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.

Environmental Stress and the Morphology of Daphnia pulex.

AbstractMorphological variation is sometimes used as an indicator of environmental stress in animals. Here, we assessed how multiple morphological traits covaried in Daphnia pulex exposed to five common forms of environmental stress (high temperature, presence of predator cues, high salinity, low food abundance, and low Ca). We measured animal body length, body width, head width, eyespot diameter, and tail spine length along with mass in animals of five different ages (3, 6, 9, 12, and 15 d). There were strong allometric relationships among all morphological traits in reference animals and strong univariate effects of environmental stress on body mass and body length. We found that environmental stressors altered bivariate relationships between select pairwise combinations of morphological traits, with effects being dependent on animal age. Multivariate analyses further revealed high connectivity among body size-related traits but that eyespot diameter and tail spine length were less tightly associated with body size. Animals exposed to natural lake water with and without supplemental food also varied in morphology, with body size differences being suggestive of starvation and other unknown nutritional deficiencies. Yet our results demonstrate that the scaling of body morphological traits of Daphnia pulex is largely invariant with possible context-dependent plasticity in eye size and tail spine lengths. The strong coordination of traits indicates tight molecular coordination of body size during development despite strong and varied environmental stress.

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.