Alimentary Tract of Kowalevskiidae (Appendicularia,Tunicata) and Evolutionary ImplicationsCarlo Brena, Francesca Cima, and Paolo Burighel*

Dipartimento di Biologia, Universita di Padova, 35131 Padova, Italy

ABSTRACT The alimentary tract of Kowalevskia tenuisand K. oceanica, the only species of the appendicularianfamily Kowalevskiidae, was studied both at the light andelectron microscope levels and compared with species be-longing to the other two families of the class. Kowa-levskids show interesting specializations: 1) the pharynxopens on both sides through two opposing spiracles, mod-ified into long ciliated fissures, and possesses an originalfiltering system of ciliated combs arranged in two pairs ofopposing longitudinal rows; 2) the endostyle is absent, itsplace being taken by a ciliated groove without any glan-dular cell; 3) posterior to the esophagus, the globularstomach and rectum form a digestive nucleus comprisinga few, large cells including two well-developed, specializedvalves, cardiac and pyloric; 4) special apical junctionsbearing characteristics of both gap and adherens junctionsare diffuse along the gut epithelium; 5) the heart is ab-sent. Our data suggest that Kowalevskiidae underwent ahigh degree of specialization for food filtering and aremore closely related to Fritillariidae, with which theyshare several characters, rather than Oikopleuridae, thelatter probably representing the most primitive family ofappendicularians. J. Morphol. 258:225–238, 2003.© 2003 Wiley-Liss, Inc.

KEY WORDS: digestive system; gut specializations; inter-cellular junction; ultrastructure; Urochordata

Kowalevskids, like other appendicularians, aresmall holoplanktonic tunicates inhabiting a gelati-nous “house,” secreted by a specialized region of theepidermis, the oikoplast (Fenaux, 1998a). Theirbody is composed of a trunk, from the ventral side ofwhich a chordate tail extends. Commonly, in appen-dicularians the house is supplied with filters, cham-bers, and channels, and used to trap food particlessuspended in a seawater flux created by the beatingof the tail. The family Kowalevskiidae has only twospecies belonging to the same genus: Kowalevskiatenuis and Kowalevskia oceanica (Fenaux, 1998b),which, in contrast with species of Oikopleuridae andFritillariidae—the other two families of appendicu-larians, very abundant in all oceans—are rare andevenly distributed in temperate and warm waters(Fenaux et al., 1998). Kowalevskids have been con-sidered of interest due to the unusual aspect of thetrunk and the house, and because they have a largepharynx which, as a unique case among tunicates,seems to lack an endostyle. However, after the orig-

inal descriptions of Fol (1872) and Lohmann (1899),nobody has studied the detailed anatomy of speciesof this family.

Stimulated by recent striking results about theecological value of appendicularians and their im-pact on marine ecosystems (see Gorsky and Fenaux,1998, for a review), we carried out a comparativestudy on the anatomy and physiology of the alimen-tary canal of these organisms. Our previous studieson Oikopleuridae and Fritillariidae had shown thatthese two families, although sharing a series of com-mon characteristics, are clearly differentiatedmainly at the level of the gut (Burighel et al., 2001;Cima et al., 2002; Brena et al., 2003). Oikopleuradioica (Burighel et al., 2001; Cima et al., 2002) pos-sesses a relatively long gut formed of an esophagus,a wide bilobate stomach, and an intestine differen-tiated into vertical, mid-, and distal, or rectum, andcharacterized by three cell types. Fritillaria (Brenaet al., 2003), instead, has a very compact gut, withan esophagus and a stomach and a rectum, bothlarge and globular in shape and dorsally connectedthrough a small, specialized proximal intestine;moreover, the entire gut is mainly formed of a fewlarge cells that cannot easily be compared with thecell types of the Oikopleura gut.

Now it appears of particular interest, within apossible evolutionary framework, to clarify variousaspects of the microscopic anatomy of kowalevskidgut, to understand the functions of the various re-gions, and also relationships with other families.The class, which has recently been reassessed for itsecological role in trophic chains, is composed of a fewliving species—around 65, according to Fenaux(1998b)—but is indeed very ancient, its fossils dat-ing to the Cambrian (Lohmann, 1923; Zhang, 1987).A comparative morphological analysis of the three

Contract grant sponsors: CNR and the EU EURAPP project; Con-tract 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: burighel@civ.bio.unipd.it

DOI: 10.1002/jmor.10145

JOURNAL OF MORPHOLOGY 258:225–238 (2003)

© 2003 WILEY-LISS, INC.

Figure 1

226 C. BRENA ET AL.

appendicularian families and the other tunicates,together with genome analysis, arouses much inter-est, particularly in the light of the recent reassess-ment of the phylogenetic place of appendiculariansat the base of the chordate radiation (Holland, 1991;Wada and Satoh, 1994; Wada, 1998), and on therevived hypothesis that the chordate ancestor was afree-swimming filtering organism (Satoh, 1995;Nishino and Satoh, 2001), in contrast with the tra-ditional “neotenic” theory (Garstang, 1928).

