Alimentary tract of kowalevskiidae (appendicularia, tunicata) and evolutionary implications

JOURNAL OF MORPHOLOGY 258:225–238 (2003)

Alimentary Tract of Kowalevskiidae (Appendicularia, Tunicata) and Evolutionary Implications Carlo Brena, Francesca Cima, and Paolo Burighel* Dipartimento di Biologia, Universita` di Padova, 35131 Padova, Italy ABSTRACT The alimentary tract of Kowalevskia tenuis and K. oceanica, the only species of the appendicularian family Kowalevskiidae, was studied both at the light and electron microscope levels and compared with species belonging to the other two families of the class. Kowalevskids show interesting specializations: 1) the pharynx opens on both sides through two opposing spiracles, modified into long ciliated fissures, and possesses an original filtering system of ciliated combs arranged in two pairs of opposing longitudinal rows; 2) the endostyle is absent, its place being taken by a ciliated groove without any glandular cell; 3) posterior to the esophagus, the globular stomach and rectum form a digestive nucleus comprising a few, large cells including two well-developed, specialized valves, cardiac and pyloric; 4) special apical junctions bearing characteristics of both gap and adherens junctions are diffuse along the gut epithelium; 5) the heart is absent. Our data suggest that Kowalevskiidae underwent a high degree of specialization for food filtering and are more closely related to Fritillariidae, with which they share several characters, rather than Oikopleuridae, the latter probably representing the most primitive family of appendicularians. J. Morphol. 258:225–238, 2003. © 2003 Wiley-Liss, Inc.

KEY WORDS: digestive system; gut specializations; intercellular junction; ultrastructure; Urochordata

Kowalevskids, like other appendicularians, are small holoplanktonic tunicates inhabiting a gelatinous “house,” secreted by a specialized region of the epidermis, the oikoplast (Fenaux, 1998a). Their body is composed of a trunk, from the ventral side of which a chordate tail extends. Commonly, in appendicularians the house is supplied with filters, chambers, and channels, and used to trap food particles suspended in a seawater flux created by the beating of the tail. The family Kowalevskiidae has only two species belonging to the same genus: Kowalevskia tenuis and Kowalevskia oceanica (Fenaux, 1998b), which, in contrast with species of Oikopleuridae and Fritillariidae—the other two families of appendicularians, very abundant in all oceans—are rare and evenly distributed in temperate and warm waters (Fenaux et al., 1998). Kowalevskids have been considered of interest due to the unusual aspect of the trunk and the house, and because they have a large pharynx which, as a unique case among tunicates, seems to lack an endostyle. However, after the orig© 2003 WILEY-LISS, INC.

inal descriptions of Fol (1872) and Lohmann (1899), nobody has studied the detailed anatomy of species of this family. Stimulated by recent striking results about the ecological value of appendicularians and their impact on marine ecosystems (see Gorsky and Fenaux, 1998, for a review), we carried out a comparative study on the anatomy and physiology of the alimentary canal of these organisms. Our previous studies on Oikopleuridae and Fritillariidae had shown that these two families, although sharing a series of common characteristics, are clearly differentiated mainly at the level of the gut (Burighel et al., 2001; Cima et al., 2002; Brena et al., 2003). Oikopleura dioica (Burighel et al., 2001; Cima et al., 2002) possesses a relatively long gut formed of an esophagus, a wide bilobate stomach, and an intestine differentiated into vertical, mid-, and distal, or rectum, and characterized by three cell types. Fritillaria (Brena et al., 2003), instead, has a very compact gut, with an esophagus and a stomach and a rectum, both large and globular in shape and dorsally connected through a small, specialized proximal intestine; moreover, the entire gut is mainly formed of a few large cells that cannot easily be compared with the cell types of the Oikopleura gut. Now it appears of particular interest, within a possible evolutionary framework, to clarify various aspects of the microscopic anatomy of kowalevskid gut, to understand the functions of the various regions, and also relationships with other families. The class, which has recently been reassessed for its ecological role in trophic chains, is composed of a few living species—around 65, according to Fenaux (1998b)— but is indeed very ancient, its fossils dating to the Cambrian (Lohmann, 1923; Zhang, 1987). A comparative morphological analysis of the three

Contract grant sponsors: CNR and the EU EURAPP project; Contract grant number: MAS3-CT98-0161 (to PB). *Correspondence to: Prof. Paolo Burighel, Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58B, 35131 Padova, Italy. E-mail: [email protected]

DOI: 10.1002/jmor.10145

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Figure 1

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appendicularian families and the other tunicates, together with genome analysis, arouses much interest, particularly in the light of the recent reassessment of the phylogenetic place of appendicularians at the base of the chordate radiation (Holland, 1991; Wada and Satoh, 1994; Wada, 1998), and on the revived hypothesis that the chordate ancestor was a free-swimming filtering organism (Satoh, 1995; Nishino and Satoh, 2001), in contrast with the traditional “neotenic” theory (Garstang, 1928). MATERIALS AND METHODS In June–July 2000, we had the chance to collect a few specimens of Kowalevskia tenuis (Fol), 1872 and K. oceanica Lohmann, 1899 in the bay of Villefranche-sur-Mer (France). Whole animals were fixed in a solution of 1.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4, plus 1.7% NaCl and 1% saccharose. Individuals were postfixed in 1.5% OsO4 in cacodylate buffer, dehydrated, and embedded in Epon for sectioning. Three individuals of Kowalevskia tenuis and two of K. oceanica were sectioned serially along various planes for detailed reconstruction of internal organs. Sections (1 ␮m thick) were cut on an LKB Ultratome, stained with 1% toluidine blue, observed under a Leica DMR light microscope, and photographed with a JVC 3CCD analogic videocamera. Thin sections (60 nm thick) were collected on copper grids, treated with uranyl acetate and lead citrate, and examined under a Hitachi H600 electron microscope (EM).

