Chytrids enhance Daphnia fitness by selectively retained chytrid‐synthesised stearidonic acid and conversion of short‐chain to long‐chain polyunsaturated fatty acids

Abstract

1. Chytrid fungal parasites convert dietary energy and essential dietary molecules, such as long‐chain (LC) polyunsaturated fatty acids (PUFA), from inedible algal/cyanobacteria hosts into edible zoospores. How the improved biochemical PUFA composition of chytrid‐infected diet may extend to zooplankton, linking diet quality to consumer fitness, remains unexplored.

2. Here, we assessed the trophic role of chytrids in supporting dietary energy and PUFA requirements of the crustacean zooplankton Daphnia, when feeding on the filamentous cyanobacterium Planktothrix.

3. Only Daphnia feeding on chytrid‐infected Planktothrix reproduced successfully and had significantly higher survival and growth rates compared with Daphnia feeding on the sole Planktothrix diet. While the presence of chytrids resulted in a two‐fold increase of carbon ingested by Daphnia, carbon assimilation increased by a factor of four, clearly indicating enhanced carbon transfer efficiency with chytrid presence.

4. Bulk carbon (δ ¹³C) and nitrogen (δ ¹⁵N) stable isotopes did not indicate any treatment‐specific dietary effects on Daphnia, nor differences in trophic position among diet sources and the consumer. Compound‐specific carbon isotopes of fatty acids (δ ¹³C_FA), however, revealed that chytrids bioconverted short‐chain to LC‐PUFA, making it available for Daphnia. Chytrids synthesised the ω‐3 PUFA stearidonic acid de novo, which was selectively retained by Daphnia. Values of δ ¹³C_FA demonstrated that Daphnia also bioconverted short‐chain to LC‐PUFA.

5. We provide isotopic evidence that chytrids improved the dietary provision of LC‐PUFA for Daphnia and enhanced their fitness. We argue for the existence of a positive feedback loop between enhanced Daphnia growth and herbivory in response to chytrid‐mediated improved diet quality. Chytrids upgrade carbon from the primary producer and facilitate energy and PUFA transfer to primary consumers, potentially also benefitting upper trophic levels of pelagic food webs.

1. INTRODUCTION

The efficiency of dietary energy transfer across the phytoplankton–zooplankton interface is crucial for upper consumers of aquatic food webs. Traditionally, phytoplankton was considered the sole functional group supplying the dietary requirements of zooplankton (Leibold, 1989) and subsequent consumers. Zooplankton growth can be limited by multiple factors such as toxic metabolites (Ger et al., 2016), but primarily by phytoplankton biomass and biochemical dietary composition (Müller‐Navarra, 2008). The elemental composition of phytoplankton constrains zooplankton growth and reproduction in particular in case of low phosphorus concentration (Elser et al., 2001). In addition, dietary lipids and their fatty acids, especially long‐chain (LC) polyunsaturated fatty acids (PUFA) can limit zooplankton fitness (Brett et al., 2009; Müller‐Navarra et al., 2000). Reduced zooplankton fitness in response to the lack of dietary LC‐PUFA (Gulati & Demott, 1997; Ruess & Müller‐Navarra, 2019) may further constrain dietary energy and essential molecule transfer towards consumers at higher trophic levels. Accordingly, a better understanding of mechanisms that enhance PUFA transfer across the phytoplankton–zooplankton interface, when phytoplankton is limited in LC‐PUFA, is important.

High dietary quality phytoplankton, containing LC‐PUFA, include diatoms rich in eicosapentaenoic acid (EPA, 20:5 n‐3) and cryptophytes rich in EPA and docosahexaenoic acid (DHA, 22:6 n‐3; Taipale et al., 2013). The physiologically required PUFA for zooplankton are linoleic acid (LIN, 18:2 n‐6), α‐linolenic acid (ALA, 18:3 n‐3), arachidonic acid (ARA, 20:4 n‐6), EPA, and DHA. Among these dietary PUFA, EPA is particularly conductive for zooplankton growth and reproduction (Becker & Boersma, 2003; Sikora et al., 2016), while DHA increases somatic growth of fish (Copeman et al., 2002) and the cognitive abilities of consumers (Pilecky et al., 2021). Zooplankton have only a limited ability to synthesise LC‐PUFA (Arts et al., 2009; Twining et al., 2021) and lack enzymes to synthesise the essential PUFA LIN and ALA (Cook & McMaster, 2004), which therefore, have to originate from the diet. LIN and ALA are also required for potential bioconversion to ARA (i.e., LIN ➔ ARA), and to EPA via stearidonic acid (SDA, 18:4 n‐3; i.e., ALA ➔ SDA ➔ EPA), and finally to DHA (i.e., EPA ➔ DHA), respectively.

Cyanobacteria, such as Planktothrix agardhii, contain no LC‐PUFA (Gerphagnon et al., 2019). Accordingly, cyanobacteria blooms can limit zooplankton growth and reproduction (Gulati & Demott, 1997; Martin‐Creuzburg et al., 2009), creating an energetic bottleneck in the grazing food chain (Havens, 2008). Diet palatability can also constrain PUFA accessibility for zooplankton. Large‐sized algae or cyanobacteria, irrespective of their dietary PUFA provision, are largely inaccessible for selective (Arndt, 1993) and non‐selective filter‐feeders (Bern, 1994; Carpenter et al., 1993). Consequently, the dominance of inedible cyanobacteria (i.e., filamentous and colonial forms) can limit zooplankton growth due to both size constraints and poor diet quality, and alternative trophic pathways become crucial.

Chytrid fungal parasites constitute a key intermediate‐level trophic group between phytoplankton and zooplankton (Rasconi et al., 2014). The free‐swimming chytrid zoospores are edible (<5 μm) and constitute an alternative trophic link between large inedible phytoplankton and zooplankton, known as mycoloop (Kagami et al., 2007, 2014). For example, during the presence of Asterionella, an inedible diatom rich in LC‐PUFA, chytrids enhanced the somatic growth of Daphnia (Kagami et al., 2007). Similarly, chytrids infecting Planktothrix, an inedible cyanobacterium with poor nutritional quality, improved the growth of Keratella (Frenken et al., 2018) and Daphnia (Agha et al., 2016). Chytrids can improve the dietary provision for zooplankton in two non‐exclusive ways. First, they fragment inedible the host into smaller, edible particles (Frenken et al., 2020; Gerphagnon et al., 2013). Second, they can enhance the biochemical dietary quality compared with the uninfected hosts (Gerphagnon et al., 2019; Taube et al., 2019). Specifically, chytrids perform trophic upgrading by converting short‐chain PUFA from inedible hosts to LC‐PUFA in the edible zoospores (Gerphagnon et al., 2019; Rasconi et al., 2020; Taube et al., 2019). Chytrids also upgrade the biochemical composition of their hosts by de novo synthesised sterols, enhancing dietary energy fluxes in pelagic food webs (Gerphagnon et al., 2019). Experiments suggest that: (1) the mycoloop constitutes a quantitatively important energy pathway at the phytoplankton–zooplankton interface (Gerphagnon et al., 2019; Rasconi et al., 2020; Taube et al., 2019); and (2) chytrids enhance zooplankton consumers' fitness (Agha et al., 2016; Frenken et al., 2018). Whether and how chytrid‐mediated carbon upgrading extends to zooplankton, providing a mechanistic link between improved diet quality and consumer performance, remains unexplored.

