Tun formation is not a prerequisite for desiccation tolerance in the marine tidal tardigrade Echiniscoides sigismundi

The so-called ‘tun’ state is best known from limno-terrestrial tardigrades and rotifers that rely on this compact body shape for anhydrobiotic survival. Little is known of tun formation in marine species and the evolutionary origin of the state is presently unknown. Here, we investigate desiccation tolerance and tun formation in the marine tidal echiniscoidean tardigrade, Echiniscoides sigismundi (M. Schultze, 1865). Groups of approximately 20 E. sigismundi sampled from Lynæs (Denmark) were dehydrated on ﬁlter paper from seawater as well as ultrapuriﬁed water and kept for 48 h at 5 ° C, after which they were rehydrated in seawater. The activity and behaviour of the tardigrades was examined under a light microscope, whereas scanning electron microscopy was used for high-resolution three-dimensional imaging. When dehydrated from seawater, E. sigismundi enters a tun, however, when exposed to ultrapuriﬁed water, the tardigrade swells and becomes incapable of movement, and thus incapable of tun formation. Nonetheless, E. sigismundi tolerates being dehydrated from ultrapuriﬁed water, revealing an exceptional and unparalleled resilience towards losing structural integrity. Our results conﬁrm previous investigations, which suggest that tun formation relies on a functional musculature. They further suggest that tun formation may have evolved as a response to elevated external pressure rather than desiccation per se.


INTRODUCTION
Tardigrades depend on having a surrounding film of water to be in their active and reproductive state. They are, nevertheless, found in a range of habitats worldwide (e.g. McInnes, 1994;Hansen, Jørgensen & Kristensen, 2001;Nelson, 2002;Convey & McInnes, 2005), including moss beds, which frequently dry out, leaving the tardigrades in a dehydrated, ametabolic state known as anhydrobiosis (e.g. Rebecchi, Altiero & Guidetti, 2007;Guidetti, Altiero & Rebecchi, 2011;Wełnicz et al., 2011). Moreover, these microscopic metazoans are well known for their ability to tolerate a range of other extreme stresses ; however, significant variations in stress tolerance between species complicate investigations into the underlying processes associated with extreme stress tolerance. Notably, the specifics of how tardigrades enter and exit states of latent life, collectively referred to as cryptobiosis (Keilin, 1959), remain elusive.
It has been a commonly held belief, based on earlier observations made in eutardigrades, that anhydrobiosis (cryptobiosis induced by desiccation) is accompanied by a morphological transition into a socalled 'tun state' in tardigrades, and that this transition is a necessary response for post-anhydrobiotic survival (e.g. Crowe & Madin, 1974;Crowe, 1975;Wright, Westh & Ramløv, 1992;Sømme, 1996). This assumption is supported by recent experimental data showing that tun formation is mediated by the musculature, and an essential process for anhydrobiotic survival in the eutardigrade Richtersius coronifer (Halberg, Jørgensen & Møbjerg, 2013). Limnoterrestrial echiniscoideans also form tuns, and we have recently shown that tun formation occurs in the arthrotardigrade Styraconyx haploceros (Jørgensen & Møbjerg, 2015). Experimental data on heterotardigrades are scarce, however, and whether tun formation is essential for anhydrobiotic survival in tardigrades in general remains to be elucidated. In the present study we present data on tun formation and desiccation tolerance in the marine tidal echiniscoidean, Echiniscoides sigismundi (M. Schultze, 1865), sampled from Lynaes, Denmark.

TARDIGRADE SAMPLING
Echiniscoides sigismundi was collected in autumn (September-November 2013) from barnacles in the tidal zone of North Sealand (Lynaes, Denmark). During sampling, the water temperature and salinity at the locality were in the ranges 9-13°C and 18-20‰, respectively. After collection, the tardigrades were kept at 5°C for 3-4 weeks on barnacle shells in seawater from the locality.

