Analysis of Encystment, Excystment, and Cyst Structure in Freshwater Eutardigrade Thulinius ru ﬀ oi (Tardigrada, Isohypsibioidea: Doryphoribiidae)

: Encystment in tardigrades is relatively poorly understood. It is seen as an adaptive strategy evolved to withstand unfavorable environmental conditions. This process is an example of the epigenetic, phenotypic plasticity which is closely linked to the molting process. Thulinius ru ﬀ oi is a freshwater eutardigrade and a representative of one of the biggest eutardigrade orders. This species is able to form cysts. The ovoid-shaped cysts of this species are known from nature, but cysts may also be obtained under laboratory conditions. During encystment, the animals undergo profound morphological changes that result in cyst formation. The animals surround their bodies with cuticles that isolate them from the environment. These cuticles form a cuticular capsule (cyst wall) which is composed of three cuticles. Each cuticle is morphologically distinct. The cuticles that form the cuticular capsule are increasingly simpliﬁed. During encystment, only one, unmodiﬁed and possibly functional buccal-pharyngeal apparatus was found to be formed. Apart from the feeding apparatus, the encysted specimens also possess a set of claws, and their body is covered with its own cuticle. As a consequence, the encysted animals are fully adapted to the active life after leaving the cyst capsule.


Introduction
In the environment, many changes may be experienced as stress factors by living organisms. Animals have developed various mechanisms to protect them from negative effects of stressors. Hypometabolic states are seen as a defense in stress responses in many metazoans [1,2]. Moreover, tardigrades developed mechanisms that allow them to withstand unfavorable environmental conditions [3][4][5]. Tardigrades are worldwide occurring micrometazoans that can be found in varied environments. They occupy marine, brackish, and freshwater (lotic and lentic) habitats. These animals can also be found in limnoterrestrial and terrestrial habitats as well. To be active they need at least a thin layer of water-even in terrestrial environments. Tardigrades have captured the attention of researchers due to their extraordinary abilities [5,6]. Today, these animals are a source of information with great potential for medicine, tissue engineering, pharmacology, astrobiological studies, etc. [7][8][9][10][11]. Cryptobiosis and diapause are known as dormancy states in tardigrades [6]. The former is a quick response to sudden changes in the environment which means that it is directly caused by the environmental stressors. In contrast, the latter is seen as controlled by exogenous and endogenous stimuli [3,6,12] however, the cyst formation is not a direct response to changes in the environment. Cryptobiosis is known from many species of tardigrades, mostly terrestrial, where they are more often exposed to unfavorable conditions. Four types of cryptobiotic states have been RT), material was embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma). The resin in which the material was embedded was polymerized at 60 °C. Semi-and ultra-thin sections were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin cross-sections of the cysts and empty cuticular capsules were stained with 1% methylene blue in 1% borax, mounted with a DPX medium and analyzed using an Olympus BX60 microscope. Ultra-thin sections were counterstained with uranyl acetate and lead citrate and analyzed using a Hitachi H500 transmission electron microscope at 75 kV.
For analysis in scanning electron microscopy, eight cysts and four empty cuticular capsules were cleaned by washing several times with distilled water and fixed in 1.5% glutaraldehyde. Material were washed three times with distilled water (3 × 10 min) and postfixed with 2% OsO4 (1 h, RT), then washed with distilled water (3 × 10 min) and dehydrated using a graded ethanol series (10%-100%, with 10% increase, 2 min each) without an acetone series. Then, material was dried in a critical point dryer (CPD-2 Pelco) and placed on stubs with adhesive carbon tabs and covered with an ultrathin layer of chromium using the sputter coater Quorum 150T ES Plus. Material was analyzed using Hitachi UHR FE-SEM SU 8010 and Phenom XL scanning electron microscopes. All used equipment is available at the Institute of Biology, Biotechnology and Environmental Protection (Faculty of Natural Sciences, University of Silesia in Katowice, Katowice, Poland).