Here we aim to disentangle the chytrid‐mediated diet quality effect, quantified by PUFA, from the increased diet quantity effect on zooplankton fitness. Using Daphnia magna as a herbivorous model organism, we performed a feeding experiment to compare the diets consisting of either the filamentous cyanobacterium Planktothrix rubescens, or Planktothrix infected with the obligate chytrid parasite Rhizophydium megarrhizum (Sønstebø & Rohrlack, 2011). We tested the hypothesis that PUFA‐induced diet quality enhancement via chytrids would improve Daphnia fitness, independent of the positive effect of improved diet palatability. We expected that the diet including chytrid parasites would enhance the consumer survival, growth and reproduction via zoospore consumption. Our hypothesis is based on the fact that PUFA assimilated from chytrid zoospores are among the most critical dietary biomolecules transferred across the phytoplankton–zooplankton interface, whereas their lack impedes carbon assimilation of the consumer (Taipale et al., 2014). The amount of assimilated rather than ingested carbon is the key driver of Daphnia fitness, which depends largely on its molecular composition, e.g. LC‐PUFA availability. The amount of assimilated carbon is the most critical in case of limiting dietary carbon availability (Khattak et al., 2018), a case highly relevant for inedible poor diet quality phytoplankton, such as the filamentous cyanobacterium Planktothrix (Gerphagnon et al., 2019).

To quantify the effect of improved dietary PUFA composition via the chytrid parasite, we investigated diet ingestion and assimilation of carbon. To identify PUFA bioconversion pathways among Planktothrix, the chytrid parasite, and Daphnia, we used compound‐specific stable carbon (δ ¹³C) isotope analysis (see Twining et al., 2020). We expected Daphnia to retain specific PUFA from the chytrid diet, and thus showing improved fitness compared to Daphnia feeding on the sole cyanobacterium.

2. MATERIAL & METHODS

2.1. Experimental cultures

The filamentous cyanobacterium P. rubescens (strain NIVA‐CYA97/1) and its chytrid fungal parasite (strain Chy‐Lys2009) were both isolated from Lake Lyseren, Norway (Sønstebø & Rohrlack, 2011). The cultures were maintained on WC Medium (Guillard & Lorenzen, 1972) in VWR cell culture flasks in a non‐axenic environment at 21°C. We applied 10.9 μmol m² s⁻¹ photosynthetically active radiation and a 16:8 light: dark cycle using two AquaLytic incubators (180 L, Liebherr, Germany). Prior to the experiment, the carbon content of uninfected and chytrid‐infected (>50% of prevalence, i.e. % of infected filaments) Planktothrix cultures were analysed to approximate a minimum of dietary c. 0.6 mg C/L during the experiment (Lampert, 1978). The high chytrid prevalence of Planktothrix was reached by weekly 1/3 V/V% medium exchange and 1/3 V/V% dense Planktothrix culture exchange. The stock cultures of D. magna grew in pre‐filtered (0.7 μm GF/F) water from Lake Lunz diluted with 10 V/V% of ADaM medium (Klüttgen et al., 1994) for multiple generations before the experiment. Daphnia magna was fed >1 mg C/L with Scenedesmus sp. and Chlamydomonas sp. (50%–50%), both grown on WC medium.

2.2. Experimental design

Two diet systems were compared: (1) Planktothrix (cyanobacterium treatment); and (2) chytrid‐infected Planktothrix (kept >50% prevalence of infection), containing the cyanobacterium, the sessile sporangia of the chytrid parasite, and free swimming chytrid zoospores (cyanobacterium–parasite–zoospore treatment). Diet treatments were run in five replicates in 1‐L Corning® Square Storage plastic bottles at 18°C. Each bottle contained 22 Daphnia neonates, all born within 48 hr. The bottles were filled up to 1 L volume (600 ml pre‐filtered LUS water + 400 ml diet containing c. 0.4 ± 0.1 μg C/L) and loosely covered with opaque plastic plates allowing air exchange. We applied a 16:8 light: dark cycle with 1.4 μmol m² s⁻¹ photosynthetically active radiation during the light phase. Food was replaced and the residuals quantified every other day to all treatments. Daphnia were checked daily for survival and egg production. An individual was considered dead if it was lying at the bottom of the bottle with no movement for 5 min. Dead individuals were removed and kept frozen at −20°C. The experiment was stopped when the first Daphnia neonates were observed (i.e., after 14 days).

2.3. Life history of Daphnia

Treatment‐specific difference in Daphnia growth rate was quantified based on dry weight (DW):

$$\mathrm{GR}=\left(\ln \left({\mathit{DW}}_{\mathit{end}}\right)-\ln \left({\mathit{DW}}_{\mathit{\text{start}}}\right)\right)/\mathrm{d},$$

where DW _end is the average per individual DW at the end of the experiment, DW _start is the average individual‐level DW of Daphnia neonates, and d is the duration of the experiment in days. The survival of Daphnia was expressed as the percentage of individuals that survived the entire experiment. The egg production rate of Daphnia was quantified based on the cumulative number of eggs produced during the entire experiment in each bottle standardised by the number of survivals and time (per individual per day).

Daphnia body length was directly measured at the end of the experiment on all survival individuals according to Bottrell et al. (1976) using the ocular micrometer of a Nikon SMZ 745 T binocular microscope.

2.4. Quantitative data on ingested diet

The fresh‐weight (FW) biomass of ingested diet was calculated as the difference between the diet concentration at time of feeding (hereafter diet) and after 48 hr (hereafter residual) in triplicates. Planktothrix growth was assumed to be negligible due to the low light and water temperature compared with the culture conditions, and, therefore, not considered. Volumetric concentration of each diet source (i.e., Planktothrix, the chytrid‐infected Planktothrix, and chytrid zoospores) was quantified by microscopic analysis from formaldehyde‐fixed samples (4% final concentration). Cell density estimation from settled 3‐ml samples using a Leica DMI 3000B inverted microscope according to Utermöhl (1958) and Lund et al. (1958), with counting in at least 2 transects. Cell biovolume were approximated based on simple geometrical forms: chytrid zoospores were considered spherical (diameter of 4 μm, corresponding to 33.5 μm³/ind), while Planktothrix cells cylindrical (diameter of 3 μm, cell length of 5 μm, corresponding to 58.9 μm³/cell). The biovolume of Planktothrix filaments was calculated by multiplying the cell biovolume with the average number of cells per filaments per ml, estimated based on 20 random filaments in each sample. Ingested diet was expressed as total FW biomass assuming a density of 1. Filament lengths of Planktothrix was compared between the diet treatments, and also between the diets and residuals at each feeding occasion (after 48 hr) based on 30 (in case of very low densities, at least 20) random filaments. We did not include sporangia maturation in the calculation of diet ingestion for two reasons: (1) due to the high prevalence rate sessile chytrid sporangia were mainly in the size of zoospores; (2) it was technically impossible to separate the sporangia and the chytrid‐infected cyanobacterium filaments to measure their biochemical profiles separately.

