ORIGINAL ARTICLE

Structure and ultrastructure of eyes of tornaria larvaeof Glossobalanus marginatus

Katrin Braun & Sabrina Kaul-Strehlow &

Esther Ullrich-Lüter & Thomas Stach

Received: 17 November 2014 /Accepted: 13 February 2015 /Published online: 1 March 2015# Gesellschaft für Biologische Systematik 2015

Abstract Enteropneusts or acorn worms are marine deutero-stomes that have retained many plesiomorphic characters.Thus, enteropneusts are of prime interest in evolutionary com-parisons between deuterostomes and protostomes. In the pres-ent study, the larval eyes of Glossobalanus marginatus werereconstructed and described based on serial sectioning fortransmission electron microscopy. The everse eyes of the lateMetschnikoff/early Krohn-stage tornaria larvae ofG. marginatus are epidermal structures consisting of two rowsof in total 13 shading pigment cells and another two rows of13 photoreceptor cells. The pigment cells form a shallow cupwith a relatively wide opening, making the cup-shaped eyeoptically unsuitable for picture generation. We demonstratethat the photosensitive cells possess numerous enlarged mi-crovilli and an unmodified apical cilium. Our ultrastructuralstudies thus corroborate the photoreceptor cells in the eye ofG. marginatus to be of a clearly rhabdomeric type. Prelimi-nary immunohistochemical experiments support those find-ings by demonstrating immunopositive reaction of the

tornarian eye photoreceptors with an antibody designedagainst rhabdomeric sea urchin photopigment (Sp-Opsin4).Observations of living animals indicate that LateMetschnikoff/early Krohn-stage tornaria larvae are negativelyphototact ic , probably concordant with imminentmetamorphosis.

Keywords Deuterostome . Enteropneust . Hemichordate .

Rhabdomeric . Ciliary photoreceptor

Introduction

The technical complexities and intricacies of optical instru-ments and their resemblance to animal eyes astonished andcontinue to interest researchers, who marvel at the diversity ofeyes found in the animal kingdom and who try to elucidate theevolutionary history of these structures (Arendt and Wittbrodt2001; Cronin and Porter 2014; Darwin 1859; Ullrich-Lüteret al. 2013).Molecular methods have been added to the toolkitof evolutionary biologists, yet structural investigations remainessential, if evolutionary transformations of structures are tobe documented and understood. Eakin (1979) was most influ-ential in emphasizing the hypothesis that photosensitive struc-tures in the animal kingdom had a single evolutionary originand diverged into two major lineages, rhabdomeric and ciliaryrespectively, based on the structure of the photosensory cells.The two types of photoreceptors were thought to broadly cor-respond to one of the major subdivisions of the animal king-dom, the one between Protostomia and Deuterostomia.Rhabdomeric eyes were thought to be predominant inProtostomia, whereas ciliary eyes were thought to be mainlyconfined to Deuterostomia. This hypothesis found consider-able support with the application of molecular techniques that

Electronic supplementary material The online version of this article(doi:10.1007/s13127-015-0206-x) contains supplementary material,which is available to authorized users.

K. Braun : T. Stach (*)Institut für Lebenswissenschaften, Vergleichende Zoologie,Humboldt-Universität zu Berlin, Philippstrasse 13, Haus 2,10115 Berlin, Germanye-mail: tstach@zoosyst-berlin.de

S. Kaul-StrehlowDepartment of Integrative Zoology, University of Vienna, Althanstr.14, 1090 Vienna, Austria

E. Ullrich-LüterMuseum für Naturkunde, Leibniz-Institut für Evolutions- undBiodiversitätsforschung, Invalidenstraße 43, 10115 Berlin, Germany

Org Divers Evol (2015) 15:423–428DOI 10.1007/s13127-015-0206-x

revealed two families of photopigments co-occurring with thetwo structural types and that were named c-opsins (for ciliaryopsins) or r-opsins (for rhabdommeric opsins), respectively(Gehring 2014; Passamaneck et al. 2011; Ullrich-Lüter et al.2013).

