Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazer

Michelle Helmer; Desiree Helmer; Elizabeth Yohannes; Jason Newton; Daniel R. Dietrich; Dominik Martin-Creuzburg

doi:10.1371/journal.pone.0306173

Abstract

Field studies suggest that changes in the stable isotope ratios of phytoplankton communities can be used to track changes in the utilization of different nitrogen sources, i.e., to detect shifts from dissolved inorganic nitrogen (DIN) uptake to atmospheric nitrogen (N₂) fixation by diazotrophic cyanobacteria as an indication of nitrogen limitation. We explored changes in the stable isotope signature of the diazotrophic cyanobacterium Trichormus variabilis in response to increasing nitrate (NO₃⁻) concentrations (0 to 170 mg L⁻¹) under controlled laboratory conditions. In addition, we explored the influence of nitrogen utilization at the primary producer level on trophic fractionation by studying potential changes in isotope ratios in the freshwater model Daphnia magna feeding on the differently grown cyanobacteria. We show that δ ¹⁵N values of the cyanobacterium increase asymptotically with DIN availability, from -0.7 ‰ in the absence of DIN (suggesting N₂ fixation) to 2.9 ‰ at the highest DIN concentration (exclusive DIN uptake). In contrast, δ ¹³C values of the cyanobacterium did not show a clear relationship with DIN availability. The stable isotope ratios of the consumer reflected those of the differently grown cyanobacteria but also revealed significant trophic fractionation in response to nitrogen utilization at the primary producer level. Nitrogen isotope turnover rates of Daphnia were highest in the absence of DIN as a consequence of N₂ fixation and resulting depletion in ¹⁵N at the primary producer level. Our results highlight the potential of stable isotopes to assess nitrogen limitation and to explore diazotrophy in aquatic food webs.

Citation: Helmer M, Helmer D, Yohannes E, Newton J, Dietrich DR, Martin-Creuzburg D (2024) Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazer. PLoS ONE 19(8): e0306173. https://doi.org/10.1371/journal.pone.0306173

Editor: Lee W. Cooper, University of Maryland Center for Environmental Science, UNITED STATES

Received: March 11, 2024; Accepted: June 12, 2024; Published: August 1, 2024

Copyright: © 2024 Helmer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The raw data supporting the results presented in this study are publicly available in the Zenodo repository at https://zenodo.org/records/11501652 (DOI: 10.5281/zenodo.11501651).

Funding: The work was financially supported by the Ministry of Science, Research, and the Arts of the Federal State Baden-Württemberg, Germany (Water Research Network Project: Challenges of Reservoir Management—Meeting Environmental and Social Requirements, to D.M.-C. and D.D.) and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 298726046/GRK2272, to D.M.-C. and E.Y.). The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Phosphorus (P) is commonly considered the most important nutrient limiting phytoplankton growth in freshwater ecosystems [1–3], despite ample evidence that freshwater phytoplankton are just as frequently nitrogen (N)- as P-limited [4, 5]. In strongly N-limited water bodies, the phytoplankton community is typically dominated by diazotrophic bloom-forming cyanobacteria [6, 7]. Diazotrophic cyanobacteria can overcome N limitation by fixing atmospheric nitrogen (N₂). N₂ fixation is an oxygen-sensitive process and therefore either temporally or spatially separated from oxygenic photosynthesis. In filamentous cyanobacteria, N₂ is often fixed in highly specialized cells, the heterocysts [8]. N₂ fixation is energetically more expensive than the assimilation of dissolved inorganic N (DIN) from nitrate (NO₃⁻) or ammonium (NH₄⁺), potentially resulting in decreased growth [9–12]. Heterocyst differentiation is thus supposed to increase with decreasing DIN concentrations [13].

Stable isotopes have been extensively used to explore carbon (C) and nitrogen fluxes as well as trophic relationships in aquatic food webs [14, 15]. ¹⁵N has been proposed to be an excellent tracer for fixed atmospheric N in ecosystems because N₂ is often depleted in ¹⁵N compared to nitrate (NO₃⁻) and other dissolved N sources [16, 17], and because diazotrophic cyanobacteria discriminate against ¹⁵N during N₂ fixation [18, 19]. Atmospheric N₂ is used as a reference gas for N isotope measurements, and the δ ¹⁵N value of N₂ is therefore 0 ‰. The discrimination against ¹⁵N during N₂ fixation typically leads to slightly negative δ ¹⁵N values (-1 to -2 ‰) in diazotrophic cyanobacteria [20]. The δ ¹⁵N values of NO₃⁻ in aquatic systems typically range between +7 and +20 ‰, depending on N sources and biological transformations [21]. Negative δ ¹⁵N values of NO₃⁻ may result from atmospheric NO₃⁻ deposition or nitrification in soils, reflecting the complexity of isotopic transformations within the nitrogen cycle [22]. Nonetheless, particulate organic matter produced with the aid of N fixation typically acquires a distinctive isotope signature that reflects the isotope signature of N₂ and the discrimination against ¹⁵N during N fixation, and this signature is traceable in the food web [23–28]. Consequently, the N isotope signature of natural seston can reveal N₂ fixation by cyanobacteria [23], while the zooplankton isotope signature can reflect the assimilation of organic matter produced by N-limited, diazotrophic cyanobacteria [24, 26, 28, 29]. This information has been used to estimate diazotrophic inputs to net N assimilation at different trophic levels of the food web [23, 25, 30]. Both N and P limitation can also influence the C isotope signature of photosynthetic organisms through changes in the activity of the CO₂ concentrating mechanism [31–35], but it remains unclear how the mode of N acquisition affects C stable isotope values of diazotrophs and their consumers.

The impact of N acquisition on the N and C isotope signature of primary producers and the traceability of this isotope change at the consumer level has rarely been studied experimentally. We cultivated the diazotrophic cyanobacterium Trichormus variabilis (formerly known as Anabaena variabilis) under controlled laboratory conditions at different dissolved nitrate (NO₃⁻) concentrations, ranging from 0 to 170 mg L⁻¹, and analyzed changes in N and C stable isotope values (δ ¹⁵N and δ ¹³C) in cyanobacterial cells. In addition, to assess the trophic transfer of the experimentally generated differences in isotope signatures, we analyzed the N and C stable isotope values of the freshwater model herbivore Daphnia after feeding on the differently grown cyanobacteria. We hypothesized that a reduction in DIN availability is associated with a switch to N₂ fixation in T. variabilis, and that this change in N acquisition is reflected both in δ ¹⁵N and δ ¹³C values of T. variabilis and Daphnia. Our data show that the availability of DIN strongly affects the N isotope values of the diazotrophic cyanobacterium T. variabilis, pointing towards increasing N₂ fixation with decreasing dissolved N availability. We also show that these differences in isotope signatures are detectable at the consumer level despite varying trophic fractionation, highlighting the potential of stable isotopes to explore the significance of diazotrophy in aquatic food webs.

Materials and methods

Experimental setup

To study the differential effects of N fixation and NO₃⁻ assimilation on stable isotope signatures of a diazotrophic cyanobacterium experimentally, we cultured Trichormus variabilis P9 (ATCC 29413) at four different dissolved N (NaNO₃) concentrations (Table 1). T. variabilis was cultured semicontinuously in 1-L flasks containing modified Woods Hole Medium without vitamins [36] with ¼ of the medium being replaced daily. The flasks (n = 4) were exposed to a light: dark cycle (16 h: 8 h) with illumination at 180 μmol quanta m⁻² s⁻¹ at 20 °C. The cultures were continuously aerated with sterile-filtered ambient air (source of N₂). Cyanobacterial cells were harvested after nine weeks of growth during their exponential growth phase (estimated from optical density measurements) and filtered onto pre-combusted GF/F filters (Whatman^™, GE Healthcare Life Science, Chicago, USA). Filters were dried at 50 °C and stored in a desiccator until subsequent stable isotope measurement.

Download:

Table 1. Sodium nitrate (NaNO₃⁻) and resulting dissolved inorganic nitrogen (DIN) concentrations as well as molar N:P ratios of the growth medium used to cultivate the diazotrophic cyanobacterium Trichormus variabilis.

The phosphorus concentration of the medium was 2.03 mg L⁻¹.

https://doi.org/10.1371/journal.pone.0306173.t001

Stock cultures of a clone of Daphnia magna [37] were maintained on filtered lake water (0.2 μm pore-sized membrane filter) and saturating concentrations of the green alga Acutodesmus obliquus (SAG 276-3a), which was cultured in Cyano Medium [38] in 5-L batch cultures under permanent illumination. The growth experiment was conducted at 20 °C with a cohort of third-clutch neonates born within 12 h which were reared on saturating amounts (2 mg C L⁻¹) of A. obliquus in glass beakers containing 200 ml of filtered lake water (0.2 μm pore-sized membrane filter). After five days of feeding on A. obliquus, the animals were transferred to beakers containing 2 mg C L⁻¹ of the cyanobacterium T. variabilis, which was cultivated at the different DIN concentrations. Food suspensions were prepared from the different DIN treatments through centrifugation and resuspension of the cells in an aliquot of filtered lake water. Carbon concentrations of the different food suspensions were estimated from photometric light extinction (480 nm) and from carbon extinction equations determined prior to the experiment. Each treatment (i.e., nitrogen concentration) initially consisted of 30 beakers with five D. magna in each beaker. The experimental animals were transferred daily into new beakers containing freshly prepared food suspensions. Before the diet switch (T₀) at day five and after the diet switch daily over a period of four days, six beakers of each treatment were randomly subsampled (5 samplings × 6 beakers = 30 beakers). The sampled animals were stored frozen in reaction tubes. After the experiment all samples were dried for 24 h, weighed on an electronic balance (Sartorius 4504MP8; ± 0.1 μg) and stored in a desiccator until subsequent stable isotope analysis.

