Studies on Phosphatase Activity in Trematoda

Abstract

Both histochemical and biochemical studies have been made on phosphatase activity in seven species of Monogenea (Entobdella hippoglossi, Calicotyle krbyeri, Polystoma integerrimum, Diplozoon paradoxum, Discocotyle sagittata, Diclidophora merlangi, and Octodactylus palmata) and eight species of Digenea (Haematoloechus medioplexus, Haplometra cylindracea, Opisthioglyphe ranae, Diplodiscus subclavatus, Gorgoderina vitelliloba, Gorgodera cygnoides, Fasciola hepatica, and Schistosoma mansoni). Results indicate that phosphatases are probably of widespread occurrence in the Trematoda but that differences exist between the two orders as to the nature and relative activity of the enzymes. In the Monogenea, alkaline phosphatase activity occurs over a fairly broad pH, with an optimum at pH 9 in vitro, and is predominantly found in the gut, the reproductive structures, and the walls of the excretory ducts. The Digenea possess both acid and alkaline phosphatases and show species differences in the relative activities of the two forms. In all cases pH optima for the enzymes, in vitro, were 4.5 to 5.0 and 9.0 to 9.5, respectively. Acid phosphatase activity is primarily localized in the microvilli of the gutt cells of all the Digenea, with the greatest activity in those species possessing an irregular and active surface to the gastrodermis. Species differences also occur in the distribution of acid and alkaline phosphatases associated with the reproductive complex, while the occurrence of alkaline phosphatase in the excretory system is common to all the species examined. Other reactive sites for phosphatases include the cuticle and subcuticular tissues, organs of attachment, and the pharyngeal gland cells in a number of species. The significance of phosphatase activity in the Trematoda is discussed in the light of its probable role in phosphorylated transfer mechanisms and carbohydrate metabolism. There are many reports of phosphatase activity in the Trematoda. In Fasciola hepatica, a fairly high degree of acid phosphatase activity has been recorded for tissue extracts (Pennoit de-Cooman and van Grembergen, 1942) and localized, histochemically, in the cuticle, subcuticular cells, and gastrodermis (Yamao and Saito, 1952; Tarazona Vilas, 1958; Saito, 1961). In addition, moderate activity for alkaline phosphatase has been found in the testes (Tarazona Vilas, 1958) and excretory duct walls (Reznik, 1963). The presence of both acid and alkaline phosphatases in F. hepatica has since been proved electrophoretically by Pantelouris (1966). Similarly, the enzymes have been reported in homogenates of Schistosoma mansoni, and again localized in the cuticle, gastrodermis, and reproductive structures (Dusanic, 1959; Robinson, 1961; Nimmo-Smith and Standen, 1963). Other species in which phosphatases have been demonstrated include Dicrocoelium lanceatum, Eurytrema sp. (Yamao, 1952a), Paragonimus westermani (Yamao, 1952b), Clonorchis sinensis (Ma, 1964), and Gastrothylax crumenifer (Goil, 1966). None of these reports include the MonogeReceived for publication 12 August 1966. * Present address: Department of Zoology, The Queen's University, Belfast, N. Ireland. nea, and for the most part they have not been comparative in nature. Because of this, and the fact that recent studies on nutrition in Trematoda (Halton and Jennings, 1965; Halton, 1967) have indicated distinct differences in phosphatase activity in the gut of the Monogenea and Digenea, both histochemical and biochemical studies have been made on phosphatase systems in a representative selection of trematodes. The investigation was primarily aimed at establishing the general pattern of phosphatase activity in the group, as well as noting species differences in the type of enzyme found. MATERIALS AND METHODS The trematodes studied were seven species of Monogenea: Entobdella hippoglossi, Calicotyle kroyeri (suborder Monopisthocotylea), Polystoma integerrimum, Diplozoon paradoxurn, Discocotyle sagittata, Diclidophora merlangi, and Octodactylus palmata (suborder Polyopisthocotylea); and eight species of Digenea: Haematoloechus medioplexus, Haplometra cylindracea, Diplodiscus subclavatus, Opisthioglyphe ranae, Gorgoderina vitelliloba, Gorgodera cygnoides, Fasciola hepatica, and Schistosoma mansoni. For details of the respective hosts and parasitic habitats see Halton and Jennings (1965) and Halton (1967). Specimens were collected alive from their hosts and after a brief wash in saline fixed for up to 8 hr at 4 C in 4% formaldehyde, phosphate-buffered to pH 7.0. Following fixation, the material was either transferred to 0.2M sucrose and frozen sec-