Spicule Formation in the Solitary Ascidian Bathypera feminalba (Ascidiacea, Pyuridae)

Spicule Formation in the Solitary Ascidian Bathypera feminalba (Ascidiacea, Pyuridae) Author(s): Gretchen Lambert Source: Invertebrate Biology, Vol. 117, No. 4 (Autumn, 1998), pp. 341-349 Published by: Wiley on behalf of American Microscopical Society Stable URL: http://www.jstor.org/stable/3227036 . Accessed: 18/06/2014 14:00 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Wiley and American Microscopical Society are collaborating with JSTOR to digitize, preserve and extend access to Invertebrate Biology. http://www.jstor.org This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/action/showPublisher?publisherCode=black http://www.jstor.org/action/showPublisher?publisherCode=amicros http://www.jstor.org/stable/3227036?origin=JSTOR-pdf http://www.jstor.org/page/info/about/policies/terms.jsp http://www.jstor.org/page/info/about/policies/terms.jsp Invertebrate Biology 117(4): 341-349. ? 1998 American Microscopical Society, Inc. Spicule formation in the solitary ascidian Bathypera feminalba (Ascidiacea, Pyuridae) Gretchen Lamberta Department of Biological Science, California State University Fullerton, Fullerton, CA 92834-6850 and University of Washington Friday Harbor Laboratories, Friday Harbor, WA Abstract. Bathypera feminalba is a solitary deep-water ascidian inhabiting rocky areas of the NE Pacific. The low mound-shaped body is sheathed by a thin fibrous tunic densely studded with calcitic spicules -150 pLm in length. Under the tunic, the ascidian body is covered by an epithelium bound to the inner edge of the tunic. New tunic and spicules form extracellularly in the epithelium inside the incurrent and excurrent siphons and around the periphery of the animal where it is attached to the substratum. Each spicule has a round flattened base, a narrow collared neck or sulcus that anchors it in the tunic, and a ring of distal spines. The central spine is the longest and usually directed outward from the central axis. A thin acellular organic envelope closely covers each spicule. A dense fibrous layer forms between the envelope and epithelium. Additional organic material remains in thin sections of demineralized spicules. The spicule base forms first; then, as the epithelial cells produce additional tunic, the spicules are pushed out of the epithelium into the ever-thickening tunic. Beyond this stage, no cells can be found in the tunic near the spicule base, and probably no further mineralization of the base occurs. As the spicule base is forming, secondary highly pseudopodial sclerocytes aggregate in the tunic overlying the distal part of the spicules. These sclerocytes are apparently responsible for production of the spines, which are comprised of numerous overlapping "shingles" of calcite. Mineralization of the spines probably continues throughout the life of the animal, be- cause secondary sclerocytes remain numerous in the distal regions of all spicules. Additional key words: biomineralization, calcite, tunicate Mineralized spicules are present in a number of as- cidian species, either in the tunic or in both tunic and body tissues depending on the species (Millar & Good- body 1974; Lowenstam & Abbott 1975; Lowenstam 1989; Lambert et al. 1990 for review). Spicule com- position and morphology are species-specific and thus are undoubtedly under cellular control, as are larval echinoderm spicules and probably all such mineralized structures (Lowenstam & Weiner 1989; Simkiss & Wilbur 1989). They always form in close association with special cells. In the ascidians studied so far, spic- ules initially form intracellularly in the colonial didem- nids (Lafargue & Kniprath 1978; Kniprath & Lafargue 1980; Ballan-Dufrancais et al. 1995) and Cystodytes spp. (Daumezon 1909; Lambert, unpubl. data) with subsequent extracellular growth. In the solitary pyurids Herdmania momus and Pyura pachydermatina, spic- ules form extracellularly inside a cellular envelope aPresent address: 12001 11th Ave. NW, Seattle, WA 98177. E-mail: [email protected] within the blood vessels or blood sinuses (Lambert 1992; Lambert & Lambert 1996b, 1997). Biominer- alization in ascidians exhibits many similarities to mol- luscs (Simkiss & Wilbur 1989), echinoderms (Markel et al. 1986; Lambert & Lambert 1987, 1996a) and even to sponge spicule formation (Jones 1970; Aizen- berg et al. 1996). All have an organic matrix integrally bound with the mineral, and matrix production pre- cedes mineral deposition. Ascidian spicules are usually composed of calcium carbonate in one or more forms, though in some pyurids they consist of a layered mix- ture of two or three mineral types including both cal- cium carbonate and phosphate (Lowenstam 1989; Lambert et al. 1990 for