The combination of biogeochemical methods and molecular techniques has the potential to uncover the black-box of the nitrogen (N) cycle in bioturbated sediments. Advanced biogeochemical methods allow the quantification of the process rates of different microbial processes, whereas molecular tools allow the analysis of microbial diversity (16S rRNA metabarcoding) and activity (marker genes and transcripts) in biogeochemical hot-spots such as the burrow wall or macrofauna guts. By combining biogeochemical and molecular techniques, we analyzed the role of tube-dwelling Chironomus plumosus (Insecta, Diptera) larvae on nitrification and nitrate reduction processes in a laboratory experiment with reconstructed sediments. We hypothesized that chironomid larvae stimulate these processes and host bacteria actively involved in N-cycling. Our results suggest that chironomid larvae significantly enhance the recycling of ammonium (80.5 ± 48.7 µmol m −2 h −1 ) and the production of dinitrogen (420.2 ± 21.4 µmol m −2 h −1 ) via coupled nitrification-denitrification and the consumption of water column nitrates. Besides creating oxygen microniches in ammonium-rich subsurface sediments via burrow digging and ventilation, chironomid larvae serve as hot-spots of microbial communities involved in N-cycling. The quantification of functional genes showed a significantly higher potential for microbial denitrification and nitrate ammonification in larvae as compared to surrounding sediments. Future studies may further scrutinize N transformation rates associated with intimate macrofaunal-bacteria associations.

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.

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Tube‐dwelling chironomid larvae are among the few taxa that can withstand and thrive in the organic‐rich sediments typical of eutrophic freshwater ecosystems. They can have multiple effects on microbial nitrogen (N) cycling in burrow environments, but such effects cease when chironomid larvae undergo metamorphosis into flying adults and leave the sediment. Here we investigated the ecological role of Chironomus plumosus by exploring the effect of its different life stages (as larva and adult midge) on microbial N transformations in a shallow freshwater lagoon by means of combined biogeochemical and molecular approaches. Results suggest that sediment bioturbation by chironomid larvae produce contrasting effects on nitrate (NO3‐)‐reduction processes. Denitrification was the dominant pathway of NO3‐ reduction (>90%), primarily fuelled by NO3‐ from bottom water. In addition to pumping NO3‐‐rich bottom water within the burrows, chironomid larvae host microbiota capable of NO3‐ reduction. However, the contribution of larval microbiota is lower than that of microbes inhabiting the burrow walls. Interestingly, dinitrogen fixation co‐occurred with NO3‐ reduction processes, indicating versatility of the larvae's microbial community. Assuming all larvae (averaging 1,800 ind./m2) leave the sediment following metamorphosis into flying adults, we estimated a displacement of 47,787 µmol of organic N/m2 from the sediment to the atmosphere during adult emergence. This amount of particulate organic N is similar to the entire N removal stimulated by larvae denitrification over a period of 20 days. Finally, the detection of N‐cycling marker genes in flying adults suggests that these insects retain N‐cycling microbes during metamorphosis and migration to the aerial and terrestrial ecosystems. This study provides evidence that chironomids have a multifaceted role in shaping the N cycle of aquatic ecosystems.

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. Author Contributions: Conceptualization, M.B., T.P.; methodology, M.B.; formal analysis, D.D., T.P.; investigation, D.D., M.B., M.Z.; resources, G.C.; data curation, T.P., D.D.; writing-original draft preparation, T.B.; writing-review and editing, D.D., G.C., M.B., M.Z.