Organization of the Epistome in Phoronopsis Harmeri (Phoronida) and Consideration of the Coelomic Organization in Phoronida

Zoomorphology (2011) 130:121–134 DOI 10.1007/s00435-011-0126-z

ORIGINAL PAPER

Organization of the epistome in Phoronopsis harmeri (Phoronida) and consideration of the coelomic organization in Phoronida

Elena N. Temereva • Vladimir V. Malakhov

Received: 16 September 2010 / Revised: 28 April 2011 / Accepted: 30 April 2011 / Published online: 17 May 2011 Ó Springer-Verlag 2011

Abstract Results provided by modern TEM methods Keywords Lophotrochozoa Á Phylogeny Á Phoronida Á indicate the existence of the lophophoral and trunk coelo- Coelom Á Ultrastructure mes but not of the preoral coelom in Phoronida. In the present work, the epistome in Phoronopsis harmeri was studied by histological and ultrastructural methods. Two Introduction kinds of cells were found in the frontal epidermis: supporting and glandular. The coelomic compartment is shown The Phoronida has since long been in focus of debates on to be inside the epistome. This compartment has a complex Metazoan evolution. The investigations of Masterman shape, consists of a central part and two lateral branches, (1898), who advanced the archicoelomate concept, stimu- and contacts the lophophoral coelom, forming two complete lated further study on the anatomy of the phoronid coelo- dissepiments on the lateral sides and a partition with many mic system. Several important works were dedicated to the holes in the center. TEM reveals that some portions of the organization and fate of the preoral coelom in the acti- incomplete partition are organized like a mesentery, with notroch larva (Menon 1902; Cowles 1904; Zimmer 1978, the two layers of cells separated by ECM. The myoepithe- 1980; Siewing 1974; Herrmann 1980, 1986) and in adult lial cells of the coelomic lining form the circular and radial phoronids (Pross 1974, 1978; Siewing 1974, 1980; Emig musculature of the epistome. Numerous amoebocytes occur and Siewing 1975). These classical studies provided evi- in the coelom lumen. The tip of the epistome and its dorso- dence phoronids possess three coelomic compartments: the lateral parts lack a coelomic cavity and are occupied by preoral coelom inside the epistome, the lophophoral coe- ECM and muscle cells. The fine structure of the T-shaped lom inside the lophophore and tentacles, and the trunk vessel is described, and its localization inside lophophoral coelom. Thus, the organization of coelomic system in coelom is demonstrated. We assert that the cavity inside the phoronids is similar to deuterostomian coelomic system, epistome is the preoral coelom corresponding to the true which also consists of three sets of coelomic compartments preoral coelom of the larva of this species. Proving this arranged along the fronto-caudal axis. Recent ultrastruc- assertion will require additional study of metamorphosis in tural studies, however, have shown that the actinotroch this species. To clarify the patterns of coelom organization larva and adult phoronids lack a preoral coelom (Bartolo- in phoronids, we discuss the bipartite coelomic system in maeus 2001 for Phoronis muelleri Selys-Longchamps, Phoronis and the tripartite coelomic system in Phoronopsis. 1903; Gruhl et al. 2005 for Phoronis ovalis Wright, 1856). These recent studies challenge the idea of a closer relation between phoronids and deuterostomes and does not longer contradict the Lophotrochozoa hypothesis, according to Communicated by T. Bartolomaeus. which the phoronids belong to the Protostomia (Halanych et al. 1995; Peterson and Eernisse 2001). This hypothesis & E. N. Temereva ( ) Á V. V. Malakhov has been supported by recent molecular studies (Dunn et al. Department of Invertebrate Zoology, Biological Faculty, Moscow State University, Moscow 119992, Russia 2008; Hejnol et al. 2009; Paps et al. 2010). However, the e-mail: [email protected] new morphological evidence that larvae of Phoronopsis 123 122 Zoomorphology (2011) 130:121–134 species have a distinct preoral coelom has been obtained by Results Temereva and Malakhov (2006). The epistome in adults of Phoronopsis species has never been studied by TEM Shape methods, so that the occurrence and characteristics of the preoral coelom in adult Phoronopsis species are unclear. The epistome is situated between the mouth or lophophoral To fill this gap, the current study provides new histo- grooves and inner rows of