Technical remarks

For many arthropods, which possess sclerotized cuticular structures, fixation without additional contrasting is sufficient for quality CT imaging (see e.g. [17, 18]). In the present study, however, we tailored the protocol to maximize the overall contrast of a soft biological specimen that is normally not amenable to tomographic imaging. To achieve this, we used a sample preparation protocol based on a combination of techniques established for transmission and scanning electron microscopy. For post-fixation and contrasting, it has been shown that for onychophorans (a related group of soft-bodied invertebrates), OsO 4 provides the highest level of overall tissue contrast compared to other contrasting agents [19]. In this regard, it is advantageous compared to selective stains, like those required for fluorescence microscopy, when visualizing the overall internal anatomy of a whole animal. We further enhanced the OsO 4 -contrasting steps of the protocol by adding ferrocyanide [20] and using the OTO technique (osmium-thiocarbohydrazide-osmium) [21].

Due to the advanced staining procedure applied in this work, the filtered backprojection (FBP) reconstruction of the tardigrade specimen (Additional file 1a) already showed a strong soft-tissue contrast. Furthermore, the introduced phase contrast, i.e., the edge enhancement caused by Fresnel diffraction, was small enough in the obtained data that it did not hamper the subsequent image segmentation steps. Consequently, it was not necessary to apply a phase-retrieval algorithm to improve the soft-tissue contrast, as had been demonstrated for the onychophoran limb [3]. This offers the advantage of no undesired image blurring, an artefact common to such phase-retrieval steps that typically must be removed by additional processing steps [3].

Instead, we used an edge-preserving statistical iterative reconstruction (SIR) that additionally sharpens the images by compensating for the source blurring during acquisition by either directly including a model for the source in the SIR [22] or by a separate deconvolution step [23]. The SIR reconstruction (Additional file 1b) clearly improved the image noise and resolution compared to the FBP reconstruction, thereby facilitating a precise segmentation and the visualization of fine details, such as the salivary glands and especially the brain, which could be more easily differentiated from the surrounding tissue (arrows in Additional file 1a, b).

On the one hand, the lack of a selective stain complicates the manual segmentation of structures that are closely associated with each other or are located in an area of high tissue density, such as the brain and associated structures in the tardigrade head as well as fine musculature. For this reason, the inner connectives could not be reliably segmented in our dataset, nor could the eyes be identified because they lie inside the contours of the brain [24]. On the other hand, lumens of structures such as the midgut and buccal tube have been well preserved during fixation and are easily recognizable in the CT scans. Further studies may focus on selectively enhancing the contrast of specific organs or structures by treating the sample with various contrasting agents [19].

Morphological analysis

The quantitative nature of the data allowed us to measure the size and volume of most of the major structures in the body and analyze their spatial relationships in an animal that was not cut, punctured, or otherwise physically disrupted. The tardigrade Hypsibius exemplaris shown herein represents a relatively small specimen at 152 μm, while adult individuals typically measure ~ 230 μm in length [25]. The presence of an ovary suggests that this specimen was at least approaching sexual maturity, although any evidence of developing eggs is absent. Therefore, we estimate that the specimen presented herein is probably a young adult, since tardigrades generally continue growing even after sexual maturity [1].

Hypsibius exemplaris is a parthenogenetic species, meaning all individuals are females and possess an ovary. The ovary occupies 1.0% TBV in the investigated specimen, but we expect the size of this organ to vary greatly depending on the reproductive cycle of the animal [26, 27]. Interestingly, our scans show that the ovary develops not directly dorsal to the midgut, but rather dorsolaterally, thereby displacing the midgut. The attachment sites of the two anterior ligaments that connect the ovary to the body wall likewise reflect this asymmetry. While the ligaments are generally presented in the literature as isolated within the body cavity [28], the right ligament in our scans appears closely associated with the midgut wall. Whether the ovary always occupies the space to the left of the midgut or whether this position varies between tardigrades has, to our knowledge, never been addressed, as the position of the ovary is usually described simply as “dorsal” or overlying the midgut [28]. Future studies using nanoCT may also aim to quantify exactly how the volume of the ovary changes throughout the reproductive cycle. The same applies to the midgut, the size of which may depend not only on the nutritional state of the animal but also on the stage of the life cycle [26]. For example, tardigrades undergoing a molt are unable to feed due to the expulsion of the entire foregut (the so-called “simplex” stage), and the midgut shrinks in size accordingly [1, 27].

