The fine structure of the miracidium of Schistosoma mansoni

JOURNAL

OF INVERTEBRATE

PATHOLOGY

The Fine Structure

36, 307-372

(1980)

of the Miracidium S. CHIA-TUNG

Department

of Tropical

Public

Health,

Harvard

School

Received

of Schistosoma

mansonil

PAN of Public

Health,

Boston,

Massachusetts

02115

June 9. 1980

location. Light microscopical studies on the morphology of the miracidium of Schistosoma mansoni reveal little of such structures that can aid in the interpretation of the biology and host location of this larval stage (Faust and Hoffman, 1934; Gordon et al., 1934; Maldonado and Acosta-Matienzo, 1947; Ottolina, 1957; Pan, 1965; Schutte, 1974). There is little doubt that investigations with the electron microscope will reveal many details of considerable significance in the interpretation of the basic biology of the miracidium of schistosomes. Studies of this nature have recently begun to appear in the literature (Brooker, 1972; Basch and DiConza, 1974; Kinoti, 1971; Lumsden, 1975; LoVerde, 1975; Wikel and Bogitsh, 1974; Wright, 1971). The cellular components of schistosome miracidia are to a large extent at or beyond the limit of resolution of the light microscope. Moreover, conventional cytologic techniques usually fail to preserve many delicate cellular elements of the miracidium that are essential for a detailed analysis of the organism. Accordingly, electron microscope techniques have been employed in order to identify cellular structures which have been poorly understood or unrecognized. It is hoped that the result of this study will provide the background for future research on the miracidium-mother sporocyst transformation, on the invertebrate host-parasite interaction at the fine structural level, on the neurobiology, and on other aspects of S. mansoni miracidium.

INTRODUCTION

It is estimated that schistosomiasis affects over 180 million people who live primarily in developing countries. The disease is spreading rapidly as irrigation schemes in these countries are expanded to accommodate agricultural needs. Yet, our knowledge of the natural history of schistosomiasis and of the basic biology of the parasite causing the disease is still far from adequate (Weller, 1975). Such knowledge is essential for a rational approach to the effective control of human schistosomiasis. The miracidium of schistosomes is the first infective stage in the complex life cycle of the parasite, and the future of the entire life cycle depends on the capacity of this larval stage to locate and enter a suitable invertebrate host (aquatic or amphibious snails) for further development. During the life in the susceptible invertebrate host, the miracidium must go through several stages of development and multiplication. In the process, the parasite produces extensive tissue damage to the host (Pan, 1963, 1965). The invertebrate host, in turn, reacts to the pathogen and exhibits severe tissue reaction that has been thoroughly studied with the light microscope (Pan, 1963, 1965). There are indications that schistosome miracidia are potent host finders (Chernin, 1974). However, little is known concerning miracidial sensory organelles that may aid the larva in host KEY WORDS: Schistosoma mansoni; miracidium; trematode; tine structure. ’ This modest work is dedicated to Dr. Thomas H. Weller who has provided encouragement and support over the years.

MATERIALS

AND METHODS

Miracidia of a Puerto Rican strain of S. mansoni were harvested from the neck of a 307 0022-201

l/80/060307-66$0

I .00/O

Copyright 0 1980 by Academic Press. Inc. All tights of reproduction in any form reserved.

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FIGS. l-64. Magnifications of electronmicrographs and photomicrographs are indicated in the appropriate legends. Letters demarcating structures are independent for each figure. All electronmicrographs and photomicrographs are from free swimming miracidia except Figures 21, 22, 25, 26. 36, 40, and 59. These electronmicrographs were from organisms (postmiracidia) within 6 112 hr after penetration into the snail host, Biomphalaria glabrata. The age of each organism is indicated in the legends.

Schisrosoma

mansoni

MIRACIDIUM

volumetric flask within 30 min after hatching (Pan, 1965) and fixed for 2 hr at 22-24°C in 3% glutaraldehyde dissolved in Chernin’s (1957) handling solution (HS) [NaCl, 2.0 g; KCl, 0.1 g; N&HP04, 0.05 g; MgSO,-7Hz0, 0.2 g; CaCl,*2H,O, 0.1 g; phenol red (0.4% aqueous solution), 5.0 ml; distilled water, 995 ml]. Fixation was carried out by mixing rapidly a volume of heavy miracidial suspension with an equal volume of 6% glutaraldehyde in double strength HS. After washing for 2 hr in four changes of ice-cold HS, the miracidia were posttixed for 1 hr at 4°C in 1% osmium tetroxide. Osmium tetroxide solution was usually prepared with HS. Occasionally potassium ferrocyanide was added (final concentration, 1.5%) to the osmium solution to improve fixation (Kamovsky, 1971). After fixation with osmium, the miracidia were washed, dehydrated rapidly in ascending concentrations of ethanol, cleared in propylene oxide, and embedded in Epon 812 (Luft, 1961) or low-viscosity epoxy resin (Spurr, 1969). The pH of fixatives and washing solutions (HS) was adjusted to 7.4-7.8 with 4.2% NaHCO, or 1 N NaOH. Thick sections for light microscopy were cut with glass knives at 0.25 to 0.5 pm and stained with 0.5% toluidine blue 0 in 1% sodium borate. Thin sections were cut with diamond knives at 500-800 A as judged from interference colors, mounted on bare copper grids (200 or 300 mesh), and stained in sequence with uranyl acetate (Huxley and Zubay, 1961) and lead citrate (Reynolds, 1963; Venable and Coggeshall, 1965). The periodic acid-silver methenamine (Rambourg, 1967) was used to demon-

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strate complex carbohydrates. Thin sections were examined in a Philips EM-300 microscope at an accelerating voltage of 60 or 80 kV. The electron micrographs were taken on Electron Image Plates (Kodak) slightly underfocused according to Fahrenbath and Kneeland (1965). GENERAL

CELLULAR

ORGANIZATION

On the basis of electron microscope findings, I am dividing the cellular organization of the miracidium of S. mansoni under eight major categories or systems: epithelial system, terebratorium, musculature, interstitial cells, penetration glands, excretory system, nervous system, and germinal cells. A schematic reconstruction of the cellular organization is shown in Figure 1. Figure 2 is a photomicrograph of a totomounted miracidium before sectioning for electron microscopy. Figure 3 is a photomicrograph of a longitudinal thick section of such a miracidium. Two scanning electron micrographs are included to show the surface structures (Figs. 4 and 5). Figures 6 through 12 are low-power electron micrographs of representative sagittal and cross sections of the miracidium. The surface of the miracidium is covered with 21 ciliated epidermal plates arranged in successive tiers of six, eight, four, and three plates, respectively, from anterior to posterior (Faust and Hoffman, 1934; Maldonado and Acosta-Matienzo, 1947; Ottolina, 1957; Schutte, 1974). These plates are separated from each other by epidermal ridges which connect their cytons (or cell bodies) with narrow cytoplasmic bridges. The cytons are

FIG. 1. A schematic reconstruction of cellular architecture of the miracidium of Schisrosoma man(primarily based on electron microscopic observations). The outline of the drawing is based on Fig. 2. The cellular architecture is, however, not drawn on exact scales: A. epidermal plate: B. epidermal ridge; C, ridge cyton; D, cytoplasmic bridge; E, cilium and its rootlet; F, terebratorium and the profile of cytoplasmic expansions; G, multiciliated, deep-pit sensory papilla; H, uniciliated sensory papilla; I, outer circular.muscle fiber; J, inner longitudinal muscle fiber; K, interstitial cyton; L, processes of interstitial cells; M, apical gland and secretory duct: N, lateral gland and secretory duct; 0, flame cell with excretory tubule; P, cyton of common excretory tubule: Q, neural mass with peripheral ganglia; R, lateral papilla; S, multiciliated saccular sensory organelle; T, multiciliated shallow-pit sensory papilla; U, uniciliated sensory papilla; V, perikaryon of the neuron to lateral papilla; W, multiciliated sensory papilla; X, perikaryon of the multiciliated sensory papilla: Y, excretory vesicle: 2, germ cell. soni

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FIG. 2. Photomicrograph of a toto-mounted miracidium (1120x): A pair of lateral papilla (arrow A) are recognizable. A bundle of sensory cilia (arrow B) is present at the anterior side of each papilla. One of the two openings of excretory vesicles is also visible (arrow C). The neural mass (D) and a lateral gland (E) are also recognizable. FIG. 3. Photomicrograph of a longitudinal thick (l/2 CL)section stained with toluidine blue 0 (1120x): A, neural mass; B, apical gland; C, lateral gland; D, ridge cell; E, germ cell.

Schistosoma

mansoni MIRACIDIUM

ULTRASTRUCTURE

FIG. 4. A scanning electon micrograph of a fixed miracidium showing dense surface cilia (1120x). FIG. 5. A scanning electron micrograph of the terebratorium showing interlaced cytoplasmic expansions of the surface (8400x): A, sensory cilium.

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FIG. 6. A sagittal section of anterior two-thirds of a miracidium (2940x): A, neural mass with peripherally arranged neurons; B, apical gland and duct filled with membrane-bound secretory droplets; C, lateral gland; D, terebratorium with profiles of cytoplasmic expansions; E, ridge cyton and its nucleus; F, perikaryon of multiciliated sensory papilla with Type D neurosecretory vesicles; G, multiciliated sensory papilla; H, lateral papilla; I, nucleus of inner longitudinal muscle fiber; J, extra-CNS neuron; K, excretory tubule; L, excretory vesicle; M. anterior nerve to terebratorium; N, process of interstitial cell.

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FIG. 8. A sagittal section of anterior area containing a ganglion (7700x): A, nerves within the ganglion, many containing neurosecretory vesicles and neurotubules; B, neuron: C, epidermal plate showing prominent pegs; D, outer circular muscle fiber nestled between pegs: E. inner longitudinal muscle; F, lateral gland; G, process of interstitial cell; H, septate desmosome joining epidermal plate with terebratorium; I, secretory duct of lateral gland; J, sensory cilia of multiciliated deep-pit sensory papilla.

Schisrasoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 9. A sagittal section of the area covered by the second and third tiers of epidermal plates (5390x): A, cyton and nucleus of epidermal ridge. The nucleus is surrounded by a zone of RER; B. cyton and nucleus of interstitial cell. The cytoplasm is crowded with a- and p-glycogen particles and lipid droplets: C, cyton and nucleus of excretory tubule: D, cyton and nucleus of common excretory tubule; E, distal portion of common excretory tubule; F, peri-flame cell space containing numerous leptotriches; G, germ cell; H, processes of interstitial cells; I, afibrillar zone of a muscle fiber containing nucleus; J, multiciliated sensory papilla; K, epidermal ridge displaying its cytoplasmic bridge (arrow); L, epidermal plate of the second tier; M, epidermal plate of the third tier.

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FIG. 10. Oblique section of posterior area covered by the third and fourth tiers of epidermal plates (5390~): A, germ cell; B, ridge cyton; C, perikaryon of excretory tubule; D, excretory tubule; E, cyton of common excretory tubule; F, distal portion of common excretory tubule near the junction with excretory vesicle; G, peri-flame cell space containing many leptotriches; H, cyton and nucleus of flame cell; I, nucleus of excretory vesicle; J, processes of interstitial cells containing numerous a- and P-glycogen particles and lipid droplets; K, epidermal plate with many membrane-bound, electrondense bodies; L, epidermal ridge.

Schisrosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. Il. Sagittal section of posterior area covered by the fourth tier of epidermal plates (5390x): A. epidermal plates of the fourth tier. Epidermal pegs are prominently displayed; B, ridge separating the fourth tier of epidermal plates. Septate desmosomes are conspicuous (arrow); C, ridge cyton and nucleus; D, processes of interstitial cells; E, outer circular muscle fiber; F, inner longitudinal muscle fiber; G, ridge; H, a group of extra-CNS neurons: I, nerve from the neurons; J, perikaryon of multiciliated sensory papilla containing many Type D neurosecretory vesicles; K, multiciliated sensory papilla.