tentacles (Figs. 1a, 2a–c). This logical and ultrastructural information on the structure of organ is very long and has a complex shape with central, the epistome in Phoronopsis harmeri. The coelomic system lateral, and dorso-lateral parts (Figs. 2c, 3a, c). In dorso- organization among phoronids is also discussed. lateral and lateral parts of the lophophore, the epistome looks like a small epidermal fold covering the lophophoral groove (Fig. 4b, c). The height of epistome is 76 ± 1 lm. Methods In the center of the lophophore, the height of the epistome considerably increases to 190 ± 2 lm (Fig. 4d–f). At the Adults of Phoronopsis harmeri Pixell, 1912 (Phoronida) lophophore median line, the epistome looks like a large lobe were collected from August to October 2005 from sandy covering the mouth opening (Fig. 3b). The central part of sediments at 3–4 m depth in the Vostok Bay, Sea of Japan, the epistome can be moved up and down whereas the lateral and from May to June 2010 in the intertidal zone in Coos and dorso-lateral parts always remain motionless (Fig. 1b). Bay (USA, Oregon). Some live animals were photographed The frontal and abfrontal sides of the epistome differ in using a Panasonic DMC-TZ10 digital camera mounted on a position relative to the mouth and also differ in epidermis binocular light microscope. Specimens extracted from organization (Figs. 3b, 4). The frontal side faces the mouth tubes were dissected at the anterior end to obtain a loph- or lophophoral groove and is covered by thick columnar ophore with tentacles and epistome. Pieces of animals were epidermis. The abfrontal side faces the inner row of ten- fixed for routine histology, scanning (SEM) and transmis- tacles and is covered by squamose epithelium. The shallow sion (TEM) electron microscope. For routine histology and SEM, a 4% paraformaldehyde solution in filtered sea water was used as a fixative. Specimens were rinsed in distilled water, dehydrated in ethanol, and embedded in Paraplast Regular (Sigma). Sagittal and cross- sections (7 lm thick) made with a Leica rotary microtome (Leica RM 2125) were stained with Caracci hematoxylin. Sections were examined with a Zeiss Axioplan2 microscope and photographed with an AxioCam HRm camera. For SEM, fixed specimens dehydrated in ethanol followed by an acetone series were critical point dried and then sputter coated with platinum-palladium alloy. Speci- mens were examined with a Camscan S2 Scan scanning electron microscope. For TEM, specimens were fixed at 4°C in 2.5% glutar- aldehyde in 0.05 M cacodylate buffer containing 21 mg/ml NaCl and then postfixed in 2% osmium tetroxide in the same buffer containing 23 mg/ml NaCl. Postfixation was followed by en bloc staining for 2 h in 1% uranyl acetate in distilled water and then specimens were dehydrated in ethanol followed by an acetone series, and embedded in Spurr resin. Semi-thin and thin sections were cut with a Reichert Ultracut E ultramicrotome. Semi-thin sections were stained with methylene blue and observed with a Reichert Polyvar light microscope. Thin sections were stained with lead citrate and then examined with a JEOL JEM 100B electron microscope. Fig. 1 Phoronopsis harmeri For all measurements, we used three different specimens The lophophore of (photographs of living animals). a The epistome (ep) is in a placid condition. b The and calculated the mean by standard formula. In the text, central part of the epistome is raised. Abbreviations: ert external row only the means are given. of tentacles, irt internal row of tentacles, m mouth 123 Zoomorphology (2011) 130:121–134 123

b Fig. 2 Schemes of the Phoronopsis harmeri anterior body part (a) and 3D reconstruction of coelom organization (b). The lophophore structure is simpliﬁed; tentacles are partly removed in a. White, gray, dark gray, and black indicate the preoral coelom, the lophophoral coelom, trunk coelom, and the coelom contour and blastocoel, respectively. In order, parts of blastocoel correspond to descending (di) and ascending (ai) branches of intestine. Hatching indicates the border between the central of the epistome cavity and the lophophoral coelom. Parts of the base and lateral sides of the epistomal cavity are hidden by the lophophoral coelom. ap anal papilla, cf collar fold, cpe epistome central part, ef epistome latero-frontal epidermal fold, ert external row of tentacles, irt inner row of tentacles, lg lophophoral groove, m mouth, np nephridiopore

