Paramecium, a ciliated protozoan, moves using thousands of cilia that cover the entire cell body. When Paramecium comes into contact with harmful substances such as ethanol, its cilia spontaneously detach and motility is substantially reduced. In the present study, Paramecium cells were shown, for the first time, to acquire tolerance to toxic ethanol concentrations after pretreatment with lower, non-toxic concentrations. The effect of pretreatment was characterized by counting numbers of non-deciliated cells. Key findings were: (1) pretreated cells retained the acquired tolerance to ethanol for at least 24 h and lost tolerance within 48 h; (2) the effect of pretreatment was attenuated by protein synthesis inhibitors; (3) the effect of pretreatment depended on duration and ethanol concentration; (4) pretreatment conferred cells the tolerance to not only a toxic concentrations of ethanol but also to lethal concentrations; and (5) cells acquired ethanol tolerance during starvation. Ethanol-induced tolerance in Paramecium may be a primitive model for cellular memory function.

Diplonemea is one of the most abundant and species-rich protist groups in marine environments. However, many lineages are still undescribed. Particularly, little is understood about members of the ‘deep-sea and pelagic diplonemids (DSPD)’ clade. The environmental DNA (eDNA) studies conducted with universal eukaryotic primers have shown that DSPDs were also abundantly distributed in natural environments, but their cultures have not yet been established. For future studies such as mitochondrial genome sequencing, culture establishment is needed. For establishing cultures, it is important to select samples containing a high quantity of diplonemids. In this study, we designed new diplonemid primers for eDNA analysis using a next-generation sequencer, testing its efficiency using eDNA that was extracted from two deep-sea water samples. Out of a total of 58,154 assembled reads, 57,633 reads (i.e. 99.1%) were affiliated with diplonemids by BLAST and reconstructed into 160 representative sequences. Phylogenetic analyses showed that many of the representative sequences (137 sequences, 85.6%) were branched within the DSPD clade and family Hemistasiidae. These findings indicate that the new primers are useful in monitoring diplonemid diversity and acquiring information for the establishment of DSPD cultures.