Encystment and Excystment
At the beginning of the incubation at tested, low temperatures, the animals were active. They were moving and eating, with the result that the midgut contents were visible. However, some of them died within a few days of transferring them to lower temperatures ( Figure 1A). Figure 1. Scheme of the general behavior pathways related to cyst dynamics. (A) Animalʹs death within a few days of being transferred to a low temperature; (B) animalʹs death after one or several reproductive cycles since being transferred to a low temperature; (C) cyst formation by specimens which have undergone one or several reproductive cycles since being transferred to low temperature; (D) cyst formation by specimens that have not reproduced since being transferred to low temperature; (E) leaving the cuticular capsule/free-living animal; (F) animalʹs death before the cuticular capsule abandonment. Period of reproduction arrest (asterisk).
Simplex forms (or in other words-animals on the simplex stage) are characterized by the lack of sclerified elements of the buccal-pharyngeal apparatus and the absence of the cuticular lining of the esophagus. These forms were observed during the active life of the animals. Single separated specimens were able to reproduce via parthenogenesis ( Figure 1B,C) and eggs within exuviae were present in further observations. The specimens reproduced once or a few times. After that, specimens died ( Figure 1B) or formed cysts ( Figure 1C). However, before cyst formation, a period when the animals did not reproduce was observed. During that time, animals were eating Figure 1. Scheme of the general behavior pathways related to cyst dynamics. (A) Animal's death within a few days of being transferred to a low temperature; (B) animal's death after one or several reproductive cycles since being transferred to a low temperature; (C) cyst formation by specimens which have undergone one or several reproductive cycles since being transferred to low temperature; (D) cyst formation by specimens that have not reproduced since being transferred to low temperature; (E) leaving the cuticular capsule/free-living animal; (F) animal's death before the cuticular capsule abandonment. Period of reproduction arrest (asterisk).
Simplex forms (or in other words-animals on the simplex stage) are characterized by the lack of sclerified elements of the buccal-pharyngeal apparatus and the absence of the cuticular lining of the esophagus. These forms were observed during the active life of the animals. Single separated specimens were able to reproduce via parthenogenesis ( Figure 1B,C) and eggs within exuviae were present in further observations. The specimens reproduced once or a few times. After that, specimens died ( Figure 1B) or formed cysts ( Figure 1C). However, before cyst formation, a period when the animals did not reproduce was observed. During that time, animals were eating intensively. This period was observed to begin either after transferring the animal to the low temperature ( Figure 1D) or after one or a few reproductive cycles ( Figure 1C). The first cysts were formed at least 14-15 days after the animals were transferred to lower temperatures. However, cyst formation often occurred after more than a month. Animals did not enter into encystment at the same time, which meant that cysts were formed after different times of incubation under low temperatures. The gut of some specimens that formed cysts was emptied by defecation before cyst formation.
During encystment, the appearance of an animal changed significantly (Figure 2A,B).
Diversity 2020, 12, 62 4 of 12 intensively. This period was observed to begin either after transferring the animal to the low temperature ( Figure 1D) or after one or a few reproductive cycles ( Figure 1C). The first cysts were formed at least 14-15 days after the animals were transferred to lower temperatures. However, cyst formation often occurred after more than a month. Animals did not enter into encystment at the same time, which meant that cysts were formed after different times of incubation under low temperatures. The gut of some specimens that formed cysts was emptied by defecation before cyst formation. During encystment, the appearance of an animal changed significantly (Figure 2A,B). At this time, discharge of the sclerified elements of the buccal-pharyngeal apparatus together with the cuticular lining of the esophagus occurred and the animals entered into the simplex stage. The animals were unable to eat anymore. The locomotor activity of the animal decreased until it stopped completely in one place. The animal surrounded its body with cuticles together with the reduction of the body size by its contraction and buccal-pharyngeal apparatus resynthesis. As a result, the animal was enclosed within the cuticular capsule ( Figure 2A). During this time, animals could not move, eat, or reproduce. Cysts with (Video S1) or without ( Figure 2A) content inside the midgut were observed. The encystment could be interrupted by the animals themselves or under laboratory conditions. The first was observed in some encysted specimens which spontaneously interrupted the encystment while still at low temperature. The second may have been caused by the transfer of the cyst from a lower to a higher temperature. Note that the old cuticle is only in the form of the remains of cuticle with claws. Anterior part of the cyst/cuticular capsule (ac); cuticle of the animal (c); claws of the animal (cl); claws of the old cuticle (clo); mummy cuticle (mc); old cuticle (oc); posterior part of the cyst/cuticular capsule (pc); pharynx (ph); sarcophagus cuticle (sc); cuticle of the hindgut (empty arrowhead); buccal-pharyngeal apparatus of the animal (filled arrowhead); increase in damage (thick empty arrow); round holes made by claws (thick filled arrow); closed mouth opening of the old cuticle (thin arrow).
