Surface sediment was taken on 24 April 2014 during low tide in the intertidal mudflat of the German Wadden Sea near Friedrichskoog (Germany). Water temperature and salinity at the sampling site were 19.9 ∘C and 31 (practical salinity units). Sediment was collected and sieved at the sampling site through 500 and 63 µm meshes to remove larger meiofauna and organic particles. In the laboratory, samples were sieved again to obtain foraminiferal specimen in the size fraction of 125–355 µm. The sediment contained high abundances of Ammonia tepida and Haynesina germanica individuals, which were picked and collected for further processing. Living individuals were identified under the microscope regarding intact protoplasma and particle accumulation around the aperture . Care was taken to achieve a homogenous size distribution within specimens in each replicate. Each species was kept separately in crystallising dishes and fed regularly with living Dunaliella tertiolecta up to the start of the experiment.

Elevated 13C values of the foraminiferal samples demonstrated a strong response to the labelled food source (Table 1). Temperature had a significant impact on pC levels in H. germanica (Table 2 and Fig. 2). An interactive effect of time and temperature caused a different time-related processing of pC at 20 ∘C compared to 25 and 30 ∘C (Table 2 and Fig. 2). The content of pC in H. germanica at 20 ∘C was significantly higher than at 25 and 30 ∘C on each day of data collection (p<0.05; no significant difference in 25 and 30 ∘C samples). Additionally, values for DIC-13C measured in water samples (day 2) at elevated temperatures were significantly higher than those from the 20 ∘C approach (Table 1). Time-related variations in cytoplasmic C (biomass normalised) in H. germanica specimens were also temperature dependent (Table 2). In contrast, there was no relation between time and temperature concerning the pC content of A. tepida. Levels of pC in A. tepida were significantly increased at 25 ∘C (25–20 ∘C: p = 0.015, 25–30 ∘C: p<0.001, pairwise t testing with pooled SDs, since no significance of time effects), revealing a higher optimum grazing temperature for A. tepida compared to H. germanica (pC, Fig. 2). Temperature had no effect on biomass C in A. tepida, while there were time-dependent variations in biomass C (Table 2). These appeared at the start and end of the experiment but there was no significant influence by temperature treatments on the other days. Individual cytoplasmic C content µg ind-1 differed between the two species and proportions of individual uptake of pC to individual C content showed similar proportions to biomass-related carbon values (Fig. 3). For detailed information about individual pC, pN, TOC and TN content, see Supplement Table S1.

Carbon uptake rates showed an exponential decrease with time for both species (Fig. 4), with a very good fit of curves except for A. tepida at 25 ∘C where a relatively low determination coefficient R2 was observed (Table 3). Mean uptake rates display a highly significant difference between species (F test: F=33.74, p=0.029). H. germanica showed a much lower efficiency in the uptake of phytodetritus derived from D. tertiolecta compared to A. tepida. Uptake rates in A. tepida can be pooled within a single function (F test; F=7.664, p=0.062). Temperature causes a strong variation in rates for H. germanica (F test; F=24.97, p=0.012). The rate of A. tepida showed a slightly faster decrease at 20 ∘C relative to the higher temperatures and the highest deviation (S) of 21.247 ng mg-1 h-1 from average uptake rates at 25 ∘C. In general, H. germanica exhibited a faster drop in the uptake rates at higher temperatures (see b, Table 3).

The δ15N signatures of all foraminiferal cytoplasm samples showed a strong increase after addition of labelled algae (Table 1). Like in pC, time and temperature showed interactive effects on the pN content in H. germanica (Table 2). Haynesina germanica showed a temperature-related variation on days 4 and 14. Cytoplasmic N showed less variation with temperature in A. tepida than in H. germanica. In contrast to the pC values, progressing time caused significant increases in pN content in both species (Fig. 2, pN). N changes in A. tepida were time dependent and increased at the start of the 25 and 30 ∘C approach. Analogous to cytoplasmic C, individual N and pN are proportional to total biomass-related values (Fig. 3).

In general, nitrogen uptake rates of H. germanica showed a steeper decline over time and at all temperatures in contrast to A. tepida (Fig. 5). While H. germanica showed the highest uptake rates at 20 ∘C, uptake rates for A. tepida were highest at 25 ∘C. At 30 ∘C, uptake rates of phytodetrital nitrogen showed fluctuating patterns for both species. In Ammonia samples, rates at 25 ∘C and 30 ∘C dropped after the fourth day. The steepest decrease at 20 ∘C can be found between day 2 and day 4. Uptake rates of nitrogen were about 10-fold higher in A. tepida compared to H. germanica.