MATERIALS AND METHODS

In June–July 2000, we had the chance to collect a few speci-mens 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% glutaraldehydein 0.2 M cacodylate buffer, pH 7.4, plus 1.7% NaCl and 1%saccharose. Individuals were postfixed in 1.5% OsO4 in cacodylatebuffer, dehydrated, and embedded in Epon for sectioning. Threeindividuals of Kowalevskia tenuis and two of K. oceanica weresectioned serially along various planes for detailed reconstructionof internal organs. Sections (1 �m thick) were cut on an LKBUltratome, stained with 1% toluidine blue, observed under aLeica DMR light microscope, and photographed with a JVC 3CCDanalogic videocamera. Thin sections (60 nm thick) were collectedon copper grids, treated with uranyl acetate and lead citrate, andexamined under a Hitachi H600 electron microscope (EM).

RESULTSGeneral Anatomy

Kowalevskia tenuis. The digestive system ofKowalevskia tenuis is greatly simplified. The round-ish mouth, without lips but endowed with tactilecilia, leads into a broad pharynx, which occupiestwo-thirds of the length of the trunk (Fig. 1A–C).The pharynx opens laterally through two spiraclesmodified in two long ciliated fissures (Fig.1A,B,D,E). The pharyngeal cavity is characterized

by the presence of an original system of ciliatedcombs, arranged in two pairs of longitudinal rows,one dorsal and one ventral, the opposing cilia ofwhich touch at several points. However, the pharynxwall does not have an endostyle or any kind of gland.In transversal section, the pharynx is more or lessrectangular in its anterior part and tends posteri-orly to enlarge into a trapezoidal shape with itslonger base upward, whereas the shorter base tendsto become more and more narrow so that the twoventral rows of ciliated combs, approaching, form agroove (Fig. 1D). Near the posterior end of the spi-racles, the pharynx floor widens and flattens and thepharyngeal cavity narrows before continuing intothe short, ventral esophagus (Fig. 1C). The latter,through a highly developed cardiac valve (Fig. 1C),is inserted ventrally into the stomach. The digestivenucleus is composed mainly of the wide, sphericalstomach, with a lumen about 120 �m in diameter onthe left side of the trunk, and the globular rectumwith a lumen of about 100 �m, on the right, con-nected to each other dorsally through a pyloric valvearranged transversally to the main axis of the trunk(Fig. 1F). The rectum opens externally through ananal papilla, situated dorsolaterally on the rightside. The digestive nucleus is kept hanging in thebody cavity thanks to contacts with the epidermis(Fig. 1C). The latter is very thin, except in the oiko-plast, which may contain one huge cell (about 150�m in its main diameter, with a nucleus up to 80 �mwide) (Fig. 1A–C,E).

Kowalevskia oceanica. In this species the phar-ynx is more reduced than in Kowalevskia tenuis,particularly when compared with the size of thetrunk (Fig. 2A–D). Unlike in K. tenuis, the spiraclesin K. oceanica extend into the central part of thepharynx and are situated ventrolaterally (Fig. 2A–C). The pharyngeal floor shows a pronounced con-vexity, clearly separated from the ventral epidermis(Fig. 2A), is densely ciliated, enlarging itself as itextends backwards (Fig. 2B–D), and lacks any traceof endostyle or ciliated groove. Thus, in the centralarea near the spiracles the pharynx has a U-shapedtransversal section; its roof rises anteriorly formingan acute angle (Fig. 2B) and enlarges posteriorly(Fig. 2C,D). Moreover, on the lateral edges of thepharyngeal floor, a pair of ciliated combs extendsalmost horizontally, in such a way as to touch thecilia of an opposing pair of combs situated on thelateral walls of the pharynx (Fig. 2B,C). The ciliatedspiracular cells are placed ventrally with respect toboth combs and median pharyngeal floor, defining achamber on each side of the trunk (Fig. 2B,C). Thesecells, which anteriorly are near the edge of the spi-racle, are posteriorly displaced more dorsally andbecome ventral again posteriorly to the closure ofthe spiracle itself. In this way, these cells form anarc in the sagittal plane (Fig. 2A). The body cavity,level with the spiracles, contains four large cells (upto 90 �m in diameter), two situated on each external

Fig. 1. Kowalevskia tenuis. A,B: Left lateral view of trunk ofwholemounted young (A) and aged specimen (B). Note series ofciliated combs (cc) within wide pharynx (ph), posteriorly, diges-tive nucleus (dn) and, in a young individual, the oikoplast in ananterodorsal position, characterized by a giant cell (goc), absentin the aged individual. Scale bar � 0.1 mm. g, gonad; m, mouth;s, spiracle; t, tail. C: Sagittal section. Wide pharynx (ph) showingthe 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 (arrow-heads) with the oikoplast (oik). The latter, characterized in thecentral area by a giant cell (goc) with a huge nucleus (n), secretesthe house in a compact form (house rudiments: hr) before inflat-ing it. Dotted lines indicate the level of the following transversesections (D–F). Scale bar � 50 �m. b, brain; g, gonad; pv, pyloricvalve. D–F: Antero-to-posterior serial transverse sections of thesame individual; anterior section (D), where teeth of four ciliatedcombs (cc) are visible in the pharynx (ph); at the level of the giantcell (goc) of the oikoplast (oik) (E) and of the digestive nucleus (F).The digestive nucleus is composed of the stomach (st) on theanimal’s right side and the rectum (r) on the left side, connecteddorsally by the pyloric valve (pv). Scale bar � 30 �m. c, cilia; ep,epidermis; g, gonad; hr, house rudiments; l, lipid droplets; s,spiracle.

