JOURNAL OF MORPHOLOGY 258:269 –283 (2003)
Larval Development of Myzostoma cirriferum (Myzostomida) Igor Eeckhaut,1* Laurence Fievez,1 and Monika C.M. Mu¨ller2 1
Marine Biology Laboratory, Natural Sciences Building, University of Mons-Hainaut, Mons, Belgium Spezielle Zoologie, Fachbereich Biologie/Chemie, Universita¨t Osnabru¨ck, Osnabru¨ck, Germany
ABSTRACT The larval development of Myzostoma cirriferum is described by means of SEM, TEM, and cLSM. It is similar to that of other myzostomids and includes three stages: the protrochophore, the trochophore, and the metatrochophore. The protrochophore is a ball-shaped larva present in culture from 18 – 48 h after egg laying. It has no internal organs and its body is made of three cell types: covering cells and ciliated cells that are external and surrounded by a cuticle, and resting cells that ﬁll the blastocoel. The trochophore is a pear-shaped larva that develops 20 –72 h after egg laying; the body includes the same three cell types as the previous stage. The metatrochophore is a pear-shaped larva that develops between 40 h and 14 days and is characterized by the presence of two bundles of four chaetae. When fully developed, the metatrochophore has a digestive system (made of a pharynx, an esophagus, and a blind digestive pouch), two pairs of protonephridia, and a nervous system composed of a supraesophageal ganglion, circumesophageal connectives, and dorsal and ventral nerves. Metamorphosis generally occurs 7 days after egg laying. At that time, the metatrochophore loses its chaetae and becomes pleated ventrally. This ultrastructural analysis suggests that chaetae and the ﬁve ventral longitudinal nerve cords of M. cirriferum metatrochophores are homologous structures to those observed in some polychaete trochophores. Coupled with recent phylogenetic analyses, where the Myzostomida are placed outside the Annelida, homologies between myzostomid and polychaete larvae support the view that a trochophore appeared early during the spiralian evolution. J. Morphol. 258:269 –283, 2003. © 2003 Wiley-Liss, Inc. KEY WORDS: Myzostomida; Annelida; trochophore; larval development; Spiralia
The taxon Myzostomida includes about 150 marine species, all of which are associated with echinoderms. Most are ectocommensals of crinoids but some are ecto- or endoparasites of crinoids, asteroids, or ophiuroids and infest the gonads, the coelom, the integument, or the digestive system of their host (Jangoux, 1990; Grygier, 2000). The association between myzostomids and echinoderms is very old, as signs of parasitic activities, similar to those induced by some extant parasitic myzostomids, are found on fossilized crinoid skeletons dating back to the Ordovician (Warn, 1974; Meyer and Ausich, 1983; Eeckhaut, 1998). Due to their long history as host-speciﬁc symbionts, myzostomids have acquired © 2003 WILEY-LISS, INC.
a unique, highly derived, adult anatomy that obscures their phylogenetic afﬁnities to other metazoans. The body plan of most of them is indeed singular, as they are parenchymous, acoelomate organisms with chaetae (see Grygier, 2000, for a recent review about myzostomid body plans). A recent study of the nervous system of the species Myzostoma cirriferum suggests that myzostomids evolved from a segmented organism, presumably an annelid (Mu¨ller and Westheide, 2000). On the other hand, phylogenetic analyses of DNA sequences including those of some myzostomid species strongly support the exclusion of the Myzostomida from the Annelida and place them close to Platyhelminthes (Eeckhaut et al., 2000; Zrzavy et al., 2001). The larval stages of metazoans can provide valuable characters for estimating the phylogenetic relationships between higher taxa. A speciﬁc larval stage, however, is often attributed to a whole taxon even if such larval stage has only been observed in a few representatives of the taxon. Another problem encountered by phylogeneticists using larval characters in their studies is the difﬁculty of assuming the homology among larvae and larval organs. For example, Rouse (1999) recently presented evidence that various ciliary bands in trochophores have had different evolutionary histories and, as a consequence, he restricted the taxa where the larval type “trochophore” is present unambiguously to the Annelida, Echiura, Entoprocta, Mollusca, and Sipuncula. Deciphering the ultrastructure of larvae is an important step to inferring homology among larval structures. In myzostomids, larval development has previously been studied using optical and/or scanning electron microscopy in the species Myzostoma