RESULTS General Anatomy Kowalevskia tenuis. The digestive system of Kowalevskia tenuis is greatly simplified. The roundish mouth, without lips but endowed with tactile cilia, leads into a broad pharynx, which occupies two-thirds of the length of the trunk (Fig. 1A–C). The pharynx opens laterally through two spiracles modified in two long ciliated fissures (Fig. 1A,B,D,E). The pharyngeal cavity is characterized

Fig. 1. Kowalevskia tenuis. A,B: Left lateral view of trunk of wholemounted young (A) and aged specimen (B). Note series of ciliated combs (cc) within wide pharynx (ph), posteriorly, digestive nucleus (dn) and, in a young individual, the oikoplast in an anterodorsal position, characterized by a giant cell (goc), absent in the aged individual. Scale bar ⫽ 0.1 mm. g, gonad; m, mouth; s, spiracle; t, tail. C: Sagittal section. Wide pharynx (ph) showing the teeth (th) of one ventral ciliated comb. The short esophagus (es) gives access to the stomach (st) through the cardiac valve (cv). Some cells of the rectum (r) protrude to make contact (arrowheads) with the oikoplast (oik). The latter, characterized in the central area by a giant cell (goc) with a huge nucleus (n), secretes the house in a compact form (house rudiments: hr) before inflating it. Dotted lines indicate the level of the following transverse sections (D–F). Scale bar ⫽ 50 ␮m. b, brain; g, gonad; pv, pyloric valve. D–F: Antero-to-posterior serial transverse sections of the same individual; anterior section (D), where teeth of four ciliated combs (cc) are visible in the pharynx (ph); at the level of the giant cell (goc) of the oikoplast (oik) (E) and of the digestive nucleus (F). The digestive nucleus is composed of the stomach (st) on the animal’s right side and the rectum (r) on the left side, connected dorsally by the pyloric valve (pv). Scale bar ⫽ 30 ␮m. c, cilia; ep, epidermis; g, gonad; hr, house rudiments; l, lipid droplets; s, spiracle.

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by the presence of an original system of ciliated combs, arranged in two pairs of longitudinal rows, one dorsal and one ventral, the opposing cilia of which touch at several points. However, the pharynx wall does not have an endostyle or any kind of gland. In transversal section, the pharynx is more or less rectangular in its anterior part and tends posteriorly to enlarge into a trapezoidal shape with its longer base upward, whereas the shorter base tends to become more and more narrow so that the two ventral rows of ciliated combs, approaching, form a groove (Fig. 1D). Near the posterior end of the spiracles, the pharynx floor widens and flattens and the pharyngeal cavity narrows before continuing into the short, ventral esophagus (Fig. 1C). The latter, through a highly developed cardiac valve (Fig. 1C), is inserted ventrally into the stomach. The digestive nucleus is composed mainly of the wide, spherical stomach, with a lumen about 120 ␮m in diameter on the left side of the trunk, and the globular rectum with a lumen of about 100 ␮m, on the right, connected to each other dorsally through a pyloric valve arranged transversally to the main axis of the trunk (Fig. 1F). The rectum opens externally through an anal papilla, situated dorsolaterally on the right side. The digestive nucleus is kept hanging in the body cavity thanks to contacts with the epidermis (Fig. 1C). The latter is very thin, except in the oikoplast, which may contain one huge cell (about 150 ␮m in its main diameter, with a nucleus up to 80 ␮m wide) (Fig. 1A–C,E). Kowalevskia oceanica. In this species the pharynx is more reduced than in Kowalevskia tenuis, particularly when compared with the size of the trunk (Fig. 2A–D). Unlike in K. tenuis, the spiracles in K. oceanica extend into the central part of the pharynx and are situated ventrolaterally (Fig. 2A– C). The pharyngeal floor shows a pronounced convexity, clearly separated from the ventral epidermis (Fig. 2A), is densely ciliated, enlarging itself as it extends backwards (Fig. 2B–D), and lacks any trace of endostyle or ciliated groove. Thus, in the central area near the spiracles the pharynx has a U-shaped transversal section; its roof rises anteriorly forming an acute angle (Fig. 2B) and enlarges posteriorly (Fig. 2C,D). Moreover, on the lateral edges of the pharyngeal floor, a pair of ciliated combs extends almost horizontally, in such a way as to touch the cilia of an opposing pair of combs situated on the lateral walls of the pharynx (Fig. 2B,C). The ciliated spiracular cells are placed ventrally with respect to both combs and median pharyngeal floor, defining a chamber on each side of the trunk (Fig. 2B,C). These cells, which anteriorly are near the edge of the spiracle, are posteriorly displaced more dorsally and become ventral again posteriorly to the closure of the spiracle itself. In this way, these cells form an arc in the sagittal plane (Fig. 2A). The body cavity, level with the spiracles, contains four large cells (up to 90 ␮m in diameter), two situated on each external

