0044-5231/03/242/02-169 $ 15.00/0

The Exceptional Blind Gut of Appendicularia sicula(Appendicularia, Tunicata)Carlo BRENA, Francesca CIMA and Paolo BURIGHEL

Dipartimento di Biologia, Universit di Padova, Padova, Italy

Abstract. In this work, we studied for the first time the histology and ultrastructure of the gut of Appendiculariasicula and demonstrated the absence of any trace of anus. Appendicularians are small holoplanktonic tunicates,characterised by very fast ingestion and quick food transfer along their gut. The high production of faecal pelletsreleased in the aqueous environment, associated with a high filtration rate, highlights their important role inmarine ecosystems. Due to the absence of an anus, in contrast with other appendicularians, A. sicula, one of thesmallest species, accumulates undigested faecal material within its body, with consequent extreme enlargement ofits rectum. The gut, the epithelium of which is generally extremely reduced, is formed of an oesophagus, a globu-lar stomach, thin proximal and mid-intestine, and a huge rectum. The latter, when filled with faecal material, mayoccupy most of the volume of the trunk in fully grown specimens. Although profoundly altered, the gut of theseanimals does show several similarities to that of Fritillaria (a genus of the same family, Fritillariidae), with whichit has in common many features such as specialised mitochondrial pump cells. In A. sicula, the structural simplifi-cations of organs seem to reach their extreme condition in comparison to other appendicularians.

Key words. Absence of anus, basal infoldings, food accumulation, Fritillariidae, mitochondrial pump cells, organreduction.

1. INTRODUCTION

Appendicularia sicula (Fritillariidae family) is a usual-ly scarce species of the class Appendicularia (Chorda-ta, Tunicata), distributed in all warm and temperateseas. Appendicularians are common, small planktonictunicates, characterised by a permanent chordate-liketail. They have a particular mode of nutrition, accom-panied by interesting specialisations in the morpholo-gy and functions of their alimentary tract and devicesfor food trapping. They use a complex gelatinous sys-tem, the house, in which they live, to collect foodparticles suspended in sea water. Particle sizes general-ly range from those of microalgal cells (200 to 20 m)to colloidal carbon particles (FLOOD et al. 1992; BEDOet al. 1993). Food is collected in the filtering nets of thehouse, and driven to the mouth by the water flow creat-ed by the ciliary beating of the spiracles. Being mainlycomposed of microalgae with a cellulose wall, the foodpasses quickly along the digestive system, and is gen-erally expelled at a high frequency in the form of fae-cal pellets (ACUA et al. 1999; LOPEZ-URRUTIA &ACUA 1999). Their great ability in both filtering large

amounts of sea water and reintroducing into the aquat-ic environment many undigested remnants availablefor other ecological compartments, highlights the keyrole of appendicularians in marine ecosystems (ALL-DREDGE 1981; DEIBEL 1988; GORSKY & FENAUX 1998).The digestive system has been demonstrated to be dif-ferently organised in the two families previously stud-ied, i.e., Oikopleuridae (BURIGHEL et al. 2001; CIMA etal. 2002) and Fritillariidae (BONE et al. 1979; BRENA etal. 2003). In particular, these results revealed interest-ing morphological specialisations of the gut, related tothe ability to process ingested food rapidly, with theproduction and expulsion of faecal pellets.In this respect, Appendicularia sicula differs from theother appendicularians described until now, in that it isconsidered to lack an anal aperture. Although in hisfirst original description FOL (1874) presented A. sicu-la as possessing an anus, ESSEMBERG (1924) reportedthe impossibility of faecal pellet evacuation and thepresence of a huge rectum filled with faecal material.After these descriptions, this species, never studied indetail, has been reported as lacking a visible anus(FENAUX 1967, 1998). Despite this exceptionality, no

Zool. Anz. 242 (2003): 169177 by Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/zoolanz

170 C. BRENA et al.

Fig. 1. Appendicularia sicula. Left lateral view of trunk of a wholemounted individual. Huge faecal pellet inside rectum (r)already visible from outside, thanks to transparency of animal trunk; tail (t) arises ventro-medially from trunk. g = gonads, m =mouth, ph = pharynx. Scale bar = 50 m.

The Exceptional Blind Gut of Appendicularia sicula 171

specific analyses of this species have been conducted.The characteristic of a possible blind gut appears tobe interesting, since it raises questions on possiblemorpho-physiological specialisations. We have anal-ysed in detail optical and ultrastructural serial sectionsof these animals, with the aim of describing the fea-tures of the digestive canal and comparing them withthose of previously studied species.