Contract grant sponsor: the F.N.R.S.; Contract grant number: 2.4.505.98.f. *Correspondence to: Igor Eeckhaut, Marine Biology Laboratory, Natural Sciences Building, University of Mons-Hainaut, 6, Av. Champ de Mars, 7000 Mons, Belgium. E-mail: [email protected]
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cirriferum (Beard, 1898; Eeckhaut and Jangoux, 1993a), M. parasiticum (Beard, 1898; Ja¨gersten, 1939), M. ambiguum (Kato, 1952), and M. alatum (Eeckhaut and Jangoux, 1992). The larval development in the four species includes three stages: the small, rounded protrochophore (Eeckhaut and Jangoux, 1993a), the pear-shaped trochophore (Eeckhaut and Jangoux, 1993a) (also called agasoma larva; Salvini-Plawen, 1980), and the pearshaped metatrochophore with chaeta (Eeckhaut and Jangoux, 1993a) (or lasiphora larva; SalviniPlawen, 1980). At metamorphosis, myzostomid larvae lose their chaetae and develop into juveniles that are always associated with echinoderms. The present study is the ﬁrst to investigate the ﬁne structure of the three larval stages of the European species, M. cirriferum. The aim is to compare myzostomid larvae to the trochophores sensu lato, especially to those of polychaete annelids to which myzostomids are often thought to be most closely related. MATERIALS AND METHODS Adults of Myzostoma cirriferum Leuckart, 1836, were collected with their host Antedon biﬁda by scuba diving at Morgat (Brittany, France) from September 1996 to January 1997. They were maintained in an open-circuit aquarium at the Observatoire Oce´anologique de Roscoff. Adult myzostomids 1–2 mm in length were separated from their hosts and placed in Petri dishes ﬁlled with 0.2 m ﬁltered seawater including 50 mg/l of streptomycin sulfate (temperature 20°C). These animals laid batches of fertilized eggs after a few hours. Cultures were maintained under slight agitation and the water was changed every day. Batches of larvae were ﬁxed every 24 h for scanning or transmission electron microscopy (SEM and TEM, respectively). For SEM observations, larvae were ﬁxed in Bouin’s ﬂuid for 24 h, dehydrated in a graded ethanol series, dried by the critical point method (using CO2 as the transition ﬂuid), mounted on aluminum stubs, coated with gold in a sputter coater, and observed with a JEOL JSM 6100 scanning electron microscope. For TEM observations, larvae were ﬁxed in a 3% solution of glutaraldehyde in cacodylate buffer (0.1 M, pH 7.8) for 3 h at 4°C. They were rinsed in the buffer and postﬁxed for 1 h with a 1% solution of osmium tetroxide in the same buffer. After a ﬁnal rinsing in buffer they were dehydrated in a graded ethanol series and embedded in Spurr’s resin. Transverse and longitudinal serial 100 nm thin sections were performed on the larvae using a Leica Ultracut UCT ultramicrotome. Sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM10 transmission electron microscope. For cLSM (confocal laser scanning microscopy) observations, individuals in different developmental stages were anesthetized for 10 min in 8% MgCl2 solution. The relaxed animals were ﬁxed on ice overnight in 4% paraformaldehyde in 0.15 M PBS (phosphate buffered saline, pH 7.4) containing 8% sucrose. After several rinses with PBS for at least 1 h, the larvae were preincubated for 1 h in PBS containing 0.1% Triton X-100, 0.25% BSA (bovine serum albumin), and 0.05% NaN3. Incubation with the primary antibody directed against acetylated ␣-tubulin (Sigma, Heidelberg; dilution 1:100 in preincubation liquid without BSA) was carried out for 12 h. Subsequently, the larvae were washed several times in PBS and incubated with a secondary FITCconjugated antibody (dilution 1:100) for 12 h. Finally, the specimens were rinsed in PBS and mounted between two coverslips in Citiﬂour (Plano, Wetzlar). Preparations were analyzed with a confocal laser scanning microscope, Zeiss LSM 410.