227GUT OF KOWALEVSKIIDAE

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

of RER cisternae and basophilic granules of varioussizes.

At the posterior end of the pharynx the ciliatedcomb cells tend to protrude less and less into thelumen until they continue with the ciliated cells ofthe esophagus. The latter is very short and its lumen

Fig. 2. Kowalevskia oce-anica. A: Left lateral view of thetrunk of a whole-mount individ-ual. Note that the pharynx (ph)is less developed in comparisonwith the trunk length and con-vexity of ciliated combs (cc). Go-nads (g) growing in a large bodycavity behind the digestive nu-cleus formed by the stomach (st)and the rectum (r). Dotted linesindicate the level of the follow-ing transverse sections (B–F).Scale bar � 0.1 mm. m, mouth;oik, oikoplast; s, spiracle; t, tail.B–F: Antero-to-posterior serialtransverse sections of the sameindividual at the level of the an-terior opening of spiracles (s)(B); in the middle part of thepharynx (ph) (C) at the level ofthe giant glandular cells (ggc);posterior to spiracles (D); at thelevel of the long cardiac valve(cv) ventrally inserted into thestomach (st) under the rectum(r) (E); stomach–rectum connec-tion by the pyloric valve (pv) (F).Note in the last section a gastriccell is protruding externally tocontact the epidermis (arrow-head). Scale bars � 40 �m inB–D, 50 �m in E–F. a, anus; c,cilia; cc, ciliated combs; ep, epi-dermis; oik, oikoplast.

228 C. BRENA ET AL.

appears to be quite depressed. It opens ventrallyinto a wide, spherical stomach through a cardiacvalve (Fig. 2E), which is highly developed. Thespherical stomach is ventral, with a lumen about220 �m in diameter, and is directly connectedthrough a well-developed pyloric valve to the widerectum, also spherical and situated dorsally (Fig.2A,E,F). The pyloric valve forms a “canal,” which istransversal with respect to the anteroposterior axisof the trunk and placed on the left side, while therectum opens externally through an anal papilla onthe right side (Fig. 2F).

Both species lack a heart. The entire digestivesystem is bathed by hemolymph, which lacks bloodcells and moves due to the active beating of the tail.

UltrastructurePharynx. This region, except for sensory cells not

analyzed in detail here, is composed of four celltypes: “unciliated cells,” very thin and simple, oftenrepresenting only a connection to the other celltypes; “ciliated cells” and, as possible modificationsof the latter, “comb cells” and “spiracle cells,” bothcharacterized by very long cilia (Fig. 3A,B). Theunciliated cells, in particular, extend in the form of athin layer on the lateral surface of both spiracle andcomb cells (Fig. 3A,B). Every comb tooth is composedof a single, elongated ciliated cell with a basal spher-ical nucleus, a great abundance of fibrous materialunderlying the lateral plasmalemma, and a markedapical bundle of cilia (Fig. 3A). The striated rootletsof the cilia are particularly extended, occupyingmost of the cytoplasm, which is filled with mitochon-dria in its basal area (Fig. 3A). A similar patterncharacterizes the spiracle cells, although, in thiscase, the cells are not so elongated and are groupedin at least three rows (Fig. 3A,B). Moreover, thestriated rootlets, besides connecting to the lateralmembranes, often reach the base of the cell. Theseciliated cells are bound to each other and to theunciliated cells by apical electron-dense junctions(inset in Fig. 3B) and numerous gap junctions. Thespiracle cells of Kowalevskia oceanica are compart-mentalized into: 1) a basal third, rich in RER; 2) acentral area, particularly rich in mitochondria andlarge vacuoles; and 3) an apical region full of stri-ated rootlets (Fig. 3B). In the apical area external tothat of the ciliary insertion of some spiracle cells, weidentified a gathering of numerous sphericalelectron-dense granules (Fig. 3B and inset).

Esophagus. In Kowalevskia tenuis the shortesophagus is characterized by ciliated cells continu-ous with the ciliated ones of the pharynx. The esoph-ageal cells are cuboid and possess a few, thin mi-crovilli and a basal spherical nucleus. The cytoplasmis rich in free ribosomes, although the endoplasmicreticulum is scarcely present, whereas mitochondriaand rare vacuoles are scattered. Cilia, directed to-wards the stomach, are inserted obliquely into the

apical cytoplasm with a large basal body providedwith short striated rootlets, which are directed to-wards the lateral plasmalemmata. The latter arealmost straight and have numerous gap junctions.

In Kowalevskia oceanica the esophagus is widerand characterized by larger ciliated cells than in K.tenuis, clearly distinct from pharyngeal cells (Fig.3C). These cells possess many characteristics com-mon to those of K. tenuis, although the striatedrootlets of the cilia are normally not so extended.