2. MATERIALAND METHODS

Specimens of Appendicularia sicula Fol, 1874 were collect-ed in the bay of Villefranche-sur-Mer (France), in summer1999 and 2000.Whole animals were selected and 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 inEpon for sectioning. Some individuals were sectioned inserial thick sections (1 m) in different planes, for detailedreconstruction of internal organs. Thin sections were collect-ed at opportune, different levels. Thick sections were cut on aLKB Ultrotome, stained with 1% toluidine blue, observedunder a Leica DMR light microscope, and photographed witha JVC 3CCD analogic videocamera. Thin sections (600 )were stained with uranyl acetate and lead citrate and exam-ined under a Hitachi H600 electron microscope (EM).

3. RESULTS

Appendicularia sicula (Fig. 1) has a short, pyriformtrunk (between 0.25 and 0.4 mm in length), dilated inits genital-digestive part. The alimentary canal has pre-viously been described by FENAUX (1967). It has asmaller pharynx than that of other appendicularians. Ahorizontal oesophagus outlets through a cardiac valve

into a globular stomach, placed on the left side of thetrunk. A narrow intestine, which arises dorsally fromthe stomach, may be divided into proximal, mid andterminal intestine or rectum. In its proximal part, itextends transversely with respect to the major axis ofthe trunk and then turns backwards to flank the huge,pyriform rectum and inserts posteriorly into it; the ter-minal part of the rectum approaches the oesophagus,so that the whole intestine forms a loop. All our speci-mens, which had no mature gonads, appeared oldenough to have the digestive tract completely alteredby the presence of faecal material, which entirely filledthe highly expanded rectal lumen. In this extreme con-dition, identification of the characteristics of all the guttracts, i.e., their shape, course and cytology, was verydifficult under the LM, even with complete series ofsections, but was possible at the ultrastructural level.Unlike other fritillarids, the presence of a heart was notrecognised in either thick or thin sections.

3.1. Mouth and pharynxThe mouth, small and roundish, without protrudinglips, gives access to the pharyngeal cavity, which isdepressed and reduced in size. In the ventral wall of thepharynx, posteriorly to the mouth, the endostyle (Fig.2) lies short and enlarged in its proximal portion. Itsextremities are slightly bent upwards. The two roundspiracle openings, one on each side, are placed posteri-orly in the pharyngeal floor.

3.2. OesophagusThe narrow oesophagus (Figs. 2, 5) (about 60 m max-imum diameter) is formed of cuboid, partially flattenedciliated cells, about 10 m thick. They possess a cen-tral spherical nucleus. In the apical region of the cells,

Fig. 2. Medial sagittal thick section of trunk. Most of trunk is occupied by faecal material within large, pyriform rectum (r);anterior extremity of rectum (arrow) lacks anus; house rudiments (hs), produced by oikoplast (oik) present outside, both dor-sally and ventrally, on front of trunk. en = endostyle, m = mouth, oe = oesophagus, ph = pharynx, t = tail. Scale bar = 30 m.Fig. 3. Transversal thick section of trunk at level of cardiac valve (cv). Stomach lumen (st) continues in proximal intestineone (pi) through pyloric valve (arrowheads), on anterior part of rectum (r). Note food debris filling whole gut lumen, morecompacted in rectum. Distal extremity of rectum (arrow) lacks any trace of aperture. MPC = mitochondrial pump cells, oik =oikoplast, t = transverse section of tail. Scale bar = 30 m.Fig. 4. Detail of Fig. 3 showing cardiac valve (cv) regulating passage towards stomach (st) and, right, epithelium of rectum (r)reduced to a thin layer (arrows) in its distal part. Scale bar = 10 m.Fig. 5. Oesophagus. Ciliated microvillar cells with a central nucleus (n). Note presence of intercellular electron-dense materi-al (arrowheads) near cardiac valve; on ventral side, these cells directly contact thin epidermis (ep). Inset: a membrane-boundgranule. Scale bars = 2 m; 0.5 m in inset.Fig. 6. Cardiac valve. A single ring of cells bearing numerous very long cilia (c) forming a sort of tube defined by electron-dense material (arrowheads). bl = basal lamina, ep = epidermis, n = nucleus, r = rectum, st = stomach. Scale bar = 3 m.

