Proteome changes in an aquatic invertebrate consumer in response to different nutritional stressors.

Nutrient imbalances in zooplankton are caused by the differences in elemental content of producers and the demand for elements in consumers, which alter the life-history traits in consumers. Changes in life-history traits are mediated through metabolic pathways that affect gene expression and the metabolome. However, less is known about proteomic changes to elemental-limitation in zooplankton. Here, we grew Daphnia pulex under high food quantity and quality (HF), low food quantity (LF), and phosphorus (P)-limited (PL) diets for six days and measured growth, elemental composition, and the proteome. Daphnids in both LF and PL diets grew less. Animals in LF diets had less carbon (C), while daphnids in PL diets had less P compared to HF fed animals. In total, we identified 1719 proteins that were used in a partial least squares regression discriminant analysis (PLS-DA). Focusing on a subset of the proteome, the PLS-DA resulted in a clear separation between animals fed HF diets and PL and LF diets. Many proteome changes in nutrient-limited diets are associated with growth, reproduction, lipid metabolism, and nutrient assimilation. Regardless of the limiting nutrient, there were less hemoglobin and small subunit processome component proteins compared to HF fed animals. Daphnids fed LF diets had less vitellogenin fused superoxide dismutase and more lipid-droplet hydrolase, whereas Daphnia fed PL diets had higher abundances of cytochrome P450 and serine protease. Our proteome results compliment other "omic" studies that could be used to study Daphnia physiology in lakes.

Biological stoichiometry and growth dynamics of a diazotrophic cyanobacteria in nitrogen sufficient and deficient conditions.

The role of nitrogen (N) fixation in determining the frequency, magnitude, and extent of harmful algal blooms (HABs) has not been well studied. Dolichospermum is a common HAB species that is diazotrophic (capable of N fixation) and thus growth is often considered never to be limited by low combined N sources. However, N fixation is energetically expensive and its cost during bloom formation has not been quantified. Additionally, it is unknown how acclimation to differing nutrient ratios affects growth and cellular carbon (C):N stoichiometry. Here, we test the hypotheses that diazotrophic cyanobacteria are homeostatic for N because of their ability to fix atmospheric N2 and that previous acclimation to low N environments will result in more fixed N and lower C:N stoichiometry. Briefly, cultures that varied in resource N:phosphorus (P) ranging from 0.01 to 100 (atom), were seeded with Dolichospermum which were previously acclimated to low and high N:P conditions and then sampled temporally for growth and C:N stoichiometry. We found that Dolichospermum was not homeostatic for N and displayed classic signs of N limitation and elevated C:N stoichiometry, highlighting the necessary growth trade-off within cells when expending energy to fix N. Acclimation to N limited conditions caused differences in both C:N and fixed N at various time points in the experiment. These results highlight the importance of environmentally available N to a diazotrophic bloom, as well as how previous growth conditions can influence population growth during blooms experiencing variable N:P.

The complexity of co-limitation: nutrigenomics reveal non-additive interactions of calcium and phosphorus on gene expression in Daphnia pulex.

Many lakes across Canada and northern Europe have experienced declines in ambient phosphorus (P) and calcium (Ca) supply for over 20 years. While these declines might create or exacerbate nutrient limitation in aquatic food webs, our ability to detect and quantify different types of nutrient stress on zooplankton remains rudimentary. Here, we used growth bioassay experiments and whole transcriptome RNAseq, collectively nutrigenomics, to examine the nutritional phenotypes produced by low supplies of P and Ca separately and together in the freshwater zooplankter Daphnia pulex. We found that daphniids in all three nutrient-deficient categories grew slower and differed in their elemental composition. Our RNAseq results show distinct responses in singly limited treatments (Ca or P) and largely a mix of these responses in animals under low Ca and P conditions. Deeper investigation of effect magnitude and gene functional annotations reveals this patchwork of responses to cumulatively represent a co-limited nutritional phenotype. Linear discriminant analysis identified a significant separation between nutritional treatments based upon gene expression patterns with the expression patterns of just five genes needed to predict animal nutritional status with 99% accuracy. These data reveal how nutritional phenotypes are altered by individual and co-limitation of two highly important nutritional elements (Ca and P) and provide evidence that aquatic consumers can respond to limitation by more than one nutrient at a time by differentially altering their metabolism. This use of nutrigenomics demonstrates its potential to address many of the inherent complexities in studying interactions between multiple nutritional stressors in ecology and beyond.

Biological Stoichiometry Regulates Toxin Production in Microcystis aeruginosa (UTEX 2385).