The esophageal cells possess a thin, dense, apicallayer resembling a terminal web adhering to theplasmalemma, and are also characterized by a re-markable fuzzy coat covering the cilia and the mi-crovilli; finely granular material, secreted by thecells, is attached to the fuzzy coat.

Cardiac valve. This specialized region is formedof the union of very long cilia, arising in great num-bers from a single ring of cells (Fig. 3C). The latterare similar to the esophageal ones but lack mi-crovilli, and their cilia closely lean upon each other,completely occupying the luminal surface of the cell(Fig. 3E). The cilia are regularly glued together toform a very compact lamina, 4–6 ciliary rows thick(Fig. 3D,F). In Kowalevskia tenuis, the precise jux-taposition of the plasma membranes of adjacent ciliaforms a honeycomb structure in transversal section(Fig. 3D) and, on the luminal surface, the lamina iscovered by a hard multilayered fibrous material,which is highly electron-dense (Fig. 3C,D). In K.oceanica, the closer adhesion of the ciliary mem-branes implies their partial union, but the lamina isnot reinforced by any extracellular material (Fig.3F). The cilia of the lamina are inserted almoststraight into the cardiac valve cells and, besideswell-developed basal bodies, have very long striatedrootlets which, crossing the cytoplasm rich in mito-chondria, reach the base of the cell. The cardiaclamina, directed towards the stomach, bends and isattached to the proximal cells of the stomachthrough an electron-dense fibrous layer (Fig.3C,D,F). The latter cells, with little cytoplasmic con-tent, are greatly reduced in thickness and in factform a thin coat supporting the cardiac lamina (Fig.3C,G). They are also connected to the stomach onesthrough extensive apicolateral junctions (Fig. 3G).At the entrance to the stomach, the lamina forms aseries of wide folds (Figs. 2E, 3G), which are partic-ularly tortuous in K. tenuis.

Stomach. In Kowalevskia tenuis this region hasfewer 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 inthickness. They lack both cilia and microvilli andthe luminal surface in K. tenuis is moderately un-dulated by a series of “bubbles.” These curious struc-tures are marked by dense, finely granular material,often inhomogeneous (Fig. 4C), resembling the con-tinuous dense fibrous layer attached to apical plas-

229GUT OF KOWALEVSKIIDAE

Figure 3

230 C. BRENA ET AL.

malemma. This layer is similar to that of esophagusand actin-like filaments, 5–7 nm in diameter, arerecognizable among its fibers. It is attached to theentire apical plasmalemma, including the lateralportion involved in the formation of the junctionalbelt (Fig. 4A). The latter, in K. oceanica, follows theentire profile of the apical interdigitations betweenthe small cells next to the pyloric valve facing therectum (Fig. 5A). This belt appears to be accompa-nied by intercellular apicolateral junctions with anunusual aspect, since they have the characteristicsof gap junctions in that the two opposing mem-branes associate closely, as occurs in typical gapjunctions. In both the cases the associated mem-branes form a sort of straight plate 6 nm thick, withthe intercellular cleft extremely reduced and diffi-cult to resolve. In addition, in both the cases thedistance between the inner layers of the two oppos-ing membranes is about 10 nm. On the other hand,these apicolateral junctions recall the adherensjunctions in that they form a belt and show accumu-lation of fibrous material on both sides of the cyto-plasm (inset a, Fig. 4A). Below these junctions arealso 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 inshape and normally in a central position. The cyto-plasm contains many free ribosomes; the RER ispoorly developed. We did not identify typical Golgicomplexes. Mitochondria are abundant and particu-

larly concentrated in the basolateral area of the cell,sometimes associated with the deep membrane in-foldings which characterize both the whole basalside and partly the basolateral one of all the gastriccells (Fig. 4A,B). These infoldings have a constantextracellular space which, in Kowalevskia tenuis, isabout 13 nm across and contains electron-densespots. Scattered, membrane-bound, roundish gran-ules, with homogeneous, strongly electron-densecontent are visible near the apical plasmalemma(Fig. 4A); however, we never observed signs of endo-cytosis. Several cells contain lipid droplets a fewmicrometers in diameter, occasionally present inhigh numbers inside the same cell (Fig. 1F), but wealso sometimes found relatively large lipid droplets.Some cells of the stomach protrude extensively intothe body cavity, making contact with the epidermalcells. The latter are reduced to a thin layer (Figs. 1C,2F), and extended gap junctions are often recogniz-able in areas of connection between them and thegastric cells (Fig. 4D).

Pyloric valve. This connects stomach and rec-tum and comprises three rings of cells (Figs. 1F, 2F,5C). The first two are smaller and irregular inshape, forming a hinge that supports the third cellring; their cells have cytoplasm similar to that of thegastric cells but they bear long cilia, which are in-serted into the cell very obliquely, are directed to-wards the rectum, and possess long striated rootletsoccupying the apical third of the cell. In Kowalevskiatenuis, on the cell surface of the first ring are thesame electron-dense bubbles recognized on the gas-tric cells.

