Fate of Planktothrix-derived toxins in aquatic food webs: A case study in Lake Mindelsee (Germany).

Blooms of the red, filamentous cyanobacterium Planktothrix rubescens occur frequently in pre-alpine lakes in Europe, often with concomitant toxic microcystin (MC) production. Trophic transfer of MCs has been observed in bivalves, fish, and zooplankton species, while uptake of MCs into Diptera species could facilitate distribution of MCs into terrestrial food webs and habitats. In this study, we characterized a Planktothrix bloom in summer 2019 in Lake Mindelsee and tracked possible trophic transfer and/or bioaccumulation of MCs via analysis of phytoplankton, zooplankton (Daphnia) and emergent aquatic insects (Chaoborus, Chironomidae and Trichoptera). Using 16 S rRNA gene amplicon sequencing, we found that five sequence variants of Planktothrix spp. were responsible for bloom formation in September and October of 2019, and these MC-producing variants, provisionally identified as P. isothrix and/or P. serta, occurred exclusively in Lake Mindelsee (Germany), while other variants were also detected in nearby Lake Constance. The remaining cyanobacterial community was dominated by Cyanobiaceae species with high species overlap with Lake Constance, suggesting a well-established exchange of cyanobacteria species between the adjacent lakes. With targeted LC-HRMS/MS we identified two MC-congeners, MC-LR and [Asp3]MC-RR with maximum concentrations of 45 ng [Asp3]MC-RR/L in lake water in September. Both MC congeners displayed different predominance patterns, suggesting that two different MC-producing species occurred in a time-dependent manner, whereby [Asp3]MC-RR was clearly associated with the Planktothrix spp. bloom. We demonstrate an exclusive transfer of MC-LR, but not [Asp3]MC-RR, from phytoplankton into zooplankton reaching a 10-fold bioconcentration, yet complete absence of these MC congeners or their conjugates in aquatic insects. The latter demonstrated a limited trophic transfer of MCs from zooplankton to zooplanktivorous insect larvae (e.g., Chaoborus), or direct transfer into other aquatic insects (e.g. Chironomidae and Trichoptera), whether due to avoidance or limited uptake and/or rapid excretion of MCs by higher trophic emergent aquatic insects.

Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia.

The dietary supply of polyunsaturated fatty acids (PUFA) crucially affects animals' performance at different temperatures. However, the underlying physiological mechanisms are still insufficiently understood. Here, we analyzed lifespan and heat tolerance of four genotypes of Daphnia magna reared on either the green alga Scenedesmus obliquus that lacks long-chain (> C18) PUFA, or the heterokont alga Nannochloropsis limnetica that contains C20 PUFA, both either at saturating and near-starvation levels. A significant genotype-by-diet interaction in lifespan was observed at saturating diets. The C20 PUFA-rich diet eliminated differences in lifespan among genotypes on the PUFA-deficient diet. Corrected for body length, acute heat tolerance was higher at low than at high food concentration, at least in the older of the two age groups analyzed. Genotypes differed significantly in heat tolerance, but there were no genotype-by-diet interactions. As predicted, the C20 PUFA-rich diet resulted in higher lipid peroxidation (LPO) and a lower mitochondrial membrane potential (ΔΨm). LPO levels averaged across clones and rearing conditions were inversely related to acute heat tolerance. Yet, heat tolerance was higher on the PUFA-rich diet than on the PUFA-deficient diet, particularly in older Daphnia, indicating that the C20 PUFA-rich diet allowed Daphnia to compensate for higher LPO. In contrast, Daphnia with intermediate levels of ΔΨm showed the lowest heat tolerance. Neither LPO nor ΔΨm explained the diet effects on lifespan. We hypothesize that antioxidants present in the PUFA-rich diet may have enabled higher heat tolerance of Daphnia despite higher LPO, which may also explain the lifespan expansion of otherwise short-lived genotypes.

A sterol-mediated gleaner-opportunist trade-off underlies the evolution of grazer resistance to cyanobacteria.