RESULTS SEM Observations The protrochophore is present in cultures 18 – 48 h after egg laying. It is spherical and measures 30 – 40 m in diameter (Fig. 1A). It has an equatorial ciliary corona that divides its body into a hyposphere and an episphere. The trochophore is 20 –72 h old. It measures 40 –50 m in length and differs from the protrochophore in having an elongated hyposphere giving the larva a pear-shaped aspect (Fig. 1B). At the top of the episphere lies an apical tuft of ca. 10 cilia (each 10 m long) (Fig. 1B). The ﬁrst metatrochophores appear 40 h after egg laying. The metatrochophore is pear-shaped and measures 50 –70 m in length, but differs from the previous stages in that it has two pairs of four lateral chaetae (Fig. 1C). Chaetae are denticulate and their length can reach up to 150 m (in 10-day-old larva). The 4-day-old metatrochophore acquires a retractile caudal appendage 5–15 m long (when retracted or extended, respectively) and an anteroventral mouth located in the episphere, above the equatorial ciliary corona that is divided into two ventral tufts (Fig. 1D). At this stage a hypospheral ciliary corona surrounds the caudal appendage (Fig. 1C). During the ﬁfth day, a tuft of 10 straight cilia 5 m long appears at the top of the caudal appendage. Metamorphosis into postmetamorphic juveniles occurs, for most of the metatrochophores, on the seventh day. It involves the loss of all chaetae and cilia and the regression of the caudal appendage. Metamorphic larvae are bean-shaped worms with their body folding back ventrally (Eeckhaut and Jangoux, 1993a). When maintained in vitro, larvae generally die when they are 12–14 days old. TEM Observations The internal morphology of both protrochophores and trochophores is similar (Fig. 2A,B). Most morphological changes occur in the metatrochophore stage. According to their internal morphology, one can distinguish young, intermediate, and late metatrochophores (Fig. 2C–E). Protrochophore and Trochophore. Both protrochophore and trochophore are larvae whose blastocoel is ﬁlled with cells (Fig. 2A,B). The body of both larval stages is composed of three cell types — covering cells, ciliated cells, and resting cells — that remain until larval development is complete (Fig. 3A–C). Covering cells and ciliated cells both lie under a cuticle that is derived from the fertilization membrane of the eggs (Fig. 3A,B). They form the future epidermis of the larva, which appears 72 h after egg laying, when a basal lamina also differentiates. The main larval surface is formed by covering cells, the ciliated cells being only present at the level of the equatorial ciliary corona and apical tuft. Rest-
LARVAL DEVELOPMENT OF MYZOSTOMA CIRRIFERUM
Fig. 1. Larval stages in Myzostoma cirriferum (SEM). A: Protrochophore. B: Trochophore. C,D: General view of a 4-day-old metatrochophore and details of its upper ventral part, respectively. AT, apical tuft; CA, caudal appendage; CH, chaeta; EC, equatorial ciliary corona; HC, hypospheral ciliary corona; M, mouth; ET, equatorial tuft.
