Metabolism of 14 C-glucose by metacercariae of Clinostomum campanulatum (Trematoda). (1967)

Metacercariae, in vivo, absorbed more glucose-U-'4C and incorporated more radioactivity into glycogen than did in vitro forms. Metacercariae, in vivo and in vitro, incorporated glucose-U-C carbon into alanine, glutamic acid, glutamine, proline, and alpha ketoglutaric, citric, fumaric, lactic, pyruvic, and succinic acids. The labeling patterns obtained indicate that the metacercariae of Clinostomum campanulatum possess the components of a TCA cycle. Available evidence indicates that the utilization of carbohydrates by helminths requires the operation of reactions of the glycolytic and pentose phosphate pathways. Whether or not the above pathways are followed by a system of organic acid oxidations as a general process in all helminths is still in doubt. Since the majority of the studies have been conducted with adult worms, little is known about the larval forms. Some authors have suggested that the metabolic patterns of parasites may vary from one stage in the life cycle to the next. As an example, Bryant and Williams (1962) investigated the metabolism of radioactive glucose and succinate in the sheep liver fluke, Fasciola hepatica, and concluded that the adult worm exhibited TCA cycle activity. However, they were unable to find any evidence of TCA cycle activity from glucose in the miracidium. Because results obtained from in vitro experimentation are somewhat questionable as to their validity when applied to in vivo situations, these experiments were designed to at least qualitatively compare in vitro and in vivo metabolism of 14C-glucose by the metacercarial larva of Clinostomum campanulatum. MATERIALS AND METHODS The parasites used in these studies were obtained from naturally infected minnows, Pimephales promelas. Infected minnows were maintained in Received for publication 20 March 1967. * From a dissertation submitted to the Graduate School of Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This investigation was supported in part by National Aeronautics and Space Administration Training Grant Ns G(T)56. t Present address: Biology Department, Chico State College, Chico, California 95926. the laboratory in aerated tanks at approximately 19 C. Preliminary studies revealed that when the host minnows were starved prior to the experiments, the amount of carbon from 14C-glucose taken up by both in vivo and in vitro parasites was greatly increased. For this reason, the host animals were starved for 3 days prior to all experiments. Larvae used in the in vitro studies were collected by placing minnows of approximately the same size in a beaker containing poikilothermic Ringer's solution at a temperature of 40 C. Clean, excysted larvae were then collected from the bottom of the beaker after about 4 min. No significant differences were noted in the uptake and incorporation of the label in worms collected in this manner when compared to worms which were manually excysted. Following collection, the larvae were washed in 10 changes (500 ml) of Ringer's solution. Flasks containing 10 ml buffered poikilothermic Ringer's solution and 1 ml of solution were equilibrated on a shake table in an environmental chamber at 19 C. The 4C-glucose solution was prepared to contain 1 Ac per 100 ,liters (Sp. Act. = 3.24 mc/mM). Fifty larvae were placed in each flask. After 1 hr incubation, the worms were removed from the flasks, washed in 10 changes (500 ml) of Ringer's solution, and dried by gentle blotting. The worms were then chilled to 0 C, rapidly weighed, and transferred to 15-ml centrifuge tubes containing 3 ml of 30% KOH. The tubes were boiled until digestion was complete and then cooled. Three 10-Aliter aliquots were removed from each tube and plated on stainless steel planchets. The aliquots were evaporated to dryness and the radioactivity was assayed in a gas flow counter. Glycogen was precipitated from the KOH digests by addition of 90% ethanol and collected by centrifugation. The glycogen pellets were dissolved in 1 ml distilled water, reprecipitated with ethanol, and redissolved in 1 ml distilled water. Three 25,liter aliquots from each sample were removed to planchets and the radioactivity determined. Minnows of approximately the same size and containing approximately the same number of encysted larvae (visual inspection) were selected for the in vivo studies. Each minnow was injected intraperitoneally (IP) with 100 gliters of the