Stable isotope analysis

The dried Daphnia samples (0.3–0.7 mg) were transferred into pre-weighed tin cups and weighed to the nearest of 0.0001 mg using a micro-balance (Sartorius 4504MP8). The dried GF/F filters loaded with cyanobacteria were also transferred into tin cups for subsequent stable isotope analysis. Encapsulated samples were combusted using a Elementar vario PYRO cube^® elemental analyzer (2013), and the resulting purified N₂ and CO₂ gases analyzed for δ ¹⁵N and δ ¹³C respectively on a Thermo Fisher Delta XP Plus Isotope Ratio Mass Spectrometer. Ratios were corrected for instrument drift and linearity using regularly interspersed internal laboratory standards (gelatin, glycine and alanine mixtures) with known stable isotope values (for details see [39]. Stable isotope ratios were expressed relative to Vienna Pee Dee Belemnite (δ ¹³C) or air (δ ¹⁵N). Precision of the measurements was 0.09 ‰ for δ ¹³C and 0.15 ‰ for δ ¹⁵N, based on the standard deviation of the most common laboratory standard used (gelatin) over the five-isotope analysis runs.

Stable isotopes data are reported (δ ¹³C and δ ¹⁵N) in parts per thousand (‰) using the δ -notation of McKinney et al. [40]:

X is ¹³C or ¹⁵N,

R_sample is the ¹³C/¹²C and ¹⁵N/¹⁴N ratios of our samples and

R_standard is that of international standards V- PDB, AIR, respectively.

A total of 60 Daphnia samples (3 replicates of each treatment and time point) and 16 phytoplankton samples (a single replicate of each time point) were subjected to stable isotope analysis.

Turnover rates.

The turnover rates were calculated from the changes in δ ¹³C and δ ¹⁵N values after the diet switch using the exponential model of Hobson and Clark [41]:

δ _t is the δ ¹³C and the δ ¹⁵N value of the daphnids at experimental time t,

δ _eq is the calculated asymptotic equilibrium with the new diet,

δ ₀ is the initial isotope value prior to the diet-switch, and

λ is the turnover rate (h⁻¹).

For the estimation of the variables in this exponential model we used the nls function in R with the self-starting Asymptotic Regression Model SSasymp based on the equation:

We expressed the turnover rates as the time period needed to achieve a 50% turnover of isotope composition of δ ¹³C and δ ¹⁵N (half-life, T_0.5) using the function of Hobson and Clark [41]:

Time depending isotope signature analysis of Daphnia.

To analyze the adaptation of Daphnia to its dietary source over time, we used the equation:

Trophic fractionation.

The trophic fractionation was determined by subtracting dietary isotope values from those of the consumer:

Statistical analysis

All statistical analyses were performed using R (version 3.6.2). The data were checked for normality (Shapiro-Wilk Test) followed by the test for homogeneity of variances (Levene’s Test). Treatment effects (n = 4) were analyzed using ANOVA followed by Tukey’s HSD post hoc test. Pearson’s method was applied to investigate correlations between C and N content (%) of the animals and their stable isotope signature. Non-linear correlation was performed using the R package “nlcor” [42]. For analyzing the dose response curve, we used the asymptotic regression model of the R package “drc” [43] with the function AR.3(). The model is a three-parameter model with the function:

c is the lower limit

d is the upper limit, and

e > 0 is determining the steepness of the increase as x.

The median effective dose (ED₅₀) was estimated using the ED function of the model.

Results

Nitrate availability and stable isotope values of T. variabilis

The δ ¹⁵N values of T. variabilis were positively correlated with the DIN concentrations in the growth medium (Fig 1a). The lowest δ ¹⁵N was found in the absence of DIN (-0.7 ‰). The asymptotic regression model revealed a maximum δ ¹⁵N of 3.0 ‰ (± 0.2; p < 0.001), a minimum δ ¹⁵N of -0.7 ‰ (± 0.2; p < 0.01) and a median effective dose (ED₅₀) of 3.6 mg L⁻¹ (± 0.7) of DIN. The residual standard error of this model fit amounts to 0.38. While the δ ¹⁵N values of T. variabilis were positively correlated with DIN, the δ ¹³C values did not show a clear relationship with DIN availability (Fig 1b). The δ ¹³C values of T. variabilis grown at a DIN concentration of 14 mg L⁻¹ were significantly lower (around 2 ‰) than the δ ¹³C values of T. variabilis grown at the other DIN concentrations (Tukey’s HSD: p < 0.001; ANOVA: F_3,11 = 110.7, p < 0.001). Carbon concentrations in the different DIN treatments (mean 0.19 ± 0.02 mg ml⁻¹) did not differ significantly (Kruskal-Wallis Test: χ² = 6.49, df = 3, p > 0.05). Molar C:N ratios of T. variabilis were consistently low (mean 3.49 ± 0.2) and did not change significantly with DIN concentrations (ANOVA, F_3,11 = 0.07, p > 0.05; Fig 1c).

Download:

Fig 1.

a Asymptotic regression model showing the relationship between dissolved inorganic nitrogen (DIN) concentrations in the growth medium and δ ¹⁵N values of T. variabilis. b Relationship between DIN concentrations and δ ¹³C of T. variabilis. c Relationship between DIN concentrations and particulate molar C:N ratios of T. variabilis. Letters show statistically significant differences between data points (Tukey’s HSD following ANOVA).

https://doi.org/10.1371/journal.pone.0306173.g001

Stable isotope values of Daphnia in relation to DIN availability

The mode of N acquisition at the primary producer level (T. variabilis) also influenced the isotope signature of Daphnia consuming the cyanobacteria that were grown at the different DIN concentrations (Fig 2). The stable isotope values of Daphnia were significantly positively correlated with those of their diet (N: r = 0.96, p < 0.001; C: r = 0.77, p = 0.002; Fig 2a and 2b).

Download:

Fig 2.

Stable isotope values (a: δ ¹⁵N and b: δ ¹³C) of the consumer Daphnia in relation to those of their cyanobacterial diet cultivated at different DIN concentrations.

https://doi.org/10.1371/journal.pone.0306173.g002

The δ ¹⁵N values of Daphnia increased with DIN concentration from 5.4 ‰ at 0 mg L⁻¹ DIN to 8.2 ‰ at 28 mg L⁻¹ DIN after the diet switch (Fig 3a). The isotope N signature of the animals (96 h after diet switch) correlated positively with the N content of the diet (Pearson: r = 0.69, p = 0.01). We did not find any correlation of isotope N signature of Daphnia and molar C:N ratio of the diet. The δ ¹³C values of Daphnia did not show a clear relationship with DIN concentration in the cyanobacterial culture medium; the obtained cosine function reflected the dietary signature (Figs 1b and 3b). We did not find any correlation between the isotope C signature of Daphnia and the elemental composition of the diet (N and C content or molar C:N ratios).

Download:

Fig 3.

Stable isotope values (a: δ ¹⁵N and b: δ ¹³C) of Daphnia in relation to the different DIN concentrations in the cyanobacterial culture medium.

https://doi.org/10.1371/journal.pone.0306173.g003

Temporal changes in stable isotope values of Daphnia

δ ¹⁵N values of Daphnia decreased over time while feeding on the cyanobacteria, most evident at DIN concentrations ≤ 7 mg L⁻¹ (Fig 4a). The N isotope turnover rates increased (decreasing T₅₀ values) with increasing DIN concentration in the growth medium (Table 2). Compared to the initial value, δ ¹⁵N values of all Daphnia were depleted in ¹⁵N at the end of the experiment in all treatments. The highest ¹⁵N depletion was found at the lowest DIN concentration (0 mg L⁻¹ = 3 ‰) and the lowest ¹⁵N depletion was found at the highest DIN concentration (28 mg L⁻¹ = 0.2 ‰). With increasing DIN concentrations, δ ¹⁵N equilibrium values of Daphnia increased incrementally from 3.7 ‰ (0 mg L⁻¹ DIN) to 8.4 ‰ (28 mg L⁻¹ DIN). Starving animals showed similar δ ¹⁵N equilibrium values (8.4 ‰) to animals fed T. variabilis cultured at the highest DIN concentrations (Table 2).

Download:

Fig 4.

Nitrogen (a) and carbon (b) isotope change over time (96 hour) in Daphnia after the diet switch to cyanobacteria and during starvation. Colored lines represent exponential model fits to the different DIN treatments (0, 7, 14, and 28 mg L⁻¹ DIN). Linear models were applied to the starvation treatment in a and b and to the 28 mg L⁻¹ DIN treatment in a.

https://doi.org/10.1371/journal.pone.0306173.g004

Download:

Table 2. Parameter estimates and standard errors of the exponential decay function fitted to the changing δ ¹⁵N values of Daphnia while feeding on the differently grown cyanobacteria for 96 h (n = 3).

