Rat11. Inst. Cat Hist. Nat., 55 (Sec. Zool., 7): 109-118. 1988

THE ULTRASTRUCTURE OF THE OCELLUS IN THE LARVA OF

CLAVELINA LEPADIFORMIS ( MOLLER ) ( TUNICATA : ASCIDIACEA)

Xavier Turon

RESUMUltrastructura de l'oceFe de la larva de Clavelina lepadiformis ( Muller)

(Tunicata: Ascidiacea)

Rebut: juny de 1986

S'estudia la ultrastructura de l'ocelle larvari d'una especie d'ascidi : Clavelina lepa-

diformis. Aquest fotoreceptor es format per tres parts : una gran cellula pigmentaria en for-

ma de copa , tres cellules que formen un sistema de lents i 15-18 cel.lules fotoreceptores.

Aquesta disposicio sembla esser la mes comuna al grup dels ascidis.

La Hum incideix damunt el sistema de tents, que probablement actua concentrant-

la sobre les membranes fotoreceptores d'origen ciliar situades a ('interior de la copa que

forma la cellula pigmentaria . Es comenta tambe I ' estructura dels fotoreceptors als grups

mes propers.

INTRODUCTION

Several studies on the anatomy of the

larx ae of some ascidian species (GRAVE,

1921; Score, 1946; BERRILL, 1948a, 1948b;

I RAsoy, 1957) have described the structu-

re of the larval occllus located in the pos-

tcro-dorsal wall of the sensory vesicle.

Chew studies agree that this photorecep-

tor is a multiccllular organ consisting of

a pigmented cup-shaped mass, a lens-sys-

tern and some photoreceptor cells. Recent-

ly, GIIRyIAy et of. (1971) have clearly de-

monstrated its photoreceptor function by

registering hvperpolarizing responses to

li'-)ht-exposure in the receptor cells of

Aplidiurn (Anlarouciunt) constellation.

Thus, the occllus is a true photoreceptor

engaged in the phototactic reactions of

ascidian larvae (GRAvIE, 1921; G RAVE &WOODBRIDGE, 1924; DILLY, 1964; MILLAR,

1971; K.u_nwARA & Yosli1DA, 1985).Other species, however, differ from this

general structural pattern in their photo-receptor organs. Some members of theStvelidae family, such as Botryllus schlos-Scr1 (GRAVE & WOODBRIDGE, 1924; GRAVE &RILEY, 1935), Stolonica socialis and Disto-11111s variolosus (BERRILL, 1949) possess acombined light-perceiving and equilibriumsense organ, the photolith, consisting ofa cup-shaped statocyte related to somephotoreceptor cells. Stycla partila has areduced occllus formed by a single cellwith a pigment-containing vesicle (GRAVE,1926; W t t ITTAKER, 1966). Other examples,such as the Molgulidae family or the ge-nus Dendrodoa, completely lack the oce-

Departament de Biologia Animal (Vertebrats). Facultat de Biologia. Universitat de Barcelona.

Avda. Diagonal, 645. 08028 Barcelona.

109

III].,, (GR(vt;, 1026; 131:RRILL, 1950; BoNE &MACKIE, 1982).

In contrast to these studies using lightmicroscopy, fine structure studies (requi-red in order to knox'v the exact nature ofthese photoreceptors), are far from nume-rous. The major literature on this topicconsists of the work of DILLY (1961, 1964);EAKIN & KtuA (1971); BARNES (1969, 1971,1974) and KAJIIVARA & YOSiiIDA (1985),which give information about four spe-cies: Aplidiunt (Antarouciunt) constella-tion, Ciona iniestinalis, Ciona sat'ignvi andDistaplia occidentalis. VoRONTSOVA & MA-

II OV (1982, 1984), on the other hand,describe the fine Structure of the photo-lith of the styclid species Cuetnidocarpaf itinttorkicnsis.

In this work, we present data on theultrastructure of the ocellus of a polyci-torid species: Clavelina lcpadiformis.

MATERIAL AND METHODS

The larvae were obtained during thenatural reproduction period of this spe-cies. Mature colonies with larvae beingincubated in the atrial cavity of zooidswere collected by SCt!IIA diving in the loca-lity of Tossa do Mar (Girona, NE of Spain)in July.

13v dissection of tunic and mantle, em-bryos were released from the atrial ca-vity and placed in Petri dishes. Within afew minutes, the already developed lar-vae broke out from their egg-membranesand began to swim actively. They werethen gently pipetted out and transferredto the fixative liquid.The fixation of the specimens was do-

ne in (1lutaraldehyde 2.5 t'o in 0.2 M phos-phate buffer at pH 7.2 for two hours,followed by several washes in buffer andpostfixation for one hour in osmium te-troxide (1 °u) in the same bolter. Dehy-dration was carried out using a graded se-ries of acetone solutions. The materialwas then embedded in Araldite (Durcu-pan ACM).