There was a noticeable peak in N uptake on the fourth day at 30 ∘C in both species. In A. tepida, this peak describes an increase of the uptake rate followed by a linear decrease. In contrast, all uptake rates showed a rapid drop in H. germanica, especially at the higher temperatures, with a recognisable negative peak at 30 ∘C on day 4 and a flattening of nitrogen uptake rates at 25 and 30 ∘C on day 4.

Haynesina germanica showed a pN enrichment in contrast to pC loss or relatively stable pC over time (Fig. 2). This enrichment of pN was detectable in both species, but was stronger in H. germanica with an almost equal increase at all temperatures by the end of the experiment. In contrast, the pN rise in A. tepida was restricted to the first week. Despite the constant enrichment of pN in H. germanica, pC levels were lower at higher temperatures. This faster loss of pC at 25 and 30 ∘C at similar levels of phytodetritus uptake results in lower pC : pN ratios at higher temperatures (Fig. 6). The interactive effect of temperature and time on the food uptake of H. germanica caused a decoupling of pC and pN (Table 2 and Fig. 7). This is visualised by a lack of correlation between phytodetrital carbon and nitrogen content in the cytoplasm of H. germanica over the course of the experiment (Fig. 7). In contrast, pC and pN were strongly coupled in A. tepida across the entire time series (Fig. 7). Cytoplasmic C : N ratios were generally similar in both species, with no significant impact of temperature (Table 4).

The metabolism of H. germanica was significantly affected by elevated temperatures. Higher temperatures reduced the amount of pC in the foraminiferal cytoplasm, while a slighter effect on pN indicates a lower impact of temperature on the general uptake of algal phytodetritus. In contrast, pC processing in A. tepida was favoured at the intermediate experimental temperature level of 25 ∘C, demonstrating different optimum phytodetritus processing temperatures in the two species. The loss of pC (Fig. 2) in H. germanica in the warmer environment or the faster decline of uptake rates at 25 and 30 ∘C (Fig. 4 and Table 3) was most likely caused by elevated respiration rates. Accordingly, increased DIC-13C content within the SSW at elevated temperatures suggests higher respiratory activities (Table 1). Elevated temperatures have been reported to increase respiration and food consumption in aquatic herbivores, detritivores and foraminifera , to lower grazing rates with simultaneously increased oxygen consumption or to raise respiration to cover the costs of maintenance of protein metabolism levels .

In general, DIC-13C values in this experiment were very high compared to foraminiferal 13C. It cannot be excluded that the foraminiferal cultures contained bacteria. Thus, a considerable amount of DIC could originate from phytodetritus remineralization by microbial activity. Several perturbation and long-term experiments on marine bacteria have also reported increased respiration due to warming . However, the higher temperatures trigger noticeable stress in H. germanica, which impacts food uptake efficiency. The interactive time/temperature effect supports this argument: the higher temperatures caused convergence of pC and pN content compared to the 20 ∘C results, particularly up to day 7. This indicates an offset of nutritional ingestion performance in the two warmer approaches. It further suggests a general preference of lower environmental temperatures. Evidently, lower temperatures are more supportive when triggering reproduction in H. germanica . Interestingly, there is no significant temperature effect on pC between 25 and 30 ∘C. This implies a critical threshold for this species between 20 and 25 ∘C. Above this level, the mineralisation of carbon increases, as the DIC-13C rises at 25 and 30 ∘C as a result of elevated respiratory activity. The decoupling of pC and pN in H. germanica over time supports this observation.

In contrast, A. tepida showed a trend for optimum uptake of carbon at 25 ∘C and the steadiest carbon uptake rates at 30 ∘C, while the pC and pN values showed similar patterns (Figs. 2 and 4). Laboratory experiments on A. tepida specimens collected at the Brouage mudflat (France) revealed an optimum temperature of grazing on bacteria at 30 ∘C . This indicates that strains of A. tepida feature adaptations to the higher average sea surface temperatures of the Brouage mudflat. In other laboratory experiments warm temperatures between 25 and 30 ∘C offered optimum conditions for reproduction, resulted in higher growth and reproductive rates and a decrease in generation time in laboratory experiments with A. tepida specimen. Accordingly, higher temperatures are likely to offer competitive advantages for A. tepida over H. germanica in terms of food uptake. Considering the high expression of stress proteins at 35 ∘C in A. tepida in relation to the results presented here, a critical temperature limit affecting physiologic performances is likely to be found between 30 and 35 ∘C in this species.

Interestingly, the higher temperature treatments caused an increase of biomass C and N in H. germanica samples on the second sampling day, followed by a quick decrease on the fourth day. A similar trend was observed in A. tepida. This effect is difficult to interpret and more detailed information about initial food uptake between day 0 and day 2 would be necessary. In summary, continued food C and N uptake appears to be a key factor in the strategies of A. tepida, according to the strong response to phytodetritus and the low effect of temperature on its uptake. Simultaneously, a high viability of the specimen in culture implies specific environmental adaptations and a strong response and sensitivity to disturbances caused by the transfer to the laboratory (Fig. 2). In contrast, H. germanica showed a comparably high robustness and overall vitality, but there was clearly a strong impact of temperature on mechanisms involved in carbon processing. This implies investment of physiological resources for survival through the cost of increased metabolic activity.