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FIG. 12. Cross section through the midlevel of the fourth tier of epidermal plates (7700x): A, epidermal plate. Many membranebound, electron-dense bodies are clearly displayed; B, epidermal ridges separating three plates. Septate desmosomes at the joints are clearly seen; C, an outer circular muscle fiber nearly entirely encircling the miracidium. Many tight junctions (arrows) between the muscle and plates are visible; D, outpocketing, afibrihar zone of circular muscle; E, neuromuscular junction. The nerve contains many neurosecretory vesicles; F, inner longitudinal muscle fiber; G, germ cell; H, cyton of ridge containing numerous membrane-bound vesicles and glycogen particles; I, processes of interstitial cells.

Schistosoma

mansoni

MIRACIDIUM

syncytial in nature and are positioned along the inner side of the longitudinal muscles at the level between the neural mass and posterior end of the organism (Figs. 1, 3, 6). Cilia appear most numerous and longest on the first tier of the epidermal plates (Figs. 2, 3, 6). The terebratorium (Reissinger, 1923) is a hemispheric structure which lacks kinocilia but is equipped with abundant sensory terminals (Figs. 1, 5, 7, 23). The epidermal plates and the terebratorium rest on a thin, continuous basal lamina (Fawcett, 1966) which in turn is bordered by the outer circular muscle fibers (Fig. 12). The neural mass (CNS) (Figs. 1, 2, 3, 6) occupies much of the area covered by the second tier of the epidermal plates. More than 20 nerves originate directly from the neural mass. Many of these nerves terminate in the peripheral sensory organelles. Anterior to the neural mass are three, flask-shaped penetration glands (an apical and two lateral). The apical gland opens through the center of the terebratorium and the lateral glands at its base (Figs. 1, 3, 23). Behind the neural ring and occupying the inner core of the miracidium are the cytons of interstitial cells which send out numerous processes to fill intercellular spaces (Fig. 1). Some 20 germinal cells with short processes are nestled among the cytons of interstitial cells. A group of extra-CNS, bipolar neurons (about 12 in number) are positioned between the neural ring and the cytons of interstitial cells. Two flame cells are located among the cytons of interstitial cells and two are on the anterolateral sides of the neural mass (Fig. 1). Four of the six major types of sensory organelles are positioned in the ridges separating the epidermal plates of adjacent tiers and the rest lie in the terebratorium. The two excretory pores open through the ridges separating the plates of the third tier. FINE STRUCTURE Epidermal

System

The epithelial system consists of 21 ciliated epidermal plates and epidermal

ULTRASTRUCTURE

319

ridges. The epidermal ridges separate plates between adjacent tiers as well as between plates within the same tier. The epidermal plates are attached to the respective ridges by septate desmosomes which line the edge of each plate (Figs. 11, 17, 33). Septate desmosomes are tight junctions that are found only in invertebrates and have structures resembling the macula adherens (Fawcett, 1966) except that two opposing membranes (150 A apart) are bridged by numerous electron-dense bands (Satir and Gillua, 1973) (Figs. 34, 35). The dense bands measure ca. 90 A and are separated from each other at 90-A intervals. In tangential sections, the arrangement of bridges appears as a hexagonal meshwork with a mesh diameter of about 90 A (Fig. 24). A layer of electron-dense material (180-200 A thick) coats the inner side of each opposing membrane (Fig. 34). Cilia The structure of kinocilia (locomotory cilia) of the miracidium is basically similar to that described for other organisms by Sleigh (1962), and therefore his nomenclature is adopted here. The kinocilia arise from the epidermal plates at a right angle to the surface. The basal body of each cilium connects at its annulus to a long tapering rootlet (about 2 pm long) that lies about a 20” angle to the plate surface with the tip pointing anteriorly (Figs. 13, 15). The shaft (7-8 pm long) of the cilium contains the characteristic nine outer double and two central single microtubules (Fig. 14). These microtubules are embedded in a fine granular matrix, and are enclosed by a membrane which is continuous with the plasma membrane of the epidermal plate (Figs. 13, 15). The two central microtubules are 250 A in diameter and are about 340 A apart from center to center. The diameter of each doublet is 390 x 265 A. The subfiber A has two arms projecting toward subfiber B of the neighboring doublet. The two central fibers terminate 50 nm above the axosome (or central granule) (Figs. 13, 15). Below

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FIG. 13. Epidermal plate (34,650x): A, epidermal plate displaying finely granular ground substance; B, cihum; C, axosome; D, cross-banded rootlet of cilium; E, membrane-bound, electron-dense body; F, mitochondrion; G, outer circular muscle fiber; H, inner longitudinal muscle fiber: I. lateral gland. FIG. 14. Cross section of cilium (100.450~): A, two central single microtubules; B. peripheral doublet microtubules. FIG. 15. High magnification of epidermal plate (60,200x): A. cihum; B, peripheral doublet microtubule: C, central single microtubule; D, axosome; E, transverse plate with central perforation; F, basal body of cilium: G, cross-banded ciliary rootlet; H, membrane-bound. electron-dense body: I. outer circular muscle fiber: J, tight junction: K, basal lamina; L, microvillus.

Schisrosoma

mansoni

MIRACIDIUM

the axosome is located the electron-dense, centrally perforated transverse plate (Fig. 15). The basal body of the cilium is embedded in the surface layer of the epidermal plate, and has a characteristic arrangement of microtubules, i.e., nine peripheral triplets without central tubules. The hollow rootlet exhibits periodic cross-banding of 650 A with four equal subunits (Figs. 13, 15, 35). The lumen of each rootlet appears to be continuous with the core of the basal body (Figs. 13, 15, 35). Scattered among the ciliary rootlets are numerous electron-dense, membranebound, round to oval bodies measuring up to 500 nm in diameter. These structures are usually uniformly electron-dense, but occasionally have a mottled or granular appearance (Figs. 13, 16, 33). Many surface cytoplasmic projections are present on the plates among the cilia. These projections or microvilh are most numerous in the first tier of epidermal plates. The microvilli are slightly tapered cylinders with dense peripheries and measure ca. 700 x 90 nm (Figs. 13, 16, 35). Numerous cristae-rich mitochondria occupy the zone between the ciliary rootlets and the basal plasma membrane of each plate (Figs. 13, 16). The cytoplasm of the epidermal plates has a finely granular texture and is moderately electron dense. Small numbers of p-glycogen particles are occasionally present. The basal surface of the epidermal plates forms numerous epidermal pegs that are most prominent in the first and last tiers (Figs. 8, 11). The basal lamina is a thin layer (450-900 A thick) that follows the basal contour of the plates and epidermal ridges (Fig. 16). The lamina has an electron-dense fibrous middle zone sandwiched between two lighter homogeneous peripheral zones. The basal surface of each plate is attached to both layers (outer circular and inner longitudinal) of muscle fibers, across the basal lamina, by many discontinuous intermediate (simple) tight junctions (macula adherens, Fawcett, 1966) (Figs. 12, 16).

ULTRASTRUCTURE

Epidermal

321

Ridge

All epidermal ridges appear continuous and connect to their respective cytons by narrow cytoplasmic bridges. The cytoplasm of the bridge resembles that of the cyton except that microtubules line the submembranous area (Figs. 17, 63). Although the cytons are syncytial in nature, individual cytons are more or less circumscribed. Each cyton appears to contain two to three round nuclei, with a prominent nucleolus per nucleus (Fig. 19). The nucleus is surrounded by a zone of granular endoplasmic reticulum (RER) with Golgi apparatus scattered among the RER (Figs. 18-20). The RER network is usually extensive and the cisternae are normally not dilated (Figs. 19, 20), but occasionally the RER cisternae may show a great deal of activity (Fig. 18). Many mitochondria with abundant prominent cristae are usually seen at the periphery of the RER zone. Small patches of RER are also found scattered along the plasma membrane. The bulk of the cytoplasm is occupied by numerous glycogen particles (primarily ,!3 particles) and membrane-bound vesicles (Figs. 18-20). Small numbers of these vesicles are sometimes seen in the cytoplasmic bridges and the areas of ridge adjacent to the bridges (Fig. 63). The membrane-bound vesicles are round or oval, contain a dense central zone and measure ca. 500 nm at the greatest diameter, The ridges are without kinocilia, and display numerous /3-glycogen particles and mitochondria. Small numbers of microvilli are also present on the ridge surface (Fig. 17). The cytoplasm of the ridge and its cyton is moderately electron dense and finely granular in texture (Figs. 17- 19,36). Comments

Very little is known about the fine structure of the epidermal system of schistosome miracidia (Lumsden, 1975). Jamuar and Lewert (1967) briefly described the fine structure of epidermal cilia of the miracidium of Schistosoma japonicum. Lee (1966, 1972) reviewed the literature and indicated

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FIG. 16. Neuromuscular junction (34,650~): A, tibrillar zone of outer circular muscle fiber displaying thick and thin myofilaments. Sarcoplasmic reticulum (+) is also recognizable near the membrane along basal lamina; B, outpocketing afibrillar zone of muscle fiber containing P-glycogen particles and electron-dense neuromuscular junctions (arrows); C, nerve terminal containing numerous Type A neurosecretory vesicles; D, mitochondrion in the afibrillar zone; E, simple tight junction; F, basal lamina; G, membrane-bound vesicle of ridge cyton; H, epidermal plate containing many mitochondria and membrane-bound, electron-dense bodies; 1, microvillus.

Schistosoma

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MIRACIDIUM

that only the fine structure of the epidermal system of Fasciola hepatica had been studied in some detail. According to Wilson (1969a) and Southgate (1970), the epidermal plates of the miracidium of F. hepatica are nucleated. In contrast to these findings, neither the present study nor that of Wikel and Bogitsch (1974) showed any nuclear structure in the epidermal plates of S. mansoni miracidium. Similarly, Wilson (1969a) and Southgate (1970) did not describe the presence of membrane-bound, electron-dense bodies in the epidermal plates of F. hepatica miracidium. Such membrane-bound structures are present in large numbers in the epidermal plates of S. mansoni miracidium, but their function is not known. The “large membrane-bound vesicles” described by Wilson (1969a) appear to differ in structure and location from the electron-dense bodies of S. mansoni. Basch and DiConza (1974) suggested that these electron-dense bodies of S. mansoni were lysosomal granules that played a role in detaching the epidermal plates after the miracidium entered the snail host. However, we found that these structures are located near the surface and do not undergo appreciable morphologic alteration as the plates detach shortly after the miracidium enters the host (Fig. 21). Perhaps the most important and interesting structures observed in the epithelial system of S. mansoni and F. hepatica miracidia are the membrane-bound vesicles present in large numbers in the ridge and its cytons. Southgate (1970) suggested that these are “rolls of stored plasma membrane . ’ ’ We observed that the membranes of these vesicles became part of the membrane of new tegument which rapidly forms around the mother sporocyst during the early stages of transformation of the S.

