Freeze-Etch and Cytochemical Studies of the Integument of Larval Acanthatrium oregonense (Trematoda)

Abstract

The integument of Acanthatrium oregonense was studied in the sporocyst and cercarial stages by means of electron microscopy, cytochemistry, and freeze-etching and was found to be cytoplasmic in nature. The sporocyst integument consists of a granular material interspersed with several unit membranes running parallel to the surface. Long slender microvilli extend from the outer surface of the sporocyst. Cytochemical evidence is given for the production of acid mucopolysaccharide in the vesicular layer of the body integument of the cercaria and its release onto the body surface. Cytochemical and freeze-etch properties of the basal lamina suggest that this intercellular space may have a filamentous substructure. The histogenesis of trematode integument from the sporocyst stage through the adult has been studied with the electron microscope by Bils and Martin (1966). They concluded that the integument of larval and adult trematodes is principally cytoplasmic and metabolically active, but they were limited in attempting to correlate histochemical data at the light microscope level with details of ultrastructure of the integument. The standard thin sectioning and related cytochemical techniques of electron microscopy have seriously limited interpretation of structure and introduced the possibility of cellular distortion and artifacts. As opposed to standard thin sectioning techniques, freezeetching does not require chemical fixatives and dehydrating agents. Tissue which has been rapidly frozen is fractured in vacuo. The fracture follows inherent planes of weakness within the tissue. One such plane of weakness separates the nonpolar bonds between methyl groups of apposing lipid layers in cellular membranes and lamellar bodies (Branton, 1966, 1969; Pinto da Silva and Branton, 1970). Since the freeze-etch technique provides information about the physical characteristics of a tissue including some of its three-dimensional aspects, it was felt that it would be a valuable adjunct to histochemical methods. Having preReceived for publication 30 July 1970. * Supported by NSF Grant GB 13646 and made possible by research leave awarded by California State College, Hayward, California. t Present address: Department of Biological Science, California State College, Hayward, California 94542. viously established (Belton and Harris, 1967) the basic ultrastructure and light microscope level histochemistry of the integument of the emerged cercaria of Acanthatrium oregonense, we have endeavored in the present study to investigate the ultrastructure of both the sporocyst and immature cercarial integuments by histochemical electron microscope techniques in conjunction with freeze-etching. MATERIALS AND METHODS Naturally infected snails, Oxytrema siliqua (Gould), were collected during February and March in Benton County, western Oregon. Emerged cercariae of Acanthatrium oregonense and infected hepatopancreas were frozen or fixed for ultrastructural and cytochemical study.