Enteropneust acorn worms are marine deuterostomes thatretain many characters that are arguably plesiomorphic forDeuterostomia, such as a basiepithelial nerve plexus, a biphas-ic life cycle with a planktonic larval stage, or a tripartite bodyorganization with an according partition of the coelomic cav-ities (Stach 2014). Thus, enteropneusts are of prime interest inevolutionary comparisons between deuterostomes andprotostomes. In a detailed study of the larval eyes of theptychoderid enteropneust Ptychodera flava Eschscholtz,1825, Brandenburger et al. (1973) argued that the photorecep-tor cells were intermediate between the ciliary andrhabdomeric type, including microvilli as well as a modifiedciliary membrane. Citing this very same study, Arendt andWittbrodt (2001) on the other hand designated the photore-ceptor cell of P. flava tornaria larvae as purely rhabdomeric. Inorder to supply new arguments to decide this discussion andpotentially add functional insight into the optical capacities ofenteropneusts, we investigated the larval eyes of anotherptychoderid enteropneust species Glossobalanus marginatusMeek, 1922 using mainly transmission electron microscopyand computer-ass is ted digi ta l three-dimensionalreconstruction.

Materials and methods

Tornaria larvae of G. marginatus Meek, 1922 were collectedusing a plankton net with a mesh size of 200 μm from 7 to20 m deep water near the Sven Lovén Centre for MarineSciences in Tjärnö (Sweden) in September 2010 (58°53′N,11°05′O) utilizing a row boat. Five individuals were investi-gated, four in late Metschnikoff/early Krohn stage. One spec-imen in an early Agassiz stage was used for additional liveobservations.

For transmission electron microscopy (TEM) and light mi-croscopy, two late Metschnikoff/early Krohn-stage larvaewere placed into ice-cold primary fixative containing 2.5 %glutaraldehyde in 0.2 mol sodium cacodylate buffer (pH 7.2),adjusted to an osmolarity of approximately 800 mosm withthe addition of NaCl. Primary fixation was stopped after45min with three buffer rinses for 10, 15, and 20min. Primaryfixation was followed by 30 min of postfixation with 2 %OsO4 in sodium cacodylate buffer. Postfixation was stoppedwith three buffer rinses (15, 30, 30 min) followed by tworinses with ddH2O (15, 30 min). After dehydration through agraded series of ethanol, specimens were embedded in Epon812 for TEM and light microscopy.

Complete transverse serial semi-thin sections 0.5 μm inthickness for light microscopy were sectioned on a LeicaUltracut S. In addition, complete transverse and longitudinalseries of sections in thickness of about 60 nm for TEM werecut. Semi-thin sections were stained with toluidine blue. Ul-trathin sections were stained with 2 % uranyl acetate and2.5 % lead citrate in an automatic stainer (NanoWlmTechnologie GmbH, Göttingen). Light microscopic imageswere recorded with a digital camera (Olympus BX-UCB)mounted on an OlympusBX51 compound microscope. Everyfifth section was recorded (distance, 2.5 μm). TEM pictureswere documented with a Philips EM 208 electron microscopeat 60 kVon Ditabis Digital Imaging Plates and subsequentlywere read by a Ditabis Micron IP-Scanner. Images werealigned using Adobe Photoshop CS3 Software. Based on theresulting stack of images, three-dimensional (3D) models of acomplete larva showing the anatomy all organ systems and ofthe right eye of another individual was created in Amira 3.0software (Mercury Computer Systems, Berlin).

Results

The tornaria larva of G. marginatus Meek, 1922 at the lateMetschnikoff/early Krohn stage is characterized by the pos-session of three ciliary bands: the circumoral neotroch (oftencalled perioral band), a perianal telotroch, and the short ventralciliary band (Fig. 1a, b). Internally, the digestive tract of thelarva is partitioned into an esophagus, stomach, and a rectumthat opens in a terminal anus. The mesoderm consists of aprotocoel that opens to the exterior through a dorsal protocoelpore. Next to the short canal that connects the protocoel to theexterior, the prospective pericardium is situated as a smallvesicle on the right side (Fig. 1c). A conspicuous muscularstrand connects the protocoel with the apical plate. On the leftand right sides of the posterior part of the stomach, theprimordia of the remaining coelomic cavities can be found(Fig. 1b).

