Copy number variation of a protease gene of Daphnia: Its role in population tolerance

Copy number variation (CNV) of genes coding for certain enzymes has been shown to be responsible for adaptation of arthropods to anthropogenic toxins. Natural toxins produced by cyanobacteria in freshwater ecosystems, that is, protease inhibitors (Pls), have been demonstrated to increase in frequency over the last decades due to eutrophication and global warming. These Pls inhibit digestive proteases of Daphnia, the major herbivore of phytoplankton and cyanobacteria. The adjustment of isoforms, differences in gene expression, and activity of gut proteases determine tolerance to dietary Pls in single Daphnia genotypes. Here, we tested whether similar mechanisms are also responsible for differences in tolerance among Daphnia population. We developed a droplet digital PCR (ddPCR) method for the analysis of CNV of Daphnia proteases. We report that one Daphnia protease gene showed CNV between populations and that CNV correlates with chymotrypsin gene expression among populations. We showed that populations of Daphnia magna differ in tolerance to cyanobacterial Pls according to the cyanobacterial background of their lake of origin, which hints at local adaptation. The tolerance of the populations correlates with IC50 values of their chymotrypsins, which is probably due to a combined effect of CNV(translating into gene expression differences) and positive selection of tolerant protease isoforms. This is the first study using ddPCR to demonstrate CNV of a gene with ecologically relevant function, and the first report of differences in tolerance to cyanobacterial Pls among Daphnia populations in combination with the assessment of underlying molecular mechanisms.