The human-caused proliferation of cyanobacteria severely impacts consumers in freshwater ecosystems. Toxicity is often singled out as the sole trait to which consumers can adapt, even though cyanobacteria are not necessarily toxic and the lack of nutritionally critical sterols in cyanobacteria is known to impair consumers. We studied the relative significance of toxicity and dietary sterol deficiency in driving the evolution of grazer resistance to cyanobacteria in a large lake with a well-documented history of eutrophication and oligotrophication. Resurrecting decades-old Daphnia genotypes from the sediment allowed us to show that the evolution and subsequent loss of grazer resistance to cyanobacteria involved an adaptation to changes in both toxicity and dietary sterol availability. The adaptation of Daphnia to changes in cyanobacteria abundance revealed a sterol-mediated gleaner-opportunist trade-off. Genotypes from peak-eutrophic periods showed a higher affinity for dietary sterols at the cost of a lower maximum growth rate, whereas genotypes from more oligotrophic periods showed a lower affinity for dietary sterols in favour of a higher maximum growth rate. Our data corroborate the significance of sterols as limiting nutrients in aquatic food webs and highlight the applicability of the gleaner-opportunist trade-off for reconstructing eco-evolutionary processes.

Comparison of sterol and fatty acid profiles of chytrids and their hosts reveals trophic upgrading of nutritionally inadequate phytoplankton by fungal parasites.

Chytrids are ubiquitous fungal parasites in aquatic ecosystems, infecting representatives of all major phytoplankton groups. They repack carbon from inedible phytoplankton hosts into easily ingested chytrid propagules (zoospores), rendering this carbon accessible to zooplankton. Grazing on zoospores may circumvent bottlenecks in carbon transfer imposed by the dominance of inedible or poorly nutritious phytoplankton (mycoloop). We explored qualitative aspects of the mycoloop by analysing lipid profiles (fatty acids, sterols) of two chytrids infecting two major bloom-forming phytoplankton taxa of contrasting nutritional value: the diatom Asterionella formosa and the filamentous cyanobacterium Planktothrix agardhii. The polyunsaturated fatty acid composition of chytrids largely reflected that of their hosts, highlighting their role as conveyors of otherwise inaccessible essential lipids to higher trophic levels. We also showed that chytrids are capable of synthesizing sterols, thus providing a source of these essential nutrients for grazers even when sterols are absent in their phytoplankton hosts. Our findings reveal novel qualitative facets of the mycoloop, showing that parasitic chytrids, in addition to making carbon and essential lipids available from inedible sources, also upgrade their host's biochemical composition by producing sterols de novo, thereby enhancing carbon and energy fluxes in aquatic food webs.

Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

Population genetic dynamics of an invasion reconstructed from the sediment egg bank.

Biological invasions are a global issue with far-reaching consequences for single species, communities and whole ecosystems. Our understanding of modes and mechanisms of biological invasions requires knowledge of the genetic processes associated with successful invasions. In many instances, this information is particularly difficult to obtain as the initial phases of the invasion process often pass unnoticed and we rely on inferences from contemporary population genetic data. Here, we combined historic information with the genetic analysis of resting eggs to reconstruct the invasion of Daphnia pulicaria into Lower Lake Constance (LLC) in the 1970s from the resting egg bank in the sediments. We identified the invader as 'European D. pulicaria' originating from meso- and eutrophic lowland lakes and ponds in Central Europe. The founding population was characterized by extremely low genetic variation in the resting egg bank that increased considerably over time. Furthermore, strong evidence for selfing and/or biparental inbreeding was found during the initial phase of the invasion, followed by a drop of selfing rate to low levels in subsequent decades. Moreover, the increase in genetic variation was most pronounced during early stages of the invasion, suggesting additional introductions during this period. Our study highlights that genetic data covering the entire invasion process from its beginning can be crucial to accurately reconstruct the invasion history of a species. We show that propagule banks can preserve such information enabling the study of population genetic dynamics and sources of genetic variation in successful invasive populations.