Ingested Planktothrix and chytrid zoospores were converted into carbon based on gravimetrically measured DW (KERN, ABT 220‐5DM) of the seston provided, the C% of diet sources and the respective % of FW biomass ingested. We also calculated ingestion efficiency for each diet source based on the ratio of the ingested and provided FW biomass (i.e. the dietary biomass % ingested).

Bacteria were also quantified in the diet sources and their residuals to estimate bacterial carbon ingestion using a Cytoflex AS18172 (Backman Coulter) flow cytometer. The formaldehyde‐fixed (4% final concentration) samples were filtered with a 2‐ μm mesh (CellTrics™) to remove Planktothrix filaments, sonicated for 20 min, 100× diluted and then stained with the fluorescent acidotropic probe LysoSensor™ Green (Invitrogen™, Fisher Scientific). The samples were run at 60 μl/min flow rate for at least 180 s with <500 events/s and analysed with the CytExpert (v.2.1) software by manual gating to detect bacterial cells with positive LysoSensor™ signal. Bacterial abundance was converted to bacteria carbon by assuming 20 fg C/cell (Agha et al., 2016; Ducklow & Carlson, 1992).

2.5. Elemental C, N, P, and stable C and N isotope analyses

The elemental C and N contents as well as the stable isotopes of carbon (δ ¹³C) and nitrogen (δ ¹⁵N) of diet sources were analysed during three occasions in triplicates (n = 9): before the experiment, immediately after the experiment, and 1 week after the end of the experiment. Previous data on the same cultures showed that the sole Planktothrix varied less in C%, N%, and lipid content compared with the chytrid zoospores (Rasconi et al., 2020). In a preliminary experiment, a large variation in C% and N% of the chytrid‐infected Planktothrix and chytrid zoospores (Appendix S1) was also observed. Here the sole Planktothrix diet was analysed only once in triplicates (n = 3) right before the experiment. Daphnia were analysed for C, N, and P in triplicates, both at the beginning (Daphnia neonates; time point zero, T₀) and at the end of the experiment (time point end, T_E).

To separate the chytrid zoospores from Planktothrix filaments, c. 100 ml culture was filtered five times through a 20‐μm mesh followed by a single filtration step through a 5‐μm mesh. Absence of Planktothrix in chytrid zoospore samples was evaluated using a Nikon Eclipse TS100 inverted microscope at 200× magnification.

Individual diet sources were collected on muffled and pre‐weighed GF/F Whatman™ filters (25 mm, 0.7 μm pore size), dried for 48 hr at 50°C (oven MMM Ecocell), weighed (KERN, ABT 220‐5DM) and folded in tin capsules (IVA, Analys. GmbH & Co. KG) with c. 0.3 mg DW seston. Daphnia were frozen at −80°C and subsequently freeze‐dried for 48 hr (VirTis benchtopK, VWR), weighed, and folded in tin capsules c. 0.3 mg DW. C, N, and bulk carbon (δ ¹³C) and nitrogen (δ ¹⁵N) stable isotopes were measured using a Thermo Fisher Scientific Flash 2000 Elemental Analyser, linked to a Delta V Advantage Mass Spectrometer. Δ ¹³C values were referenced to the Vienna PeeDee Belemnite (¹³C:¹²C = 0.01118), using USGS standards, applying the formula:

$${\delta }^{13}{C}_{\mathit{FA}}=\left(\frac{{}^{13}C{/}^{12}{C}_{\mathit{\text{Sample}}}}{{}^{13}C{/}^{12}{C}_{\mathit{\text{VPDB}}}}-1\right)\times 1000$$

To rule out a potential dietary effect of P over PUFA in Daphnia, we quantified POP in Daphnia at T₀ and T_E based on the ascorbic acid colorimetric method following persulfate digestion (American Public Health Association, 1998). P was not measured in diet sources during the experiment, but P was never limiting in culture conditions (i.e. C:P < 50 in all diet sources, independent measure of the experiment, data not shown). Elemental ratios expressed as molar ratios were C:N for diet sources, and C:N, C:P and N:P for Daphnia.

2.6. Fatty acid and compound‐specific carbon isotope analyses

The fatty acid composition and their compound‐specific carbon isotopes (δ ¹³C_FA) were analysed in the diet treatments in line with the elemental composition. Individual diet sources (c. 400 ml of culture, c. 1 mg seston; DW) were collected on muffled and pre‐weighed GF/F Whatman™ filters (47 mm, 0.7 μm pore size). Daphnia were analysed for FA and δ ¹³C_FA profiles at the beginning (T₀) and at the end (T_E) of the experiment in triplicates. Daphnia were freeze‐dried (see above) and c. 1 mg DW was transferred into tin caps.

Lipids were extracted from the freeze‐dried, homogenised samples using chloroform–methanol mix (2:1), following the protocol described in Heissenberger et al. (2010). FA were derivatised to methyl esters by incubation with 1% H₂SO₄ in methanol at 50°C for 16 hr. FA methyl esters (FAMEs) were dried under N₂ and dissolved in hexane. For quantification via flame ionisation, FAMEs were separated using a GC (Trace™ 1,310 Thermo Specific, Italy) equipped with a Supelco™ SP‐2560 column (100 m × 0.25 mm × 0.2 μm). ¹³C isotope analysis was performed using a Thermo Trace 1,310 GC (ThermoFisher Scientific), connected via a ConFlo IV (Thermo Co.) to an isotope ratio mass spectrometer (DELTA V Advantage, Thermo Co.). FAMEs were separated using a VF‐WAXms 60‐m column, 0.25 mm ID, film thickness 0.25 μm following oxidation to CO₂ in a combustion reactor, filled with Ni, Pt, and Cu wires, at a temperature of 1,000°C. The temperature started at 80°C, which was kept for 2 min, after which the temperature was raised by 30°C/min to 175°C, by 5°C/min to 200°C and finally by 2.4°C/min to 250°C, which was maintained for 30 min. Samples were run against certified Me‐C20:0 standards (USGS70: δ¹³C = −30.53‰, USGS71: δ¹³C = −10.5‰, and USGS72: δ¹³C = −1.54‰), which were used for drift and linear correction. All peaks were validated and corrected manually if necessary.

2.7. Carbon transfer efficiency

Carbon transfer efficiency was calculated as the ratio of carbon accrual by Daphnia (i.e., the total weight gained in carbon) to the total ingested carbon (Müller‐Navarra et al., 2000), and expressed as %: , where DDWC is the average DW of Daphnia expressed in carbon in each replicate at the end (T_E) and start of the experiment (T₀), FDWC _TOT is the total DW of ingested diet in carbon. Total ingested carbon was calculated from ingestion rates, standardised by the number of surviving Daphnia individuals at each feeding occasion (i.e. accounting for the change in abundances per replicate). Compound‐specific ¹³ C PUFA pathways. We quantified each δ ¹³C PUFA value to follow dietary PUFA sources for Daphnia by calculating the difference in δ ¹³C of each FA between the consumer (δ ¹³C_C) and its respective diet sources (δ ¹³C_D), i.e. Δ¹³C_FA, following Chiapella et al. (2021): Δ¹³C_FA = δ¹³C_C − δ¹³C_D. In a similar way, we calculated Δ¹³C_FA differences within Daphnia between LIN ➔ ARA, ALA ➔ SDA, ALA ➔ EPA, and SDA ➔ EPA conversion pathways.