Between the apical loops of the neotroch, a pair of darklypigmented eyes is visible as part of the apical plate (Fig. 1;supplementary material SM3, SM4). The eyes are approxi-mately ovoid, cup-shaped, with the openings directed lateral-ly, slightly obliquely, and apically. Along their long axis, theeyes measure approximately 20 μm externally, their short axisis about 12 μm in width, and the eye is around 10–12 μm inheight (Figs. 2, 3). These measurements circumscribe the out-er periphery of the eye that is constituted by the pigment cells.The opening into the chamber that contains the photosensitiveparts of the photoreceptor cells is oval and measures 7–8 μmalong the longer axis and about 3 μm along the shorter axis.The chamber containing the photosensitive parts of the pho-toreceptor cells is again ovoid and measures 10 μm along the

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long axis and around 6 μm along the shorter axis, and 3–4 μmin height.

The eye inG. marginatus is composed of two different celltypes: photoreceptor cells and pigment cells. Thirteen cells ofeach type constitute one eye. The eyecup is formed by 13pigment cells. These cells are part of the epidermis andepithelially organized with a single apical cilium, somemicro-villi, and apical adherens junctions (Fig. 2). The cilium showsthe familiar 9*2+2 arrangement of microtubules, an accessorycentriole, and a short striated rootlet fiber. The pigment cellsare roughly cuboidal and measure about 7 μm in height. The

nucleus is situated in the basal third of the pigment cells andmitochondria are found predominantly in the cytoplasm sur-rounding the nucleus. The most characteristic feature of thepigment cells are the numerous clear pigment vesicles. Thesevesicles are bound by a single membrane, are around 0.2 μmin diameter, and appear electron lucent in transmission elec-tron microscopic aspect. The pigment vesicles are situatedabove the nucleus. The area of neighboring pigment cellscontaining the pigment vesicles forms an almost continuoussheet that is only perforated by the narrow neck-like connec-tion between the perikaryon area of the photoreceptor cellsand the photosensitive apical parts of the photoreceptor cellsinside the photosensitive chamber of the eye.

The photoreceptor cells are clearly divided into two partsthat are connected by a narrow, waist-like neck (see Figs. 2,3). The basal part of the photoreceptor cell contains theconspicuous nucleus and measures approximately 4 μm indiameter. A single axon projects from the basal part of thephotoreceptor cells into the nervous system. The narrowneck, barely measuring 0.5 μm, connects this basal part ofthe cell with the apical part. The apical part of the photo-receptor cells is roughly pear shaped to a more narrowhorseradish shape with the widest part measuring approxi-mately 2–4 μm in diameter. All along the lateral sides ofthe Bpears,^ numerous microvilli project into the photosen-sitive chamber, filling it up entirely. At the apical narrowpole of the photoreceptor cells, a single cilium projects intothe exterior through the opening of the cup-shaped eye(Fig. 3d). This cilium has a straight membrane withoutmodifications and shows the regular 9*2+2 pattern of mi-crotubules. It possesses an accessory centriole and a longrootlet fiber that anchors the cilium in the basal perikaryonarea of the photoreceptor cells. The photoreceptor cells arearranged in two rows: one row comprising six photorecep-tor cells, the other seven.

When freely swimming, the late Metschnikoff/early Krohnstage larvae are mainly oriented horizontally with the apicalpole pointing forwards in the direction of movement but oc-casionally the apical pole is directed towards the water surface(see supplementary materials: movies SM3 & SM4). The lar-vae are propelled by the telotroch. They are usually swimmingwithout rotating along their longitudinal axis, although occa-sionally a slow rotation occurs. Preliminary experiments dem-onstrate that in the majority of cases the larvae were found inthe dark half of the petri dish (see supplementary materialSM5). The early Agassiz-stage larva still utilized the telotrochcilia for locomotion, yet the behavior drastically differed fromthe late Metschnikoff/early Krohn stage larvae (see supple-mentary materials: movies SM3 & SM4). The Agassiz-stagelarva moved over the substrate, with the ventral side down andfrequently probing the substrate with the anterior tip of thehighly movable proboscis. At the tip of the proboscis, thetwo pigment spots of the eye were still visible.

