Chytrids alleviate the harmful effect of heat and cyanobacteria diet on Daphnia via PUFA-upgrading

Abstract

Chytrid fungal parasites increase herbivory and dietary access to essential molecules, such as polyunsaturated fatty acids (PUFA), at the phytoplankton–zooplankton interface. Warming enhances cyanobacteria blooms and decreases algae-derived PUFA for zooplankton. Whether chytrids could support zooplankton with PUFA under global warming scenarios remains unknown. We tested the combined effect of water temperature (ambient: 18°C, heat: +6°C) and the presence of chytrids with Daphnia magna as the consumer, and Planktothrix rubescens as the main diet. We hypothesized that chytrids would support Daphnia fitness with PUFA, irrespective of water temperature. Heating was detrimental to the fitness of Daphnia when feeding solely on the Planktothrix diet. Chytrid-infected Planktothrix diet alleviated the negative impact of heat and could support Daphnia survival, somatic growth and reproduction. Carbon stable isotopes of fatty acids highlighted a ~3x more efficient n-3 than n-6 PUFA conversion by Daphnia feeding on the chytrid-infected diet, irrespective of temperature. The chytrid diet significantly increased eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6) retention in Daphnia. The EPA retention remained unaffected, while ARA retention increased in response to heat. We conclude that chytrids support pelagic ecosystem functioning under cyanobacteria blooms and global warming via chytrids-conveyed PUFA toward higher trophic levels.

INTRODUCTION

Recent evidence suggests that chytrid fungal parasites perform vital functions at the phytoplankton–zooplankton interface. Chytrids enhance herbivory by fragmenting poorly edible algal/cyanobacteria hosts (Frenken et al., 2020). They also improve the dietary supply of polyunsaturated fatty acids (PUFA; Gerphagnon et al., 2019; Rasconi et al., 2020) and sterols (Kagami et al., 2007; Gerphagnon et al., 2019), as essential molecules, for zooplankton. Chytrids are ubiquitous, invading various phytoplankton hosts (Gleason et al., 2015) and producing edible (~2–5 μm) free-swimming zoospores for subsequent consumers, such as zooplankton. Chytrids thus provide an alternative dietary energy pathway between phytoplankton and zooplankton, called the “mycoloop” (Kagami et al., 2014). Chytrids upgrade dietary carbon from the algal/cyanobacteria hosts in terms of omega-3 PUFA and sterols (Gerphagnon et al., 2019; Rasconi et al., 2020), which positively affects the fitness of Daphnia as a subsequent consumer (Abonyi et al., 2023). Whether and how the mycoloop can support zooplankton with enhanced PUFA availability under global warming (IPCC, 2021) remains largely unexplored.

The efficient transfer of dietary energy and essential molecules across the phytoplankton–zooplankton trophic interface is crucial for consumers at higher trophic levels. The energy transfer can be constrained quantitatively and qualitatively. A quantitative limitation may occur in response to reduced primary production, e.g. during oligotrophication (Jeppesen et al., 2002; Jochimsen et al., 2013). Alternatively, increased primary production in response to eutrophication and global warming may result in the more frequent proliferation of harmful phytoplankton (Paerl and Huisman, 2008; O’Neil et al., 2012). Bloom-forming cyanobacteria are typically filamentous or colonial, which hinders zooplankton grazing and creates an energetic bottleneck between pelagic primary producers and herbivorous consumers (Havens, 2008). Qualitative limitation of energy transfer may also be linked to phytoplankton blooms. Cyanobacteria especially lack essential compounds for zooplankton, such as PUFA and sterols, so their proliferation truncates the trophic transfer of highly required dietary compounds.

Zooplankton cannot synthesize PUFA de novo (Demott and Müller-Navarr, 1997; Gulati and Demott, 1997). The omega-3 PUFA eicosapentaenoic acid (EPA, 20:5n-3) and the omega-6 PUFA arachidonic acid (ARA, 20:4n-6) are the two conditionally indispensable molecules for zooplankton growth and reproduction (see, e.g. Müller-Navarra et al., 2000; Sikora et al., 2016; Ilić et al., 2021). They can be obtained directly from the diet or converted from their respective precursors, linoleic acid (LIN, 18:2n-6) and alpha-linolenic acid (ALA, 18:3n-3), respectively (Pilecky et al., 2021). A key source of PUFA for herbivorous zooplankton in aquatic food webs is phytoplankton, which vary widely in PUFA composition (Taipale et al., 2013). Diatoms are rich in EPA; cryptophytes contain EPA and docosahexaenoic acid (DHA, 22:6n-3); thus, both groups are considered high-quality diets for zooplankton. Green algae contain mostly LIN and ALA and are referred to as intermediate diet quality. Cyanobacteria generally lack long-chain PUFA and sterols (but see the gamma-linoleic acid, GLA, 18:3n-6, content of Microcystis sp.; Strandberg et al., 2020) and are thus low-quality diets for zooplankton (Martin-Creuzburg et al., 2009).