ing cells ﬁll the larval body (Fig. 3B). They progressively transform into the various internal cell types that will form the body of late metatrochophores. The structure of the cuticle is similar in protrochophores, trochophores, and metatrochophores. It is 200 nm thick in protrochophores and reaches 500 nm thick 72 h after laying (Figs. 3A–C, 4D). It is made of three ﬁbrillar layers: an upper electrondense layer 50 –100 nm thick, a middle, less electron-dense layer, 100 –200 nm thick, and an inner electron-lucent layer 50 –200 nm thick (Figs. 3C, 4D). The cuticle is crossed by the microvilli of both covering cells and ciliated cells (Fig. 3A,C). Covering cells and resting cells are polyhedral and measure ca. 5–10 m in diameter (Fig. 3B). The cytoplasmic fringe that borders their nucleus is thin, generally 1 m thick, and includes a few mitochondria, poorly developed rough endoplasmic reticulum and Golgi apparatus, and electron-dense yolk droplets 100 nm to 2 m in diameter (Fig. 3B,C). Covering cells and resting cells differ from each other by their position (peripheral and internal, respectively) and by the number of yolk droplets they include (on average, 10 and 2 per section, respectively) (Fig. 3B). In addition, covering cells have microvilli 150 – 600 nm long and they are joined together by zonulae adherentes and septate junctions. Ciliated cells are cylindrical and measure ca. 10 m high (Fig. 3A). Each cell has 10 –25 cilia ca. 10 m long. Each basal body is prolonged by two ciliary rootlets separated from each other by an angle of 6° (Fig. 3A). Their cytoplasm
includes a few yolk droplets and they bear numerous microvilli 150 – 600 nm long (Fig. 3A). Young metatrochophore. The young metatrochophores are 40 –96 h old. They differ from protrochophores and trochophores in having a pair of chaetal follicles, located on the right and left sides of the hyposphere. Each chaetal follicle is composed of two new cell types, i.e., primary chaetoblasts and lateral cells. Seventy-two hours after egg laying, an epidermal basal lamina appears as a thin electrondense matrix 20 nm thick that surrounds the chaetal follicles. At the same time, some muscle cells begin to differentiate from resting cells and their apices contact the basal lamina of chaetal follicles. A basal lamina is still not obvious under the covering cells. Chaetal follicles are ovoid cell masses 25 m long and 15 m wide (Fig. 4A,B). Each is made of four primary chaetobasts and four to ﬁve lateral cells. Both cells are grossly ovoid, 5 m in diameter, and include numerous mitochondria. Primary chaetoblasts are the innermost cells of the follicles. They do not contact the cuticle and each of them synthesizes one chaeta (Fig. 4C). The chaetoblast cell membrane is depressed in such a way that each chaetoblast has a ﬁnger-like invagination where a chaeta is synthesized (Fig. 4F). Electron-dense material 10 –100 nm in diameter is secreted by chaetoblats into the extracellular spaces (Fig. 4C). Lateral cells contact the cuticle and form a sheath around chaetae that reach the external medium (Fig. 4B). They enclose the chaetae by
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Fig. 2. Schematic drawings of the larval stages in Myzostoma cirriferum. A: Protrochophore. B: Trochophore; C,D,E: Young, intermediate, and late metatrochophores, respectively. CA, caudal appendage; CF, chaetal follicle; CIC, ciliated cell; COC, covering cell; DP, digestive pouch; E, esophagus; PE, pharyngeal epithelium; PL, pharyngeal lumen; PM, pharyngeal muscle sheath; PN, protonephridium; RC, resting cell; SC secondary chaetoblast; SP, sensory process.
means of cell processes that are tightly joined together through septate junctions and zonula adherentes (Fig. 4E). In transverse section, each chaeta is circular, 2 m in diameter (Fig. 4B,E). It is made of an electron-dense granular material arranged around one large, central, lucent channel 500 nm in diameter and ca. 20 small peripheral channels (Fig. 4E). Intermediate metatrochophore. The intermediate metatrochophores are 66 –144 h old. They differ from the last stage in having a caudal appendage that appears at the top of the hyposphere, a differentiating digestive system, and a pair of protonephridia (Fig. 2D). Nerve cells and nerve processes are now clearly visible and a basal lamina completely underlies the epidermis. The caudal appendage includes an axial core of resting cells crossed by muscle ﬁbers and nerve processes. Muscle ﬁbers, ca. 50 m long each, are located ventrally and dorsally in the caudal appendage. They anchor to the basal lamina of epidermal cells in the mid part of the larval body and at the top
of the caudal appendage. Nerve processes are dendrites of nerve cells whose bodies lie in the brain in the larval episphere (Fig. 5A,D). The brain is a cell mass, 20 m in diameter, located in front of the posterior part of the pharynx. It is made of nerve cells, each with a large nucleus and a small fringe of cytoplasm (Fig. 5D). The cells are surrounded by nerve processes that include two types of vesicles: large, dense-cored vesicles 70 –140 nm in diameter (Fig. E), and small, clear vesicles 10 –30 nm in diameter (Fig. 5F). Nerve processes have been observed around the muscle sheath associated with the pharynx (Fig. 5A) and between the pharyngeal muscle sheet and the pharyngeal epithelium (Fig. 5B). Nerve processes end in sensory ciliated processes at the level of the mouth and the caudal appendage (Fig. 5C). Nerve processes contain mitochondria and the dense-cored vesicles (Fig. 5C). Each sensory ciliated process bears one to three straight cilia that cross the cuticle. Cilia have the usual microtubular arrangement (9 ⫻ 2 ⫹ 2). Be-
LARVAL DEVELOPMENT OF MYZOSTOMA CIRRIFERUM
Fig. 3. Trochophore of Myzostoma cirriferum (TEM) A: Ciliated cell and cuticle. B: Covering cells and resting cells. C: Cuticle. BB, basal body; CC, covering cell; CU, cuticle; CR, ciliary rootlet; MI, mitochondria; RC, resting cell; VI, microvilli; Y, yolk droplet. Arrows 1, 2, 3 indicate the 1st, 2nd, and 3rd layer of the cuticle, respectively.
