in the survival of Chara contraria in the eutrophic Curonian Lagoon. The gammarid facilitates C. contraria via active grazing on the macroalgaeassociated epiphytes combined with ammonium excretion, thus supporting the growth of the characeans

Metabolic Rates of Rainbow Trout Eggs in Reconstructed Salmonid Egg Pockets

In situ evaluations of the metabolic rates (i.e., respiration and excretion) of salmonid eggs are mostly indirect, focusing on the sampling of hyporheic water from wild or artificial nests. Comparatively, experimental studies carried out under controlled, laboratory conditions are less abundant due to methodological difficulties. This study presents a novel experimental setup aimed to address this issue and enable the measurement of oxygen and dissolved inorganic nitrogen fluxes in simulated rainbow trout (O. mykiss) egg pockets. The experimental setup consists of reconstructed egg pockets in cylindrical cores under flow-through conditions. Live and dead eyed-stage eggs were incubated in a natural, sterilised gravel substrate. Hyporheic water circulation was ensured using peristaltic pumps, with the possibility to collect and analyse inflowing and outflowing water for chemical analyses. Microcosm incubations, with closed respirometry of eggs in water alone, were also carried out in order to infer the importance of microbial respiration in the simulated egg pockets. The results show an increasing trend in oxygen demand, due to the development of biofilm in the reconstructed egg pockets and increased egg respiration rates. Moreover, egg pockets showed positive ammonium net fluxes connected with the advancing developmental egg stage, while nitrate removal peaked during the last phase of the experiment, mainly due to the formation of oxic-hypoxic interfaces, leading to couple nitrification–denitrification processes. The suggested approach enables to test a number of in situ situations, including the effects of extreme hydrological conditions, sediment clogging and sudden changes in water chemistry or temperature on the survival and metabolic performances of nests, at different egg development stages

Estuarine macrofauna affects benthic biogeochemistry in a hypertrophic lagoon

Coastal lagoons display a wide range of physico-chemical conditions that shape benthic macrofauna communities. In turn, benthic macrofauna affects a wide array of biogeochemical processes as a consequence of feeding, bioirrigation, ventilation, and excretion activities. In this work, we have measured benthic respiration and solute fluxes in intact sediment cores with natural macrofauna communities collected from four distinct areas within the Sacca di Goro Lagoon (NE Adriatic Sea). The macrofauna community was characterized at the end of the incubations. Redundancy analysis (RDA) was used to quantify and test the interactions between the dominant macrofauna species and solute fluxes. Moreover, the relevance of macrofauna as driver of benthic nitrogen (N) redundancy analysis revealed that up to 66% of the benthic fluxes and metabolism variance was explained by macrofauna microbial-mediated N processes. Nitrification was stimulated by the presence of shallow (corophiids) in combination with deep burrowers (spionids, oligochaetes) or ammonium-excreting clams. Deep burrowers and clams increase ammonium availability in burrows actively ventilated by corophiids, which creates optimal conditions to nitrifiers. However, the stimulatory effect of burrowing macrofauna on nitrification does not necessarily result in higher denitrification as processes are spatially separated

Data from: Direct contribution of invertebrate holobionts to methane release from coastal sediments

In this study, we incubated 103 animals specimens equivalent to 19 macrofaunal species to quantify holobiont-associated methane (CH4) fluxes and metabolic processes [oxygen (O2) - respiration and ammonium (NH4+) - excretion rates]. The specific goals were to quantify holobionts CH4 production/uptake and to establish correlations between CH4 fluxes and environmental factors (e.g., salinity). Invertebrates were collected in 4 coastal systems and incubated in 22 mL glass microcosms filled with 0.22 µm twice-filtered in situ water. Individual and Mass-standardized CH4 Production Rates (IPR and MPR, respectively), O2 Respiration Rates (IRR and MRR) and NH4+ Excretion Rates (IER and MER) were measured in 103 animals’ incubations. 1. IRR were calculated from linear regression analysis of the solute (O2) versus time equation: $IRR=(Reg.Slope × V)/N$ where IRR (µmol O2 ind.−1 day−1) is the respiration of the chemical species O2; Reg.Slope is the slope of the regression (µmol O2 L−1 day−1); V (L) is the water volume in the glass microcosm; N is the number of incubated animals per microcosm. 2. IER and IPR were calculated from the difference in concentrations (NH4+ and CH4) in the water using the equation: $IER and IPR = ((C_f-C_i )×V)/(N×t)$ where IER and IPR (µmol ind.−1 day−1 and nmol ind.−1 day−1) are the excretion or production of the chemical species (NH4+ or CH4); Cf and Ci (µmol or nmol L−1) are the final and initial concentrations of the chemical species; V (L) is the water volume in the glass microcosm; N is the number of incubated animals per microcosm; and t (days) is the incubation time. Positive values represent productions while negative values represent uptake. Same equations were used to calculated mass-standardized rates, but instead of N the total animal biomass (gdw) was used. Animals’ biomass was determined as dry weight (DW) or as dry weight shell free (DWSF) for bivalves, after the desiccation at 70°C until constant mass. Water temperature and salinity were measured in situ with a multiple probe (556 MPS, YSI). Rates are reported as average ± standard error.This work was supported financially by project INBALANCE funded by the European Social Fund (Grant No. 09.3.3-LMT-K-712-01-0069). Stefano Bonaglia was additionally supported by the Swedish Research Council Formas (Grants No. 2017-01513 and No. 2022-00546)

Food sources for benthic grazers in trophic networks of macrophyte habitats in a transitional Baltic ecosystem

