Dissolved organic matter transformations in a freshwater rivermouth.

River-to-lake transitional areas are biogeochemically active ecosystems that can alter the amount and composition of dissolved organic matter (DOM) as it moves through the aquatic continuum. However, few studies have directly measured carbon processing and assessed the carbon budget of freshwater rivermouths. We compiled measurements of dissolved organic carbon (DOC) and DOM in several water column (light and dark) and sediment incubation experiments conducted in the mouth of the Fox river (Fox rivermouth) upstream from Green Bay, Lake Michigan. Despite variation in the direction of DOC fluxes from sediments, we found that the Fox rivermouth was a net sink of DOC where water column DOC mineralization outweighed the release of DOC from sediments at the rivermouth scale. Although we found DOM composition also changed during our experiments, alterations in DOM optical properties were largely independent of the direction of sediment DOC fluxes. We found a consistent decrease in humic-like and fulvic-like terrestrial DOM and a consistent increase in the overall microbial composition of rivermouth DOM during our incubations. Moreover, greater ambient total dissolved phosphorus concentrations were positively associated with the consumption of terrestrial humic-like, microbial protein-like, and more recently derived DOM but had no effect on bulk DOC in the water column. Unexplained variation indicates that other environmental controls and water column processes affect the processing of DOM in this rivermouth. Nonetheless, the Fox rivermouth appears capable of substantial DOM transformation with implications for the composition of DOM entering Lake Michigan. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-01000-z.

Characterizing nutritional phenotypes using experimental nutrigenomics: Is there nutrient-specificity to different types of dietary stress?

The ability to directly measure and monitor poor nutrition in individual animals and ecological communities is hampered by methodological limitations. In this study, we use nutrigenomics to identify nutritional biomarkers in a freshwater zooplankter, Daphnia pulex, a ubiquitous primary consumer in lakes and a sentinel of environmental change. We grew animals in six ecologically relevant nutritional treatments: nutrient replete, low carbon (food), low phosphorus, low nitrogen, low calcium and high Cyanobacteria. We extracted RNA for transcriptome sequencing to identify genes that were nutrient responsive and capable of predicting nutritional status with a high degree of accuracy. We selected a list of 125 candidate genes, which were subsequently pruned to 13 predictive potential biomarkers. Using a nearest-neighbour classification algorithm, we demonstrate that these potential biomarkers are capable of classifying our samples into the correct nutritional group with 100% accuracy. The functional annotation of the selected biomarkers revealed some specific nutritional pathways and supported our hypothesis that animal responses to poor nutrition are nutrient specific and not simply different presentations of slow growth or energy limitation. This is a key step in uncovering the causes and consequences of nutritional limitation in animal consumers and their responses to small- and large-scale changes in biogeochemical cycles.

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.

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.

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.

Variable silver nanoparticle toxicity to Daphnia in boreal lakes.

Variable sensitivity of organisms to silver nanoparticles (AgNPs) caused by changes in physico-chemical variables in aquatic ecosystems is receiving increasing attention. Variables such as dissolved organic carbon, pH, light, the presence of algae and bacteria, dissolved oxygen and different ions have all been studied individually, but it is still unclear how these variables in combination alter AgNP toxicity in natural ecosystems. Here we examined AgNP toxicity on survival of wild-caught Daphnia using AgNP suspensions placed in water from several different lakes at the IISD-Experimental Lakes Area, which span a gradient of water quality parameters. The partitioning of AgNPs between particulate and dissolved organic matter fractions was also assessed due to the potential for algal sequestration and detoxification of AgNPs. We found that toxicity varied between lakes with LC50 values ranging between 34 and 292µg AgL-1. Time of year in terms of days since ice-off and carbon to nitrogen ratios of particulate matter were the major predictors of toxicity between ecosystems. Total dissolved phosphorus, dissolved organic carbon, and particulate carbon to phosphorus ratios also played minor roles in influencing survival of Daphnia between water types. We found variable partitioning of silver into the particulate fraction within lakes and no significant differences between lakes. Silver associated with particulate organic matter increased with increasing concentrations of AgNPs in the ecosystem. Overall, we found strong evidence that AgNP toxicity is highly context dependent in natural lake ecosystems.

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.

Exploited and excreting: parasite type affects host nutrient recycling.