In both species, the relation of pC to pN (pC : pN ratio) within foraminiferal cytoplasm decreased compared to their phytodetrital C : N ratio of ∼ 10 (Table 4) as time progressed. In theory, a comparably high ratio of detrital-derived pC : pN would approach or undercut somatic consumer C : N ratio to retain homeostasis or ensure appropriate nutrition . The growing content of pN within foraminiferal cytoplasm over time with a remarkable increase of 15N values (Table 1 and Fig. 2) could be explained by the integration of a high amount of food-derived nitrogen into amino acids and metabolic consumption of pC. Benthic foraminifera inhabiting Antarctic sediments have been reported to use a large amount of algae-derived carbon sources to synthesise nitrogen-rich proteins in relation to a lower generation of other products with a lower nitrogen content . Such effects could be responsible for the relatively slight decrease in nitrogen uptake rates over time compared to the strong decrease in carbon uptake rates (Figs. 4 and 5). A strong decline of continued food uptake or degradation-induced change of phytodetrital C : N could likewise be responsible for this effect.

Both tested species showed similar demands for food-derived carbon and nitrogen, since at moderate temperatures (20 ∘C) pC : pN ratios showed a similar trend over time (Fig. 6). Again, minor temperature influences were demonstrated within the overall linear relationship of pC to pN in A. tepida across the samples (Fig. 7). In contrast, the rising variation and highly significant difference in pC : pN (lower ratios at higher temperatures, Fig. 6), resulting in the diffuse relationship of pC : pN (Fig. 7) in H. germanica, reflect the thermal stress on the level of increased carbon expenditure at steady feeding progress (barely influenced pN levels between treatments). Therefore, sediments with a high dominance of H. germanica like river inlets or estuaries could possibly experience additional temperature induced nutrient flux variations, boosting C losses while triggering N retention.

An in situ experiment, monitoring population shifts of near-shore benthic foraminifera to artificially heated sediments, proposed migrations to deeper (colder) regions as a response to elevated environmental temperatures . Marine species are known to migrate depending on temperature changes . To maintain an optimum energy budget, expenses of metabolic maintenance could therefore be compensated by a shift of H. germanica populations towards the subtidal zone. Foraminiferal C : N ratios are known from bathyal foraminifera of the Sagami Bay and from oxygen minimum zones of the Arabian Sea, where they range from 2.6 to 6.4 . In our study, both species showed similar C : N ratios and natural 13C and 15N signatures (Tables 1 and 4), indicating equivalent trophic levels and similar nutritional demands. During the course of the experiment, the temperature stress in H. germanica was even noticeable on the level of cytoplasmic stoichiometry. The remarkable drop of the C : N ratio on the fourth day at 30 ∘C (Table 4) and the subsequent recovery and rapid increase of pC and pN after the fourth day (Fig. 2) could denote a metabolic adaptation process. However, the strong temperature influence on carbon uptake persisted.

In A. tepida, the occasionally significant (at 25 ∘C) fluctuations in the C : N ratio can be related to the high uptake of phytodetritus containing high C : N ratios and it is therefore an influence of food source C : N on the grazer C : N (Fig. 3 and Table 4). Generally, considerations of C : N ratios and intracellular pC / pN coupling also reflect the lower effects of high temperature exposure on A. tepida and the high impact on the feeding behaviour of H. germanica. These observations reflect the different strategies of the two coexisting intertidal foraminiferal species on the level of nutrient balance. It further implies shifts in population distributions of A. tepida and H. germanica at events of persisting food source and/or temperature changes. This study verifies a strong involvement of foraminifera in the turnover of intertidal POM. Depending on the POM source or dominant foraminiferal species, this turnover can be strongly influenced by increased temperature, causing a decoupling of carbon and nitrogen cycling. However, when relating these findings to natural environments, considerations of cascading effects of temperature change, e.g. switches in trophic conditions and related shifts in nutrient coupling of primary producers, have to be included.

The very high uptake rates and levels of phytodetrital carbon and nitrogen in Ammonia tepida (day 2 ∼ 14% pC : C; ∼ 10 % pN : N, comp. Figs. 3 and 4) indicate a strong response to the food source, in contrast to H. germanica which showed a clear, but much lower uptake (day 2: ∼ 2% pC : C; ∼ 1.5 % pN : N). The observed decline of carbon uptake rates over time in both species suggests the highest uptake within the first 2 days of the experiment and indicates a fast processing of the phytodetritus source. During an in situ experiment, also observed a rapid response of Ammonia sp. to green algae (Chlorella sp.) within hours, together with the much weaker ingestion of such a food source by Haynesina sp.