mouth, cells are up to 35 ± 1 lm high (Fig. 4e, f), whereas they measure merely 17 ± 1 lm in height in the dorso- lateral and lateral parts (Fig. 4b). Two kinds of cells were found in the frontal epidermis: supporting cells and glandular cells (Fig. 5a). The apical cell membrane extends into numerous thin, 2-lm-long microvilli bearing a glycocalix; one cilium emanates from the apical cell membrane of the supporting cells (Fig. 5a). The axoneme has the usual 9 9 2 ? 2 pattern of microtubule arrangement. The basal structure of the cilium includes vertical and horizontal striated rootlets and an accessory centriole that points toward the mouth opening (Fig. 5b). The Golgi complex is situated next to the vertical rootlet. Because of a high concentration of free ribosomes, the cytoplasm appears electron-dense. Several large granules occur in the apical cytoplasm (Fig. 5a, c). These have an irregular shape and a complex structure with electron-transparent inclusions in the electron-dense material (Fig. 5c). The elongate nucleus occupies the middle part of the cell and contains a large nucleolus (Fig. 5a, c). Many specialized retort-shaped gland cells reside between the other epidermal cells (Fig. 5a, c, d). The expanded basal part of the glandular cell contains numerous granules, vesicles, and a small spherical nucleus (Fig. 5d). Most vesicles are rough endoplasmic reticulum cisterns (460 ± 2 nm in diameter) that contain the electron-transparent material (Fig. 5c, d). The cytoplasm contains electron-dense granules and large inclusions with intermediate electron density. A concentration of neural processes is located basally in the frontal epidermis (Fig. 6a). Most of these processes are 1.5 ± 0.1 lm in diameter and contain numerous vesicles (74 ± 2 nm in diameter), each with a dense core surrounded by a lighter halo. The frontal epithelium rests on a 1.4 ± 0.1-lm-thick ECM composed of three layers: a ciliated groove runs along the abfrontal side of the epi- 50-nm-thick basal lamina underneath the epidermal cells, a stome (Fig. 3a). 25-nm-thick basal lamina the coelothelial cells rest on, and a central ﬁbrillar layer (Fig. 6a) that has a thickness of Epidermis and extracellular matrix (ECM) about 1 lm and consists of collagen ﬁbers. The abfrontal epidermis is 7 ± 1 lm high (Fig. 6b). The frontal epidermis is composed of a very high epithe- The cells are slightly prismatic, with one cilium each. The lium (Fig. 3b). In the center of the epistome under the microvilli are 0.8 lm long. The basal structure of the