Hardly palatable cyanobacteria of poor diet quality are deleterious for zooplankton (Bednarska et al., 2014), representing a worst-case scenario in the trophic transfer of energy and essential dietary molecules. Recent evidence suggests that chytrid fungal parasites can buffer this effect by enhancing trophic transfer from cyanobacteria to zooplankton during cyanobacteria blooms (Agha et al., 2016; Abonyi et al., 2023). In previous studies, the presence of chytrids enhanced the somatic growth and reproduction of cladocerans (Kagami et al., 2007; Agha et al., 2016), copepods (Kagami et al., 2011) and rotifers (Frenken et al., 2018) when growing on a poor quality diet. Mechanisms potentially underlying the positive dietary effects of chytrids are (1) enhanced herbivory by fragmenting otherwise inedible cyanobacteria (Agha et al., 2016; Frenken et al., 2020), and (2) increased nutritional quality in terms of PUFA (Gerphagnon et al., 2019; Taube et al., 2019; Rasconi et al., 2020). Chytrids can upgrade short-chain to long-chain PUFA from the host, i.e. convert the omega-3 PUFA ALA to stearidonic acid (SDA) and then further to long-chain PUFA, such as EPA (Rasconi et al., 2020). Recent evidence suggests that SDA synthesized by chytrids is selectively retained by Daphnia with a concomitant increase in the fitness of the consumer (Abonyi et al., 2023). How the mycoloop and the conveyed PUFA interact with heat remains a crucial issue, especially to understand better the combined effects of warming and increased occurrence of phytoplankton blooms.

Warming amplifies the deleterious effect of reduced diet intake on ectothermic organisms (Huey and Kingsolver, 2019). Hardly edible PUFA-deprived cyanobacteria blooms thus may help collapse the populations of herbivorous consumers, especially under water temperature increase. In light of ongoing global warming (IPCC, 2021) and the more frequent occurrence of cyanobacteria blooms in aquatic ecosystems, it is crucial to test the strength of the mycoloop in warmer conditions. If improved dietary PUFA provision via chytrids enhances Daphnia fitness on top of increased herbivory (Abonyi et al., 2023), it may also be able to provide insurance against increasing water temperature.

Here we ask whether and how chytrids can support Daphnia fitness under water temperature increase and stress induced by the detrimental interaction of heat and hardly available dietary carbon and PUFA. Masclaux et al. (2009) showed a trade-off in Daphnia growth rate along with diet quality and water temperature. If a high-quality diet was available ad libitum, water temperature increase enhanced Daphnia growth, while reduced diet quality or quantity alleviated the positive temperature effect (Masclaux et al., 2009). On the other hand, multiple observations showed that Daphnia growth rate decreased at water temperatures >20°C (Giebelhausen and Lampert 2001; Masclaux et al., 2009), but it remained an essentially optimal condition for growth under non-limiting diet conditions (Müller et al., 2018). Daphnia adjust FA content to the water temperature, decreasing PUFA toward higher temperatures (Zeis et al., 2019). Dietary EPA limitation was also shown to be more critical at lower than higher water temperatures (Sperfeld and Wacker, 2012). The fitness of Daphnia magna is already constrained at 18°C when feeding on poor diet quality and hardly edible Planktothrix rubescens (Abonyi et al., 2023). We thus performed a feeding experiment where D. magna was supplied with Planktothrix rubescens alone—a filamentous bloom-forming cyanobacterium—or with chytrid-infected Planktothrix. We combined the two alternative diets with two water temperature treatments, i.e. ambient (18°C) and heat (ambient +6°C). Applying a 24°C heat treatment enabled us to study the PUFA transfer via the mycoloop with water temperature increase without directly inducing Daphnia mortality by heat-mediated metabolic stress (Zeis et al., 2019; Im et al., 2020).

We hypothesized that a +6°C water temperature increase in combination with a filamentous cyanobacterium diet would be detrimental to Daphnia fitness, also mediated by PUFA-deprived conditions. We expected that diets including chytrids would support Daphnia fitness due to increased PUFA transfer even under increased water temperature. We examined survival, somatic growth and reproduction of Daphnia as well as PUFA accrual of Daphnia among the treatments. In a ¹³C-labeling approach, we also aimed at identifying the trophic carbon pathways via which physiologically functional PUFA were transferred to and converted by Daphnia.