Fig. 1 Tornaria larva ofG. marginatus. a Picture of living tornaria in lateMetschnikoff/early Krohn stage, about 2 mm in length. Ventral view,posterior is at the bottom. White arrows show eyes, which can be distin-guished as dark spots, lying apical. b–d 3D reconstruction of anatomy oftornaria in late Metschnikoff/early Krohn stage. b Ventral view, posterioris at the bottom. The ciliary band of the neotroch runs along the grooves ofthe oral field (of) around the mouth opening. The protocoel (pc) attachesto the basal side of the apical organ (ao) by a muscular buccal band (bb). cApical view onto the eyes in the apical organ (ao). The protocoel opening,i.e., the hydropore (hp) is seen next to the pericardium (pe). The arrowspoint to the eye, enlarged in d. d 3D reconstruction of eyecup. Darkmauve-colored cells are pigment cells (ec), forming the eyecup, cream-colored cells are processes of photoreceptor cells (ppc). Dashed orangelines indicate maximum reconstructed light cone, restricted by pigmentcells, entering the eye: af, aboral field; ao, apical organ; bb, buccal band;ec, pigment cells forming the eye cup; hp, hydropore; in, intestine; mo,mouth opening; ms, mesoderm; oe, esophagus; of, oral field; pe, pericar-dium; pob, postoral ciliary band; ppc, processes of photoreceptor cells;prb, preoral ciliary band; st, stomach; tt, telotroch; vb, ventral ciliary band

Ultrastructure of tornaria eyes 425

Discussion

The photoreceptors ofG. marginatus are of a rhabdomeric celltype

The eye of G. marginatus as revealed in the present study issimilar to the one described from another ptychoderid larva,P. flava by Brandenburger et al. (1973), although the larva ofG.marginatus is considerably older and larger. Like inP. flava,the eye is simple and cup-shaped and consists of two differentcell types: pigment cells and photoreceptor cells. The cells arealso similarly arranged and of similar shape, appearance, andstructure. In the photoreceptor cells, however, we could notdetect modifications in the apical cilium, such as a ballooningof the cilium or a ruffling of the ciliary membrane, as

described for P. flava. The enlarged numerous microvilli onthe other hand originate from the apical surface of the cellsadjacent to the central cilium. The membrane of the cilia isunmodified. The microvilli are filling the chamber that isenclosed by the pigment cells except for the apical openingto the exterior. Despite the presence of an unmodified apicalcilium, we therefore conclude that the photoreceptor cells areof the rhabdomeric type and not intermediary between a cili-ary condition and a rhabdomeric condition. These structuralobservations are supported by a preliminary immunohisto-chemical experiment that showed immunopositive reac-tion of an antibody designed against the rhabdomericphotopigment Sp-Opsin 4 (supplementary material SM1).Cases of rhabdomeric photoreceptor cells bearing an addition-al cilium are widespread in the animal kingdom and are, e.g.,

Fig. 2 Transmission electron micrographs of longitudinal sections of thetornarian eye of G. marginatus. a Ultrastructure of a pigment cell. Theapical cilium (ci) is surrounded by few microvilli (mv). Note thenumerous pigment vesicles (pv). Arrowhead points to the short ciliaryrootlet. b Ultrastructure of a photoreceptor cell (prc). A thin neck (np)connects the cell body with the apical process (apc). Note the ciliaryrootlet (cr), which runs through the thin neck region into the basal cellregion. Arrowheads demarcate apical adherens junctions. c Highermagnification of apical photoreceptor cell region. Arrowheads apical

adherens junctions interconnect receptor cells and pigment cells. dCilium (ci) of a photoreceptor cell (prc) without modifications of cellmembrane along the cilium. e Processes of photoreceptor cells areclosely adjoined in the photosensitive chamber of the eyecup. Note thelong microvilli (mv) oriented perpendicularly to incoming light. apc,apical process of photoreceptor cell; bb, basal body; ci, cilium; cr,ciliary rootlet; (mi) mitochondria; mv, microvilli; np, neck region ofphotoreceptor cell; nu, nucleus; pc, pigment cell; prc, photoreceptorcell; pv, pigment vesicle

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known from deuterostomes such as amphioxus (Ruiz andAnadón 1991) and protostomes such as polychaetes (Eakinet al. 1977; Randel et al. 2013; Smith 1984) or insects(Wachmann et al. 1983). The sites of cell surface magnifica-tion inG. marginatuswhere the photopigments are most prob-ably located, however, are strictly microvilli and therespective photoreceptor cell type should be classified asmicrovillar or rhabdomeric as has been done by Arendt and

Wittbrodt (2001) and not intermediate between a ciliary andmicrovillar type as proposed in Brandenburger et al. (1973).Thus, the present study not only clarifies this question but alsoreveals that the relegation of ciliary versus rhabdomericphotoreceptors to the taxa Deuterostomia versus Protostomiais obviously not as clear cut as assumed based on Eakin’shypothesis (Eakin 1979), thereby confirming previous studies(Passamaneck et al. 2011; Wachmann et al. 1983).

