A Self-Cleaning Mechanism in the Operculum of <I>Serpula Vermicularis</I> L. (Polychaeta: Serpulidae)

BULLETIN OF MARINE SCIENCE, 48(2): 412-419, 1991

A SELF-CLEANING MECHANISM IN THE OPERCULUM OF SERPULA VERMICULARIS L. (pOL YCHAET A: SERPULIDAE)

Clifford H. Thorp, Fran M. Sewell and Peter R. Bond

ABSTRACT Mucus-secreting cells are described in the opercular plate epithelium of Serpula vermicularis. At the light-microscope level the cells are characterized by large granular inclusions identified at the electron-microscope level as electron-dense vesicles. Histochemical analysis of the mucous cells gives positive reactions for both neutral glycoproteins and non-sulfated acid mucopolysaccharides. The presence of mucous cells in the opercular plate of S. vermicularis is assumed to reflect the homology of the opercular filament with the branchial filaments from which they evolved. It is suggested that mucus is secreted across the substantial opercular plate cuticle to form a thin investing layer. The significance of a mucous layer on the opercular plate is discussed and both mechanical and antibiotic functions are implicated in a self-cleaning (antifouling) role. It is further suggested that "advanced" serpulids have abandoned mucus secretion as a protective mechanism in favor of increasing sclerotization/ keratinization and eventual calcification. It is suggested that opercular mucous cells could be used as an additional tool in resolving serpulid phylogeny.

Serpulids are sedentary marine polychaetes which secrete a calcareous tube. The prostomium carries, as its only appendages, two hemicirclets of pinnulate, ciliated branchial filaments which serve as respiratory, feeding and sensory organs (Hanson, 1949). One or more modified filaments form an occlusive structure, the operculum (Zeleny, 1905; Segrove, 1941; Thorp and Segrove, 1975). The opercular filament comprises a distal operculum borne on a peduncle (Zeleny, 1905; Zibrowius, 1968). The distal surface of the operculum is prone to the accumulation of "fouling," both living (bacteria, microalgae, protozoans, ro- tifers etc.) and non-living (McIntosh, 1923; 1926; Thorp and Segrove, 1975). Observations of Serpula vermicularis L. revealed that the opercula of this species remained free from fouling. In addition, the functional operculum in the live animal is very difficult to handle and it seems likely that this is due to a mucous layer on its surface. The structure of the opercular plate of S. vermicularis has been investigated to determine what mechanism is operating to prevent fouling. This paper reports preliminary observations of the structure of the opercular filament, particularly of mucus-secreting cells, and discusses the possible significance of these cells in the phylogeny of serpulids.

MATERIALS AND METHODS

Apart from a few specimens from Millbay Docks, Plymouth, England, Serpula vermicularis were collected from Killary Harbour, Ireland. For light microscopy and histochemical studies whole specimens or excised opercula were fixed in Bouin, seawater Bouin or 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 with 3% NaCI. Serial longitudinal sections were prepared at 6, 8 and 10 /tm and stained with Ehrlich's hematoxylin and eosin or Masson's trichrome stain (Drury and Wallington, 1967). To identify the granules in the mucus-secreting cells the histochemical methods employed included: periodic acid Schiff(PAS); alcian blue (AB) pH 2.5 and 1.0; and combined AB/PAS. For transmission electron microscopy opercula were cut longitudinally into small pieces, fixed in 3% gluteraldehyde in 0.1 M phosphate buffer at pH 7.2 with 3% NaCl and post fixed in 0.1 % osmium tetroxide in 0.1 M phosphate buffer (pH 7.2). After dehydration in alcohol and embedding in Spurr's resin, ultrathin sections were cut on an LKB III Ultrotome using glass knives. Sections were stained in uranyl acetate and lead citrate and observed in a Philips EM 300 electron microscope.

412 THORP ET AL.: OPERCULAR MUCOUS CELLS IN SERPULA 413

Figure I. S. vermicularis. Longitudinal section of the plate region ofthe opercular filament: B. blood capillary; C. cuticle; CT. connective tissue; E. epithelial layer. Scale bar 0.25 mm.