neath each cilium lies a basal body lacking a ciliary rootlet (Fig. 5C). The digestive system is made of two separate parts: a blind pharynx (20 m long) that opens to the exterior through the blastopore, and a differentiating digestive pouch (Fig. 2D). The pharyngeal epithelium is composed of covering cells with cuticle similar to those of the epidermis (Fig. 5A,B). It is surrounded by a muscle sheath ca. 5 m thick (Fig. 5A,B). The muscle sheath includes circular and radial muscle ﬁbers that are isolated from the surrounding resting cells by a basal lamina 20 nm thick (Fig. 5A). In the middle of the hyposphere, some resting cells transform into digestive cells that will progressively form the epithelium of a digestive pouch (Fig. 5I). Developing digestive cells are irregular, approximately 10 m in diameter. Their cytoplasm includes many large vacuoles 500 nm to 3 m in diameter that often include electron-dense bodies (Fig. 5I). The content of these vacuoles is gradually secreted into the forming lumen of the digestive pouch, thus increasing its volume. The developing digestive cells gradually form an epithelium that surrounds an ovoid digestive lumen: they are joined by septate junctions and zonula adhaerentes and separate from the surrounding resting cells by a basal lamina (Fig. 5G). Progressively, they acquire microvilli and become ﬂattened (Fig. 5G).
Two pairs of protonephridia appear during metatrochophore development (Fig. 2E). The protonephridia that appear ﬁrst are located in the right and left sides of the hyposphere during the third day of development. They are curved ducts ca. 15 m long that open to the exterior through inconspicuous slits below the chaetal follicles. Protonephridia of the second pair, only observed with cLSM, appear in the episphere 1 day later and open on either side of the mouth. Each protonephridium is made of one terminal cell and three duct cells that contact epidermal cells (Fig. 5H). All these cells are joined together by septate junctions and zonula adherentes (Fig. 5H). They are separated from the surrounding resting cells by a basal lamina ca. 50 nm thick. The terminal cell is ovoid, measures ca. 7 m in diameter and has a heterochromatic nucleus (Fig. 5H). Microvilli and six cilia, whose axonemes have a 9 ⫻ 2 ⫹ 2 microtubular arrangement, extend from the terminal cell into the protonephridial lumen. The lumen is ca. 10 m long and is ﬁlled with cilia and microvilli. The duct cells are ca. 7 m in diameter and reach the epidermis where they contact the base of covering cells. Duct cells have no microvilli, or cilia, and their structure is very similar to the surrounding resting cells (Fig. 5H). Late metatrochophore. Most of the late metatrochophores appear on the sixth day of devel-
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Fig. 4. Young metatrochophore of Myzostoma cirriferum (TEM). A: Frontal section through a larva showing the episphere and the two chaetal follicles (left is indicated by arrows). B: Transversal section through a chaetal follicle (broad and thin arrows show the chaetal follicle and chaetae, respectively) C: Section through a chaetoblast and a forming chaeta. D: Cuticle and microvilli. E: Transversal section through a chaeta. F: Longitudinal section through the base of a chaeta. CCH, central channel; CH, chaeta; CU, cuticle; DM, dense material; E, episphere; H, hyposphere; MI, mitochondria; NU, nucleus; PCH, peripheral channel; RC, resting cell; SJ, septate junction; VI, microvillus; ZA, zonula adhaerens.