Parasite-induced changes in the nutrient balance of hosts could alter the availability of nutrients in ecosystems by changing consumer-driven nutrient recycling. While these effects on host nutrient use are mediated by host physiology, they likely depend on characteristics of the parasite and host diet quality. We examined this possibility by measuring nutrient release rates of uninfected Daphnia and conspecifics infected by two microparasites (the bacterium Pasteuria ramosa and the microsporidium Hamiltosporidium tvaerminnensis) from daphnid hosts fed food that varied in phosphorus content. We found that infection type and diet affected host nutrient release rates, but the strength of these effects varied among parasite treatments. To improve our understanding of these effects, we examined whether two separate aspects of host exploitation (parasite-induced reductions in host fecundity and parasite load) could account for variation in Daphnia nutrient release, ingestion, and elemental ratios caused by our infection and diet treatments. Regardless of whether we compared individuals across infection type or diet treatment, Daphnia fecundity described variation in multiple aspects of host nutrient use better than infection, diet, or spore load. Our results suggest that parasite-induced changes in host nutrient use are both parasite and diet specific, and that host fecundity could be a useful parameter for predicting the magnitude and direction of these changes.

Human activities cause distinct dissolved organic matter composition across freshwater ecosystems.

Dissolved organic matter (DOM) composition in freshwater ecosystems is influenced by the interactions among physical, chemical, and biological processes that are controlled, at one level, by watershed landscape, hydrology, and their connections. Against this environmental template, humans may strongly influence DOM composition. Yet, we lack a comprehensive understanding of DOM composition variation across freshwater ecosystems differentially affected by human activity. Using optical properties, we described DOM variation across five ecosystem groups of the Laurentian Great Lakes region: large lakes, Kawartha Lakes, Experimental Lakes Area, urban stormwater ponds, and rivers (n = 184 sites). We determined how between ecosystem variation in DOM composition related to watershed size, land use and cover, water quality measures (conductivity, dissolved organic carbon (DOC), nutrient concentration, chlorophyll a), and human population density. The five freshwater ecosystem groups had distinctive DOM composition from each other. These significant differences were not explained completely through differences in watershed size nor spatial autocorrelation. Instead, multivariate partial least squares regression showed that DOM composition was related to differences in human impact across freshwater ecosystems. In particular, urban/developed watersheds with higher human population densities had a unique DOM composition with a clear anthropogenic influence that was distinct from DOM composition in natural land cover and/or agricultural watersheds. This nonagricultural, human developed impact on aquatic DOM was most evident through increased levels of a microbial, humic-like parallel factor analysis component (C6). Lotic and lentic ecosystems with low human population densities had DOM compositions more typical of clear water to humic-rich freshwater ecosystems but C6 was only present at trace to background levels. Consequently, humans are strongly altering the quality of DOM in waters nearby or flowing through highly populated areas, which may alter carbon cycles in anthropogenically disturbed ecosystems at broad scales.

Does infection tilt the scales? Disease effects on the mass balance of an invertebrate nutrient recycler.

While parasites are increasingly recognized as important components of ecosystems, we currently know little about how they alter ecosystem nutrient availability via host-mediated nutrient cycling. We examined whether infection alters the flow of nutrients through hosts and whether such effects depend upon host diet quality. To do so, we compared the mass specific nutrient (i.e., nitrogen and phosphorus) release rates, ingestion rates, and elemental composition of uninfected Daphnia to those infected with a bacterial parasite, P. ramosa. N and P release rates were increased by infection when Daphnia were fed P-poor diets, but we found no effect of infection on the nutrient release of individuals fed P-rich diets. Calculations based on the first law of thermodynamics indicated that infection should increase the nutrient release rates of Daphnia by decreasing nutrient accumulation rates in host tissues. Although we found reduced nutrient accumulation rates in infected Daphnia fed all diets, this reduction did not increase the nutrient release rates of Daphnia fed the P-rich diet because infected Daphnia fed this diet ingested nutrients more slowly than uninfected hosts. Our results thus indicate that parasites can significantly alter the nutrient use of animal consumers, which could affect the availability of nutrients in heavily parasitized environments.

Phosphorus Availability Alters the Effects of Silver Nanoparticles on Periphyton Growth and Stoichiometry.

