Ultrastructure of the Metacercarial Cyst of Posthodiplostomum minimum (Maccallum, 1921)

Abstract

The 2-layered cyst consists of an outer fibrous cellular capsule composed of endothelial cells and an inner noncellular primary cyst wall of homogeneous and moderately electron-dense texture. An osmiophilic interface zone separates these layers. Adjacent to the primary cyst wall is a cyst cavity filled with flocculent material and membrane-bound vesicles. These vesicles are found in close proximity to the cyst wall and resemble similar vesicles in teguminal extrusions of the metacercaria, suggesting that cystogenous materials are formed by the parasite, released into the cyst cavity, and eventually deposited onto the cyst wall. The endothelial layers of the outer fibrous capsule contain numerous intracellular microfilaments that may function in providing additional tensile strength, protecting the metacercaria during ingestion by the definitive host. Micropinocytotic vesicles and extracellular deposits of collagen are characteristic of loose fibroblasts which surround the periphery of the cyst. Necrotic areas are designated the interface zone. It is suggested that they may be attributed to mechanical compression or isolation from oxygen and important nutrients. The metacercariae of the trematode Posthodiplostomum minimum utilize numerous species of freshwater fish as second intermediate hosts, particularly sunfish of the family Centrarchidae. The cercariae penetrate epidermal tissues and enter the general systemic circulation. The larval trematode forms a thin cyst wall which appears as a frail baglike structure loosely covering the metacercariae after reaching those visceral organs which are usually associated with a plentiful blood supply, e.g., liver, heart, or kidney. The process of encystation has been studied by electron microscopy in certain families of digenetic trematodes in order to elucidate the structure, function, host response, and relative contributions of the host and parasite during elaboration of the cyst wall. Electron microscopy of Fasciola hepatica metacercariae which openly encyst in an external environment has revealed that cyst components form a fourlayer wall which is solely contributed by cystogenous gland secretions from the parasite. The quinone-tanned, keratinlike proteinaceous layers are rapidly deposited, providing protection against desiccation and fungal and bacterial injury (Dixon and Mercer, 1964, 1967; Mercer and Dixon, 1967). The cyst of Notocotylus attenuatus has been described by Southgate (1971) as three-layered, originating in a similar manner from parenchymal gland Received for publication 1 May 1973. 67 cell secretions. Cyst wall ultrastructure within a second intermediate host has been described in one psilostomatid (Macy et al., 1968) and in two microphallid species (Strong and Cable, 1972; Stanier et al., 1968). These cysts, protected internally by their hosts from the environment, complete differentiation after periods of up to 4 weeks in which as many as four layers may be present. There is evidence in various heterophyid species of Ascocotyle that the cyst wall is derived from tegumental extrusions which form one or more layers. Host involvement is limited to peripheral infiltration of fibroblasts with extracellular reticular collagen (Lumsden, 1968; Stein and Lumsden, 1971, 1972). This paper reports on the ultrastructure of Posthodiplostomum minimum metacercarial cyst wall organization as an initial step in describing the process of encystation in strigeids. MATERIALS AND METHODS Metacercarial cysts were obtained from naturally infected sunfish (Lepomis sp.) which were seined from ponds in the vicinity of Marion, Ohio, and the Bonnet Carre' Spillway, Norco, Louisiana. Fish were maintained in aquaria and fed a diet of commercial fish food, mealworms, and earthworms. Cysts were excised from liver parenchyma and processed for electron microscopy. Cysts were fixed for 6 hr (1 to 4 C) in 3% glutaraldehyde buffered at pH 7.3 in 0.12 mI monobasic sodium phosphate-NaOH, containing 1% CaCl2 and 3% sucrose. These were washed overnight in cold phosphate buffer, postfixed for 1/, hr with 1% phosphate-buffered OsO4, and This content downloaded from 207.46.13.15 on Fri, 26 Aug 2016 05:32:13 UTC All use subject to http://about.jstor.org/terms 68 THE JOURNAL OF PARASITOLOGY, VOL. 60, NO. 1, FEBRUARY 1974 washed in several rinses of veronal acetate buffer at pH 5.3. Tissue was stained en bloc in 1% uranyl acetate (in veronal acetate buffer, pH 5.3) for 1 hr, and dehydrated with ethanol before gradual embedment in low viscosity epoxy resin mixture (Spurr, 1969). Sections were cut on glass knives with a Sorvall MT-2 ultramicrotome, collected on uncoated copper grids, and stained with 1% aqueous uranyl acetate and with lead citrate. Electron micrographs were made with a Siemens Elmiskop IA operating at an accelerating voltage of 80 kv. Cysts were cut open during fixation allowing access of the fixatives and embedding media into both inner and outer surfaces of the cyst wall.