At this time, discharge of the sclerified elements of the buccal-pharyngeal apparatus together with the cuticular lining of the esophagus occurred and the animals entered into the simplex stage. The animals were unable to eat anymore. The locomotor activity of the animal decreased until it stopped completely in one place. The animal surrounded its body with cuticles together with the reduction of the body size by its contraction and buccal-pharyngeal apparatus resynthesis. As a result, the animal was enclosed within the cuticular capsule ( Figure 2A). During this time, animals could not move, eat, or reproduce. Cysts with (Video S1) or without ( Figure 2A) content inside the midgut were observed. The encystment could be interrupted by the animals themselves or under laboratory conditions. The first was observed in some encysted specimens which spontaneously interrupted the encystment Diversity 2020, 12, 62 5 of 12 while still at low temperature. The second may have been caused by the transfer of the cyst from a lower to a higher temperature.
Based on the possibility of inducing encystment termination (excystment), it was possible to study this process. In encysted animals which were transferred to room temperature, movements inside the cuticular capsule were observed (Video S1). Movements of the body, including the movement of the legs with their claws caused the breaking of the cuticular capsule ( Figure 2C-E). The claws could pierce the cuticles making characteristic perforations. Then, the increase in cracks could cause greater damage weakening the capsule, making it easier for the animal to leave the capsule ( Figure 2E). After about 2-3 days, at room temperature, the animals started to abandon the cuticular capsule or were already moving freely in the environment after leaving the capsule ( Figures 1E and 2C,D). Encysted specimens that after their permanent transfer to room temperature did not abandon the cuticular capsule (for more than a week) were considered dead ( Figure 1F).

Cuticular Capsule
Animals surrounded their bodies with three cuticles that formed a cyst capsule ( Based on the possibility of inducing encystment termination (excystment), it was possible to study this process. In encysted animals which were transferred to room temperature, movements inside the cuticular capsule were observed (Video S1). Movements of the body, including the movement of the legs with their claws caused the breaking of the cuticular capsule ( Figure 2C-E). The claws could pierce the cuticles making characteristic perforations. Then, the increase in cracks could cause greater damage weakening the capsule, making it easier for the animal to leave the capsule ( Figure 2E). After about 2-3 days, at room temperature, the animals started to abandon the cuticular capsule or were already moving freely in the environment after leaving the capsule ( Figure  1E and Figure 2C,D). Encysted specimens that after their permanent transfer to room temperature did not abandon the cuticular capsule (for more than a week) were considered dead ( Figure 1F).

Cuticular Capsule
Animals surrounded their bodies with three cuticles that formed a cyst capsule (     sarcophagus cuticle (middle cuticle) with the remains of the old cuticle with claws, ventro-lateral view; (C) mummy cuticle (inner cuticle). Scale bars 10 μm. Old cuticle (oc); claws of the old cuticle (clo); mummy cuticle (mc); sarcophagus cuticle (sc). Note that A-P indicates anterior to posterior direction.   Additionally, the encysted animal possessed its own cuticle ( Figure 4A,B and Figure 5C,D). The cyst capsule (from outside to inside) was formed by an old cuticle (outer cuticle), sarcophagus cuticle (middle cuticle), and mummy cuticle (inner cuticle). This structure of the cuticular capsule was observed in the 24-h old cysts (Figure 2A). Each of these three cuticles was different from each other (Figure 3). In the old cuticle, the mouth opening was closed ( Figure 2A) and shedding of the hindgut cuticle could be observed (Figure 2A). The legs of the old cuticle ended with claws (Figure 2A,C,D, Figure 3A and Figure 4C). The legs of the sarcophagus cuticle were developed as cuticular cones without claws; however, a small hole was present at the end of each cone ( Figure 4C-F). These cones were located in the regions where the legs of the old cuticle had been ( Figure 4C-F). On the surface of this cuticle numerous folds and wrinkles were observed ( Figure 2E and Figure 3B). The mummy cuticle did not possess legs ( Figure 3C). Between the old cuticle and the sarcophagus cuticle, a network of numerous cuticular connections of different thickness was observed ( Figure 5). A similar network of connections was also noted between the sarcophagus and mummy cuticles ( Figure 5D). Cysts without the continuous layer of the old cuticle were also observed. When the old cuticle