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Fig. 2. Kowalevskia oceanica. A: Left lateral view of the trunk of a whole-mount individual. Note that the pharynx (ph) is less developed in comparison with the trunk length and convexity of ciliated combs (cc). Gonads (g) growing in a large body cavity behind the digestive nucleus formed by the stomach (st) and the rectum (r). Dotted lines indicate the level of the following transverse sections (B–F). Scale bar ⫽ 0.1 mm. m, mouth; oik, oikoplast; s, spiracle; t, tail. B–F: Antero-to-posterior serial transverse sections of the same individual at the level of the anterior opening of spiracles (s) (B); in the middle part of the pharynx (ph) (C) at the level of the giant glandular cells (ggc); posterior to spiracles (D); at the level of the long cardiac valve (cv) ventrally inserted into the stomach (st) under the rectum (r) (E); stomach–rectum connection by the pyloric valve (pv) (F). Note in the last section a gastric cell is protruding externally to contact the epidermis (arrowhead). Scale bars ⫽ 40 ␮m in B–D, 50 ␮m in E–F. a, anus; c, cilia; cc, ciliated combs; ep, epidermis; oik, oikoplast.

side (Fig. 2C) and two ventrally, in contact with the epidermis. These cells seem to lack any recognizable baso-apical axis, either towards the pharynx or towards the outside; their function is difficult to interpret, although they are probably secreting cells, due to the presence in their cytoplasm of large amounts

of RER cisternae and basophilic granules of various sizes. At the posterior end of the pharynx the ciliated comb cells tend to protrude less and less into the lumen until they continue with the ciliated cells of the esophagus. The latter is very short and its lumen

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appears to be quite depressed. It opens ventrally into a wide, spherical stomach through a cardiac valve (Fig. 2E), which is highly developed. The spherical stomach is ventral, with a lumen about 220 ␮m in diameter, and is directly connected through a well-developed pyloric valve to the wide rectum, also spherical and situated dorsally (Fig. 2A,E,F). The pyloric valve forms a “canal,” which is transversal with respect to the anteroposterior axis of the trunk and placed on the left side, while the rectum opens externally through an anal papilla on the right side (Fig. 2F). Both species lack a heart. The entire digestive system is bathed by hemolymph, which lacks blood cells and moves due to the active beating of the tail. Ultrastructure Pharynx. This region, except for sensory cells not analyzed in detail here, is composed of four cell types: “unciliated cells,” very thin and simple, often representing only a connection to the other cell types; “ciliated cells” and, as possible modifications of the latter, “comb cells” and “spiracle cells,” both characterized by very long cilia (Fig. 3A,B). The unciliated cells, in particular, extend in the form of a thin layer on the lateral surface of both spiracle and comb cells (Fig. 3A,B). Every comb tooth is composed of a single, elongated ciliated cell with a basal spherical nucleus, a great abundance of fibrous material underlying the lateral plasmalemma, and a marked apical bundle of cilia (Fig. 3A). The striated rootlets of the cilia are particularly extended, occupying most of the cytoplasm, which is filled with mitochondria in its basal area (Fig. 3A). A similar pattern characterizes the spiracle cells, although, in this case, the cells are not so elongated and are grouped in at least three rows (Fig. 3A,B). Moreover, the striated rootlets, besides connecting to the lateral membranes, often reach the base of the cell. These ciliated cells are bound to each other and to the unciliated cells by apical electron-dense junctions (inset in Fig. 3B) and numerous gap junctions. The spiracle cells of Kowalevskia oceanica are compartmentalized into: 1) a basal third, rich in RER; 2) a central area, particularly rich in mitochondria and large vacuoles; and 3) an apical region full of striated rootlets (Fig. 3B). In the apical area external to that of the ciliary insertion of some spiracle cells, we identified a gathering of numerous spherical electron-dense granules (Fig. 3B and inset). Esophagus. In Kowalevskia tenuis the short esophagus is characterized by ciliated cells continuous with the ciliated ones of the pharynx. The esophageal cells are cuboid and possess a few, thin microvilli and a basal spherical nucleus. The cytoplasm is rich in free ribosomes, although the endoplasmic reticulum is scarcely present, whereas mitochondria and rare vacuoles are scattered. Cilia, directed towards the stomach, are inserted obliquely into the