MATERIAL AND METHODS

Experimental cultures

The filamentous cyanobacterium Planktothrix rubescens (strain NIVA-CYA97/1) was cultured and maintained alone, and together with its specific chytrid fungal parasite (strain Chy-Lys2009) on WC Medium (Guillard and Lorenzen, 1972). We maintained a high chytrid prevalence rate (>50%) in the chytrid-infected Planktothrix culture by 1/3 V/V% medium- and 1/3 V/V% dense uninfected Planktothrix culture-exchange once a week (Abonyi et al., 2023). We used the chytrid-infected Planktothrix culture as a diet without separating chytrid zoospores and Planktothrix filaments. Cell culture flasks (600 mL polystyrene with 0.22 μm hydrophobic PP vented cap; VWR International™, Radnor, PA, USA) were kept under non-axenic conditions at 21°C, applying 10.9 μmol m² s⁻¹ PAR and a 16:8 light:dark cycle in AquaLytic incubators (180 L, Liebherr, Germany). D. magna was grown in pre-filtered (0.7 μm GF/F) lake water from Lake Lunz diluted with 10 V/V % of ADaM medium (Klüttgen et al., 1994). Daphnia were fed with 50% Scenedesmus sp. and 50% Chlamydomonas sp. >1 mg C L⁻¹; both feeding cultures were also grown in WC medium.

¹³C-labeling of diet sources

Planktothrix and chytrid-infected Planktothrix diet cultures were labeled with ¹³C using NaH¹³CO₃ (98 atom%, Sigma-Aldrich, USA) in the WC medium. Before each feeding occasion, diet cultures were diluted (1:1 v/v) with either non-labeled or labeled WC medium using SIMAX 2000 mL glass bottles filled up completely to avoid air space under the caps and kept under the culturing conditions for 24 h. We did not manipulate water temperature for the diets, which would have modified their ¹³C values and made it thus impossible to compare Daphnia response among the treatments to diet versus heat. We expressed the ¹³C uptake efficiency based on all the pairwise differences in δ¹³C_FA between the labeled and the non-labeled replicates for each FA (i.e. Δ¹³C_FA = δ¹³C_FA-labeled-δ¹³C_non-labeled).

Experimental design

The two diet treatments were: (1) Planktothrix (“P”) and (2) chytrid-infected Planktothrix (“PC+”), containing also free-swimming chytrid zoospores (“Z”). Two water temperature treatments were applied: (1) keeping the experimental bottles at 18°C (“T-”; temperature set in the experimental climate room), or (2) in a 24 ± 1°C water bath (“T+”) using an aquarium heater (AniOne, 25 W, MultiFit Tiernahrung GmbH, Germany). The water temperature in the experimental bottles (Corning^® International, 1 L square polycarbonate storage flasks) varied by ±0.5°C during the entire experiment. The Daphnia were either fed with non-labeled or with ¹³C-labeled cultures. All treatments—applying a full factorial design—were performed in triplicates, resulting in 24 experimental units.

At the start of the experiment, bottles contained 30 Daphnia neonates that were <48 h old. The volume was constantly kept at 1 L, and the bottles were covered with a non-transparent plastic plate in a non-hermetic way. A 16:8 light:dark cycle with 1.4 μmol m² s⁻¹ PAR during the day-light phase was applied. We replaced the medium and provided new diets to Daphnia every other day, corresponding to 1 ± 0.01 mg C L⁻¹ in all treatments. First, the cultures’ carbon content was analyzed prior to the experiment (Abonyi et al., 2023). Subsequently, we approximated the carbon content based on dry weight per volume for each feeding occasion (i.e. adding different diet volumes to meet the 1 mg C L⁻¹). Daphnia were observed daily for survival and egg production. Individuals lying at the bottom of the bottles with no movement for 5 min were considered dead and removed. To reliably compare the PUFA content of Daphnia across treatments, ambient and heat treatments were stopped separately at the onset of the first successful hatching.

Life history of Daphnia

We quantified Daphnia survival as the % of surviving individuals. The somatic growth rate (GR) of the animals was calculated as

DW_end was the average individual dry weight in each experimental bottle at the end of the experiment,

DW_start was the average individual dry weight of Daphnia neonates at the start of the experiment (average of 3 × 20 individuals)

and d was the duration of the experiment in days. We quantified the number of eggs produced by Daphnia (ind⁻¹) and egg production rates calculated from the total number of eggs produced over time (day⁻¹).

Elemental C, N, P compositions of diet and Daphnia

The elemental C, N and P contents of diets and chytrid zoospores were analyzed in triplicates at the beginning of the experiment (Day 1), every second time of feeding (Day 4, Day 8, Day 12) and right after the experiment (n = 15). Daphnia were analyzed for C, N and P in triplicates at the start (T₀) and the end of the experiment (T_E). We separated chytrid zoospores from Planktothrix filaments by filtering ~100 mL culture five times through a 20 μm mesh, followed by a single filtration through a 5 μm mesh. The absence of Planktothrix in chytrid zoospore samples was evaluated using a Nikon Eclipse TS100 inverted microscope at 200× magnification. Diets were collected on muffled and pre-weighed GF/F Whatman™ filters, dried for 48 h, weighed and folded in tin capsules. Daphnia were frozen (−80°C), then freeze-dried for 48 h (VirTis benchtopK, VWR International), weighed and put in tin capsules. C and N were measured using an elemental analyzer (Flash 2000, Thermo Fisher Scientific International; Waltham, MA, USA), linked to a mass spectrometer (Delta V Advantage, Thermo Fisher Scientific International; for more details, see  Abonyi et al., 2023). Using the USGS standards, the δ¹³C values were referenced to Vienna PeeDee Belemite (¹³C:¹²C = 0.01118). P was measured as particulate organic phosphorus based on the ascorbic acid colourimetric method following persulfate digestion (APHA, 1998). Elemental C:N, C:P and N:P ratios were expressed as molar ratios.

