Epidermal ultrastructure of Anoplodium stichopi (Plathelminthes, Dalyellioida), a flatworm endosymbiotic in Stichopus tremulus (Holothurioida)

Zoomorphology

Zoomorphology (1986) 106: 254-259

© Springer-Verlag1986

Epidermal ultrastructure of Anoplodium stichopi (Plathelminthes, Dalyellioida), a flatworm endosymbiotic in Stichopus tremulus (Holothurioida) Ulf Jondelius Department of Zoology, University of Göteborg, Box 25059, S-40031 Göteborg, Sweden

Summary. The epidermal ultrastructure of Anoplodium stichopi Bock 1925 (Platyhelminthes, Dalyellioida, Umagillidae) was studied using transmission and scanning electron microscopy. The species lives in the perivisceral coelom of the aspidochirote holothurian Stichopus tremulus Gunnerus 1767. Two types of cells were observed in the epidermis of A. stichopi: ciliated cuboidal epithelial cells and nonciliated pear-shaped cells. The surface of the ciliated epidermal cells is folded into anastomosing ridges. Numerous coated vesicles are subjacent to the surface folds and mitochondria are abundant just below them. Observations indicate that A. stichopi takes up nutrients pinocytically from the coelomic fluid of the host. The ciliation of A. stichopi is sparse.

A. Introduction

In a phylogenetic system of the Platyhelminthes put forward by Ehlers (1984, 1985) the subordinate taxon Dalyellioida is the adelphotaxon of the Neodermata which consists of the exclusively parasitic taxa Trematoda, Monogenea and Cestoda. The Dalyellioida contains both freeliving and endosymbiotic species. The proposed phylogenetic position of the Dalyellioida, close to the exclusively parasitic groups of the Platyhelminthes, make the relationships of endosymbiotic Dalyellioida to their hosts interesting to study. The nature of symbioses in the Dalyellioida might shed light on the evolution of parasitism within the Platyhelminthes. The family Umagillidae of the Dalyellioida contains the largest number of endosymbiotic flatworm species outside the Neodermata. The approximately 50 species of the Umagillidae are all endosymbionts of echinoderms or sipunculans (Cannon 1982). The majority of the Umagillidae are reported to live wholly or mainly in the intestine of the host, but eight species of the genus Anoplodium are reported only from the perivisceral coelom of holothurians (Shinn 1985; and references therein). The purpose of this study is to search the epidermis of Anoplodium stichopi Bock 1925, by transmission and scanning electron microscopy for ultrastructural adaptations to parasitism such as absorption of nutrients through the epidermis. A. stichopi is commonly found in the perivisceral coelom of the aspidochirote holothurian Stichopus tremulus Gunnerus 1767.

B. Materials and methods

Specimens of Stichopus tremulus were collected by dredging at a depth of about 40 m in the Koster area of the Swedish west coast on several occasions between October 1984 and December 1985. They were kept in running seawater until dissection. Collection of holothurian coelomic fluid was through a small dorsolateral incision, thus avoiding damage to the intestine. Coelomic fluid was pipetted into dishes. The dorsal side was cut open with scissors. The intestine and respiratory tree were removed in one piece, placed in a separate dish and examined under a dissecting microscope, as was the inside of the coelomic cavity. Flatworms were found only in the coelomic cavity of the holothurians. Bright field and phase-contrast microscope observations were made of living specimens kept in coelomic fluid on slides with or without coverglasses. All flatworms examined belonged to the species Anoplodium stichopi. The species was identified with the aid of Bock's (1925) description of A. stichopi and by comparison with Westblad's descriptions (1926, 1930) of other umagillid endosymbionts of S. tremulus.

Transmission electron microscopy. Specimens for transmission electron microscopy were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate buffer with 10% sucrose at pH 7.3. Fixation was at 3° C for at least 48 h. After rinsing in sodium cacodylate buffer the worms were postfixed in 1% osmium tetroxide at 3°C for 1 h, dehydrated in an acetone series and embedded in Epon. An LKB Ultrotome III was used for sectioning. Thin sections, 1-2 tam thick, were stained with toluidine blue and examined with a light microscope for orientation. Ultrathin sections, with gold-silver interference colours (40-50 nm thick), were collected on 200 or 300 mesh copper grids with or without Formvar coating, or on Formvar-coated copper hole-grids. They were stained with uranyl acetate for 90 s, followed by lead citrate for 4 min, and examined in a Zeiss EM 109 electron microscope operated at 50 kV. Sections from 11 specimens were studied. Seanning electron microscopy. Specimens for scanning electron microscopy were rinsed in filtered seawater (mesh 0.45 lam) before fixation. Fixation was either in 1% osmium tetroxide in filtered seawater or in 3% glutaraldehyde in

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Fig. 1. Scanning electron micrograph of the dorsal surface of a whole specimen of Anoplodium stichopi. The anterior end of the animal points upward. The specimen is slightly contracted. Primary fixation in osmium tetroxide Fig. 2. Scanning electron micrograph of the epidermal surface of Anoplodium stichopi. The cilia (e) are few. The surface of the ciliated epidermal cells is folded into anastomosing ridges (f). Primary fixation in osmium tetroxide Fig. 3. Scanning electron micrograph of the epidermal surface folds (f) between the cilia (c) of Anoplodium stichopi. Primary fixation in osmium tetroxide