The cells of the third ring (main or distal cells) aremuch wider than those of the first two and also havethin microvilli (Fig. 5C). Their cilia are insertedvertically and the striated rootlets, althoughshorter, interconnect with each other and extendlike an upside-down fan (Fig. 5B). The sphericalnucleus is central. The pyloric valve cells havestraight lateral membranes and their cytoplasmcontains abundant ribosomes and a highly dense,subapical layer. Only in Kowalevskia tenuis, afterthe pyloric valve, is there a short ring of a few, verysmall cells, poor in cytoplasmic organelles and ex-tensively interdigitated, both between each otherand, apically, with the first cells of the rectum (Fig.5C). They do not possess microvilli or cilia and lackthe subapical dense layer. All along the contactingsurface between cells wide gap junctions and partic-ular, diffuse adherens-like junctions are present.

Rectum. In Kowalevskia tenuis, this region iscomposed of about 20 cells and in K. oceanica ofabout 30. They are morphologically uniform in thesame species, large and comparable in size withthose of the stomach in K. tenuis, and larger (up to110 �m), albeit always of limited thickness, in K.oceanica (Figs. 1F, 2E,F, 5D). The rectal cells resem-ble the gastric ones in their cytoplasmic features,but differ due to the presence of long cilia among

Fig. 3. A,B,E–G: Kowalevskia oceanica. C,D: Kowalevskiatenuis. 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 arenext to spiracle cells (SC), both having cilia with long abundantstriated rootlets (sr), and are laterally covered by a monolayer ofcontiguous unciliated cells (uc and arrows). Scale bar � 2 �m. bc,body cavity; ph, pharynx. B: Spiracle ring in transverse section iscomposed 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 ininset) are present apically, lateral to striated rootlets. Lateralcovering unciliated cells (uc) are attached to the spiracular cellsby 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 microvillarcells (EC), with cilia showing developed striated rootlets (sr) (C).The cardiac valve lamina (arrows in C) is formed of unions of 4–8rows of cilia (c) (D–F), arising from a single cell ring (CVC) andexhibiting long striated rootlets (st) associated with mitochondria(mt). Cilia keep their membranes distinct to form a honeycombstructure in K. tenuis (D) but partially fuse in K. oceanica (ar-rowheads in F). Lamina in K. tenuis is reinforced on the luminalside by multilayered, electron-dense fibrous material (arrow-heads and D). In both species, laminae are attached through adense layer (arrows in D and F) to thin proximal gastric cells(PGC); the latter are attached to normal gastric cells (GC) bydeveloped apical junctions (arrow in G). Within the lumen of thecardiac valve lamina (cvl), food particles (fp) are recognizable asfar 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 �min F, 2 �m in G. bc, body cavity; ph, pharynx.

231GUT OF KOWALEVSKIIDAE

Fig. 4. Stomach. A,D: Kowalevskia oceanica. B,C: Kowalevskia tenuis. The stomach is composed of large cells each bearing a centralnucleus (n), many scattered mitochondria (mt), some associated with basolateral membrane infoldings (bmi in A,B), dense granulesnear the apical plasmalemma (arrows in A), and an electron-dense layer on the apical surface. This layer connects and surroundsparticular apical junctions between contiguous cells with characteristics common to both gap and adherens junctions (arrowheads ininset a of A and in C). The apical cell surface exhibits characteristic electron-dense bubbles (arrows in C) in K. tenuis. Contiguousgastric cells (GC) are interconnected by extended and numerous gap junctions (arrowheads in A and inset b of A), also present betweensome 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.

232 C. BRENA ET AL.

scarce, rod-like microvilli. The cilia, inserted verti-cally into the cell, have a well-developed basal bodyand short striated rootlets (inset, Fig. 5D). The dis-tribution pattern of the basal membrane infoldingsis similar to that of the stomach, with similar extra-cellular space of about 13 nm between infoldings andthe association with the mitochondria. In the cyto-plasm, we observed some multivesicular bodies andvesicles. Moreover, in the rectal cells small vesiclesfrom about 0.1–0.4 �m in diameter and containingelectron-dense, granular material inside a uniformmatrix often occur. In K. oceanica the giant dorsalrectal cells contain numerous mitochondria, veryelongated and parallel to the lateral cell walls (Fig.5D). The contiguous cells form numerous gap junc-tions all along the lateral plasmalemmata.

The rectal lumen is packed with the fecal pellet, inwhich several microalgal cells are recognizable, stillpartly undigested and varying greatly in size, up to110 �m maximum diameter (Fig. 2F).

At the end of the rectum the few anal papilla cells(Fig. 2F) contain large amounts of intracellular fi-bers and extensive, convoluted interdigitations,marked by both dense adherens-like junctions andrare gap junctions (Fig. 5F).

Stomach–Rectum Connection. The gastric andrectal cells contact each other with their basal sur-face in the medial part of the digestive nucleus (Figs.1F, 2E,F, 5E). Here, the thickness of the opposingcells may be very low, sometimes down to 3.5 �m. Allalong the contacting surface the opposing mem-branes interdigitate, forming a wide loop in Kowa-levskia tenuis but not in K. oceanica (Fig. 5E). Inboth species the opposing cells are in communicationwith each other through numerous gap junctions(Fig. 5E), sometimes remarkably extended. Overmost of the contact the surface devoted to the gapjunction is greater than the nonjunctional one. In K.tenuis, we also identified some adherens-like junc-tions.

