Peters-Didier J and Sewell MA (2019), The role of the hyaline spheres in sea cucumber...

ECB-ART-47151

Evodevo 2019 Jan 01;10:8. doi: 10.1186/s13227-019-0119-4.

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The role of the hyaline spheres in sea cucumber metamorphosis: lipid storage via transport cells in the blastocoel.

Peters-Didier J , Sewell MA .

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Background: For echinoderms with feeding larvae, metamorphic and post-settlement success may be highly dependent on larval nutrition and the accumulation of energetic lipids from the diet. In contrast to the sea urchins, starfish and brittle stars within the Phylum Echinodermata, sea cucumber metamorphosis does not involve formation of a juvenile rudiment, but instead there is a rearrangement of the entire larval body. Successful metamorphosis in sea cucumbers is often associated with the presence in the late auricularia stage of an evolutionary novelty, the hyaline spheres (HS), which form in the base of the larval arms. Known since the 1850s the function of these HS has remained enigmatic-suggestions include assistance with flotation, as an organizer for ciliary band formation during metamorphosis and as a nutrient store for metamorphosis. Results: Here using multiple methodologies (lipid mapping, resin-section light microscopy, lipid and fatty acid analyses) we show definitively that the HS are used to store neutral lipids that fuel the process of metamorphosis in Australostichopus mollis. Neutral lipids derived from the phytoplankton diet are transported by secondary mesenchyme cells ("lipid transporting cells", LTC), likely as free fatty acids or lipoproteins, from the walls of the stomach and intestine through the blastocoel to the HS; here, they are converted to triacylglycerol with a higher saturated fatty acid content. During metamorphosis the HS decreased in size as the triacylglycerol was consumed and LTC again transported neutral lipids within the blastocoel. Conclusion: The HS in A. mollis functions as a nutrient storage structure that separates lipid stores from the major morphogenic events that occur during the metamorphic transition from auricularia-doliolaria-pentactula (settled juvenile). The discovery of LTC within the blastocoel of sea cucumbers has implications for other invertebrate larvae with a gel-filled blastocoel and for our understanding of lipid use during metamorphosis in marine invertebrates.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC115919910

