Flow cytometric analysis of the encystment process induced by paraquat exposure in Haematococcus pluvialis (Chlorophyceae)

Abstract

The freshwater microalga Haematococcus pluvialis exhibits a unique morphological response to environmental stress, accumulating carotenoid pigment during encystment. The complexity of characterizing the different cell stages and monitoring the pigment cell content during the life cycle of this microalga is one of the main problems reported when assessing astaxanthin accumulation and degradation. Therefore, with the aim of studying the potential encystment response in this microalga by means of flow cytometry (FCM), we induced oxidative stress in cultures of vegetative growing cells by treating them with paraquat, a known generator of superoxide anion radicals. Two flow cytometric approaches were successfully used to monitor the effect of oxidative stress on morphological changes and genesis of carotenoids in H. pluvialis: (1) a cytometric characterization of different cell types based on analysis of the fluorescence of chlorophyll a vs the fluorescence of astaxanthin, and (2) staining with the fluorochromes hydroethidium (HE) and dihydrorhodamine 123 (DHR), in order to measure the in vivo intracellular levels of reactive oxygen species (ROS). FCM data showed that astaxanthin accumulation during encystment hampers the production of ROS. Furthermore, the cell content of astaxanthin seems to be a good indicator of the extent to which H. pluvialis cells undergo oxidative stress, and also of how the cells defend themselves under stress conditions.