diversity Article Analysis of Encystment, Excystment, and Cyst Structure in Freshwater Eutardigrade Thulinius ruffoi (Tardigrada, Isohypsibioidea: Doryphoribiidae) Kamil Janelt * and Izabela Poprawa * Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, Bankowa 9, 40-007 Katowice, Poland * [email protected] (K.J.); [email protected] (I.P.) Received: 20 December 2019; Accepted: 2 February 2020; Published: 4 February 2020 Abstract: 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 ruffoi 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. Keywords: Thulinius ruffoi; diapause; encystment; cyst 1. 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 [3–5]. 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. [7–11]. 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 Diversity 2020, 12, 62; doi:10.3390/d12020062 www.mdpi.com/journal/diversity Diversity 2020, 12, 62 2 of 12 described for tardigrades: Anhydrobiosis, anoxybiosis, cryobiosis, and osmobiosis [5]. Diapause is represented by encystment and cyclomorphosis [12]. In spite of quite well known cryptobiotic states in tardigrades (especially anhydrobiosis and cryobiosis), knowledge about encystment is poor [3,13,14]. Encystment in tardigrades is also seen as a kind of epigenetic phenotypic plasticity, closely linked to the molting process that evolved to withstand unfavorable environmental conditions [6,12]. An ability to form cysts has been noted in some freshwater, bryophilous, and soil tardigrades [14]. Recently, Clausen et al. [15] described the cysts in the marine heterotardigrade Echiniscoides sigismundi. Within the order Isohypsibioidea, an ability to form cysts has been reported in several representatives [16–23] including in Thulinius ruffoi [24]. In this paper, we analyze the encystment and the excystment process in the freshwater tardigrade T. ruffoi and describe the structure and morphology of the cysts using varied methods and techniques. Moreover, a comparative, morphological analysis of the buccal-pharyngeal apparatus between encysted and nonencysted animals was performed. 2. Materials and Methods 2.1. Material Specimens of T. ruffoi (Thu.ruf_PL.014) that were used in this study were kindly provided by the Michalczyk Lab (Jagiellonian University, Cracow, Poland). Specimens were cultured under laboratory conditions on Petri dishes and plastic 24-well plates (Institute of Biology, Biotechnology and Environmental Protection, University of Silesia in Katowice, Katowice, Poland). Before placing animals on the Petri dishes or 24-well plates, the bottoms were scratched, washed with distilled water, and flooded with a culture medium (distilled water and Zywiec˙ Zdrój mineral water, 1:1). The animals were fed with a mixture (1:1) of Chlorella sp. and Chlorococcum sp. ad libitum and kept at 19 ◦C and room temperature (RT about 25 ◦C). Cysts of T. ruffoi used in this study were obtained under laboratory conditions as follows. Scratched, plastic 24-well plates were washed with distilled water, then flooded with culture medium and a mixture of algae added. Randomly selected, nonencysted, adult animals were then added, one specimen per well. Plates were transferred to lower temperatures with a mean of 6.5 and 8 ◦C. At both temperatures, cysts were formed. Observations were made using a stereoscopic microscope (Olympus SZ40) and at room temperature for the shortest possible time. To interrupt the encystment artificially, selected cysts were permanently transferred to a higher temperature (RT). 2.2. Methods An Olympus BX60 microscope was used for analysis of the structure of the cyst and cuticular capsules using brightfield (BF) and differential interference contrast (DIC). Moreover, filters for detecting UV autofluorescence according to Perry et al. [25] were used in the analysis. Permanent slides were prepared using Hoyer’s mounting medium (prepared according to Morek et al. [26] and closed with a coverslip. Nonencysted specimens were investigated using the same microscope and techniques. Encysted and nonencysted animals (in vivo), as well as empty cuticular capsules were also observed in a drop of water between a slide and a coverslip. Additionally, some cysts were dissected by rolling the cyst between the slide and a coverslip carefully until the cuticular capsule broke and the animals were pulled out. Ten empty cuticular capsules, 20 encysted and 20 nonencysted animals were used. Material for semi- and ultra-thin sections was prepared as follows. Eight cysts and four empty cuticular capsules of T. ruffoi were washed a few times with distilled water and fixed with 2.5% glutaraldehyde (4 C). Then, the material was washed three times (3 30 min each, RT) in 0.1M ◦ × phosphate buffer (pH = 7.4), postfixed in 2% osmium tetroxide in phosphate buffer (2 h, RT), and washed in phosphate buffer (3 10 min each, RT). Then, material was dehydrated in a graded ethanol × series (30%, 2 50%, 70%, 90%, 96%, 4 100%, 15 min each, RT), transferred to a solution of 100% × × ethanol and acetone (1:1, 15 min, RT), washed in acetone (2 10 min, RT), and then transferred to × a solution of epoxy resin and acetone (1:1, 1.5 h, RT). After the evaporation of acetone (overnight, RT), Diversity 2020, 12, 62 3 of 12 Diversity 2020, 12, 62 3 of 12 RT),material material was embeddedwas embedded in epoxy in epoxy resin (Epoxyresin (Epoxy Embedding Embedding Medium Medium Kit; Sigma). Kit; Sigma). The resin The in resin which in whichthe material the material was embedded was embedded was polymerized was polymerized at 60 ◦C. at Semi- 60 °C. and Semi ultra-thin‐ and ultra sections‐thin were sections cut on were a Leica cut onUltracut a Leica UCT25 Ultracut ultramicrotome. UCT25 ultramicrotome. Semi-thin Semi cross-sections‐thin cross of‐sections the cysts of andthe cysts empty and cuticular empty cuticular capsules capsuleswere stained were with stained 1% methylene with 1% bluemethylene in 1% borax,blue in mounted 1% borax, with mounted a DPX medium with a andDPX analyzed medium using and analyzedan Olympus using BX60 an microscope.Olympus BX60 Ultra-thin microscope. sections Ultra were‐thin counterstained sections were with counterstained uranyl acetate with and uranyl lead acetatecitrate andand analyzedlead citrate using and aanalyzed Hitachi using H500 transmissiona Hitachi H500 electron transmission microscope electron at 75microscope kV. at 75 kV. ForFor analysis inin scanning scanning electron electron microscopy, microscopy, eight eight cysts cysts and and four four empty empty cuticular cuticular capsules capsules were werecleaned cleaned by washing by washing several several times withtimes distilled with distilled water and water fixed and in 1.5%fixed glutaraldehyde.in 1.5% glutaraldehyde. Material Materialwere washed were three washed times three with times distilled with water distilled (3 water10 min) (3 × and 10 min) postfixed and postfixed with 2% OsO with 2%(1 h, OsO RT),4 (1 then h, × 4 RT),washed