Histochemical Studies of Carboxylic Esterase Activity in Fasciola hepatica

Abstract

A histochemical study has been made of the distribution of carboxylic esterase activity in adult Fasciola hepatica. By the use of specific substrates and inhibitors three distinct esterases have been localized in various tissues within the worm. In general, acetylcholinesterase activity is found in the tegument and neuromuscular tissues, nonspecific esterases in the reproductive structures, and a resistant-type indoxyl esterase occurs in the gut epithelium. The results are compared with biochemical findings and the role of carboxylic esterases in the liver fluke is discussed. The occurrence of carboxylic esterases in adult Fasciola hepatica was first reported by Bacq and Oury (1937) who demonstrated the presence of cholinesterase activity in extracts of the worm. Using whole fluke homogenates, Pennoit-de Cooman and van Grembergen (1942) found lipase activity and, in addition, confirmed the presence of a cholinesterase which has since been identified as a true (acetyl) cholinesterase (Chance and Mansour, 1953; Sekardi and Ehrlich, 1962; Frady and Knapp, 1967). Pantelouris (1967), using electrophoretic separation techniques, found a single main band of esterase activity in extracts of F. hepatica which was capable of hydrolyzing a number of carboxylic esters, and two slower bands when a naphthol ester was used as substrate. From spectrophotometric measurements the esterases were found to hydrolyze esters of long chain fatty acids more readily than those of either short chain fatty acids or of choline, and, further, the main band of activity was found to be resistant to known inhibitors of cholinesterase, such as eserine sulphate, as well as to a number of organophosphate compounds. It would seem therefore that F. hepatica contains at least three distinct carboxylic esterases, the most active of which is an organophosphate-resistant type. The present work represents a histochemical examination of carboxylic esterases in F. hepatica, including a study of the effect of inhibitors, in an attempt to locate sites of specific esterase activity within the worm and to relate the findings, as far as possible, to the known biochemical evidence. Received for publication 7 September 1967. MATERIALS AND METHODS Live specimens of F. hepatica were collected from the bile ducts of freshly slaughtered sheep, washed in Hedon-Fleig solution, and fixed for 2 hr at 4 C in 10% formalin containing 0.05 Mi sucrose and held at pH 7.2 with 0.15 M phosphate buffer. In most cases the worms were bisected transversely in fixative to aid penetration of the latter and facilitate subsequent sectioning. Following fixation each half worm was washed in three changes of buffer and frozen in 5% sucrose on a thermoelectric (Pelcool) freezing stage fitted to a standard rotary microtome. Sections (10 to 15 u thick) were cut using a mounted steel razor blade and transferred to glass slides and allowed to dry in air (1 to 2 hr). Alternatively, a number of sections were transferred directly to the incubating medium and free-floated. Sections were processed for esterase activity using standard techniques described in Pearse (1960). The following substrates, together with details of the incubation times and temperatures, were used: naphthyl acetate, propionate (30 min, 20 C), caprylate (12 hr, 20 C), naphthol ASacetate (6 hr, 20 C), and 5-bromoindoxyl acetate (3 hr, 37 C) for nonspecific esterases; naphthyl laurate, stearate (12 hr, 20 C), Tween 60 and Tween 80 (24 hr, 37 C) for lipases; and acetyl or butrylthiocholine iodide (6 hr, 37 C) for cholinesterases. In the case of the long chain fatty acid esters, where extended periods of incubation were needed, sections were transferred to freshly prepared media at 3-hr intervals. The effect of inhibitors on esterase activity (see Pearse, 1960) was studied by preincubating sections for 1 hr in a buffered solution of inhibitor followed by incubation in the standard test medium containing that inhibitor. The inhibitors are listed in Table I along with details of their molarity in the incubation mixture. Control slides consisted of sections exposed to distilled water at 60 C for at least 3 min prior to incubation or sections incubated in test media without the substrate. In all other details the inhibitor and control media were identical to the corresponding test series.