Fatty acids and compound-specific carbon isotope analyses

The fatty acids (FA) and their compound-specific carbon isotopes (i.e. CSI data on δ¹³C_FA) were analyzed in the diets (i.e. Planktothrix, chytrid-infected Planktothrix excluding chytrid zoospores, chytrid zoospores) and Daphnia. Individual diet sources were collected on muffled and pre-weighed GF/F Whatman™ filters (47 mm, 0.7 μm pore size), stored at −80°C then freeze-dried (VirTis benchtopK, VWR International). Daphnia were also freeze-dried and weighted ~1 mg dry-weight into tin caps. Lipids were extracted from the freeze-dried samples using a mix of chloroform-methanol (2:1), following the protocol described in Heissenberger et al. (2010). Fatty acids were derivatized to methyl esters by incubation with 1% H₂SO₄ in methanol at 50°C for 16 h. Fatty acid methyl esters (FAME) were dried under N₂ and dissolved in hexane. For quantification via flame ionization, FAME were separated using a GC (Trace™ 1 310, Thermo Fisher Scientific International) equipped with a Supelco™ SP-2560 column (100 m × 0.25 mm × 0.2 μm). The analytical setup allowed us to separate cis and trans isomers and components with different double bond positions. We, however, did not report trans-FA as they were scarce, if at all present. ¹³C CSI analysis was performed using a GC (Trace™ 1 310, Thermo Fisher Scientific International), connected via a ConFlo IV (Thermo Fisher Scientific International) to an Isotope Ratio Mass Spectrometer (IRMS, DELTA V Advantage, Thermo Fisher Scientific International). 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 (for more details, see Abonyi et al., 2023).

Quantitative PUFA retention by Daphnia

We compared the retention of LIN, GLA, ARA, ALA, SDA and EPA by Daphnia among treatments in two complementary ways: first, by comparing FA mass fractions per unit biomass (i.e. μg FA mg⁻¹), and, second, based on the pairwise difference in δ¹³C_FA values between the labeled and the non-labeled replicates for each FA (i.e. Δ¹³C_FA = δ¹³C_FA-labeled-δ¹³C_non-labeled). The higher the value, the more efficient the PUFA retention, including dietary uptake and endogenous bioconversion sources. Furthermore, for comparing the efficiency of the endogenous conversion of short-chain (LIN, ALA) with long-chain PUFA (ARA, EPA) by Daphnia, we expressed the ratios of FA retentions between the pairs, i.e. Δ¹³C_ARA/Δ¹³C_LIN and Δ¹³C_EPA/Δ¹³C_ALA. The higher the value, the more efficient the endogenous FA conversion.

Statistical analyses

We visualized Daphnia survival (%) and egg production (eggs ind⁻¹) by fitting linear (LM) and non-linear (GAM; Wood 2011, 2017) regressions to time trends. We selected the best model based on Akaike’s information criterion (AIC). We tested for significant differences in fitness parameters (i.e. survival, somatic growth rates, eggs.ind⁻¹, egg production rate) and PUFA retention efficiencies among treatments using Kruskal–Wallis rank sum tests (Hollander and Wolfe, 1973). In case of significant differences among treatments, we ran a pairwise comparison using the Wilcoxon rank sum test (Bauer, 1972). We controlled P-values for multiple testing by applying the false discovery rate approach (Benjamini and Hochberg, 1995). We tested for significant differences in FA content between two groups (ambient and heat) using the Wilcoxon rank sum test. We compared heat versus diet effects by calculating Wilcoxon effect sizes with the wilcox_effsize() function in the rstatix R package (Kassambara, 2023). The FA data were ln(x + 1) transformed for data visualization and statistical analyses. The level of statistical significance was set at p < 0.05 in all cases. All data analyses and visualizations were performed in R with RStudio Version 1.1.383 (R Core Team, 2019).

RESULTS

Daphnia fitness

Daphnia feeding on the chytrid-infected diet under heat reproduced successfully on Day 11, i.e. the end of the heat treatment. Daphnia on the ambient water temperature reproduced on Day 15, i.e. the end of the ambient water temperature treatment. Daphnia feeding on the sole Planktothrix diet did not develop eggs during the entire experiment, and they started to die off from Day 2 (Fig. 1A,B). The decline in survival was non-linear under both the heat and ambient water temperatures treatments (GAM, p < 0.001, in both cases). The survival decline was more pronounced with heat, indicated by a significantly lower survival under heat already at Day 2 (pairwise Wilcoxon, p < 0.01; r > 0.8 for heat effect).

Fig. 1.