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apical cytoplasm with a large basal body provided with short striated rootlets, which are directed towards the lateral plasmalemmata. The latter are almost straight and have numerous gap junctions. In Kowalevskia oceanica the esophagus is wider and characterized by larger ciliated cells than in K. tenuis, clearly distinct from pharyngeal cells (Fig. 3C). These cells possess many characteristics common to those of K. tenuis, although the striated rootlets of the cilia are normally not so extended. The esophageal cells possess a thin, dense, apical layer resembling a terminal web adhering to the plasmalemma, and are also characterized by a remarkable fuzzy coat covering the cilia and the microvilli; finely granular material, secreted by the cells, is attached to the fuzzy coat. Cardiac valve. This specialized region is formed of the union of very long cilia, arising in great numbers from a single ring of cells (Fig. 3C). The latter are similar to the esophageal ones but lack microvilli, and their cilia closely lean upon each other, completely occupying the luminal surface of the cell (Fig. 3E). The cilia are regularly glued together to form a very compact lamina, 4 – 6 ciliary rows thick (Fig. 3D,F). In Kowalevskia tenuis, the precise juxtaposition of the plasma membranes of adjacent cilia forms a honeycomb structure in transversal section (Fig. 3D) and, on the luminal surface, the lamina is covered by a hard multilayered fibrous material, which is highly electron-dense (Fig. 3C,D). In K. oceanica, the closer adhesion of the ciliary membranes implies their partial union, but the lamina is not reinforced by any extracellular material (Fig. 3F). The cilia of the lamina are inserted almost straight into the cardiac valve cells and, besides well-developed basal bodies, have very long striated rootlets which, crossing the cytoplasm rich in mitochondria, reach the base of the cell. The cardiac lamina, directed towards the stomach, bends and is attached to the proximal cells of the stomach through an electron-dense fibrous layer (Fig. 3C,D,F). The latter cells, with little cytoplasmic content, are greatly reduced in thickness and in fact form a thin coat supporting the cardiac lamina (Fig. 3C,G). They are also connected to the stomach ones through extensive apicolateral junctions (Fig. 3G). At the entrance to the stomach, the lamina forms a series of wide folds (Figs. 2E, 3G), which are particularly tortuous in K. tenuis. Stomach. In Kowalevskia tenuis this region has fewer than 30 cells and, in K. oceanica, about 50 (Figs. 1C,F, 2E,F, 4A). They are globular in shape, prominent on the basal side, and variable in size, occasionally reaching 83 ␮m in width and 30 ␮m in thickness. They lack both cilia and microvilli and the luminal surface in K. tenuis is moderately undulated by a series of “bubbles.” These curious structures are marked by dense, finely granular material, often inhomogeneous (Fig. 4C), resembling the continuous dense fibrous layer attached to apical plas-

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Figure 3

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malemma. This layer is similar to that of esophagus and actin-like filaments, 5–7 nm in diameter, are recognizable among its fibers. It is attached to the entire apical plasmalemma, including the lateral portion involved in the formation of the junctional belt (Fig. 4A). The latter, in K. oceanica, follows the entire profile of the apical interdigitations between the small cells next to the pyloric valve facing the rectum (Fig. 5A). This belt appears to be accompanied by intercellular apicolateral junctions with an unusual aspect, since they have the characteristics of gap junctions in that the two opposing membranes associate closely, as occurs in typical gap junctions. In both the cases the associated membranes form a sort of straight plate 6 nm thick, with the intercellular cleft extremely reduced and difficult to resolve. In addition, in both the cases the distance between the inner layers of the two opposing membranes is about 10 nm. On the other hand, these apicolateral junctions recall the adherens junctions in that they form a belt and show accumulation of fibrous material on both sides of the cytoplasm (inset a, Fig. 4A). Below these junctions are also numerous gap junctions with usual features (inset b, Fig. 4A), but sometimes extended up to 1 ␮m. In the gastric cells the nucleus is subspherical in shape and normally in a central position. The cytoplasm contains many free ribosomes; the RER is poorly developed. We did not identify typical Golgi complexes. Mitochondria are abundant and particu-

Fig. 3. A,B,E–G: Kowalevskia oceanica. C,D: Kowalevskia tenuis. A: Each tooth of the ciliated combs is composed of a single, elongated ciliated cell, reinforced by a cortical layer of microfibers (arrowheads) and exhibiting a basal nucleus (n). These cells are next to spiracle cells (SC), both having cilia with long abundant striated rootlets (sr), and are laterally covered by a monolayer of contiguous unciliated cells (uc and arrows). Scale bar ⫽ 2 ␮m. bc, body cavity; ph, pharynx. B: Spiracle ring in transverse section is composed of three ciliated cells, very rich in long striated rootlets (sr) on the apex, mitochondria (mt) in the middle, and microfibers (arrows) in the basal area. Several dense granules (arrowheads in inset) are present apically, lateral to striated rootlets. Lateral covering unciliated cells (uc) are attached to the spiracular cells by apical dense junctions (arrow in inset). Scale bars ⫽ 2 ␮m, 0.5 ␮m in inset. bc, body cavity. C–G: Esophagus and cardiac valve. Esophagus is very short and is formed of a few ciliated microvillar cells (EC), with cilia showing developed striated rootlets (sr) (C). The cardiac valve lamina (arrows in C) is formed of unions of 4 – 8 rows of cilia (c) (D–F), arising from a single cell ring (CVC) and exhibiting long striated rootlets (st) associated with mitochondria (mt). Cilia keep their membranes distinct to form a honeycomb structure in K. tenuis (D) but partially fuse in K. oceanica (arrowheads in F). Lamina in K. tenuis is reinforced on the luminal side by multilayered, electron-dense fibrous material (arrowheads and D). In both species, laminae are attached through a dense layer (arrows in D and F) to thin proximal gastric cells (PGC); the latter are attached to normal gastric cells (GC) by developed apical junctions (arrow in G). Within the lumen of the cardiac valve lamina (cvl), food particles (fp) are recognizable as far as the narrow entrance of the lamina into the gastric lumen (gl) (G). Scale bars ⫽ 2 ␮m in C, 0.2 ␮m in D, 0.5 ␮m in E, 0.2 ␮m in F, 2 ␮m in G. bc, body cavity; ph, pharynx.