The nervous system in tardigrades consists of a dorsal brain and a ganglionated ventral nerve cord (reviewed in ref. [29]). Our volume measurements of the brain revealed that the size of this organ relative to body volume is approximately double that of honeybees and even greater compared to larger insects, such as diving beetles (ditystids; [30]). Our results therefore seem to follow the trend described by Haller’s Rule, by which a reduction in body size accompanies an increase in the relative brain volume, at least in insects [31]. However, direct comparisons between the brains of tardigrades and insects should be made with caution, as several species of featherwing beetles (ptilids), which are 380–630 μm in length, have brain volumes of 2.9–4.3% TBV [32], i.e., much larger than that of the tardigrade presented herein. Brain size may also depend in part on the innervation of any sensory organs on the head, as the specialized brain regions associated with these structures may be enlarged [33]. In this regard, it would be interesting to compare the brain sizes across different heterotardigrades, which possess an array of head appendages that are not present in eutardigrades like H. exemplaris.

Although we did not measure the volumes of the ventral trunk ganglia, analyzing these structures in 3D demonstrates that most of the cell bodies of each ganglion lie dorsal to the neuropil (i.e., connectives) [34]. This is different from onychophorans and pycnogonids, where most of the somata occupy the ventral space of the nerve cord and ganglion, respectively [35, 36]. The ganglia in arthropods have a more complex organization with neuropils at several levels [37] but share with tardigrades an anterior position relative to the corresponding leg pair of the same segment [38, 39]. This anterior shift, known as “parasegmental” (summarized in ref. [29]), is clearly visible in our scans.

The storage cells of tardigrades are cells of the body cavity that are involved primarily in nutritional maintenance [40, 41] but also play a role in vitellogenesis [42, 43] and possibly immune function [44]. These cells may therefore display variable internal morphologies and ultrastructure relating to their roles [41, 45], a hypothesis that our data support based on the different internal gray values and textures seen in the storage cells in our CT scans. The sizes of storage cells have been measured in several studies in a number of different species [40, 42, 45, 46]. Reuner et al. [40] performed the most comprehensive comparison (although H. exemplaris was not one of the species studied) and found that the number and size of storage cells varies greatly depending on the species. Assuming a spherical shape, the average diameter of the storage cells in our specimen is ~ 4.5 μm, thereby most closely resembling those of Macrobiotus sapiens in size, which measured 4.4–8.3 μm in diameter [40].

Previous studies have also investigated how different physiological processes affect storage cells, for example oogenesis [41,42,43, 47], cryptobiosis [46], and starvation [40]. Based on these studies, it is evident that many parameters affect different species differently. For example, anhydrobiosis (desiccation-induced cryptobiosis) has a significant effect on the average size of storage cells in Milnesium tardigradum, and Richtersius coronifer but not in Paramacrobiotus tonollii or Macrobiotus sapiens [40, 46]. On the other hand, starvation led to a decrease in the size of storage cells in all species investigated, supporting the hypothesis that the primary role of these cells is nutritional maintenance [40, 41].

The specimen presented herein has a smaller number and smaller size of storage cells than any species previously investigated using other techniques, as well as a relatively small body size [40]. Although the data of Reuner et al. [40] argue against a positive correlation between body size and the number of storage cells, our results seem to suggest otherwise (at least for H. exemplaris). It should also be noted that the diameter estimates of storage cells presented herein are based on volume measurements; we did not directly measure diameters as has generally been done in previous studies. Because a large proportion of storage cells tend to be irregular in shape [46], calculations based solely on diameter may be imprecise compared to volume measurements. The different techniques may therefore lead to significant differences in size estimates. For example, while critical point drying as performed in the present study may affect the absolute sizes of all structures, our data indicate that, even if structures did indeed shrink, the degree of shrinkage was similar for all structures and spatial relationships were thus unaffected [48]. Hence, our measurements based on % TBV are intended to account for this potential artifact. Although future studies may aim to specifically address the effects of different methods on final size estimates, we propose that CT scans may be a more accurate method of analyzing size distributions of storage cells. An additional advantage is that the scanned specimens remain fully intact, minimizing the chance that any storage cells may have leaked out of the animal.