Exposure to silver nanoparticles (AgNPs) may alter the structure and function of freshwater ecosystems. However, there remains a paucity of studies investigating the effects of AgNP exposure on freshwater communities in the natural environment where interactions with the ambient environment may modify AgNP toxicity. We used nutrient diffusing substrates to determine the interactive effects of AgNP exposure and phosphorus (P) enrichment on natural assemblages of periphyton in three Canadian Shield lakes. The lakes were all phosphorus poor and spanned a gradient of dissolved organic carbon availability. Ag slowly accumulated in the exposed periphyton, which decreased periphyton carbon and chlorophyll a content and increased periphyton C:P and N:P in the carbon rich lakes. We found significant interactions between AgNP and P treatments on periphyton carbon, autotroph standing crop and periphyton stoichiometry in the carbon poor lake such that P enhanced the negative effects of AgNPs on chlorophyll a and lessened the impact of AgNP exposure on periphyton stoichiometry. Our results contrast with those of other studies demonstrating that P addition decreases metal toxicity for phytoplankton, suggesting that benthic and pelagic primary producers may react differently to AgNP exposure and highlighting the importance of in situ assays when assessing potential effects of AgNPs in fresh waters.

Nutrient loading associated with agriculture land use dampens the importance of consumer-mediated niche construction.

The linkages between biological communities and ecosystem function remain poorly understood along gradients of human-induced stressors. We examined how resource provisioning (nutrient recycling), mediated by native freshwater mussels, influences the structure and function of benthic communities by combining observational data and a field experiment. We compared the following: (1) elemental and community composition (algal pigments and macroinvertebates) on live mussel shells and on nearby rocks across a gradient of catchment agriculture and (2) experimental colonisation of benthic communities on live vs. sham shells controlling for initial community composition and colonisation duration. We show that in near pristine systems, nutrient heterogeneity mediated by mussels relates to greater biodiversity of communities, which supports the notion that resource heterogeneity can foster biological diversity. However, with increased nutrients from the catchment, the relevance of mussel-provisioned nutrients was nearly eliminated. While species can persist in disturbed systems, their functional relevance may be diminished or lost.

Phosphorus nutrition alters herbicide toxicity on Daphnia magna.

We examined the effects of algal phosphorus (P) content on the toxicity of a common herbicide, WeatherMAX Roundup (WMR), to Daphnia magna. The growth, reproduction, and survival of D. magna were assessed with animals consuming different food P content and exposed to different concentrations of WMR. While the effects of WMR on mortality increased with time of exposure, we found no interactive effects between food P content and WMR on daphnid survival over any of time periods examined (4, 10, or 20 days). In contrast, we found interactive effects of WMR and dietary P content on Daphnia juvenile growth (measured after 6 days) with the greatest effects of WMR on animals consuming P-rich food. Interactive effects of WMR and food P content were also found on some aspects of Daphnia's reproduction (number of broods and total offspring production) with P-deprived animals most affected by WMR exposure. Our results demonstrate that P-nutrition can alter the toxicity of WMR on key life-history traits of D. magna but that the nature and strength of these effects differ among the traits examined. The effects of P-nutrition on WMR-toxicity likely reflect changes in the exposure to and/or incorporation of WMR into animal bodies associated with changes in growth resulting from poor nutrition and the ability of animals to repair ensuing damage. Given the widely variable nutritional state of animals in nature, this differential toxicity of WMR with food quality warrants further study and should be incorporated in future risk assessments of this widely used chemical.

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.

Watershed discharge modulates relationships between landscape components and nutrient ratios in stream seston.

In order to understand the environmental controls of the elemental composition of suspended particles in streams and rivers, we examined relationships between the stoichiometry of suspended particles and catchment characteristics (e.g., topography and land cover) under contrasting hydrological conditions in streams from the Upper Peninsula of Michigan, USA. Particulate C:N, N:P, and C:P ratios varied considerably among streams on each of 10 separate sampling dates and within most streams through time. When averaged across the study period by stream, particulate C:N:P ratios were strongly related to several catchment properties. For example, particulate C:P and N:P ratios were negatively related to the percentage of catchment used for agriculture but positively related to the percentage of catchment found as wetlands. These relationships reflected, in part, variable concentrations of mineral particles, which were strongly related to stream length, agriculture, and upstream lake area. In addition, the strength of the relationships between particulate elemental composition and catchment properties changed depending upon hydrologic condition of the Ontonagon watershed. For example, periods of wetness, as indicated by high river discharge, were found to increase the strength of seston C:N ratio-percentage of wetlands relationships but to reduce the strength of seston C:P ratios-percentage of wetlands relationships. Our results thus demonstrate that the balance of elements fluxing through forested streams is strongly affected by catchment properties but that these effects are sensitive to watershed discharge. At times of high discharge, there are strong connections to catchment sources while in-stream processes appear to predominate at time of low discharge.

