Absence of sterols constrains carbon transfer between cyanobacteria and a freshwater herbivore (Daphnia galeata) A key process in freshwater plankton food webs is the regulation of the efficiency of energy and material transfer. Cyanobacterial carbon (C) in particular is transferred very inefficiently to herbivorous zooplankton, which leads to a decoupling of primary and secondary production and the accumulation of cyanobacterial biomass, which is associated with reduced recreational quality of water bodies and hazards to human health. A recent correlative field study suggested that the low transfer efficiency of cyanobacterial C is the result of the absence of long-chain polyunsaturated fatty acids (PUFA) in the diet of the zooplankton. By supplementation of single-lipid compounds in controlled growth experiments, we show here that the low C transfer efficiency of coccal and filamentous cyanobacteria to the keystone herbivore Daphnia is caused by the low sterol content in cyanobacteria, which constrains cholesterol synthesis and thereby growth and reproduction of the herbivore. Estimations of sterol requirement in Daphnia suggest that, when cyanobacteria comprise more than 80% of the grazed phytoplankton, growth of the herbivore may be limited by sterols and Daphnia may subsequently fail to control phytoplankton biomass. Dietary sterols therefore may play a key role in freshwater food webs and in the control of water quality in lakes dominated by cyanobacteria.

In past decades, a considerable amount of research has been conducted to elucidate the factors that affect the carbon transfer across the cyanobacteria-Daphnia interface. It is well accepted that cyanobacteria are a nutritionally inadequate food source for cladocerans, but the underlying mechanisms are still controversial. Morphological properties, toxicity, and the absence of essential lipids, i.e., polyunsaturated fatty acids (PUFAs) and sterols, are discussed as the most important factors that account for this nutritional inadequacy. Here, we conducted standardized growth experiments with the herbivore Daphnia magna feeding on coccal, filamentous, and putatively toxic cyanobacterial strains comprising the genera Synechococcus, Anabaena, Aphanizomenon, and Microcystis and on a cyanobacterial mixture containing strains of these genera. The relative importance of PUFAs and sterols in determining the food quality of cyanobacteria for Daphnia was assessed by supplementation of eicosapentaenoic acid-and/or cholesterol-containing liposomes to the cyanobacterial carbon. We provide evidence that somatic growth of daphnids on coccal as well as on filamentous cyanobacteria is primarily constrained by the absence of sterols, provided that the cyanobacterial carbon is readily ingested and nontoxic. The absence of PUFAs in cyanobacteria appears to be of minor importance for somatic growth but potentially affects egg production in Daphnia. Thus, the absence of sterols has to be considered a major food-quality constraint that potentially affects the efficiency of carbon transfer across the cyanobacteria-Daphnia interface.

Empirical data providing evidence for a colimitation of an herbivore by two or more essential nutrients are scarce, particularly in regard to biochemical resources. Here, a graphical model is presented, which describes the growth of an herbivore in a system with two potentially limiting resources. To verify this model, life-history experiments were conducted with the herbivore Daphnia magna feeding on the picocyanobacterium Synechococcus elongatus, which was supplemented with increasing amounts of cholesterol either in the presence or the absence of saturating amounts of eicosapentaenoic acid (EPA). For comparison, D. magna was raised on diets containing different proportions of S. elongatus and the cholesterol-and EPA-rich eukaryotic alga Nannochloropsis limnetica. Somatic and population growth of D. magna on a sterol-and EPA-deficient diet was initially constrained by the absence of sterols. With increased sterol availability, a colimitation by EPA became apparent and when the sterol requirements were met, the growth-limiting factor was shifted from a limitation by sterols to a limitation by EPA. These data imply that herbivores are frequently limited by two or more essential nutrients simultaneously. Hence, the concept of colimitation has to be incorporated into models assessing nutrient-limited growth kinetics of herbivores to accurately predict demographic changes and population dynamics.

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.

Kankaala, P. (2014). Differing Daphnia magna assimilation efficiencies for terrestrial, bacterial, and algal carbon and fatty acids. Ecology, 95 (2), 563-576. doi:10.1890/13-0650.1 Retrieved from http://www.esajournals.org/doi/abs/10.1890/13-0650.1 2014Publisher's PDF Ecology, 95(2), 2014Ecology, 95(2), , pp. 563-576 Ó 2014 Abstract. There is considerable interest in the pathways by which carbon and growthlimiting 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 (x-3 and x-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 x-6 and x-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 x-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.