review). Surprisingly, the ami- no acid composition of the soluble matrix in the amor- phous calcium carbonate (ACC) body spicules of P. pachydermatina is very similar to that of the spicular ACC layer in the sponge Clathrina (Aizenberg et al. 1996). Most Bathypera species live at bathyal or abyssal depths and are thus difficult to collect and study (Low- This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp Lambert enstam 1989). An exception is the recently described species Bathypera feminalba YOUNG & VAZQUEZ 1995 from Vancouver Island, British Columbia, Canada. B. feminalba is a small ( Spicule formation in a solitary ascidian Fig. 1. a. Living Bathypera feminalba on rock, collected by Dr. Wm. Austin in Barkley Sound, Vancouver Is., Canada, with the PISCES submersible at 70 m during dive #1284 off Dixon Island, May 8, 1983, station KML 70/83 [KML= Khoyatan Marine Laboratory] and photographed on board ship. Scale bar, 4 mm. b. SEM of a tunic spicule. Base (B), long spine (LS), short lateral spines (SS), sulcus (Su). Scale bar, 20 jLm. c. SEM of a spicule base. Note sulcus with a large collar (Su). Scale bar, 10 Jim. d-e. SEM details of the short spines, made up of "shingles" of overlapping calcite . Scale bars: (d) 10 jLm, (e) 5 jLm. f. SEM of the fractured tip of the long spine, showing concentrically layered pattern of mineral deposition. Scale bar, 1 KLm. 343 This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp Lambert T I 0 -w ,^TP -. , . IA ." W9 ,' ' ># ' I Fig. 2. a-b. Spicule fractured at the level of the sulcus. Arrows indicate location of an abrupt change in the pattern of mineral deposition. Scale bars: (a) 10 ULm, (b) 5 pLm. c-d. Light micrographs of 2 uim sections through the tunic and demineralized spicules; c: contracted tunic, d: relaxed tunic. Toluidine blue staining. Note spicule bases sectioned obliquely (B), epithelium (E), tunic (T), tunic papilla (TP), secondary sclerocytes (arrowheads). Scale bars, 50 uxm. e. Contracted tunic and its cuticle between two spicules. TEM. Note demineralized spicules with remnants of intraspicular organic matrix (S), tunic (T), tunic cuticle (TC). Scale bar, 2 wim. f. Tunic lining a siphon, showing the progressive stages of growth of tunic spicules. DIC illumination. Scale bar, 100 iLm. 344 I This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp Spicule formation in a solitary ascidian in the same discrete areas as new tunic production, namely the epithelium inside the oral and atrial si- phons at the basal edge of the siphonal lining (Fig. 2f). They also originate at the outer tunic periphery at- tached to the substrate. Both new tunic and spicules form extracellularly. The new tunic is extremely thin in these regions. Each spicule forms in an extracellular compartment surrounded by epithelial cells bounded by tight junctions (Figs. 3a-c). Between the epithelium and the mineralizing spicule is a thick, fibrous organic layer (Fig. 3c). Well-developed Golgi, free ribosomes, RER with expanded cisternae filled with a fibrous elec- tron-dense material, and numerous mitochondria all in- dicate that the epithelial cells are highly secretory. As the epithelium produces more tunic material, the tunic becomes thicker. The growing spicules either break out of or are pushed out of the epithelium, presumably by the continuing production of new tunic material behind them. The spicules remain embedded in a very super- ficial layer of tunic just under the cuticle (Fig. 2c). There are no cells near the spicule base after it is no longer surrounded by epithelium (Fig. 2d). Spicule growth continues, however, even after they are no longer covered by epithelium. Numerous pseu- dopodial cells, designated here as secondary sclero- cytes, aggregate in the thin layer of tunic that covers the spinous region of each spicule distal to the neck (Figs. 2c-d, 3d). Though spine production begins while the spicule is still contained within the epithe- lium (Fig. 3b), these secondary sclerocytes are prob- ably responsible for continued addition and elongation of spines. Figure 3e is a highly magnified view of the small mass of acellular organic material in Fig. 3a that re- mains at the center of demineralized spicules. Rem- nants of additional insoluble organic matrix that was bound to the mineral inside the spicule also remain after demineralization in EDTA (Figs. 3b, c, f). Like all other ascidian spicules, those of B. feminalba are covered by an acellular organic sheath or envelope (Figs. 3d, f). Even after demineralization in EDTA, the sheath retains the outline of all the surface details of the spicule, including the microcrystalline "shingles" (Fig. 3f). Discussion Bathypera feminalba is a member of the Pyuridae, and like Herdmania momus and Pyura pachyderma- tina, the other pyurids studied