Food quality shapes gradual phenotypic plasticity in ectotherms facing temperature variability.

Organisms exhibit reversible physiological adjustments as a response to rapidly changing environments. Yet such plasticity of the phenotype is gradual and may lag behind environmental fluctuations, thereby affecting long-term average performance of the organisms. By supplying energy and essential compounds for optimal tissue building, food determines the range of possible phenotypic changes and potentially the rate at which they occur. Here, we assess how differences in the dietary supply of essential lipids modulate the phenotypic plasticity of an ectotherm facing thermal fluctuations. We use three phytoplankton strains to create a gradient of polyunsaturated fatty acid and sterol supply for Daphnia magna under constant and fluctuating temperatures. We used three different fluctuation periodicities to unravel the temporal dynamics of gradual plasticity and its long-term consequences for D. magna performance measured as juvenile somatic growth rate. In agreement with gradual plasticity theory, we show that in D. magna, fluctuation periodicity determines the differential between observed growth rates and those expected from constant conditions. Most importantly, we show that diet modulates both the size and the direction of the growth rate differential. Overall, we demonstrate that the nutritional context is essential for predicting ectotherm consumers' performance in fluctuating thermal environments.

Quantifying the energetic cost of food quality constraints on resting metabolism to integrate nutritional and metabolic ecology.

Consumer metabolism controls the energy uptake from the environment and its allocation to biomass production. In natural ecosystems, available energy in food often fails to predict biomass production which is also (co)limited by the relative availability of various dietary compounds. To date, the link between energy metabolism and the effects of food chemical composition on biomass production remains elusive. Here, we measured the resting metabolic rate (RMR) of Daphnia magna along ontogeny when undergoing various (non-energetic) nutritional constraints. All types of dietary (co)limitations (Fatty acids, Sterols, Phosphorus) induced an increase in mass-specific RMR up to 128% between highest and lowest quality diets. We highlight a strong negative correlation between RMR and growth rate indicating RMR as a promising predictor of consumer growth rate. We argue that quantifying the energetic cost imposed by food quality on individual RMR may constitute a common currency enabling the integration of nutritional and metabolic ecology.

Stable isotopes of fatty acids: current and future perspectives for advancing trophic ecology.

To understand consumer dietary requirements and resource use across ecosystems, researchers have employed a variety of methods, including bulk stable isotope and fatty acid composition analyses. Compound-specific stable isotope analysis (CSIA) of fatty acids combines both of these tools into an even more powerful method with the capacity to broaden our understanding of food web ecology and nutritional dynamics. Here, we provide an overview of the potential that CSIA studies hold and their constraints. We first review the use of fatty acid CSIA in ecology at the natural abundance level as well as enriched physiological tracers, and highlight the unique insights that CSIA of fatty acids can provide. Next, we evaluate methodological best practices when generating and interpreting CSIA data. We then introduce three cutting-edge methods: hydrogen CSIA of fatty acids, and fatty acid isotopomer and isotopologue analyses, which are not yet widely used in ecological studies, but hold the potential to address some of the limitations of current techniques. Finally, we address future priorities in the field of CSIA including: generating more data across a wider range of taxa; lowering costs and increasing laboratory availability; working across disciplinary and methodological boundaries; and combining approaches to answer macroevolutionary questions. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

U-shaped response Unifies views on temperature dependency of stoichiometric requirements.

Temperature and nutrient availability, which are major drivers of consumer performance, are dramatically affected by global change. To date, there is no consensus on whether warming increases or decreases consumer needs for dietary carbon (C) relatively to phosphorus (P), thus hindering predictions of secondary production responses to global change. Here, we investigate how the dietary C:P ratio optimising consumer growth (TERC:P : Threshold Elemental Ratio) changes along temperature gradients by combining a temperature-dependent TERC:P model with growth experiments on Daphnia magna. Both lines of evidence show that the TERC:P response to temperature is U-shaped. This shape indicates that consumer nutrient requirements can both increase or decrease with increasing temperature, thus reconciling previous contradictive observations into a common framework. This unified framework improves our capacity to forecast the combined effects of nutrient cycle and climatic alterations on invertebrate production.