δ₀ is the initial isotope ratio, δ_eq the asymptote (plateau), λ the incorporation rate of δ ¹⁵N, AIC is the Akaike Information Criterion, and T₅₀ is the half-life isotope turnover rate. For the highest DIN concentration, λ and T₅₀ were not calculated because of the lack of differences between δ₀ and δ_eq.

https://doi.org/10.1371/journal.pone.0306173.t002

δ ¹³C values of Daphnia continuously decreased over time after the diet switch to cyanobacteria in all DIN treatments; most pronounced at 14 mg L⁻¹ DIN, resulting in the fastest carbon isotope turn-over rate at this DIN concentration (Fig 4b; Table 3). In animals starving after the diet switch at day 5 of the experiment, δ ¹³C values increased over time while δ ¹⁵N values remained constant (Fig 4a and 4b).

Download:

Table 3. Parameter estimates and standard errors of exponential decay function fitted to the changing δ ¹³C values of Daphnia while feeding on the differently grown cyanobacteria for 96 h (n = 3).

δ₀ is the initial isotope ratio, δ_eq the asymptote (plateau), λ the incorporation rate of δ ¹³C, AIC is the Akaike Information Criterion, and T₅₀ is the half-life isotope turnover rate.

https://doi.org/10.1371/journal.pone.0306173.t003

Elemental composition of Daphnia

After the diet switch at day 5, the relative N content (%) of the animals decreased over time until the end of the experiment (t₀-t₄), significantly in the starvation treatment (3.6%; Tukey’s HSD, p = 0.004) and slightly in the various DIN treatments (Fig 5a). The relative N content of starving animals differed significantly from the relative N content of animals reared on the different cyanobacterial DIN treatments (ANOVA, F_5,15 = 17.41, p < 0.001. The relative C content (%) of the animals also decreased over time in most treatments, significantly in the starvation treatment (14.1%; ANOVA, F_5,15 = 13.17, p < 0.001) but less pronounced and non-uniformly in the various DIN treatments (Fig 5b). The molar C:N ratio of starving animals and that of animals of the lowest cyanobacterial DIN treatment (0 mg L⁻¹) increased over time, whereas the molar C:N ratio of animals of all other DIN treatments did not change significantly (Fig 5c; ANOVA, F_5,15 = 5.07, p = 0.006).

Download:

Fig 5.

Change in relative nitrogen (a) and carbon (b) content (% of dry weight) as well as molar C:N ratios of Daphnia after the diet switch to cyanobacteria at day 5 of the experiment and during starvation. Colored lines represent exponential model fits to the different DIN treatments (0, 7, 14, and 28 mg L⁻¹ DIN).

https://doi.org/10.1371/journal.pone.0306173.g005

The δ ¹⁵N values of Daphnia reared on the different DIN treatments did not correlate with body N and C content (%) or molar C:N ratio (Pearson: p > 0.05). In contrast, δ ¹⁵N values of starving animals were positively correlated with N (Pearson: r = 0.63, p = 0.012) and C (Pearson: r = 0.61, p = 0.017) and negatively with the molar C:N ratio (Pearson: r = -0.67, p = 0.007). The δ ¹³C values of Daphnia of the highest DIN treatment (28 mg L⁻¹) were positively correlated with relative body C content (Pearson: r = 0.57, p = 0.02) and molar C:N ratio (Pearson: r = 0.51, p = 0.044). In all other DIN treatments, the δ ¹³C values of Daphnia did not correlate significantly with either body N or C content or C:N ratio. In contrast, δ ¹³C of starving animals were negatively correlated with body N (Pearson: r = -0.90, p < 0.001) and C content (Pearson: r = -0.92, p < 0.001) and positively with C:N ratios (Pearson: r = 0.83, p < 0.001).

Trophic fractionation

In general, δ ¹⁵N of daphnids of the different DIN treatments were enriched by 5.1 ‰ (± 0.6) compared to their diet (Fig 6a). The Δ¹⁵N_cons-diet values were non-linearly correlated with the DIN concentration in the cyanobacterial culture medium (Pearson: r = 0.91, p = 0.03); the values first decreased with increasing DIN concentration until 14 mg L⁻¹ and then increased again at 28 mg L⁻¹ (Fig 6a). The ¹⁵N trophic enrichment was thus highest (5.8 ‰) in the absence of DIN in the cyanobacterial culture medium. The Δ¹⁵N_cons-diet values of Daphnia showed an inverse relationship with the nitrogen content of their diet, but this was not significant (Pearson: r = -0.55, p = 0.053; Fig 6c). Additionally, there was no relationship between the Δ¹⁵N_cons-diet values of Daphnia and the molar C:N ratios of their diet (Pearson: r = 0.11, p > 0.05; Fig 6c). However, when considering the trophic N fractionation (Δ¹⁵N_cons-diet) in comparison to the elemental composition of the animals, there was a significant negative correlation with the molar C:N body ratio of the animals (Pearson: r = -0.60, p = 0.037; Fig 6i). We did not find any relationship between trophic fractionation of N and body N content (%) (Fig 6g).

Download:

Fig 6.

Trophic fractionation of ¹⁵N and ¹³C. Δ¹⁵N_cons-diet and Δ¹³C_cons-diet values versus DIN concentration in the cyanobacterial culture medium (a and b), N and C content of the diet (c and d), molar C:N ratio of the diet (e and f), body N and C content of Daphnia (% of dry weight; g and h), and molar C:N ratios of Daphnia (i and j).

https://doi.org/10.1371/journal.pone.0306173.g006

The Δ¹³C_cons-diet values increased with increasing DIN availability in the cyanobacterial culture medium (Pearson: r = 0.8, p < 0.001; Fig 6b); the trophic enrichment thus increased from 3.3 ‰ at 0 mg L⁻¹ DIN to 4.2 ‰ at 28 mg L⁻¹ DIN. The Δ¹³C_cons-diet values of Daphnia showed a positive trend with the carbon content of their diet, which was not significant (Pearson: r = -0.55, p = 0.052; Fig 6d). The molar C:N ratios of the diet did not correlate with the Δ¹³C_cons-diet values of Daphnia (Pearson: r = 0.15, p > 0.05; Fig 6f). However, when considering the trophic C fractionation (Δ¹³C_cons-diet) of Daphnia in comparison to the elemental composition of the Daphnia, there was no significant relationship to the C content (Pearson: r = -0.23, p > 0.05; Fig 6d) and the molar C:N ratio (Pearson: r = -0.07, p > 0.05; Fig 6f).

Body dry weight of D. magna

The dry weight of the animals did not change significantly during the experimental exposure to the cyanobacteria (from t₀ to t₄) in none of the DIN treatments and the observed dry weight changes did not differ among treatments (ANOVA, F_4,11 = 2.028, p > 0.05). The starving animals lost weight from t₀ to t₄ (on average 49.9% of the initial body dry weight at t₀).

Discussion

In our experiment, the δ ¹⁵N values of the diazotrophic cyanobacterium T. variabilis were positively correlated with the availability of DIN in the culture medium, suggesting a successive change in N acquisition from N₂ fixation at low DIN concentrations to the uptake of DIN at high DIN concentrations. In other words, the increasing depletion of δ ¹⁵N from 3.1 to -0.7 ‰ with decreasing DIN concentrations likely reflects increasing N₂ fixation to compensate for decreasing DIN availability. The strong depletion in δ ¹⁵N values in the absence of DIN corroborates previous findings. Bauersachs et al. [17] reported even lower values of δ ¹⁵N (-1.85 ‰) for the same strain of T. variabilis cultivated with N₂ gas. A depletion in δ ¹⁵N values (up to -0.5 ‰) at low DIN concentrations was also observed in field studies conducted in both eutrophic and oligotrophic lakes [44–46]. Anabaena variabilis has been reported to discriminate against ¹⁵N during N fixation, resulting in δ ¹⁵N values of approximately -1 ‰ [18, 19]. The non-linear relationship we observed between δ ¹⁵N and DIN availability suggests a switch to N₂ fixation at a DIN concentration of <14 mg L⁻¹ (NaNO₃ <85 mg L⁻¹). Based on bottle incubation experiments conducted in P-rich reservoirs, Bradburn et al. [47] concluded that the efficiency of N₂ fixation increases with decreasing DIN availability and is highest at DIN concentrations of <50 μg L⁻¹ and low irradiance (due to photoinhibition). In a mesocosm study, N₂ fixation has been found to increase with decreasing N:P [12]. Whether this translates to lower thresholds for N₂ fixation in oligotrophic lakes remains unclear. The relationship between nutrient loading, productivity and biological N transformations (N₂ fixation versus denitrification) is complex [48] and requires further investigation, also in regard to environmental thresholds [49] potentially allowing to assess the onset of N₂ fixation and to explore seasonal changes in the balance between biological N transformations.

