Resting cysts can not only be spontaneously generated in nature (Ricci et al., 1985; Olendzenski, 1999), but also be induced to form under laboratory conditions (Arroyo-Begovich and Cárabez-Trejo, 1982). It has been suggested that no single inducer is effective to all ciliates (Corliss and Esser, 1974), and one species may require multiple factors to form cysts (with exceptions, see below; Barker and Taylor, 1931; Johnson and Evans, 1941; van Wagtendonk, 1955).

Nutritional deficiency is a dominant factor in inducing cyst formation. This has been evident by the intracristal inclusions of mitochondria (Vickerman, 1960; Bowers and Korn, 1969), nucleolar fusion (Raikov, 1982; Frenkel, 1992), and decrease of RNA synthesis (Gutiérrez et al., 1990), found in both cysted and starved vegetive cells, indicating the connection between encystment and starvation. It is noteworthy that not only general food insufficiency can induce cyst formation, lack of specific food source, such as vitamins, can also lead to encystment (Garnjobst, 1947; van Wagtendonk, 1955; Corliss and Esser, 1974). Interstingly, in Pelagostrombidiurn fallax, excess food (Rhodomonas sp.) besides starvation can lead to cyst formation (Müller, 1996).

Other conditions, including unfavorable changes in temperature (Müller and Wünsch, 1999; Kim et al., 2002), freezing (Uspenskaya and Lozina-Lozinsky, 1979), humidity (Gutiérrez et al., 2001), salinity (Li et al., 2017), ultraviolet irradiation (Uspenskaya and Lozina-Lozinsky, 1979; Matsuoka et al., 2017), dehydration (Corliss and Esser, 1974; Gutiérrez et al., 2001), and population density (Corliss and Esser, 1974; Matsuoka, 2013; Chen et al., 2018; Sogame et al., 2019) have also been reported to be involved in inducing encystment. Additionally, change in pH (Sogame et al., 2011), concentration of Ca²⁺ or K⁺ (Yamaoka et al., 2004; Matsuoka, 2013) and oxygen (Corliss and Esser, 1974) may induce cyst formation in some species as well.

Generally speaking, cells tend to excyst when external environment conditions become more favorable (Zhao et al., 2009; Verni and Rosati, 2011). It has been suggested that sufficient food, optimal temperature and high oxygen concentration would prompt the cells to excyst (van Wagtendonk, 1955; Müller, 2002). The optimal temperature of different species ranges widely from 19.5°C (Gastrostyla stein) to 35°C (Woodrufia metabolica) (Jeffries, 1956). Moreover, cells exposed to low light condition tend not to excyst compared to those under sufficient light (Kamiyama et al., 1995).

On one hand, certain conditions might only work for some species but not others. For instance, increased pH extends the time required for excystment in Stylonychia pustulata and Pleurotricha lanceolate, while it does not influence the process in species such as Colpoda duodenaria and Didinium nasutum (Jeffries, 1956). Whereas, in most cases, exctsyment could be induced by re-feeding (Rawlinson and Gates, 1985; Benčat’ová et al., 2020). However, the concentration and source of food is important. For example, the addition of a moderate concentration of phytoplankton facilitates excystment in tintinnid ciliates (Kamiyama, 1994), while excessive concentrations of phytoplankton inhibit excystment (Kamiyama, 1997). As for the impact of food source, Jeffries (1956) suggested that dilute plant infusions are often the best for excystment.

The process of encystment involves changes not only in shape and the cortex (e.g., CW secretion and ciliature resorption) but also inside the cell (e.g., macronuclear aggregation, organelles clustering) among other characteristic cell recycling and remodeling processes (Walker and Maugel, 1980; Verni et al., 1984; Figures 4, 5).

Encystment generally begins with decreasing cell volume as well as transforming into spherical body shape, either due to cytoplasmic dehydration (Gutiérrez et al., 2001, 2003), or direct excretion of cytoplasmic contents (Zhang and Pang, 1981). This process leads to vast cell surface area reduction, organelle clustering (Martín-González et al., 2001), strong autophagic activity and decrease in the metabolic rate (Gutiérrez et al., 2001). In addition, Ricci et al. (1985) suggested that smaller volume requires less and thinner CW, which may be beneficial for fast CW formation and rapid response to the changing environment.