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Fig. 3 The epistome of Phoronopsis harmeri SEM. a General front diaphragm (d). c The lophophore with the entire epistome (arrow- view including the broad central part (cpe) and the narrow lateral heads) on the right side. d The epistomal cavity (ec) is crossed by parts (lpe). Tentacles around the mouth (m) have broken down and muscle cells. ai ascending branch of intestine, d diaphragm, ert been removed. b Sagittal section through the anterior body part with external row of tentacles, irt inner row of tentacles, lc lophophoral external (et) and internal (it) tentacles; epistome (ep), mouth (m), and coelom, T T-branches of the median blood vessel, tc trunk coelom cilium includes only one, vertical striated rootlet (Fig. 6c). extending into the basal part of cell form the circular The electron-transparent cytoplasm contains a few organ- musculature of the epistome (Fig. 7b). Basally, myoepi- elles: Golgi complex, mitochondria, multivesicular bodies, thelial cells adhere to the ECM by electron-dense hemi- vesicles, and large electron-dense granules. The nucleus is desmosomes (Fig. 7b). Some coelothelial cells form the irregular in shape and basally located (Fig. 6b, c). The radial musculature of the epistome (Fig. 9a–c) and cross abfrontal epidermis rests on a 1.1 ± 0.1-lm-thick ECM the coelomic cavity from the frontal to abfrontal side (Fig. 5d). (Figs. 3d, 4f). Cell ends are transformed into long processes that are procumbent on the basal lamina (Fig. 9b). Epistomal cavity The processes of different cells adhere to each other via desmosomes. The cell body is spindle-shaped and contains The cavity is completely lined with an epithelium con- a nucleus and radial myoﬁlaments that adhere to the ECM sisting of myoepithelial cells that are interconnected via by hemidesmosomes (Fig. 9b, c). median adherens junctions (Figs. 6d, 7b). The cells rest on The ECM forms large protrusions (1.8 ± 0.6 lmin the ECM. The cell middle and basal parts form long pro- diameter) underneath the myoepithelial lining that bulge cesses bearing desmosomes (Fig. 7b). The enlarged apical into the epistomal cavity on both the frontal and abfrontal part appears to be free and contains a large nucleus that sides (Figs. 2b, 6e). They pass within the radial muscular contains an acentric nucleolus (Fig. 6d). Myoﬁlaments bunches (see below) of the epistome. The protrusions of the

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Fig. 4 Serial sagittal histological sections through the anterior body (in comparison with b). The dorsal (abfrontal) side of the body (at the part of Phoronopsis harmeri. The distance between sections: left) has a compound lophophoral coelom (lc) with several parts, a a–b 14 lm, b–c 21 lm, c–d 21 lm, d–e 28 lm, e–f 14 lm. a A nephridial funnel (nf), one of two T-branches (T) of the median blood section through the right side of the body through the lateral part of vessel, the descending/efferent lophophoral blood vessel (dlv), and the epistomal cavity (borders are indicated by arrowheads), epistome diaphragm (d). d–f Sections through the mouth (m); there is large (ep), lophophoral groove (lg), and lophophoral coelom (lc) with cavity inside epistome (central part of epistomal cavity). d In the ascending/afferent (alv) and descending/efferent (dlv) lophophoral dorsal (abfrontal) side of the body (at the left), the descending/efferent blood vessels. b The epistomal cavity is divided into dorso-lateral (at lophophoral blood vessel disappears and there are only two parts of the left, arrowheads) and central (medial line, arrowheads) parts by the lophophoral coelom (lc). e The dorsal (abfrontal) side of the body the lophophoral coelom. The trunk coelom (tc) is under the (at the left) contains only one part of the lophophoral coelom (lc). diaphragm (d). c The dorso-lateral part of the epistomal cavity f Large central part of the epistomal cavity (arrowheads) and the disappears; the central part (arrowheads) becomes more voluminous diaphragm (d), which is perforated by the median blood vessel (mv)

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Fig. 5 Phoronopsis harmeri epistome, details of frontal epidermis central cell parts with large granules, apical ﬁlaments (arrows), and organization (TEM, longitudinal sections). a Epidermis sector, nucleus (n). d Part of gland cell (gc) with rough endoplasmic general view. b Ciliary basal structures: basal body (bb), accessory reticulum (rr) and electron-dense granules (gr). G Golgi complex, ec centriole (ac), vertical (vr), and horizontal (hr) rootlets. c Apical and epistomal cavity (preoral coelom), mi microvilli