(A) Daphnia survival (% from 30 individuals per bottle) at ambient water temperature (18°C); and (B) Daphnia survival in the heat water temperature treatment (24°C); (C) the number of eggs produced per Daphnia at 18°C; and (D) the number of eggs produced per Daphnia at 24°C. The empty circles are Daphnia feeding on Planktothrix; the full circles represent Daphnia feeding on chytrid-infected Planktothrix. The vertical dashed lines indicate Day 11, the onset of the first successful reproduction of Daphnia feeding on the chytrid-infected diet under heat (i.e. when the heat treatment was stopped). Trend lines are based on non-linear regression analyses (GAM), except a linear regression (LM) for the chytrid-infected diet on (A).

Survival (%) differed significantly among the treatments at Day 11 (S1A, Kruskal–Wallis, chi-squared = 20.405, df = 3, p < 0.001). Heat significantly reduced survival only in Daphnia feeding on the sole Planktothrix diet (ambient: 58.9 ± 10.9% (mean ± SD), heat: 13.3 ± 10.1%; pairwise Wilcoxon, p < 0.01), while survival of Daphnia feeding on chytrid-infected diet remained unaffected by heat (ambient: 97.8 ± 1.7%, heat: 96.7 ± 2.1%; pairwise Wilcoxon, p > 0.05). The heat effect on Daphnia survival was large if animals were feeding on the sole Planktothrix diet (r > 0.8) and small in the case of the chytrid-infected diet (r < 0.3).

Somatic growth rates differed significantly among the treatments (S1B, Kruskal–Wallis, chi-squared = 17.561, df = 3, p < 0.001). It remained unaffected by heat (pairwise Wilcoxon, p > 0.05), but it was significantly enhanced by chytrid-infected diet (p < 0.01). Effect sizes indicated strong diet effects (r > 0.8) but small heat effects (r < 0.3) on Daphnia growth rates.

Daphnia feeding on the chytrid-infected diet started to produce eggs on Day 7 under heat, while at the ambient water temperature on Day 10 (Fig. 1C,D). Heat significantly reduced the number of eggs produced per Daphnia when it was compared at the day of the first successful reproduction (Day 11 under heat: 0.33 ± 0.16 eggs.ind-1 (mean ± SD), Day 15 under the ambient condition: 0.89 ± 0.23 eggs.ind⁻¹, Wilcoxon, W = 36, p < 0.01). Egg production rate differed significantly among the treatments (S1C, Kruskal–Wallis, chi-squared = 20.341, df = 3, p < 0.001). It significantly increased in Daphnia feeding on chytrid-infected Planktothrix (pairwise Wilcoxon, p < 0.01), which positive effect was significantly reduced by heat (pairwise Wilcoxon, p < 0.01). The diet effect on the Daphnia egg production rate was large (r > 0.8), as well as the heat effect reducing the egg production rates of animals feeding on the chytrid-infected diet (r = 0.693).

Individual PUFA transfer to Daphnia

Due to the high mortality of Daphnia feeding on the sole Planktothrix diet, it was only possible to analyze the PUFA content in Daphnia feeding on the chytrid-infected diet. The individual PUFA contents in Daphnia feeding on chytrid-infected Planktothrix did not differ significantly between the ambient and heat treatments (Wilcoxon rank sum tests, p > 0.05 in all cases, Fig. 2). ALA had the highest mass fractions of the identified short-chain PUFA in Daphnia (ambient: 11.2 ± 8.7 μg mg⁻¹ (mean ± SD), heat: 6.0 ± 0.6 μg mg⁻¹) followed by LIN (ambient: 2.0 ± 1.5 μg mg⁻¹, heat: 1.5 ± 0.1 μg mg⁻¹) and SDA (ambient: 1.2 ± 0.9 μg mg⁻¹, heat: 0.7 ± 0.1 μg mg⁻¹) and GLA (ambient: 0.1 ± 0.08 μg mg⁻¹, heat: 0.1 ± 0.02 μg mg⁻¹). EPA had the highest mass fractions of the identified long-chain PUFA in Daphnia (ambient: 0.9 ± 0.5 μg mg⁻¹, heat: 1.3 ± 0.2 μg mg⁻¹), followed by ARA (ambient: 0.5 ± 0.3 μg mg⁻¹, heat: 0.8 ± 0.1 μg mg⁻¹) and DHA (ambient: 0.1 ± 0.07 μg mg⁻¹, heat: 0.05 ± 0.01 μg mg⁻¹).

Fig. 2.

PUFA content of Daphnia feeding on the chytrid-infected cyanobacterium Planktothrix and chytrid zoospores (C+) at the time of the first successful reproduction. T- stands for ambient water temperature, while T+ stands for heat. (A) linoleic acid (18:2n-6, LIN); (B) gamma-linoleic acid (18:3n-6, GLA); (C) arachidonic acid (20:4n-6, ARA); (D) alpha-linolenic acid (18:3n-3, ALA); (E) stearidonic acid (18:4n-3, SDA); (F) eicosapentaenoic acid (20:5n-3, EPA). Different ranges of the y-axes are color-coded. The letters indicate significant differences between water temperature treatments based on Wilcoxon rank sum tests (n = 6 in all cases; different letters indicate significant differences).

