Figure 1.
Rearrangement of organs and cells during anhydrobiotic tun formation in Richtersius coronifer.
Light microscopy of A. active, hydrated animal (lateral view) and B. a tun (ventral view) showing the rearrangement of major anatomical structures during tun formation. Note the compact body shape of the tun. Dashed circles indicate areas of the midgut (mg), gonads (go) and pharyngeal bulb (pb), respectively. The degree of longitudinal contraction is ultimately limited by the length of the rigid stylets (st). The pharyngeal bulb is for the most part repositioned in the dorsomedian plane. Maximum projection image of a confocal z-series of C. hydrated DAPI stained specimen (lateral view), and D. DAPI stained tun (ventral view) demonstrating the reposition of cell nuclei during tun formation. Scanning electron micrograph of E. a hydrated specimen (lateral view) and F. a tun (dorsal view) revealing the extensive changes in external morphology associated with formation of the tun. A↔P, anterior-posterior axis; br, brain; gI-gIV, ventral ganglia; mo, mouth. Scale bars = 100 μm.
Figure 2.
Effect of DNP and phalloidin on anhydrobiotic survival.
A. Pre-incubation in DNP prior to dehydration, and attempting to induce anhydrobiosis (D→A→W), significantly reduces survival to 4 ± 9 % (0.1 mM) and 2 ± 4 % (1.0 mM). B. Incubating tardigrades in phalloidin (P→W) did not decrease survival at 0.01 mg/ml (97 ± 7 %), 0.1 mg/ml (96 ± 4 %) and 0.5 (95 ± 5 %) mg/ml. At 1 mg/ml, survival was significantly reduced to 26 ± 7 % (P<0.001; Table S2). Pre-incubation in phalloidin (P→A→W) reduced post-anhydrobiotic survival at concentrations of 0.1 mg/ml (82 ± 6 %), 0.5 mg/ml (77 ± 6 %) and 1.0 mg/ml (0 ± 0%) (P<0.001; Table S2). Significant differences between treatments are indicated by asterisks, with the significance levels P>0.05 (not significant) and P≤0.001 (significant, ***).
Figure 3.
Myoanatomical changes in Richtersius coronifer during tun formation.
A-D. 3D reconstructions of the tardigrade musculature as visualized by fluorescent phalloidin. A. Lateral view of active (hydrated) state showing details of the dorsal, lateral and leg musculature. B. Ventral view showing details of the ventral longitudinal musculature in the active state. C. Dorsal view of the myoanatomy of the tun (dehydrated) state D. Ventral view of the myoanatomy of the tun. E-F. Scanning electron microscopy (SEM) of animals in the tun state showing the corresponding external morphology of the myoanatomy presented in C and D. E. Dorsal view of the tun. F. Ventral view of the tun. G. SEM of an animal incubated in 1.0 mg/ml phalloidin for 24 h and subsequently dehydrated. The animal failed to form a tun upon dehydration, and collapsed into a flattened shape. A similar collapse was seen in DNP exposed animals upon dehydration. H. Corresponding maximum projection image of a confocal z-series of the musculature of a specimen incubated in 1.0 mg/ml phalloidin for 24 h before dehydration. A↔P, anterior-posterior axis; A-W, dorsal attachment sites; τ0-τ4, lateral attachment sites; la-g, ventral intermediate attachment sites; 1-7, ventromedian attachment sites; pb, pharyngeal bulb; ω1-Ω, attachment sites of muscles associated with the pharyngeal bulb; stm, stylet muscles. Solid circles indicate lateral attachment sites, solid squares show ventral intermediate attachment sites, while dashed circles indicate areas of the legs. Scale bars = 100 μm.
Figure 4.
Schematic representation of the muscles involved in tun formation in Richtersius coronifer.
Schematic representation illustrating contraction of muscles during the transition from the active (hydrated) to the tun (dehydrated) state. A. Ventral longitudinal musculature. B. Dorsal longitudinal musculature. C. Leg musculature. The dorsal longitudinal, ventral longitudinal, as well as lateral musculature are involved in reshaping the whole body during anhydrobiosis, and are consequently responsible for generating the compact body shape of the tun. Tun formation is moreover characterized by the withdrawal of the legs into the body cavity. Letters and numbers indicate specific muscle attachment sites (see Figure 3).