fibrillar layer are formed by numerous thick electron-dense diverse (Fig. 10c). There are areas with high density of fibers (Fig. 6e). collagen, with powdery collagen, and without collagen The cavity in the epistome is restricted to the central and fibers. Last areas contain amorphous material and seem lateral parts, i.e., the cavity is absent in its dorso-lateral electron transparent (Fig. 10c). There is no evident con- parts (Fig. 2b). The cavity has a complex shape and con- nection between the ECM of the partition and the ECM of sists of a central part and two lateral branches. The cavity the epidermis (Fig. 10b). In frontal sections through lies above the lophophoral coelom and contacts it, forming the central part of the epistome, the partition between the two complete dissepiments on the lateral sides and one epistomal cavity and lophophoral coelom is very thin and incomplete partition in the central part (Figs. 4, 8). In incomplete (Fig. 8a, b). sagittal sections, the dissepiment between the central part Cross-sections reveal two complete dissepiments of the epistomal cavity and lophophoral coelom appears as between the lateral sides of the epistomal cavity and the a thin partition composed of two cellular layers without a lophophoral coelom (Fig. 8c, d). These dissepiments con- thick fibrillar layer between (Fig. 4a–e). In semi-thin sec- sist of a continuous central ECM layer the myoepithelial tion, this partition appears to consist of one layer of cells monociliary cells rest on (Fig. 7a). The ECM fibrillar layer (Fig. 10a). TEM, however, reveals that the incomplete is composed of two parts: the first, which is 0.5 ± 0.1 lm partition is organized like mesentery, with two layers of thick, is formed by circular collagen fibers and is situated cells separated by ECM (Fig. 10b). The ECM is penetrated near the preoral coelom; the second, which is 1.5 ± 0.3 lm by processes of muscle cells and does not form a single thick, is formed by longitudinal collagen fibers and is sit- layer (Fig. 10b). The density of ECM fibrillar layer is uated near the lophophoral coelom (Fig. 7a).

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Fig. 6 Phoronopsis harmeri epistome (TEM, longitudinal sections). thick collagen ﬁbers (cf)) on frontal (fs) and abfrontal (afs) sides. cc a Basal part of frontal epidermis with neural processes (np) and ECM coelothelial cell (lining of epistomal cavity), G Golgi complex, mb (ECM). b Abfrontal epidermis sector, general view. c Apical cell part multivesicular body, mi microvilli, mf myoﬁlaments, vr vertical of abfrontal epidermis. d ECM (ECM) of abfrontal epidermis and rootlet. The desmosomes between coelothelial cells are marked by lining of epistomal cavity (ec). e Protrusions of ECM (with numerous arrowheads

Numerous amoebocytes occur in the coelom lumen The tip of the epistome and its dorso-lateral parts lack a (Fig. 9d). Large phagosomes, electron-dense granules, coelomic cavity. Here, the ECM occupies the space inclusions with intermediate electron density, vesicles, and between the frontal and abfrontal epidermis (Fig. 10a). The glycogen granules are found in their cytoplasm. The ﬁbrillar layer of the ECM lacks collagen ﬁbrils and con- nucleus is small and irregular in shape (Fig. 9d). tains solitary muscle cells that are not connected to each 123 128 Zoomorphology (2011) 130:121–134

radially (Fig. 11b). The basal cell membrane forms 0.4 ± 0.1 lm long protrusions surrounded by the ECM (Fig. 11b). These basal protrusions are connected to the ECM by hemidesmosomes. ECM is represented by a 0.25–1-lm-thick layer of electron-dense amorphous material (Fig. 11a). The third layer consists of endothelial cells that form a solid lining. Desmosome-like contacts between endothelial cells are sometimes evident (Fig. 11b). The apical cell part is free and expanded and contains the nucleus. The middle part of the cell is narrow and occupied by mitochondria. The basal cell membrane forms a small number of processes that extend into the ECM. Erythrocytes and amoebocytes occur in the vessel lumen (Fig. 11a).

