Methods.

A number of methods were tested for artificially inducing the expulsion of internal organs. The use of 1 part 7N ammonia in 800 parts of sea water as described by Kille (1931) for Thyone was unsatisfactory for S. mollis. Some specimens died without expelling viscera, while in the remainder autotomy did not occur in less than several hours and the specimens died within 14 days. A series of specimens were induced to undergo autotomy by placing them in just sufficient sea-water to cover the specimen. This method was effective within from 8–24 hours, without further treatment. It was successful in 85% of the specimens treated in this way, but had the disadvantage that autotomy would frequently occur during the night and specimens would then remain for several more hours in a small volume of polluted sea-water. This contributed to a high mortality rate.

When injecting distilled water into the body cavity of holothurians as a control in experiments for testing the effect of drugs on autotomy and behaviour, Domantay (1931) found that it produced no effect in small amounts. Four specimens of Holothuria sanguinolenta injected with 10–14 cc. of distilled water, however, expelled viscera. These results suggested the use of distilled water for inducing autotomy in S. mollis. The injection of 1 cc. per 10 gm. body weight induced autotomy in all specimens treated. The response occurred within 30 seconds to 5 minutes after injection, which greatly facilitated observations on the process of autotomy, the weighing of specimens before sea-water was drawn into the body cavity, and the rapid transference of autotomised specimens to fresh sea-water.

It was not found possible to maintain autotomised specimens under the available aquarium conditions. They could be kept satisfactorily in wooden crates with perforated sides and with rocks, fine sand and mud in the bottom. The boxes were submerged to a depth of 1 ½ metres in “Te Aro” salt-water baths, in Wellington Harbour. Sea-water from the harbour circulated freely through the boxes, which were nevertheless protected from the effect of heavy seas. While many specimens died in less than six weeks after autotomy, sufficient numbers remained in good condition up to 145 days, to permit studies in regeneration.

Specimens for examination were anaesthetised by adding magnesium sulphate to the sea-water. When extended and unresponsive, Bouin's fluid was injected into the body cavity to fix and harden mesenteries and regenerating tissue sufficiently to prevent any tearing on opening the specimen. An incision was made along the right dorsal interambulacrum, and the flaps of the body wall were pinned out. The specimen was then immersed in fresh Bouin's fluid, which completed fixation and rendered the structures opaque for closer examination with a dissecting microscope. Before fixation, the regenerating tissues and mesenteries are very soft and transparent, which makes it difficult to determine their limits accurately.