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323

mansoni miracidium in Biomphalaria glabrata and that the contents of the vesicles were emptied into snail tissues (Fig. 22). Therefore, it is concluded that the membrane-bound vesicles are devices for storing membrane in the ridge cytons prior to use in the formation of new tegument of mother sporocysts. The contents of the vesicles may protect this stage from attack by the host amebocytes by preventing their attachment to the surface of the newly formed tegument (Fig. 22). The abundance of RER, ribosomes, Golgi apparatus, and complex carbohydrate in ridge cytons suggests that these cells are active in production of exportable proteins. Such activity is particularly prominent shortly after the miracidium enters the snail host (Fig. 36). During the first several hours after the miracidium penetrates the snail, cisternae of the RER in the ridge cytons are dilated and filled with electron-dense material (Pan, 1972). Terebratorium

The terebratorium (Reissinger, 1923) is frequently referred to as the anterior papilla by other authors. However, we prefer to use the term terebratorium in order to distinguish this structure from the several sensory papillae that will be described later. The terebratorium is a semispherical, complex structure containing several sensory endings as well as three openings of glandular ducts (Figs. 1, 2, 5, 7, 23). The surface covering (epithelial sheet) may be regarded as a modified epidermal plate without kinocilia (Fig. 23). The surface presents a network of interlaced cytoplasmic expansions (Figs. 5, 23-25). The profiles of these expansions usually appear as more or less straight filopodia (ca. 1 pm long) consisting of two closely apposed

FIG. 17. Epdiermal ridge and connecting cytoplasmic bridge (21,210~): A, ridge containing pglycogen particles and mitochondria; B, cytoplasmic bridge connecting ridge with its cyton. Microtubules (arrows) line submembraneous area; C. ridge cyton; D, membrane-bound vesicle; E, outer circular muscle fiber; F, inner longitudinal muscle fiber; G, septate desmosome joining ridge to epidermal plate; H, microvillus.

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FIG. 18. Ridge cyton displaying hyperactivity of organelles (17,360~): A, nucleus of ridge cyton; B, nuclear envelope with outer membrane forming RER; C, mitochondrion with many long cristae; D, membrane-bound vesicles, some of which containing electron-dense core; E, /3-glycogen particles; F, RER with dilated cisternae: G, Golgi complex with dilated cistemae and vesicles; H, neuron in the CNS; I, nerves with neurosecretory vesicles; J, fibrillar zone of inner longitunal muscle fiber; K, afibrillar zone of longitudinal muscle fiber containing /3-glycogen particles and mitochondria; L, outer circular muscle fiber and its sarcoplasmic reticulum (arrow): M, epidermal plate: N. basal lamina.

Schistosoma

FIG. 19. Ridge cyton B, prominent nucleolus; drion; F, membrane-bound interstitial cell; H. lipid

mansoni

MIRACIDIUM

ULTRASTRUCTURE

displaying three nuclei and perinuclear RER network (13,650~): A, nucleus; C, RER. The cisternae are not distended; D, Golgi complex: E. mitochonvesicle. Many of these vesicles have an electron-dense core: G. cyton of droplet.

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FIG. 20. Extensive granular endoplasmic reticulum network around nucleus of ridge cyton (34,650~): A, nucleus of ridge cell; B, nucleolus; C, Golgi complex; D, membrane-bound vesicle with electron-dense core: E, coil of RER; F, P-glycogen particles.

Schisrosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 21. Postmiracidium within 3 l/2 hr after entering snail host, Biomphalaria glabrara (13,650~): This electron micrograph is intended to show early stages of reorganization of miracidium after successfully entering the snail host. The epidermal plates detach from the basal lamina, exposing the outer muscular layer to the host amebocytes and other defense elements. A, detached epidermal plate with cilia, mitochondria, and membrane-bound electron-dense bodies; B, cilia from epidermal plates of the parasite; C, snail amebocyte; D, cilia of epidermal plates within amebocyte phagosome; E, portion of epidermal plate still attached to ridge with septate desmosome; F, outer circular muscle fiber of the parasite; G, inner longitudinal muscle fiber; H, epidermal ridge containing mitochondria and membrane-bound vesicles; I, ridge cyton containing numerous membrane-bound vesicles and pglycogen particles; J, nerve; K, degenerating neuron: L, precipitated snail hemolymph.

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FIG. 22. Postmiracidium within 6 l/2 hr after entering snail host, Biomphalaria glabrata (26,950~): The electron micrograph is intended to show reorganization of the miracidium after infection and participation of membrane-bound vesicles of ridge cyton in the formation of new tegument. A. newly formed tegument of the parasite; B, membrane-bound vesicle in juxtaposition with surface membrane of the tegument; C, membrane-bound vesicle discharging electron-dense content into snail tissue and its own membrane becoming part of tegument membrane; D, discharged electron-dense content of the vesicle; E, outer circular muscle fiber of the parasite; F, pseudopodia of snail amebocyte interdigitating with the surface of parasite tegument; G, cilium of epidermal plate within phagosome of amebocyte; H, pseudopodium of another amebocyte; I, cilium of epidermal plate free in the snail tissue: J, cross sections of snail collagenous fibers.

Schisrosoma

mansoni MIRACIDIUM

ULTRASTRUCTURE

FIG. 23. Terebratorium showing duct openings of penetration glands and ciliated sensory papillae (17,360x): All these organelles except duct opening of apical gland are attached to the terebratorium with septate desmosomes (arrows). A, duct and its opening of apical gland. The opening is covered with a membrane, and the duct is lined with microtubules along the submembraneous area; B, duct and its opening of lateral gland. The opening is covered with a membrane, and duct membrane is also lined with microtubules; C, multiciliated, deep-pit sensory papilla; D, uniciliated sensory papilla; E, muscle tiber: F, nerve to a uniciliated sensory papilla: G, profile of cytoplasmic expansions of terebratorium: H. epidermal plate of the first tier.

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FIG. 24. Detail structure of multiciliated deep-pit sensory papilla and of septate desmosome (34,650~): A, nerve containing Type A neurosecretory vesicles and neurotubules; B, wall of multiciliated deep-pit sensory papilla with Type A neurosecretory vesicles; C, cross section of sensory cilium lacking central single microtubules; D, septate desmosome joining the papilla to terebratorium; E, muscle fibers; F, duct of lateral gland; G, oblique section of septate desmosome joining glandular duct to terebratorium; H, septate desmosome joining epidermal plate of the first tier to terebratorium; I, profile of cytoplasmic expansion of terebratorium; J, epidermal plate of the first tier: K, duct of apical gland; L, microtubules lining the duct membrane.

Schistosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 25. Terebratorium of a miracidium within 1 l/2 hr after penetration into snail host, Biomglabrafa (30,380~): At this stage of infection the miracidium essentially retains its normal fine structure. Arrows indicate septate desmosomes. A, uniciliated sensory papilla containing Type B neurosecretory vesicles; B, multiciliated, deep-pit sensory papilla containing Type A neurosecretory vesicles; C, nerve with Type A neurosecretory vesicles and/or neurotubules; D, secretory duct of lateral gland; E, cross sections of microtubules lining duct membrane; F, epidermal plate of the first tier: G, outer circular muscle fiber; H, profile of the cytoplasmic expansion of terebratorium; I, collagenous fibers of snail connective tissue; J, pseudopodium of snail amebocyte.

phalariu

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FIG. 26. Cross sectron of terebratorium from a postmiracidium within 6 l/2 hr after the parasite entered the snail, Biomphalaria glabrata (17,360x): At this stage of infection most epidermal plates on the miracidium have detached but terebratorium may still remain attached. Septate desmosomes are indicated with arrows. A, cross section of duct of apical gland lined with microtubules along the duct membrane; B, circular muscle fiber forming a sphincter muscle; C, multiciliated, deep-pit sensory papilla; D, profile of cytoplasmic expansion of terebratorium; E, muscle fiber; F, nerve to sensory papilla containing neurotubules.

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mansoni

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ULTRASTRUCTURE

333

membranes (Fig. 34). The network is ap- Comments parent only in tangential sections (Fig. 27). Studies with the light microscope have When viewed from the surface, the network appears as the septa of numerous pit-like revealed little of the detailed structure of its surface may appear depressions (ca. 1 pm deep) on the semi- the terebratorium; spherical terebratorium (Fig. 5). The thin smooth (Kinoti, 1971) or appear to be cytoplasm of the surface covering is finely equipped with minute spines in sectioned granular, similar to that of the epidermal material (Pan, 1965). With the help of the electron microscope, Kinoti (197 1) deplates, and contains a few P-glycogen particles (Figs. 24, 25). The cytoplasmic expanscribed these spine-like structures as sions in many sections appear extremely “ramifying microvilli.” However, these electron dense, partly because two closely “microvilli’‘-like structures do not have the apposed plasma membranes are frequently organization of true microvilli as no circular viewed in overlapping positions (Figs. 5, cross sections of microvilli were seen in 24, 25, 27). The epithelial sheet rests on the sections cut at various angles. They are basal lamina and is attached by septate des- actually the profiles of interlacing cytomosomes to the first tier of epidermal plasmic expansions. Thus, tangential secplates (Fig. 25). The base of the epithelial tions and scanning electron micrographs sheet of terebratorium forms many thin reveal thin, interlaced cytoplasmic expan“pegs” which extend into the miracidial sions that enclose numerous pits on the body (Fig. 25). Beneath the basal lamina lie surface of the terebratorium. Similar structures have been described for miraseveral outer circular and inner longitudinal muscle fibers. These muscle fibers are at- cidia of S. japonicum, Schistosoma haematached to the base of the epithelial sheet by tobium, and related species (Blankesimple tight junctions (macula adherens) spoor and van der Schalie, 1976; Koie (Figs. 26, 34). and Frandsen, 1976; LoVerde, 1975). These The secretory duct of the apical gland is pits may act as microsuckers and aid the funnel shaped as it opens to the outside miracidium to attach onto the surface of the through the center of the terebratorium snail as postulated by Wright (1971). How(Figs. 7, 23). The rim of the opening is at- ever, it is doubtful that effective suction can tached to the terebratorium without desmobe exerted by these pits, since no muscle somes (Fig. 23). The circular muscle fibers fibers are present within the wall to create a form a sphincter around the “neck” of the negative pressure, and the rim of each pit is funnel-shaped excretory duct, which is uneven. Probably the most important funcusually flattened dorsoventrally (Fig. 26). tion of the terebratorium is sensory, as Four multiciliated, deep-pit nerve endings there are at least 12 ciliated nerve endings (see section on nervous system) are posidistributed on the surface, and miracidia tioned at the four corners of the flattened are frequently observed probing the surface opening of the apical gland (Fig. 26). At of the snail host with the terebratorium. least eight uniciliated nerve endings lie The surface of the terebratorium of S. manbetween the multiciliated, deep-pit nerve soni miracidium, as described here, appears endings (Fig. 23). The rims of these 12 to differ from that of F. hepatica miracidium (Wilson, 1969a) which is “corrugated.” nerve endings are attached to the tereHowever, the nature of this corrugation is bratorium by septate desmosomes (Figs. 23-26). poorly understood. Five glandular openings The duct openings of the two lateral and five pairs of sensory nerve endings are glands are located at the base of the tere- present in the terebratorium of F. hepatica bratorium adjacent to the first tier of epidermiracidium (Wilson, 1970, 1971), as comma1 plates (Fig. 23). Septate desmosomes pared with three glandular ducts and six line the rims of the openings. pairs of ciliated nerve endings for the S.

334

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PAN

FIG. 27. Tangential section of terebratorium showing interlacing cytoplasmic expansions (21,210~): Septate desmosomes are indicated with arrows. A, mesh work formed by cytoplasmic expansion of terebratorium; B, multiciliated deep-pit sensory papilla; C, uniciliated sensory papilla. One of the papillae contains Type C neurosecretory vesicles; D, nerve to sensory papilla: E, muscle fiber: F, duct opening of lateral gland. FIG. 28. Muscle fiber showing arrangement of thin and thick myofibrils in the fibrillar zone, and afibrillar zone (21,210~): A, epidermal plate; B, basal lamina; C, afibrillar zone of muscle fiber containing numerous P-glycogen particles; D, nucleus: E, mitochondrion; F, thick myofibril: G, thin myofibrils.

Schistosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 29. Nuclear area of muscle fiber (34,650x): A, nucleus; B, cluster of dense granules (inclusions?): C, fibrillar zone with thick and thin myofibrils. Double arrow indicates sarcoplasmic reticulum; D, atibrillar zone containing numerous P-glycogen particles; E, mitochondrion; F. nerve containing many Type A neurosecretory vesicles. Neuromuscularjunction is indicated by single arrow; G, epidermal ridge; H, cytoplasmic bridge of ridge: I, microvillus; J. process of interstitial cell containing numerous Q- and P-glycogen particles; K. basal lamina. FIG. 30. Cross section of muscle fiber showing arrangement of thick and thin myotibrils (67,410x).