The molar C:N ratios of T. variabilis cells did not differ significantly among the different DIN treatments, but tended to correlate negatively with increasing DIN availability (not considering the C:N ratio at 0 mg L⁻¹ DIN; Fig 1C). The small peak in C:N ratios at 7 mg L⁻¹ DIN may reflect a stoichiometric response to the switch to N₂ fixation. Overall, however, C:N ratios of T. variabilis were consistently low, ranging from 3.3 to 3.6 (mean values), suggesting that T. variabilis did not experience N limitation in any of the treatments. Thus, the switch to N₂ fixation presumably allowed T. variabilis to overcome N limitation at low DIN concentrations. This also indicates that T. variabilis switches to energetically more expensive N₂ fixation only if DIN becomes limiting [9–11, 50]. Stoichiometric N homeostasis has been reported already from other diazotrophic cyanobacteria. In Dolichospermum, stoichiometric N homeostasis has been found to come at the expense of reduced biomass production during N₂ fixation [13, 51]. In contrast, Aphanizomenon flos-aquae has been found to exhibit flexible C:N stoichiometry and no growth trade-off due to N-fixation, potentially reflecting a high diversity in strategies to cope with N deficiencies among diazotrophs [52]. In our experiment, biomass production (i.e., carbon concentrations at the end of the experiment) did not differ among DIN treatments, suggesting that T. variabilis is able to maintain stoichiometric N homeostasis during N₂ fixation without compromising growth.

Nutrient limitation, i.e., N limitation [31, 34] and P limitation [32, 33, 35], as well as the relative availability of CO₂ and HCO₃⁻ [34] can also influence the C isotope signature of photosynthetic organisms through changes in the activity of the CO₂ concentrating mechanism (CCM). In our experiment, the δ ¹³C values of T. variabilis did not show a clear relationship with DIN availability (Fig 1B). The 2.1 ‰ depletion in δ ¹³C values at a DIN concentration of 14 mg L⁻¹ (δ ¹³C = -29.7 ‰ compared to an average of -27.6 ‰ in the other treatments) may be related to changes in C acquisition. However, our cyanobacteria cultures were consistently aerated (CO₂ supply) and additionally supplied with HCO₃⁻ via the growth medium, making C limitation rather unlikely. Thus, the switch to N₂ fixation probably had no effect on the uptake of carbon in our experiment.

Field studies suggest that the stable isotope signature of consumers can reflect the assimilation of organic matter produced by N-limited, diazotrophic cyanobacteria [24, 26–29]. However, this has rarely been explored under controlled laboratory conditions [26]. We show here experimentally that the mode of N acquisition at the primary producer level (i.e., N₂ fixation versus DIN uptake) not only affects the stable isotope values of the primary producers but also those of their consumers (i.e., Daphnia). δ ¹⁵N values of Daphnia decreased over time while feeding on the differently grown cyanobacteria, most evident at DIN concentrations ≤7 mg L⁻¹ in the growth medium (Fig 2A). After 96 h of feeding, the highest ¹⁵N depletion was found in Daphnia in the absence of DIN (0 mg L⁻¹ = 3 ‰) and the lowest ¹⁵N depletion was found in Daphnia at the highest DIN concentration (28 mg L⁻¹ = 0.2 ‰) in the cyanobacterial growth medium, reflecting the switch from N₂ fixation to DIN uptake at the primary producer level. The δ ¹⁵N values of Daphnia were positively correlated with the δ ¹⁵N values of T. variabilis, i.e., N₂ fixation was clearly detectable also at the consumer level. The N isotope turnover rates, expressed as the time it takes for 50% of the stable isotopes in a consumer’s tissue to be replaced by the stable isotopes in the diet (T₅₀ values), decreased with increasing DIN concentration in the growth medium. Thus, T₅₀ values of Daphnia were highest in the absence of DIN as a consequence of N₂ fixation and resulting depletion in ¹⁵N at the primary producer level. The larger the isotope differences between consumer and diet, the longer it takes until the isotope signature of the consumer reflects that of its diet. In our setting, it took 65.4 h until 50% of the stable isotopes in Daphnia tissue were replaced by stable isotopes derived from T. variabilis acquiring its N via N₂ fixation. The ¹⁵N depletion caused by N₂ fixation at the primary producer level should thus be detectable also at the consumer level (zooplankton) within a few days (2–3) after the switch to N₂ fixation. The δ ¹⁵N values of Daphnia (96 h after diet switch) increased asymptotically with DIN concentration in the cyanobacterial growth medium, also reflecting the switch from N₂ fixation to DIN uptake at the primary producer level and the establishment of consumer-food isotope equilibria.

The Δ¹⁵N_cons-diet values of Daphnia showed a U-shaped relationship with DIN concentration in the cyanobacterial growth medium, suggesting that ¹⁵N trophic fractionation changed non-linearly with DIN availability–a finding that requires further investigations. On average (i.e., across DIN treatments), Daphnia were enriched in ¹⁵N by 5.1 ‰ (±0.6 ‰, Δ¹⁵N_cons-diet) compared to its diet. Δ¹⁵N values are known to vary considerably across trophic levels, because most consumers feed on a variety of different food sources and often in varying proportions [53, 54]. Adams and Sterner [55] showed already that even within a consumer-resource pair (Daphnia-green algae), strong differences in Δ¹⁵N values from 1 to 6 ‰ can occur due to differences in N content of the diet. The strong positive relationship between ¹⁵N enrichment (Δ¹⁵N_cons-diet) and C:N ratio of the algal diet reported by Adams and Sterner [55] was not expected in our study because we used a diazotrophic cyanobacterium as food, capable of switching to N₂ fixation at low DIN concentrations, thus avoiding N limitation. The δ ¹⁵N values of the cyanobacterium increased significantly with the switch from N₂ fixation to DIN uptake, potentially also affecting the trophic enrichment of ¹⁵N. Adams and Sterner [55] reported that the δ ¹⁵N values of Daphnia and the consumer-diet isotope fractionation factor (Δ¹⁵N_cons-diet) were inversely related to the N content of the algae. We found a significant relationship between (Δ¹⁵N_cons-diet) and C:N body ratio of the animals, but no relationship with the C:N ratio or N content of their cyanobacterial diet.

The carbon and nitrogen content of Daphnia exposed to the cyanobacterium T. variabilis for four days decreased slightly, but much less than in the starvation treatment, indicating that the animals were able to ingest the T. variabilis filaments and maintain their carbon and nitrogen content by consuming T. variabilis. Cyanobacteria are unsuitable as food for Daphnia and other invertebrate consumers for a number of reasons, including their potential toxicity and the lack of essential lipids [56–58]. Daphnia feeding on T. variabilis (ATCC 29413, formerly Anabaena variabilis) have been shown to struggle primarily with sterol limitation, resulting in very low somatic growth rates [57, 59]. Also in our experiment, the dry mass of Daphnia feeding on T. variabilis increased only slightly. However, the stable isotope data showed that the animals incorporated and processed the carbon and nitrogen provided by the cyanobacterium, suggesting that the animals were able to at least meet their basic energy requirements by consuming the cyanobacterium. δ ¹⁵N and δ ¹³C values of consumers can change due to food quantity- and quality-related isotope fractionation [60]. During periods of nutritional stress, enrichment in ¹⁵N may result from the segregation of ¹⁴N, i.e., the excretion of isotopically light urea [61, 62]. In the present study, δ ¹⁵N values of starving Daphnia decreased only slightly (by 0.5 ‰), whereas the N weight fraction (% of body weight) strongly decreased (by 43.3%), suggesting similar excretion of δ ¹⁵N and δ ¹⁴N during starvation. Doi et al. [63] conducted a meta-analysis to evaluate the effects of starvation on δ ¹⁵N of consumers and found that the δ ¹⁵N values of most consumers increased with the length of starvation. The four-day starvation period applied in our study might have been too short to affect the δ ¹⁵N values of Daphnia.

In contrast to δ ¹⁵N values, the δ ¹³C values of starving Daphnia increased over time, which has been reported previously [60, 64]. Enrichment in ¹³C during starvation may result from increased metabolization of ¹³C-depleted lipids in order to meet energetic demands [63]. The samples obtained in our study were not subjected to lipid extraction prior to stable isotope analysis. After the diet switch to cyanobacteria, δ ¹³C values of Daphnia continuously decreased in all DIN treatments, with no clear relationship with DIN concentration. Decreasing δ ¹³C values after a diet switch to cyanobacteria have been reported previously [60]. One possible explanation could be that the animals incorporated ¹³C-depleted lipids from the cyanobacterial food but were unable to grow and reproduce due to sterol limitation and thus did not further metabolize the accumulating lipids and also could not allocate them to reproduction. At high food quantity and low dietary sterol supply (as in the present study), Daphnia have been reported to increase excretion and respiration of excess bulk C [65], which should lead to an enrichment in bulk ¹³C, i.e., increasing δ ¹³C values. Decreasing δ ¹³C values in consumers in response to declining food quality should therefore be further investigated to elucidate the underlying mechanisms. The ¹³C enrichment (Δ¹³C_cons-diet) in Daphnia was in the range of 3.1 to 4.1 ‰ compared to its cyanobacterial diet, and thus much larger than the commonly assumed trophic enrichment of ≤1 ‰ for C [54, 66, 67], potentially also reflecting nutritional constraints. The C stable isotope data at the consumer level generally did not show a clear relationship with the DIN concentration provided at the primary producer level.