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	Fig. 1. Late auricularia of A. mollis. a Light microscope view of general anatomy of 14-day larva showing ciliated band (CB), stomach (ST), axohydrocoel (AX, position shown by ), left somatocoel (LS, position shown by ) and granular mass (GM). Scale bar 100 µm. b 14-day larva stained with Nile Red under blue light excitation and polarized light. Stomach fluoresces green; one of many red blastocoel spherules (BS) shown with white circle; granular mass (GM, position shown by *) also fluoresces red. Scale bar 100 µm. c 16-day auricularia stained with Nile Red under blue light excitation. Some blastocoel spherules (BS) are highlighted with white circles. Scale bar 200 µm. d 16-day auricularia stained with Nile Red under blue light excitation. Stomach (S) and blastocoelic spherule (arrow) carrying both neutral lipids (yellow) and more polar lipids (red). Scale bar 25 µm. e 16-day auricularia larvae stained with Nile Red and Hoechst nuclear stain under simultaneous blue light excitation and light microscopy. Stomach (S), left somatocoel (LS) and blastocoelic spherule (BS), with likely location of the nucleus (thin arrow). Black arrow indicates a cell of similar size to the BS that appears to differentiate from the stomach epithelium. White arrow indicates a smaller cell type lining the stomach epithelium. Scale bar: 25 µm. f Blastocoelic spherule within the blastocoel (Bl) of a 14-day larva stained with Nile Red under Nomarski light microscopy. Spherical inclusions (SI) in the blastocoelic spherule are indicated by arrow. Blastocoel also contains a fibroblast-type cell (Fb). Scale bar: 12.5 µm. g Blastocoelic spherule amoeboid movement in a 14-day auricularia larva stained with Nile Red under light microscopy. Arrows indicate sequence of movement of one blastocoelic spherule (white arrow, white circle) in a time lapse of 40 min. Scale bar: 50 µm
	Fig. 2. Late auricularia of A. mollis. a Confocal image of 14-day auricularia stained with Hoechst nuclear dye and LipidTOX™ green neutral lipid stain. Full digestive system is shown: mouth (M), oesophagus (O), stomach (S), intestine (I), anus (A). Only stomach stains green for neutral lipids. Ciliary band (CB) and left somatocoel (LS, position shown by *) are also shown. Scale bar: 50 µm. b 16-day auricularia stained with Nile Red under blue light excitation showing multiple red-staining LTC within the blastocoel. Scale bar: 25 µm. c Confocal image of 20-day auricularia stained with Hoechst nuclear dye, LipidTOX™ green neutral lipid stain and LipidTOX™ red phospholipid stain. Lipid transporting cells (LTC, white circle) and hyaline spheres (HS) stain green for neutral lipid; some LTC (white arrows) and one granular mass (GM) show mixed fluorescence between green and red. Ciliary band (CB) and stomach (S) are also shown. Scale bar: 100 µm. d Longitudinal section of digestive system of 14-day auricularia. Oesophagus (O), stomach (S), intestine (I), blastocoel (Bl), lipid transporting cell (LTC, black circle). Collagen/elastin fibres are present in the blastocoel. Scale bar: 60 µm. e Longitudinal section through 16-day auricularia. Lipid transport cells (LTC) and progenitor type of cells (CII) suspended in the blastocoel. Stomach (S) and intestine (I) are both lined internally with pink-staining mucus layer. Lipid deposits (LD) in the stomach wall are circled (white circle). Scale bar: 25 µm. f LTC emerging from the digestive epithelium of the intestine (I) and into the blastocoelic space (Bl) between the stomach (S) and the intestine. Blastocoelic space is filled with collagen/elastin fibres. Scale bar: 25 µm
	Fig. 3. Late auricularia of A. mollis with detail of hyaline spheres. a Light microscope image of 21-day auricularia showing numerous hyaline spheres (HS and ) and granular mass (GM, position shown by ) in the posterolateral larval lobes. Scale bar: 200 µm. b 19-day auricularia stained with Nile Red under blue light excitation; larva gently pressed against the glass slide using a coverslip. Hyaline spheres (HS) in all lobes stain yellow for neutral lipid. Lipid transporting cells (LTC; white circle) and granular mass (GM) stain red for a more polar lipid. Digestive system includes mouth (M), oesophagus (O), stomach (S). Scale bar: 200 µm. c Section through a hyaline sphere (HS) within blastocoel (Bl), adjacent to the ciliated band (CB) of 16-day auricularia. Macrophage-type cells (MC, white circle) are present within the HS. Arrow indicates collagen/elastin fibres inside the HS. Scale bar: 25 µm. d Hyaline sphere (HS) next to a granular mass (GM), abutting the ciliated band (CB) in one of the posterolateral larval lobes of 16-day auricularia. Arrows indicate collagen/elastin fibres in the blastocoel (Bl) and inside the HS. Scale bar: 25 µm