DISCUSSIONPharynx

First observed in vivo and described in its generalmorphology by Fol (1872), the genus Kowalevskiahas not been described in detail since then. Themain difference that characterizes the general as-pect of these animals in comparison with other ap-pendicularians is the specialization of the pharynx.The latter stimulates particular interest in under-standing its functions mainly because, for example,it lacks an endostyle. In comparison with other tu-nicates, the endostyle of Oikopleuridae and Fritil-lariidae is generally very reduced, in both number ofcell types and dimension. In oikopleurids the en-dostyle has been described by Olsson (1965) and itsfunctional activity in producing the pharyngeal se-cretion for particle agglutination has been demon-strated (Fenaux, 1968, 1989; Deibel and Powell,

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 com-pacted (Martini, 1909) and its opening into the phar-ynx floor is short, almost punctiform. Nevertheless,the presence of both ciliated and secreting cells im-plies an important role in collecting food particles atthe entrance to the pharynx. In contrast, our anal-ysis of complete sequences of thick sections, togetherwith observations at the ultrastructural level, didnot allow us to identify any glandular structure be-longing to or directly opening into the pharynx, sothat, in Kowalevskia, food particles cannot be agglu-tinated by a mucous-like substance. As no secretoryduct can be seen near the large gland cells externalto the pharynx in K. oceanica, they may release theirbasophilic granules in hemolymph or through theepidermis, with which they are closely in contact, asin the oral glands of Oikopleura (Flood and Deibel,1998). Thus, pharyngeal filtering of food seems todepend only on the mechanical activity of the cilia ofthe comb cells, while swallowed water is, as in otherappendicularians, driven outside by the spiraclecells.

Although distinct, the elements of the pharynxmay be compared with those of other appendicular-ians. The monocellular teeth of the ciliated combprobably represent a deep modification of the peri-coronal arches of other tunicates, and the oblongspiracles result from the enlargement of the spi-racles of the commonest appendicularians. It is note-worthy that very large, ellipsoid spiracles have ap-peared more than once in the evolutionary history oflarvaceans, as they are also present in oikopleurids,particularly in the genus Mesochordaeus.

Our ultrastructural data reveal great activity ofthe cilia of both ciliated combs and spiracles, as thecilia are densely packed and their long striated root-lets are associated with abundant mitochondria.Moreover, the ciliated comb cells are supported lat-erally by laminar unciliated cells, with which theyare connected apically by electron-dense junctions.All these features suggest that these cells supportstrong mechanical stress. Following the observa-tions of Fol (1872) and Fenaux (1968) on living ani-mals, when food particles reach the pharyngeal cav-ity they are probably trapped by the comb system,since the cilia of opposing comb teeth, coming intocontact, make a sort of continuous filtering barrier.In this way, as water is driven outside by the ciliaryactivity of the spiracles, food particles tend to accu-mulate on the floor of the pharynx, which has theform of a groove in Kowalevskia tenuis, and are thentransported backwards to the esophagus, thanks tothe action of the ciliated cells. In K. oceanica, thepharynx has a quite different shape, although itsmain elements are clearly comparable with those ofthe other species; in particular, we may consider itsaspect as the result of a simple elevation of its floor,causing lateral translation of the combs.

233GUT OF KOWALEVSKIIDAE

Figure 5

234 C. BRENA ET AL.

Esophagus and Cardiac Valve

The large volume of the pharynx, together withthe abundance of ciliated cells, accompanies thegreat reduction of the esophagus, which has fewciliated cells and represents a simple channel con-necting the pharynx and the well-developed cardiacvalve. Fenaux (1968) ascribed the function of ag-glomeration of food particles to the esophagus as aconsequence of the forward and backward move-ment caused by the contrasting action of the cilia ofboth spiracles and esophagus. The cardiac valve,composed of very long cilia joined to each other toform a lamina and arising from a single ring of cells,seems to represent a variation on the theme of thecardiac valves of fritillarids: the reinforcement onthe luminal side of the lamina of Kowalevskia tenuismay be homologous to the far less developed situa-tion seen in both Fritillaria formica (Brena et al.,2003) and F. borealis (unpubl. data). The cilia areclosely compacted together—particularly in K. oce-anica, where the lateral plasmalemmata betweencilia seem to disappear. Both the compactness andthe considerable length of this valve suggests fun-damental functions in regulating access by food par-ticles to the digestive nucleus and also in preventingreflux of food to the pharynx, since the laminae couldcollapse together in the event of reverse flow.

Digestive Nucleus

Gastric cells completely lack both cilia and mi-crovilli and do not show recognizable features ofendocytosis or secretion. The role played by theelectron-dense bubbles observed on the apical sur-face of the gastric cells of Kowalevskia tenuis is notevident. They do not seem to be an artifact, since wehave frequently found them in various specimens of

this species and only in that specific region. Consid-ering their size, they may represent a system toexpand the surface for absorption, instead of themicrovilli, rather than a simple ornamentation ofthe epithelial surface.