Conclusion

We show here experimentally that the δ ¹⁵N values of the diazotrophic cyanobacterium T. variabilis are positively related to the concentration of DIN in the growth medium, indicating a switch from N₂ fixation to DIN uptake with increasing DIN availability. Thus, N₂ fixation occurs only at low and potentially limiting DIN concentrations (<14 mg L⁻¹), most likely because N₂ fixation is energetically more costly than DIN uptake. C:N ratios of T. variabilis were consistently low, suggesting that N₂ fixation allowed T. variabilis to overcome N limitation at low DIN concentrations. The δ ¹³C values of T. variabilis did not show a clear relationship with DIN availability. The mode of N acquisition at the primary producer level also influenced the stable isotope values of the consumer Daphnia. The δ ¹⁵N values of T. variabilis were positively correlated with the δ ¹⁵N values of Daphnia and the estimated N isotope turnover rates suggest that a switch to N₂ fixation at the primary producer level should be detectable at the consumer level within a few days (2–3). The diet switch to cyanobacteria strongly influenced the δ ¹⁵N and δ ¹³C values of Daphnia due to food quality-related isotope fractionation, but this did not affect the detectability of diazotrophic N in the consumer. We conclude that stable isotope analysis of bulk N at the phytoplankton-zooplankton interface can provide valuable information about N limitation and the fate of diazotrophic N in lake food webs.