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larly concentrated in the basolateral area of the cell, sometimes associated with the deep membrane infoldings which characterize both the whole basal side and partly the basolateral one of all the gastric cells (Fig. 4A,B). These infoldings have a constant extracellular space which, in Kowalevskia tenuis, is about 13 nm across and contains electron-dense spots. Scattered, membrane-bound, roundish granules, with homogeneous, strongly electron-dense content are visible near the apical plasmalemma (Fig. 4A); however, we never observed signs of endocytosis. Several cells contain lipid droplets a few micrometers in diameter, occasionally present in high numbers inside the same cell (Fig. 1F), but we also sometimes found relatively large lipid droplets. Some cells of the stomach protrude extensively into the body cavity, making contact with the epidermal cells. The latter are reduced to a thin layer (Figs. 1C, 2F), and extended gap junctions are often recognizable in areas of connection between them and the gastric cells (Fig. 4D). Pyloric valve. This connects stomach and rectum and comprises three rings of cells (Figs. 1F, 2F, 5C). The first two are smaller and irregular in shape, forming a hinge that supports the third cell ring; their cells have cytoplasm similar to that of the gastric cells but they bear long cilia, which are inserted into the cell very obliquely, are directed towards the rectum, and possess long striated rootlets occupying the apical third of the cell. In Kowalevskia tenuis, on the cell surface of the first ring are the same electron-dense bubbles recognized on the gastric cells. The cells of the third ring (main or distal cells) are much wider than those of the first two and also have thin microvilli (Fig. 5C). Their cilia are inserted vertically and the striated rootlets, although shorter, interconnect with each other and extend like an upside-down fan (Fig. 5B). The spherical nucleus is central. The pyloric valve cells have straight lateral membranes and their cytoplasm contains abundant ribosomes and a highly dense, subapical layer. Only in Kowalevskia tenuis, after the pyloric valve, is there a short ring of a few, very small cells, poor in cytoplasmic organelles and extensively interdigitated, both between each other and, apically, with the first cells of the rectum (Fig. 5C). They do not possess microvilli or cilia and lack the subapical dense layer. All along the contacting surface between cells wide gap junctions and particular, diffuse adherens-like junctions are present. Rectum. In Kowalevskia tenuis, this region is composed of about 20 cells and in K. oceanica of about 30. They are morphologically uniform in the same species, large and comparable in size with those of the stomach in K. tenuis, and larger (up to 110 ␮m), albeit always of limited thickness, in K. oceanica (Figs. 1F, 2E,F, 5D). The rectal cells resemble the gastric ones in their cytoplasmic features, but differ due to the presence of long cilia among

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Fig. 4. Stomach. A,D: Kowalevskia oceanica. B,C: Kowalevskia tenuis. The stomach is composed of large cells each bearing a central nucleus (n), many scattered mitochondria (mt), some associated with basolateral membrane infoldings (bmi in A,B), dense granules near the apical plasmalemma (arrows in A), and an electron-dense layer on the apical surface. This layer connects and surrounds particular apical junctions between contiguous cells with characteristics common to both gap and adherens junctions (arrowheads in inset a of A and in C). The apical cell surface exhibits characteristic electron-dense bubbles (arrows in C) in K. tenuis. Contiguous gastric cells (GC) are interconnected by extended and numerous gap junctions (arrowheads in A and inset b of A), also present between some protruding gastric cells (GC) and the thin epidermis (ep) (arrowhead in D). bl, basal lamina; gl, gastric lumen. Scale bars ⫽ 2 ␮m in A and 0.2 ␮m in both insets, 0.4 ␮m in B, 0.2 ␮m in C, 0.2 ␮m in D.

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scarce, rod-like microvilli. The cilia, inserted vertically into the cell, have a well-developed basal body and short striated rootlets (inset, Fig. 5D). The distribution pattern of the basal membrane infoldings is similar to that of the stomach, with similar extracellular space of about 13 nm between infoldings and the association with the mitochondria. In the cytoplasm, we observed some multivesicular bodies and vesicles. Moreover, in the rectal cells small vesicles from about 0.1– 0.4 ␮m in diameter and containing electron-dense, granular material inside a uniform matrix often occur. In K. oceanica the giant dorsal rectal cells contain numerous mitochondria, very elongated and parallel to the lateral cell walls (Fig. 5D). The contiguous cells form numerous gap junctions all along the lateral plasmalemmata. The rectal lumen is packed with the fecal pellet, in which several microalgal cells are recognizable, still partly undigested and varying greatly in size, up to 110 ␮m maximum diameter (Fig. 2F). At the end of the rectum the few anal papilla cells (Fig. 2F) contain large amounts of intracellular fibers and extensive, convoluted interdigitations, marked by both dense adherens-like junctions and rare gap junctions (Fig. 5F). Stomach–Rectum Connection. The gastric and rectal cells contact each other with their basal surface in the medial part of the digestive nucleus (Figs. 1F, 2E,F, 5E). Here, the thickness of the opposing cells may be very low, sometimes down to 3.5 ␮m. All along the contacting surface the opposing membranes interdigitate, forming a wide loop in Kowalevskia tenuis but not in K. oceanica (Fig. 5E). In both species the opposing cells are in communication with each other through numerous gap junctions (Fig. 5E), sometimes remarkably extended. Over most of the contact the surface devoted to the gap junction is greater than the nonjunctional one. In K. tenuis, we also identified some adherens-like junctions. DISCUSSION Pharynx First observed in vivo and described in its general morphology by Fol (1872), the genus Kowalevskia has not been described in detail since then. The main difference that characterizes the general aspect of these animals in comparison with other appendicularians is the specialization of the pharynx. The latter stimulates particular interest in understanding its functions mainly because, for example, it lacks an endostyle. In comparison with other tunicates, the endostyle of Oikopleuridae and Fritillariidae is generally very reduced, in both number of cell types and dimension. In oikopleurids the endostyle has been described by Olsson (1965) and its functional activity in producing the pharyngeal secretion for particle agglutination has been demonstrated (Fenaux, 1968, 1989; Deibel and Powell,