336

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mansoni miracidium. It appears that the structure of the terebratorium of S. mansoni and of F. kepatica differ considerably and this may indicate differences in their functional capacities.

PAN

scription of neuromuscular junctions is given in the section on the nervous system. The thick myofilaments are about 250 A and the thin about 60 A in thickness (Figs. 28, 30). In cross section, each thick myofilament is surrounded (or orbitted) by 6- 10 Musculature thin myofilaments. The thick myofilament The musculature consists of outer circu- appears to be composed of ca. 10 subunits. lar and inner longitudinal layers (Figs. 1, 12, The distance between two neighboring 33). Muscle fibers (or myofibrils) are not thick filaments varies from 500 to 1000 A. grouped into bundles as in the striated mus- There is suggestive evidence that thick and cles of vertebrates. Each layer of musculathin filaments may be crosslinked with ture is only one cell (fiber) thick, and each oblique lines. Dense bodies (about 800 A thick) are spindle-shaped myofibril forms a muscle that runs parallel to others within the layer. scattered randomly in the muscle fibers and In cross section, circular fibers appear al- appear to be connected to the thin myofilamost to encircle the entire miracidium (Fig. ments (Fig. 31). 12). The muscle fiber may measure 4 pm Agranular endoplasmic reticulum (sarthick at the level of the nucleus. The circu- coplasmic reticulum) is distributed primarlar muscle fibers lie close to the basal ily along the periphery of myofibrils close lamina and are frequently observed nestling to the sarcolemma (less than 300 A from the between adjacent epidermal pegs (Figs. 8, membrane), but are also found in the center 11). Both circular and longitudinal myofiof myofibrils (Fig. 32). The profiles of the brils are attached, across the basal lamina, to sarcoplasmic reticulum are more readily the base of the epidermal plates or epiderrecognized in cross section of muscle tibers, giving an impression that the long axis mal ridges with discontinuous intermediate tight junctions (macula adherens, Fawcett, tends to run parallel to that of myofibrils. 1966) (Figs. 15, 16, 34). The macula adherThe oval nucleus (2 x 4 pm) contains a ens for inner longitudinal fibers is usually small nucleolus, is usually poor in heteroseen attached to the epidermal pegs via the chromatin, and frequently holds a cluster of gaps between adjacent circular fibers (Fig. dense granules (up to 20 in number and ca. 1). Occasional tight junctions between cir- 100 nm in diameter). Each of the granules is cular and longitudinal myofibrils are also surrounded by ribosomes (Fig. 29). Similar present. Each muscle fiber consists of a dense granules are also found in the nuclei major outer tibrillar zone containing longiof neurons in the neural mass (see below). tudinally oriented thick and thin myofilaInvagination of the nuclear envelope is ments, and a minor inner afibrillar zone (or sometimes observed, probably caused by outpocket) containing the oval nucleus, contraction of fibers. Numerous mitochonmany mitochondria, lipid droplets, and dria surrounding the nucleus contain abunabundant glycogen particles (mostly p par- dant long cristae. ticles) (Fig. 29). Small numbers of pComments glycogen particles are also seen scattered among the myofilaments (Fig. 16). Despite the orderly arrangement of the The plasma membrane of the fibrillar thick and thin myofilaments in the horizone occasionally forms inpocketings or in- zontal plane in the muscle fibers of S. manvaginations that may be associated with soni miracidia, their vertical orientation dense bodies. The plasma membrane of the does not result in recognizable A and I afibrillar zone frequently forms outpockbands, nor Z discs. In these respects, the etings that are associated with neuromusmuscle fibers of S. mansoni miracidia share cular junctions (Fig. 16). The detailed de- some properties with both striated and

Schisrosoma

mansoni MIRACIDIUM

ULTRASTRUCTURE

FIG. 31. Longitudinal section of muscle fiber (34,650): A, dense body; B, sarcoplasmic reticulium: C, a-glycogen particle. FIG. 32. Cross section of muscle fiber displaying abundant sarcoplasmic reticula in the center of fibrillar zone (42,350x): A, sarcoplasmic reticulum; arrow indicates cross section of thick myofibril.

338

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smooth muscle fibers of the vertebrates. Lumsden and Foor (1968) described “striated myofibrils” in the longitudinal caudal fibers of S. mansoni cercariae. They showed that the striations were formed by vertical and lateral “register” of dense bodies. These authors postulated that the “striations” formed by such “cross register” of dense bodies are equivalent to the Z discs in skeletal muscles of vertebrates and that such “striations” are necessary for the longitudinal caudal fibers because of the rapid contractions required. The dense bodies in the muscle fibers of S. mansoni miracidia are few and far apart, and are not arranged to form cross-bandings. This arrangement may be a reflection of the slower contraction rate of miracidial muscles, since the mobility of miracidia depends primarily on ciliary motions. The dense bodies in the muscle of S. mansoni miracidium do not appear to be always associated with sarcolemma as is the case for platyhelminths in general (Lumsden and Byram, 1967; Lumsden and Foor, 1968; MacRae, 1963). However, this discrepancy may largely be due to the angle of sectioning in our preparations. Muscle fibers of platyhelminths thus far studied (MacRae, 1963, 1965; Lumsden and Byram, 1967; Lumsden and Foor, 1968) are nucleated with the exception of those of F. hepaticu miracidium (Wilson, 1969b) whose muscle fibers are reported to be anucleate. Except for the presence of nuclei in the muscle fibers, the fine structure of the musculature of S. munsoni miracidium resembles closely that of F. heputica miracidium. On the other hand, the difference in the muscular structure of S. munsoni miracidium and that of Dugesia tigrinu (a planarian) is in the site of neuromuscular junctions, which in the former is in the granular zone and in the latter in the fibrillar zone (MacRae, 1963). Although the nature of thick and thin myofilaments in S. mansoni miracidium is not established, their structural arrangements closely resemble those of vertebrate muscles in which thick and thin myofibrils

PAN

contain myosin and actin, respectively. This structural resemblance suggests that miracidial myofilaments may also contain myosin and actin, and that contraction of miracidial muscle may depend on the advocated sliding mechanism between thick and thin myofilaments (Huxley, 1957; Hanson and Lowy, 1960). MacRae (1963) also suggested that such a mechanism may be operating in the pharyngeal muscle of the planarian. So far, transverse (T) tubule systems have not been found in the muscles of platyhelminths. However, some investigators (MacRae, 1963; Lumsden and Byram, 1967; Lumsden and Foor, 1968; Wilson, 1969b) indicated that the close association of sarcoplasmic reticulum with sarcolemma suggests a function similar to that of T tubules of vertebrate muscle in the efficient conduction of stimuli to the myotilaments. In S. munsoni miracidium, sarcoplasmic reticulum is not only closely associated with surface membranes, but is also found deep in the myofibrils. Thus, the sarcoplasmic reticulum of S. mansoni miracidium may function similarly to the T tubules of vertebrate muscle fibers in the conduction of stimuli. interstitial

Cells

There are approximately 20 interstitial cells in the miracidium of S. mansoni, which have not been previously described. These cells fill intercellular spaces (hence their name) and apparently serve as an energy store. The small, irregularly shaped cytons occupy the core area of the miracidium posterior to the neural ring, and are surrounded by the cytons of ridge cells (Figs. 9, 37). From the cytons of interstitial cells extend many long processes that fill the intercellular spaces of the miracidium. In sections, these processes appear as elongated, oval, round, or irregularly shaped structures (Fig. 37). The small irregular nucleus with a relatively large nucleolus has little heterochromatin (Fig. 37) and is surrounded by a thin layer of granular endoplasmic reticulum and ribosomes. Adjacent

Schistosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 33. Tangential section of epidermal plates and ridge (5390x): Arrow indicates septate desmosome. A, epidermal plate of the second tier; B, epidermal plates of the third tier: C, ridge separating the second and third tier of epidermal plates; D, ridge separating plates of the third tier; E, outer circular muscle fiber; F, inner longitudinal muscle fiber; G, multiciliated sensory papilla; H, processes of interstitial cell.

340

S. CHIA-TUNG

PAN

FIG. 34. Uniciliated sensory papilla of terebratorium (67,410x): A, sensory papilla containing type C neurosecretory vesicles; B, sensory cilium; C, basal body of cilium without rootlet; D, axosome of cilium; E, septate desmosome; F, basal lamina; G, simple tight junction (macula adherens): H, muscle fiber; I, profile of cytoplasmic expansion of terebratorium. FIG. 35. Filopodium of ridge around uniciliated sensory papilla located at the base of lateral papilla (34,650~): A, filopodium B, septate desmosome; C, basal body of cilium joining its cross-banded rootlet; D, rootlet showing hollow central canal; E, membrane-bound electron-dense body of epiderma.l plate; F, mitochondrion; G, cross section of cilium.

Schistosoma

mnnsoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 36. Ridge cyton displaying hyperactivity of RER and Golgi complex (11,550X): The organism g/&rata. The epidermal plates have was within 6 l/2 hr after entering the snail host, Biomphalaria already detached and a thin tegument has been formed around the postmiracidium. A, nucleus of ridge cell; B, hyperactive RER with dilated cisternae; C, hyperactive Golgi complex with dilated vesicles and cisternae; D, mitochondrion; E, a- and P-glycogen particles in ridge cytoplasm; F; cyton of interstitial cell containing numerous (Y- and /3-glycogen particles and lipid droplets: G, perikaryon of multiciliated sensory papilla. The cytoplasm is crowded with Type D neurosecretory vesicles; H, dilated agranular endoplasmic reticulum; I, inner longitudinal muscle fiber: J, outer circular muscle fiber; K, newly formed tegument; L, cilia from detached epidermal plate.

341

342

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to the RER layer are usually numerous mitochondria with abundant cristae. Small patches of RER are scattered throughout the cytons and their processes, frequently along the cell membrane (Fig. 37). These patches of RER are often masked by (Y- and /3-glycogen particles which till much of the cytoplasm (Figs. 9, 10). Lipid droplets are scattered among the glycogen particles (Figs. 9, 10,37). Germinal cells, flame cells, excretory tubules, and the cytons of “chemoreceptor” neurons (see below) are all surrounded by the processes of interstitial cells (Figs. 10, 36, 63). These processes are also found around the neural ring (Figs. 48, 49) and along many nerves throughout the miracidium . Comments Kinoti (1971) mentioned the presence of “loose connective tissue” in the intercellular spaces of S. mansoni miracidium, but gave no description of it. We did not recognize in the miracidium of S. mansoni a connective tissue similar in nature to that present in the vertebrates. Similarly, other investigators, thus far, have not described connective tissue in platyhelminths. Based on the presence of abundant glycogen particles and lipid droplets, it appears that the primary function of the interstitial cells is storage of sources of energy for the essentially nonfeeding miracidium. Since germinal cells, which are poor in glycogen particles and lipid droplets (see below) are embedded among the processes of interstitial cells, they may also serve as “nurse cells” for the germinal elements, at least during the first few days after the miracidium enters the snail host. At the same time the interstitial cells may provide room for germinal elements to increase as their glycogen and lipid content is utilized. The role of interstitial cells as “connective tissue” (sensu stricto) is not clear. The processes of these cells are present in many intercellular spaces, particularly around the nervous tissue. However, collagenous fibers are not present and the processes are not attached to other cells.