2.8. Data analyses

Treatment‐specific differences in Daphnia growth rates, egg production rates, survival, and carbon transfer efficiency were tested with one‐way ANOVA, or Wilcoxon in the case of unequal variance. Ingested diet among sources, as well as the C:N, C:P and N:P ratios in Daphnia were compared with two‐way ANOVA with multiple comparisons of means (function glht using Tukey's contrasts in the multcomp R package). The statistically significant value was set at p < 0.05.

To visualise ingestion efficiency of diet sources by Daphnia over time, linear [LM] and non‐linear (generalised additive models [GAMs] with smooth splines, Wood, 2011, 2017) regressions were fitted to the time trends. The best model was selected according to the Akaike information criterion. Pearson's product–moment correlation analysis was also performed to test how the number of eggs produced by Daphnia was related to body length (size) at the end of the experiment.

Individual FA, total FAME, and PUFA among diet sources and Daphnia were analysed by ANOVA, reporting Tukey's HSD. The statistically significant value was set at p < 0.05. Fatty acid data were based on mass fractions (μg FA/mg DW) and log (x + 1) transformed to improve normality.

A redundancy analysis ordination was applied to relate the FA profiles of Daphnia to the FA profile of diet sources in a quantitative way, i.e. using diet FA profiles as independent constraining variables. To this end, we weighted the FA matrix of Daphnia by their DW gained (DW_TE – DW_T0), and the FA matrix of diet sources by their respective total amount of DW ingested. The FA matrices were ln (x + 1) transformed, and significant dietary FA selected in an a priori way based on combined backward and forward selection (function ordistep in vegan; Oksanen et al., 2019). The final significance of models was tested by 999 Monte Carlo permutations for each term in full models (function ANOVA; by = terms). All data analyses and visualisations were performed in R (R Core Team, 2019).

3. RESULTS

3.1. Life history of Daphnia

The survival of Daphnia was significantly higher in the chytrid‐infected diet treatment (Figure 1a, Wilcoxon, W = 2.5, n = 5, p < 0.05). The somatic growth rate of Daphnia feeding on chytrid‐infected Planktothrix was also significantly higher compared with Daphnia feeding on the sole Planktothrix diet (Figure 1b, ANOVA, F _1,4.5 = 404.6, n = 5, p < 0.001). Daphnia produced eggs only in the chytrid‐infected Planktothrix diet treatment (Figure 1c, Wilcoxon, W = 0, n = 5, p < 0.01). The number of eggs produced by Daphnia in the chytrid‐infected Planktothrix diet treatment correlated moderately, but significantly with body lengths (Pearson, r[104] = 0.43, p < 0.001).

FIGURE 1.

(a) Survival of Daphnia (in %) during the experiment; (b) somatic growth rates of Daphnia based on dry weight; (c) egg production rates of Daphnia. D_CY: Daphnia feeding on the cyanobacterium (Planktothrix), D_CPZ: Daphnia feeding on the chytrid‐infected cyanobacterium (Planktothrix, chytrid parasites and chytrid zoospores). Letters indicate the level of significance between diet treatments based on ANOVA (n = 5, at p < 0.05).

3.2. Filament length of Planktothrix

The length of Planktothrix did not differ significantly between uninfected (range: 30–1,000 μm, median: 530 μm) and infected (range: 30–6,000 μm, median: 460 μm) filaments between the supplied diet treatments (Wilcoxon, W = 11,158, p > 0.05). The length of Planktothrix supplied as diet did not vary over time (GAM, $R_{\mathit{Adj}}^2$ = −0.0008, p > 0.05, Appendix S1), while it significantly decreased with time in its residual (GAM, $R_{\mathit{Adj}}^2$ = 0.12, p < 0.001). The length of chytrid‐infected Planktothrix supplied as diet decreased slightly over time (GAM, $R_{\mathit{Adj}}^2$ = 0.05, p < 0.05; Appendix S1), while in the residual, it decreased considerably (GAM, $R_{\mathit{Adj}}^2$ = 0.26, p < 0.001). The residual of the chytrid‐infected Planktothrix diet (range: 10–1,300 μm, median: 400 μm) was significantly shorter compared with the residual of the uninfected Planktothrix diet (range: 15–10,000 μm, median: 200 μm; Wilcoxon, W = 180,637, p < 0.001).

The prevalence of chytrids in the chytrid‐infected Planktothrix diet stayed >50% during the experiment, and it decreased linearly with time in the residual of diet collected after 48 hr (Appendix S1). The difference in the prevalence of chytrids between the diet provided and its residual increased over time, i.e. a decreasing negative trend in ∆ (LM, $R_{\mathit{Adj}}^2$ = 0.715; p < 0.001, Appendix S1).

3.3. Ingestion rates

The ingestion rate of FW diets per Daphnia differed significantly among diet sources (Figure 2a). Daphnia ingested c. 2× more FW biomass of the chytrid‐infected Planktothrix (25 ± 12 μg ind⁻¹ day⁻¹) than of the uninfected Planktothrix (14 ± 7 μg ind⁻¹ day⁻¹), and comparatively, much less biomass of chytrid zoospores compared with of the chytrid‐infected and non‐infected Planktothrix (0.2 ± 0.3 μg ind⁻¹ day⁻¹). Daphnia ingested 6.4 ± 2.8 μg C ind⁻¹ day⁻¹ of chytrid‐infected Planktothrix (median: 7.3 μg C ind⁻¹ day⁻¹), 4.4 ± 2.2 μg C ind⁻¹ day⁻¹ of Planktothrix only (median: 3.8 μg C ind⁻¹ day⁻¹), and 0.07 ± 0.004 μg C ind⁻¹ day⁻¹ of chytrid zoospores (median: 0.02 μg C ind⁻¹ day⁻¹), with significant differences among diet sources (Figure 2b, p _AP–A < 0.05, p _A–Z < 0.001, p _AP–Z < 0.001). Daphnia ingested 7.2*10⁵ ± 2.3*10⁵ bacteria ind⁻¹ day⁻¹ in the Planktothrix diet treatment, corresponding to 0.37 ± 0.12 μg C ind⁻¹ day⁻¹. Daphnia in the chytrid‐infected Planktothrix diet treatment ingested 1.5*10⁶ ± 6.7*10⁵ bacteria ind⁻¹ day⁻¹, corresponding to 0.69 ± 0.31 μg C ind⁻¹ day⁻¹.

FIGURE 2.