References

1. Hecky RE, Kilham P. Nutrient limitation of phytoplankton in freshwater and marine environments: A review of recent evidence on the effects of enrichment. Limnol Oceanogr. 1988; 33(4part2): 796–822.
- View Article
- Google Scholar
2. Correll D. Phosphorus: a rate limiting nutrient in surface waters. Poult Sci. 1999; 78(5):674–82. pmid:10228963
- View Article
- PubMed/NCBI
- Google Scholar
3. Schindler DW. Evolution of phosphorus limitation in lakes: Natural mechanisms compensate for deficiencies of nitrogen and carbon in eutrophied lakes. Science. 1977; 195(4275): 260–2.
- View Article
- Google Scholar
4. Elser JJ, Bracken MES, Cleland EE, Gruner DS, Harpole WS, Hillebrand H, et al. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol Lett. 2007; 10(12): 1135–42. pmid:17922835
- View Article
- PubMed/NCBI
- Google Scholar
5. Lewis WM, Wurtsbaugh WA. Control of lacustrine phytoplankton by nutrients: Erosion of the phosphorus paradigm. Int Rev Hydrobiol. 2008; 93(4–5): 446–65.
- View Article
- Google Scholar
6. Wood SA, Prentice MJ, Smith K, Hamilton DP. Low dissolved inorganic nitrogen and increased heterocyte frequency: precursors to Anabaena planktonica blooms in a temperate, eutrophic reservoir. J Plankton Res. 2010; 32(9): 1315–25.
- View Article
- Google Scholar
7. Higgins SN, Paterson MJ, Hecky RE, Schindler DW, Venkiteswaran JJ, Findlay DL. Biological nitrogen fixation prevents the response of a eutrophic lake to reduced loading of nitrogen: Evidence from a 46-year whole-lake experiment. Ecosystems. 2018; 21(6): 1088–100.
- View Article
- Google Scholar
8. Kumar K, Mella-Herrera RA, Golden JW. Cyanobacterial heterocysts. Cold Spring Harbor Perspectives in Biology. 2010; 2(4): a000315–a000315. pmid:20452939
- View Article
- PubMed/NCBI
- Google Scholar
9. Blomqvist P, Pettersson A, Hyenstrand P. Ammonium-nitrogen: A key regulatory factor causing dominance of non-nitrogen-fixing cyanobacteria in aquatic systems. Arch Hydrobiol. 1994; 132(2): 141–64.
- View Article
- Google Scholar
10. Flores E, Frías JE, Rubio LM, Herrero A. Photosynthetic nitrate assimilation in cyanobacteria. Photosynth Res. 2005; 83(2): 117–33. pmid:16143847
- View Article
- PubMed/NCBI
- Google Scholar
11. Bergman B, Gallon JR, Rai AN, Stal LJ. N₂ fixation by non-heterocystous cyanobacteria. FEMS Microbiol Rev. 1997; 19(3): 139–85.
- View Article
- Google Scholar
12. Baker BC, Wilson AE, Scott JT. Phytoplankton N₂-fixation efficiency and its effect on harmful algal blooms. Freshw Sci. 2018; 37(2): 264–75.
- View Article
- Google Scholar
13. Grover JP, Scott JT, Roelke DL, Brooks BW. Dynamics of nitrogen-fixing cyanobacteria with heterocysts: a stoichiometric model. Mar Freshwater Res. 2020; 71(5): 644.
- View Article
- Google Scholar
14. Nielsen JM, Clare EL, Hayden B, Brett MT, Kratina P. Diet tracing in ecology: Method comparison and selection. Methods Ecol Evol. 2018; 9(2): 278–91.
- View Article
- Google Scholar
15. Glibert PM, Middelburg JJ, McClelland JW, Jake Vander Zanden M. Stable isotope tracers: Enriching our perspectives and questions on sources, fates, rates, and pathways of major elements in aquatic systems. Limnol Oceanogr. 2019; 64(3): 950–81.
- View Article
- Google Scholar
16. Owens NJP. Natural variations in 15N in the marine environment. In: Advances in Marine Biology. Editors: Blaxter JHS, Southward AJ. Academic Press 1988; 24: 389–451.
17. Bauersachs T, Schouten S, Compaoré J, Wollenzien U, Stal LJ, Sinninghe Damsteé JS. Nitrogen isotopic fractionation associated with growth on dinitrogen gas and nitrate by cyanobacteria. Limnol Oceanogr. 2009; 54(4): 1403–11.
- View Article
- Google Scholar
18. Rowell P, James W, Smith WL, Handley LL, Scrimgeour CM. ¹⁵N discrimination in molybdenum- and vanadium-grown N₂-fixing Anabaena variabilis and Azotobacter vinelandii. Soil Biol Biochem. 1998; 30(14): 2177–80.
- View Article
- Google Scholar
19. Zhang X, Sigman DM, Morel FMM, Kraepiel AML. Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia. Proc Natl Acad Sci USA. 2014; 111(13): 4782–7. pmid:24639508
- View Article
- PubMed/NCBI
- Google Scholar
20. Montoya JP. Nitrogen stable isotopes in marine environments. In: Nitrogen in the marine environment. Editors: Capone DG, Bronk DA., Mulholland MR, Carpenter EJ. Academic Press 2008: 1277–302.
21. Soto DX, Koehler G, Wassenaar LI, Hobson KA. Spatio-temporal variation of nitrate sources to Lake Winnipeg using N and O isotope (δ¹⁵N, δ¹⁸O) analyses. Sci Total Environ. 2019; 647: 486–93.
- View Article
- Google Scholar
22. Matiatos I, Wassenaar LI, Monteiro LR, Venkiteswaran JJ, Gooddy DC, Boeckx P, et al. Global patterns of nitrate isotope composition in rivers and adjacent aquifers reveal reactive nitrogen cascading. Commun Earth Environ. 2021; 2(1): 52.
- View Article
- Google Scholar
23. Montoya JP, Carpenter EJ, Capone DG. Nitrogen fixation and nitrogen isotope abundances in zooplankton of the oligotrophic North Atlantic. Limnol Oceanogr. 2002; 47(6): 1617–28.
- View Article
- Google Scholar
24. Sommer F, Hansen T, Sommer U. Transfer of diazotrophic nitrogen to mesozooplankton in Kiel Fjord, Western Baltic Sea: a mesocosm study. Mar Ecol Prog Ser. 2006; 324: 105–12.
- View Article
- Google Scholar
25. Reynolds SE, Mather RL, Wolff GA, Williams RG, Landolfi A, Sanders R, et al. How widespread and important is N₂ fixation in the North Atlantic Ocean? Global Biogeochemical Cycles. 2007; 21(4): GB4015.
- View Article
- Google Scholar
26. Loick-Wilde N, Dutz JR, Miltner A, Gehre M, Montoya JP, Voss M. Incorporation of nitrogen from N₂ fixation into amino acids of zooplankton. Limnol Oceanogr. 2012; 57(1): 199–210.
- View Article
- Google Scholar
27. Karlson AML, Gorokhova E, Elmgren R. Nitrogen fixed by cyanobacteria is utilized by deposit-feeders. PLoS ONE. 2014; 9(8): e104460. pmid:25105967
- View Article
- PubMed/NCBI
- Google Scholar
28. Motwani NH, Duberg J, Svedén JB, Gorokhova E. Grazing on cyanobacteria and transfer of diazotrophic nitrogen to zooplankton in the Baltic Sea. Limnol Oceanogr. 2018; 63(2): 672–86.
- View Article
- Google Scholar
29. McClelland JW, Holl CM, Montoya JP. Relating low δ¹⁵N values of zooplankton to N₂-fixation in the tropical North Atlantic: insights provided by stable isotope ratios of amino acids. Deep Sea Research Part I: Oceanographic Research Papers. 2003; 50(7): 849–61.
- View Article
- Google Scholar
30. Patoine A, Graham MD, Leavitt PR. Spatial variation of nitrogen fixation in lakes of the northern Great Plains. Limnol Oceanogr. 2006; 51(4): 1665–77.
- View Article
- Google Scholar
31. Kaplan A, Badger MR, Berry JA. Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO₂ concentration. Planta. 1980; 149(3): 219–26.
- View Article
- Google Scholar
32. Beardall J, Johnston A, Raven J. Environmental regulation of CO₂-concentrating mechanisms in microalgae. Can J Bot. 1998; 76(6): 1010–7.
- View Article
- Google Scholar
33. Beardall J, Giordano M. Ecological implications of microalgal and cyanobacterial CO₂ concentrating mechanisms, and their regulation. Functional Plant Biol. 2002; 29(3): 335.
- View Article
- Google Scholar
34. Badger MR, Price GD, Long BM, Woodger FJ. The environmental plasticity and ecological genomics of the cyanobacterial CO₂ concentrating mechanism. J Exp Bot. 2006; 57(2): 249–65.
- View Article
- Google Scholar
35. Giordano M, Beardall J, Raven JA. CO₂ concentrating mechanisms in algae: Mechanisms, environmental modulation, and evolution. Annu Rev Plant Biol. 2005; 56(1): 99–131.
- View Article
- Google Scholar
36. Guillard RRL, Lorenzen CJ. Yellow-green algae with chlorophyllide C. J Phycol. 1972; 8(1): 10–4.
- View Article
- Google Scholar
37. Lampert W. The dynamics of Daphnia magna in a shallow lake. SIL Proceedings, 1922–2010. 1991; 24(2): 795–8.
- View Article
- Google Scholar
38. Jüttner F, Leonhardt J, Möhren S. Environmental factors affecting the formation of mesityloxide, dimethylallylic alcohol and other volatile compounds excreted by Anabaena cylindrica. Microbiology. 1983; 129(2): 407–12.
- View Article
- Google Scholar
39. Jones KA, Ratcliffe N, Votier SC, Newton J, Forcada J, Dickens J, et al. Intra-specific niche partitioning in Antarctic fur seals, Arctocephalus gazella. Sci Rep. 2020; 10(1): 3238.
- View Article
- Google Scholar
40. McKinney CR, McCrea JM, Epstein S, Allen HA, Urey HC. Improvements in mass spectrometers for the measurement of small differences in isotope abundance ratios. Rev Sci Instrum. 1950; 21(8): 724–30. pmid:14781446
- View Article
- PubMed/NCBI
- Google Scholar
41. Hobson KA, Clark RG. Assessing avian diets using stable isotopes I: Turnover of ¹³C in tissues. Condor. 1992; 94(1): 181–8.
- View Article
- Google Scholar
42. Chitta Ranjan. Package „nlcor“: Compute nonlinear correlations. 2020; http://rgdoi.net/10.13140/RG.2.2.33716.68480
43. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS ONE. 2015; 10(12): e0146021. pmid:26717316
- View Article
- PubMed/NCBI
- Google Scholar
44. MacGregor BJ, Van Mooy B, Baker BJ, Mellon M, Moisander PH, Paerl HW, et al. Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan. Environ Microbiol. 2001; 3(3): 205–19. pmid:11321537
- View Article
- PubMed/NCBI
- Google Scholar
45. Vuorio K, Meili M, Sarvala J. Taxon-specific variation in the stable isotopic signatures (δ¹³C and δ¹⁵N) of lake phytoplankton. Freshw Biol. 2006; 51(5): 807–22.
- View Article
- Google Scholar
46. Gu B. Variations and controls of nitrogen stable isotopes in particulate organic matter of lakes. Oecologia. 2009; 160(3): 421–31. pmid:19352718
- View Article
- PubMed/NCBI
- Google Scholar
47. Bradburn MJ, Lewis WM, McCutchan JH. Comparative adaptations of Aphanizomenon and Anabaena for nitrogen fixation under weak irradiance. Freshw Biol. 2012; 57(5): 1042–9.
- View Article
- Google Scholar
48. Scott JT, McCarthy MJ, Paerl HW. Nitrogen transformations differentially affect nutrient-limited primary production in lakes of varying trophic state. Limnol Oceanogr Letters. 2019; 4(4): 96–104.
- View Article
- Google Scholar
49. Moutinho FHM, Marafão GA, Calijuri MDC, Moreira MZ, Marcarelli AM, Cunha DGF. Environmental factors and thresholds for nitrogen fixation by phytoplankton in tropical reservoirs. Internat Rev Hydrobiol. 2021; 106(1): 5–17.
- View Article
- Google Scholar
50. Meeks JC, Wycoff KL, Chapman JS, Enderlin CS. Regulation of expression of nitrate and dinitrogen assimilation by Anabaena species. Appl Environ Microbiol. 1983; 45(4): 1351–9.
- View Article
- Google Scholar
51. Osburn FS, Wagner ND, Taylor RB, Chambliss CK, Brooks BW, Scott JT. The effects of salinity and N: P on N-rich toxins by both an N-fixing and non-N-fixing cyanobacteria. Limnol Oceanogr Letters. 2023; 8(1): 162–72.
- View Article
- Google Scholar
52. Wagner ND, Osburn FS, Taylor RB, Back JA, Chambliss CK, Brooks BW, et al. Diazotrophy modulates cyanobacteria stoichiometry through functional traits that determine bloom magnitude and toxin production. Limnol Oceanogr. 2023; 68(2): 348–60. pmid:36819961
- View Article
- PubMed/NCBI
- Google Scholar
53. Minagawa M, Wada E. Stepwise enrichment of ¹⁵N along food chains: Further evidence and the relation between δ¹⁵N and animal age. Geochim Cosmochim Acta. 1984; 48(5): 1135–40.
- View Article
- Google Scholar
54. Post DM. Using stable isotopes to estimate trophic position: Models, methods, and assumptions. Ecology. 2002; 83(3): 703–18.
- View Article
- Google Scholar
55. Adams TS, Sterner RW. The effect of dietary nitrogen content on trophic level ¹⁵N enrichment. Limnol Oceanogr. 2000; 45(3): 601–7.
- View Article
- Google Scholar
56. Wilson AE, Sarnelle O, Tillmanns AR. Effects of cyanobacterial toxicity and morphology on the population growth of freshwater zooplankton: Meta-analyses of laboratory experiments. Limnol Oceanogr. 2006; 51(4): 1915–24.
- View Article
- Google Scholar
57. Martin-Creuzburg D, von Elert E, Hoffmann KH. Nutritional constraints at the cyanobacteria—Daphnia magna interface: The role of sterols. Limnol Oceanogr. 2008; 53(2): 456–68.
- View Article
- Google Scholar
58. Basen T, Rothhaupt KO, Martin-Creuzburg D. Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve. Oecologia. 2012; 170(1): 57–64. pmid:22398861
- View Article
- PubMed/NCBI
- Google Scholar
59. von Elert E, Martin-Creuzburg D, Le Coz JR. Absence of sterols constrains carbon transfer between cyanobacteria and a freshwater herbivore (Daphnia galeata). Proc R Soc Lond B. 2003; 270(1520): 1209–14.
- View Article
- Google Scholar
60. Helmer M, Helmer D, Martin-Creuzburg D, Rothhaupt KO, Yohannes E. Toxicity and starvation induce major trophic isotope variation in Daphnia individuals: A diet switch experiment using eight phytoplankton species of differing nutritional quality. Biology. 2022; 11(12): 1816.
- View Article
- Google Scholar
61. Ambrose SH, DeNiro MJ. The isotopic ecology of East African mammals. Oecologia. 1986; 69(3): 395–406. pmid:28311342
- View Article
- PubMed/NCBI
- Google Scholar
62. Hobson KA, Alisauskas RT, Clark RG. Stable-nitrogen isotope enrichment in avian tissues due to fasting and nutritional stress: Implications for isotopic analyses of diet. Condor. 1993; 95(2): 388.
- View Article
- Google Scholar
63. Doi H, Akamatsu F, González AL. Starvation effects on nitrogen and carbon stable isotopes of animals: an insight from meta-analysis of fasting experiments. R Soc Open Sci. 2017; 4(8): 170633. pmid:28879005
- View Article
- PubMed/NCBI
- Google Scholar
64. Oelbermann K, Scheu S. Stable isotope enrichment (δ¹⁵N and δ¹³C) in a generalist predator (Pardosa lugubris, Araneae: Lycosidae): effects of prey quality. Oecologia. 2002; 130(3): 337–44.
- View Article
- Google Scholar
65. Lukas M, Wacker A. Acclimation to dietary shifts impacts the carbon budgets of Daphnia magna. J Plankton Res. 2014; 36(3): 848–58.
- View Article
- Google Scholar
66. DeNiro MJ, Epstein S. Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta. 1978; 42(5): 495–506.
- View Article
- Google Scholar
67. Fry B, Sherr EB. δ¹³C measurements as indicators of carbon flow in marine and freshwater ecosystems. In: Stable Isotopes in Ecological Research. Editors: Rundel PW, Ehleringer JR, Nagy KA. Ecological Studies; vol 68. Springer New York; 1989: 196–229.