Discussion

Epidermis

The epistomal epidermis consists of ciliated cells. The position of accessory centrioles in these cells supports the direction of the effective stroke. On the frontal side, the cilia beat in the direction of the mouth opening, while the abfrontal cilia create water flow toward the epistomal tip (see Gilmour 1978; Riisga˚rd 2002). The cilia contribute to Fig. 7 Phoronopsis harmeri (TEM, longitudinal sections). a Dissep- the epistome’s function as a filter that defines the upper size iment between lateral part of epistomal cavity (ec) and lophophoral of particles taken in after capture by the lophophore ten- coelom (lc). b Lining of preoral coelom (ec), sector with desmosomes tacles (Temereva and Malakhov 2010). (arrowheads), hemidesmosome (double arrow), and myofilaments Apical granule organization in the frontal epidermis (mf). cf circular collagen fibers, lf longitudinal collagen fibers. Basal body and accessory centriole in coelothelial cell of lophophoral cells is similar to phagosome organization in digestive cells coelom are marked by white arrows (e.g., see Vandermeulen 1970). The existence of phagosomes and numerous specialized gland cells may indicate other via desmosomes (Fig. 9e). These cells form processes that digestive activity occurs in the epistome frontal epi- containing myofilaments and are attached to the basal dermis. This inference is consistent with previous research lamina by hemidesmosomes. The nucleus occupies the cell indicating that esterasic and phosphotasic activities are center and contains a nucleolus (Fig. 9e). very high in the upper portion of the digestive tract (Vandermeulen and Reid 1969; Vandermeulen 1970). Blood vessel Epistomal cavity and coelomic lining The two branches of the median blood vessel form a T-shaped structure at the base of the epistome underneath The existence of the coelomic cavity in the epistome has the epistomal cavity and close to the mouth (Figs. 8a, b, been shown for Phoronis muelleri (Siewing 1974; Herr- 10a). The walls of the vessels are composed of two layers: mann 1980, 1986), Phoronis psammophila Cori, 1889 the ECM and the inner lining of the blood vessel (Emig and Siewing 1975), and Phoronis ijimai Oka, 1897 (Fig. 11a). The blood vessel is surrounded by coelothelial (Pross 1974, 1978). These studies do not provide strong epithelium of lophophoral cavity (Fig. 10a). This epithe- evidence that the epistomal cavity is coelomic in nature, lium is formed by myoepithelial cells with numerous ves- because they lack clear documentation of an epithelial icles and cisterns of rough endoplasmic reticulum lining. Bartolomaeus (2001) failed to recognize the true (Fig. 11b). The cell apex is free and contains the nucleus coelomic epithelium at the ultrastructural level in Phoronis with a large nucleolus. The middle cell forms processes muelleri. His study revealed that both larval and adult P. bearing desmosomes (Fig. 11). The myofilaments extend- muelleri lack an epistome cavity with such a lining. ing into the basal cell cytoplasm are directed circularly or According to data of Gruhl and coauthors (2005), there is 123 Zoomorphology (2011) 130:121–134 129

Fig. 8 Histological section through the anterior body part of epistomal cavity (ec) and lophophoral coelom (lc). c, d Cross- Phoronopsis harmeri. a, b Frontal sections through the epistomal sections. c General view of the section through the collar fold (cf), cavity (ec), which is crossed by numerous cells and bordered with nephridial duct (nd), epistomal cavity (ec), lophophoral coelom, and lophophoral coelom (lc) by T-branches (T) of median blood vessel lophophoral blood vessels (dlv). d Left side of the cross-section and thin partition (arrows). The diaphragm between lophophoral and through the dissepiment between the epistomal cavity (ec) and trunk coeloms is indicated by arrowheads. a General view of section. lophophoral coelom (lc) b The left side of the section shows a thin partition between the

also no coelomic cavity inside the epistome of Phoronis that are interconnected by apical adherens junctions. The ovalis. In this species, this part of the body is ﬁlled with myoepithelial cells of the coelomic lining rest on an ECM. myoepithelial cells, which are continuous with the epithe- Some of the myoepithelial cells of the epistome lining in P. lial lining of the lophophore cavity. These cells form a harmeri are spindle shaped. These cells resemble muscles lumenless bilayer and possess long, tiny myoﬁlamentous cells in the mouth shield of pterobranchs and in the pro- processes, which are completely embedded in an extra- boscis of enteropneusts (Benito and Pardos 1997). In both cellular matrix. Based on their data, Grobe (2008) con- cases, cells with the nucleus in the central part are attached cluded that two coelomic compartments are plesiomorphic at two ends and span the coelomic cavity. in Phoronida. The epistome cavity of the adult P. harmeri, thus, is a According to data presented here, there is a coelomic true coelom. The present data are in accordance with pre- cavity inside the epistome of Phoronopsis harmeri which is vious observations of larvae in Phoronopsis species. Zim- completely lined by a true epithelium with polarized cells mer (1978) noted that larvae of Phoronopsis californica 123 130 Zoomorphology (2011) 130:121–134