PAN

Penetration

Glands

These large glandular cells, one apical and two lateral, occupy much of the area anterior to the neural ring (Figs. 1, 3, 6). Each cell is comprised of a cyton and a long secretory duct (Figs. 1, 6, 7). The apical gland (sometimes called the primitive gut) (Faust et al., 1934; Ottolina, 1957) is shaped like a volumetric flask with its cyton located immediately anterior to the neural ring. The long secretory duct opens through the center of the terebratorium (Figs. 6, 7, 23,26). In the basal third of the cyton of the apical gland are clustered four irregularly shaped nuclei, each containing a relatively large nucleolus but little chromatin (Fig. 38). The nuclei are surrounded by a layer of free ribosomes and granular endoplasmic reticulum. The rest of the cytoplasm, including that of the duct, is more or less crowded with membrane-bound, round secretory droplets of various sizes (up to 2 pm in diameter) and electron densities (Figs. 6, 7, 38). The spaces between droplets are occupied by ribosomes, RER, CXand /?-glycogen particles and occasional Golgi apparatus. The cisternae of RER are usually distended and frequently contain low to moderately electron-dense granular material which resembles the contents of secretory droplets. Many mitochondria with long cristae are also scattered in the cytoplasm. The duct is lined with a parallel array of microtubules along the plasma membrane (Fig. 23). The rim of the duct opening is attached to the surface layer of the terebratorium without desmosomes. The “opening” is sealed by a plasma membrane with occasional breaks (Fig. 23). On each side of the apical gland is a pair of retort-shaped, lateral gland cells, sometimes referred to as the penetration glands (Faust and Hoffman, 1934; Maldonado and Acosta-Matienzo, 1947). The ducts of the lateral glands which are lined with microtubules open at the base of the terebratorium (Fig. 23). The rim of each duct opening is lined with septate desmosomes. The line structure of the lateral glands is

Schisrosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 37. Perikarya of interstitial cells and common excretory tubule (16,800~): The complex carbohydrate in the cyton and processes of interstitial cell was not well preserved due to poor fixation. However, this condition made the cytoplasmic boundaries of these elements stand out clearly. A, nucleus of interstitial cell surrounded by a thin zone of ribosomes and RER; B, patch of ribosomes along membrane; C, mitochondrion; D, section of processes of interstitial cell; E, perikaryon of common excretory tubule; F, nucleus of common excretory tubule; G, excretory tubule; H, septate desmosome; I, lipid droplet. FIG. 38. Perikaryon of apical gland (6860x): A, nucleus with a relatively large nucleolus surrounded with a zone of ribosomes; B, membrane-bound secretory droplet.

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PAN

FIG. 39. Perikaryon of lateral gland (21,210x): A, chromatin-poor nucleus with a large nucleolus; B, RER with greatly dilated cistemae filled with amorphous material; C, Golgi complex with dilated vesicles and cisternae; D, fully formed membrane-bound secretory droplet; E, developing secretory droplet; F, neuron in the CNS; G, mitochondrion.

Schismsoma

mansoni

MIRACIDIUM

essentially similar to that of the apical gland with the following exceptions. The cyton of the lateral glands contain but one round nucleus and their secretory droplets are generally smaller and less electron dense (Fig. 39). Comments The glandular structure of S. munsoni miracidium has been described by several investigators (Faust and Hoffman, 1934; Maldonado and Acosta-Matienzo, 1947; Pan, 1965; Kinoti, 1971). However, the descriptions to date present little detail as to the structure of the glands and their openings in the terebratorium. Our fixation procedures appeared to have preserved the glandular cells and, thus, have enabled us to study their fine structures in more detail than could Kinoti (1971) and Wikel and Bogitsh (1974). The fine structure of the apical and lateral glands of S. mansoni miracidium appear to have the general characteristics of active secretory cells, i.e., presence of abundant ribosomes, RER, glycogen particles, and Golgi apparatus. The miracidium of S. mansoni maintains its glandular cells for several days and discharges secretory droplets into snail tissues after entering the snail host (unpubl.). The apical and accessory glands of the miracidium of F. hepatica (Wilson, 1971) have fine structures resembling those of the three glandular cells of S. munsoni miracidium. Wilson (1971) suggested that the secretory droplets of F. heputicu miracidium may contain zymogen. He also observed that the miracidium retained its glandular cells for some time after it entered the snail host. Based on Wilson’s (1971) and our observations, it seems probable that the apical and lateral glands of S. munsoni miracidia have the dual function of aiding the miracidium to enter the snail host and of preparing sites for intramolluscan development. It is to be noted that the secretory droplets appear to be formed at first in the cistemae of RER and are closely associated with the Golgi apparatus, and are probably protein in nature.

345

ULTRASTRUCTURE

Excretory

System

Based on light microscopical studies, Ottolina (1957) divided the excretory system into four elements: the flame cell, excretory canal (tubule) excretory ampulla (vesicle), and excretory pore. Our electron microscope study supports this classification. The flame cell consists of three distinct parts, i.e., a cyton, a hollow cylinder (barrel), and numerous cilia within the cylinder (Fig. 40). The stellate cyton sends many thin processes into intercellular spaces nearby, probably as anchoring devices. A flattened, peripherally located nucleus occupies about a third of the cyton (Fig. 41). The cytoplasm contains abundant free ribosomes, granular and smooth endoplasmic reticula, mitochondria, small membranebound vesicles, and microtubules. The cytoplasm opposite the nucleus expands into a thin sheet which is rolled into a gradually tapering hollow cylinder with its margins joined by septate desmosomes (Fig. 43). As many as 100 cilia originating from basal bodies that are embedded in the cyton are present within the cylinder (Figs. 40,41,43, 44). Each basal body is attached to a short, banded rootlet (Fig. 41). The tapered distal end of the cylinder joins the proximal end of the excretory tubule. The proximal quarter of the barrel forms a “grille-work” which contains an outer “row” of thick “bars” and an inner “row” of thin “bars” (Fig. 44). In cross section, thick “bars” (160 nm across) alternate with thin “bars” (100 nm across) forming a zigzag line. Each bar is connected to its neighbors by simple tight junctions. The cytoplasm of thick “bars” of the grille-work resembles that of the cylinder (see below) and the cytoplasm of the thin “bars” is similar in appearance to that of the flame cell cyton. Numerous leptotriches (Kiimmel, 1964) (elongate projections resembling microvilli and ca. 80 nm in diameter) (Figs. 40, 44) extend from thick and thin “bars.” Leptotriches originating from thick “bars” are long and in large numbers and extend into a relatively large

346

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PAN

FIG. 40. Flame cell (11,550~): This flame cell is from a postmiracidium within 6 112 hr after entering snail host, Biomphalaria glabrata. The general fine structure, however, appears to be well preserved. A, a small section of flame cell nucleus; B, perikaryon of flame cell; C, cylinder wall; D, cytoplasmic process of flame cell cyton; E, cilia in the cylinder; F, peri-flame cell space displaying many leptotriches; G, leptotriches within cylinder; H, grille-work of cylinder; I, neuropiles in the CNS; J. cyton of lateral gland; K, longitudinal muscle fiber.

Schisrosomn

mnnsoni

MIRACIDIUM

ULTRASTRUCTURE

of flame cell (42,350x): A, nucleus; B, nucleolus: C, cilia in the cylinder; D, FIG. 41. Perikaryon cross-banded rootlet: E, microtubule; F, mitochondrion; G, RER. FIG. 42. Excretory pore (25,200~): A, lumen of the pore; B, diaphragm sealing the opening; C, microvillus on the diaphragm: D, septate desmosome joining pore wall to ridge: E, process of pore cell; F, wall of excretory vesicle; G, septate desmosome joining pore with excretory vesicle; H, epidermal ridge; I, outer circular muscle fiber.

348

S. CHIA-TUNG

PAN

FIG. 43. Cross section of the flame cell cylinder at midlevel (26,950~): A, cylinder wall containing circular fibrils (arrow); B, cross sections of cilia within the cylinder; C, septate desmosome joining the margins of cylinder wall; D, lateral gland. FIG. 44. Cross section of flame cell cylinder at the level of grille-work (25,200): A, cylinder wall; B, thick bar of the grille-work; C, thin bar of the grille-work; D, cross sections of cilia within the cylinder; E, septate desmosome joining the margins of cylinder wall; F, leptotriches in the peri-flame cell space; G, process of interstitial cell.

Schistosoma

mnnsoni

MIRACIDIUM

ULTRASTRUCTURE

349

intercellular space around the flame cell granular and contains a few membrane(Fig. 40). Leptotriches originating from thin bound small vesicles. Several cytoplasmic bars are short and few in number and ex- processes extend from the pore into spaces tend into the flame cell cylinder (Fig. 40). between nearby muscle layers (Fig. 46). Except for the presence of circular fibrils in The nucleus of the “pore” cell was not observed. The lumen surface of the pore is the inner zone, the cytoplasm of the cylinder wall (Fig. 43) appears to have a struc- irregular but forms no prominent projections or deep folds (Figs. 42, 47). A ture similar to that of the flame cell body. structure (Fig. 42) covers The presence of circular fibrils makes that diaphragm-like the pore, similar to that present in F. hepart of cytoplasm appear more electron dense than the rest (Fig. 42). putica (Wilson, 1969~). Several short miTwo excretory tubules lie on each side of crovilli extend from the diaphragm to the the miracidium adjacent to the flame cells outside (Fig. 42). and consist of elongated cells whose cytoplasm forms a continuous tubule (Figs. 9, Discussion 10, 45). Both tubules on each side join a The excretory system of many species of common tubule which is formed from a trematodes has been studied for many years separate cell (Fig. 37). Cytoplasmic projecby light microscopy. However, apparenttions are present in the lumen of all tubules ly only the excretory system of the miraand are more numerous distally. The indi- cidium of F. hepatica had been studied vidual tubule is formed from flattened cyto- extensively with the electron microscope plasm which is rolled up and joined at the (Wilson, 1969~). The fine structure of the edges by a continuous septate desmosome excretory system of S. mansoni miracidium (Figs. 37, 45). The irregularly shaped nu- generally resembles that of the miracidium cleus contains many heterochromatin of F. hepatica (Wilson, 1969~) and that of patches. The cytoplasm resembles that of the daughter sporocysts of S. munsoni the flame cell cyton except for the absence (Meulman, 1972). Unlike the flame cell of microtubules. The terminal segment of body of F. heputicu miracidium (Wilson, each common tubule empties into the 1969c), the flame cell cyton of S. munsoni proximal end of the excretory vesicle. The miracidium is anchored by cytoplasmic vesicle is formed from a single cell in the processes to the neighboring cells. Wilson shape of an elongate sac. The cytoplasm of (1969~) and Meulman (1972) believed that the thick wall resembles that of the flame the outer leptotriches were an anchoring cell except that the former contains numerdevice for the flame cell. In S. munsoni ous large mitochondria with abundant long miracidia, the outer leptotriches appear to cristae. The lumen of the vesicle appears be suspended in the relatively large space irregular because of many deep cytoplasmic around the flame cell. They may function to folds and long projections (Fig. 46). The keep fluid in motion around the flame cell oval nucleus is located peripherally at the and to absorb metabolic by-products for midpoint of the sac and contains a fair discharge through the flame cell and exnumber of heterochromatin patches. The cretory duct. The inner leptotriches may excretory vesicle joins the excretory also have a function similar to that of the “pore” with a continuous septate desmoflame cell barrel. Although the function of some (Fig. 46). The circular excretory pore the flame cell has not been determined in (Fig. 47) appears to be formed by a separate trematodes, most investigators believe that cell which is attached by a continuous sep- the flame cell functions as a primitive kidtate desmosome to the epidermal ridge ney and that filtration of waste metabolic separating the third and fourth tiers of epi- products may take place across the reladermal plates. The sparse cytoplasm is tively thin area of tight junctions between

350

S. CHIA-TUNG

PAN

FIG. 45. Perikaryon of excretory tubule (25,200x): A, perikaryon of excretory tubule; B, nucleus; C. septate desmosome joining margins of tubule wall; D, agranular endoplasmic reticulum; E, cytoplasmic process in the tubule lumen; F, process of interstitial cell. FIG. 46. Excretory vesicle (I 1,550~): A. excretory vesicle displaying many deep folds; B, nucleus of excretory vesicle; C, excretory pore: D, septate desmosome joining excretory vesicle with excretory pore: E, septate desmosome joining excretory pore with ridge: F, cytoplasmic process of excretory pore: G, epidermal ridge: H. epidermal plate; I, outer circular muscle fiber; 5. cytoplasmic process of interstitial cell; K, nerve; L, leptotriche in the peri-flame cell space.