0.2 M sodium cacodylate buffer with 10% sucrose at p H 7.3 or in Karnovsky's fixative in sodium cacodylate buffer with a final concentration o f 10% sucrose. Primary fixation was at 3 ° C for at least 14 h. Specimens fixed in osmium tetroxide were rinsed in filtered seawater and transferred to 70% ethanol. The specimens fixed in glutaraldehyde or Karnovsky's fixative were rinsed in sodium cacodylate buffer and transferred to 70% ethanol, except for some of the glutaraldehyde fixed worms which were first postfixed in 1% osmium tetroxide at 3 ° C for 4 h. The 21 specimens were dehydrated in an ethanol series, critical point dried in a Balzers Union F L 9496 critical point drier, mounted on brass stubs and coated with gold/palladium in a Polaron SEM coating unit E 5000. The worms were examined in a Bausch and L o m b Nanolab 2000 scanning electron microscope operated at 20 kV. C. Results

A. stichopi were found in 17 of the 20 specimens of S. tremulus. The worms were moving slowly amongst the gonadal tubules or the longitudinal muscle bands of the host. The specimens were 1.0-1.4 m m long and 0.3-0.4 m m wide. As

mentioned, no other umagillid was found in the body cavity or the intestine of the host specimens. A. stichopi has a convex dorsal surface and a slightly concave ventral surface. The anterior end is narrower than the broadly rounded posterior end (Fig. 1). Scanning electron microscopy showed that all of the body is sparsely ciliated; there are only about 0.2 cilia per ttm 2. Between the cilia the surface o f the epidermal cells is covered with numerous anastomosing folds, creating an irregular, somewhat honeycomb-like pattern (Figs. 2 and 3). The surface folds were present in specimens fixed in each of the three fixatives. The epidermal cells of the convex dorsal part of the worm and those o f the concave ventral part are similar. Two types of cells were observed in the epidermis.

a) Ciliated cuboidal epidermal cells The surface of the ciliated epidermal cells is enlarged by irregular folds about 1.5 ~tm high (Figs. 4, 5). Numerous vesicles with a diameter of 0.14).3 ~tm were observed in the folds and in the subjacent 0.5 lain thick surface layer of the ciliated epidermal cells (Fig. 5). The membrane o f the vesicles possesses a glycocalyx on the extracellular sur-

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Fig. 4. Transmission electron micrograph of ciliated epidermal cells of Anoplodium stichopi in cross section. The cell surface is enlarged by folds ( f ) . Cilia (c) are sparse. Mitochondria (m) are a b u n d a n t just below the cell surface. The nucleus (n) with nucleolus (no) of a ciliated epidermal cell can be seen. The lateral cell membranes (cm) of ciliated epidermal cells adjacent to each other are interdigitated and continuous from the basal lamina (b/) to the cell surface. In the basal portion of the cells several cytoplasmic inclusions (i) can be seen Fig. 5. Transmission electron micrograph of the apical part of a ciliated epidermal cell of Anoplodium stichopi in cross section. Cilia (c) are sparse. Numerous vesicles (v) are present in the surface folds ( f ) . Mitochondria (m) are abundant in a zone just below the folds Fig. 6. Transmission electron micrograph of the apical part of a ciliated epidermal cell of Anoplodium stichopi in tangential section. Some of the vesicles (v) present in the surface folds ( f ) are connected with the outside, forming pits (p) in the cell surface. Mitochondria (m) are present just below the vesicles

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Fig. 7. Transmission electron micrograph of pits (p) in the surface folds of a ciliated epidermal cell of Anoplodium stichopi. Note the thick and electron dense membrane of the pits with bristles (br) on the cytoplasmic side

Fig. 8. Transmission electron micrograph of pear-shaped epidermal cell in longitudinal section of Anoplodium stichopi. Rough endoplasmic reticulum (re) is prominent in the basal part. Mitochondria (m) occur perinuclearly. Cytoplasmic inclusions (i) of different electron densities can be seen in the mid regions. The lateral cell membranes (cm) are continuous from the cell surface to the basal lamina (b/). The nucleus (n) is about 4 l~m by 3 I~m. In adjacent ciliated epidermal cells ciliary rootlets (cr) can be seen