¹³C-labeling of dietary PUFA and their retention by Daphnia based on CSI analysis

The ¹³C uptake via LIN, ALA and SDA differed significantly among diet sources (Kruskal–Wallis rank sum tests, chi-squared_LIN = 415.27 (df = 2), chi-squared_ALA =  277.47 (df = 2), chi-squared_SDA = 295.48 (df = 1), p < 0.001 in all cases). ¹³C uptake via LIN was the highest in Planktothrix (438 ± 229‰), significantly lower in chytrid-infected Planktothrix (284 ± 104‰), and again, significantly lower in chytrid zoospores (34 ± 26‰)(Fig. 3A). ¹³C labeling of ALA was the highest in chytrid-infected Planktothrix (139 ± 57‰), significantly lower in the cyanobacterium diet (121 ± 60‰), and chytrid zoospores (43 ± 33‰)(Fig. 3B). SDA was absent in Planktothrix but present in the chytrid-infected cyanobacterium and the chytrid zoospores (Fig. 3C). ¹³C uptake via SDA was significantly higher in chytrid-infected Planktothrix (56 ± 39‰) than in the chytrid zoospores (6 ± 5‰). Diet sources did not contain ARA or APA (see also S3A).

Fig. 3.

¹³C uptake via key PUFA in the diet sources based on all pairwise differences between labeled and non-labeled FA (n = 225 for each pair) (A) linoleic acid (18:2n-6, LIN); (B) alpha-linolenic acid (18:3n-3, ALA); (C) stearidonic acid (18:4n-3, SDA). The letters stand for significant differences in ¹³C uptake via key PUFA among diet sources based on Pairwise Wilcoxon rank sum tests.

Due to the high mortality of Daphnia feeding only on Planktothrix under heat, Daphnia from this treatment could not be analyzed. Among the other treatments, ¹³C retention in Daphnia significantly differed via LIN, ALA, SDA, ARA and EPA (Fig. 4; Kruskal–Wallis rank sum tests, p_LIN < 0.01, p_ALA < 0.001, p_SDA < 0.001, p_ARA < 0.001, p_EPA < 0.01). At ambient water temperature, ¹³C-labeling indicated significantly enhanced LIN, ALA, SDA, ARA and EPA retention in Daphnia feeding on the chytrid-infected Planktothrix diet (Pairwise Wilcoxon rank sum tests, p_LIN < 0.001, p_ALA < 0.01, p_SDA < 0.001, p_ARA < 0.001, p_EPA < 0.05). The heat treatment did not affect PUFA retention significantly in Daphnia feeding on the chytrid-infected diet (Pairwise Wilcoxon rank sum tests, p > 0.05, in all cases), except for ARA, in which case heat significantly enhanced the retention (Fig. 4D, Pairwise Wilcoxon rank sum tests, p < 0.01). Effect sizes indicated large effects of the diet type on the retention of individual FA (r > 0.6 in all cases), while heat only affected ARA retention largely (r > 0.6). In all other cases, heat only had a small effect on the retention of individual FA (r < 0.3).

Fig. 4.

¹³C retention via key PUFA in Daphnia at the onset of the first successful reproduction among treatments (all pairwise differences between labeled and non-labeled FA, n = 9 for each pair) (A) linoleic acid (18:2n-6, LIN); (B) alpha-linolenic acid (18:3n-3, ALA); (C) stearidonic acid (18:4n-3, SDA); (D) arachidonic acid (20:4n-6, ARA); and (E) eicosapentaenoic acid (20:5n-3, EPA). The letters indicate significant differences in FA retention of Daphnia among treatments based on Pairwise Wilcoxon rank sum tests.

Endogenous conversion of short-chain to long-chain PUFA along with the n-3 chain (i.e. ALA to EPA) occurred with a ~3x higher conversion efficiency than the n-6 chain (i.e. LIN to ARA, Fig. 5). The chytrid-infected diet combined with heat significantly increased the LIN to ARA conversion (Fig. 5A, Pairwise Wilcoxon rank sum tests, p < 0.001, in all cases). Chytrid-infected diet had a large impact on LIN to ARA conversion (r > 0.7), which was also largely impacted by heat (r > 0.8). Along with the ALA to EPA chain, the chytrid diet alone slightly but significantly (p < 0.05), and the chytrid diet together with the heat highly significantly (p < 0.001) enhanced the endogenous conversion (Fig. 5B, Pairwise Wilcoxon rank sum tests). Effect sizes, however, indicated only small effects of chytrids and heat on the ALA to EPA conversion efficiency (r < 0.3 in all cases).

Fig. 5.