Thick serial sections (1 micron) werecut with a glass knife and stained with1 °o methylene blue solution untill thelocalization of the desired area was achic-vecl with the light microscope. Thin sec-tions were obtained with a Reichert Om-U2 microtomc with a diamond knife. The-se were stained with uranvl acetate andlead citrate (REYNOLDS, 1963). Some of thegrids, however, were stained followingThicry's procedure (Tit AIRY, 1967) in or-der to test the polvsaccharid nature ofsome elements which will be discussedlater.

Fic. 1. Lateral view of the trunk region of the larva of Clavelitta lepadiforntis. The two blackspots are the eyespot (E) and the statocyte (S) within the sensory vcsiclc. x 130.Vista lateral do la redo del tronc do la larva de Clavelina lcpudi%onnis. Les dues taqucs ncgres curlesponen a 11)celle (E) i a I'estatucist (S) dins de la vesicula sensitiva. x 130.

FiG. 2. Section of the ocellus shun tug its general structure. E: epidermis; L: lens cell; M: mcm-branous processes; PC: pigment cell; SC: sensory cell; SV: sensory vesicle; T: tunic. Arrow marksthe wall of the sensory vesicle. x 2200.Seccio de 1'ocetle most ant la Seca estructura general. E: epidermis; L: cctlula de la lent;; M: piocessos mcinhta-nosos; PC: cellula pigmentaria; SC: ccllula sensitiva; SV: vesicula sensitiva; 1: tunica. La tletxa indica la parctdo la vesicula scnsitiva. x 2200.

Fic. 3. Nucleus (N) of the pigment cell. Note the deep invagination of its membrane (arrowhead).x 6200.Nucli (N) de Ia cctlula pigmentaria. Observeu la profunda imaginaciu a la membrana (ptutta de tletxa), x 6200.

FIG. 4. Pigment xesicles (PV) of the pigment cell. Arrowheads indicate the limiting membrane.M: mitochondrion. x 22140.Vesicules de pigment (PV) de la cctlula pigmentaria. Les pontes de nctxa marquen la membiaua limitant. M: mito-condri. x 22140.

FIG. 5. Sensory cell showing its soma (S), neck region (N) and receptor processes (RP). LC: lenscell; M: receptor membranes; N: nucleus; PC: pigment cell. x 6190.Cctiula sensitiva most ant el seu soma (S), Ia regio del cull (N) i cis processes receptors (RP). LC. cellula do la lent;M: membranes receptores; N: nucli; PC: cctlula pigmentaria. x 6190.

FIG. 6. Basal region of a sensory cell with its nucleus (N), nucleolus (arrowhead) and endoplas-mic reticulum (ER). x 6200.Part basal d'una ccllula scnsitiva amb el seu nucli (N), nuclcol (punta de tletxa) i reticle enduplasmatic (ER).x 6200.

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111

The sections were examined under aPhilips EM 301 electron microscope inthe Microscopy Service of the Universityof Barcelona.

RESULTS

The ocellus lies anchored to the rightpostcro-dorsal wall of the cavity whichforms the sensory vesicle (fig. 1). It ismade up of three parts (fig. 2): a pigmen-ted cup-shaped cell (U-shaped in section),whose aperture points anteriorly andslightly ventrally, a lens system consistingof three lenses placed in front of the con-cavity of the « U», and a group of sensorycells, the cell bodies of which lie outsidethe dorsal aim of the pigmented cup.Within the cup cavity there are manypiled membranous structures that arepart of the sensory cells.The pigment cup is made up of a single

cell which is the largest of the cells in theocellus. Its section displays a cellular so-ma and two arms that give it its charac-teristic shape (fig. 2). The length of thecell is about 45-50 gym, its largest diameterbeing 21 atm. The «U» aperture reaches 14ttm in diameter.The nucleus is at the base of the cell. It

is irregular in shape and a deep invagina-tinon of the nuclear membrane is oftenobserved (fig. 3).The most conspicuous feature of this

cell is the large number of electron densepigment granules which are uniformly dis-tributed in the cytoplasm, except in thebasal zone. These granules (fig. 4) are

Iitembrane-bounded and their size xaricsbetween 0.5 and 1.8 in in diameter. Nu-merous vesicles and mitochondria are tobe seen in the cytoplasm between thegranules.The position of these pigment vesicles

prevents light from entering the cavity ofthe eyecup except from the directionwhich passes through the lens system.MIyGANTI (1951) demonstrated that the

substance filling the granules is a mela-nin. The mclanogenesis process in asci-dian embryos was accurately studied byW 1I Lr rAKEiz (1966, 1976, 1979).The basal region of the cell is a part of

the wall of the larva's sensory vesicle(fig. 2). It is quite free of pigment granu-les and features some yolk vesicles withassociated rough encfoplasmic reticulum.The sensory cells lie dorsally to the pig-

ment cup cell. Peripheral to them is thelarval epidermis and, outside this, thetunic.