Fig. 9 Phoronopsis harmeri epistome (TEM, longitudinal sections). lamina. Radial myoﬁlaments (mf) adhere to ECM by hemidesmosome A–C. Radial musculature: a Radial protrusions of coelothelial cells (double arrow). d Amoebocyte with large phagosomes (ph) in the with myoﬁlaments (mf). The desmosomes are indicated by arrow- epistomal cavity lumen. e Muscle cells (mc) in the epistome tip. ac heads. b Cell body with nucleus (n) and nucleolus. The desmosomes amoebocyte, afs epistome abfrontal side, bl basal lamina, ec preoral are indicated by arrowheads. c Long process procumbent on the basal coelom, fs epistome frontal side

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Fig. 10 The partition between the epistomal cavity and the loph- The desmosomes in the coelomic lining of the epistomal cavity (ec) ophoral coelom. a Semi-thin parasagittal section along the epistome. are indicted by arrowheads. c The section of partition’s ECM. The The partition (pt) appears as a thin border between the epistomal density of ﬁbrillar layer is diverse; there are areas with high density of coelom (ec) and the lophophoral coelom (lc). The tip of the epistome collagen (da), with powdery collagen (pa), and without collagen does not contain the coelomic cavity and is occupied by solitary ﬁbers (ta). The arrows indicate hemidesmosome. afs epistome muscle cells (mc). b Thin longitudinal section through the epistome abfrontal side, bv blood vessel—T-shaped vessel, d diaphragm, er and partition. The ECM (ECM) is penetrated by muscle cells (mc). erythrocyte, fs epistome frontal side tc trunk coelom

Hilton, 1930 and Ph. harmeri possess a ‘‘diminutive pro- epistomal and trunk cavities (Gruhl et al. 2009). This sit- tocoel’’ that looks like a cylinder under the apical plate. uation can be explained in two ways: ﬁrst, the epistomal Moreover, a clearly visible cylinder-like protocoel is a cavity might be formed as a protrusion of the trunk coelom distinctive feature of Phoronopsis species larvae (Temer- (in bryozoans) or of the lophophoral coelom (in phoronids); eva 2009). According to a recent electron microscopic second, the epistome might be a true coelomic cavity. Our study, the lining of the larval protocoel of Ph. harmeri is own and other studies provide strong evidence for the latter formed by monociliary cells interconnected by apical evidence, because a preoral coelom occurs in larvae of all adherens junctions and resting on ECM (Temereva and Phoronopsis species (Zimmer 1978; Temereva and Mal- Malakhov 2006). akhov 2006; Temereva 2009). In addition, the partition It is interesting that the central part of the epistomal between lophophoral and epistomal coeloms is organized cavity has an incomplete partition in Ph. harmeri. The like true mesentery with two cellular layers with ECM same is known for other Lophophorates. In phylactola- between them. Determining whether the epistomal cavity ematan bryozoans, for example, the dissepiments between arises from the larval preoral coelom in Phoronopsis spe- the lophophoral and trunk coeloms are incomplete, too cies will require detailed study of the fate of the larval (Hyman 1959). Moreover, in some species of phylactola- preoral coelom during metamorphosis from larva to adult. ematan bryozoans there is no true partition between Moreover, our previous work provides photographic

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evidence for the presence of a preoral coelom during some stages of Phoronopsis harmeri metamorphosis (see Temereva 2010: 1165–1166, Figs. 43, 44).

