The Nervous System of Larval Schistosoma mansoni as Revealed by Acetylcholinesterase Staining

Abstract

A complex AChE-positive neural network is described in miracidia and cercariae of Schistosoma mansoni. Longitudinal tracts in miracidia connect the anterior and posterior regions of the body to the central ganglia. A fine network of circular fibers was also found in the posterior region of the miracidium. In freshly shed cercariae only external papillae and regions with natural openings were positive for AChE staining. Staining after the alteration of the cercarial coat revealed a network of longitudinal and circular fibers near the surface, as well as nerve trunks and commissures in the interior of the organism. Extracts of adult Schistosoma mansoni have been demonstrated to contain acetylcholinesterase (AChE), choline acetylase activities (Bueding, 1952), and to contain a substance with properties similar to those of acetylcholine (ACh) (Barker et al., 1966). Histochemical evidence for the occurrence of AChE in adult worms was reported by Bueding et al. (1967). The occurrence of ACh in larval forms of S. mansoni has been inferred through histochemical demonstrations of AChE. Pepler (1958) and Lewert and Hopkins (1965) found AChE staining activity to be confined to the central neural mass of miracidia and cercariae. Fripp (1967) demonstrated trueand pseudo-cholinesterase activity to occur in adult worms but detected only true cholinesterase activity to occur in larval stages. Fripp described AChE distribution in greater detail than Pepler, and Lewert and Hopkins, but much of his description was based upon the use of nonspecific substrates. In this paper, the staining results in miracidia and cercariae, using acetylthiocholine iodide (ASChI) and butyrylthiocholine iodide (BSChI) as substrates, are described. MATERIALS AND METHODS AChE histochemical localization was carried out according to the methods of Koelle and Friedenwald (1949) and Gomori (1952) as described by Bueding et al. (1967) following further modifications. Organisms were individually collected and placed in watch glasses for incubation, Received for publication 12 November 1973. * Supported in part by Research Grant AI11107-01 from the NIH, U. S. Public Health Service, Bethesda, Maryland. using glass tubing drawn out to a fine point. The maximum number of organisms in any one incubation was 50. Inhibitors were added 60 min before the addition of substrates and maintained until completion of incubation. Substrate concentrations and their incubation periods varied with the experiment. After completion of incubation, organisms were washed twice in 20% Na2SO4 for 5 min each. During the last minute of the second wash, distilled water was added to the Na2SO4 solution. Organisms were transferred to distilled water for 5 min, reacted with H2S-saturated 70% ethanol for 45 sec, transferred to 50% ethanol, and finally water. For slide preparations, organisms (in water) were added to a drop of PVP in 30% glacial acetic acid and covered with a cover slip for examination. After reaction with H2S-saturated ethanol, some organisms were transferred to 70% ethanol and dehydrated through 80, 95, and 100% ethanol. These organisms were mounted in Spurr medium (Spurr, 1969). All organisms were of the Puerto Rican strain of Schistosoma mansoni. ASChI, BSChI, and physostigmine salicylate were obtained from Sigma Chemical Co., St. Louis, Mo.