[ref1] 1. Hecky RE, Kilham P. Nutrient limitation of phytoplankton in freshwater and marine environments: A review of recent evidence on the effects of enrichment. Limnol Oceanogr. 1988; 33(4part2): 796–822.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Correll D. Phosphorus: a rate limiting nutrient in surface waters. Poult Sci. 1999; 78(5):674–82. pmid:10228963
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Schindler DW. Evolution of phosphorus limitation in lakes: Natural mechanisms compensate for deficiencies of nitrogen and carbon in eutrophied lakes. Science. 1977; 195(4275): 260–2.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref4] 4. Elser JJ, Bracken MES, Cleland EE, Gruner DS, Harpole WS, Hillebrand H, et al. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol Lett. 2007; 10(12): 1135–42. pmid:17922835
View Article
PubMed/NCBI
Google Scholar

[12] View Article

[13] PubMed/NCBI

[14] Google Scholar

[ref5] 5. Lewis WM, Wurtsbaugh WA. Control of lacustrine phytoplankton by nutrients: Erosion of the phosphorus paradigm. Int Rev Hydrobiol. 2008; 93(4–5): 446–65.
View Article
Google Scholar

[16] View Article

[17] Google Scholar

[ref6] 6. Wood SA, Prentice MJ, Smith K, Hamilton DP. Low dissolved inorganic nitrogen and increased heterocyte frequency: precursors to Anabaena planktonica blooms in a temperate, eutrophic reservoir. J Plankton Res. 2010; 32(9): 1315–25.
View Article
Google Scholar

[19] View Article

[20] Google Scholar

[ref7] 7. Higgins SN, Paterson MJ, Hecky RE, Schindler DW, Venkiteswaran JJ, Findlay DL. Biological nitrogen fixation prevents the response of a eutrophic lake to reduced loading of nitrogen: Evidence from a 46-year whole-lake experiment. Ecosystems. 2018; 21(6): 1088–100.
View Article
Google Scholar

[22] View Article

[23] Google Scholar

[ref8] 8. Kumar K, Mella-Herrera RA, Golden JW. Cyanobacterial heterocysts. Cold Spring Harbor Perspectives in Biology. 2010; 2(4): a000315–a000315. pmid:20452939
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref9] 9. Blomqvist P, Pettersson A, Hyenstrand P. Ammonium-nitrogen: A key regulatory factor causing dominance of non-nitrogen-fixing cyanobacteria in aquatic systems. Arch Hydrobiol. 1994; 132(2): 141–64.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref10] 10. Flores E, Frías JE, Rubio LM, Herrero A. Photosynthetic nitrate assimilation in cyanobacteria. Photosynth Res. 2005; 83(2): 117–33. pmid:16143847
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref11] 11. Bergman B, Gallon JR, Rai AN, Stal LJ. N₂ fixation by non-heterocystous cyanobacteria. FEMS Microbiol Rev. 1997; 19(3): 139–85.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref12] 12. Baker BC, Wilson AE, Scott JT. Phytoplankton N₂-fixation efficiency and its effect on harmful algal blooms. Freshw Sci. 2018; 37(2): 264–75.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref13] 13. Grover JP, Scott JT, Roelke DL, Brooks BW. Dynamics of nitrogen-fixing cyanobacteria with heterocysts: a stoichiometric model. Mar Freshwater Res. 2020; 71(5): 644.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref14] 14. Nielsen JM, Clare EL, Hayden B, Brett MT, Kratina P. Diet tracing in ecology: Method comparison and selection. Methods Ecol Evol. 2018; 9(2): 278–91.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref15] 15. Glibert PM, Middelburg JJ, McClelland JW, Jake Vander Zanden M. Stable isotope tracers: Enriching our perspectives and questions on sources, fates, rates, and pathways of major elements in aquatic systems. Limnol Oceanogr. 2019; 64(3): 950–81.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref16] 16. Owens NJP. Natural variations in 15N in the marine environment. In: Advances in Marine Biology. Editors: Blaxter JHS, Southward AJ. Academic Press 1988; 24: 389–451.

[ref17] 17. Bauersachs T, Schouten S, Compaoré J, Wollenzien U, Stal LJ, Sinninghe Damsteé JS. Nitrogen isotopic fractionation associated with growth on dinitrogen gas and nitrate by cyanobacteria. Limnol Oceanogr. 2009; 54(4): 1403–11.
View Article
Google Scholar

[52] View Article

[53] Google Scholar

[ref18] 18. Rowell P, James W, Smith WL, Handley LL, Scrimgeour CM. ¹⁵N discrimination in molybdenum- and vanadium-grown N₂-fixing Anabaena variabilis and Azotobacter vinelandii. Soil Biol Biochem. 1998; 30(14): 2177–80.
View Article
Google Scholar

[55] View Article

[56] Google Scholar

[ref19] 19. Zhang X, Sigman DM, Morel FMM, Kraepiel AML. Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia. Proc Natl Acad Sci USA. 2014; 111(13): 4782–7. pmid:24639508
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref20] 20. Montoya JP. Nitrogen stable isotopes in marine environments. In: Nitrogen in the marine environment. Editors: Capone DG, Bronk DA., Mulholland MR, Carpenter EJ. Academic Press 2008: 1277–302.

[ref21] 21. Soto DX, Koehler G, Wassenaar LI, Hobson KA. Spatio-temporal variation of nitrate sources to Lake Winnipeg using N and O isotope (δ¹⁵N, δ¹⁸O) analyses. Sci Total Environ. 2019; 647: 486–93.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref22] 22. Matiatos I, Wassenaar LI, Monteiro LR, Venkiteswaran JJ, Gooddy DC, Boeckx P, et al. Global patterns of nitrate isotope composition in rivers and adjacent aquifers reveal reactive nitrogen cascading. Commun Earth Environ. 2021; 2(1): 52.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref23] 23. Montoya JP, Carpenter EJ, Capone DG. Nitrogen fixation and nitrogen isotope abundances in zooplankton of the oligotrophic North Atlantic. Limnol Oceanogr. 2002; 47(6): 1617–28.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref24] 24. Sommer F, Hansen T, Sommer U. Transfer of diazotrophic nitrogen to mesozooplankton in Kiel Fjord, Western Baltic Sea: a mesocosm study. Mar Ecol Prog Ser. 2006; 324: 105–12.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref25] 25. Reynolds SE, Mather RL, Wolff GA, Williams RG, Landolfi A, Sanders R, et al. How widespread and important is N₂ fixation in the North Atlantic Ocean? Global Biogeochemical Cycles. 2007; 21(4): GB4015.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref26] 26. Loick-Wilde N, Dutz JR, Miltner A, Gehre M, Montoya JP, Voss M. Incorporation of nitrogen from N₂ fixation into amino acids of zooplankton. Limnol Oceanogr. 2012; 57(1): 199–210.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref27] 27. Karlson AML, Gorokhova E, Elmgren R. Nitrogen fixed by cyanobacteria is utilized by deposit-feeders. PLoS ONE. 2014; 9(8): e104460. pmid:25105967
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref28] 28. Motwani NH, Duberg J, Svedén JB, Gorokhova E. Grazing on cyanobacteria and transfer of diazotrophic nitrogen to zooplankton in the Baltic Sea. Limnol Oceanogr. 2018; 63(2): 672–86.
View Article
Google Scholar

[85] View Article

[86] Google Scholar

[ref29] 29. McClelland JW, Holl CM, Montoya JP. Relating low δ¹⁵N values of zooplankton to N₂-fixation in the tropical North Atlantic: insights provided by stable isotope ratios of amino acids. Deep Sea Research Part I: Oceanographic Research Papers. 2003; 50(7): 849–61.
View Article
Google Scholar

[88] View Article

[89] Google Scholar

[ref30] 30. Patoine A, Graham MD, Leavitt PR. Spatial variation of nitrogen fixation in lakes of the northern Great Plains. Limnol Oceanogr. 2006; 51(4): 1665–77.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref31] 31. Kaplan A, Badger MR, Berry JA. Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO₂ concentration. Planta. 1980; 149(3): 219–26.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref32] 32. Beardall J, Johnston A, Raven J. Environmental regulation of CO₂-concentrating mechanisms in microalgae. Can J Bot. 1998; 76(6): 1010–7.
View Article
Google Scholar

[97] View Article

[98] Google Scholar

[ref33] 33. Beardall J, Giordano M. Ecological implications of microalgal and cyanobacterial CO₂ concentrating mechanisms, and their regulation. Functional Plant Biol. 2002; 29(3): 335.
View Article
Google Scholar

[100] View Article

[101] Google Scholar

[ref34] 34. Badger MR, Price GD, Long BM, Woodger FJ. The environmental plasticity and ecological genomics of the cyanobacterial CO₂ concentrating mechanism. J Exp Bot. 2006; 57(2): 249–65.
View Article
Google Scholar

