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 Lynaes (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.

Little is known about ionic and osmotic stress tolerance in tardigrades. Here, we examine salt stress tolerance in Ramazzottius oberhaeuseri and Echiniscus testudo from Niv a (Denmark) and address whether limno-terrestrial tardigrades can enter a state of quiescence (osmobiosis) in the face of high external osmolyte concentrations. Direct transfers into NaCl solutions showed an upper tolerance level of around 600 mOsm kg À1 in R. oberhaeuseri and 200 mOsm kg À1 in E. testudo. During salt exposures, R. oberhaeuseri contracted into a 'tun', whereas E. testudo remained active leaving it more susceptible to acute effects of the ions. Further experiments focused on the more resilient R. oberhaeuseri, which entered a tun and readily regained activity when directly exposed to polyethylene glycol and sucrose of up to 872 AE 0 and 813 AE 3 mOsm kg À1 , respectively, revealing a higher tolerance towards non-ionic osmolytes as compared to NaCl. Ramazzottius oberhaeuseri furthermore readily regained activity following gradual increases in non-ionic osmolytes and NaCl of up to 2434 AE 28 and 1905 AE 3 mOsm kg À1 , respectively, showing that short-term acclimation promoted salt stress tolerance. Our results suggest that the limno-terrestrial R. oberhaeuseri enters a state of quiescence in the face of high external osmotic pressure and that it, in this state, is highly tolerant of ionic and osmotic stress.

Tardigrades are microscopic metazoans that withstand environmental extremes by entering dormant states, such as cryptobiosis (latent life). In addition, they may also form cysts. Here, we present the first report of cyst formation in a marine heterotardigrade, i.e., Echiniscoides sigismundi, which constitutes a cryptic species complex present worldwide in tidal zones. The cysts were initially discovered during experimental series constructed to investigate osmotic stress tolerance. The animals, which eventually formed cysts, showed signs of an imminent molt at the beginning of experimentation. We use the term ''cyst'' for stages, where a total of three or more cuticles have been synthesized. Our observations show that encystment in E. sigismundi involves synthesizing of at least two new cuticle layers. Legs with discharged claws are present in connection with the first outer cuticle, as well as the second cuticular layer. In the most developed cyst, a third cuticle lacking claws seems to surround the animal, which is delineated by a fourth cuticle. Many features are shared with the well-studied cysts of eutardigrades. The cysts of E. sigismundi, however, lack pigmentation and have an extra set of claws, and the animal inside retains buccopharyngeal sclerified parts, until discharging the third cuticle. The finding of cysts in a marine heterotardigrade is novel and confirms that encystment also occurs within this major evolutionary lineage.

Tardigrades belong to the most radiation tolerant animals on Earth, as documented by a number of studies using both low-LET and high-LET ionizing radiation. Previous studies have focused on semi-terrestrial species, which are also very tolerant to desiccation. The predominant view on the reason for the high radiation tolerance among these semi-terrestrial species is that it relies on molecular mechanisms that evolved as adaptations for surviving dehydration. In this study we report the first study on radiation tolerance in a marine tardigrade, Echiniscoides sigismundi. Adult specimens in the hydrated active state were exposed to doses of gamma radiation from 100 to 5000 Gy. The results showed little effect of radiation at 100 and 500 Gy but a clear decline in activity at 1000 Gy and higher. The highest dose survived was 4000 Gy, at which ca. 8% of the tardigrades were active 7 days after irradiation. LD50 in the first 7 days after irradiation was in the range of 1100–1600 Gy. Compared to previous studies on radiation tolerance in semi-terrestrial and limnic tardigrades, Echiniscoides sigismundi seems to have a lower tolerance. However, the species still fits into the category of tardigrades that have high tolerance to both desiccation and radiation, supporting the hypothesis that radiation tolerance is a by-product of adaptive mechanisms to survive desiccation. More studies on radiation tolerance in tardigrade species adapted to permanently wet conditions, both marine and freshwater, are needed to obtain a more comprehensive picture of the patterns of radiation tolerance.