High food quality increases infection of Gammarus pulex (Crustacea: Amphipoda) by the acanthocephalan parasite Pomphorhynchus laevis.

Parasitism is an important process in ecosystems, but has been largely neglected in ecosystem research. However, parasites are involved in most trophic links in food webs with, in turn, a major role in community structure and ecosystem processes. Several studies have shown that higher nutrient availability in ecosystems tends to increase the prevalence of parasites. Yet, most of these studies focused on resource availability, whereas studies investigating resource quality remain scarce. In this study, we tested the impact of the quality of host food resources on infection by parasites, as well as on the consequences for the host. Three resources were used to individually feed Gammarus pulex (Crustacea: Amphipoda) experimentally infected or not infected with the acanthocephalan species Pomphorhynchus laevis: microbially conditioned leaf litter without phosphorus input (standard resource); microbially conditioned leaf litter enriched in phosphorus; and microbially conditioned leaf litter without phosphorus input but complemented with additional inputs of benthic diatoms rich in both phosphorus and eicosapentaenoic acid. During the 110 day experiment, infection rate, parasite load, host survival, and parasite-mediated behavioral traits implicated in trophic transmission were measured (refuge use, geotaxis and locomotor activity). The resources of higher quality, regardless of the infection status, reduced gammarid mortality and increased gammarid growth. In addition, higher quality resources increased the proportion of infected gammarids, and led to more cases of multi-infections. While slightly modifying the geotaxis behavior of uninfected gammarids, resource quality did not modulate the impact of parasites on host behavior. Finally, for most parameters, consumption of algal resources had a greater impact than did phosphorus-enriched leaf litter. Therefore, manipulation of resource quality significantly affected host-parasite relationships, which stressed the need for future research to investigate in natura the relationships between resource availability, resource quality and parasite prevalence.

Interactive Impacts of Silver and Phosphorus on Autotrophic Biofilm Elemental and Biochemical Quality for a Macroinvertebrate Consumer.

Autotrophic biofilms are complex and fundamental biological compartments of many aquatic ecosystems. In particular, these biofilms represent a major resource for many invertebrate consumers and the first ecological barrier against toxic metals. To date, very few studies have investigated the indirect effects of stressors on upper trophic levels through alterations of the quality of biofilms for their consumers. In a laboratory study, we investigated the single and combined effects of phosphorus (P) availability and silver, a re-emerging contaminant, on the elemental [carbon (C):nitrogen (N):P ratios] and biochemical (fatty acid profiles) compositions of a diatom-dominated biofilm initially collected in a shallow lake. We hypothesized that (1) P and silver, through the replacement of diatoms by more tolerant primary producer species, reduce the biochemical quality of biofilms for their consumers while (2) P enhances biofilm elemental quality and (3) silver contamination of biofilm has negative effects on consumers life history traits. The quality of biofilms for consumers was assessed for a common crustacean species, Gammarus fossarum, by measuring organisms' survival and growth rates during a 42-days feeding experiment. Results mainly showed that species replacement induced by both stressors affected biofilm fatty acid compositions, and that P immobilization permitted to achieve low C:P biofilms, whatever the level of silver contamination. Gammarids growth and survival rates were not significantly impacted by the ingestion of silver-contaminated resource. On the contrary, we found a significant positive relationship between the biofilm P-content and gammarids growth. This study underlines the large indirect consequences stressors could play on the quality of microbial biomass for consumers, and, in turn, on the whole food web.

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.

A microcalorimetric approach for investigating stoichiometric constraints on the standard metabolic rate of a small invertebrate.