172 C. BRENA et al.

cilia are abundant and inserted obliquely inside thecytoplasm through an electron-dense basal body, towhich striated rootlets are associated and directedtowards the lateral plasmalemma. The cytoplasm isrich in small, scattered RER vesicles; occasionally,some smooth vesicles and round granules with homo-geneous contents are present (inset in Fig. 5). Severalmitochondria are widespread in the cytoplasm. Thesub-apical plasmalemma is reinforced by a thin layerof electron-dense material. Contiguous cells are con-nected by wide, irregular interdigitations, with fre-quent gap junctions along the lateral surfaces. Near thecardiac valve, the intercellular space is filled withirregular deposits of highly electron-dense material.This material penetrates between cells, mainly at thebasal level. Some flat cisternae in the cytoplasm alsocontain similar electron-dense material. Thus, the latterprobably originates from the oesophageal cells, but noaspects of secretion were identified. In the ventral side,the oesophageal cells contact the epidermic cells,which are about 0.51.5 m thick.

3.3. Cardiac valveThe cardiac valve (Figs. 3, 4, 6) is constituted of 45cells, which are very similar to the oesophageal ones,except for that they lack microvilli and are endowedwith numerous, much longer cilia, directed towards thelumen of the stomach. These cilia are arranged in rowsand form a sort of tube, but they do not adhere to eachother to form laminae. Straight, short, striated rootletsare associated with the basal body. Golgi complexeswere recognised in the cells, although with few cister-nae. The external cilia of the valve tube lie on verysmall cells, interposed before the proximal stomachcells; the base of the ciliary tube is externally coveredby thin supporting cells, and a highly electron-denselayer lies between these cells and the cilia (Fig. 6).

3.4. StomachThe stomach is roundish in younger individuals, buttends to be crushed and pushed towards the trunkperiphery by the huge rectum in older individuals. It iscomposed of a few, flattened, large cells, which appearto be the thickest ones in the post-cardiac gut (about 45m thick) (Figs. 3, 4, 7). They lack cilia but possesslong microvilli. A thin, electron-dense, continuousfibrous layer extends, associated with the inner face ofthe apical plasmalemma. It is also in continuity withmicrofilaments penetrating the microvilli. The latter aredifferently distributed on the cell surfaces, being moreabundant in some gastric areas which, however, do notseem to show any other particular specialisations. In thecytoplasm, mitochondria are scattered and, in the basalarea, tend to associate with groups of basal membraneinfoldings; the latter possess an external cleft space ofabout 10 nm and characterise the main part of this cellarea, although they are not distributed according to aregular pattern. Some gastric cells are also characterisedby diffuse vesicles. The lateral plasmalemmata are quitestraight, and present frequent gap junctions betweencontiguous cells. In their apical region, the cells are alsojoined by highly electron-dense junctions, on the cyto-plasmic side of which a sub-apical fibrous layer isattached. The cells of the external wall of the stomachamply contact the epidermic cells, reciprocally inter-mingling their basal lamina (Fig. 7).

3.5. Pyloric areaAlthough the large amounts of gastric contents and tis-sue deformation do not allow us to identify a true valveat the passage between stomach and intestine (Fig. 3),ultrastructural observations do reveal a ring of flat-tened cells (from 5 to 0.5 m thick), which follow thegastric cells, lack microvilli, and are provided with

The Exceptional Blind Gut of Appendicularia sicula 173

Fig. 7. Stomach. Microvillar, flattened cells with scattered mitochondria (mt), some of which associated with irregular basalmembrane infoldings (arrowheads); the basal laminae (bl) of gastric and epidermic (ep) cells intermingle with each other; mi= microvilli. Scale bar = 0.5 m.Fig. 8. Pyloric area. Cells endowed with long cilia, inserted obliquely and characterised by electron-dense striated rootlets(arrowheads); lumen engulfed by food debris, in which microalgal cells are common (arrows). Scale bar = 2 m.Figs. 910. Mitochondrial pump cells. Microvillar cells characterised by dense association of several basal membrane infold-ings (bmi) associated with mitochondria (mt). Apical connections through electron-dense junctions (arrowheads, and detail ininset). bl = basal lamina, il = intestinal lumen, mi = microvilli, oik = oikoplast. Scale bars = 1 m in Fig. 9; 0.5 m in inset; 0.3m in Fig. 10.Figs. 1112. Rectum. Rectal cells (RC), flat in proximal part (Fig. 11), become very thin (arrows) in distal area (Fig. 12),where faecal pellet (fp) is tightly compacted and characterised by an external layer of fibrous material (fm) and an electron-dense core rich in debris of digested cells (arrowhead). ep = epidermis, mt = mitochondrion, oik = oikoplast, rl = rectal lumen.Scale bars = 1 m in Fig. 11; 0.4 m in Fig. 12.

abundant, long cilia (Fig. 8). In these cells, the cilia areinserted obliquely with their basal bodies and possesswell-developed striated rootlets which extend towardsthe lateral plasmalemmata.