In this study, we provide insights into that characteristics of two sites representing different conditions of productivity and salinity impact on trophic network structures of macrophyte habitats and diet of benthic grazers at the active vegetation period in the Curonian Lagoon (southeastern Baltic Sea). Regarding the epiphytic growth, macrophytes were more overgrown in the relatively less productive (northern) site with a muddy bottom and more frequent marine water inflow than in the (southern) site with higher productivity and freshwater sandy habitat. Stable isotope analysis revealed that organisms’ samples from the northern site were more enriched with the heavier carbon isotopes, but depleted in the heavier nitrogen isotopes than those from the southern site. Gastropods and amphipods mainly consumed sedimentary organic matter in the southern site, while they grazed epiphytes together with sedimentary organic matter in the northern site. Although to a low extent, gastropods consumed more charophytes than pondweeds in the southern site. This study contributes to a better understanding of the functioning and structure of lagoonal systems, highlighting the importance, often overlooked, of the benthic compartment, which, however, may have a relevant influence on the productivity of the whole system

Active DNRA and denitrification in oxic hypereutrophic waters

Since the start of synthetic fertilizer production more than a hundred years ago, the coastal ocean has been exposed to increasing nutrient loading, which has led to eutrophication and extensive algal blooms. Such hypereutrophic waters might harbour anaerobic nitrogen (N) cycling processes due to low-oxygen microniches associated with abundant organic particles, but studies on nitrate reduction in coastal pelagic environments are scarce. Here, we report on 15N isotope-labelling experiments, metagenome, and RT-qPCR data from a large hypereutrophic lagoon indicating that dissimilatory nitrate reduction to ammonium (DNRA) and denitrification were active processes, even though the bulk water was fully oxygenated (> 224 µM O2). DNRA in the bottom water corresponded to 83 % of whole ecosystem DNRA (water + sediment), while denitrification was predominant in the sediment. Microbial taxa important for DNRA according to the metagenomic data were dominated by Bacteroidetes (genus Parabacteroides) and Proteobacteria (genus Wolinella), while denitrification was mainly associated with proteobacterial genera Pseudomonas, Achromobacter, and Brucella. The metagenomic and microscopy data suggest that these anaerobic processes were likely occurring in low-oxygen microniches related to extensive growth of filamentous cyanobacteria, including diazotrophic Dolichospermum and non-diazotrophic Planktothrix. By summing the total nitrate (NO3 −) fluxes through DNRA and denitrification, it results that DNRA retains approximately one fifth (19 %) of the fixed N that goes through the NO3 − pool. This is noteworthy as DNRA represents thus a very important recycling mechanism for fixed N, which sustains algal proliferation and leads to further enhancement of eutrophication in these endangered ecosystems

Zebra mussel holobionts fix and recycle nitrogen in lagoon sediments

Bivalves are ubiquitous filter-feeders able to alter ecosystems functions. Their impact on nitrogen (N) cycling is commonly related to their filter-feeding activity, biodeposition, and excretion. A so far understudied impact is linked to the metabolism of the associated microbiome that together with the host constitute the mussel’s holobiont. Here we investigated how colonies of the invasive zebra mussel (Dreissena polymorpha) alter benthic N cycling in the shallow water sediment of the largest European lagoon (the Curonian Lagoon). A set of incubations was conducted to quantify the holobiont’s impact and to quantitatively compare it with the indirect influence of the mussel on sedimentary N transformations. Zebra mussels primarily enhanced the recycling of N to the water column by releasing mineralized algal biomass in the form of ammonium and by stimulating dissimilatory nitrate reduction to ammonium (DNRA). Notably, however, not only denitrification and DNRA, but also dinitrogen (N2) fixation was measured in association with the holobiont. The diazotrophic community of the holobiont diverged substantially from that of the water column, suggesting a unique niche for N2 fixation associated with the mussels. At the densities reported in the lagoon, mussel-associated N2 fixation may account for a substantial (and so far, overlooked) source of bioavailable N. Our findings contribute to improve our understanding on the ecosystem-level impact of zebra mussel, and potentially, of its ability to adapt to and colonize oligotrophic environments

Spatiotemporal patterns of N2 fixation in coastal waters derived from rate measurements and remote sensing

Coastal lagoons are important sites for nitrogen (N) removal via sediment burial and denitrification. Blooms of heterocystous cyanobacteria may diminish N retention as dinitrogen (N2) fixation offsets atmospheric losses via denitrification. We measured N2 fixation in the Curonian Lagoon, Europe's largest coastal lagoon, to better understand the factors controlling N2 fixation in the context of seasonal changes in phytoplankton community composition and external N inputs. Temporal patterns in N2 fixation were primarily determined by the abundance of heterocystous cyanobacteria, mainly Aphanizomenon flos-aquae, which became abundant after the decline in riverine nitrate inputs associated with snowmelt. Heterocystous cyanobacteria dominated the summer phytoplankton community resulting in strong correlations between chlorophyll a (Chl a) and N2 fixation. We used regression models relating N2 fixation to Chl a, along with remote-sensing-based estimates of Chl a to derive lagoon-scale estimates of N2 fixation. N2 fixation by pelagic cyanobacteria was found to be a significant component of the lagoon's N budget based on comparisons to previously derived fluxes associated with riverine inputs, sediment–water exchange, and losses via denitrification. To our knowledge, this is the first study to derive ecosystem-scale estimates of N2 fixation by combining remote sensing of Chl a with empirical models relating N2 fixation rates to Chl a