(a) Ingestion rates of diet sources expressed as fresh weight (FW): CY—Cyanobacterium (Planktothrix), CP—Chytrid‐infected cyanobacterium, Z—Chytrid zoospores; (b) ingestion rates of diet expressed as carbon; (c) ingestion efficiency (% of diet biomass ingested) by Daphnia for Planktothrix with fitted linear trend line (LM); (d) ingestion efficiency of the chytrid‐infected Planktothrix and chytrid zoospores by Daphnia with fitted non‐linear trend lines (generalised additive model). Letters indicate the level of significance among diet sources based on ANOVA, multiple comparison of means, Tukey (n = 18, p < 0.05).

The ingestion efficiency of Planktothrix increased significantly and linearly with time in the Planktothrix‐only treatment (Figure 2c, LM, $R_{\mathit{Adj}}^2$ = 0.5622, p < 0.001), while it increased exponentially, levelling off towards the end of the experiment in the chytrid‐infected Planktothrix treatment (Figure 2d, GAM, $R_{\mathit{Adj}}^2$ = 0.881, p < 0.001). The chytrid zoospore density supplied in chytrid‐infected diet ranged between 253 and 1,214 ind/ml (median: 678 ind/ml), which was grazed with varying efficiency. The ingestion efficiency of chytrid zoospores first dropped from c. 40% to no grazing (0%) for the period from day 5 to 9, then increased with time, where its maxima (c. 70%–80%) occurred at the end of the experiment (Figure 2d, GAM, $R_{\mathit{Adj}}^2$ = 0.929, p < 0.001).

3.4. Stoichiometry of diet sources and Daphnia

Chytrid zoospores had significantly higher C content than the chytrid‐infected Planktothrix, while C in chytrid‐infected and uninfected Planktothrix did not differ significantly (Appendix S1, ANOVA, multiple comparisons of means, Tukey, p _Z–AP <0.01, and p > 0.05 in all other cases). The N content of chytrid zoospores was significantly higher compared with the uninfected (p _Z–A < 0.01) and chytrid‐infected Planktothrix (p _Z–AP <0.001), while it did not differ significantly between chytrid‐infected and uninfected Planktothrix (ANOVA, multiple comparisons of means, p _A–AP >0.05, in all other cases). The C:N ratio among diet sources differed only significantly between chytrid‐infected Planktothrix and chytrid zoospores (ANOVA, multiple comparisons of means, Tukey, p _Z–AP <0.05).

In Daphnia, the C, N, and P contents did not differ significantly among Daphnia at T₀ and T_E, nor at T_E between diet treatments (Appendix S1, multiple comparisons of means, Tukey, p > 0.05, in all cases). The C:N ratio did not differ significantly between Daphnia feeding on Planktothrix and chytrid‐infected Planktothrix diets (ANOVA, Tukey, p > 0.05), while it was significantly lower in Daphnia feeding on both experimental diets compared to Daphnia neonates (ANOVA, Tukey, p _N–A < 0.01, p _N–APZ <0.05). The C:P ratio did not differ significantly among Daphnia neonates or adults with any treatment (ANOVA, Tukey, p > 0.05, in all cases). The N:P ratio was significantly higher in Daphnia feeding on the Planktothrix diet compared to Daphnia neonates (ANOVA, Tukey, p _N–A < 0.05, in all cases), while all the other pairwise comparisons were non‐significant (ANOVA, Tukey, p > 0.05, in all cases).

3.5. Bulk δ ¹³C and δ ¹⁵ N values of diet sources and Daphnia

The δ ¹³C values differed only significantly between Planktothrix and chytrid zoospores, with zoospores having significantly lower values (Appendix S1, Wilcoxon, W = 0, p < 0.05), and between Daphnia neonates and chytrid zoospores, with zoospores having significantly lower values (Wilcoxon, W = 0, p < 0.05). The δ ¹⁵N did not differ significantly among diet sources or among diet sources and Daphnia consumers (Wilcoxon, p > 0.05 in all cases). The SD of both δ ¹³C and δ ¹⁵N values were high, especially in the chytrid‐infected diet, and in Daphnia feeding on the chytrid‐infected diet.

3.6. Fatty acid contents of diet sources and Daphnia

Total lipids in Daphnia, both at the beginning and end of the experiment, were significantly higher than in diet sources (ANOVA, n = 30, F _1,5 = 37, p < 0.001). Total PUFA content was also highest in Daphnia neonates (107 ± 8 μg/mg, Tukey's HSD; p ≤ 0.01), higher than in Daphnia feeding on chytrid‐infected Planktothrix (17 ± 3 μg/mg) and Planktothrix alone (16 ± 0.5 μg/mg).

Dietary PUFA content was identical between Planktothrix and chytrid‐infected Planktothrix (11 ± 0.7 μg/mg and 11 ± 3 μg/mg, respectively), and significantly lower in chytrid zoospores (Tukey's HSD; p < 0.01). Diet sources were identical in terms of short‐chain PUFA content (Figure 3a), except chytrid zoospores having the smallest content of ALA (Tukey's HSD; p < 0.001). Among dietary LC‐PUFA, the SDA content was the highest in chytrid zoospores (0.4 ± 0.16 μg/mg), followed by chytrid‐infected Planktothrix (0.1 ± 0.03 μg/mg), while it was absent in Planktothrix. Diet sources did not contain LC‐PUFA, such as ARA, EPA, or DHA.

FIGURE 3.

(a) Polyunsaturated fatty acids (PUFA) content of diet sources. CY: Cyanobacterium (Planktothrix), CP: Cyanobacterium‐chytrid parasite, Z: Chytrid zoospores; (b) PUFA content of Daphnia at the beginning of the experiment (D_N: Daphnia neonates) and at the end of the experiment for only Planktothrix diet (D_CY) and for chytrid‐infected Planktothrix diet (D_CPZ) including zoospores. Letters denote the level of significance among diet sources, Daphnia neonates, and Daphnia in the different diet treatments based on ANOVA, multiple comparison of means, Tukey (p < 0.05).

Short‐chain PUFA in Daphnia decreased significantly between neonates and adults at the end of the experiment, while LC‐PUFA increased (Figure 3b). Daphnia feeding on chytrid‐infected Planktothrix had significantly higher SDA (0.9 ± 0.2 μg/mg) compared with Daphnia feeding on uninfected Planktothrix (0.2 ± 0.03 μg/mg, Tukey's HSD; p < 0.05). Daphnia adults at the end of the experiment contained ARA, EPA, and DHA. ARA was significantly higher in Daphnia feeding on chytrid‐infected Planktothrix (1.4 ± 0.1 μg/mg) compared to Daphnia feeding on Planktothrix (1 ± 0.04 μg/mg). The EPA content was significantly higher in Daphnia adults irrespective of diet treatments compared with Daphnia neonates (Tukey's HSD; p < 0.001). The DHA content was significantly higher in Daphnia feeding on the sole Planktothrix diet (Tukey's HSD; p < 0.001) compared with Daphnia feeding on the chytrid‐infected diet.