As many as eight sensory cells havebeen found in one section, and their esti-mated number is between 15 and 18, si-milar to that found in other species (EA-KIN d: KtnA, 1971; B:xRNES, 1974). Thesecells have Four parts: a basal axon, a cel-lular soma, a narrow section or neck tra-versing the dorsal arm of the eyecup anda laminar process which develops in thelumen of the cup (fig. 5).The soma is conical or pear-shaped. It

contains the rounded nucleus, with a cons-picuous nucleolus. The cytoplasm is filledwith vesicles, mitochondria, encloplasmicreticulum and yolk droplets (fig. 6).Each cell has a tapering basal process

FIG. 7. Bundle of basal axonal processes (AP) surrounded by the membranes of the sensory cells(arrowheads). x 30000.Grup ('axons basals (AP), envoltats per les membranes de les e ttuIes sensitives (puntes de fletua). x 30000.

FIG. 8. Receptor process of a sensory cell showing its proximal (PS) and distal (DS) segments. BB:basal body; LC: lens cell; PC: pigment cell. Double arrowheads mark a desmosome-like intercellularjunction . Single arrowheads indicate a hemi-desmosome-like junction. x 13110.Proecs receptor d'una o:•llula sensitiva mostrant el segment proximal (PS) i distal (DS). 1313: corpuscle basal; LC:cellula de la lent PC: cclula pigmentaria. Les puntes do fletxa dobles marqucn aim unio cellular de Iipus des-mosoma; les puntes de flora senzillcs n'indiqucn una de tipus hemidesmosoma . x 13110.

FIG. 9. Arrays of lamellae (AL) derived from three receptor processes (asterisks) inside the cavityof the pigment cell (PC). LC: lens cell. x 10025.Formacions lamcilars (AL) dcrivades do tres processor receptors (asteriscs) dins la cavitat de la ciitlula pigmen-taria (PC). LC: ccilula lenticular. x 10025.

FIG. 10 to 12. Three sections of the same photoreceptor process. BB: basal body; C: accessory cen-triole; L: lamellae; PC: pigment cell. Arrowheads mark the narrow circumeiliary space. x25700.Tres sections del mateix proccs fotoreccptor. BB: corpuscle ha,al: C: centriol; I.: lanuldes; 1'l: ccllula pignxm-taria. Els caps de fletxa marquen I'estret espai ciicumciliar. x 25700.

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However, no continuity has been foundbetween these profiles and the discsthemselves. The width of the discs is ab-out 200 A (fig. 16).There are three lens cells, which are

arranged in a row in the anterior part ofthe eyecup (fig. 2). They are in a linewith the direction of light passing into thephotoreceptors through the open end ofthe pigment cup.The lens cells are oval in shape, roughly

28 pm long and 14 p.m wide. Most of thecytoplasm in each cell is occupied by amassive aggregate of electron dense gra-nules (average 300-450 A in diameter) ofirregular shape; between them there is aslight reticular net (fig. 17). There is ahigher concentration of these elements atthe centre than on the periphery of thecentral body. This aggregate is limited bya single layer of mitochondria, whose longaxis is usually parallel to the granulousmass. Some tiny gaps in the line of mito-chondria allow contact between the subs-tance of the central mass and the rest ofthe cytoplasm. The distally-placed nucleus(fig. 18) is round-shaped and displays aconspicuous nucleolus. As for the rest ofthe cytoplasm, it is pushed away to theperiphery of the cell. It is electron denseand features numerous mitochondria, ri-bosomes and endoplasmic reticulum.

DISCUSSION

The ultrastructure of the ocellus in Cla-velina lepadifornris does not differ in anymajor way from that found in Apliditim(Awaroucium) constellation, Ciona intes-tinalis and Dislaplia occidentalis (Du.E),,1961, 1964; EAKIN & Kum, 1971 and BAR-NES, 1969, 1971, 1974).The working of the eye, as inferred from

its morphology, is a simple one: the con-figuration of the pigment cup shields thereceptor membranes from all light exceptthat which comes through the lens sys-tem. Thus, the eye gives the larva directio-nal information which is important forexplaining the photic orientation featuredin the behaviour of ascidian larvae (GRA-VE, 1921, 1944; GRAVE & WOODBRIDGE, 1924;Du.i.yy, 1964; KviiwARA & Yosii IM, 1985).

