Cytochemical and Biochemical Observations on the Digestive Tracts of Digenetic Trematodes. X. Starvation Effects on Megalodiscus temperatus

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The gastrodermis of the frog rectal fluke, Megalodiscus temperatus, was examined with the electron microscope for acid phosphatase activity following starvation periods of 12 and 24 hr. After 12 hr starvation, enzyme activity was observed associated primarily with a saccular membrane system that appears to be associated with the basal plasma membrane of the gastrodermis. Reaction product is also observed in the cisternae of the nuclear envelope, the rough endoplasmic reticulum, and, to a lesser extent, associated with the microvilli of the brush border. A number of vacuoles are also observed in the gastrodermis. These often contain recognizable organelles such as mitochondria. After 24 hr starvation, enzyme activity is observed only in these vacuoles, and no enzyme activity is seen at other sites. The difference in localization of enzyme activity is believed to reflect an increase in autophagy following prolonged starvation. In a report on the cytochemistry of the gastrodermis of the trematode, Megalodiscus temperatus, Bogitsh (1972) observed nonspecific acid phosphatase (AcPase) activity associated with the brush border and with infoldings of the basal plasma membrane. A change in localization of the enzyme activity occurred when no food was present in the lumen of the gut. The purpose of the present investigation was to determine the localization of nonspecific AcPase activity in the gastrodermis of M. temperatus following starvation. MATERIALS AND METHODS Adult Megalodiscus temperatus were removed from the rectal areas of naturally infected Rana pipiens and placed in 0.69% saline at room temperature. The digestive tracts of worms at the time of removal from their hosts were completely filled with blood. One group of these well-fed worms was removed from the saline after 12 hr, at which time the deep red color was virtually absent from the lumen of their guts, although subsequent Received for publication 18 July 1972. * Supported, in part, by a research grant from NIH (AI-08058). microscopic examination did reveal small quantities of blood. The other group of worms was kept in the saline for 24 hr. Subsequent examination of the digestive tracts of these worms revealed no blood in the gut lumen. At this time, the worms were considered completely starved. Both groups of worms were fixed under coverslip pressure either in 3% distilled glutaraldehyde-cacodylate (0.1 M, pH 7.2) for 3 hr at 4 C for morphological studies or in 2% distilled glutaraldehyde-cacodylate (0.1 Wi, pH 7.2) for 20 min at 4 C for cytochemical studies. Each group of fixed worms was sectioned at 40 Aum on a Sorvall TC-2 Tissue Sectioner and rinsed in cacodylate buffer (0.1 M, pH 7.2) overnight at 4 C. The latter group of sections was incubated for 30 min at 37 C either in the medium of Novikoff (1963) for acid phosphatase localization using cytosine monophosphate (CMP) as a substrate at pH 5.0 or in the medium of Novikoff and Goldfischer (1961) for nucleoside diphosphatase (NDPase) or thiamine pyrophosphatase (TPPase) localization using inosine diphosphate and thiamine pyrophosphate, respectively, as substrates at pH 7.2. Control sections were incubated either in a medium from which the substrate was omitted and, in the case of acid phosphatase, in a medium to which sodium fluoride (NaF) (0.01 M) was added. Following incubation, the sections were rinsed for 10 min in the buffer. These sections and those to be used for morphological studies were postFIGURES 1, 2. Gastrodermis of Megalodiscus temperatus starved for 24 hr. 1. Morphology of gastrodermis showing distribution of autophagic vacuoles. Arrows are pointing to several. Section stained with lead citrate and uranyl acetate. X 7,800. 2. Gastrodermis reacted for acid phosphatase. Reaction product is observed in autophagic vacuoles (arrows), and, to a lesser extent, associated with microvilli of brush border (b). X 13,200. Inset. Higher magnification view of autophagic vacuole with reaction product. X 30,000.