The total bacterial fatty acid content differed significantly among diet sources (ANOVA, F _1,2 = 23.91, p < 0.001). The chytrid‐infected Planktothrix diet contained >2× more bacterial fatty acids (10.2 ± 2.4 μg/mg; measured in the chytrid zoospore fraction) than the uninfected Planktothrix diet (3.9 ± 1.5 μg/mg). The total bacterial fatty acid content in Daphnia did not differ significantly between diet treatments (ANOVA, multiple comparisons of means, Tukey, p > 0.05), and decreased significantly in Daphnia feeding on both diet treatments (Planktothrix diet: 5.2 ± 0.2 μg/mg, chytrid‐infected Planktothrix diet: 7.3 ± 1.8 μg/mg) compared with Daphnia neonates (19.3 ± 1.3 μg/mg; ANOVA, multiple comparisons of means, Tukey, p < 0.001, in both cases).

3.7. Carbon transfer efficiency related to PUFA

The carbon transfer efficiency was c. 4× higher in Daphnia feeding on chytrid‐infected Planktothrix (19.2 ± 2.9%) compared with Daphnia feeding on the sole Planktothrix diet (4.9 ± 0.4%, Figure 4a, ANOVA, F _1,4.1 = 119, n = 5, p < 0.001).

FIGURE 4.

(a) Carbon transfer efficiency to Daphnia based on the ratio of carbon accrual and ingested carbon from diet sources (D_CY: Daphnia feeding on the cyanobacterium (Planktothrix), D_CPZ: Daphnia feeding on chytrid‐infected Planktothrix, n = 5). Letters indicate the level of significance between Daphnia in the different diet treatments (ANOVA; p < 0.05); (b) redundancy analysis (RDA) ordination predicting the FA profile of Daphnia (weighted by carbon accrual) from the FA profiles of diet sources (weighted by their respective carbon weight digested). Full model: p < 0.05; axes: p _RDA1 < 0.05, p _RDA2: n.s.

The fatty acid composition of diet sources, weighted by their respective DW ingested, explained 99.1% variance in the FA profile of Daphnia, weighted by their DW gained (Figure 4b, redundancy analysis, p[ANOVA] < 0.05). Two dietary FA affected the FA profile of Daphnia significantly: SDA and 20:3 n‐3, both originating from chytrid zoospores (p[adonis] < 0.01).

3.8. Compound specific carbon isotopes of PUFA in Daphnia

The δ ¹³C_LIN values differed significantly among diets and Daphnia (Figure 5a). Among the diets, LIN was isotopically the most enriched in Planktothrix, significantly lighter in the chytrid‐infected Planktothrix (p < 0.001), and significantly heavier again in chytrid zoospores (p < 0.05, ANOVA, multiple comparisons of means, Tukey). The δ ¹³C_LIN values did not differ significantly in Daphnia between the two diet treatments, while they were significantly heavier in Daphnia feeding on the chytrid‐infected diet compared with the diet itself (p < 0.01, ANOVA), but not compared with chytrid zoospores (p > 0.05, ANOVA). The δ ¹³C_ALA values did not differ significantly either among diet sources, or among diet sources and Daphnia from the diet treatments, or in Daphnia between the two diet treatments (p > 0.05 in all cases, ANOVA, Figure 5b). The δ¹³C_SDA values were significantly lighter in chytrid zoospores (p < 0.01, ANOVA), in the chytrid‐infected Planktothrix (p < 0.05, ANOVA), and in Daphnia feeding on the chytrid‐infected Planktothrix (p < 0.01, ANOVA) compared with Daphnia neonates (Figure 5c). The δ ¹³C_ARA values did not differ significantly in Daphnia among diet treatments, but δ ¹³C_EPA did differ significantly with higher values in Daphnia feeding on the chytrid‐infected diet (ANOVA, multiple comparisons of means, Tukey, p < 0.01, Figure 5d,e).

FIGURE 5.

Mean ± SD of the δ ¹³C signal in polyunsaturated fatty acids (PUFA) in the CY—Cyanobacterium (Planktothrix), in the chytrid‐infected Planktothrix (CP—Cyanobacterium‐chytrid parasite), chytrid zoospores (Z), and in Daphnia neonates (D_N), Daphnia feeding on cyanobacterium (D_CY) and the on the chytrid‐infected cyanobacterium (D_CPZ); (a) the short‐chain ω‐6 PUFA linoleic acid (LIN); (b) the short‐chain ω‐3 PUFA alpha‐linoleic acid (ALA); (c) the long‐chain ω‐3 PUFA stearidonic acid (SDA); (d) the long‐chain ω‐6 PUFA arachidonic acid (ARA); (e) the long‐chain ω‐3 PUFA eicosapentaenoic acid (EPA). Letters indicate the level of significance among diet sources, Daphnia neonates, and Daphnia in the different diet treatments based on ANOVA, multiple comparison of means, Tukey (p < 0.05). Dotted vertical lines separate the fatty acid profiles of diets and Daphnia.

The Δ¹³C discrimination factor of individual PUFA indicated that Daphnia feeding on Planktothrix were significantly depleted in δ¹³C_LIN (−7.8‰), while slightly enriched in δ¹³C_ALA (+0.2‰). Daphnia feeding on the chytrid‐infected Planktothrix were enriched in both δ¹³C_LIN (+6.9‰) and δ¹³C_ALA (+2.8‰)_, although the difference was only significant for LIN. Comparing Daphnia feeding on chytrid zoospores, the consumer was slightly enriched in all δ¹³C_LIN (+1.2‰), δ¹³C_ALA (+0.8‰) and δ¹³C_SDA (+1.2‰) from the zoospores, but the differences were not significant.

Since SDA was missing in the Planktothrix diet, as well as ARA and EPA in Daphnia neonates, we calculated the Δ¹³C discrimination factor to compare δ ¹³C of SDA, ARA, AND EPA with δ ¹³C of their precursors: LIN, ALA, and SDA (Table 1). Δ¹³C_ARA indicated slightly depleted δ ¹³C in the consumer compared with its precursor LIN, while the difference was not significant for either diet treatments. Consumers’ SDA was enriched with δ ¹³C compared to δ ¹³C_ALA in both diet treatments, while it was only significant in Daphnia feeding on Planktothrix. Daphnia feeding on the Planktothrix diet was largely depleted in δ ¹³C_EPA compared to δ ¹³C_SDA, while compared to both δ ¹³C_ALA and δ ¹³C_SDA in the chytrid‐infected diet treatment.

TABLE 1.

Discrimination factors (Δ¹³C) between arachidonic acid (ARA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) and their precursors (linoleic acid [LIN], α‐linolenic acid [ALA], SDA, respectively) within Daphnia in each diet treatment.

PUFA in Daphnia	Δ13CARA	Δ13CSDA	Δ13CEPA
Planktothrix diet
LIN	−1.5 ‰ (n.s.)	–	–
ALA	–	+ 6.3 ‰ (*)	+ 0.7 ‰ (n.s.)
SDA	–	–	−5.6 ‰ (n.s.)
Chytrid‐infected Planktothrix diet
LIN	−1.0 ‰ (n.s.)	–	–
ALA	–	+ 2.9 ‰ (n.s.)	−2.7 ‰ (n.s.)
SDA	–	–	−3.8 ‰ (n.s.)