3.6. Proximal intestineThe proximal intestine follows the pyloric area and ischaracterised by laminar cells, lacking cilia andmicrovilli (Fig. 3). These cells, about 4 m thick at thelevel of the nucleus, are generally extremely flat,sometimes reaching 80 nm in thickness. In severalareas, they tend to form thin, laminar protrusions,which bend onto the surface of the same cell. The foodin the lumen of this tract is distributed loosely.

3.7. Evagination of mitochondrial pump cellsIn the region in which the proximal intestine bendsbackwards, there is a roundish evagination of the walldefining a lumen (Fig. 3), the confluence of which withthe intestinal lumen was not unequivocally identified.This evagination is composed of a few cells, less than 4 m thick, devoid of cilia and provided with a few,small microvilli. These cells are characterised by greatnumbers of baso-lateral membrane infoldings, closelyassociated with each other and with abundant mito-chondria, clearly resembling the mitochondrial pumpcells described in the genus Fritillaria (BRENA et al.2003) (Figs. 9, 10). Contiguous cells are interconnect-ed sub-apically by extensive, highly electron-densejunctions (inset of Fig. 9).

3.8. Mid-intestine and rectumPosteriorly, the proximal intestine continues with themid-intestine, which forms a short passage charac-terised by cells lacking cilia and microvilli and havingreduced cytoplasmic contents. Generally, these cellshave an aspect with wide protrusions, variable thick-ness, and wide, loose interdigitations, in which theintercellular space is still evident (data not shown).Along these interdigitations, in the areas of contactbetween contiguous cells, extensive, highly electron-dense junctions are present, provided with long,fibrous extensions into the cytoplasm, close to thejunctional membrane. On the whole, these cells form asort of passive valve. The remaining mid-intestine ischaracterised by cells reaching, in some areas, a thick-ness similar to that of the gastric cells. They shareother analogies with the latter, such as the presence ofmicrovilli, the abundance of cytoplasmic organelles (inparticular, RER cisternae), and numerous bundles ofbasal membrane infoldings, sometimes associated withmitochondria. However, as a features distinguishing

them from gastric cells, they possess cilia, scattered inthe whole luminal surface and mainly inserted oblique-ly into the cell with dense basal bodies provided withshort striated rootlets. Posteriorly, where the lumennarrows, these cells reduce their thickness and presentmore and more rarefied cilia and microvilli, approach-ing the boundary with the rectum. The latter has wide,thin cells (about 1 m thick), with rare cilia andmicrovilli, and with cytoplasmic characteristics similarto those of the contiguous mid-intestine cells (Fig. 11).In the posterior area of the trunk, the whole luminalvolume of the rectum is occupied by the faecal pellet,which is more or less compact, with still clearly recog-nisable contents of debris and entire cells of microal-gae in an electron-dense mass of heterogeneous mate-rial. Anteriorly, where the rectum extends under theother gut tracts, crushing them (Fig. 3), the rectalepithelium tends to deteriorate and become reduced,reaching a minimum thickness of 60 nm in the terminalarea (Fig. 12). In this area, the faecal pellet is so dense-ly compacted that a terminal pellet may be identi-fied, distinct from the remaining main mass, by beingcharacteristically covered by many fibrous layers, likea peritrophic membrane (Fig. 12). However, at the endof the rectal lumen, no traces of opening and/or an analpapilla could be found.

