Ma KCK et al. (2025), Diversity and distribution of extracellular mic...

ECB-ART-54306

R Soc Open Sci 2025 Sep 17;129:250514. doi: 10.1098/rsos.250514.

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Diversity and distribution of extracellular microcrystals in holothuroid echinoderms.

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Biomineralization research in echinoderms has been focused on skeletal structures, which provide strength and protection. Other minerals, specifically extracellular microcrystals, are described here for the first time in echinoderms. Six morphotypes were isolated from a holothuroid and classified into three chemical compounds. Uric acid crystals were associated with fluids from the hydrovascular system, perivisceral coelom and respiratory tree, while calcium carbonate crystals were detected in the epithelium of the respiratory tree and calcium oxalate dihydrate crystals were found in tissues of the cloaca, integument, Polian vesicle and tentacle. Uric acid crystals were mostly non-encapsulated in tissues, whereas, in fluids, they were often encapsulated by phagocytes, alone or in groups, suggesting that they are waste products. Calcium carbonate crystals in the respiratory tree and calcium oxalate dihydrate crystals in the cloaca suggest that they are being expelled from the body. Insofar as calcium oxalate dihydrate crystals could be involved in calcium regulation, we speculate that they might be crystallized and retained in ossicle-associated integumentary tissues and tentacles, which are susceptible to damage and could use them as a calcium reserve to synthesize and repair ossicles. Observations of microcrystals in different holothuroid species examined suggest their ubiquity in the class Holothuroidea and more generally, deuterostomes.

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	Figure 1. . Microcrystals in C. frondosa: (A–B) cuboidal crystal (arrow indicates the line similar to barrel-shaped crystals), (C) barrel-shaped crystal with line along the length of the structure, (D) rhombic crystal, (E) multi-layered rhombic crystal, (F–G) other variants of the rhombic crystal, (H–I) rhombohedral crystal, (J) small rosette-shaped crystal, (H) large rosette-shaped crystal, (I) cylindrical crystal, (M) cylindrical crystal with visible internal structure (arrow), (N) cylindrical crystal with faceted ends (arrow), (O) unusually elongated cylindrical crystal, (P) cylindrical crystal with notches (arrows), (Q) cylindrical crystal with a lateral outgrowth (arrow), (R) three-pointed crystal, (S–T) cross-shaped crystal, (U–V) octahedral crystal and (W–X) dodecahedral crystal. Microcrystals in panels N, V and X imaged using a scanning electron microscope and all other microcrystals imaged under a light microscope. Scale bars represent 10 µm.
	Figure 2. . Raman spectroscopy spectra of five morphologies of microcrystals isolated from C. frondosa. Morphologies: (A) cuboidal, (B) rhombic, (C) cylindrical, (D) octahedral and (E) dodecahedral. Green bands highlight diagnostic peaks characteristic of uric acid in A and B, of calcium carbonate in C, and of calcium oxalate dihydrate in D and E.
	Figure 3. . Energy-dispersive X-ray spectroscopy spectra of three morphologies of microcrystals isolated from C. frondosa. Morphologies: cylindrical (green line), octahedral (black line) and dodecahedral (red line). C = carbon, O = oxygen, Na = sodium, Cl = chloride, Ca = calcium.
	Figure 4. . Corporeal distribution of microcrystals in C. frondosa comprising multiple morphologies grouped into six morphotypes and three chemical compounds (uric acid, calcium carbonate and calcium oxalate dihydrate). Low = prevalence of ≤33% of the samples examined per body component, moderate = prevalence of ˃33−66% of the samples, high = prevalence of ˃66% of the samples.
	Figure 5. . Microcrystals encapsulated by phagocytic coelomocytes of C. frondosa: (A) one cuboidal microcrystal, (B) two cuboidal microcrystals, (C) multiple cuboidal microcrystals, (D) one rhomb-shaped microcrystal, (E) two rhomb-shaped microcrystals, (F) orthographic view of a rhomb-shaped microcrystal and oblique view of a another, (G–H) rhomb-shaped microcrystal inside a phagocytic coelomocyte with filopodia (arrow), (I) rhomb-shaped microcrystal packaged with other materials around the distal ends of the crystal (arrows) and (J) several microcrystals packaged with other material (arrows). Black arrows in panels A–F indicate the phagocyte, white arrows in panels C–E indicate a foreign particle. Scale bars represent 10 µm.
	Figure 6. . Morphologies of microcrystals in C. frondosa (this study) compared with non-echinoderm or laboratory-grown examples from the literature. Images from the literature were adapted from Mundt and Shanahan [14], Patel et al. [15], Neuendorf [16], Lee et al. [48], Daudon et al. [53,54], Bird [64], Free [67], Marickar and Salim [68], Chatterjee et al. [69], Shastri et al. [75], Didymus et al. [80], Weber et al. [85], Pan et al. [87], Laipnik et al. [88], Thurgood et al. [91], Hartl et al. [94]. NB: barrel-shaped crystals described by Marickar and Salim [68] were classified as cuboidal to better fit this morphological comparison.
	Figure 7. . Suggested pathways of crystallization and excretion of uric acid and calcium oxalate dihydrate microcrystals in C. frondosa. A pathway for calcium carbonate microcrystals was not apparent due to their limited distribution within the body.