Note: Positive numbers indicate δ ¹³C enrichment in polyunsaturated fatty acids relative to the precursor. The larger the number, the larger the isotopic fractionation. The level of significance is based on ANOVA, multiple comparisons of means, Tukey. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, significant data are in bold.

4. DISCUSSION

The results supported our hypothesis that chytrids, as an intermediate‐level functional group, would enhance the fitness of the key herbivore zooplankton, Daphnia. Increased fitness in Daphnia could not result solely from the quantitative increase of ingested diet with chytrids. Rather, diet including chytrids unproportionally enhanced carbon yield in Daphnia, indicating the importance of the biochemical composition of ingested carbon. The increased availability of required PUFA due to the chytrid‐mediated carbon upgrading (Gerphagnon et al., 2019; Kagami et al., 2007; Rasconi et al., 2020; Taube et al., 2019) thus extends to Daphnia, underlying the enhanced fitness of the consumer.

4.1. Quantitative trophic transfer

Chytrids as an intermediate‐level functional group enhanced the dietary energy acquisition of Daphnia as evidenced by the recorded twofold increase in diet ingestion rate of the consumer. The chytrid infection itself, however, did not shorten the length of Planktothrix filaments (i.e., data on chytrid‐infected Planktothrix culture—diet), as has been argued and reported (Agha et al., 2016; Gerphagnon et al., 2013), which mechanistic fragmentation (Gerphagnon et al., 2013) would enhance cyanobacteria ingestion and so herbivory (Frenken et al., 2020). Rather, our chytrid‐infected Planktothrix culture contained both long and short filaments in the diet supplied, and the fragmentation of long filaments seemed to occur after the mechanical interference with Daphnia (i.e., grazing effect observed in the residual of diet). Enhanced grazing with chytrid infection could reflect either the increased edibility of shorter filaments or the improved physiological status of consumers (Frenken et al., 2020). The disproportional increase in carbon yield relative to ingested carbon by Daphnia suggests that chytrids support Daphnia fitness. It may then result in a positive feedback loop between: (1) increased somatic growth of the consumer in response to enhanced ingestion of the higher quality diet; and (2) the better performing Daphnia being able to graze more efficiently and so further ingest the diet of higher quality. Increased diet ingestion, therefore, also resulted from enhanced Daphnia fitness when feeding on the chytrid‐infected diet. The existence of a positive feedback loop is supported by:

1. the enhanced efficiency in diet ingestion with chytrids by the consumer over time (i.e. exponential vs. linear increase for chytrid‐infected and uninfected Planktothrix, respectively). Whether and how far the enhanced grazing efficiency could also result from a suppressed physiological status of the host with chytrid infection, or it was mainly related to the increased fitness of Daphnia, remains to be explored.

2. more efficient breaking‐up of Planktothrix by the Daphnia over time (i.e., significantly shorter filaments in the residual of the chytrid‐infected diet).

3. the increasing efficiency of Daphnia to control the chytrid infection with time (i.e., increasing efficiency to reduce chytrid prevalence). How proportionally this reduction resulted from the breaking‐up of the filaments, i.e. grazing of the chytrid‐infected end of the filaments, compared with the effect of increasing grazing rate on the zoospores, also remains to be explored.

The reproduction of Daphnia in the chytrid‐infected diet treatment was also related to Daphnia size, where the chytrids' positive dietary effect may manifest in a hierarchic way, i.e., first enhanced survival, then enhanced growth, and finally successful reproduction. Such hierarchy during somatic Daphnia development might also be visible in Agha et al. (2016), where diet‐related treatment effects in Daphnia appeared mainly on reproduction, while survival and body size of adults were less affected. This might be related to the overall higher dietary C supplied via shorter dietary Planktothrix filaments in their experiment, and so higher dietary carbon provision. Chytrids ensuring Daphnia reproduction under a cyanobacterium bloom (Planktothrix) have already been suggested experimentally. Agha et al. (2016) showed that c. 2,000–2,500 chytrid zoospores per ml enhanced Daphnia fitness and reproduction. Our chytrid zoospore density was considerably lower (c. 600 ind/ml) but could already enhance Daphnia fitness substantially. This may also have direct relevance to observational data where natural chytrid zoospore density can reach c. 3,000 ind/ml (see Rasconi et al., 2012; Sime‐Ngando, 2012). Sustained cladoceran growth has been evidenced during cyanobacteria blooms (Davis et al., 2012; Moustaka‐Gouni et al., 2006), suggesting the existence and importance of alternative energy pathways. As trophic transfer efficiency between phytoplankton and zooplankton decreases with cyanobacteria dominance (McCauley & Kalff, 1981; Selmeczy et al., 2019) and also the dominance of other large‐sized colonial and filamentous algae (Kagami et al., 2007, 2014), our results also support the mycoloop hypothesis of parasitic chytrids providing the energetic needs of zooplankton for survival, growth, and reproduction.

4.2. Qualitative trophic transfer

Dietary phosphorus could have potentially driven the treatment‐specific differences in life history traits of Daphnia. Enhanced Daphnia fitness, however, may not be explained by the mere elemental composition of the diet sources since Daphnia did not differ significantly in their respective C:P, N:P, and C:N ratios between diet treatments. Furthermore, the C:P ratio of Daphnia remain well‐below the potentially limiting C:P c. 155 (Khattak et al., 2018), further emphasising that Daphnia were not P limited in any of the diet treatments. The N:P ratio of chytrids is expected to follow the ratio of the host, which affects zoospore production (Frenken et al., 2017). We did not measure dietary N:P, but lower N:P in Daphnia feeding on the chytrid‐infected diet might indicate higher P availability relative to N, yet no significant differences in N availability between diet treatments. The C:N ratios reported for diet sources (c. 5) vary little and are similar to those reported by Frenken et al. (2017). In line with Barranco et al. (2020), chytrids might have upgraded diet quality in terms of N, resulting in significantly lower C:N ratios in chytrid zoospores compared with the host. However, lowering C:N did not extend further to Daphnia, suggesting that Daphnia adjusted its C:N ratio irrespective of dietary supply. Organic matter leakage linked to chytrid infection and related higher bacterial abundance could also lead to lower C:N ratio and so diet quality increase for zooplankton (Barranco et al., 2020). Our results confirmed a higher bacterial abundance linked to chytrid‐infection (Agha et al., 2016; Klawonn et al., 2021; Senga et al., 2018). Ingested bacterial carbon, however, remained only a minor fraction of total dietary carbon. The fact that the Daphnia fatty acid profile did change irrespective of the diet treatments is in line with the observation that ingested bacterial carbon does not provide dietary energy for growth and certainly not for reproduction (Taipale et al., 2012). Using a similar Planktothrix‐chytrid‐Daphnia experimental setup, Agha et al. (2016) showed that bacterial diet reduced somatic growth, fecundity and offspring size compared with the sole Planktothrix and chytrid‐infected Planktothrix diets. Accordingly, functional carbon that affected Daphnia fitness may have been originated from the cyanobacteria and chytrids, while the diet response observed did not depend on bacteria. Furthermore, the significant C:N decrease in Daphnia compared with the neonates may suggest that dietary carbon rather than nitrogen availability was constrained, especially for Daphnia feeding on the sole Planktothrix diet (i.e. C:N < 5). The eventual C limitation of Daphnia therefore emphasises the paramount importance of the molecular composition of diet, and thus, the quality of the C transferred to zooplankton.