REsULTS Serpula vermicularis possesses two opercular filaments, one large and functional and the other normally small and rudimentary. The rim of the opercular plate is characteristically crenulated; the crenulations are normally rounded or bluntly pointed. The upper and lower surfaces are radially grooved, the grooves originating in the troughs of the marginal crenulations. Histology. - The opercular filament comprises a single layer of epithelial cells enclosing a loose connective tissue core and invested by a substantial cuticle (Fig. 1). The core also contains muscle fibers, which terminate at the operculo-peduncle junction, three opercular nerves and a single blood vessel which, after repeated branching in the opercular connective tissue, terminates in swollen peripheral smuses. The opercular epithelial cells are cuboidal (30-50 ~m), vacuolated, with elon- gated apical nuclei. In 18 (53%) out of 34 animals sectioned an additional cell type has been observed in the opercular epithelium. These cells are apical in the epithelial layer, oval in shape and their rounded, granular contents stain deeply with the Light Green component of Masson's. These cells are regularly distributed and largely restricted to the opercular plate epithelium, although occasional cells occur just below the opercular rim on the lower surface of the plate. While their frequency, size and intensity of staining reactions were variable, the variations could neither be correlated with size (age) and coloring of the animal nor to the time of year or source of material. At the electron-microscope level the granular cell contents are observed as aggregations of electron-dense vesicles. In the majority of cells the vesicles exhibit extremely electron-dense contents (Fig. 2a) but other cells were observed where the contents were of medium to low electron density and the vesicles appreciably 414 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

Figure 2A. S. vermicularis. Electron micrograph of opercular plate goblet cells with electron-dense vesicles: ac. apical cytoplasm; ev. electron-dense vesicles. Scale bar 1.0 Itm. 2B. S. vermicularis. Electron micrograph of opercular plate goblet cell with medium electron-dense vesicles. Scale bar 0.5 Itm. 2C. S. vermicularis. Electron micrograph of opercular plate goblet cell with low electron-dense vesicles. Scale bar 1.0 Itm. 2D. S. vermicularis. Electron micrograph of opercular plate goblet cell with small vesicles of1ow electron density. Scale bar 0.25 Itm. smaller (Fig. 2b, c, and d). The granular cells exhibit apical microvilli which, like those of normal plate cells, penetrate the complex cuticle to emerge at the surface as epicuticular projections. The origin of the vesicles has not been determined. Few prominent Golgi complexes have been observed but, when present, are fre- quently associated with multivesicular bodies. From the limited material exam- ined there is no evidence of a secretory route for the vesicle contents. Histochemistry. -A series of histochemical tests was performed to identify the contents of the granular cells. The granules gave a positive reaction with PAS both before and after diastase digestion and were positive with alcian blue (AB) at pH 2.5 but negative at pH 1.0. These results suggested that the cOlitents are acidic but not sulfated since, at pH 1.0, carboxyl groups are not ionized and do THORP ET AL.: OPERCULAR MUCOUS CELLS IN SERPULA 415 not stain, whereas sulfate groups are demonstrated. At pH 2.5 carboxyl groups stain well. As substances with the above reactions could be sialomucins (Culling, 1974), neuraminidase digestion followed by AB/PAS staining (Pearse, 1972) was tried with inconclusive results. AB/PAS staining demonstrated both neutral (red) and acid (blue) staining granules. It was also noted that, in opercular sections stained with PAS and AB both alone and combined, the outer layer ofthe cuticle stained more deeply. With AB/PAS combined the blue (acid) concentrated in the outer (?epicuticular) layer.

DISCUSSION At the light-microscope level the granular cells in the operculum are similar to mucus-secreting cells in the branchial filaments, collar and some of the mucus- secreting cells of the thoracic ventral shield, hence they too are probably mucus- secreting. Preliminary electron-microscope studies support this assumption in that the electron-dense vesicles within the mucus-secreting cells are similar to those in other serpulids (Bubel, 1973; 1983b) and oligochaetes (Richards, 1977). Wheth- er the lower electron density of the vesicle contents and the different sized vesicles observed in some cells indicates more than one type of mucous cell or different stages in the formation/secretory process has not been resolved. The origin of the vesicles and their contents remains unclear. The small number of observations ofGolgi complexes associated with multivesicular bodies suggests that the vesicles may be Golgi-derived, as might be expected from the studies of Richards (1974; 1975; 1977) in oligochaetes and Bubel (1973; 1983b) in Spirorbis spirorbis (as S. borealis) and Pomatoceros lamarckii respectively. Culling (1974) observed that combined AB/PAS staining demonstrated mucin and the degree to which it was composed of neutral (red) and acid (blue) components. The presence of both red and blue granules in the opercular mucous cells of S. vermicularis suggests that they contain both a mucin (neutral glycoprotein), which stains positively with PAS both before and after diastase digestion, and a non-sulfated acid mucopolysaccharide. The presence of acid mucopolysaccharides would also be expected from the ultrastructural studies, as the appearance of the electron-dense vesicles closely resembles that of acid mucus-secreting cells in oligochaetes (Richards, 1977). The observation of mucous cells in the opercular filament of S. vermicularis contrasts markedly with their absence from the opercula of other serpulids and spirorbids studied (Bubel, 1973; 1983a; 1983b; Bubel and Thorp, 1985; Bubel et aI., 1977; Bubel et aI., 1985; Fitzsimons, 1981; Thorp, 1975; Thorp, unpubI.). The observation of opercular mucous cells might be considered unusual, but. a consideration of the probable evolution of the opercular filament suggests that the absence of opercular mucous cells from other species studied is the more unusual. It is widely held that the opercular filament has been derived by branchial filament modification (Zeleny, 1905; Thomas, 1940; Hanson, 1949; Thorp and Segrove, 1975) and that branchial filaments possess mucous cells to facilitate feeding (Thomas, 1940; Jargensen, 1966). Elaboration of the opercular filament has involved the gradual loss of branchial pinnules and it is reasonable to assume that mucous cells also disappeared gradually as the opercular filament lost its respiratory function and assumed a purely occlusive role. Mucous cells in the opercular filament, therefore, could represent the retention ofa branchial filament 416 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991

