Nitrous oxide emission by aquatic macrofauna

Results and Discussion

The field survey covered 21 different invertebrate taxa sampled at 7 field sites that included several creeks and streams, a lake, and the seashore. Rates of nitrous oxide emission differed markedly between taxa; emission rates were, however, not correlated with taxonomic affiliation or habitat type of the invertebrates but rather with the feeding guild to which the species belonged (Fig. 1). Filter- and deposit-feeders were grouped into 1 feeding guild (FD-feeders) because many of their representatives are able to switch between the 2 feeding modes (9). FD-feeders emitted the highest amounts of nitrous oxide per individual, shredders and grazers took an intermediate position, and predators played an insignificant role (Fig. 1). This overall pattern was also evident when the rate of nitrous oxide emission was expressed per milligram dry weight of the specimens (Table S1). Because FD-feeding species ingest greater numbers of microbes than predators do (10), this observation suggests microbial nitrogen conversions in the gut of the invertebrates as the source of nitrous oxide. Given anoxic conditions and the availability of easily degradable organic carbon in the gut of terrestrial and aquatic invertebrates (8, 11), we hypothesized that nitrous oxide production was due to the activity of denitrifying bacteria in the animal gut.

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Fig. 1.

Nitrous oxide emission by various aquatic invertebrate taxa. Animals are grouped into 4 different feeding guilds and sorted according to emission rate. Error bars indicate SEM (n = 3–23). Line drawings of animal taxa are stylized and not to scale. See Table S1 for details on sampling locations and taxonomy.

To test this hypothesis, we carried out detailed experiments with the midge larvae C. plumosus. These worm-shaped larvae often dominate benthic invertebrate communities of freshwater ecosystems and can reach levels of abundance from several hundred to thousands of individuals per square meter (12). The larvae live in U-shaped burrows in the sediment and feed on suspended and settled particulate organic matter, thereby ingesting large numbers of bacteria (13). Our investigation of larvae, dissected guts, dissected gut epithelia, and pupae of C. plumosus unambiguously demonstrated nitrate-dependent denitrification activity and nitrous oxide production associated with bacteria coingested with particulate organic matter (Fig. 2). In the absence of oxygen, larvae and dissected guts showed identical emission rates, and thus denitrification and the production of nitrous oxide by C. plumosus larvae were exclusively associated with the gut (Fig. 2). More specifically, they were associated with anaerobic processes in the gut contents, given that only complete guts but not isolated gut epithelia denitrified and emitted nitrous oxide (Fig. 2). Pupae, possessing a nonfunctional gut, showed very low rates of nitrous oxide emission and denitrification (Fig. 2). Further evidence for a dietary origin of denitrifying bacteria active in the larval gut was provided by the analysis of expressed denitrification genes in the gut content of C. plumosus. Transcripts of nitrate and nitrous oxide reductase genes (narG and nosZ), coding for the first and last step in denitrification, had high sequence identities (76–99%) with diverse genes retrieved from freshwater sediments and soils [supporting information (SI) Fig. S1], suggesting that the denitrifiers had indeed been coingested with the food of the larvae. These collective results demonstrate that the nitrous oxide emission of C. plumosus is not due to specific symbionts in the animal gut; although we cannot fully exclude symbiotic nitrous oxide production for the other invertebrate species tested, the strict dependence of the nitrous oxide emission on the feeding guild and the very low emission rates of animals with empty guts are strong arguments against such a scenario.

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Fig. 2.

Nitrogen gas emissions from C. plumosus. Nitrous oxide emission and total denitrification (i.e., nitrous oxide plus dinitrogen emission) measured in live larvae, dissected complete guts, gut epithelia, and pupae with or without nitrate in the sediment–water microcosms and with or without oxygen in the incubation vials. Error bars indicate SEM (n = 4–18). b.d., below detection limit.

