Holothuria Lab Demonstration

Instructor- José E. Garcia-Arrarás, University of Puerto Rico

I. Echinoderm groups and regenerative abilities II. Sea cucumbers (Holothuria)

Evisceration Wound Healing Tentacles

III. Intestinal Regeneration Process- Anatomy Histological Process

IV. Cellular Processes Muscle de-differentiation Extracellular Matrix degradation Blastema formation

Evisceration- Members of the group Holothuria (H. glaberrima) eviscerate through the cloaca. Usually most of the viscera except one or two respiratory trees are ejected. Members of the group Dendrochirotida (Athyonidium, Thyonella, Thyone) eviscerate anteriorly, including calcareous nerve ring and tentacles.

In the laboratory, evisceration is usually done using KCl injections. Other investigators have used distilled water and electricity to induce evisceration in other species. In the workshop- We will induce evisceration with 0.35 KCl. In our case we have used 3-5mls of 0.35MKCl injected into the coelomic cavity. Usually the animals will complete evisceration in 1-3 minutes. The animals are eviscerated in a dish containing enough sea water to cover them up. Once evisceration occurs, animals are taken out of the dish, rinsed in sea water and transferred to the aquarium where they will undergo regeneration. Preliminary experiments with Athyonidium chilensis suggest that this species is not prone to eviscerate following KCl or distilled water injections. We will attempt to eviscerate animals with a larger dose of KCl. Otherwise animals will be sectioned in the anterior end (just posterior to the calcareous ring and tentacles), and the viscera will be removed. Sea cucumber anatomy- Dissection - Place animals in ice-cold water for 30min-1 hr. With a sharp knife or blade cut the anterior part of the animal removing the tentacles and nerve ring. Dissect the animal lengthwise and pin- down on dissecting dish. Animals can be immersed in seawater to prevent tissues from drying. Determine the organs present in normal versus regenerating animals. Use the following diagrams to identify the internal structure.

Holothuria glaberrima

Thyone briaereus (after Coe,1912, in Hyman 1955)

Histological Process- Slides from various regenerating stages of H. glaberrima stained with toluidene blue will be available for observation. Identify mesentery and regenerating structure Identify three tissue layers:

Mesothelium (includes coelomic epithelium and muscle layer)- (The coelomic epithelium is similar to the vertebrate serosa)

Internal connective tissue- similar to submucosa Luminal epithelium- similar to mucosa.