feature. As acid mucins have been ascribed a role in respiratory as well as anti- dehydration functions in oligochaetes (Richards, 1977), the presence of acid mucus components within the mucous cells of S. vermicularis could be taken as sup- porting their branchial filament ancestry. It is suggested that mucus is secreted across the substantial opercular plate cuticle to form a thin layer on the outer surface. It was not possible to determine whether mucus is secreted by way of discrete pores, as in the oligochaetes Lum- bricil/us georgiensis (L.) and L. mirabilis (L.) (Richards, 1977) or the thoracic mucus-secreting cells in the serpulid Pomatoceros lamarckii (Bubel, 1983b). In the absence of evidence of distinct secretory channels through the cuticle it is unclear how the mucus reaches the outer surface. Goffinet and Compere (1986), however, reported the retraction of microvilli from the cuticle of the shore crab, Carcinus maenas (L.), during molting, to facilitate both resorption and secretory processes. Microvillus withdrawal has also been observed in serpulids prior to cuticle calcification (Bubel, 1983a; 1983b; Bubel et a1., 1980; 1985). It remains possible, therefore, that mucus could be secreted through microvillar channels after temporary withdrawal of the microvilli. Jergensen (1966) considered that mucus secretion on the branchial filaments of serpulids fulfilled a cleaning role and was not directly involved in feeding. Nicol (1960) and Simkiss and Wilbur (1977), however, supported the view that mucous secretions playa major part in ciliary feeding, cleaning and transport systems of tubicolous polychaetes and molluscs respectively. If a mucous layer on the operculum of S. vermicularis functions in a similar manner to that on the branchial filaments it will both trap settling material and be sloughed off. Jergensen (1966) also suggested that mucus secretion was a continuous process, whereas the present observation that mucous cells are not always observed in the operculum of S. vermicularis suggests the existence of an "all-or-none" mechanism which is both phased and synchronized. Jakowska (1963), Fletcher (1978; 1982) and Ingram (1980) ascribed both an- tiseptic and antibiotic roles to fish mucus and Pickering (1976) reported increased mucus production in infected fish. It is possible that mucus in serpulids has similar additional functions, and circumstantial evidence from opercular regeneration experiments with S. vermicularis supports this possibility. In normal circum- stances there is little visible evidence of mucus production by the opercular filament. In one series of experiments, however, a succession of mucous caps de- veloped on both regenerating and mature opercula (Thorp and Stapleton, unpub1.). These animals were maintained in Petri dishes in a constant temperature facility shared with bacteria cultures and it is possible that production of opercular mucous caps was a protective reaction to accidental cross-contamination by bacteria from adjacent Petri dishes. If mucus production is of value in a cleaning/protective role, why and how has it been abandoned in other (more advanced) serpulids? In Serpula the substantial opercular cuticle is unspecialized whereas, in Hydroides and Ficopomatus, the opercular plate cuticle is reinforced and waterproofed by sclerotization (Zibrqwius, 1978; Thorp et a1., 1987; Thorp, unpub1.) and in Pomatoceros the operculum is further strengthened through calcification (Bubel, 1983a; 1983b; Bubel et a1., 1980; 1985). Such reinforcement confers protection from microbial attack but, at the same time, results in the opercular plate becoming prone to fouling. One must assume, therefore, that loss of an apparent antifouling, and possibly antibiotic, mechanism in more advanced serpulids is outweighed by the advantage of a more substantial protective covering. It is interesting to note a similar trend from mucus THORP ET AL.: OPERCULAR MUCOUS CELLS IN SERPULA 417 secretion to sclerotization (keratinization) and calcification in fish epidermis (Mit- tal and Bannerjee, 1980). That the operculum can increase in diameter to accom- modate the increasing tube diameter without being replaced (Thorp and Segrove, 1975) suggests that a fouled operculum is not a disadvantage. Finally, one must consider the significance of opercular mucous cells in relation to serpulid phylogeny. Zeleny (1905) suggested a possible phylogeny through an opercular transformation series. He used as his starting point the genera Protula and Protis with no apparent modification to any ofthe branchial