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1987): it accomplishes the last phases of filtering, which start in the wide, complex filters of the house. In fritillarids the endostyle is smaller and compacted (Martini, 1909) and its opening into the pharynx floor is short, almost punctiform. Nevertheless, the presence of both ciliated and secreting cells implies an important role in collecting food particles at the entrance to the pharynx. In contrast, our analysis of complete sequences of thick sections, together with observations at the ultrastructural level, did not allow us to identify any glandular structure belonging to or directly opening into the pharynx, so that, in Kowalevskia, food particles cannot be agglutinated by a mucous-like substance. As no secretory duct can be seen near the large gland cells external to the pharynx in K. oceanica, they may release their basophilic granules in hemolymph or through the epidermis, with which they are closely in contact, as in the oral glands of Oikopleura (Flood and Deibel, 1998). Thus, pharyngeal filtering of food seems to depend only on the mechanical activity of the cilia of the comb cells, while swallowed water is, as in other appendicularians, driven outside by the spiracle cells. Although distinct, the elements of the pharynx may be compared with those of other appendicularians. The monocellular teeth of the ciliated comb probably represent a deep modification of the pericoronal arches of other tunicates, and the oblong spiracles result from the enlargement of the spiracles of the commonest appendicularians. It is noteworthy that very large, ellipsoid spiracles have appeared more than once in the evolutionary history of larvaceans, as they are also present in oikopleurids, particularly in the genus Mesochordaeus. Our ultrastructural data reveal great activity of the cilia of both ciliated combs and spiracles, as the cilia are densely packed and their long striated rootlets are associated with abundant mitochondria. Moreover, the ciliated comb cells are supported laterally by laminar unciliated cells, with which they are connected apically by electron-dense junctions. All these features suggest that these cells support strong mechanical stress. Following the observations of Fol (1872) and Fenaux (1968) on living animals, when food particles reach the pharyngeal cavity they are probably trapped by the comb system, since the cilia of opposing comb teeth, coming into contact, make a sort of continuous filtering barrier. In this way, as water is driven outside by the ciliary activity of the spiracles, food particles tend to accumulate on the floor of the pharynx, which has the form of a groove in Kowalevskia tenuis, and are then transported backwards to the esophagus, thanks to the action of the ciliated cells. In K. oceanica, the pharynx has a quite different shape, although its main elements are clearly comparable with those of the other species; in particular, we may consider its aspect as the result of a simple elevation of its floor, causing lateral translation of the combs.

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Figure 5

GUT OF KOWALEVSKIIDAE

Esophagus and Cardiac Valve The large volume of the pharynx, together with the abundance of ciliated cells, accompanies the great reduction of the esophagus, which has few ciliated cells and represents a simple channel connecting the pharynx and the well-developed cardiac valve. Fenaux (1968) ascribed the function of agglomeration of food particles to the esophagus as a consequence of the forward and backward movement caused by the contrasting action of the cilia of both spiracles and esophagus. The cardiac valve, composed of very long cilia joined to each other to form a lamina and arising from a single ring of cells, seems to represent a variation on the theme of the cardiac valves of fritillarids: the reinforcement on the luminal side of the lamina of Kowalevskia tenuis may be homologous to the far less developed situation seen in both Fritillaria formica (Brena et al., 2003) and F. borealis (unpubl. data). The cilia are closely compacted together—particularly in K. oceanica, where the lateral plasmalemmata between cilia seem to disappear. Both the compactness and the considerable length of this valve suggests fundamental functions in regulating access by food particles to the digestive nucleus and also in preventing reflux of food to the pharynx, since the laminae could collapse together in the event of reverse flow. Digestive Nucleus Gastric cells completely lack both cilia and microvilli and do not show recognizable features of endocytosis or secretion. The role played by the electron-dense bubbles observed on the apical surface of the gastric cells of Kowalevskia tenuis is not evident. They do not seem to be an artifact, since we have frequently found them in various specimens of