4. DISCUSSION

Appendicularians are able to filter continuously largeamounts of sea water and food during their lifetime andfood, concentrated in the house filters, reaches the indi-vidual and passes along the alimentary canal in a veryshort time, down to about 8 min in Oikopleura dioica(ALLDREDGE 1981). Their gut seems to be regulated toprocess ingested food very quickly. However, intactmicroalgal cells have been found in the faecal pellets ofpreviously analysed species, i.e., O. dioica (DEIBEL &TURNER 1985; BURIGHEL et al. 2001; CIMA et al. 2002)and Fritillaria pellucida and Fritillaria formica(BRENA et al. 2003). It is particularly due to the con-stant emission of numerous sinking faecal pellets aswell as discarded houses that appendicularians pass onavailable nutrients to other trophic levels of the sea(GORSKY & FENAUX 1998; GONZALES et al. 2000).From this point of view, Appendicularia sicula is acurious exception, because it cannot expel faecal pel-lets owing to the absence of an anal aperture, asdemonstrated in an examination of complete series ofsections. The absence of an anus is not exceptional in the animalkingdom, particularly not in cnidarians and flatworms.There are also several cases of secondary evolutionaryclosure of the end of a functioning gut, e.g., in ophiu-

174 C. BRENA et al.

rans, articulate brachiopods, and some acari (Para-sitengona; EVANS 1992) and transitory blind guts occurin some insect larvae, as in the genera Myrmeleon andApis. According to ESSEMBERG (1924), Appendiculariasicula progressively accumulates and compacts thematerial ingested throughout its lifetime. This continu-ous accumulation of bulky faecal material implies anincrease in the volume of the rectum and progressivetissue shrinkage and dislocation of some gut tracts,especially intestine and posterior stomach, while phar-ynx, oesophagus and anterior part of the stomach areonly slightly altered.Almost nothing is known about the structure and filter-ing functions of the house in this species, except for itsapparently simplified architecture in comparison withthat of oikopleurids. In any case, particles may beselected by size at the mouth, which is particularly nar-row and rich in cilia. The only glandular structureobservable in the pharynx is the endostyle, which iscommonly involved in producing a pharyngeal filterenhancing food collection. When water enters thepharynx, thanks to the action of the spiracular cilia,food entrapped by endostylar secretions is driventowards the oesophagus. The main role of the latter,very narrow, is to transfer the food from the pharynx tothe stomach, although secretion of material synthe-sised in the RER and accumulating in membrane-bound granules may signal enzyme contents for extra-cellular digestion. The texture of these granules indi-cates that they contain proteins and resemble thezymogen-like granules previously described in thestomach of other appendicularians (BURIGHEL et al.2001; CIMA et al. 2002) and ascidians (BURIGHEL &CLONEY 1997), and, in particular, the oesophagus ofthe brittle star, Ophiuroiderma panamensis, whichlacks an anal aperture (SCHECHTER & LUCERO 1968).Access to the stomach is regulated by the cardiacvalve, similar to that described in Fritillaria (BRENA etal. 2003) both as regards cell type and arrangement in asingle cell ring, although in Appendicularia the cilia donot adhere to form well-compacted laminae. It is note-worthy that i) the cilia of the cardiac valve form a tubewhich, although not well-organised as in other fritillar-ids, has its cilia arranged in crowded rows and isenveloped at its base by supporting cells with a layer ofdense material; ii) the cardiac cells possess few mito-chondria, so that they are not involved in intense cil-iary activity; iii) the cells themselves and the cilia arearranged in such a way that they cannot find space forany kind of change permitting alternative ciliary beat-ing in opposite directions, as occurs in the pyloricvalve of the genus Fritillaria (FENAUX 1961; BRENA etal. 2003). All these observations suggest that a refluxof food from stomach to pharynx via the oesophagus isnot possible.