filaments. From Salmacina dysteri, where each branchial filament has a terminal enlargement which collectively serve as a tube plug, Zeleny traced a path of increasing opercular filament elaboration. Zeleny's scheme encompassltd the progressive loss of branchial pinnules, elaboration ofthe operculum, restriction of opercular development to a particular filament and loss of the ability to develop opercula on other filaments when the functional one is damaged or destroyed. The most recent attempt at rationalizing serpulid phylogeny is that often Hove (1984) whose hypothetical transformation series of serpulid branchial crowns is not too far removed from Zeleny's earlier ideas. In ten Hove's scheme the evolutionary line Protula-Salmacina-Apomatus is largely retained. Filograna, however, is placed as an offshoot from the main line because its two opercula develop from the most dorsal radiole on each side in contrast to the second dorsal in other bi-operculate species. The next evolutionary step according to ten Hove (1984) could have been distal reinforcement of the operculum by "horny" (?sclerotized) and calcareous structures. Josephella, therefore, is separated from Apomatus (Ze- leny's Group 4) by ten Hove (1984) on the basis of the horny operculum in Josephella. Ifnon-reinforcement is the primitive (plesiomorphous) character-state and reinforcement the more advanced (apomorphous) character-state, it is interesting to note the horny opercula in Filograna (ten Hove, 1984). The presence of an apomorphic character in what could be considered to be a primitive serpulid supports the action often Hove in placing Filograna as an offshoot from the main evolutionary line. Filograna may well be a relic of a line of evolutionary development where the dorsal radiole on each side was selected for opercular development. That all other serpulids with restricted opercular development have their opercula in place of the second dorsal radiole implies that this arrangement confers some evolutionary advantage. If non-reinforcement ofthe operculum is the plesiomorphic character-state the position of Serpula in ten Hove's scheme deserves discussion. Placing Serpula, with a thick, but otherwise non-reinforced opercular plate cuticle, after Josephella and Vermiliopsis, both of which have horny opercula (ten Hove, 1984), suggests that Serpula could have undergone an evolutionary regression by re-adopting the plesiomorphic character-state. Similarly, the presence of mucous cells (plesiomorphic) in the operculum of Serpula, if they are absent from Josephella and/or Vermiliopsis, would also suggest regression. The retention of pinnules on the peduncle in Josephella, the plesiomorphic character-state, however, supports the positioning of this genus before Serpula. Until histologicaVhistochemical studies have resolved whether so-called "horny" structures are reinforced (sclerotized) or not and the presence/absence of mucus-secreting cells investigated in the relevant species, however, the possibility of regression must remain conjectural. Ventral thoracic blood vessel patterns were used by ten Hove and Pantus (1985) to resolve the taxonomic problem of the validity of Apomatus and Protula as distinct taxa. It was further emphasized by ten Hove (1984) that the arrangement of his transformation series was still unresolved. He further suggested that new, 418 BULLETIN OF MARINE SCIENCE. VOL. 48. NO.2. 1991 and perhaps better, characters remained to be discovered to facilitate the reso- lution of serpulid taxonomy. We suggest that the presence or absence of mucous cells could be such a new character.

ACKNOWLEDGMENTS

We wish to thank H. ten Hove for valuable criticism of the phylogenetic aspects of the paper. We are also indebted to B. O'Connor, Y. Leahy, J. Brodie (University College, Galway) and W. Farnham (Portsmouth Polytechnic), for the collection and transport oflive specimens of S. vermicularis; without their help this study would not have been possible. We would also like to record the participation of undergraduates, K. Stapleton, W. Hodgson and M. Horrobin in aspects of this study. We thank also J. Hepburn and C. Derrick for photographic assistance.

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DATEACCEPTED: March 19, 1990.

ADDRESSES: (C.H. T.) School o/Biological Sciences, Portsmouth Polytechnic, Marine Laboratory, Ferry Road, Hayling Island, Hampshire, PO]] ODG, United Kingdon; (F.M.S. and P.R.B.) School oj Bio- logical Sciences. Portsmouth Polytechnic, King Henry Building, King Henry] Street, Portsmouth, POI 2DY, United Kingdom.