Well-fed or nearly dead? Using quantitative PCR to detect dietary stress in Daphnia pulex.

Nutrition is at the center of interactions between organisms and their environment. During this time of unprecedented anthropogenic environmental change, biological indicators of nutritional changes and their effects on ecosystems are crucial if we are to understand and mitigate these changes. In this study, we developed a qPCR assay to examine nutritional responsiveness of ten genes identified as potential indicators of nutritional state in the freshwater zooplankton Daphnia pulex. We grew animals in six ecologically relevant treatments: nutrient replete, low carbon (food), low phosphorus, low nitrogen, low calcium, and high Cyanobacteria. We selected ten nutrient sensitive genes, two per limiting nutrient, and two reference genes from an RNA sequencing dataset. We found that using the nutrient-specific pairs of genes for single nutrient limitation assays showed mixed results with some treatments (calcium, nitrogen, carbon) with high levels of treatment discrimination, but gene responses designed for other treatments (phosphorus and Cyanobacteria) showed little to no treatment discrimination. However, when we used all ten genes as a single nutritional state assay, we could discriminate between our six nutritional states with high levels of accuracy. These results represent a compelling proof of concept for the use of gene-based nutritional biomarkers, paving the way for the use of genetically based nutritional state biomarkers in the study of ecology