Compound-specific δ(13)C analyses reveal sterol metabolic constraints in an aquatic invertebrate.

RATIONALE: Dietary sterol deficiencies can have severe life history consequences for consumers. Compound-specific stable isotope analysis (CSIA) was applied to the exploration of the sterol metabolic constraints and bioconversion capacities of the amphipod Gammarus roeselii. Evaluating structural sterol requirements has great potential to improve our understanding of the ecological relevance of sterols as limiting nutrients. METHODS: Juvenile G. roeselii were reared on food mixtures consisting of different ratios of the two algae Scenedesmus obliquus (cultivated with (13)C-labeled NaHCO3) and Nannochloropsis limnetica (unlabeled), which have been shown previously to differ in food quality. We measured the sterol content and composition using a gas chromatograph equipped with a flame ionization detector and the δ(13)C values of sterols using compound-specific isotope ratio mass spectrometry to examine potential sterol-mediated nutritional constraints of G. roeselii. RESULTS: In the food mixtures, δ(13)C values of cholesterol, synthesized by N. limnetica, were -25‰ and those of the Δ(7)-phytosterols, chondrillasterol and fungisterol, synthesized by S. obliquus, were 7 and 18‰, respectively. Although the cholesterol concentrations in G. roeselii decreased with increasing proportion of dietary S. obliquus, the δ(13)C values remained constant at -25‰. Lathosterol, which appeared in G. roeselii at high dietary proportions of S. obliquus, had a δ(13)C value of 35‰. CONCLUSIONS: We provide evidence that the the Δ(7)-phytosterols present in S. obliquus cannot be metabolized to cholesterol in G. roeselii, resulting in the accumulation of lathosterol in the animals and potentially in sterol-limited growth. These findings emphasize the advantage of CSIA in revealing the physiological mechanisms associated with nutritional constraints.

Differing Daphnia magna assimilation efficiencies for terrestrial, bacterial, and algal carbon and fatty acids.

There is considerable interest in the pathways by which carbon and growth-limiting elemental and biochemical nutrients are supplied to upper trophic levels. Fatty acids and sterols are among the most important molecules transferred across the plant-animal interface of food webs. In lake ecosystems, in addition to phytoplankton, bacteria and terrestrial organic matter are potential trophic resources for zooplankton, especially in those receiving high terrestrial organic matter inputs. We therefore tested carbon, nitrogen, and fatty acid assimilation by the crustacean Daphnia magna when consuming these resources. We fed Daphnia with monospecific diets of high-quality (Cryptomonas marssonii) and intermediate-quality (Chlamydomonas sp. and Scenedesmus gracilis) phytoplankton species, two heterotrophic bacterial strains, and particles from the globally dispersed riparian grass, Phragmites australis, representing terrestrial particulate organic carbon (t-POC). We also fed Daphnia with various mixed diets, and compared Daphnia fatty acid, carbon, and nitrogen assimilation across treatments. Our results suggest that bacteria were nutritionally inadequate diets because they lacked sterols and polyunsaturated omega-3 and omega-6 (omega-3 and omega-6) fatty acids (PUFAs). However, Daphnia were able to effectively use carbon and nitrogen from Actinobacteria, if their basal needs for essential fatty acids and sterols were met by phytoplankton. In contrast to bacteria, t-POC contained sterols and omega-6 and omega-3 fatty acids, but only at 22%, 1.4%, and 0.2% of phytoplankton levels, respectively, which indicated that t-POC food quality was especially restricted with regard to omega-3 PUFAs. Our results also showed higher assimilation of carbon than fatty acids from t-POC and bacteria into Daphnia, based on stable-isotope and fatty acids analysis, respectively. A relatively high (>20%) assimilation of carbon and fatty acids from t-POC was observed only when the proportion of t-POC was >60%, but due to low PUFA to carbon ratio, these conditions yielded poor Daphnia growth. Because of lower assimilation for carbon, nitrogen, and fatty acids from t-POC relative to diets of bacteria mixed with phytoplankton, we conclude that the microbial food web, supported by phytoplankton, and not direct t-POC consumption, may support zooplankton production. Our results suggest that terrestrial particulate organic carbon poorly supports upper trophic levels of the lakes.

