Well-fed or nearly dead? Using quantitative PCR to detect dietary stress
in Daphnia pulex
Abstract
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 measured the growth rate and elemental
composition of these animals and extracted their RNA. We then selected
ten nutrient sensitive genes, two per limiting nutrient, and two
reference genes from an RNA sequencing dataset of Daphnia pulex grown
under the same six nutritional treatments. We then designed and
validated qPCR primers for the ten indicator and two reference genes. We
ran qPCR using these primers on cDNA from our experimental animals and
found that the differential expression patterns of these genes 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