The pyloric valve is very similar to that in fritil-larids, although not so structurally complex. As infritillarids (Brena et al., 2003), it comprises threerings of cells, of which the distal one (main or distalpyloric valve cells) has the longest and the mostabundant cilia. As reported for living specimens byFol (1872), this valve probably operates as in Fritil-laria pellucida (Fenaux, 1961): the long cilia of themain pyloric valve cells collect food by brushing thegastric lumen and, after complete rotation on theadjacent cell rings, which act like a pivot, transfer ittowards the rectal lumen. In Kowalevskia tenuis,this valve is followed by a short ring of cells withelectron-dense apical junctions. This pattern clearlyrecalls that of the distal part of the proximal intes-tine of Fritillaria (Brena et al., 2003), to which thiscell ring may therefore be considered homologous. K.oceanica completely lacks this short tract, so thatthe connection between stomach and rectum onlyoccurs through the pyloric valve.

As in the fritillarid rectum, we suggest that, alsoin kowalevskids, this may be the gut region in whichfood stays longest. It is known that in Fritillariafood is removed from the gastric lumen and accumu-lated in the rectum every 1–2 min, thanks to theactivity of the pyloric valve cilia (Fenaux, 1961).Thus, the globular rectum should be the main placefor both digestive and absorptive processes. Thepresence of microvilli supports the hypothesis of theabsorption capability of small molecules and ions bythe rectal cells on the contents of fecal pellets, inwhich extracellular digestion probably occurs. In ad-dition, more than in fritillarids, stomach and rectumare interconnected medially by very long, abundantgap junctions, which allow the exchange of smallmolecules between the two compartments. This typeof communication is indeed frequent in many con-tacting cells of the gut epithelium, and also in somebasally protruding gut cells and the epidermis. Theefficiency of the absorptive function of the kowa-levskid gut is also indicated by the fact that it canaccumulate large quantities of lipid droplets—anaspect common to other appendicularians (Burighelet al., 2001; Cima et al., 2002; Brena et al., 2003)—and which may allow these organisms to withstandbrief periods of food shortage. The fact that we neverfound signs of endocytosis and a considerable lipidstorage was detected may be explained by the par-ticular physiological stage of the specimens we ex-amined. On the other hand, the membrane-boundgranules found in the apical region of the gastriccells may participate to a merocrine secretion ofdigestive enzymes, as suggested by their morpholog-ical similarity with the zymogen-like granules insidethe gastric cells, previously observed in both oiko-

Fig. 5. A,B,D-E: Kowalevskia oceanica. C: Kowalevskiatenuis. A: Gastric cells near the pyloric valve are characterized byapical interdigitations associated with well-developed apicaljunctions (arrows). Scale bar � 0.2 �m. B,C: The pyloric valve iscomposed of three cell rings, of which the main or distal one (DPCin C) is widest, with a central nucleus (n) and very long ciliaamong microvilli that are characterized by a remarkable basalbody (bb in B) and fan-shaped striated rootlets (sr in B). In K.tenuis, a few very small cells (arrow in C), extensively interdigi-tating with each other, amid distal pyloric cells and the mostproximal rectal cells (RC in C). Scale bars � 0.4 �m in B, 2 �m inC. bc, body cavity; gl, gastric lumen. D: The rectum is composedof 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 evidentbasal bodies (bb) and poorly developed striated rootlets (sr). Scalebars � 2 �m and 0.4 �m in inset. ep, epidermis. E: Abundant gapjunctions (arrowheads) are evident along contacting surfaces be-tween gastric (GC) and rectal cells (RC). Scale bar � 0.2 �m. gl,gastric lumen. F: Anal papilla cells are characterized by numer-ous intracellular fibers (black arrowheads) and deep interdigita-tions with frequent, electron-dense junctions (arrows) and occa-sional gap junctions (white arrowhead). Scale bar � 0.4 �m.

235GUT OF KOWALEVSKIIDAE

pleurids (Burighel et al., 2001) and fritillarids(Brena et al., 2003). These organisms might alter-nate periods of digestive enzyme synthesis and se-cretion with periods of nutrient absorption and stor-age; in this respect, it is noteworthy that gastriccells in a different state of digestive enzyme activityhave been found in various specimens of F. pellucida(Brena et al., 2003).

Comparative Considerations

As noted by Fenaux (1968), kowalevskids areunique among all tunicates in their feeding mecha-nism, due to the absence of endostyle and any se-creting pharyngeal organs, the great development ofthe pharynx, and of the complex filtering systemassociated with it, i.e., the ciliated combs. In partic-ular, the uniqueness of the pharynx seems to repre-sent an apomorphy of this family. However, as re-gards the digestive nucleus, the kowalevskids showsimilarities with fritillarids, e.g., stomach and rec-tum are the two major cavities in both Fritillaria(Brena et al., 2003) and Kowalevskia (Fig. 6) and areconnected by a well-developed pyloric valve. Thisindicates that the two families are evolutionarilyclose and separated for a long time from the oiko-pleurids, the latter probably being the most primi-tive extant family, which shows distinctive charac-teristics, such as the presence of well-developed guttracts 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 gutloop disposition, considered both fritillarids andkowalevskids as modified secondarily with respectto oikopleurids. Indeed, kowalevskids and fritillar-ids share some signs of apomorphy, like the similarreduction of both gut regions and cell types, theoccurrence of species with no heart, and the extremethinning of the epidermis in some areas of the trunk(Bone et al., 1977; Brena et al., 2003).