CW generation is key to the encystment process, which usually initiates after the body shape change (Figure 4). The two most intriguing questions are: (1) which part of the cell generates the CW? and (2) how is this achieved? A series of delicate works by Foissner and co-authors provided a very detailed and systematic description of CW formation in the planktonic ciliate, Meseres corlissi (Foissner, 2005; Foissner et al., 2005, 2006; Foissner and Pichler, 2006). The main components of the CW are the various protein complexes and carbohydrates, which usually exist in the form of chitin (Xu et al., 2007). CWs not only derive from various sources among species (e.g., precursors generated de novo; organelles present in the vegetative cell; combination of precursors and organelles present in the vegetative cell; without recognizable, membrane-bound precursors) (Foissner et al., 2006), but also show different origins for distinct CW layers. That’s to say, for cysts of some species, formation of the multi-layer barrier might depend on more than one secretory organelle, such as stacks of disks for ectocyst, long fibrous bodies for mesocyst, small dense bodies for the granular layers in Oxytricha fallax (Gutiérrez et al., 1983).

Dedifferentiation is another main feature of encystment, which is represented by resorption of the ciliature (Corliss and Esser, 1974). It is widely accepted that ciliate resting cysts are divided into three types based on the extent of ciliature resorption (Li et al., 2017): (a) non-kinetosome-resorbing cysts (NKR), with ciliary shafts (i.e., ciliary structure above kinetosomes) partially dedifferentiated (Chen et al., 2014), which appears in euplotids, nassophorean (Rawlinson and Gates, 1985), and colpodean ciliates (Tibbs, 1968); (b) partial-kinetosome-resorbing (PKR) cysts, found in urostylids and Dileptus visscheri whose oral and somatic ciliature disappear, while some single kinetosomes remain intact (Calvo et al., 2003); (c) kinetosome-resorbing (KR) that appears in the cysts of oxytrichids, with the absence of cilia and even of basal bodies (Grimes, 1973b). Interestingly, there is no apparent connection with ciliates’ phylogenetic position and type/extent of ciliature resorption. Among closely related taxa, ciliature resorption can be particularly different. As seen in Australocirrus cf. australis, disassembled ventral cirri were observed first, followed by the dorsal bristles (Li et al., 2017), while in Sterkiella histriomuscorum, dorsal kineties were found resorbed before its ventral cirri (Adl and Berger, 1997).

In addition to the changes on or above the cortex, various rearrangements have been reported to take place in the macro- and micronuclei. Besides macronuclear fusion, which is described in several species (Kamra and Sapra, 1991; Benčat’ová and Tirjaková, 2017), changes in the macronuclei include the condensation, fusion (Gutiérrez et al., 1998; Popenko et al., 1998; Martín-González et al., 2001) and extrusion (Jeffries, 1956) of chromatin, as well as the segregation, extrusion and fusion of nucleoli (Popenko et al., 1998; Martín-González et al., 2001).

Compared with macronuclei, detailed investigations of micronuclei are challenging, as micronuclei are usually too small to be clearly observed and illustrated during encystment, although Gutiérrez et al. (1998) have described that degradation and chromatin condensation occurs in micronuclei of several species. Furthermore, the numerous envelopes encapsulating micronuclei in the mature cysts may play a role in protecting the micronuclei from autophagy. It has been suggested that the extensive changes in the nuclei serve to reinforce gene-silencing and genome preservation (Gutiérrez et al., 1998).

Expendable organellar materials, such as mitochondria (Funatani et al., 2010) and ribosomes (Grimes, 1973a), can be recycled through autophagy, and new secretory organelles emerged (McArdle et al., 1980), facilitating encystment related functions. It’s suggested that the organelles and other structures in autophagic vacuoles are digested into smaller elements, so as to provide energy and materials for biosynthetic during the E-E cycle.

Contrary to encystment, the process of excystment aims to bring encysted cells back to their vegetative forms when facing favorable environment (Figures 4, 5).