The absence of essential biochemical nutrients, such as polyunsaturated fatty acids or sterols, has been considered as a mechanism determining trophic interactions between the herbivore Daphnia and its phytoplankton food source. Here, we experimentally quantify the sensitivity of two Daphnia species to decreasing amounts of dietary sterols by measuring variations in life history traits. The two species Daphnia magna and D. galeata were fed different mixtures of the sterol-containing green alga Scenedesmus obliquus and the sterolfree cyanobacterium Synechococcus elongatus; a higher proportion of Synechococcus in the food is equivalent to a decrease in dietary sterols. To address the significance of sterol limitation, the Daphnia species were also fed Synechococcus supplemented with cholesterol. In both species, somatic and population growth rates, maternal dry mass, the number of viable offspring, and the probability of survival were significantly reduced with the lower availability of sterols. A high correlation between the sterol content of the mixed diet and the somatic and population growth rates was found, and growth on cholesterol-supplemented Synechococcus fitted well into this correlation. Somatic growth of first-clutch neonates grown on 100% Synechococcus exhibited a pattern similar to that of somatic growth of their mothers grown on the different food regimes, which demonstrated the significance of maternal effects for sterol-limited population growth. Daphnia galeata had a twofold higher incipient limiting sterol level than D. magna, which indicated interspecific differences in sterol requirements between the two Daphnia species.The results suggest a strong impact of dietary sterols on life history traits and therefore, population dynamics of the keystone species Daphnia.

Summary 1.In nature, food conditions change temporally and force consumers into trade-offs during resource allocation. In particular, under poor food conditions, for example during cyanobacterial blooms, herbivores have to optimize their resource allocation to maximize fitness, and face two decisions: (i) an individual might attempt to allocate acquired essential resources to reproductive tissues or use them for its own maintenance; and (ii) an individual might decide to optimize the chemical quality of its eggs. 2. As cyanobacteria feature a deficiency in some essential lipids that leads to a decline in the growth and fecundity of Daphnia , an important freshwater herbivore, we investigated Daphnia magna 's Strauss allocation of lipids such as polyunsaturated fatty acids (PUFAs) and cholesterol during an experimental non-toxic cyanobacterial bloom. 3. Generally, we found a substantial maternal investment of the particularly important omega-3 (n-3) PUFAs, in particular eicosapentaenoic acid (EPA), into the eggs. The concentration of EPA was 2·4-fold higher in eggs than in somatic tissue, a cumulative effect, which was not significantly changed under cyanobacterial food conditions. 4. Under poor conditions, D. magna not only decreased the number of eggs produced but, in principle, reduced the previously high concentrations of EPA in both eggs and somatic tissues to a similar degree. In contrast to EPA, the concentrations of α -linolenic acid and cholesterol, although lower than EPA, were more homeostatic in eggs than in somatic tissues, in which concentrations decreased. 5. When food quality was improved, D. magna were able to recover completely the fatty acid concentrations in their somatic tissues and eggs. 6. This study shows that the content of particular lipids in its food clearly affects resource allocation in D. magna , and suggests that cholesterol is important for somatic growth, while PUFAs are primarily needed for reproduction. As a decreasing investment of essential lipids into eggs implies a reduced fitness of the animals' progeny under poor food conditions, this could have a strong impact on population dynamics, which might also be valid for other species.

The quality of heterotrophic bacteria as food for metazoan grazers has been investigated poorly. We conducted growth experiments with juvenile Daphnia magna feeding on different strains of heterotrophic bacteria that represent typical pelagic bacteria of five phylogenetically distinct groups. The bacterial food suspensions were supplemented with cholesterol and/or the polyunsaturated fatty acid eicosapentaenoic acid (EPA), two essential nutrients that are either absent or scarcely represented in bacteria. Our data imply that the selected heterotrophic bacteria are of poor food quality for D. magna, which was indicated either by very low somatic growth rates or by high mortality. However, with four out of six bacterial strains tested, the somatic growth rates increased significantly upon supplementation with cholesterol, which shows that the lack of sterols in bacteria is a major food quality constraint. We did not find clear evidence for a limitation by EPA on bacterial diets within our growth experiments. High mortality was observed when D. magna was fed with Hydrogenophaga sp. or Pseudomonas sp., which indicates that these two bacterial strains are toxic to D. magna. Our findings highlight the limitations of bacteria as a carbon source for Daphnia and point to a so far underestimated diversity of interactions between grazers and its bacterial food.