	Fig. 4. Doliolaria of A. mollis. a Confocal image showing outer structure with three ciliated bands (CB) of 23-day doliolaria stained with Hoechst nuclear dye, LipidTOX™ green neutral lipid stain and LipidTOX™ red phospholipid stain. Scale bar: 50 µm. b Inner structures of same larvae as in Panel A highlighting ciliated band (CB), hyaline spheres (HS) and lipid transporting cells (LTC, white circle). Scale bar: 50 µm. c Doliolaria larva (23-day) stained with Nile Red under Nomarski light microscopy with ciliated band (CB), stomach (S), hyaline spheres (HS), granular mass (GM), area containing the 5 oral tentacles (OT, white circle), and new oral opening (Or, position shown by *). Scale bar: 100 µm. d Same larva as in Panel C under blue light excitation highlighting lipid transporting cells (LTC, white circle). Scale bar: 100 µm. e 25-day doliolaria larva with protruding oral tentacles stained with Nile Red under blue light excitation. Hyaline spheres (HS), granular mass (GM). Scale bar: 100 µm. f Section through 23-day doliolaria larva. Blastocoel (Bl); ciliary band (CB); hyaline spheres (HS) containing lipid transporting cells (white circle); oral tentacles (T); fibrillar matrix (F). Arrows indicate collagen/elastin fibres in the blastocoel pulling the HS towards the centre of the larva. Scale bar: 100 µm
	Fig. 5. A. mollis pentactula (early juvenile). a Pentactula (27-day) stained with Nile Red under blue light excitation. Note minimal external lipid staining. Granular mass (GM), hyaline spheres (HS), oral tentacles (OT, circled). Scale bar: 75 µm. b Confocal image of pentactula (27-day) stained with Hoechst nuclear dye, LipidTOX™ green neutral lipid stain and LipidTOX™ red phospholipid stain showing formation of juvenile digestive system (DS) and oral tentacles (OT, circled). Scale bar: 50 µm. c Section through pentactula (27-day) with degrading hyaline spheres (HS) and multiple lipid transporting cells (LTC, white circle) between blastocoelic fibres (F). Oral tentacles (T) and water vascular system (WVS). Scale bar: 50 µm. d Pentactula (27-day) with degrading hyaline spheres (HS), developing juvenile digestive system (DS) and lipid transporting cells (LTC, white circle) in blastocoel (Bl) and within the HS. Scale bar: 35 µm. e Pentactula (27-day) longitudinal section showing hyaline spheres (HS) close to developing juvenile digestive system (DS). Oral tentacles (T), fibrillar matrix (F) and granular mass (GM). Scale bar: 80 μm. f Pentactula (27-day) longitudinal section showing hyaline spheres (HS) close to developing juvenile digestive system (DS). Oral tentacles (T), granular mass (GM). Scale bar: 120 μm
	Fig. 6. Lipid and fatty acid composition of A. mollis. a Neutral lipid content of triacylglycerol (TAG), free fatty acid (FFA) and diacylglycerol (DAG) in auricularia, auricularia with hyaline spheres (HS) and through metamorphosis via doliolaria to pentactula. (N = 5). b Total structural lipids (phospholipids and sterol) as in Panel A, but with age of larvae (days post-fertilization) as X-axis. c TLC plate developed in a neutral solvent system containing hexane–diethyl ether–acetic acid (80:20:1, by vol.) followed by staining with iodine vapours. Lipid extracts of A. mollis auricularia with HS (A-HS), doliolaria (D) and lipid standards (STD). Grey line shows origin. d Re-extracted TAG band from TLC plate stained with Nile Red shows yellow colour under blue light excitation. e Re-extracted FFA band from TLC plate stained with Nile Red shows red colour under blue light excitation. f Fatty acid composition of auricularia and auricularia with hyaline spheres (HS) divided into saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (N = 3)
	Fig. 7. Summary diagram of size and arrangement of lipid transporting cells (LTC, orange circles), hyaline spheres (HS, yellow) and granular masses (GM, red) during larval development and metamorphosis of A. mollis. Green colour of digestive system indicates active feeding on phytoplankton and lipid accumulation. Grey colour of digestive system indicates lipid use during the non-feeding period. Images of: a early auricularia, b late auricularia, c doliolaria and d pentactula; drawn at the same scale based on photographic images. In the early auricularia the GM and a few small LTC are observed. The late auricularia is a much larger larva, with an increase in the number and size of the LTC (one group circled) that have transported neutral lipids to multiple HS in the base of the larval arms. During the metamorphic transition to the doliolaria the HS release LTC to fuel the reorganization of the gut and the development of the water vascular system (primary tentacles shown in white). In the pentactula the primary tentacles are now external and HS remain in the juvenile stage to provide further nutrients for the peri-metamorphic period. The function of the GM remains unknown

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