Thresholds for sterol-limited growth of Daphnia magna: a comparative approach using 10 different sterols.

Arthropods are incapable of synthesizing sterols de novo and thus require a dietary source to cover their physiological demands. The most prominent sterol in animal tissues is cholesterol, which is an indispensable structural component of cell membranes and serves as precursor for steroid hormones. Instead of cholesterol, plants and algae contain a variety of different phytosterols. Consequently, herbivorous arthropods have to metabolize dietary phytosterols to cholesterol to meet their requirements for growth and reproduction. Here, we investigated sterol-limited growth responses of the freshwater herbivore Daphnia magna by supplementing a sterol-free diet with increasing amounts of 10 different phytosterols and comparing thresholds for sterol-limited growth. In addition, we analyzed the sterol composition of D. magna to explore sterol metabolic constraints and bioconversion capacities. We show that dietary phytosterols strongly differ in their potential to support somatic growth of D. magna. The dietary threshold concentrations obtained by supplementing the different sterols cover a wide range (3.5-34.4 µg mg C(-1)) and encompass the one for cholesterol (8.9 µg mg C(-1)), indicating that certain phytosterols are more efficient in supporting somatic growth than cholesterol (e.g., fucosterol, brassicasterol) while others are less efficient (e.g., dihydrocholesterol, lathosterol). The dietary sterol concentration gradients revealed that the poor quality of particular sterols can be alleviated partially by increasing dietary concentrations, and that qualitative differences among sterols are most pronounced at low to moderate dietary concentrations. We infer that the dietary sterol composition has to be considered in zooplankton nutritional ecology to accurately assess potential sterol limitations under field conditions.

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

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

Consumer biodiversity increases organic nutrient availability across aquatic and terrestrial ecosystems.

Human land-use intensification threatens arthropod (for example, insect and spider) biodiversity across aquatic and terrestrial ecosystems. Insects and spiders play critical roles in ecosystems by accumulating and synthesizing organic nutrients such as polyunsaturated fatty acids (PUFAs). However, links between biodiversity and nutrient content of insect and spider communities have yet to be quantified. We relate insect and spider richness to biomass and PUFA-mass from stream and terrestrial communities encompassing nine land uses. PUFA-mass and biomass relate positively to biodiversity across ecosystems. In terrestrial systems, human-dominated areas have lower biomass and PUFA-mass than more natural areas, even at equivalent levels of richness. Aquatic ecosystems have consistently higher PUFA-mass than terrestrial ecosystems. Our findings reinforce the importance of conserving biodiversity and highlight the distinctive benefits of aquatic biodiversity.

Dietary supply with polyunsaturated fatty acids and resulting maternal effects influence host--parasite interactions.

BACKGROUND: Interactions between hosts and parasites can be substantially modulated by host nutrition. Polyunsaturated fatty acids (PUFAs) are essential dietary nutrients; they are indispensable as structural components of cell membranes and as precursors for eicosanoids, signalling molecules which act on reproduction and immunity. Here, we explored the potential of dietary PUFAs to affect the course of parasitic infections using a well-established invertebrate host - parasite system, the freshwater herbivore Daphnia magna and its bacterial parasite Pasteuria ramosa. RESULTS: Using natural food sources differing in their PUFA composition and by experimentally modifying the availability of dietary arachidonic acid (ARA) and eicosapentaenoic acid (EPA) we examined PUFA-mediated effects resulting from direct consumption as well as maternal effects on offspring of treated mothers. We found that both host and parasite were affected by food quality. Feeding on C20 PUFA-containing food sources resulted in higher offspring production of hosts and these effects were conveyed to a great extent to the next generation. While feeding on a diet containing high PUFA concentrations significantly reduced the likelihood of becoming infected, the infection success in the next generation increased whenever the maternal diet contained PUFAs. We suggest that this opposing effect was caused by a trade-off between reproduction and immunity in the second generation. CONCLUSIONS: Considering the direct and maternal effects of dietary PUFAs on host and parasite we propose that host - parasite interactions and thus disease dynamics under natural conditions are subject to the availability of dietary PUFAs.