so far, the spicules form extracellularly within an epithelium (Lambert 1992; Lambert & Lambert 1996b, 1997). Unlike the other pyurids, however, B. feminalba produces only tunic spicules, and these do not form within tunic blood ves- sels but in special regions of the mantle epithelium. This is not an unusual difference, however. Tunic blood vessel walls are outgrowths of the epithelium (Burighel & Cloney 1997), but B. feminalba has a thin tunic that lacks obvious blood vessels (unpubl. data; E. Vazquez, pers. comm.). The mantle epithelium also manufactures new tunic (Welsch 1984; Burighel & Cloney 1997); it thus covers and connects the entire body residing inside the tunic with the inner lining of the expanding tunic. New epithelium originates at the base of the siphonal openings at the level of the ten- tacular fold (see Buencuerpo 1988 for a description of the relationship of epithelium and tunic in ascidian si- phons). The entire epithelium may produce some new tunic throughout the animal's life, but most of the new tunic and all of the new spicules form at the inner bases of the siphonal lining and around the periphery of the animal where it attaches to the substratum. Many re- searchers have noted that numerous spicules line the siphonal openings of all bathyperids, where the tunic is inrolled (Michaelsen 1904; Ritter 1907; Kott 1969; Millar & Goodbody 1974; Herdman 1923; Young & Vaizquez 1995), but only Michaelsen (1904) remarked on the rapid diminution in their size at the inner basal edge of the inflected tunic. Young & Vazquez (1995) followed the post-metamorphic growth of young in- dividuals and noted that the first spicules formed around the siphons but did not specify the exact lo- cation. Their Fig. 3E illustrates a region of new spicule production but it is probably the thin flange of new tunic that forms along the outer peripheral border of the animal. The arrow indicating direction of tunic growth can only be true for the tunic periphery, be- cause at the inner base of the siphons the tunic is ex- panding outward or upward, in the direction of the enlarging spicules and toward the siphon apertures. This process is well described by T. Newberry (pers. comm.), who states that "Bathypera is the beast that convinces me that the outermost layer of the stolidob- ranch tunic is extruded from the apertures, like water from fountains." Tight junctions join the epithelial cells surrounding the growing spicules as in other ascidian epithelial cells (Georges 1979; Burighel & Cloney 1997), espe- cially in their apical region, and serve to isolate the mineralizing compartment. All spicules in a given re- gion of tunic appear to be the same size and thus are presumed to be the same age. The base of the spicule forms first (Young & Vaizquez 1995), while it is still surrounded by epithelium. In the light micrograph tak- en by Young & Vaizquez (1995, their Fig. 3G) of very small immature spicules, they point out a dark unmin- eralized center in the middle of each young spicule 345 This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp S ' . . Fig. 3. a. One pLm section of tissue at the inner base of a siphon, showing three young spicules forming in the epithelium. Toluidine blue staining. Scale bar, 10 pLm. b. Young spicule forming in siphonal epithelium. TEM. Basal lamina (BL), epithelium (E), remnants of organic matrix remaining after demineralization (OM), short spines (SS). Scale bar, 5 Bm. c. Two epithelial cells bordering a young spicule. TEM. Basal lamina (BL), nucleus (N), organic layer between epithelium and spicule (OL), demineralized spicule with remnants of organic matrix (S), tight junctions (arrowheads). Scale bar, 1 im. d. Longitudinal thin section through the tip of the long spine covered by its tunic papilla. Note pseudopodia of one or more 346 Lambert This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp Spicule formation in a solitary ascidian base. This is probably the same as the small organic remnant (stained with toluidine blue) visible in the center of demineralized spicules in my Figs. 3a and 3e, which I interpret as the site of initial mineraliza- tion. A similar organic mass may have been present in the spicule shown in my Figs. 2a and 2b before treat- ment with sodium hypochlorite. Note the numerous small empty compartments in Fig. 3e that probably contained crystallites before demineralization. Figure 3G of Young & Vazquez (1995) shows the radial ar- rangement of the crystals in a very young spicule, which correlates with the radial ultrastructure at the center of a fractured spicule in my Figs. 2a and 2b. Their Figs. 3F and 3G show that the organic matrix is first laid down radially before deposition of calcite be- gins, an example of Lowenstam's matrix-mediated biomineralization (for review see Lowenstam & Wei- ner 1989). The small mineralizing compartments in my Fig. 3e appear to have been cut in cross section, thus obscuring the radial arrangement of the crystals. After the initial stage of spicule formation, second- ary sclerocytes aggregate in the thin layer of tunic overlying the distal region of the spicule where the spines form (Figs. 2a-b, 3d). These clusters of sec- ondary sclerocytes are probably what Ritter (1907) saw when he demineralized pieces of Bathypera ovo- ida tunic and hypothesized the cellular origin of the spicules. The origin of these cells is not known, but they are probably one of the many types of blood cells that migrate into the tunic and are responsible for a variety of functions in the tunic (Welsch 1984; Bur- ighel & Cloney 1997). Presumably this second stage of mineralization continues throughout the life of the animal and perhaps is responsible for repair as well as continued elongation of the spines. The spines are composed of many overlapping "shingles" of calcite, similar to lichenoporid bryozoans (Taylor et al. 1995) in which the skeletal walls are composed of tiny flat calcite plates called crystallites that are seeded onto pre-existing crystallites. They define crystallites as "the products of discrete seeding events." As mineral is added to the crystallites they become irregularly lobed as in B. feminalba. It is not clear at this time exactly how the B. feminalba secondary sclerocytes enlarge the spines, but organic and crystalline com- ponents present in the numerous sclerocyte pseudo- podia (not shown) are apparently laid down over the enlarging spines. Further evidence for the genetic basis of spine formation is the fact that Bathypera spp. can be readily identified by the species-specific morphol- ogy and arrangement of spines on the adult spicules (Millar & Goodbody 1974; Lowenstam 1989). As new tunic is added by the epithelial cells, it also accumulates around the spicule bases and pushes them out of the epithelium until they are embedded only in tunic (Figs. 2c, d). The bases appear to be fully formed by the time they are ejected from the epithelium. Pre- sumably no further mineralization of the base takes place, because there are no secondary sclerocytes in this region of the tunic. Labelling studies of living B. feminalba will be needed to verify this, however, using the fluorescent marker calcein (Lambert & Lambert 1996b, 1997). Young and Vaizquez (1995) describe the spicules as forming inside a capsule. In the present study, micro- graphs of sectioned demineralized tissue show that this capsule is actually not a discrete structure but merely a thin layer of tunic overlying the projecting spicules and clearly continuous with the rest of the tunic, as is the cuticle (Figs. 2e, 3d). The tunic is rigid and pro- longed over each spicule to form a papilla (see Herd- man 1923); if a spicule is removed manually with for- ceps, a capsule-like space remains in the tunic. Similarly, dissolution of spicules in situ with a dilute acid leaves spicule-shaped spaces in the tunic. Possible functions of the spicules While their function has not yet been clearly de- fined, they are most likely utilized for structural sup- port (Koehl 1982) and/or defense (Young 1986; Lam- bert & Lambert 1987; Lowenstam 1989). Unlike other pyurids (Lambert et al. 1990; Lambert 1992, Lambert & Lambert 1996b, 1997), B. feminalba and apparently all other bathyperids possess only tunic spicules and lack body spicules (Lowenstam 1989; Young & Vaiz- quez 1995). B. feminalba may not need body spicules, whose function appears to be supportive rather than defensive (Koehl 1982; Lambert & Lambert 1987, 1996b, 1997), even though it lives subtidally and often in regions of strong current. The species is much smaller than other pyurids with body spicules, is more secondary sclerocytes cut in cross section (arrowhead). Organic envelope (OE), demineralized spicule (S), tunic (T), tunic cuticle (TC). Scale bar, 1 pLm. e. Dense mass of organic material that may be the initial mineralizing area of a young spicule. Higher magnification of the center of the same spicule shown in a. TEM. Scale bar, 1 0m. f. Organic envelope (OE), remnants of intraspicular matrix (OM) of demineralized spicule, and fibrous organic layer between epithelium and spicule (OL). TEM. Scale bar, 0.5 am. 347 This content downloaded from 62.122.79.78 on Wed, 18 Jun 2014 14:00:53 PM All use subject to JSTOR Terms and Conditions http://www.jstor.org/page/info/about/policies/terms.jsp Lambert flattened, has very short siphons and adheres to the rocky substrate by a broad expanse of tunic (our Fig. la; Young & Vaizquez 1995). The soft-body tissues are thus probably less stressed by strong currents than are other spicule-bearing