Experimental procedures
Prior to experimentation, E. sigismundi was isolated from the barnacle shells under a microscope (Zeiss Stemi 2000). Only clearly active animals were used in the experiments. The animals were pooled into groups of approximately 20 specimens and placed in watch glasses with either filtered seawater from the locality or ultrapurified water (Barnstead EASYpure UV/UF; Dubuque, IA, USA) for 1 h. The tardigrades were subsequently transferred to a piece of filter paper in a small volume of appropriate water and desiccated under ambient conditions (22-23°C). The point of complete desiccation was defined as the moment when E. sigismundi exhibited an easily visible colour change, i.e. the animals would go from transparent to rusty yellow. Following complete desiccation the tardigrades were kept dehydrated on filter paper for 48 h at 5°C, after which they were rehydrated in seawater. Their activity was monitored at 5 min, 30 min, 2 h, 24 h, and 48 h post rehydration. The tardigrades were considered active and alive if they exhibited clear movement or responded to a tactile stimulus. Dehydration was performed on two different dates, with three replicates in both seawater and ultrapurified water on each date. Relative humidity was 62% during dehydration and 56% during rehydration in the first experiment, whereas it was 39% and 44%, respectively, in the second experiment. Survival rates did not vary between the two dates.

Scanning electron microscopy
For scanning electron microscopy (SEM), fully hydrated specimens were fixed for 2 h at room temperature in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). The specimens were subsequently washed in ultrapurified water, dehydrated through a graded series of ethanol, critical-point dried (Autosamdri-815; Tousimis, Rockville, USA), and mounted on aluminium stubs. In addition, unfixed specimens dried on filter paper were also mounted on stubs. The stubs were subsequently coated for 80 s with platinum-palladium using a JFC-2300HR sputter coater (Jeol, Tokyo, Japan). SEM images were obtained using a JSM-6335F scanning electron microscope (Jeol, Tokyo, Japan).

Statistics
Data are expressed as means AE standard errors of the mean (SEM). For statistical analysis, OriginPro 9.1 (OriginLab, Northampton, MA, USA) was used to compute a one-tailed, two-sample Student's t-test at a significance level of P ≤ 0.05. Data sets were tested for normality and variance before computing the t statistic.

RESULTS AND DISCUSSION
During dehydration, limnoterrestrial eutardigrades contract along the anterior-posterior axis, while at the same time retracting their legs, thereby forming the body shape termed a tun. This tun state seems to be a prerequisite for anhydrobiotic survival in eutardigrades (Crowe & Madin, 1974;Crowe, 1975;Wright et al., 1992;Sømme, 1996;Halberg et al., 2013).
Our present study reveals that the marine heterotardigrade E. sigismundi dehydrated from its natural habitat (seawater) contracts into a tun that is comparable with the well-known eutardigrade tuns (Fig. 1A, B). When exposed to ultrapurified water, E. sigismundi swell and become incapable of contraction, and when subsequently transferred to filter paper and dehydrated, they collapse rather than form tuns (Fig. 1C).
Echiniscoides sigismundi thus seems to handle short-term desiccation from seawater and ultrapurified water equally well. Yet there is a difference in the recovery rates, with animals dried from seawater recovering significantly faster than those dried from ultrapurified water (Fig. 2), i.e. at 30 min and 2 h post-rehydration there is a significant difference in activity between seawater and ultrapurified water (P = 0.02 and 0.001, respectively). At 48 h the postrehydration activity of animals exposed to seawater and ultrapurified water did not significantly differ (P = 0.19). Hence, this species seems less dependent on maintaining structural integrity compared with limnoterrestrial eutardigrades.
Our results suggest that the processes underlying desiccation tolerance in echiniscoideans are different from those of eutardigrades. A possible implication is that anhydrobiosis has evolved several times within tardigrades or, alternatively, that the fundamental processes underlying cryptobiosis are an inherent feature of tardigrades and that these processes subsequently have been modified and refined in the different evolutionary lineages. The latter hypothesis is supported by the observation of desiccation tolerance and tun formation within marine arthrotardigrades (Jørgensen & Møbjerg, 2015). The presence of tun formation among all extant lineages, including marine echiniscoideans and arthrotardigrades, implies that the tun is an ancient and homologous trait, which evolved within the marine environment. If this is the scenario, the tun evolved as an adaptation to something other than anhydrobiosis. A characteristic of the marine environment is the presence of salts that build osmotic pressure. Tun formation may thus have evolved as a response to increases in salt concentrations associated with fluctuating marine habitats near land. Notably, E. sigismundi only forms tuns when dehydrated from seawater, supporting the hypothesis that tun formation originally was a response to elevated external osmotic pressure rather than desiccation per se. This hypothesis would further explain the ability of the tardigrade tun to withstand extraordinarily high hydrostatic pressures (Seki & Toyoshima, 1998).