was falling apart, the remains of this cuticle and claws could still be observed on the surface of the sarcophagus cuticle ( Figure 2E, Figure 3B and Figure 4G). The cuticle that covered the body of the Additionally, the encysted animal possessed its own cuticle ( Figure 4A,B and Figure 5C,D). The cyst capsule (from outside to inside) was formed by an old cuticle (outer cuticle), sarcophagus cuticle (middle cuticle), and mummy cuticle (inner cuticle). This structure of the cuticular capsule was observed in the 24-h old cysts (Figure 2A). Each of these three cuticles was different from each other (Figure 3). In the old cuticle, the mouth opening was closed ( Figure 2A) and shedding of the hindgut cuticle could be observed (Figure 2A). The legs of the old cuticle ended with claws (Figure 2A,C,D, Figures 3A and 4C). The legs of the sarcophagus cuticle were developed as cuticular cones without claws; however, a small hole was present at the end of each cone ( Figure 4C-F). These cones were located in the regions where the legs of the old cuticle had been ( Figure 4C-F). On the surface of this cuticle numerous folds and wrinkles were observed (Figures 2E and 3B). The mummy cuticle did not possess legs ( Figure 3C). Between the old cuticle and the sarcophagus cuticle, a network of numerous cuticular connections of different thickness was observed ( Figure 5). A similar network of connections was also noted between the sarcophagus and mummy cuticles ( Figure 5D). Cysts without the continuous layer of the old cuticle were also observed. When the old cuticle was falling apart, the remains of this cuticle and claws could still be observed on the surface of the sarcophagus cuticle ( Figures 2E, 3B and 4G). The cuticle that covered the body of the encysted animals was developed as in nonencysted animals. The animals enclosed within the cuticular capsule also possessed, in addition to their own cuticle, a set of claws what was confirmed using optical microscopy for analysis of the cysts in toto ( Figure 2C,D and Figure 6A), as well as on the cross-sections through the cyst ( Figure 4B) and based on the specimens obtained by the cysts' dissection and encystment interruption. Anterior part of the cyst/cuticular capsule (ac); buccal tube (bt); condyle of the furca (cf); claws of the animal (cl); claws of the old cuticle (clo); stylet coat (co); esophagus cuticle (ec); first band of teeth (fb); three macroplacoids in a row (m1,m2,m3); mummy cuticle (mc); old cuticle (oc); pharyngeal apophysis (pa); posterior part of the cyst/cuticular capsule (pc); pharynx (ph); peribuccal lamellae (pl); peribuccal lobes (plo); piercing stylet (ps); rod-shaped thickening (r); stylet (s); second band of teeth (sb); sarcophagus cuticle (sc); stylet furca (sf); stylet sheath (ss); buccal tube margin (tm); putative modified mouth part of the sarcophagus cuticle (asterisk); cuticular legs of the sarcophagus cuticle (empty arrowhead); stylet support (filled arrowhead); buccal crown (star); buccal-pharyngeal apparatus of the encysted animal (thick filled arrow); closed mouth opening of the old cuticle (thin arrow).
The cuticular capsule isolated the animals from the environment. In nondamaged cysts (where the continuity of all cuticles of the cuticular capsule was not broken) the bacteria from the environment were observed outside the old cuticle, as well as in the space between the old cuticle and sarcophagus cuticle ( Figure 5B,D). Bacteria were not observed in the spaces between the animal Anterior part of the cyst/cuticular capsule (ac); buccal tube (bt); condyle of the furca (cf); claws of the animal (cl); claws of the old cuticle (clo); stylet coat (co); esophagus cuticle (ec); first band of teeth (fb); three macroplacoids in a row (m1,m2,m3); mummy cuticle (mc); old cuticle (oc); pharyngeal apophysis (pa); posterior part of the cyst/cuticular capsule (pc); pharynx (ph); peribuccal lamellae (pl); peribuccal lobes (plo); piercing stylet (ps); rod-shaped thickening (r); stylet (s); second band of teeth (sb); sarcophagus cuticle (sc); stylet furca (sf); stylet sheath (ss); buccal tube margin (tm); putative modified mouth part of the sarcophagus cuticle (asterisk); cuticular legs of the sarcophagus cuticle (empty arrowhead); stylet support (filled arrowhead); buccal crown (star); buccal-pharyngeal apparatus of the encysted animal (thick filled arrow); closed mouth opening of the old cuticle (thin arrow). The cuticular capsule isolated the animals from the environment. In nondamaged cysts (where the continuity of all cuticles of the cuticular capsule was not broken) the bacteria from the environment were observed outside the old cuticle, as well as in the space between the old cuticle and sarcophagus cuticle ( Figure 5B,D). Bacteria were not observed in the spaces between the animal and the most inner cuticle of the capsule, the mummy cuticle.