pyurids. Especially when the an- imal contracts its tunic and the spicules are brought very close together as in Fig. 2c, tunic flexion is great- ly modified by limiting further compressibility, as is the case with tightly packed surface sclerites in gor- gonians (Lewis & Von Wallis 1991). Possibly the closely packed protruding spicules, with their elongat- ed spines, modify the boundary layer of seawater im- mediately surrounding the animal, lowering the water velocity and allowing the animal to feed more effi- ciently. The spicules may also have a defensive function. Lowenstam (1989) found that closure of the siphons brought the siphonal spicules into even closer prox- imity, with the long spines interdigitated across the depression over the closed contracted siphon (his Fig. 1:145). My own observations on living B. feminalba confirmed that when a relaxed animal with open si- phons was touched, the siphons immediately closed and pulled inward, causing all the spicular spines to point inward into the siphonal opening and interdigi- tate as the tunic contracted. The asymmetric placement of the long spines may facilitate this interdigitation. Lowenstam (1989) hypothesized that this might keep out small predators like amphipods that are often found inside ascidians, eating the branchial mucous sheet. It would certainly exclude flatworms that prey on other solitary ascidians (Lambert 1968). Young and Vaizquez (1995) found that in their animals prepared for SEM the spines pointed away from the closed si- phons (their Figs. 3A, B), so it is possible that the spicules may be capable of changing their orientation. The unmineralized but stiff tunic spines of Halocyn- thia igaboja effectively deter predation by the gastro- pod Fusitriton oregonensis (Young 1986). Spicules of a given animal vary in the degree of offset shown by the long central spine. It is not known what, if any, significance this individual variation has to their function, or whether this morphology changes as continued tunic formation moves the spicules away from the siphons. In its natural environment B. fem- inalba appears to maintain its tunic completely unfou- led, though how it does this is unknown. There is no evidence that it periodically sheds the outer layer of tunic (along with its load of spicules) as does Pyura pachydermatina (Lambert & Lambert 1996b, 1997). Herdmania momus, another pyurid with spinous tunic spicules, tolerates and perhaps even encourages a large amount of fouling (Lambert & Lambert 1987), which sometimes renders it nearly invisible. Nevertheless, the tips of H. momus tunic spicules break off easily and can penetrate human skin, causing irritation and in- flammation (Lambert & Lambert 1987). Though I have not experienced this with B. feminalba, it could be a possible defense mechanism, and the abundant sec- ondary sclerocytes would then be responsible for con- tinued spine formation and repair. Acknowledgments. This paper is dedicated to the memory of Heinz Lowenstam, who analyzed the biominerals of many phyla but took a particular delight in the many forms of ascidian spicules, especially those of Bathypera spp. Special thanks go to Gary Freeman for dredging the Bathypera and kindly giving me a few live specimens for this study, and to Bill Austin who generously lent the slide from which Fig. la was made. Charles Lambert provided invaluable help with the light microscopy and photography; Steve Karl per- formed the thin sectioning. I thank Dr. Dennis Willows and the staff of the Friday Harbor Laboratories for providing space and facilities for part of this research. References Aizenberg J, Lambert G, Addadi L, & Weiner S 1996. 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[341] p. 342 p. 343 p. 344 p. 345 p. 346 p. 347 p. 348 p. 349 Issue Table of Contents Invertebrate Biology, Vol. 117, No. 4 (Autumn, 1998), pp. 261-349 Volume Information Front Matter A Redescription of Rhabdamoeba marina, an Inconspicuous Marine Amoeba from Benthic Sediments [pp. 261-270] Embryogenesis, Planulae Longevity, and Competence in the Octocoral Dendronephthya hemprichi [pp. 271-280] The Infection Mechanism of the Cystophorous Cercariae of Halipegus occidualis (Digenea: Hemiuridae) [pp. 281-287] Extracellular Matrix and Muscle Fibers in the Gills of Freshwater Bivalves [pp. 288-298] External Gestation in Exogonine Syllids (Annelida: Polychaeta): Dorsal Egg Attachment by Means of Epitokous Chaetae [pp. 299-306] Ultrastructural Evidence of Detoxification in the Alimentary Canal of Urechis caupo [pp. 307-317] Cuticular Scales of Spiders [pp. 318-330] The Ecology of an Assemblage Dominant: The Encrusting Bryozoan Fenestrulina rugula [pp. 331-340] Spicule Formation in the Solitary Ascidian Bathypera feminalba (Ascidiacea, Pyuridae) [pp. 341-349] Back Matter