Blood vessel

In Ph. harmeri, the two branches of the median vessel blood, which form a T-branch, are located in the lophophoral coelom under the epistomal cavity close to the mouth. The epistomal cavity and the blood vessel are separated by a partition with many holes. The position of the median vessel and its branches was described by Selys- Longchamps (1907), who was convinced that the T-shaped vessel is situated in the lophophoral coelom. In contrast, Emig and Siewing (1975) stated that ‘‘the two basic branches of median blood vessel lie free in the epistome- coelom’’ in Phoronis psammophila. The vessel wall in the epistome consists of two layers: the ECM and the inner lining of the blood vessel. The blood vessel is surrounded by the coelothelial epithelium of the lophophoral cavity. This sort of organization is typical for the median vessel in the head region of Ph. harmeri (see Temereva and Malakhov 2004). The presence of ‘‘continuous’’ endothelial lining in the distal part of Pho- ronis psammophila median blood vessels was discovered by Emig in 1977. Usually, the endothelial cells do not connect to each other via desmosomes, but desmosome- like structures sometimes occur between the endothelial cells (see Temereva and Malakhov 2004). These are not true desmosomes, and the endothelium does not form a true epithelium inside the vessels. In addition, the vessel lumen always contains the erythrocytes, a feature that clearly distinguishes the vessel lumen from a coelomic cavity.

Conclusion

Our results show the presence of three coelomic compartments in Phoronopsis harmeri: the epistomal coelom, the lophophoral coelom, and the trunk coelom. These data corroborate our previous results, which were obtained with Phoronopsis harmeri embryos (Temereva and Malakhov 2007) and larvae (Temereva and Malakhov 2006). As Fig. 11 Sagittal sections of the T-branch of median blood vessel in shown in these earlier papers, the preoral coelom, which Phoronopsis harmeri epistome basis (TEM). a The sector of forms in embryo, is the first coelom, and the larva of this vascular wall and lumen (bl) with erythrocyte (er) and amoebocyte species has a well-developed preoral coelom that is located (am). The blood wall is formed by the ECM (ECM) and the endothelium (enc) and is surrounded by coelothelium (cc). b Three in the preoral lobe under the apical plate. As mentioned layers of blood wall at high magnification. Small white arrowheads above, some preliminary information is available con- indicate the basal protrusions and hemidesmosomes. lc lophophoral cerning the fate of the larval preoral coelom during meta- coelom, m mitochondria, mf myofilaments, rr rough endoplasmic morphosis (see Temereva 2010). This preliminary reticulum. Arrows show desmosome-like contacts between endothelial cells. The black large arrowheads indicate desmosomes information indicates that the epistomal cavity of adult between coelothelial cells Phoronopsis harmeri corresponds to the preoral coelom of

123 Zoomorphology (2011) 130:121–134 133 the larva of this species. Moreover, given that the coelomic number of phoronids that differ in size. Specifically, it will be cavity has been found in the larvae of other Phoronopsis very interesting to study the epistome organization in large species (Zimmer 1978; Temereva 2009), we suggest that representatives of the genus Phoronis, for example, in all Phoronopsis species probably have a protocoel. Phoronis australis Haswell, 1883. On the other hand, the results of some authors’ investigations (Bartolomaeus 2001; Gruhl et al. 2005; Grobe Acknowledgments ENT is grateful to Svetlana Maslakova for 2008) have cast doubt on the existence of a three coelomic hosting her at the Oregon Institute of Marine Biology (Coos Bay) in 2010. We appreciate an anonymous reviewer for his criticisms and compartments in phoronids. According to these results, improving of manuscript. We are most grateful to Dr. Mikhailov, larvae of Phoronis species do not have a preoral coelom Dr. Jaffee for the help with manuscript preparation. The project was (Bartolomaeus 2001; Temereva 2006), and the epistome of funded by the Russian Foundation for Basic Research (project 11-04- adult Phoronis species does not have a cavity (Bartolo- 00690), Russian Ministry of Education and Science and Grant Council of the President of Russia (#4456.2010.4, #02.740.11.0280, maeus 2001; Gruhl et al. 2005). #P727, # MD-2892.2011.4). Thus, among phoronids there are two patterns of organization for the coelomic system. The first pattern—the bipartite coelom—is found in specimens of Phoronis, which References have two coelomic compartments: the mesocoel (the ten- tacular or lophophoral coelom) and the metacoel (the trunk Bartolomaeus T (2001) Ultrastructure and formation of the body coelom). The second pattern—the tripartite coelom—is cavity lining in Phoronis muelleri (Phoronida, Lophophorata). 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