The gut of FD-feeding invertebrates is obviously the distinct microsite of denitrification and production of nitrous oxide, as confirmed with microsensors on dissected guts of the mayfly larvae E. danica (Fig. 3). When incubated under close-to-anoxic conditions in an experimental flow cell (11), the concentration of nitrous oxide increased toward the center of the gut (Fig. 3A). In the presence of acetylene, an inhibitor of the last step of denitrification (14), nitrous oxide concentrations were higher than in the absence of acetylene, indicating complete denitrification activity in the gut (Fig. 3A). Oxygen inhibited denitrification activity in the gut in proportion to the concentration that was maintained in the medium surrounding the dissected gut (Fig. 3B). The in vivo concentration of oxygen in the hemolymph of E. danica larvae is not known, but our microsensor measurements indicate that at a concentration of 90 μmol L⁻¹ or lower the gut will be completely anoxic and thus a favorable microsite for denitrification (Fig. 3C). From the concentration profiles in Fig. 3A (without acetylene), an average rate of nitrous oxide production of 189 pmol gut⁻¹ h⁻¹ was calculated using a diffusion-reaction model for radial geometry (11). This was very close to the rate that was measured for living larvae (Fig. 1). Hence, just as with C. plumosus larvae, the gut of E. danica seems to be the exclusive production site of nitrous oxide in these FD-feeding animals.

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Fig. 3.

Distribution of nitrous oxide (A and B) and oxygen (C) in dissected guts of E. danica larvae as measured with microsensors. Guts were incubated in a flow cell with physiologic salt solution adjusted to 50 μmol L⁻¹ NO₃⁻ and various O₂ concentrations and with or without 10% acetylene to inhibit the last step of denitrification. Microsensor profiles were measured perpendicular to the gut and are plotted relative to the surface of the gut content (dotted line). In detail, the conditions were (A) 17 μmol L⁻¹ O₂, no and 10% acetylene, (B) 17–252 μmol L⁻¹ O₂, 10% acetylene, and (C) 5–135 μmol L⁻¹ O₂, no acetylene. Error bars indicate SEM (n = 4–9, A) and range (n = 2, B and C).

A striking feature of gas emissions by larvae of C. plumosus and E. danica was the incomplete denitrification with nitrous oxide accounting for 43–68% and 15–29% of the nitrogen gas flux, respectively (Figs. 2 and 3A), as compared with only 1% normally observed in aquatic sediments (15, 16). However, similarly high N₂O-to-N₂ ratios have been reported from pure cultures (17) and soils (18) after sudden shifts from oxic to anoxic conditions, and from earthworms ingesting oxic soil into their anoxic guts (19). Aquatic FD-feeders ingest bacteria-colonized particles that normally are exposed to oxic, and thus non-denitrifying, conditions at the sediment surface and in the water column. The transfer of bacteria into the anoxic animal gut apparently leads to a transient imbalance of gene expression during the onset of denitrification (20, 21), with accumulation of intermediates and emission of nitrous oxide as the unavoidable consequence (Fig. 4C). The detection of only 7 actively expressed phylotypes of nosZ in the gut contents compared with 26 of narG (Table 1) may indicate that the gut residence time in C. plumosus larvae of 2 to 3 h at 15 °C is for some denitrifiers too short to induce the full set of denitrification genes. Other phylotypes of narG might originate from nondenitrifying, dissimilatory nitrate-reducing bacteria, which only convert nitrate to nitrite and not further to gaseous products. These bacteria might be indirectly involved in nitrous oxide production via the accumulation of nitrite in the animal gut. Nitrite has been shown to stimulate nitrous oxide production in estuarine sediment (22) and the earthworm gut (23) more effectively compared with nitrate.

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Fig. 4.

Effect of C. plumosus larvae on sediment environment and nitrous oxide emission from the lake bottom. The larvae ventilate their U-shaped burrows, resulting in oscillating concentrations of oxygen (A) and nitrate (B) inside the burrows. Dotted lines indicate concentrations in the water column. (C) Conceptual model of nitrous oxide emission. The larvae feed on particles with attached bacteria (black), which inside the anoxic gut turn on nitrate reduction and denitrification (yellow), resulting in nitrous oxide emissions (yellow plumes). Also illustrated are point emissions from a filter-feeding bivalve and a deposit-feeding snail and the diffusive emission from anoxic, nitrate-containing sediment layers. (D) Effect of density of C. plumosus larvae on nitrous oxide emission from the sediment. Error bars indicate SEM (n = 6).