Schisrosoma mansoni MIRACIDIUM

the “bars” of the “grille.” In the miracidiurn of F. hepatica, Wilson (1969~) suggested that selective reabsorption of filtered material within the excretory system probably takes place in the distal portion of the excretory tubule and in the vesicle, where many long cytoplasmic projections are present. The terminal portion of the excretory system of S. mansoni miracidium also contains many long cytoplasmic projections in the lumen and probably functions in selective reabsorption. Since the opening of the pore is covered by a membrane, and since there seems to be no anatomical provision for fluid intake in the miracidium of S . mansoni, the contents of the excretory vesicle cannot be emptied directly to the outside. Excretion probably takes place through the membrane by permeation or transport. Based on line structural studies, the excretory system of different trematodes exhibit little variation between species or between stages in the life cycle (Gallagher and Threadgold, 1967; Kummel, 1958, 1959,1964; Meulman, 1972; Pantelouris and Threadgold, 1963; Senft et al., 1961; Wilson, 1969~). Nervous System

Little is known about the nervous system of the miracidium of S. mansoni and of other trematodes. Recent studies by Wilson (1970) and Brooker (1972) indicate that trematode miracidia possess a relatively well-developed, complex nervous system that may aid the larvae in their host location (Chemin, 1974) and other functions. The nervous system of S. mansoni miracidium consists primarily of a neural ring (central nervous system orCNS), at least six types of peripheral sensory organelles that are connected to CNS, and nerves innervating the musculature. Neural Ring The neural ring is a spherical

structure

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351

that may comprise nearly 10% of the miracidial volume. The neurons are located peripherally, encircling numerous axons and dendrites that are commonly referred to as neuropiles (Figs. 1, 3, 6, 48). Except for small areas where excretory tubules come in direct contact, the surface of the neural ring is covered with the processes of interstitial cells (Fig. 48). These processes also extend superficially into interneuronal spaces but seldom reach into the neuropiles (Fig. 49). The neurons (3-6 pm in diameter), which form a layer one to two cells thick, are fitted between each other and, thus, usually appear irregularly shaped. The nuclei are also generally irregular, appearing oval, kidney-shaped, elongated, or lobulated (Figs. 48, 50). The nuclei contain a small nucleolus, patches of heterochromatin, and occasionally one or two clusters of electron-dense granules (Fig. 51). Each cluster appears to be an aggregate of ribosomes within which are embedded several larger and denser granules (300450 A). The nature of these granules is not clear, but perhaps they represent inclusions. Similar granules are also present in muscle nuclei (Fig. 29). The granular cytoplasm is moderately electron dense and contains some or all of the following structures: (Y (more numerous) and /3-glycogen particles, lipid droplets, Golgi complex, mitochondria, granular and agranular endoplasmic reticula, free ribosomes, myelin figures, and four types of membrane-bound “neurosecretory” vesicles (see below) (Figs. 49-51). The granular cytoplasm of some neurons is less electron dense than that of the rest (Fig. 51). Neuropiles appear as oval, sausageshaped or elongated membrane-bound structures that are closely fitted together in the central region of the CNS (Fig. 48). The numerous neuropiles and nerves have cytoplasm and cytoplasmic inclusions similar to their perikaryons (Figs. 49, 53). In addi-

FIG. 47. Cross section of excretory pore (19,600x): A, excretory pore joined to ridge with septate desmosome; B, epidermal ridge: C, septate desmosome joining epidermal ridge with epidermal plate: D, epidermai plate.

352

S. CHIA-TUNG

FIG. 48. neuropiles tubule; F, muscle; I,

PAN

Neural mass (CNS) (5390x): A, neurons located at the periphery of neural mass; 9, (axons) in the center of CNS; C, processes of interstitial cells; D, nerve; E, excretory ridge cyton containing numerous membrane-bound vesicles; G, lateral gland; H, circular epidennal plate; J, epidermal ridge.

Schistosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 49. Detailed structure of neural mass (17,360x): cY-Glycogen particles are indicated with arrows. A, nucleus of neuron at the periphery; B, perikaryon; C, mitochondrion; D, myelin figure: E, axon encircled with several concentric membranes (primitive myelin sheath); F, axon containing Type C neurosecretory vesicles; G, axon containing Type A neurosecretory vesicles; H, process of interstitial cell containing numerous a- and P-glycogen particles: I. nerve leaving CNS: J. excretory tubule; K. ridge cyton; L. membrane-bound vesicle in ridge cyton.

353

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FIG. 50. Neuron in neural mass (42,350~): Arrows indicate wglycogen particles. A, nucleus of neuron: B, perikaryon; C, agranular endoplasmic reticulum; D, mitochondrion; E, axon containing Type A neurosecretory vesicles; F, axon containing Type B neurosecretory vesicles.

Schistosoma

mansoni

MIRACIDIUM

ULTRASTRUCTURE

FIG. 5 1. Neurons in the neural mass displaying different densities of ground substances (26,950~ ): Arrows indicate o-glycogen particles. A, nucleus: B, “inclusion granules” of unknown nature; C, perikaryon having electron-lucent cytoplasm: D, perikaryon having electron-dense cytoplasm: E, mitochondrion; F & G, Type B neurosecretory vesicles; H, axon containing Type B neurosecretory vesicles; I, axon having electron-lucent cytoplasm; J, axon containing Type C neurosecretory vesicles: K, ridge cyton; L, membrane-bound vesicle.

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FIG. 52. Neuropiles and synapses (26,950~): A, synapsis; B. axon containing Types A and B neurosecretory vesicles; C, axon containing nemotubules; D. mitochondrion. FIG. 53. Axon enclosed by several concentric membranes (primitive myelin sheath) (34,650x): A, axon; B, primitive myelin sheath; C, process of interstitial cell; D, perikaryon of a neuron: E, excretory tubule.

Schistosoma

mansoni

MIRACIDIUM

tion, neurotubules are frequently seen in neuropiles and nerves (Figs. 52, 55). The synapses of neuropiles usually appear as a unilateral electron-dense zone along one of the two opposing membranes, but bilateral synaptic structures are also present (Fig. 52). Many synaptic vesicles are commonly present near the synapses. Some neuropiles may occasionally be encircled by four or more layers of unit membrane suggesting a primitive form of myelination (Figs. 49, 53).

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357

sory organelle (Fig. 57), and in the nerves at the neuromuscular junctions (Figs. 16, 56). Type B vesicles. This type of circular vesicle is relatively uniform in size, measuring 1000 A in diameter. The center of vesicle (ca. 5/7 of the total area) is occupied by a round, strongly electron-dense material. A narrow, distinctly electron-lucent rim separates the membrane from the central material (Fig. 25). Type B vesicles are present in some uniciliated nerve endings in the terebratorium (Fig. 25), in some nerve endings at neuromuscular junctions (Fig. Peripheral Nerves 56), and in some neurons and neuropiles of Peripheral nerves are essentially similar CNS (Fig. 52). in fine structure to neuropiles but may Type C vesicles. This type of round vesicontain more abundant neurosecretory cles measure ca. 1200 A in diameter. They vesicles particularly at the terminals. Usuare tilled with moderately dense material ally only one type of synaptic vesicle is and are found in some uniciliated nerve endpresent in each nerve but occasionally two ings in the terebratorium (Fig. 34) and types may be seen (Fig. 56). Peripheral some nerve endings at the neuromuscular nerves are frequently accompanied by sev- junctions. eral ganglion cells which resemble neurons Type D vesicles. These vesicles are the in the neural ring. Larger nerves, such as largest (2000 A in diameter) and most conthat extending to the terebratorium (Fig. spicuous of the four types. They are uni58), contain more ganglion cells than do form in size and are usually filled with modsuch smaller ones as those going to the lat- erate to strongly electron-dense material. eral papilla (Fig. 57). Rarely, a thin, electron-lucent space may be seen along the membrane (Fig. 63). Neurosecretory Vesicles These vesicles are seen only in a group of At least four types of membrane-bound, bipolar ganglions behind the CNS, their (synaptic)” vesi- axons, and ciliated nerve endings (Figs. 55, round, “neurosecretory cles are present in the nervous system of 62, 63) (see below). the miracidium. These vesicles are classiJunctions fied into Types A, B, C, and D according to Neuromuscular The fine structure of neuromuscular size and-electron-dense material within. junctions is generally similar to that of Type A vesicles. These vesicles usually synapses between neuropiles in the CNS measure 700 A in diameter and are generally electron lucent in the center. The inner except that the junctions are usually unilateral in nature. The nerve endings usually side of the membrane is coated with slightly or “depressions” on electron-dense material (Figs. 54, 57, 59, attach to “pockets” the muscle surface on the inner granular 61). Occasionally, the entire vesicle may appear to be filled with the similar, slightly zone (Figs. 16, 56). Mitochondria are freelectron-dense material (Fig. 16). Type A quently present in this region of the muscle. neurosecretory vesicles are seen in some Sometimes more than three-quarters of a neurons and neuropiles of CNS (Fig. 52), in nerve ending may be surrounded by the plasma membrane of the invaginated musthe lateral papilla and its nerves and ganglicle surface. The opposing membranes of ons (Fig. 54), in the multiciliated deep-pit nerve endings of the terebratorium (Fig. both the muscle and nerve ending are dens25), in the wall of the ciliated sacular sen- er than normal, and are separated by a

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FIG. 54. Lateral sensory papilla (17,360x): A, bulbular enlargement of the sensory nerve terminal (papilla) containing numerous Type A neurosecretory vesicles and neurotubules (arrow); B, nerve containing neurotubules (arrow) and mitochondria; C, ridge; D, septate desmosome joining ridge with epidermal plate: E, basal iamina, Note strongly electron-dense area (double arrows) of ridge covering the papilla. FIG. 55. Multiciliated sensory papilla with multiciliated axoneme (26,950~): A, sensory papilla containing numerous Type D neurosecretory vesicles; B, multiciliated axoneme; C, basal body of individual cilium connected to a short rootlet; D. electron-dense particles around rootlets; E, septate desmosome joining papilla to ridge; F, epidermal ridge; G, circular muscle; H, basal lamina.