Fig. 3 a–c, e 3D reconstructions of the eye of the tornaria larva ofG. marginatus based on complete serial sectioning for transmissionelectron microscopy (TEM). Microvilli of receptor cells are not shown.a Oblique apical view of entire eyecup, ventral is to the right. Pigmentcells (pc) form the eyecup, into which receptor cells extend.Section planes of schematic drawings in d and f are indicated. b Twopigment cells (pc, dark mauve-colored) and two photoreceptor cells (prc,light cream-colored) in detail. The cilia of photoreceptor cells passthrough the apical process (apc) and neck region of the photoreceptorcells (np) and reach into the basal cell body (see arrowheads). Incontrast the cilia of the pigment cells are restricted to the apical areas ofthe pigment cells (see respective arrowheads). Black arrow shows wherethe neck of the receptor cell passes through pigment cells. Orange arrow

indicates the direction of incoming light. c Apical processes ofphotoreceptor cells are closely adjoined in the photosensitive chamberof the eyecup. d Interpretative schematic of a sagittal section throughthe eyecup based on TEM sections. Two rows of pigment cells and tworows of receptor cells can be distinguished. e Digitally isolatedphotoreceptor cells (prc) of complete eye in detail; note ciliary rootlets.f Interpretative schematic of a longitudinal section through the eyecupbased on TEM sections. Microvilli of receptor cells are orientatedperpendicularly to incoming light. apc, apical process of photoreceptorcell; ax, axon emerging from base of photoreceptor cell; np, neck regionof photoreceptor cell; pc, pigment cell; prc, photoreceptor cell; pv,pigment vesicle; orange arrow indicates direction of incoming light

Ultrastructure of tornaria eyes 427

Tornaria larvae of G. marginatus do not see focused images

From the optical properties of the cup-shaped eye ofG. marginatus that can be inferred from the three-dimensional structure of the eye, we can deduct that a sharpimage is not formed in the eye. There is no lens and we couldapply basic formulae proper for the physical geometric opticsof a camera obscura. With the approximate dimensions of thephotosensitive chamber of the cup-shaped eye ofG. marginatus of 10 μm length and 6 μm in breadth and aheight of 4 μm, the optimal opening of the chamber can becalculated according to Schmidt-Ploch (2001) to be 0.07 μmin diameter. Our detailed three-dimensional reconstruction ofthe eye ofG.marginatus reveals however that the real openingdiameter of the optical opening is about 100 times larger,around 7 μm. Thus based on physical optical geometry, thecup-shaped eye cannot form a focused image. However,because of the pigment cells forming a cup around the photo-sensitive chamber and with the oval opening, the eyes have anopening angle of about 120°, pointing obliquely apically.Therefore, the two eyes, directed apically at slightly differentdirections in relation to the body axis of the animal, areperfectly well set up to accurately identify the direction ofincoming light.

Behavior changes from positive to negative phototaxis

The lateMetschnikoff/early Krohn stage larvae investigated inthe current study were negatively phototactic. Previous studieshave argued that light plays only a minor role in the behaviorof tornaria larvae (Ritter and Davis 1904) but other studiesobserved positively phototactic behavior in tornariae larvaeof the ptychoderid species P. flava (Brandenburger et al.1973) and Balanoglossus clavigerus (Stiasny 1914). Consid-ering the depictions of the respective larval stages, the posi-tively phototactic period precedes the negatively phototacticone. This is congruent with the observation that the earlyAgassiz stage monitored in the present study crawled on thesurface of the petri dish, seemingly probing the substrate per-manently. If given the opportunity, this animal readily buriedinto the sand. In nature, this behavioral switch from positive tonegative phototaxis is probably co-occurring simultaneouslyto the transition period between the planktonic phase to thebenthic phase.

Acknowledgments We thank Maria I. Arnone for kindly providing theanti-Sp-Opsin4 antibody. Financial support by the DeutscheForschungsgemeinschaft (DFG) is gratefully acknowledged.

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