258 face. There are projecting bristles on the cytoplasmic surface of the vesicles. Thus the vesicle membrane is thicker and more electron-dense than that of surrounding areas of the cell (Figs. 6, 7). The sparse cilia are about 8-10 lam long. Each has two striated rootlets, one almost perpendicular to the cell surface and the other almost parallel to it. The nucleus of a ciliated epidermal cell is rounded, about 6 I~m by 4 lam and has a spherical nucleolus of about 1.5 pm diameter. Mitochondria are abundant in an approximately 3 ~tm deep zone just below the apical folds and vesicles. Mitochondria were also observed close to the lateral cell membranes of ciliated epidermal cells adjacent to each other. The mitochondria are peanut-shaped, about 3 ~tm long and 1 pm wide. Basally in the ciliated epidermal cells there are numerous spherical inclusions of higher electron density than the surrounding cytoplasm (Fig. 4). The lateral cell membranes are continuous from the basal lamina to the cell surface. They are highly convoluted and have desmosomes. The basal cell membranes of the ciliated epidermal cells are not convoluted. Subjacent to the basally situated cell membrane is a 60-80 nm thick basal lamina consisting of electron-dense extracellular material.

b) Non ciliated pear-shaped epidermal cells On every cross section of a worm a few pear-shaped cells can be seen between the ciliated epidermal cells (Fig. 8). These pear-shaped cells are about 6-8 pm wide basally and 2-2.5 Ixm wide apically. The rounded nucleus is situated basally and measures about 4 Ixm by 3 txm in longitudinal section. The nucleolus is circular and has a diameter of about 1 Ixm. Neither surface-enlarging folds nor cilia were observed in these cells. Golgi complexes and rough endoplasmic reticulum are prominent features in the basal region. The lateral cell membranes are not as convoluted as those of ciliated epidermal cells adjacent to each other. D. Discussion

The enlargement of the surface of the ciliated epidermal cells of A. stichopi is striking. The numerous vesicles and pits below the surface folds are similar to the coated vesicles a¢tive in adsorptive micropinocytosis. As described by e.g. Fawcett (1981) absorptive micropino¢ytosis is a selective uptake of protein in specialized areas characterized by a thickening of the ¢ell membrane which has projecting bristles on the cytoplasmic side. The coated vesicles are formed from invaginations of this specialized membrane. The extensive enlargement of the epidermal surfa¢e and the presence of coated vesicles below the surface folds in A. stichopi suggest that this species absorbs nutrients from the coelomi¢' fluid of the host. Thus proteins, known to be present in echinoderm coelomic fluid (Endean 1966), might be taken up by the epidermis of this flatworm. The abundance of Golgi complexes and rough endoplasmic reticulum suggests that the pear-shaped epidermal ¢ells have a secretory function. This does not, of course, exclude other functions, e.g. a sensory one. Two species of the family Graffillidae of the Dalyellioida, Paravortex cardi Hallez 1909 and P. karlingi Pike and Burt 1981 are both endosymbionts of the ¢o¢kle Cardium edule Linnaeus 1758. Ma¢Kinnon et al. (1981) suggested that interdigitations of adjacent cells in the epidermis of these species is asso¢iated with

metabolic interaction and transfer of nutrients between the ceUs. Mitochondria in the interdigitations of the epidermal ceUs of A. stichopi seems to suggest a similar process. Since interdigitation of the lateral cell membranes of epidermal cells also occurs in free-living flatworms (Hendelberg and Hedlung 1974) it does not necessarily indicate transfer of nutrients through the epidermis. A cell web that gives mechanical support (Rieger 1981a) is absent in A. stichopi. Therefore the interdigitations of the lateral cell membranes could also be an adaptation for increased mechanical strength. The epidermal surface enlargement in A. stichopi greatly exceeds that of two intestine inhabiting species of the Umagillidae (Lyons 1973; Holt and Mettrick 1975). It is, however, comparable to that of Kronborgia amphipodicola Christensen and Kanneworff 1964 of the family Fecampidae of the Dalyellioida. K. amphipodicola is parasite in the body cavity of amphipods. Since it lacks a pharynx and intestine, it is thought to absorb nutrients through the epidermis, which possesses numerous branched microvilli (Bresciani and Koie 1970). It is tempting to suggest that the surface enlargement present in A. stichopi and K. amphipodicola is an adaptation to parasitism in the body cavity of the host, through which nutrients could easily be taken up from the perivisceral fluid. A. stichopi has both pharynx and intestine; it seems reasonable to expect that some food is ingested this way. Host coelomocytes sometimes seen adhering to the surface of A. stichopi might be ingested through the pharynx. Possible pharyngeal food uptake will be the subject of a separate study. There are only about 0.2 cilia per pm 2 in the epidermis of A. stichopi. Cilia in free-living flatworms occur at a density of 3-6 per ~tm2 (Rieger 1981b). Reduction of the number of cilia in this species may be an adaptation to endosymbiotic life in the perivisceral coelom of the host. There seems to be little need for rapid ciliary locomotion for an endosymbiont of the body cavity. Living specimens of A. stichopi moved slowly on the inside of the body wall or among the gonadal tubules of the host; they were never seen swimming. Sparse ciliation may also facilitate epidermal absorption of nutrients since the surface available for pinocytosis increases when cilia are lost.

Acknowledgements. The scanning electron microscope studies were performed at the department of Zoology, Lund University. Dr. Jan Hendelberg critically read the manuscript and gave valuable comments. The work was supported by the Swedish Natural Sciences Research Council grant no B-Bu 2819-105 to Jan Hendelberg.

References

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