Buccal-Pharyngeal Apparatus
The cyst formation was preceded by the simplex stage where the buccal-pharyngeal apparatus together with the cuticle lining of the esophagus was ejected outside the body and the mouth opening was closed ( Figure 2B). However, during cyst formation this feeding apparatus and esophagus lining was resynthesized together with a new set of claws (Figure 2A,C,D, Figures 4B and 6A-F). Only one buccal-pharyngeal apparatus was observed in the cysts of T. ruffoi despite the use of various techniques of optical microscopy (Figures 2A and 6). In 24-h old cysts, the buccal-pharyngeal apparatus appeared completely formed (Figure 2A). Moreover, in the older cysts ( Figure 6A-F), a complete, single buccal-pharyngeal apparatus was observed. In the animals closed inside the cyst, peribuccal structures around the mouth such as peribuccal lobes and peribuccal lamellae were observed ( Figure 6D,E). The buccal armature (oral cavity armature) was composed of two bands of teeth where the teeth of the second band were larger than those which formed the first band ( Figure 6D). The buccal crown surrounded externally the wall of the anterior part of the buccal tube ( Figure 6E). The most posterior end of the buccal tube was located within the pharynx. The buccal tube ended with an enlarged margin ( Figure 6B,E). Within the pharynx, three pharyngeal apophyses that alternated with three rows of symmetrical placoids were located. In each row of placoids, three macroplacoids were present, the middle one being the smallest ( Figure 6E). On both sides of the buccal tube, elements of the stylet system were observed. This part of the buccal-pharyngeal apparatus was formed by two stylets and two stylet supports ( Figure 6B,E). Each stylet was composed of a piercing stylet located inside a stylet coat. Anteriorly, the stylet coat was formed as a stylet sheath ( Figure 6E) and posteriorly as a stylet furca ( Figure 6B). Each stylet sheath was laterally reinforced by a thin, rod-shaped cuticular thickening that was anteriorly connected to the buccal crown ( Figure 6D,E). The stylet furca was branched and each branch ended with a condyle. Two stylet supports located on both sides of the buccal tube connected the posterior part of the stylet coat with the buccal tube. The distal extremities of the stylet supports were bifurcated and fused to the arc between two condyles of the stylet furca. However, the proximal end was connected with the buccal tube ( Figure 6B,E). Comparative, morphological analysis of the buccal-pharyngeal apparatus between nonencysted and encysted animals showed that the feeding apparatus of the cysts had no morphological modifications. The apparatus did not show any signs of simplification in its morphology ( Figure 6A-E). The buccal-pharyngeal apparatus was located inside the animal in a typical place (Figure 2A,C,D and Figure 6A,B,F). Morphological analysis ( Figure 6A-E), as well as stylet system movements of this apparatus (Video S1) indicated that this apparatus was functional and not modified. No additional buccal-pharyngeal apparatuses were found between the cuticles of the cuticular capsule despite the use of various techniques of optical microscopy (Figures 2A and 6A,B,F-I).