Bacterial infection changes the elemental composition of Daphnia magna.

1. An animal's elemental composition can be an important indicator of its physiological state and role in ecosystem nutrient cycling. We examined the interactive effects of bacterial (Pasteuria ramosa) infection and phosphorus (P)-poor food on the body stoichiometry of Daphnia magna. Daphnia were exposed to or held free of a bacterial parasite and fed algal food of different C:P ratios (100-500) over a 28-day period. 2. To assess the effects of exposure and infection on Daphnia stoichiometry, we measured their whole body content of carbon (C), nitrogen (N), and P on four different days (4, 8, 15, and 28) during the experiment. 3. We found strong effects of infection, food quality, and/or their interactions on the C, N, and P content of Daphnia, especially as the infectious disease progressed. At the end of the experiment, infected animals had significantly more C and less P in their bodies than uninfected conspecifics. Body N content of Daphnia consuming P-rich food was reduced by bacterial infection whereas Daphnia consuming P-poor algae showed increased body N content from infection. 4. Using a mass-balance model, we found that changes in N and P content of host bodies were largely accountable by disease-induced alterations to Daphnia reproduction (i.e. bacterial induced sterility) and the accumulation of Pasteuria spores in the body cavity. Our calculations also show that the observed increase in host C content could not be accounted for by loss of eggs or accumulation of bacterial spores. This instead must result from unidentified changes to underlying daphnid tissue C content. 5. These results demonstrate that intraspecific variation in zooplankton body stoichiometry can be caused by exposure to and infection by bacterial parasites. In addition, these effects were found to depend both upon the stage of the disease and upon the food quality consumed by the host.

Responses of a bacterial pathogen to phosphorus limitation of its aquatic invertebrate host.

Host nutrition is thought to affect the establishment, persistence, and severity of pathogenic infections. Nutrient-deficient foods possibly benefit pathogens by constraining host immune function or benefit hosts by limiting parasite growth and reproduction. However, the effects of poor elemental food quality on a host's susceptibility to infection and disease have received little study. Here we show that the bacterial microparasite Pasteuria ramosa is affected by the elemental nutrition of its aquatic invertebrate host, Daphnia magna. We found that high food carbon:phosphorus (C:P) ratios significantly reduced infection rates of Pasteuria in Daphnia and led to lower within-host pathogen multiplication. In addition, greater virulent effects of bacterial infection on host reproduction were found in Daphnia-consuming P-deficient food. Poor Daphnia elemental nutrition thus reduced the growth and reproduction of its bacterial parasite, Pasteuria. The effects of poor host nutrition on the pathogen were further evidenced by Pasteuria's greater inhibition of reproduction in P-limited Daphnia. Our results provide strong evidence that elemental food quality can significantly influence the incidence and intensity of infectious disease in invertebrate hosts.

Threshold elemental ratios of carbon and phosphorus in aquatic consumers.

Inadequate supply of one or more mineral elements can slow the growth of animal consumers and alter their physiology, life history and behaviour. A key concept for understanding nutrient deficiency in animals is the threshold elemental ratio (TER), at which growth limitation switches from one element to another. We used a stoichiometric model that coupled animal bioenergetics and body elemental composition to estimate TER of carbon and phosphorus (TER(C:P)) for 41 aquatic consumer taxa. We found a wide range in TER(C:P) (77-3086, ratio by atoms), which was generated by interspecific differences in body C : P ratios and gross growth efficiencies of C. TER(C:P) also varied among aquatic invertebrates having different feeding strategies, such that detritivores had significantly higher threshold ratios than grazers and predators. The higher TER(C:P) in detritivores resulted not only from lower gross growth efficiencies of carbon but also reflected lower body P content in these consumers. Supporting previous stoichiometric theory, we found TER(C:P) to be negatively correlated with the maximum growth rate of invertebrate consumers. By coupling bioenergetics and stoichiometry, this analysis revealed strong linkages among the physiology, ecology and evolution of nutritional demands for animal growth.