Fig. 5. A,B,D-E: Kowalevskia oceanica. C: Kowalevskia tenuis. A: Gastric cells near the pyloric valve are characterized by apical interdigitations associated with well-developed apical junctions (arrows). Scale bar ⫽ 0.2 ␮m. B,C: The pyloric valve is composed of three cell rings, of which the main or distal one (DPC in C) is widest, with a central nucleus (n) and very long cilia among microvilli that are characterized by a remarkable basal body (bb in B) and fan-shaped striated rootlets (sr in B). In K. tenuis, a few very small cells (arrow in C), extensively interdigitating with each other, amid distal pyloric cells and the most proximal rectal cells (RC in C). Scale bars ⫽ 0.4 ␮m in B, 2 ␮m in C. bc, body cavity; gl, gastric lumen. D: The rectum is composed of large ciliated microvillar cells, each with a central nucleus (n), basal membrane infoldings (arrows), and scattered mitochondria (mt). Among the microvilli (mv in inset), are cilia (c) with evident basal bodies (bb) and poorly developed striated rootlets (sr). Scale bars ⫽ 2 ␮m and 0.4 ␮m in inset. ep, epidermis. E: Abundant gap junctions (arrowheads) are evident along contacting surfaces between gastric (GC) and rectal cells (RC). Scale bar ⫽ 0.2 ␮m. gl, gastric lumen. F: Anal papilla cells are characterized by numerous intracellular fibers (black arrowheads) and deep interdigitations with frequent, electron-dense junctions (arrows) and occasional gap junctions (white arrowhead). Scale bar ⫽ 0.4 ␮m.

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this species and only in that specific region. Considering their size, they may represent a system to expand the surface for absorption, instead of the microvilli, rather than a simple ornamentation of the epithelial surface. The pyloric valve is very similar to that in fritillarids, although not so structurally complex. As in fritillarids (Brena et al., 2003), it comprises three rings of cells, of which the distal one (main or distal pyloric valve cells) has the longest and the most abundant cilia. As reported for living specimens by Fol (1872), this valve probably operates as in Fritillaria pellucida (Fenaux, 1961): the long cilia of the main pyloric valve cells collect food by brushing the gastric lumen and, after complete rotation on the adjacent cell rings, which act like a pivot, transfer it towards the rectal lumen. In Kowalevskia tenuis, this valve is followed by a short ring of cells with electron-dense apical junctions. This pattern clearly recalls that of the distal part of the proximal intestine of Fritillaria (Brena et al., 2003), to which this cell ring may therefore be considered homologous. K. oceanica completely lacks this short tract, so that the connection between stomach and rectum only occurs through the pyloric valve. As in the fritillarid rectum, we suggest that, also in kowalevskids, this may be the gut region in which food stays longest. It is known that in Fritillaria food is removed from the gastric lumen and accumulated in the rectum every 1–2 min, thanks to the activity of the pyloric valve cilia (Fenaux, 1961). Thus, the globular rectum should be the main place for both digestive and absorptive processes. The presence of microvilli supports the hypothesis of the absorption capability of small molecules and ions by the rectal cells on the contents of fecal pellets, in which extracellular digestion probably occurs. In addition, more than in fritillarids, stomach and rectum are interconnected medially by very long, abundant gap junctions, which allow the exchange of small molecules between the two compartments. This type of communication is indeed frequent in many contacting cells of the gut epithelium, and also in some basally protruding gut cells and the epidermis. The efficiency of the absorptive function of the kowalevskid gut is also indicated by the fact that it can accumulate large quantities of lipid droplets—an aspect common to other appendicularians (Burighel et al., 2001; Cima et al., 2002; Brena et al., 2003)— and which may allow these organisms to withstand brief periods of food shortage. The fact that we never found signs of endocytosis and a considerable lipid storage was detected may be explained by the particular physiological stage of the specimens we examined. On the other hand, the membrane-bound granules found in the apical region of the gastric cells may participate to a merocrine secretion of digestive enzymes, as suggested by their morphological similarity with the zymogen-like granules inside the gastric cells, previously observed in both oiko-

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Fig. 6. Sketch of the digestive tracts of representative species of the three appendicularian families. Drawn from the dorsal side. Note that the proportions among the three species are not respective. a, anus; cc, ciliated combs; en, endostyle; es, esophagus; ls, left gastric lobe; m, mouth; mi, mid-intestine; pi, proximal intestine; r, rectum; rs, right gastric lobe; sp, spiracle; st, stomach; vi, vertical intestine.

pleurids (Burighel et al., 2001) and fritillarids (Brena et al., 2003). These organisms might alternate periods of digestive enzyme synthesis and secretion with periods of nutrient absorption and storage; in this respect, it is noteworthy that gastric cells in a different state of digestive enzyme activity have been found in various specimens of F. pellucida (Brena et al., 2003). Comparative Considerations As noted by Fenaux (1968), kowalevskids are unique among all tunicates in their feeding mechanism, due to the absence of endostyle and any secreting pharyngeal organs, the great development of the pharynx, and of the complex filtering system associated with it, i.e., the ciliated combs. In particular, the uniqueness of the pharynx seems to represent an apomorphy of this family. However, as regards the digestive nucleus, the kowalevskids show similarities with fritillarids, e.g., stomach and rectum are the two major cavities in both Fritillaria (Brena et al., 2003) and Kowalevskia (Fig. 6) and are connected by a well-developed pyloric valve. This indicates that the two families are evolutionarily close and separated for a long time from the oikopleurids, the latter probably being the most primitive extant family, which shows distinctive characteristics, such as the presence of well-developed gut tracts and a variety of cell types (Burighel et al.,