Cysts were formed by the animals after variable incubation times at low temperature. During active life at lower temperature animals are able to move, eat, and reproduce, which is not possible during encystment. Some specimens did not reproduce at the beginning of incubation at low temperature and after some time, during which they ate intensively, they formed cysts. Moreover, some animals that reproduced at these low temperatures also formed cysts. However, similarly to the previous case, before cyst formation they stopped reproducing and ate intensively. The reason for these behaviors may be related to the high energetic cost of reproduction and cyst formation. In addition, eggs have never been found in the encysted specimens of T. ruffoi. Before cyst formation, animals accumulated the reserve materials to be a source of energy during encystment, which has also been observed for other species [16,44,45]. The reproductive arrest observed before cyst formation in T. ruffoi could possibly allow accumulation of larger amounts of reserve material that would be a source of energy during ongoing encystment. As an adaptation to survive for many months without food supplied from the environment, the energy consumption must be limited. Encystment is a process in which energetic costs seem to be reduced by formation of simplified nonfunctional structures [6]. The formation of the simplified structures, together with the lowering of the metabolic rate of the encysted animals [48], reduces the energy consumption during encystment.
During cyst formation, specimens of T. ruffoi surround their body with three cuticles, which together form the cuticular capsule of the cyst. These cuticles (from outside to inside) are named the old cuticle, sarcophagus cuticle, and mummy cuticle, using terminology proposed by Hansen and Katholm [47] and Guidetti et al. [14]. The cuticles of the cyst capsule are increasingly simplified (from outside to inside). The most external cuticle of the cuticular capsule is the old cuticle. Here, the closed mouth opening, shed hindgut cuticle, and the legs terminating with claws can be observed. The sarcophagous cuticle has a folded structure. The legs of this cuticle are visible as clawless cones under the legs of the old cuticle and they terminate with small holes. The innermost cuticle of the cuticular capsule has no legs. Cuticles of the cyst capsule are connected by a network of cuticular connections.
An interesting aspect of the encystment process concerns the changes related to the buccal-pharyngeal apparatus. Studies by Guidetti et al. [14] showed that in encysted specimens of D. parthenogeneticus after the simplex stage, a modified buccal-pharyngeal apparatus is formed two times and seems to be nonfunctional. After discharging them, an unmodified feeding apparatus is formed. In species of Bertolanius (B. nebulosus, B. volubilis, and B. weglarskae) two cyst types were noted [14,[41][42][43]47] but only in B. volubilis and only in "type 2" cysts was the synthesis of one modified buccal-pharyngeal apparatus after the simplex stage described. After discharging this modified one, another unmodified feeding apparatus is formed. The "type 1" cysts of B. volubilis do not synthesize the modified buccal-pharyngeal apparatus but only a single, unmodified buccal-pharyngeal apparatus is formed after the simplex stage [14]. Similarities between the cysts of D. parthenogeneticus and the "type 2" cysts of B. volubilis were observed, although with the reservation that the cyst of this first one is more complex [14]. In contrast to data presented by Guidetti et al. [14] for cysts of D. parthenogeneticus or "type 2" cysts of B. volubilis, in T. ruffoi only one buccal-pharyngeal apparatus is synthesized during encystment. Its morphological analysis showed that it looks like ones found in nonencysted animals. The presence of modified buccal-pharyngeal apparatuses was not found in cysts of this species despite using various investigative methods. Moreover, its movements after transferring the cysts to higher temperatures indicated functional efficiency and suggests that this structure is helpful in the interruption of the cyst capsule cuticles' continuity during excystment, as well as being useful after leaving the cuticular capsule. Therefore, not only body movements including movements of the legs terminated with claws but also movements of the feeding apparatus may be useful during breaking of the cyst capsule.
In T. ruffoi, formation of the normally developed cuticle, claws, and a single, functional, nonmodified feeding apparatus seems to reduce energetic costs to the necessary minimum and the animal is fully prepared for life after encystment termination and leaving the cuticular capsule of the cyst. As a result, T. ruffoi can immediately start eating and replenish its energy. Still many aspects of the encystment process are not known or clear. More research on this process is needed to better understand the encystment in tardigrades.

Conclusions
Our conclusions from this study are as follows: (1) T. ruffoi is able to form cysts. (2) Cysts may be obtained under laboratory conditions. (3) The cyst capsule inside which the animal is closed is composed of three cuticles. (4) Cuticles of the cyst capsule are increasingly simplified (from outside to inside). (5) The animal inside the cyst is covered with a cuticle which develops like nonencysted specimens of this species and possesses claws. (6) The animal produces only one, functional and unmodified buccal-pharyngeal apparatus.
Author Contributions: Conceptualization, investigation, data analysis, methodology, writing-original draft preparation, and review and editing, K.J.; visualization, K.J. and I.P.; writing-review and editing and supervision, I.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.