Schistosoma

mnnsoni

MIRACIDIUM

space of ca. 150 A. The inner side of muscle membrane is coated with electron-dense material (ca. 300 A wide) which may also be present in the gap between the membrane (Figs. 15, 56). The nerve ending is crowded with many small synaptic vesicles of Type A (Fig. 16) and occasionally also contains both Types A and B (Fig. 56). Peripheral Sensory Organelles Of the six types of peripheral sensory organelles that were recognized, two are distributed in the terebratorium and five are in the ridges between the epidermal plates. Ciliated sensory organetles in the terebratorium. Multiciliated, Deep-Pit Nerve Ending (Sensory Papilla). There are four multiciliated, deep-pit nerve endings in the terebratorium. These nerve endings are positioned at the level between the openings of the apical and lateral glands, encircling the terebratorium and equally spaced from each other (Figs. 23, 25, 26). Each sensory organelle is formed by a nerve ending that is shaped like a pit or goblet. The pit opens to the outside with its rim attached to the tegument of the terebratorium with a continuous septate desmosome (Figs. 23-25). About 12 sensory cilia arising from the inner surface of the pit protrude slightly beyond the opening of the pit. Each cilium originates from a basal body which is embedded in the pit wall (Fig. 24). The basal bodies have no rootlets and lack the usual two central microtubules. The pit wall and its connecting nerve contain numerous Type A neurosecretory vesicles and neurotubules (Figs. 24, 25). Uniciliated Nerve Ending (Sensory Papilla). There are at least eight nerve endings of this type in the terebratorium. Four of these are distributed between and slightly anterior and the other four posterior to the four multiciliated deep-pit nerve end-

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359

ings (Figs. 23, 25). Each structure has a slightly enlarged, rounded tip which is attached to the tegument with a continuous septate desmosome. A single cilium arises from the basal body which has no rootlet and lacks the two central microtubules (Fig. 34). The four anteriorly positioned uniciliated nerve endings contain Type B neurosecretory vesicles (Fig. 25) and the other four contain Type C vesicles (Fig. 34). All 12 nerves appear to originate from a ganglion located in the anterodorsal side of the apical gland cyton (Figs. 8, 58). A thick, short nerve trunk connects the ganglion with the CNS (Figs. 8, 58). Lateral papilla (sensory papilla). There is a pair of bulbous sensory organelles which are located, one on each side, in the ridge separating the first and second tiers of the epidermal plates (Figs. 1, 2, 54, 59). Each papilla is formed by spherical enlargement of a nerve ending and is enveloped by a thin layer of ridge cytoplasm. The bulb and its connecting nerve are separated from the ridge by basal lamina (Fig. 54). The papillae usually protrude conspicuously from the general body outline of the miracidium (Fig. 2). Many Type A neurosecretory vesicles and neurotubules are present in the papillae and their connecting nerves (Fig. 54). Numerous large mitochondria are present in the nerves in addition to a few lipid droplets (Fig. 57). The mitochondria are distributed close to the base of the bulb but not within it. The surface zone of the ridge cytoplasm covering each papilla is more electron dense than the zone adjacent to the basal lamina (Figs. 54, 59). The nerve is formed by a bipolar ganglion cell that connects with the CNS (Fig. 57). Multiciliated, saccular sensory organelle. This pair of sensory organelles has the most complex structure of all the peripheral sensory nerve endings. Each is

FIG. 56. Neuromuscularjunction (42,350~): A, nerve terminal containing numerous Types A and B neurosecretory vesicles; B, atibrillar zone of muscle fiber where neuromuscular junction (arrow) is located. The membranes of muscle and/or nerve are coated with electron-dense material: C, tibrillar zone of muscle containing thick and thin myoftbrils; D, epidermal plate; E, basal lamina; F, simple tight junction attaching muscle fiber to epidermal plate.

360

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FIG. 57. Lateral nerves and their sensory terminals (11,550~): A, nucleus and perikaryon of the nerve (B) to lateral papilla; C, nucleus and perikaryon of the nerve (D) to multiciliated, shallow-pit sensory papilla (E); F, septate desmosome joining (E) to epidermal ridge; G, nerve (axon) to multiciliated saccular sensory organelle (H); I, nerve (axon) to the unicihated sensory papilla (J); K, tilopodium; L, septate desmosome joining uniciliated sensory papilla to ridge; M, septate desmosome joining epidermal ridge (N) with plate (0); P, ridge cyton and connecting cytoplasmic bridge (Q); R, circular muscle; S, lateral gland; T, septate desmosome attaching multiciliated saccular sensory organelle to ridge. FIG. 58. Anterior nerve and ganglion (5390x): A, neural mass; B, nerve; C, ganglion; D, neuron: E, lateral gland; F, apical gland.

Schisrosoma

mansoni

MIRACIDIUM

located along the anterior side of the nerve to the lateral papilla (Fig. 57). The organelle is an elongate pouch (10 x 2.5 pm) formed at the terminus of an axon of a bipolar ganglion cell (Fig. 60). The other axon terminates in the CNS. The rim of the pouch opening is attached to the ridge with septate desmosomes (Figs. 57, 59). Although we did not observe the actual opening, there is suggestive evidence that the “pouch” may open to the outside (Fig. 59). Many Type A synaptic vesicles are present in the wall of along with mitochondria, the “pouch” glycogen particles, and neurotubules (Figs. 57, 59, 61). The basal wall of the pouch contains an extensive network of agranular endoplasmic reticulum (Fig. 60). The lumen of the pouch contains at least four coils of concentric lamellae. Each lamella is formed by two closely apposed (300-A wide gap) plasma membranes (Figs. 60, 61), and is connected to at least one cilium (Figs. 60, 61). The cilium originates from a basal granule embedded in the sac wall and has the “9 + 0” arrangement of microtubules.

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361

vesicles (Fig. 59). The ridge cytoplasm around the rim of the nerve ending expands to form several tilopodia-like projections (nearly 3 nm long). The projections (Figs. 35, 57, 59) have the texture of ridge cytoplasm but without any visible cytoplasmic organelles or inclusions, except for a few P-glycogen particles. The layer beneath the unit membrane is strongly electron dense. Multiciliated sensory papilla. There are about 12 sensory nerve endings of this type that girdle the miracidium in the ridge separating the second and third tiers of epidermal plates. Each papilla is spaced at about 12 Km (Fig. 33) and is the terminus of one of the two axons of a bipolar ganglion cell (Fig. 1). The other axon extends into the CNS at its posterior side. The perikaryons of ganglia are clustered behind the neural ring and are surrounded by processes of interstitial cells and ridge cytons (Figs. 6, 36, 63). The papillae are directly exposed to the outside and are attached to the ridge by septate desmosomes (Figs. 55, 63). Each papilla contains about 12 sensory Multiciliated, shallow-pit nerve ending cilia (Fig. 33). In some papillae, cilia form (sensory papilla). This shallow-pit nerve individual axonemes (Fig. 63), while in ending is located between the base of the others, cilia are bundled together and are lateral papilla and the attachment of the enveloped in a plasma membrane (mulciliated saccular sensory organelle to the ticiliated axoneme) (Fig. 55). The basal ridge (Figs. 57, 59). There are about six rel- body of each cilium continues into a short, atively long sensory cilia originating from banded, tapered rootlet. The tip of each basal granules embedded in the pit wall. rootlet extends into a cluster of dense partiThe rim of the pit is attached to the ridge cles (850 A) (Fig. 55). Electron-dense with septate desmosomes. The connecting synaptic vesicles of Type D occupy much nerve is one of the two axons of a bipolar of the individual perikaryon and its axons neuron running between the nerves con- including the papilla (Figs. 55, 62, 63). necting the lateral papilla and the ciliated Some perikarya may also contain numerous saccular sensory organelle. These three membrane-bound vesicles which are either nerves along with the nerve for the uni- partially filled with moderately electronciliated papilla (see e below) run side by dense material or are entirely electron luside and form a short nerve trunk (Fig. 57). cent. In the latter instance, the vesicles apUniciliated sensory nerve endings (pa- pear to be “collapsed” (Fig. 62). The cytopilla). A pair of this type of nerve ending plasm of the ganglion cells is granular and is present, one at the posterior side of the contains ribosomes, P-glycogen particles base of each lateral papilla (Fig. 57). The mitochondria, neurotubules, and Golgi fine structure of these nerve endings is es- complex (Figs. 62, 63). Functionally active sentially similar to that present in the tere- Golgi complexes are frequently associated bratorium, except that the nerve endings with an unidentified membranous structure here contain Type A membrane-bound (Fig. 63). The structure appears to consist

362

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FIG. 59. Lateral papilla, multiciliated shallow-pit sensory papilla and multiciliated saccular sensory organelle (21,210~): A postmiracidium within 1 l/2 hr after entering snail, Biomphuluria glabraru. A, lateral papilla containing neurotubules and Type A neurosecretory vesicles, and covered with a thin ridge cytoplasm (B); C, nerve to lateral papilla containing numerous neurotubules; D, uniciliated sensory papilla; E, multiciliated shallow-pit sensory papilla; F, septate desmosome attaching the papilla to ridge; G, multiciliated saccular sensory organelle; H, septate desmosome attaching the organelle to ridge. Arrow indicates possible opening of the saccular sensory organelle to the outside; I, ridge; J. septate desmosome joining ridge to epidermal plate (K); L. snail tissue.

Schistosoma

mansoni

MIRACIDIUM

of a stack of distended cisternae whose membranes form parallel wavy lines. These cisternae may be a modified agranular endoplasmic reticulum. Each perikaryon may measure 5 pm at the widest dimension and contains a chromatin-rich, irregularly shaped nucleus (Figs. 6, 36, 63) which sometimes appears lobed. Comments The most important function of the trematode miracidium is the capacity to find and enter a suitable snail host. Since the infectivity of “self-contained” S. mansoni miracidia is less than 12 hr after hatching (Chernin, 1968; Ottolina, 1957), they must within this time frame find a suitable snail host in bodies of water which are immense in comparison to their small size. Yet, Chernin and Dunavan (1962), and Upatham (1972a, b, 1973) reported that S. mansoni miracidia can readily locate B. glabrata against tremendous spatial odds and, that some sensory mechanisms may be involved in miracidial host location (Chernin, 1970). Our study on the fine structure of the nervous system indicates that S. munsoni miracidium has a well-developed nervous system and that its surface is provided with abundant nerve endings of several types. Although there is littie experimental evidence to indicate that these surface nerve endings are sensory in nature, the resemblance of their fine structure to that of sensory endings in man and other animals (Barber and Wright, 1969; Porter and Bonneville, 1968) suggests that their function may be sensory. Similar suggestions have been made by various investigators (Wilson, 1970; Brooker, 1972; Lyons, 1972; Dixon and Mercer, 1965). Brooker (1972) similarly described both uni- and multiciliated nerve endings in the terebratorium, but did not mention any neurosecretory vesicles in the nerve endings.

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Brooker (1972) suggested that these nerve endings may function as tangoreceptors as well as chemoreceptors. The fact that three types of neurosecretory vesicles are present in different nerve endings suggests that different functions are served by these sensory organelles. Wilson (1970) described several multiciliated pit nerve endings in the terebratorium of F. heputicu miracidium. The nerves contain neurosecretory vesicles equivalent to our Type B vesicles. He suggested that these sensory organelles were either chemo- or tangoreceptors. The walls of the multiciliated, deep-pit nerve ending and its nerve in the terebratorium of S. munsoni miracidium contain many clear vesicles of Type A, while uniciliated nerve endings contain either Type B or C vesicles. These observations suggest that the elaborate sensory apparatus in the terebratorium of S. munsoni miracidium is involved in host location, directly or indirectly. The lateral papilla of S. munsoni miracidium is a single bulbous enlargement of a nerve ending and is different from paired bulbous nerve endings in F. heputicu miracidium (Wilson, 1970). Wilson (1970) suggested that the paired bulbous nerve endings may have a secretory function or represent the developmental phase of a sensory structure. Brooker (1972), on the other hand, believes that the lateral papilla, in conjunction with the multiciliated nerve ending at its base, may act as a “depth sensor.” Based on its fine structure and its flexibility in live organisms, we also suggest that the lateral papilla of S. munsoni miracidium may function as a “depth sensor.” The water pressure at different depths obviously exerts varying stimuli to the flexible, bulbous lateral papilla which in turn transmits through its nerve different impulses to the neural ring. It is interesting to speculate on the function of the ciliated saccular sensory or-

FIG. 60. Multiciliated saccular sensory organelle (17,360~): A, concentric lamellae. Each lamella is formed by the membrane of a cilium (B) originated from sac wall (0; D, extensive network of agranular endoplasmic reticulum at the base of the sac.

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FIG. 61. Cross section of multiciliated saccular sensory organelle (26,950~): A, wall of saccular sensory organelle containing Type A neurosecretory vesicles, neurotubules, and mitochondrion; B, cilium originating from the wall and tapering to form a circular lamella. Cilium has no central microtubules; C, concentric lamellae; D, rim of multiciliated shallow-pit sensory papilla: E. epidermal ridge; F, circular muscle. Fro. 62. Perikaryon of multiciliated sensory papilla (26,950~): A, nucleus; B, Golgi complex; C, Type D neurosecretory vesicles; D, empty and/or collapsed neurosecretory vesicles: E. mitochondrion.