Together with the oesophagus, the stomach is appar-ently the only tract which seems capable of variousfunctions in the specimens analysed. The size of thistract is comparable with that of small fritillarids likeFritillaria formica (BRENA et al. 2003), and showssimilar morphological characteristics, i.e., microvillarcells, also suggesting similar functions, i.e., digestionand absorption, although typical aspects of secretionand endocytosis were not detected. As suggested forFritillaria (BRENA et al. 2003), the basal membraneinfoldings, although much less highly developed andorganised, may be implicated in fluid and/or ion trans-port towards body fluids.Most of the intestinal wall is so altered by the physicalpressure of the material engulfing the lumen and soreduced in thickness, that the main role of the intestineseems to be food accumulation, since ultrastructuralsigns of digestive and absorbing functions are difficultto identify. However, some correlations with the guttracts of Fritillaria (BRENA et al. 2003) may be pro-posed. In particular, as regards food transfer, the cellswith well-developed cilia of the pyloric area appear tocorrespond for their position to the complex pyloricvalve of Fritillaria (FENAUX 1961; BRENA et al. 2003),thus suggesting that they represent a vestige of apyloric valve. Moreover, referring to BRENA et al.(2003), who recognised only two intestinal tracts inFritillaria, on the basis of ultrastructural data and posi-tion, it can be proposed that the proximal intestine andcontiguous part of the mid-intestine of Appendiculariasicula correspond to the proximal intestine of Fritillar-ia, whereas the remaining part of the mid-intestine andrectum of A. sicula correspond to the rectum of Fritil-laria. Noteworthy is the evagination of the mitochon-drial pumps cells in the proximal intestine. This evagi-nation appears very similar in position and cytology tothe mitochondrial pump cells of Fritillaria (BRENA etal. 2003). In these cells, the conspicuous extension ofbasal infoldings associated with mitochondria may beconsidered to play the same osmoregulatory and excre-tion functions hypothesised for Fritillaria, throughactive transport of substances between the gut lumenand the haemocoele (BRENA et al. 2003). In this tract, asin most of the whole gut epithelium, the contiguouscells are joined apically by a sort of tight junction asso-ciated with dense cytoplasmic fuzz. As previously sug-gested for oikopleurids (MARTINUCCI et al. 1990) andascidians (MARTINUCCI et al. 1988), for A. sicula too theprobable functions of these tight junctions are those oflimiting fluid and/or ion passage between cells andkeeping the cells joined together. The latter functionseems to be particularly important in this species,which lacks, like other appendicularians, desmosomesin the gut epithelium and which has its epithelia partic-ularly distended owing to faeces retention.

The Exceptional Blind Gut of Appendicularia sicula 175

According to ESSEMBERG (1924), the accumulation offood in the rectal lumen occurs throughout the lifetimeof the animal and no signs of evacuation wereobserved, not even when living specimens were exper-imentally compressed. Our observations show that i)the lumen contents are heterogeneous and loose,except in the distal tract, where a thick envelope, simi-lar to the peritrophic membrane around the faecal pel-lets of other appendicularians (BURIGHEL et al. 2001) isrecognisable; ii) the rectal wall is extremely thin andsimple; iii) aspects of lipid droplets, often reported inother appendicularians (BURIGHEL et al. 2001; CIMA etal. 2002; BRENA et al. 2003) are never detected in thegut of Appendicularia sicula. Thus, it appears that themain function of the rectum is to store the food, whichmay undergo slow extracellular digestion there, withthe production of small molecules then absorbed by thecells and transferred to body fluids. The latter, in theabsence of the heart although very voluminous inFritillaria and erroneously reported as present by FOL(1874) in A. sicula may be moved by body move-ments caused by the muscular tail.The presence of particularly high amounts of organicmatter in the gut lumen is indicative of slow assimila-tion efficiency, as a difference with the reports on otherappendicularians (DEIBEL 1998). It is known that, inappendicularians, the final phases of gonad growth andmaturation are accompanied by progressive regressionof the gut and recycling of its constituents (FOL 1872).Appendicularia sicula probably presents a system inwhich the source of exogenous energy represented bythe ingested food is accumulated in the huge rectumbefore being progressively transformed for gonaddevelopment.In conclusion, in consideration of the small size of theanimal trunk, simplification of organ structures, andthe extremely reduced genome size estimated to 20.5Mbp, near that of a unicellular organisms such as Sac-charomyces cerevisiae (16 Mbp) (GASSER et al. 2000) it seems that Appendicularia sicula has reached highspecialisation in guaranteeing sexual reproductionwith maximum of energy savings.

Acknowledgements. The authors wish to thank Dr. G.Gorsky of the Station Zoologique of Villefranche-sur-Merfor facilities in providing specimens. This work was support-ed by grants from the C.N.R. and the EU EURAPP project(contract MAS3-CT98-0161) to P. Burighel. The English textwas revised by G. Walton.

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Authors address: Prof. Paolo BURIGHEL, Dipartimento diBiologia, Universit di Padova, Via Ugo Bassi 58B, 35131Padova, Italy; Tel.: +39 049 8276185, Fax: +39 049 8276199,e-mail: burighel@civ.bio.unipd.it

Received: 15. 08. 2002Revised: 07. 01. 2003Accepted: 18. 02. 2003Corresponding Editor: E. GITTENBERGER

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