Understanding the determinants of metabolism is a core ecological topic since it permits to link individual energetic requirements to the ecology of communities and ecosystems. Yet, besides temperature, metabolic responses to environmental factors remain poorly understood. For example, it is commonly assumed that dietary stoichiometric constraints increase metabolism of small invertebrates despite scarce experimental support. Here, we used microcalorimetric measurements to determine the standard metabolic rate (SMR) of Daphnia magna fed stoichiometrically balanced (C/P: 166) or imbalanced (C/P: 1439). Daphnids fed imbalanced maintained their stoichiometric homeostasis within narrow boundaries. However, they consistently increased their SMR while decreasing their growth rate. Our measurements demonstrate that homeostatic regulation implies higher metabolic costs, thereby reducing available energy for growth. We demonstrate that microcalorimetry is a powerful and precise tool for measuring small-sized organisms' metabolic rate, thus opening promising perspectives for understanding how environmental factors, such as nutritional constraints, affect organismal metabolism.

[Strengths, weaknesses, and opportunities of French research in trophic ecology]. / Forces, failles et opportunités de la recherche française en écologie trophique.

The French National Institute of Ecology and Environment (INEE) aims at fostering pluridisciplinarity in Environmental Science and, for that purpose, funds ex muros research groups (GDR) on thematic topics. Trophic ecology has been identified as a scientific field in ecology that would greatly benefit from such networking activity, as being profoundly scattered. This has motivated the seeding of a GDR, entitled "GRET". The contours of the GRET's action, and its ability to fill these gaps within trophic ecology at the French national scale, will depend on the causes of this relative scattering. This study relied on a nationally broadcasted poll aiming at characterizing the field of trophic ecology in France. Amongst all the unique individuals that fulfilled the poll, over 300 belonged at least partly to the field of trophic ecology. The sample included all French public research institutes and career stages. Three main disruptions within the community of scientist in trophic ecology were identified. The first highlighted the lack of interfaces between microbial and trophic ecology. The second evidenced that research questions were strongly linked to single study fields or ecosystem type. Last, research activities are still quite restricted to the ecosystem boundaries. All three rupture points limit the conceptual and applied progression in the field of trophic ecology. Here we show that most of the disruptions within French Trophic Ecology are culturally inherited, rather than motivated by scientific reasons or justified by socio-economic stakes. Comparison with the current literature confirms that these disruptions are not necessarily typical of the French research landscape, but instead echo the general weaknesses of the international research in ecology. Thereby, communication and networking actions within and toward the community of trophic ecologists, as planned within the GRET's objectives, should contribute to fill these gaps, by reintegrating microbes within trophic concepts and setting the seeds for trans- and meta-ecosystemic research opportunities. Once the community of trophic ecologists is aware of the scientific benefit in pushing its boundaries forwards, turning words and good intentions into concrete research projects will depend on the opportunities to obtain research funding.

Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits.

Gammarus fossarum is an often-abundant crustacean detritivore that contributes importantly to leaf litter breakdown in oligotrophic, mainly heterotrophic, headwater streams. This species requires large amounts of Ca to moult, thus allowing growth and reproduction. Because resource quality is tightly coupled to the organism's growth and physiological status, we hypothesised that low Ca concentration [Ca] and low food resource quality (low phosphorus [P] and/or reduced highly unsaturated fatty acid [HUFA] contents) would interactively impair molecular responses (gene expression) and reproduction of G. fossarum. To investigate the effects of food resources quality, we experimentally manipulated the P content of sycamore leaves and also used diatoms because they contain high amounts of HUFAs. Three resource quality treatments were tested: low quality (LQ, unmanipulated leaves: low P content), high quality 1 (HQ1; P-manipulated leaves: high P content), and high quality 2 (unmanipulated leaves supplemented with a pellet containing diatoms: high P and HUFA content). Naturally, demineralised stream water was supplemented with CaSO4 to obtain three Ca concentrations (2, 3.5, and 10.5 mg Ca L-1). For 21 days, pairs of G. fossarum were individually exposed to one of the nine treatments (3 [Ca] × 3 resource qualities). At the individual level, strong and significant delays in moult stage were observed in gammarids exposed to lower [Ca] and to lower resource quality, with additive effects lengthening the duration of the reproductive cycle. Effects at the molecular level were investigated by measuring expression of 12 genes involved in energy production, translation, or Ca or P homeostasis. Expression of ATP synthase beta (higher in HQ2), calcified cuticle protein (higher in HQ1 and HQ2), and tropomyosin (higher in HQ2 compared to HQ1) was significantly affected by resource quality, and significant additive effects on Ca transporting ATPase expression were induced by [Ca] and resource quality (higher for low [Ca] and higher resource quality). These results highlight the potential drastic deleterious effects of water [Ca] depletion on G. fossarum physiology, populations, and ecosystem functioning, especially in oligotrophic environments.