Rates of gut denitrification and nitrous oxide production increased in the presence of nitrate and decreased in the presence of oxygen (Fig. 2). By adding ¹⁵N-nitrate to sediment–water microcosms, it was proven that nitrate from the water column was the precursor of nitrous oxide produced in the gut of C. plumosus (data not shown). Favorable conditions for gut production of nitrous oxide in the form of high nitrate availability and periodic anoxia were measured directly within the burrows with microsensors (Fig. 4 A and B). Measurements in sediment–water microcosms also showed a profound effect of C. plumosus larvae on the emission of nitrous oxide from lake sediment, reaching an 8-fold enhancement at the highest abundance (Fig. 4D). This enhancement, however, was 5 times higher than computed from larval abundance and the average emission rate of separated larvae in anoxic and oxic incubations (Figs. 2 and 4D). The discrepancy might be due to additional nitrous oxide emission from stimulated denitrification in the burrow walls and in fecal pellets, or suboptimal conditions in the incubations of isolated larvae. Notably, the emission of dinitrogen due to denitrification also increased with animal density, but to a slightly lower degree than that of nitrous oxide (data not shown). Thereby, the N₂O-to-N₂ ratio of the sedimentary effluxes of gaseous nitrogen compounds more than doubled from 0.51% to 1.25% with 0 and 3,450 C. plumosus larvae per square meter, respectively.

In a previous study of C. plumosus, an increase of sedimentary nitrous oxide flux was also observed but was attributed to nitrification in the sediment and not to denitrification in the gut of the larvae (24). For burrowing animals, the surrounding sediment acts as an additional sink or source of nitrous oxide, which may weaken the relative impact of gut denitrification (Fig. 4C). This is not the case for animals colonizing hard substrates or the sediment surface (e.g., snails and bivalves on rocky shores), and we suggest that the very high nitrous oxide fractions and emissions measured at intertidal hard substrate sites (25) could be due to gut denitrification of the epifauna. The rate of nitrous oxide emission from benthic invertebrates may thus not only depend on the rate of nitrous oxide production in their gut but also on the site of the animal habitat relative to the sediment–water interface (5).

Our findings prove valuable for predicting the effect of local management of nitrate pollution and eutrophication on nitrous oxide emission. The main conclusion is that animal-enhanced nitrous oxide emission is both nitrate dependent and feeding guild dependent. Thus, the contribution of the invertebrate community to the total emission of nitrous oxide of a given, nitrate-polluted aquatic habitat can be estimated using the rates typical of certain species or feeding guilds. In the hypertrophic lake Großer Binnensee, representative of a human-impacted aquatic ecosystem, C. plumosus reaches an abundance of 1,600 larvae per square meter and is the dominant benthic invertebrate species (26). During summer, nitrate concentration and temperature can exceed 100 μmol L⁻¹ and 20 °C, respectively (27). In this setting, gut denitrification of C. plumosus increases the sedimentary nitrous oxide emission by 32–104% (according to data presented in Figs. 2 and 4). Restoration measures that reduce the nitrate concentration will decrease the overall emission of nitrous oxide. Additionally, restoration measures that remove the numeric dominance of FD-feeders (28), which according to our findings are the principal nitrous oxide emitters, will decrease the animal-associated emission of nitrous oxide.

Nitrous oxide emission by aquatic invertebrates will be quantitatively important in environments with extensive nitrate inputs from agriculture, like lakes, streams, and coastal marine ecosystems (4, 29, 30) or from nutrient-rich subsurface water in coastal upwelling zones (3, 31, 32). In such eutrophic systems, FD-feeders are often the numerically dominant feeding guild of the benthic invertebrate community (28), which may further enhance animal-associated nitrous oxide emission. In deep-sea environments with naturally high nitrate concentrations, such as hydrothermal vents (33), nitrous oxide might be produced by symbiotic microbes of the hot-vent fauna. Some tropical aquatic ecosystems will receive much more nitrate over the next few decades because of agricultural intensification (34), and nitrous oxide emission is already several times higher for tropical mangroves than for temperate estuaries and intertidal flats (35). Thus, the rates of nitrous oxide emission associated with tropical aquatic invertebrate species could also be particularly high, a hypothesis that needs to be confirmed in future studies.