Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Aoao Yang,Fengting Li,Ying Zhong Tang,Siheng Lin,Yunyan Deng,Zhangxi Hu

doi:10.3390/ijms22147325

Abstract

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the β subunit of ATP synthase gene (β-F1-ATPase), a key indicator for the status of cell’s energetic metabolism. The full-length cDNA of β-F1-ATPase gene from S.trochoidea (Stβ-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stβ-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stβ-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stβ-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

Highlights

Harmful algae blooms (HABs) are ecological phenomenon that pose serious impacts on ecosystems, economy, and public health and have been increasing globally [1]
In the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, 57 sequences were annotated into metabolic pathways, accounting for 70% of all sequences that could be annotated in the KEGG database, and 20% of these 57 sequences could be annotated into the energy metabolism (Figure 1)
Due to the central role played by ATPase in the energetic metabolism, a 159 bp fragment annotated as gene encoding “β subunit of Adenosine triphosphate (ATP) synthase” was selected for further characterizations

Summary

Introduction

Harmful algae blooms (HABs) are ecological phenomenon that pose serious impacts on ecosystems, economy, and public health and have been increasing globally [1]. The encystment (i.e., formation of resting cysts) for most, if not all, species of dinoflagellates is associated with a sexual process whereby the fusion of two motile gametes yields a planozygote that can eventually develop into a non-motile resting cyst and sink into sediments [8,9] Such cysts have been shown to have essential functions in the biology and ecology of dinoflagellates, especially for HABs-causative species, since they are related with recombination of genes, generation and termination of blooms, expansion of biogeographical distribution, and self-protection from viruses, grazers, or parasite attacks [6,7,8,9,10,11,12,13,14,15,16,17]. Resting cyst production was described in many species and considered for being a key adaptation or survival strategy of dinoflagellates, relatively little information had been obtained about the physiology of dormancy in resting cysts, about the energetic metabolism

Methods

Results

Conclusion