Schistosoma

mansoni

MIRACIDIUM

ganelle. Brooker (1972) was inclined to ascribe the role of photoreception to this organelle. A similar organelle was observed in the miracidium of F. hepatica by Wilson (1970) who suggested that the organelle is a gravity receptor. It is possible that the ciliated saccular sensory organelles in S. mansoni miracidium are primitive photoreceptors because of the presence of ciliaassociated lamellae. However, no apparent pigment or pigment-containing cells are associated with the organelles, and the sac most likely opens to the outside (Fig. 60). It seems more likely that these organelles are vibration sensors, because the lamellae may act as amplifiers of vibration for the attached cilia. For aquatic animals, such as S. munsoni miracidium, vibration-sensing organelles seem to be important for host location, as previously suggested (Hot-ridge, 1966). A possible function for the multiciliated, shallow-pit nerve endings and the nearby uniciliated nerve endings cannot easily be ascribed. It is interesting to note that all these four types of organelles and their associated nerves contain similar neurosecretory vesicles, i.e., Type A. The last type of sensory papillae is connected to a group of conspicuous, extraCNS ganglion cells which are crowded with Type D “neurosecretory vesicles.” Both Ottolina (1957) with the light microscope and Brooker (1972) with the electron microscope observed the ciliated papillae on the surface of S. mansoni miracidium but did not describe the conspicuous perikarya. The abundance of neurosecretory vesicles in the papillae and their perikarya suggests that these sensory papillae may perform extraordinary functions. In addition, the presence of two kinds of axonemes may indicate that all papillae in this group do not perform exactly the same functions. Chernin (1970) has experimentally shown that some species of snails emit small molecular substances which S. mansoni miracidia are able to “perceive.” Ottolina (1957) suggested that this group of sensory papillae may be chemoreceptors. We have no

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365

experimental evidence to suggest that these papillae are chemoreceptors. However, it would be interesting to experimentally identify the functions of these papillae as well as others described in this study. The four types of membrane-bound vesicles in the nervous tissue of S. mansoni miracidia resemble several neuronal and neuroendocrine vesicles present in mammals (Cooper et al., 1970). In mammals, the vesicles are suspected to store acetylcholine, catecholamines, and other neurosecretory hormones. Some of these chemicals have been indirectly or directly demonstrated in the miracidia, cercariae, and adults of S. mansoni (Bueding, 1952); Bennett et al., 1969; Bennett and Bueding, 1971; Bruckner and Voge, 1974). Dixon and Mercer (1965) described three types of membrane-bound vesicles (Types a, b, and d) in the nervous system of F. hepatica cercariae. These vesicles appear to resemble closely our Types A, B, and D. “Type c inclusion” in the nerve of cercaria as described by Dixon and Mercer (1965) are “stellate granules” without membranes. We have observed similar dense stellate granules (particles) in the neuropiles and neurons of the neural rings, and in some nerve endings in S. mansoni miracidia. These particles appear to be complex carbohydrate granules. In mammals, glycogen is not present in adult nervous tissue in histochemically demonstrable amounts (Bloom and Fawcett, 1972). The nervous tissue of S. mansoni miracidium appears to contain abundant glycogen particles. Although we occasionally observed in the neural mass a few neuropiles which were surrounded by several concentric layers of membranes (Figs. 49, 53), these membranous sheaths are not as elaborate as the true myelin sheath. The concentric membranes do not have true myelin structure, i.e., fusing of closely apposed membrane leaflets (Porter and Bonneville, 1968). These membranous structures in the neural ring may possibly be regarded as a “primitive myelin” sheath in

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FIG. 63. Multiciliated sensory papilla (26,950~): A, sensory papilla containing uniciliated axonemes and Type D neurosecretory vesicles; B, basal body of cilium continuing into short rootlet; C, septate desmosome attaching papilla to epidermal ridge; D, perikaryon of sensory papilla containing numerous Type D neurosecretory vesicles; E, Golgi apparatus; F, agranular endoplasmic reticulum; G, mitochondrion; H, nerve; I, cytoplasmic bridge of ridge lined with microtubules along the membrane (arrow); J, ridge cyton; K, membrane-bound vesicle; L, epidermal ridge; M, circular muscle.

Schistosoma

mansoni

MIRACIDIUM

lower animals. Another interesting feature of the nervous tissue (especially of the CNS) of the miracidium is the absence of cells equivalent to neuroglia in mammals. The processes of interstitial cells superficially penetrate interneuronal spaces and, thus, may function as “glial cells.” Germinal

Cells

As far as we could determine, there are about 20 germinal cells in a miracidium of S. munsoni. These cells are located behind the clustered cytons of the multiciliated papillae of the presumed “chemoreceptors” (Figs. 1, 10) and at the level of the third and fourth tiers of epidermal plates. Germinal cells are in general irregularly shaped and contain several thin processes that extend into the intercellular spaces, possibly as anchoring devices (Fig. 3). Their cytons may measure 5 pm at the widest diameter and are surrounded by processes of the interstitial cells (Fig. 64). The granular cytoplasm of germinal cells is crowded with numerous ribosomes, many mitochondxia, rare Golgi apparatus, granular endoplasmic reticulum, and a few pglycogen particles. The usually oval nucleus is large (3 pm), in relation to the cyton, contains a prominent nucleolus (1.5 Fm), and is poor in heterochromatin. Comments The germinal cell is easily recognized by a large nucleus which occupies nearly onehalf of the cyton, and by the conspicuous nucleolus. The large number of ribosomes in the cytoplasm is an indication of its capability to synthesize large amounts of proteins for self-consumption (Porter and Bonneville, 1968). The scarcity of complex carbohydrate in the cytoplasm is probably compensated by the presence in juxtaposition of glycogen-rich processes of the interstitial cells. Olivier and Mao (1949) described an average of 53 germinal cells in a miracidium, and 200-400 germ balls in the lCday-old mother sporocysts. Their observations suggest that the germinal cells multiply rapidly in the mother sporocyst after

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the miracidium enters a suitable snail host, and that the numerous ribosomes in the cytoplasm function actively in protein synthesis, necessary for cell division. SUMMARY

An electron microscope study has been carried out to document the cellular composition of the miracidium of S. mansoni. The cellular organization of the miracidium has been divided into eight major categories: epidermal system, terebratorium, musculature, interstitial cells, penetration glands, excretory system, nervous system, and germinal cells. The surface of the miracidium is covered with 21 ciliated, anucleate epidermal plates which are separated by epidermal ridges. The plates are attached to the ridges with septate desmosomes, and contain numerous mitochondria and membrane-bound, electron-dense structures. Many microvilli are present on the surface of epidermal plates among the cilia. The epidermal ridges connect to the syncytial cytons by narrow cytoplasmic bridges. Numerous membrane-bound vesicles are present in ridge cytons besides abundant RER, Golgi apparatus, and complex carbohydrate particles. These vesicles are believed to be stored membranes, and were observed to participate in rapid formation of the tegument of the mother sporocyst during the first 24 hr after the miracidium enters the snail host. The terebratorium is a hemispheric structure bearing at least 12 ciliated sensory organelles. Its surface is invested with a network of interlaced cytoplasmic expansions. The secretory ducts of both apical and lateral glands open to the outside at the terebratorium. The outer circular and inner longitudinal muscle fibers are situated immediately beneath the basal lamina, and are attached to the epidermal plates with simple tight junctions. Only smooth muscle fibers are present, and these have thick and thin myofibrils as well as dense bodies, similar to those

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FIG. 64. Germ cell (17,360x): A, nucleus of germ cell; B, nucleolus; C, cytoplasm filled with ribosomes and mitochondria; D, Golgi apparatus; E, RER; F, cytoplasmic process of another germ cell; G, perikaryon of multiciliated sensory papilla; H, process of interstitial cell.

Schistosoma

monsoni

MIRACIDIUM

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cytoplasmic organelles, the neurons and of smooth muscle fibers of other invertetheir neuropiles contain numerous complex brates . The intercellular spaces of the miracarbohydrate granules. Both peripheral nerves and neuropiles are essentially simicidium are largely occupied by cytoplasmic processes of interstitial cells which play a lar in structure, and appear as oval, or elongated structures role equivalent to that of fibroblasts in high- sausage-shaped, that are fitted together snugly forming cords er animals. The interstitial cells contain abundant complex carbohydrates and lipid or masses of various shapes. Membranedroplets and, thus, may also serve as re- bound, neurosecretory vesicles are abunserve food stores. dant in the nerve endings and neuropiles. An apical and two lateral glandular cells Four types of membrane-bound, neuroseoccupy much of the area anterior to the cretory vesicles are recognized. These are neural mass. Except for the number of nu- classified according to size (700-2000 A> clei (four in apical gland and one in lateral and electron density. The appearance of gland) and the electron density (denser in these membrane-bound vesicles closely reapical gland than in lateral gland) of con- semble those present in the nervous system tained secretory droplets, the basic cellular of vertebrates. Neuromuscular junctions architecture appears to be similar in both are structurally similar to synapses between glands. The glandular cells contain abun- neuropiles. The nerve ending is separated from the muscle by a space of ca. 150 A. dant RER, (Y- and P-glycogen particles, ribosomes, and Golgi apparatus. Most of The opposing membranes are thicker than these cellular components are gradually re- normal and are coated with electron-dense placed by secretory droplets. material. Many synaptic vesicles are presThe excretory system consists of two ent in the nerve endings. There are four pairs of flame cells, their excretory tubules multiciliated, deep-pit nerve endings and at and vesicles, and excretory pores. Each least eight uniciliated nerve endings distribflame cell is composed of a cyton and a uted in the terebratorium. At the base of each of two lateral papillae is a mulhollow cylinder enclosing numerous cilia. The stellate cyton sends many processes ticiliated, shallow-pit nerve ending, a uniinto intercellular spaces as anchoring de- ciliated nerve ending, and a ciliated, sacvices. Cilia in the cylinder originate in the cular sensory organelle. In the epidermal cyton and usually have the “9 + 2” mi- ridge between the second and third epidercrotubule arrangement. Parts of the cylinder ma1 plates are distributed around the wall form a “grille-work” through which miracidium about a dozen multiciliated senfluid may pass more freely than through the sory papillae. All these peripheral sensory rest of the wall. Each excretory tubule is organelles except lateral papillae are diformed from a separate cell whose flattened rectly exposed to the outside and are nerve cytoplasm is rolled up and joined at the terminals that are modified into various edge by septate desmosomes. The excre- structures, presumably to suit their inditory vesicle is formed from a single cell in vidual functions. The cytoplasm of all the the shape of an elongate sac, and joins the sensory organelles contains abundant neuexcretory pore with septate desmosomes. rosecretory vesicles of one or rarely two The nervous system consists of a neural types. The multiciliated sensory papillae mass (CNS), six types of peripheral sensory have conspicuous extra-CNS bipolar neurons. organelles with their connecting nerves, and nerves for muscle fibers. The neurons Germinal cells number about 20 and are in the neural ring are positioned peripherlocated in the posterior third of the ally, encircling neuropiles which occupy miracidium. They are situated among the much of the CNS mass. Besides various interstitial cell meshes and contain large

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conspicuous nucleoli. The scarce cytoplasm is literally filled with ribosomes, RER, and small mitochondria, but contains few organelles of other types. ACKNOWLEDGMENTS These studies were supported in part by Research Grant AI-00513 and Training Grant AI46 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service. The author is especially grateful to Mary Anne Chillingworth, Mrs. Lucija Kaulins, and Mr. Alfred Pan for their technical assistance. We are very grateful to the Irene Heinz Given and John LaPorte Given Foundation for donating the Philips EM-300 electron microscope used in this study. Credits and thanks are also given to Dr. Susumu Ito of Harvard Medical School for providing the scanning electron micrographs in Figures 4 and 5. Finally, I am very grateful to the William F. Milton Fund of Harvard University for a grant providing additional support for this work.

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