In addition, a ,shadow reaction>> hasbeen demonstrated (GRAVE, 1921; GRAVE &

WOODBRIncr, 1924; Dii.t.),, 1964): when

light falling on the pigment cup is inter-rupted, the larva begins to swiin immedia-tely. Consequently, during the rotatingmovement made by ascidian larvae whenthey swim actively, the receptors are illu-minated (i.e. when the axis of the evecuppoints towards the surface of water) andimmediately shaded once in each rota-tion, thereby stimulating further locomo-tive activity.The gradient in density from the cen-

tre to the periphery of the central bodiesof the lens cells probably enables them toconcentrate light on the receptoral mem-branes. EAKIN & KUDA (1972) indicated af-ter cvtochemical studies that the granu-les in the <<lenses>> are particles of glyco-gen. The positive reaction of these gra-nules found here to Thierv's staining me-thod (fig. 17) supports this conclusion.The flattened membranous discs orien-

ted perpendicularly to the path of theincoming light are presumably the siteof the photopigment. Numerous micro-villi and cellular junctions (figs. 8, 13, 14)give rigidity to these membranous units.EAKIV & KuD, (1971) found microvilli de-rived from the pigmented cell, whereasBAm' es (1971, 1974) described microvillias arising from the sensory cell, near thebasal bodies. Both types exist in Clar'eli-na lepadifornris (figs. 13, 14). Their exactfunction is unknown. B: RNLS (1974) indi-cates the possibility of them having a pho-toreceptor funcion. However, their poordevelopment compared to the other re-ceptor membranes suggests a supporting,and perhaps also a nutritive function (EA-KIN & KuDn, 1971). The deep interdigita-tion between the microvilli and the recep-tor processes as well as the thickening orunion observed between their membranes(fig. 14) supports this latter interpreta-tion.The transmission of the impulse is,

most probably, via the neck of the sen-sory cell and then through its basal axon.The bundles of axons presumably runbackwards and end at the larval cerebralganglion (BARNES, 1971). No synapses havebeen observed between the axons and ad-jacent cells.Two types of photoreceptors exist anion;,

the invertebrates: one of these dcri-ves the sensitive organelles from inodifi-cations of a ciliary membrane. In the se-cond type, called rhabdomeric, the rc-

116

ceptur organelles det-ivc Crum microvilli

(EAKIN, 1965) .The ocellus of the ascidians

is ut the ciliary type. Its possible homo-

logy with the vertebrate photoreceptors

was already suggested by SALENSKY (1893).

Electron microscopists (DILLY, 1964; EA-

KIN & Kt u:A, 1971; BARNES, 1971) also poin-

ted Out that the line structure of the sen-

sitiyc scunlcnt bears a remarkable like-

ness to the structure of vertebrate reccp-

tors (MooDY, 1964; COLLIN, 1969).As fur the related groups, the hemichor-

date tornarian larva has two eyespots

with both cilia and nlicrovilli. It remainsunclear which of the structures, if not

both, acts as a photoreceptor (BRANDEN-

lit R(ER et al., 1973).In 13ranchioslonta (Cephalochordata)

two types of photoreceptors have so far

been described: the Hesse cells (EAKIN &

WLs Ir.vI.t., 1962) and the Joseph cells

(Wrls( i f , 1968; AN:IDON, 1976), both rela-

ted to the rhabdoinet is type. However,

near the Joseph cells, in the dorsal wall

of the cerebral ycsicle, there are other

cells With Inenlbranous appendages deri-

ved from cilia which are strikingly simi-

lar to the Outer segment of the ascidiansensory cells and which may also act as

photoreccptors (E,1KIN & WESILALL, 1962;

.Nit A i s, 1972).Other groups of tunicates, such as ap-

pendicularia and doliulids, do not have anocellus (Bust: & MAcKIF, 1982; HOLy1BERG,1984). Salps possess a nlicrovillous recep-

tor (13:vRNt:s et al., 1970; Gouylyy et al.,

1971). Finally, some adult ascidians like

C'iorta iuteslirtalis have rhabdomeric occlli

at the border of the siphons (DILLY &

WOLKEN, 1973).

ACKNOWLEDGEMENTS

Thanks are due to Dr. M. Durfort, ofthe University of Barcelona for her wel-

come advice, criticism and encourage-I1leilt.

I am indebted to the team in the Mi-croscopy Service of the University of Bar-celona for their help in solving the techni-cal problems in this study.

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