Development of a Real-Time PCR assay for quantitative assessment of uncultured freshwater zoosporic fungi.

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi. Although they are known as saprobes and algal parasites in freshwater systems, zoosporic fungi have been neglected in microbial food web studies. Recently, it has been suggested that zoosporic fungi, via the consumption of their zoospores by zooplankters, could transfer energy from large inedible algae and particulate organic material to higher trophic levels. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of fungal zoospores in the field. Hence, quantitative data on fungal zoospores in natural environments is missing. We have developed a quantitative PCR (qPCR) assay for the quantification of fungal zoospores in lakes. Specific primers were designed and qPCR conditions were optimized using a range of target and non-target plasmids obtained from previous freshwater environmental 18S rDNA surveys. When optimal DNA extraction protocol and qPCR conditions were applied, the qPCR assay developed in this study demonstrated high specificity and sensitivity, with as low as 100 18S rDNA copies per reaction detected. Although the present work focuses on the design and optimization of a new qPCR assay, its application to natural samples indicated that qPCR offers a promising tool for quantitative assessment of fungal zoospores in natural environments. We conclude that this will contribute to a better understanding of the ecological significance of zoosporic fungi in microbial food webs of pelagic ecosystems.

From aquatic to terrestrial food webs: decrease of the docosahexaenoic acid/linoleic acid ratio.

Fatty acid composition of the adipose tissue of six carnivorous mammalian species (European otter Lutra lutra, American mink Mustela vison, European Mink Mustela lutreola, European polecat Mustela putorius, stone marten Martes foina and European wild cat Felis silvestris) was studied. These species forage to differing degrees in aquatic and terrestrial food webs. Fatty acid analysis revealed significant differences in polyunsaturated fatty acid composition between species. More specifically, our results underline a gradual significant decrease in the docosahexaenoic acid (DHA)/linoleic acid (LNA) ratio of carnivore species as their dependence on aquatic food webs decreases. In conclusion, the use of the DHA/LNA ratio in long-term studies is proposed as a potential proxy of changes in foraging behaviour of semi-aquatic mammals.

Supplementation with sterols improves food quality of a ciliate for Daphnia magna.

Experimental results provide evidence that trophic interactions between ciliates and Daphnia are constrained by the comparatively low food quality of ciliates. The dietary sterol content is a crucial factor in determining food quality for Daphnia. Ciliates, however, presumably do not synthesize sterols de novo. We hypothesized that ciliates are nutritionally inadequate because of their lack of sterols and tested this hypothesis in growth experiments with Daphnia magna and the ciliate Colpidium campylum. The lipid content of the ciliate was altered by allowing them to feed on fluorescently labeled albumin beads supplemented with different sterols. Ciliates that preyed upon a sterol-free diet (bacteria) did not contain any sterols, and growth of D. magna on these ciliates was poor. Supplementation of the ciliates' food source with different sterols led to the incorporation of the supplemented sterols into the ciliates' cells and to enhanced somatic growth of D. magna. Sterol limitation was thereby identified as the major constraint of ciliate food quality for Daphnia. Furthermore, by supplementation of sterols unsuitable for supporting Daphnia growth, we provide evidence that ciliates as intermediary grazers biochemically upgrade unsuitable dietary sterols to sterols appropriate to meet the physiological demands of Daphnia.