No or slight differences in bulk δ ¹³C and δ ¹⁵N among the host, chytrid‐infected host, and Daphnia have been reported before, suggesting a large variation in isotopic enrichment across host‐parasitic systems (Barranco et al., 2020, and references therein). Despite the high variation in our data, the results may highlight the limitations of bulk stable isotopes to estimate trophic positions and basal energy sources. As reported on a diatom–chytrid–Keratella system (Barranco et al., 2020), here we also showed that the host together with the chytrids tended to be at the highest trophic position, while the zooplankton at a lower level. Grey (2006) suggested that inversed δ ¹⁵N chains might occur if the consumers were not in isotopic equilibrium with the diet, and the ¹⁵ N value was taken up faster by the primary producer than by the subsequent consumer. Thus, temporal variations in uptake and assimilation also have to be taken into account. Alternatively, an inversed δ ¹⁵N chain can also indicate starvation of the consumer and reflect changes in the internal use of N (Barranco et al., 2020). Due to the low dietary carbon provided via the hardly palatable Planktothrix, energy limitation of Daphnia was evident, especially on the sole Planktothrix diet. Non‐significant differences in Daphnia bulk δ ¹³C may also suggest that diet sources overlapped isotopically, or multiple isotopic fractionations might have occurred across the tropic levels, being further constrained by differences in the palatability of diet sources.

The n‐3 PUFA EPA is key for Daphnia growth and reproduction (Becker & Boersma, 2003; Sikora et al., 2016), but was absent in the diet. When dietary PUFA compositions do not meet the physiological requirements of consumers, bioconversion of dietary precursors for target PUFA is necessary. Chytrid‐synthesised SDA (Gerphagnon et al., 2019; Rasconi et al., 2020), was selectively retained by Daphnia, leading to a significant treatment‐specific difference in SDA contents. We provide isotopic evidence that SDA was bioconverted and made available from ALA of Planktothrix (Gerphagnon et al., 2019; Rasconi et al., 2020) for Daphnia. The fact that Daphnia efficiently retained dietary SDA when feeding on the chytrid‐infected diet might point at its potential physiological importance for Daphnia. Interestingly, we did not find evidence that chytrids also performed upgrading to the ω‐6 PUFA ARA, suggesting the preference of chytrids for n‐3 PUFA upgrading, for reasons that require further studies. The lack of conversion from SDA to EPA may be due to the relatively short test time and/or that these chytrids are unable to convert SDA to longer‐chain n‐3 PUFA. Alternatively, we suggest that EPA was bioconverted from ALA by the Daphnia itself without any intermediate conversion support.

4.3. Compound specific ¹³C sheds light on carbon assimilation

The carbon limitation of Daphnia feeding on the sole Planktothrix diet and the higher carbon yield in Daphnia feeding on the chytrid‐infected diet demonstrate the paramount importance of the composition in dietary PUFA carbon. The higher carbon content assimilated with the chytrid‐infected diet evidences chytrids' positive dietary effect, also mediated by PUFA. Increased dietary PUFA provision as SDA, on top of the slightly more carbon ingested, may have a beneficial effect on Daphnia fitness. The selective retention of dietary SDA also coincided with an increased SDA:C ratio in Daphnia feeding on the chytrid‐infected diet (data not shown). The increased PUFA:C ratio indicates higher PUFA than C retention in Daphnia, while the dietary effects of chytrids in supporting the bioconversion to EPA by Daphnia still remains to be elucidated.

The bioconversion of ALA to EPA by Daphnia was supported by CSI data that suggested that the chytrid parasite also synthesised LIN, an essential PUFA known to be synthesised mainly by algae (Taipale et al., 2013). The isotopically significantly heavier LIN (and also ALA) in chytrid zoospores, as well as in Daphnia relative to the chytrid‐infected Planktothrix, further suggests the metabolic use of these PUFA by which isotopically heavier LIN and ALA were retained by Daphnia. The isotopically lighter δ ¹³C_SDA values also suggest SDA synthesis in the chytrid zoospore, which was subsequently selectively retained by Daphnia. While we did not find treatment‐specific differences in ARA retention of Daphnia, the isotopically lighter ARA compared to LIN also suggests the synthesis of these essential PUFA, irrespective of the two diet treatments. Similarly, the isotopically lighter EPA compared to ALA also suggests the endogenous EPA synthesis by Daphnia.

5. CONCLUSIONS

Our experiment provides isotopic evidence of trophic upgrading by chytrid fungal parasites, resulting in improved dietary PUFA provision and subsequently higher fitness of the herbivorous consumer Daphnia. This study demonstrates that carbon ingestion by Daphnia was only slightly enhanced with chytrid‐infected diet, but the carbon transfer efficiency increased disproportionally, suggesting that chytrid‐mediated trophic PUFA upgrading is crucial for trophic carbon transfer. The selective retention of the chytrid‐synthesised SDA together with the enhanced carbon yield suggests that dietary SDA may support the bioconversion to the LC‐PUFA EPA in Daphnia. Compound‐specific stable carbon isotope analysis outperformed the power of bulk δ ¹³C data in identifying the trophic pathways of key PUFA. LIN and ALA were also synthetised by the chytrid parasite, and directly assimilated by Daphnia. We further demonstrated the endogenous bioconversion of LIN to ARA and ALA to EPA, respectively, by the Daphnia. The quantitative role of chytrids in supporting the innate conversion of short chain to LC‐PUFA in Daphnia remains to be further elucidated, e.g. via ¹³C‐labelling of key functional PUFA. We note that in addition to PUFA, chytrids also produce sterols de novo (Gerphagnon et al., 2019; Kagami et al., 2007), mediating dietary sterol provision and thus positive dietary effects of chytrids on consumers. We conclude that chytrid‐mediated improved dietary PUFA extends to Daphnia, which enhances its performance when exposed to poor nutritional quality and hardly palatable cyanobacteria.

AUTHOR CONTRIBUTIONS

Conceptualisation: S.R., M.J.K., R.P. Developing methods: S.R., A.A., M.J.K., R.P. Conducting the research: A.A. Data analysis. A.A. Data interpretation: A.A., M.J.K., S.R., R.P., M.P. Preparation of figures and tables: A.A. Writing: A.A. wrote the original draft and then all authors edited and commented on the drafts.

FUNDING INFORMATION

This work was supported by the Austrian Science Fund (FWF Project P 30419‐B29).

Supporting information

Appendix S1

Abonyi, A. , Rasconi, S. , Ptacnik, R. , Pilecky, M. , & Kainz, M. J. (2023). Chytrids enhance Daphnia fitness by selectively retained chytrid‐synthesised stearidonic acid and conversion of short‐chain to long‐chain polyunsaturated fatty acids. Freshwater Biology, 68, 77–90. 10.1111/fwb.14010

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.