Harmful algal blooms (HABs) are increasing in magnitude, frequency, and duration globally. Even though a limited number of phytoplankton species can be toxic, they are becoming one of the greatest water quality threats to public health and ecosystems due to their intrinsic toxicity to humans and the numerous interacting factors that undermine HAB forecasting. Here, we show that the carbon:nitrogen:phosphorus (C:N:P) stoichiometry of a common toxic phytoplankton species, Microcystis, regulates toxin quotas during blooms through a tradeoff between primary and secondary metabolism. Populations with optimal C:N (< 8) and C:P (< 200) cellular stoichiometry consistently produced more toxins than populations exhibiting stoichiometric plasticity. Phosphorus availability in water exerted a strong control on population biomass and C:P stoichiometry, but N availability exerted a stronger control on toxin quotas by regulating population biomass and C:N:P stoichiometry. Microcystin-LR, like many phytoplankton toxins, is an N-rich secondary metabolite with a C:N stoichiometry that is similar to the optimal growth stoichiometry of Microcystis. Thus, N availability relative to P and light provides a dual regulatory mechanism that controls both biomass production and cellular toxin synthesis. Overall, our results provide a quantitative framework for improving forecasting of toxin production during HABs and compelling support for water quality management that limit both N and P inputs from anthropogenic sources.

The threshold elemental ratio of carbon and phosphorus of Daphnia magna and its connection to animal growth.

The growth of animal consumers is affected by the balance of elements in their diet with the transition between limitation by one element to another known as the threshold elemental ratio (TER). Precise estimates of TERs with known levels of uncertainty have yet to be generated for most zooplankton consumers. We determined the TER for carbon (C) and phosphorus (P) in for a common lake zooplankter, Daphnia magna, using experimental measurements and theoretical considerations. Daphnia growth responses to food C:P ratios across a relatively narrow range (80-350) generated an empirical estimate of TERC:P of 155 ± 14. While this TER matched our modelled estimate of TERC:P (155 ± 16), it was lower than previous estimates of this dietary transition point. No threshold was found when we examined daphnid body C:N or C:P ratios in response to changing food C:P ratios, which indicates P-limitation at even lower food C:P ratios. Our results provide strong evidence that D. magna is likely to experience acute P-limitation when food C:P ratios exceed even relatively low ratios (~155). Our model further demonstrated that while physiological adjustments may reduce the likelihood of P-limitation or reduce its intensity, these changes in animal material processing would be accompanied by reduced maximum growth rates.

Interactive effects of genotype and food quality on consumer growth rate and elemental content.

Consumer body stoichiometry is a key trait that links organismal physiology to population and ecosystem-level dynamics. However, as elemental composition has traditionally been considered to be constrained within a species, the ecological and evolutionary factors shaping consumer elemental composition have not been clearly resolved. To this end, we examined the causes and extent of variation in the body phosphorus (P) content and the expression of P-linked traits, mass specific growth rate (MSGR), and P use efficiency (PUE) of the keystone aquatic consumer Daphnia using lake surveys and common garden experiments. While daphnid body %P was relatively constrained in field assemblages sampled across an environmental P gradient, unique genotypes isolated from these lakes showed highly variable phenotypic responses when raised across dietary P gradients in the laboratory. Specifically, we observed substantial inter- and intra-specific variation and differences in daphnid responses within and among our study lakes. While variation in Daphnia body %P was mostly due to plastic phenotypic changes, we documented considerable genetic differences in daphnid MSGR and PUE, and relationships between MSGR and body P content were highly variable among genotypes. Overall, our study found that consumer responses to food quality may differ considerably among genotypes and that relationships between organismal life-history traits and body stoichiometry may be strongly influenced by genetic and environmental variation in natural assemblages.

Responses of alkaline phosphatase activity in Daphnia to poor nutrition.

The use of biochemical and molecular indices of nutritional stress have recently been promoted for their potential ability to assess the in situ nutritional state of zooplankton. The development and application of these indicators should at least consider the cross-reactivity with other nutritional stressors. We examined the potential usefulness of body alkaline phosphatase activity (APA) as an indicator of dietary phosphorus (P) stress in Daphnia. We measured growth rate, body P-content, and body APA of two species of Daphnia (D. magna, D. pulex) grown for different periods under diverse dietary conditions. We found P-poor food reduced daphnid growth rates and body P-content, while body APA increased in both species. However, body APA increased in P-sufficient D. magna and D. pulex that were feeding on cyanobacterial compared to green algal food, despite no differences in animal body P content. Body APA increased in D. magna fed P-poor food whether cyanobacterial or algal. Body APA also varied with age and other nutritional stresses (low food quantity, nitrogen-poor algae) in both daphnid species. Our results demonstrate that whole body homogenate APA in Daphnia is not singularly responsive to P-poor food, which will complicate or limit its future usefulness and application as an indicator of dietary P-stress in metazoans.