A HISTOCHEMICAL STUDY OF DIGESTION AND DIGESTIVE ENZYMES IN THE RHYNCHOCOELAX LINEUS RUBER (O. F. MULLER) J. B. JENNINGS Department of Zoology, The University of Leeds, England It has been shown in a previous account (Jennings, 1960) that digestion in the rhynchocoelan Linens ruber is the result of both extracellular and intracellular processes. The food, which consists of animals such as small annelids and crus-taceans captured by means of the eversible proboscis, is swallowed whole and, after being killed by acid secretions poured on to it during its passage through the foregut, is broken down into a semi-fluid mass within minutes of arrival in the intestine. The enzyme bringing about this initial, extracellular, breakdown is produced by gland cells scattered throughout the intestinal gastrodermis and operates at a pH of 5.0-5.5. The fragmenting food is phagocytosed by other gas-trodermal cells and digestion completed intracellularly. In the present work the course of digestion has been examined in greater detail and an attempt made to identify some of the enzymes concerned in both the breakdown of the food and the general metabolic activity of the gastrodermis. MATERIALS AND METHODS Individual Linens ruber were isolated and starved for seven days to clear the gut of all traces of previous meals. Individual isolation was necessary since after five or six days without food cannibalism often occurs. To study the course of digestion, and to locate and identify the enzymes con-cerned, starved Linens were fed upon inert test foods such as clotted frog blood, either alone or mixed with cooked beef fat or starch paste. These foods were used in preference to the natural living food to eliminate any possibility of enzymes contained in the latter being mistaken for those produced by the Linens gut. Series of Linens were killed for examination after seven days' starvation and at progressive intervals up to 48 hours after an observed meal on one or other of the test foods. In earlier experiments the Lineus were killed by freezing in isopentane cooled by liquid nitrogen to -160 C. and subsequently dehydrated for 48 hours at -40 C. under a vacuum of 10~ 3 mm. mercury. Such specimens were embedded in paraffin wax (melting point 42 C.), sectioned at 8 p. and examined by the histochemical techniques listed below. During the course of the work, however, it was found that specimens fixed for 12 hours at 4 C. in 10% formalin in sea water, buffered to pH 7.0, and then rapidly dehydrated in absolute acetone at the same temperature, cleared in xylol at room temperature and embedded in 42 C. wax showed no significant decrease in enzyme activity when compared with the freeze dried specimens. The duration of dehydration, clearing and 63
BioStor
