<p class="HeadingRunIn"><strong>Combined visualization of echinoderm hard and soft parts using contrast-enhanced micro-computed tomography</strong></p>

Abstract

Recent studies have shown that micro-computed tomography (µCT) must be considered one of the most suitable techniques for the non-invasive, three-dimensional (3D) visualization of metazoan hard parts. In addition, µCT can also be used to visualize soft part anatomy non-destructively and in 3D. In order to achieve soft tissue contrast using µCT based on X-ray attenuation, fixed specimens must be immersed in staining solutions that include heavy metals such as silver (Ag), molybdenum (Mo), osmium (Os), lead (Pb), or tungsten (W). However, while contrast-enhancement has been successfully applied to specimens pertaining to various higher metazoan taxa, echinoderms have thus far not been analyzed using this approach. In order to demonstrate that this group of marine invertebrates is suitable for contrast-enhanced µCT as well, the present study provides results from an application of this technique to representative species from all five extant higher echinoderm taxa. To achieve soft part contrast, freshly fixed and museum specimens were immersed in an ethanol solution containing phosphotungstic acid and then scanned using a high-resolution desktop µCT system. The acquired datasets show that the combined visualization of echinoderm soft and hard parts can be readily accomplished using contrast-enhanced µCT in all extant echinoderm taxa. The results are compared with µCT data obtained using unstained specimens, with conventional histological sections, and with data previously acquired using magnetic resonance imaging, a technique known to provide excellent soft tissue contrast despite certain limitations. The suitability for 3D visualization and modeling of datasets gathered using contrast-enhanced µCT is illustrated and applications of this novel approach in echinoderm research are discussed.

Novel insights into early life stages of finned octopods (Octopoda: Cirrata)

“…Such an approach-based on MRI-was recently shown to be effective in studying the anatomy of a prematurely hatched cirrate embryo (Shea et al, 2018), but was later also used to gather anatomical data suitable for a full species description using an adult cirrate specimen collected in the northwestern Pacific Ocean (Werner et al, 2016;Ziegler, 2021;Ziegler & Sagorny, 2021). The present study extends this approach by applying MRI to intact cirrate egg capsules as well as contrast-enhanced µCT to cirrate embryos (Ziegler, 2019;Ziegler et al, 2018). However, while the isotropic voxel resolution achieved here both using MRI and µCT may be sufficient to differentiate between major egg capsule structures as well as embryonic organ systems (Figs.…”

“…A single cirrate egg containing an embryo and subsequently the individual, extracted embryo as well as two larger embryos were analyzed in 3D using contrastenhanced µCT (Table 1L, N, O). Samples were stained for 6 weeks each with 0.3% phosphotungstic acid (PTA) Photography, illustration Boletzky (1978Boletzky ( -1979Boletzky ( , 1980Boletzky ( , 1982Boletzky ( , 1985) Galathea, 1950Galathea, -1952 Laccadive Sea, Indian Ocean, 01°00'N 76°17'E, 4425 m Illustration Boletzky (1980Boletzky ( , 1982 Written report Verrill (1885) in 70% ethanol (Ziegler, 2019;Ziegler et al, 2018)…”

Methods to study organogenesis in decapod crustacean larvae II: analysing cells and tissues

“…12, 13). In the field of anatomy, µCT has been demonstrated to be a useful analytical tool applied in a rapidly growing variety of metazoan taxa, such as cnidarians [100], plathelminths [101], nematomorphs [99], nematodes [102], polychaetes [103,104], molluscs [98,105], echinoderms [106,107] as well as arthropods [108][109][110][111][112][113]. Modern, lab-based µCT-scanners deliver high resolution allowing the investigation of tiny specimens with body sizes of free-living crustacean larvae ranging from 75-195 µm in Tantulocarida [114], the smallest arthropods in the world, up to 5 cm in length, e.g., in Stomatopoda [115].…”

Schizasterid Heart Urchins Host Microorganisms in a Digestive Symbiosis of Mesozoic Origin

“…Three-dimensional (3D) analysis of internal organ systems was conducted using contrast-enhanced µCT. Three intact specimens of B. townsendi (21, 22, 23 mm TL) were formalin-fixed (4% in seawater) and, after several weeks of storage in fixative, were transferred to 70% ethanol through a graded series, and finally contrasted with 0.3% phosphotungstic acid (PTA) in 70% EtOH for 4 weeks (Ziegler et al, 2018;Ziegler, 2019). A single, ethanol-fixed specimen of A. cordatus (42 mm TL) as well as two formalin-fixed museum specimens (Protenaster rostratus, 28 mm TL and Tripylus excavatus, 37 mm TL) were treated in the same way.…”