On the other hand, kowalevskids show furtherspecialization at the ultrastructural level. In com-parison with fritillarids, they have the proximal in-testine reduced that lacks the mitochondrial pumpcells which, in the genus Fritillaria, are consideredimportant for transmembrane transport and os-motic regulation of body fluids owing to their fre-quent association with mitochondria, as well as ami-nopeptidase M, 5�-nucleotidase, and Mg2�-ATPaseactivities (Brena et al., 2003). However, Kowalevski-idae, like Fritillariidae, are rich in basolateral in-foldings along both the stomach and the intestine,which greatly increase the membrane surface andare often associated with mitochondria. These char-acteristics are typical of ion-transporting epitheliaas described in the gill of crabs (Luquet et al., 2002),in vertebrate salt glands (Pease, 1956), and in themammalian vestibular system (Pitovski and Kerr,2002). In appendicularians, three kinds of structuralmorphologies were found indicating the capacity of

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, leftgastric lobe; m, mouth; mi, mid-intestine; pi, proximal intestine; r, rectum; rs, right gastric lobe; sp, spiracle; st, stomach; vi, verticalintestine.

236 C. BRENA ET AL.

gut epithelium to regulate ion or fluid transports, allbased on membrane extensions commonly associ-ated with mitochondria. Increase of membrane sur-face occurs 1) in oikopleurids, through basolateralcell interdigitations; 2) in fritillarids, through basalinfoldings and mitochondrial pump cells; and 3) inkowalevskids, through basal infoldings. Thus, fritil-larids and kowalevskids share the presence of dif-fuse basal infoldings; it remains difficult to establishif, in kowalevskids, the complete absence of mito-chondrial pump cells is an apomorphy, or if theynever developed them. In this family, osmoregula-tion may also be supported by their exclusive, spe-cial apical intercellular junctions, particularly de-veloped in gastric cells. The abundant fibrousmaterial surrounding the junctions suggests a con-comitant function of cell linkage, as previously hy-pothesized for the shorter, punctiform, apical tightjunctions of Oikopleura (Martinucci et al., 1990;Burighel et al., 2001). Similar to these latter junc-tions, the particular junctions of kowalevskids pre-sumably play a sealing role separating the body fluidfrom the gut lumen content. Although it is difficultto establish the true characteristics of these junc-tions in the absence of freeze-fracture studies, wethink that these junctions resemble a kind of gapjunction observed in ascidians, which is accompa-nied by dense fibers associated with its junctionalcytoplasmic surfaces, and which is considered toplay a double role in allowing exchange of ions andsmall molecules and mechanical adhesion between

contiguous cells (Lane et al., 1995). The function ofmechanical adhesion is evidenced by the fact thatthese junctions are particularly extended along ar-eas of higher mechanical stress throughout the ali-mentary canal, i.e.: i) along the contact of proximalgastric cells with the adjacent main gastric cells,where mechanical stress is caused by the transit offood from the cardiac valve; ii) along the interdigi-tations of the cells preceding the pyloric valve,where mechanical stress may be caused by the ex-tensive movements of the pyloric cells; and iii)among the anal papilla cells, where stretching iscaused by the passage of the fecal pellet as it isevacuated.

The whole of the characteristics of the gut of thethree families of appendicularians, as shown in Fig-ure 6 and Table 1, suggest that kowalevskids aremore closely related to fritillarids than to oikopleu-rids. However, the special adaptations in kowa-levskids, probably linked to the modality of foodparticle filtering, required new specializations,which caused a clear-cut evolutionary parting fromtheir common ancestor.

ACKNOWLEDGMENTS

The authors thank Dr. P. Flood of the Bathybio-logica A/S (Norway) and Dr. G. Gorsky of the StationZoologique of Villefranche-sur-Mer for facilities incollecting kowalevskids. The English text was re-vised by G. Walton.

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

Gut region Oikopleuridae Fritillariidae Kowalevskiidae

Esophagus �� �� �CMC CMC CMC

ft, sc, or ft, sc, dg, ab ft, scCardiac valve �� �� ��

CC CC CCftr (passive) ftr (active) ftr (active ?)

Stomach �� (bilobed) �� ��CMC, GC, GBC MC UCft, ab, dg, ns, or ab, dg, ns, or ab(?), dg(?), ns,

orPyloric valve � �� ��

CC CC, CMCftr (active) ftr (active)

Proximal intestine �� � �CMC UC, MPC UC

ft, fpa, ns, or orMid-intestine �� � �

CMCft, ab, (ns), or

Rectal valve �� � �CC, UC CMC

ftr (active) dg, ftr (passive)Rectum �� �� ��

CMC, GC CMC CMCft, ab, dg, ns, or ft, ab, dg, ns, fpa, or ft, ab, dg, fpa, or

� absent; � poorly extended; � present; �� relatively well extended. CC, ciliated cells; CMC, ciliated microvillar cells; GBC, gastricband 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, osmoticregulation, sc, granular material secretion.

237GUT OF KOWALEVSKIIDAE

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