Ciliary structures degenerate during encystment reappear when excysting. For those species with complicated and specialized ciliature (e.g., hypotrichs), the order of ciliary recovery in different cell area varies among species (Grimes, 1973a; Adl and Berger, 1997). Additionally, in some species, the ciliature is fully restored prior to the very first division after escaping from the CW [e.g., Sterkiella histriomuscorum (Adl and Berger, 1997)], while it can take several divisions to return to the original vegetative pattern in other species [e.g., Parentocirrus hortualis (Benčat’ová et al., 2016)].

Similar process occurs inside the cell. Macronuclei that have twisted or merged during encystment restore their original organization, which includes recovering the shape of the nuclei, [e.g., Euplotes encysticus (Rawlinson and Gates, 1985)], and the number of nuclei [e.g., Histriculus similis (Calvo et al., 1988) and Stylonychia mytilus (Zhang and Pang, 1981)]. In addition, chromatin extrusion has been observed in Colpoda inflata, suggesting potential changes in the macronuclear genome after excystment as well (Chessa et al., 2001). The number and distribution of mitochondria and other organelles also return to the vegetative form. For example, in Oxytricha fallax, the mitochondria will swell and disperse subsequently (Grimes, 1973a).

Along with the dynamic changes within the cell, the cell body eventually escapes from the CW. The escape is assisted either by a preformed apparatus (i.e., emergence pore with a removable “plug” or “operculum”) and/or a rupture generated by the pressure of cell movement inside the CW and excystation vacuole, which has been described as the contractile vacuole in Pleurotricha lanceolata (Jeffries, 1956). This process is typically initiated by the active rotation of the cell within the cyst, followed by lifting the plug or operculum, or generation of a rupture of a small pore (at uncertain position) on the CW. Subsequently, the cell body squeezes out through the opening and eventually regains the vegetative form after a period of free swimming.

The fate of the CW after excystment varies greatly among species: (1) the CW as a whole is left behind in the environment; (2) the encysted cell is still enclosed by the inner CW layer when breaking through the outer CW layer, afterward the inner CW layer will be ruptured and/or resorbed later, like in Pleurotricha lanceolate (Jeffries, 1956), Sterkiella histriomuscorum (Adl and Berger, 1997), Coniculostomum monilata (Kamra and Sapra, 1991), and Histriculus similis (Calvo et al., 1988); 3) all CW layers are left behind with well as the material of ruptured inner membrane (Phacodinium metchnikoffi, Benčat’ová and Tirjaková, 2018) in the empty resting cyst (Figure 4). The fate of the CW is not restricted to a single mode in one species. For example, both first and second types exist in Rigidohymena quadrinucleata (Benčat’ová and Tirjaková, 2017) and Stylonychia mytilus cysts (Zhang and Pang, 1981), and both second and third types have been found in Parentocirrus hortualis (Benčat’ová et al., 2016).

Breakdown of CW is an important step during excystment process. Ubiquitin, ubiquitin carboxyl terminal hydrolase family protein and cullin family protein are speculated to participate in the degradation process (Wang B. et al., 2017). Besides, cysteine protease with different functions may also involve in the CW breakdown process or signal transduction pathway that is sensitive to ambient pH (Villalobo et al., 2003).

The homologs of dead box RNA helicases, which regulate transcription and mRNA turnover, were detected 10 min after the induction of excystment in Colpoda (Sogame et al., 2013), indicating the recover of transcription. Comparative transcriptome analyses of Colpoda aspera showed that the synthesis of palmitic acid and its synthetic substrate hexadecanoyl-CoA are both up-regulated when forming resting cysts. Palmitic acid acts as precursor to synthesize other longer fatty acids during vegetative periods, and was speculated to accumulate as fat storage to be used during excystment (Jiang et al., 2019).

Elongation factor 1α (EF-1α) is upregulated or downregulated during encystment process, and downregulated in the excystment process (Sogame et al., 2012b,2013; Chen et al., 2018). As a multifunctional protein, EF-1α may be involved in protein synthesis, proteasomal degradation, nuclear export, as well as actin or microtubule bundling activity (Sogame et al., 2013; Chen et al., 2018), which can partially explain the contradictory results and imply the relationship between EF-1α and the structural changes in the excystment process.