The quick slowdown of food uptake rates could indicate levels of saturation especially after a strong response following the food pulse. In addition, a cellular release of the label is likely to exceed uptake as time progresses. Other reasons for the decrease in food uptake with increasing time (reflected in decreasing uptake rates of C and N as well as in pC and pN) could be the stage of phytodetrital decay or decreasing food availability on the subsequent sampling days. Aging or phytodetrital degeneration and its change in quality in marine benthic environments is a result of bacterial colonisation and subsequent transformation and mineralisation of the algal material . Through bacterial mineralisation, the degrading detritus decreases in quality with an increase in C : nutrient ratios. However, microorganisms colonising patches of degrading detritus are likely to be incorporated simultaneously with foraminiferal phytodetritus grazing. Therefore, a fraction of the isotope label within the foraminiferal cytoplasm could be a result of indirect label intake with phytodetritus-associated microorganisms, especially towards the end of the experiment. However, a visible layer of phytodetritus particles persisted until the end of the experiment. The influence of food availability on feeding, where high food concentrations support increased feeding rates, have been reported for foraminifera and also in other organismic groups such as macrofauna or bacteria . Fast incorporation of food pulses seems to be necessary to cover the high energy demand for reproduction and growth, since food concentrations (Dunaliella) sp. of less than 112 cells mm2 (0.40 µg C cm-2; ) do not permit growth or reproduction in A. tepida, while growth rate and reproductive activity increase with additional food . stated that Ammonia beccarii specimen preferred to feed on “new” (Fig. 1. 10 days old) cultures of living Chlorococcum sp. over “old” (up to 40 days old) cultures. Following these observations, food limitation resulting from the aging of the phytodetrital food source could be present in this study. This proceeding degradation of phytodetritus appears to be particularly problematic to A. tepida specimen, which showed an increasing fraction of dead individuals with time (Fig. 1). On the other hand, the health condition of the specimens could be a response to the conditions of the laboratory incubation. Compared to a previous feeding experiment by with A. tepida fed with D. tertiolecta phytodetritus (614 mg C m-2), uptake rates of carbon of 1899 pg ind-1 h-1 were much higher in this study after 48 h than in the other feeding experiment (149 pg ind-1 h-1). This might be related to general differences in feeding of the tested strains.

The low affinity of H. germanica to D. tertiolecta phytodetritus could be explained by a generally lower carbon demand or, more likely, a specialisation on other food sources, for instance diatoms or secondary products of microphytobenthic biofilms (e.g. extracellular polymeric substances). The latter assumption corresponds to the preference of a diatom diet over sewage-derived POM , the presence of a diatom cracking mechanism , the sequestration of chloroplasts derived exclusively from diatoms or the correlation of the distribution of H. germanica populations with a high abundance of Nitzschia sp. . This coherence with food availability or organic matter accumulation respectively, was used to explain population distributions of a H. germanica/A. beccarii assemblages in muddy sediments of Spain . The different patterns of pC accumulation over time in the two species also reflect a different feeding behaviour. The onset of declining health conditions in H. germanica specimen at the end of the experiment (Fig. 1) was likely caused by starvation due to the absence of an appropriate food source. In general, the quality of organic carbon or foraminiferal food sources oscillates throughout the year and includes allochthonous detritus , while the main source of primary production in intertidal environments are microphytobenthic diatoms, accompanied by chlorophytes or other autotrophic microorganism, which seasonally suppress diatom dominance . In this experiment, D. tertiolecta was used as a food source, because this species is easy to maintain in culture, was previously used in several feeding experiments with benthic foraminifera and serves as a representative for allochthonous detrital carbon sources. Ratios of A. tepida and H. germanica abundances are considered to be related to organic matter quality or environmental variability due to their different feeding specialisations or environmental adaptations . High amounts of pC (max. 30 % pC to C) in A. tepida compared to H. germanica (max. 2.6 % pC to C), together with the low influence of temperature on the feeding behaviour of A. tepida, prove an opportunistic feeding behaviour and generalist temperature adaptations in A. tepida. These findings will help to interpret oscillations in the abundances of these two common intertidal foraminiferal species in relation to organic carbon quality and ambient temperatures. These data report varying strategies in phytodetritus feeding by means of source exploitation and time dependence. However, laboratory studies do not always reflect natural behaviour accurately and might be biased regarding the suppression or alteration of natural behaviours in the investigated organisms. This work is meant to help solve ecological problems and the patterns presented here of foraminiferal feeding behaviour can be useful for the careful interpretation or explanation of successions in species dominance by means of food source availability, and it complements data from field surveys.