RESUMO
The increasing inflow of nitrogen (N) substrates into marine nearshore ecosystems induces proliferation of harmful algal blooms (HABs) of dinoflagellates, such as potentially toxic invasive species Prorocentrum minimum. In this study, we estimated the influence of NO3-, NH4+ and urea on transcription levels and urea transporter dur3 and nitrate transporter nrt2 genes expression in these dinoflagellates. We identified dur3 and nrt2 genes sequences in unannotated transcriptomes of P. minimum and other dinoflagellates presented in MMETSP database. Phylogenetic analysis showed that these genes of dinoflagellates clustered to the distinct clade demonstrating evolutionary relationship with the other known dur3 and nrt2 genes of microalgae. The evaluation of expression levels of dur3 and nrt2 genes by RT-qPCR revealed their sensitivity to input of the studied N sources. Dur3 expression levels were downregulated after the supplementation of additional N sources and were 1.7-2.6-fold lower than in the nitrate-grown culture. Nrt2 expression levels decreased 1.9-fold in the presence of NH4+. We estimated total RNA and DNA synthesis rates by the analysis of incorporation of 3H-thymidine and 3H-uridine in batch and continuous cultures. Addition of N compounds did not affect the DNA synthesis rates. Transcription levels increased up to 12.5-fold after the N supplementation in urea-limited treatments. Investigation of various nitrogen sources as biomarkers of dinoflagellate proliferation due to their differentiated impact on expression of dur3 and nrt2 genes and transcription rates in P. minimum cells allowed concluding about high potential of the studied parameters for future modeling of HABs under global N pollution.