Omega-3 versus Omega-6: Are We Underestimating the Ecological Significance of Arachidonic Acid in Aquatic Systems?

The long-chain polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA, ω-3, or n-3) and arachidonic acid (ARA, ω-6 or n-6) are known to have distinct physiological functions, yet can both support growth and reproduction of consumers, raising the question of whether EPA and ARA are ecologically substitutable dietary resources. We explored the relative importance of EPA and ARA for the growth and reproduction of the freshwater keystone herbivore Daphnia in a life-history experiment. Both PUFA were supplemented in a concentration-dependent manner to a PUFA-free diet, separately and in combination (50% EPA: 50% ARA mixture). The growth-response curves obtained with EPA, ARA, and the mixture were virtually congruent and the thresholds for PUFA limitation did not differ, indicating that EPA (n-3) and ARA (n-6) were substitutable dietary resources under the applied experimental conditions. The actual requirements for EPA and ARA might change with growth conditions, e.g., under the influence of parasites or pathogens. The higher retention of ARA in Daphnia suggests that EPA and ARA are subject to different turnover rates, which also implies different physiological functions. Studies on the ARA requirements of Daphnia could provide valuable information on the presumably underestimated ecological importance of ARA in freshwater food webs.

Trophic transfer of polyunsaturated fatty acids across the aquatic-terrestrial interface: An experimental tritrophic food chain approach.

Aquatic and their adjacent terrestrial ecosystems are linked via the flux of organic and inorganic matter. Emergent aquatic insects are recognized as high-quality food for terrestrial predators, because they provide more physiologically relevant long-chain polyunsaturated fatty acids (PUFA) than terrestrial insects. The effects of dietary PUFA on terrestrial predators have been explored mainly in feeding trials conducted under controlled laboratory conditions, hampering the assessment of the ecological relevance of dietary PUFA deficiencies under field conditions. We assessed the PUFA transfer across the aquatic-terrestrial interface and the consequences for terrestrial riparian predators in two outdoor microcosm experiments. We established simplified tritrophic food chains, consisting of one of four basic food sources, an intermediary collector gatherer (Chironomus riparius, Chironomidae), and a riparian web-building spider (Tetragnatha sp.). The four basic food sources (algae, conditioned leaves, oatmeal, and fish food) differed in PUFA profiles and were used to track the trophic transfer of single PUFA along the food chain and to assess their potential effects on spiders, that is, on fresh weight, body condition (size-controlled measurement of nutritional status), and immune response. The PUFA profiles of the basic food sources, C. riparius and spiders differed between treatments, except for spiders in the second experiment. The PUFA α-linolenic acid (ALA, 18:3n-3) and É£-linolenic acid (GLA, 18:3n-6) were major contributors to the differences between treatments. PUFA profiles of the basic food sources influenced the fresh weight and body condition of spiders in the first experiment, but not in the second experiment, and did not affect the immune response, growth rate, and dry weight in both experiments. Furthermore, our results indicate that the examined responses are dependent on temperature. Future studies including anthropogenic stressors would deepen our understanding of the transfer and role of PUFA in ecosystems.

Stream degradation affects aquatic resource subsidies to riparian ground-dwelling spiders.

