Control of Swimming in Cercariae of Proterometra macrostoma (Digenea)

Abstract

A specific acetylcholinesterase (AChE) was localized histochemically in Proterometra macrostoma cercariae. This esterase activity was inhibited by eserine, but was resistant to tetraisopropyl pyrophosphoramide. A transverse band of intense AChE activity was observed in the posterior region of the tail. Suction electrode recordings from the tail showed that the action potentials associated with swimming activity were conducted from the transverse band region toward the anterior end of the tail. Removal of the body of the cercaria had no effect on the cyclical electrical activity. 5-Hydroxytryptamine had a strong excitatory effect on the electrical activity whereas acetylcholine and eserine were inhibitory. These observations suggested that swimming behavior was initiated in the transverse band of the tail and that the neuromuscular activity underlying this behavior may be modulated by acetylcholine. Swimming behavior of Proterometra macrostoma cercariae is characterized by alternating periods of swimming and sinking (Prior and Uglem, 1979). Aided by a pair of flat furcae, alternating lateral contractions of the tail pull the cercaria up through the water. A single, biphasic, action potential precedes each contraction of the tail. Following a burst of swimming the cercaria drifts passively downward until touching some object. Once it contacts something another sequence of swimming is initiated. Although the duration of a swimming period is quite regular, a burst of swimming can be initiated at any time during a period of sinking. The body of this large furcocystocercous cercaria is attached to the anterior end of the tail, and is usually withdrawn into the tail at the time of emergence from the snail. One of the most striking aspects of the electrical pattern underlying swimming activity is that the pattern continues unaltered after detachment of the body from the tail. Furthermore, the potentials are conducted from the posterior to the anterior end of the tail. These observations suggested that swimming activity is generated in the posterior region of the tail. We have localized a band of intense, acetylcholinesterase activity in the posterior region of the tail using the histochemical method of Gomori (1952). Our report describes the transverse band and also the effects of various drugs that suggest that the neuromuscular activity underlying swimming behavior may be modulated by acetylcholine. Received 14 October 1982; revised 28 December 1982; accepted 6 January 1983. MATERIALS AND METHODS Snails of the genus Goniobasis Lea infected with Proterometra macrostoma were collected from local, springfed streams and placed in shallow pans of well-aerated natural spring water. Cercariae emerged from the snails after several hours in the laboratory. Some snails were cracked open and the cercariae carefully teased from the tissues. The cercariae were transferred to artificial spring water (ASW; 0.5 mM NaCl, 0.05 mM KC1, 0.4 mM CaCl2, and 0.025 mM MgCl) and held for 2 hr at 25 C prior to use in experiments. Acetylcholinesterase (AChE) was localized histochemically according to the method of Gomori (1952). Worms were incubated (37 C) in the reaction medium for 3 to 6 hr. The reaction medium contained as substrate acetylthiocholine iodide (2 mg/ml; Sigma, St. Louis, Missouri), and in some cases cholinesterase inhibitors, 10-3 M eserine sulfate or 10-4 M tetraisopropyl pyrophosphoramide (isoOMPA; Sigma). The stained cercariae were dehydrated, cleared and prepared as whole mounts for photomicrography. The electrophysiological recordings were made by applying thin plastic tubing as suction electrodes to the tails of cercariae that had been pinned to the bottom of a recording chamber filled with ASW (18-22 C). The signals were amplified and recorded with a Grass P79 polygraph or kymograph camera. Drugs were applied to cercariae by replacing the ASW in the recording chamber (approximate volume = 2.0 ml) using a perfusion system. The effect of each drug on the swimming rhythm was based on observations of at least 25 bursts of swimming. Results are expressed as the x + SD. Differences were analyzed using the Student's t-test; a value of P < 0.05 was considered significant. Acetylcholine, 5-hydroxytryptamine (5-HT), carbamylcholine chloride (carbachol) and eserine sulfate were obtained from Sigma. All chemicals were reagent grade.