Maas-Hernández LA et al. (2025), Description of the embryonic development of Hol...

ECB-ART-54067

PLoS One 2025 Jul 03;207:e0325564. doi: 10.1371/journal.pone.0325564.

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Description of the embryonic development of Holothuria floridana (Pourtalès, 1851) to produce juveniles for aquaculture and restocking.

Maas-Hernández LA , Olvera-Novoa MA , Rogers A , Felaco L , Macal-López K , Colas-Marrufo T .

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Overexploitation has severely affected the populations of Holothuria floridana, the second most valuable species of sea cucumber in the Gulf of Mexico and Greater Caribbean. Knowledge about its reproductive biology is limited, so this study aimed to update particularities about its reproduction and embryonic development that supports aquaculture technologies. Adults were collected in coastal waters off Celestún, Yucatán, Mexico, and transferred to the laboratory to induce spawning by thermal shock. Oocytes were obtained from individual females and fertilized artificially. Samples were incubated under controlled conditions to assess embryonic development using standard histology and by applying optical and scanning electron microscopy techniques. Fertilized oocytes settle and adhere to the substrate through a gelatinous cover. This species exhibits a lecithotrophic embryonic development, with a non-ciliated vitellaria larva that does not go through the auricularia and doliolaria stages, hatching as pentactula. At 1.5 dpf, the primordia of the oral tentacles appeared; for the 2 dpf, the structures of the oral tentacles were apparent, and a primary intestinal tubule was also distinguished, with a link with the anus at 3 dpf when the ring canal of the water vascular system appeared. At 3.5 dpf, the intestinal tubule enlarged and took on a rounded shape, hatching as a pentactula at 4 dpf, with the intestine connected to the mouth and anus, confirming that the digestive tube is complete and functional at this stage showing feeding behavior. The early juvenile stage was attained between 5 and 7 dpf, a much shorter time when compared with planktotrophic species. Its direct embryonic development, the absence of planktotrophic larvae, and the feasibility of inducing spawning through thermal shocks conferred H. floridana high potential for aquaculture in the Tropical Western Atlantic. Considering its benthic development, these results give practical information for developing hatchery infrastructure and protocols for rearing in captivity.

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	Fig 1. Holothuria floridana ovaries (A, B) and histological sections of the tubules (C, D).The gonad fixed for histology (B) shows its structuring in long and short-branched tubules. (C) Female 1 mature ovary tubule with the lumen filled with vitellogenic oocytes (vtg). (D) Female 2 developing ovary tubule with degrading oocytes (do), a few mature oocytes (mo), and primary oocytes (po) lining the tubule.
	Fig 2. Embryonic development of H. floridana from fertilization to morula [23]: A) fertilization envelope (f) and fertilized one-cell stage embryo (e). B) adherent gelatinous capsule (c) and oocyte protuberance (op). C) adherent gelatinous capsule (c). D) Extrusion of the polar body (pb). E) First cell division until the formation of the two blastomeres (2.5 hpf); F) Second cell division with four blastomeres (3 hpf); G) Third cell division (4 hpf); H-I: Morula at 5 hpf.
	Fig 3. Embryonic development of H. floridana from 8 hpf to 3.5 dpf.A) Blastula: blastoderm (bd). B) Blastula, characterized by the blastocele (bc). C) 24-h gastrula: blastopore (bp), archenteron (ac), and ectoderm (ec). D) 1.5 dpf vitellaria larva showing rounded edges on the opposite side of the blastopore, indicating the primordia of the primary oral tentacles. E) 2-dpf larva with early differentiation of primary oral tentacles (ot). F) 2.5 dpf vitellaria larva with further differentiation of oral tentacles. G) Larva vitellaria of 3 dpf with defined oral tentacles. H) Front view of 3.5 dpf vitellaria larva with five oral tentacles. I) Initiation of hatching at 3.5 dpf with the rupture of the fertilization envelope (f) by the oral tentacles.
	Fig 4. Pentactula and juvenile development of H. floridana.A) Pentactula at 4 dpf with oral tentacles (ot) and main podium (mp). B) 5 dpf pentactula larva: five oral tentacles and main podium. C) Pentactula with 6 dpf: sigmoidal intestine (i). D) Juvenile at 7 dpf with a secondary podium (sp), ossicles (os), and intestine. E) Juvenile of 10 dpf with two secondary podia and intestine. F) Juvenile of 20 dpf: sigmoid intestine and anus (a). G) Juvenile with 30 dpf showing main podium and three rows of podia, each containing three podia: stomach (st). H) Juvenile of 45 dpf showing six oral tentacles, main podium, and 10 secondary podia. I) Juvenile of 60 dpf showing eight oral tentacles.
	Fig 5. Biscuit ossicles of H. floridana at five (A), seven (B), and nine days (C) post-fertilization.
	Fig 6. SEM images of H. floridana.A) Oocytes showing spherical shape and details of the broken fertilization envelope. B) Details of the vitellaria with a rough surface. C) 5.5 dpf pentactula. D) view of the juvenile’s body at 45 dpf partially embedded in agarose. At this time, it is possible to distinguish E) ossicles (os), F) oral tentacle (ot), and G) oral tentacle bud (bu). At 60 dpf, the entire juvenile’s body is observed (H) as well as the I) ossicles (os), J) podia (p), K) oral tentacles (ot), and L) peltate buds of the oral tentacle (bu).
	Fig 7. Histological sections of H. floridana embryos.A) The morula in the process of blastulation, and blastomeres (bl) are set on the periphery. B) Blastula with blastoderm (bd) and blastocele (bc). C) Gastrulating blastula: blastopore (bp), blastocele, archenteron (ac), endoderm (ed) and ectoderm (ec). D) 24 hpf gastrula. E) Embryo at 1.5 dpf with primordia of the oral tentacle (ot). F) 2 dpf embryo with developing oral tentacles and primitive gut tube (pgt).
	Fig 8. Histological sections of vitellaria, pentactula, and juvenile of H. floridana.A) 2.5 dpf larva with intestinal tubule (it) and oral tentacles (ot). B) Larva of 3 dpf with oral tentacles, intestinal tubule connected to the anus (a), and ring canal of the water vascular system (rc). C) larva of 3.5 dpf with stomach (st), anus, and ring canal. D) 4 dpf pentactula larva; the connection between the mouth and the stomach is observed. E-F) Pentactula at 4.5 and 5 dpf with sigmoidal intestine (si). G) 5.5 dpf pentactula larva with the mouth (m), anus, and ring canal. H) Pentactula of 6 dpf, showing the digestive system consisting of the esophagus(es), stomach, and anus. I) Juvenile of 7 dpf with esophagus and stomach.
	Fig 9. Histological sections of H. floridana juveniles at 9 dpf (A): stomach (st) and anus (a).B) Juvenile of 10 dpf: esophagus (es), stomach, and anus. C) Juvenile of 15 dpf: esophagus, stomach, and anus. D) Juvenile of 20 dpf with oral tentacles (ot), esophagus, stomach, anus, and respiratory tree (rt). E) Juvenile of 30 dpf: esophagus, stomach, intestine (i), and anus. F) Juvenile of 45 dpf: esophagus, stomach, intestine, anus, pelted oral tentacles, and podia (p).