[103] View Article

[104] Google Scholar

[ref35] 35. Giordano M, Beardall J, Raven JA. CO₂ concentrating mechanisms in algae: Mechanisms, environmental modulation, and evolution. Annu Rev Plant Biol. 2005; 56(1): 99–131.
View Article
Google Scholar

[106] View Article

[107] Google Scholar

[ref36] 36. Guillard RRL, Lorenzen CJ. Yellow-green algae with chlorophyllide C. J Phycol. 1972; 8(1): 10–4.
View Article
Google Scholar

[109] View Article

[110] Google Scholar

[ref37] 37. Lampert W. The dynamics of Daphnia magna in a shallow lake. SIL Proceedings, 1922–2010. 1991; 24(2): 795–8.
View Article
Google Scholar

[112] View Article

[113] Google Scholar

[ref38] 38. Jüttner F, Leonhardt J, Möhren S. Environmental factors affecting the formation of mesityloxide, dimethylallylic alcohol and other volatile compounds excreted by Anabaena cylindrica. Microbiology. 1983; 129(2): 407–12.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref39] 39. Jones KA, Ratcliffe N, Votier SC, Newton J, Forcada J, Dickens J, et al. Intra-specific niche partitioning in Antarctic fur seals, Arctocephalus gazella. Sci Rep. 2020; 10(1): 3238.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref40] 40. McKinney CR, McCrea JM, Epstein S, Allen HA, Urey HC. Improvements in mass spectrometers for the measurement of small differences in isotope abundance ratios. Rev Sci Instrum. 1950; 21(8): 724–30. pmid:14781446
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref41] 41. Hobson KA, Clark RG. Assessing avian diets using stable isotopes I: Turnover of ¹³C in tissues. Condor. 1992; 94(1): 181–8.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref42] 42. Chitta Ranjan. Package „nlcor“: Compute nonlinear correlations. 2020; http://rgdoi.net/10.13140/RG.2.2.33716.68480

[ref43] 43. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS ONE. 2015; 10(12): e0146021. pmid:26717316
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref44] 44. MacGregor BJ, Van Mooy B, Baker BJ, Mellon M, Moisander PH, Paerl HW, et al. Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan. Environ Microbiol. 2001; 3(3): 205–19. pmid:11321537
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref45] 45. Vuorio K, Meili M, Sarvala J. Taxon-specific variation in the stable isotopic signatures (δ¹³C and δ¹⁵N) of lake phytoplankton. Freshw Biol. 2006; 51(5): 807–22.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref46] 46. Gu B. Variations and controls of nitrogen stable isotopes in particulate organic matter of lakes. Oecologia. 2009; 160(3): 421–31. pmid:19352718
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref47] 47. Bradburn MJ, Lewis WM, McCutchan JH. Comparative adaptations of Aphanizomenon and Anabaena for nitrogen fixation under weak irradiance. Freshw Biol. 2012; 57(5): 1042–9.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref48] 48. Scott JT, McCarthy MJ, Paerl HW. Nitrogen transformations differentially affect nutrient-limited primary production in lakes of varying trophic state. Limnol Oceanogr Letters. 2019; 4(4): 96–104.
View Article
Google Scholar

[147] View Article

[148] Google Scholar

[ref49] 49. Moutinho FHM, Marafão GA, Calijuri MDC, Moreira MZ, Marcarelli AM, Cunha DGF. Environmental factors and thresholds for nitrogen fixation by phytoplankton in tropical reservoirs. Internat Rev Hydrobiol. 2021; 106(1): 5–17.
View Article
Google Scholar

[150] View Article

[151] Google Scholar

[ref50] 50. Meeks JC, Wycoff KL, Chapman JS, Enderlin CS. Regulation of expression of nitrate and dinitrogen assimilation by Anabaena species. Appl Environ Microbiol. 1983; 45(4): 1351–9.
View Article
Google Scholar

[153] View Article

[154] Google Scholar

[ref51] 51. Osburn FS, Wagner ND, Taylor RB, Chambliss CK, Brooks BW, Scott JT. The effects of salinity and N: P on N-rich toxins by both an N-fixing and non-N-fixing cyanobacteria. Limnol Oceanogr Letters. 2023; 8(1): 162–72.
View Article
Google Scholar

[156] View Article

[157] Google Scholar

[ref52] 52. Wagner ND, Osburn FS, Taylor RB, Back JA, Chambliss CK, Brooks BW, et al. Diazotrophy modulates cyanobacteria stoichiometry through functional traits that determine bloom magnitude and toxin production. Limnol Oceanogr. 2023; 68(2): 348–60. pmid:36819961
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref53] 53. Minagawa M, Wada E. Stepwise enrichment of ¹⁵N along food chains: Further evidence and the relation between δ¹⁵N and animal age. Geochim Cosmochim Acta. 1984; 48(5): 1135–40.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref54] 54. Post DM. Using stable isotopes to estimate trophic position: Models, methods, and assumptions. Ecology. 2002; 83(3): 703–18.
View Article
Google Scholar

[166] View Article

[167] Google Scholar

[ref55] 55. Adams TS, Sterner RW. The effect of dietary nitrogen content on trophic level ¹⁵N enrichment. Limnol Oceanogr. 2000; 45(3): 601–7.
View Article
Google Scholar

[169] View Article

[170] Google Scholar

[ref56] 56. Wilson AE, Sarnelle O, Tillmanns AR. Effects of cyanobacterial toxicity and morphology on the population growth of freshwater zooplankton: Meta-analyses of laboratory experiments. Limnol Oceanogr. 2006; 51(4): 1915–24.
View Article
Google Scholar

[172] View Article

[173] Google Scholar

[ref57] 57. Martin-Creuzburg D, von Elert E, Hoffmann KH. Nutritional constraints at the cyanobacteria—Daphnia magna interface: The role of sterols. Limnol Oceanogr. 2008; 53(2): 456–68.
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref58] 58. Basen T, Rothhaupt KO, Martin-Creuzburg D. Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve. Oecologia. 2012; 170(1): 57–64. pmid:22398861
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref59] 59. von Elert E, Martin-Creuzburg D, Le Coz JR. Absence of sterols constrains carbon transfer between cyanobacteria and a freshwater herbivore (Daphnia galeata). Proc R Soc Lond B. 2003; 270(1520): 1209–14.
View Article
Google Scholar

[182] View Article

[183] Google Scholar

[ref60] 60. Helmer M, Helmer D, Martin-Creuzburg D, Rothhaupt KO, Yohannes E. Toxicity and starvation induce major trophic isotope variation in Daphnia individuals: A diet switch experiment using eight phytoplankton species of differing nutritional quality. Biology. 2022; 11(12): 1816.
View Article
Google Scholar

[185] View Article

[186] Google Scholar

[ref61] 61. Ambrose SH, DeNiro MJ. The isotopic ecology of East African mammals. Oecologia. 1986; 69(3): 395–406. pmid:28311342
View Article
PubMed/NCBI
Google Scholar

[188] View Article

[189] PubMed/NCBI

[190] Google Scholar

[ref62] 62. Hobson KA, Alisauskas RT, Clark RG. Stable-nitrogen isotope enrichment in avian tissues due to fasting and nutritional stress: Implications for isotopic analyses of diet. Condor. 1993; 95(2): 388.
View Article
Google Scholar

[192] View Article

[193] Google Scholar

[ref63] 63. Doi H, Akamatsu F, González AL. Starvation effects on nitrogen and carbon stable isotopes of animals: an insight from meta-analysis of fasting experiments. R Soc Open Sci. 2017; 4(8): 170633. pmid:28879005
View Article
PubMed/NCBI
Google Scholar

[195] View Article

[196] PubMed/NCBI

[197] Google Scholar

[ref64] 64. Oelbermann K, Scheu S. Stable isotope enrichment (δ¹⁵N and δ¹³C) in a generalist predator (Pardosa lugubris, Araneae: Lycosidae): effects of prey quality. Oecologia. 2002; 130(3): 337–44.
View Article
Google Scholar

[199] View Article

[200] Google Scholar

[ref65] 65. Lukas M, Wacker A. Acclimation to dietary shifts impacts the carbon budgets of Daphnia magna. J Plankton Res. 2014; 36(3): 848–58.
View Article
Google Scholar

[202] View Article

[203] Google Scholar

[ref66] 66. DeNiro MJ, Epstein S. Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta. 1978; 42(5): 495–506.
View Article
Google Scholar

[205] View Article

[206] Google Scholar

[ref67] 67. Fry B, Sherr EB. δ¹³C measurements as indicators of carbon flow in marine and freshwater ecosystems. In: Stable Isotopes in Ecological Research. Editors: Rundel PW, Ehleringer JR, Nagy KA. Ecological Studies; vol 68. Springer New York; 1989: 196–229.

Figures

Abstract

Introduction

Materials and methods

Experimental setup

Stable isotope analysis

Turnover rates.

Time depending isotope signature analysis of Daphnia.

Trophic fractionation.

Statistical analysis

Results

Nitrate availability and stable isotope values of T. variabilis

Stable isotope values of Daphnia in relation to DIN availability

Temporal changes in stable isotope values of Daphnia

Elemental composition of Daphnia

Trophic fractionation

Body dry weight of D. magna

Discussion

Conclusion

References