2001). This hypothesis fits that of Garstang (1928), who, on the basis of oikoplast organization and gut loop disposition, considered both fritillarids and kowalevskids as modified secondarily with respect to oikopleurids. Indeed, kowalevskids and fritillarids share some signs of apomorphy, like the similar reduction of both gut regions and cell types, the occurrence of species with no heart, and the extreme thinning of the epidermis in some areas of the trunk (Bone et al., 1977; Brena et al., 2003). On the other hand, kowalevskids show further specialization at the ultrastructural level. In comparison with fritillarids, they have the proximal intestine reduced that lacks the mitochondrial pump cells which, in the genus Fritillaria, are considered important for transmembrane transport and osmotic regulation of body fluids owing to their frequent association with mitochondria, as well as aminopeptidase M, 5⬘-nucleotidase, and Mg2⫹-ATPase activities (Brena et al., 2003). However, Kowalevskiidae, like Fritillariidae, are rich in basolateral infoldings along both the stomach and the intestine, which greatly increase the membrane surface and are often associated with mitochondria. These characteristics are typical of ion-transporting epithelia as described in the gill of crabs (Luquet et al., 2002), in vertebrate salt glands (Pease, 1956), and in the mammalian vestibular system (Pitovski and Kerr, 2002). In appendicularians, three kinds of structural morphologies were found indicating the capacity of

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TABLE 1. Comparative characteristics of the post-pharyngeal tract in three appendicularian families Gut region

Oikopleuridae

Fritillariidae

Kowalevskiidae

Esophagus

⫹⫹ CMC ft, sc, or ⫹⫹ CC ftr (passive) ⫹⫹ (bilobed) CMC, GC, GBC ft, ab, dg, ns, or

⫹⫹ CMC ft, sc, dg, ab ⫹⫹ CC ftr (active) ⫹⫹ MC ab, dg, ns, or

⫺

⫹⫹ CC ftr (active) ⫹ UC, MPC or ⫺

⫹ CMC ft, sc ⫹⫹ CC ftr (active ?) ⫹⫹ UC ab(?), dg(?), ns, or ⫹⫹ CC, CMC ftr (active) ⫾ UC

Cardiac valve Stomach

Pyloric valve Proximal intestine Mid-intestine Rectal valve Rectum

⫹⫹ CMC ft, fpa, ns, or ⫹⫹ CMC ft, ab, (ns), or ⫹⫹ CC, UC ftr (active) ⫹⫹ CMC, GC ft, ab, dg, ns, or

⫹ CMC dg, ftr (passive) ⫹⫹ CMC ft, ab, dg, ns, fpa, or

⫺ ⫺ ⫹⫹ CMC ft, ab, dg, fpa, or

⫺ absent; ⫾ poorly extended; ⫹ present; ⫹⫹ relatively well extended. CC, ciliated cells; CMC, ciliated microvillar cells; GBC, gastric band cells; GC, globular cells; MC, microvillar cells; MPC: mitochondrial pump cells; UC: unciliated, not microvillar cells. ab, absorption; dg, digestion; fpa, fecal pellet assembling; ft, food transport; ftr, food transit regulation; ns, nutrient storage; or, osmotic regulation, sc, granular material secretion.

gut epithelium to regulate ion or fluid transports, all based on membrane extensions commonly associated with mitochondria. Increase of membrane surface occurs 1) in oikopleurids, through basolateral cell interdigitations; 2) in fritillarids, through basal infoldings and mitochondrial pump cells; and 3) in kowalevskids, through basal infoldings. Thus, fritillarids and kowalevskids share the presence of diffuse basal infoldings; it remains difficult to establish if, in kowalevskids, the complete absence of mitochondrial pump cells is an apomorphy, or if they never developed them. In this family, osmoregulation may also be supported by their exclusive, special apical intercellular junctions, particularly developed in gastric cells. The abundant fibrous material surrounding the junctions suggests a concomitant function of cell linkage, as previously hypothesized for the shorter, punctiform, apical tight junctions of Oikopleura (Martinucci et al., 1990; Burighel et al., 2001). Similar to these latter junctions, the particular junctions of kowalevskids presumably play a sealing role separating the body fluid from the gut lumen content. Although it is difficult to establish the true characteristics of these junctions in the absence of freeze-fracture studies, we think that these junctions resemble a kind of gap junction observed in ascidians, which is accompanied by dense fibers associated with its junctional cytoplasmic surfaces, and which is considered to play a double role in allowing exchange of ions and small molecules and mechanical adhesion between

contiguous cells (Lane et al., 1995). The function of mechanical adhesion is evidenced by the fact that these junctions are particularly extended along areas of higher mechanical stress throughout the alimentary canal, i.e.: i) along the contact of proximal gastric cells with the adjacent main gastric cells, where mechanical stress is caused by the transit of food from the cardiac valve; ii) along the interdigitations of the cells preceding the pyloric valve, where mechanical stress may be caused by the extensive movements of the pyloric cells; and iii) among the anal papilla cells, where stretching is caused by the passage of the fecal pellet as it is evacuated. The whole of the characteristics of the gut of the three families of appendicularians, as shown in Figure 6 and Table 1, suggest that kowalevskids are more closely related to fritillarids than to oikopleurids. However, the special adaptations in kowalevskids, probably linked to the modality of food particle filtering, required new specializations, which caused a clear-cut evolutionary parting from their common ancestor. ACKNOWLEDGMENTS The authors thank Dr. P. Flood of the Bathybiologica A/S (Norway) and Dr. G. Gorsky of the Station Zoologique of Villefranche-sur-Mer for facilities in collecting kowalevskids. The English text was revised by G. Walton.

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