Efficiency in the endogenous bioconversion of (A) LIN to ARA (n-6 chain); and (B) ALA to EPA (n-3 chain) in Daphnia at the onset of the first successful reproduction among treatments (boxplots show all pairwise ratios between Δ¹³C_ARA/Δ¹³C_LIN and Δ¹³C_EPA/Δ¹³C_ALA; n = 81, for each treatment). The letters indicate significant differences in FA conversion efficiencies of Daphnia among treatments based on Pairwise Wilcoxon rank sum tests.

DISCUSSION

The high mortality of Daphnia neonates exposed to heat after 2 days confirmed our hypothesis that a +6°C water temperature increase would harm the zooplankton consumer feeding on the sole Planktothrix diet. Our data also confirmed that the chytrid-infected diet could support Daphnia fitness under stress conditions induced by the combined impact of heat and limiting diet (i.e. organic matter quantity, nutritional quality). Chytrids could enhance the dietary supply and subsequent retention of key PUFA by Daphnia at both water temperature treatments. Our study thus provides the first experimental evidence for the positive diet quality effect of chytrids in terms of PUFA as a possible key mechanism providing insurance against the detrimental effect of heat under cyanobacteria blooms in nature.

PUFA transfer in the mycoloop under heat

Chytrids can perform trophic upgrading along with the n-3 and n-6 PUFA chains (Rasconi et al., 2020), resulting in enhanced PUFA retention in Daphnia (Abonyi et al., 2023). Here we show that enhanced PUFA retention by Daphnia with the chytrid-infected diet remains largely unaffected by a +6°C water temperature increase if the diet conditions are limiting in terms of both organic matter quantity and nutritional quality. Due to the lack of ARA and EPA in the diet sources and the quantitative limitation in diet, LIN and ALA are converted into ARA and EPA by the Daphnia, respectively (Bec et al., 2003; Abonyi et al., 2023). The compound-specific fatty acid data clearly show that the endogenous bioconversion of ALA to EPA is more efficient than the conversion of LIN to ARA in Daphnia. While the conversion rate is low and the EPA content produced may still limit somatic growth (Ilić et al., 2021 and herein references), here we see that it is strongly enhanced by chytrids, providing a plausible mechanistic link between chytrid-mediated enhanced n-3 PUFA retention and Daphnia fitness. The n-3 conversion pathway producing EPA, a critical functional LC-FA for growth and reproduction (Becker and Boersma, 2003), remains unaffected by heat if chytrids are present in the diet. The sustained retention under heat also holds for the n-3 PUFA SDA, as yet, however, with unknown energetic benefits (Abonyi et al., 2023).

Surprisingly, while the chytrid diet enhances n-3 over n-6 PUFA retention (i.e. in response to enhanced endogenous conversion), the heat significantly enhances the n-6 PUFA ARA retention via a more efficient LIN to ARA conversion. While this may suggest a crucial physiological role for ARA, the diet quality effect of chytrids in the conversion was weak. Thus, its potential benefits against heat conditions remain to be elucidated. The more efficient endogenous n-3 PUFA conversion and more stable EPA content in Daphnia may show that EPA is the limiting factor over ARA. Accordingly, as soon as a certain amount of EPA is acquired, Daphnia may reduce its conversion rate and start reproducing (Pilecky et al., 2021). This might be supported by the fact that D. magna preferentially store EPA, which appears in higher mass fractions than ARA in the eggs (Becker and Boersma, 2005). If this is the case, high conversion rates from LIN to ARA might be an unnecessary by-product—process for the animal; in case of its high need, ARA levels would also remain more stable (Pilecky et al., 2021).

On dietary PUFA, however, a recent study suggested that Daphnia could be even more susceptible to the lack of ARA than EPA (Ilić et al., 2021). As both ARA and EPA were lacking in diets, our results highlighting a preferential n-3 PUFA over n-6 PUFA conversion may not support the view of Ilić et al. (2021). Moreover, the potential effect of the chytrid-synthesized SDA on the endogenous conversion pathway of ALA to SDA remains unknown. The preferential n-3 PUFA conversion by the Daphnia might have also been enhanced by the favored n-3 PUFA upgrading of chytrids (Rasconi et al., 2020; Abonyi et al., 2023), which would provide a mechanistic link between increased dietary quality for Daphnia and the enhanced performance of the animals.

The chytrid-mediated PUFA insurance against the heat

Ectothermic organisms can genetically adapt to higher temperatures (Geerts et al., 2015). Also, the physiological plasticity of animals allows changes in biochemical characteristics to counter the negative impact of heat. For example, Daphnia adjust its FA content with decreasing PUFA toward higher temperatures (Zeis et al., 2019). Masclaux et al. (2009) argued that the unsaturation level of fatty acids increases toward lower temperature in membrane lipids, which makes the animal more susceptible to heat-induced oxidative stress (Zeis et al., 2019). Here we show that Daphnia acclimated at 18°C for >1 year can successfully cope with a sudden +6°C water temperature increase and counteract energy and PUFA limitation by Planktothrix if chytrids are also available in the diet.