Freshwater systems have undergone drastic alterations during the last century, potentially affecting cross-boundary resource transfers between aquatic and terrestrial ecosystems. One important connection is the export of biomass by emergent aquatic insects containing omega-3 polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), that is scarce in terrestrial systems. Because of taxon-specific differences in PUFA content and functional traits, the contribution of different insect groups should be considered, in addition to total biomass export. In this context, one important trait is the emergence mode. Stoneflies, in contrast to other aquatic insects, crawl to land to emerge instead of flying directly from the water surface, making them accessible to ground-dwelling predators. Because stoneflies are especially susceptible to environmental change, stream degradation might cause a mismatch of available and required nutrients, particularly for ground-dwelling predators. In this study, we estimated emergent biomass and EPA export along two streams with different levels of habitat degradation. The EPA content in aquatic insects did not differ with different degrees of habitat degradation and total biomass export in spring was with 7.9 ± 9.6 mg m-2 day-1 in the degraded and 7.3 ± 8.5 mg m-2 day-1 in the natural system, also unaffected. However, habitat degradation substantially altered the contribution of crawling emergence to the total export in spring, with no biomass export by stoneflies at the most degraded sites. The EPA content in ground-dwelling spiders was correlated with emergent stonefly biomass, making up only 16.0 ± 6.2 % of total fatty acids at sites with no stonefly emergence, but 27.3 ± 3.0 % at sites with highest stonefly emergence. Because immune function in ground-dwelling spiders has been connected to EPA levels, reduced crawling emergence might impact spider fitness. Functional traits, like emergence mode as well as nutritional quality, should be considered when assessing the effects of stream degradation on adjacent terrestrial ecosystems.

Export of dietary lipids via emergent insects from eutrophic fishponds.

Fishponds, despite being globally abundant, have mainly been considered as food production sites and have received little scientific attention in terms of their ecological contributions to the surrounding terrestrial environment. Emergent insects from fishponds may be important contributors of lipids and essential fatty acids to terrestrial ecosystems. In this field study, we investigated nine eutrophic fishponds in Austria from June to September 2020 to examine how Chlorophyll-a concentrations affect the biomass of emergent insect taxa (i.e., quantity of dietary subsidies; n = 108) and their total lipid and long-chain polyunsaturated fatty acid content (LC-PUFA, i.e., quality of dietary subsidies; n = 94). Chironomidae and Chaoboridae were the most abundant emergent insect taxa, followed by Trichoptera, Ephemeroptera, and Odonata. A total of 1068 kg of emergent insect dry mass were exported from these ponds (65.3 hectares). Chironomidae alone exported 103 kg of total lipids and 9.4 kg of omega-3 PUFA. Increasing Chl-a concentrations were associated with decreasing biomass export and a decrease in total lipid and LC-PUFA export via emergent Chironomidae. The PUFA composition of emergent insect taxa differed significantly from dietary algae, suggesting selective PUFA retention by insects. The export of insect biomass from these eutrophic carp ponds was higher than that previously reported from oligotrophic lakes. However, lower biomass and diversity are exported from the fishponds compared to managed ponds. Nonetheless, our data suggest that fishponds provide crucial ecosystem services to terrestrial consumers by contributing essential dietary nutrients to consumer diets via emergent insects. Supplementary Information: The online version contains supplementary material available at 10.1007/s10750-022-05040-2.

Multiple resource limitation theory applied to herbivorous consumers: Liebig's minimum rule vs. interactive co-limitation.

There is growing consensus that the growth of herbivorous consumers is frequently limited by more than one nutrient simultaneously. This understanding, however, is based primarily on theoretical considerations and the applicability of existing concepts of co-limitation has rarely been tested experimentally. Here, we assessed the suitability of two contrasting concepts of resource limitation, i.e. Liebig's minimum rule and the multiple limitation hypothesis, to describe nutrient-dependent growth responses of a freshwater herbivore (Daphnia magna) in a system with two potentially limiting nutrients (cholesterol and eicosapentaenoic acid). The results indicated that these essential nutrients interact, and do not strictly follow Liebig's minimum rule, which consistently overestimates growth at co-limiting conditions and thus is not applicable to describe multiple nutrient limitation of herbivorous consumers. We infer that the outcome of resource-based modelling approaches assessing herbivore population dynamics strongly depends on the applied concept of co-limitation.

Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve.

The accumulation of cyanobacterial biomass may severely affect the performance of aquatic consumers. Here, we investigated the role of sterols in determining the food quality of cyanobacteria for the invasive clam Corbicula fluminea, which has become a common benthic invertebrate in many freshwater ecosystems throughout the world. In standardized growth experiments, juvenile clams were fed mixtures of different cyanobacteria (Anabaena variabilis, Aphanothece clathrata, Synechococcus elongatus) or sterol-containing eukaryotic algae (Cryptomonas sp., Nannochloropsis limnetica, Scenedesmus obliquus). In addition, the cyanobacterial food was supplemented with different sterols. We provide evidence that somatic growth of C. fluminea on cyanobacterial diets is constrained by the absence of sterols, as indicated by a growth-enhancing effect of sterol supplementation. Thus, our findings contribute to our understanding of the consequences of cyanobacterial mass developments for benthic consumers and highlight the importance of considering sterols as potentially limiting nutrients in aquatic food webs.

Dietary lipid quality affects temperature-mediated reaction norms of a freshwater key herbivore.

Temperature-mediated plasticity in life history traits strongly affects the capability of ectotherms to cope with changing environmental temperatures. We hypothesised that temperature-mediated reaction norms of ectotherms are constrained by the availability of essential dietary lipids, i.e. polyunsaturated fatty acids (PUFA) and sterols, as these lipids are involved in the homeoviscous adaptation of biological membranes to changing temperatures. A life history experiment was conducted in which the freshwater herbivore Daphnia magna was raised at four different temperatures (10, 15, 20, 25°C) with food sources differing in their PUFA and sterol composition. Somatic growth rates increased significantly with increasing temperature, but differences among food sources were obtained only at 10°C at which animals grew better on PUFA-rich diets than on PUFA-deficient diets. PUFA-rich food sources resulted in significantly higher population growth rates at 10°C than PUFA-deficient food, and the optimum temperature for offspring production was clearly shifted towards colder temperatures with an increased availability of dietary PUFA. Supplementation of PUFA-deficient food with single PUFA enabled the production of viable offspring and significantly increased population growth rates at 10°C, indicating that dietary PUFA are crucial for the acclimation to cold temperatures. In contrast, cumulative numbers of viable offspring increased significantly upon cholesterol supplementation at 25°C and the optimum temperature for offspring production was shifted towards warmer temperatures, implying that sterol requirements increase with temperature. In conclusion, essential dietary lipids significantly affect temperature-mediated reaction norms of ectotherms and thus temperature-mediated plasticity in life history traits is subject to strong food quality constraints.

The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: a gene expression approach.

Nutritional ecology of the aquatic model genus Daphnia has received much attention in past years in particular with regard to dietary polyunsaturated fatty acids (PUFAs) which are crucial for growth and reproduction. Besides their significant role as membrane components, C20 PUFAs serve as precursors for eicosanoids, hormone-like mediators of reproduction, immunity and ion transport physiology. In the present study we investigate transcriptomic changes in Daphnia magna in response to different algal food organisms substantially differing in their PUFA composition using quantitative real-time PCR and relate them to concomitantly documented life history data. The selection of target genes includes representatives that have previously been shown to be responsive to the eicosanoid biosynthesis inhibitor ibuprofen. The beneficial effect of C20 PUFA-rich food on reproduction and population growth rates was accompanied by an increased vitellogenin (DmagVtg1) gene expression in D. magna. Additionally, genes involved in eicosanoid signaling were particularly influenced by dietary C20 PUFA availability. For example, the cyclooxygenase gene (Cox), coding for a central enzyme in the eicosanoid pathway, was highly responsive to the food treatments. Our results suggest that dietary PUFAs are fundamental in D. magna physiology as substrate for eicosanoid synthesis and that these eicosanoids are important for D. magna reproduction.