Our results suggest that trophic upgrading within the mycoloop is a crucial and strong mechanism supporting Daphnia fitness under a significant water temperature increase, e.g. during heatwaves. Extreme temperatures may amplify the deleterious effect of warming on the performance and fitness of ectothermic organisms (Kingsolver et al., 2013), especially under reduced diet availability (Huey and Kingsolver, 2019). This certainly holds for late summer phytoplankton in the temperate region, where large-sized, hardly palatable phytoplankton often dominate under eutrophic conditions (Sommer et al., 1986; Ptacnik et al., 2008; O’Neil et al., 2012).

Although our data suggest a positive diet quality effect of trophic upgrading by chytrids between Planktothrix and Daphnia, cyanobacteria lack growth-supporting sterols (Martin-Creuzburg et al., 2010). Thus, trophic upgrading is limited to somatic effects of fatty acids for Daphnia and, importantly, subsequent consumers. Since we lack experimental evidence for the combined diet quality effect of sterol and PUFA-upgrading at the phytoplankton–zooplankton interface, including chytrids, we speculate that trophic upgrading by chytrids remains a valid mechanism, irrespective of the type of algae (Rasconi et al., 2020). The relative importance of sterol- versus PUFA-upgrading and different host types in supporting Daphnia performance under heat remains to be elucidated.

The elemental composition of phytoplankton can also constrain zooplankton fitness under low phosphorus concentrations (Elser et al., 2001). Daphnia feeding on chytrids, however, remained unchanged in their C:P ratios, irrespective of water temperature, even though the chytrid zoospores contained significantly more phosphorus than Planktothrix and the chytrid-infected Planktothrix (Fig. S2). Instead, lipids and their FA, such as EPA and ARA, were the key molecules that improved Daphnia fitness (Müller-Navarra et al., 2000; Brett et al., 2009). While Daphnia can adapt EPA to higher water temperatures to sustain membrane fluidity if the diet is neither limiting in quantity nor quality (Schlechtriem et al., 2006), they may function differently in limiting diet conditions of organic matter quantity and nutritional quality. We argue that Daphnia preferentially convert along with the n-3 chain (ALA to EPA) to sustain first survival, then growth and reproduction, which remains largely unaffected by a +6°C water temperature increase.

While zooplankton fitness decreases in response to the dietary lack of ARA and EPA (Gulati and Demott, 1997), here we show that chytrids can support Daphnia fitness via sustained endogenous EPA and ARA conversion, even under heat. Global warming further intensifies harmful, largely inedible PUFA-deprived phytoplankton blooms (Paerl and Huisman, 2008; Wagner and Adrian, 2009). Thus, the alternative energy and PUFA pathway via chytrid parasites may become increasingly crucial to ensure pelagic food web functioning in the future.

The strength of diet quality versus heat effects

The positive effect of the chytrid diet on Daphnia fitness was systematically strong. In contrast, the negative heat effect was systematically small, except for large heat effects if the animals were feeding on the sole cyanobacterium diet. This emphasizes that chytrids provide a strong insurance for sustained dietary energy transfer toward higher trophic levels and can efficiently buffer PUFA requirements of Daphnia under heat.

CONCLUSIONS

The presence of chytrids in an otherwise deleterious diet may strongly improve growth and reproduction in the keystone herbivore, Daphnia. The positive effect of chytrids on Daphnia growth and reproduction was particularly strong in heated conditions, underling the potential relevance of such effects under global warming. Chytrids support and improve PUFA accrual in Daphnia via increased dietary access to the n-3 PUFA SDA (preferential n-3 upgrading by the chytrids), which co-occurs with higher n-3 (ALA to EPA) than n-6 (LIN to ARA) PUFA upgrading by the animals. Though both were significant, chytrids' positive diet quality effect on the n-3 PUFA conversion was large but small on the n-6 PUFA conversion path. We observed that the chytrid-synthesized SDA, retained selectively by Daphnia, remained quantitatively unchanged with heat. Heat significantly enhanced LIN to ARA bioconversion by the consumer, but the effect was small, with unknown benefits for the consumer. We conclude that the positive dietary effect of chytrids in terms of PUFA is crucial for Daphnia fitness under heat and long-chain PUFA-deprived cyanobacteria blooms. Thus, chytrids likely help function pelagic food webs by improving PUFA availability for higher trophic levels under global warming.

ACKNOWLEDGEMENT

We thank Samuel-Karl Kämmer, Katharina Winter and WasserCluster Lunz for their help with preparations for the experiment and laboratory analyses. We thank Thomas Rohrlack, Norwegian University of Life Sciences (NMBU), for providing the Planktothrix and chytrid isolates.

FUNDING

Austrian Science Fund (FWF Project P 30419-B29); National Research, Development and Innovation Office, Hungary (Project FK 142485 to A.A.); the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (to A.A.). Funding for open access charge: NRDI Office, Hungary (Project FK142485).

DATA AVAILABILITY

The data of this study are available from the corresponding author upon request.