opment, but some are present from the fourth day. The main structural differences in these metatrochophores compared to the last stage are the presence of a fully formed larval integument, a complete larval digestive system, a second pair of nephridia, and a second pair of chaetoblasts (Fig. 2E). The late metatrochophores have no coelom but a blastocoel largely ﬁlled by resting cells. The blastocoel appears as a small cavity at the bottom of the hyposphere. It often includes cytoplasmic sheets of degenerated cells, including yolk droplets (Fig. 6D). The integument of late metatrochophores is made of an epidermis with cuticle and a parenchyma that ﬁlls the blastocoel between internal organs (Fig. 6A). The epidermis includes covering cells between which end sensory processes of nerve cells. The cuticle is now 700 nm to 1 m thick. Covering cells are similar to those observed in earlier stages but the
number of yolk droplets has decreased signiﬁcantly. The parenchyma is made of resting cells between which occur muscle cells (Fig. 6A). Muscle cells do not form a continuous layer under the epidermis. Retractor muscle cells occur in the caudal appendage and at the level of chaetal follicles. The contractions of the former induce the retraction of the caudal appendage; those of the latter force the chaetae to move up or down. Circular muscle cells also occur around the digestive system. Within the parenchyma lie the chaetal follicles and a pair of newly formed chaetoblasts, i.e., the secondary chaetoblasts. The latter cells appear in the hyposphere, on the right and left sides of the larval body, just below the epidermis (Fig. 6C–E). They give rise to the ﬁrst two chaetae in postmetamorphic juveniles. Both cells are separated from the parenchyma by a basal lamina. They are large, ovoid, and measure 10 m in diameter (Fig.
LARVAL DEVELOPMENT OF MYZOSTOMA CIRRIFERUM
Fig. 5. Intermediate metatrochophore of Myzostoma cirriferum (TEM). A: Transversal section through the pharynx. B: Detail of the pharyngeal muscle sheath. C: Sensory ciliated cells found at the apex of the caudal appendage. D: Nerve cell located in the episphere. E,F: Large dense-cored vesicles 100 nm in diameter and small clear vesicles 10 nm in diameter, respectively. G,I: Mature and developing digestive cells, respectively. H: Terminal and duct cells of a protonephridium. BB, basal body; BL, basal lamina; CI, cilium; CM, circular muscle cell; CU, cuticle; DB, dense body; DL, lumen of the digestive pouch; DU, duct cell; DV, large dense-cored vesicles; MI, mitochondria; MS, muscle sheath; NL, nephridial lumen; NP, nerve process; NU, nucleus; PE, pharyngeal epithelium; PL, pharyngeal lumen; RC, resting cell; RM, radial muscle cell; SJ, septate junction; TE, terminal cell; V, vacuole; VI, microvilli; ZA, zonula adhaerens.
6D,E). Their cytoplasm includes numerous mitochondria, many Golgi cisternae, and a welldeveloped rough endoplasmic reticulum (Fig. 6E). Their main characteristic is the presence in each cell of one intracytoplasmic ﬁbrillar object, 1 m in diameter, that is the core of a forming chaeta (Fig. 6E). The digestive pouch is now formed and connects to the pharynx through a short esophagus. No anus was observed during larval development. The pharynx of the late metatrochophore is similar to that of the previous stage. The esophagus is a short duct 10 m long with a lumen 6 m in diameter (Fig. 6B). The lumen is bordered by an epithelium without cuticle (Fig. 6B). The esophageal cells are ﬂat cells, 5 m high, and
lacking microvilli. The digestive pouch is ovoid, 30 m high and 20 m wide (Fig. 6A). Its lumen includes a few degenerated cells that are packed together (Fig. 6A). The epithelium bordering the lumen is also made of ﬂat cells 5 m in height (Fig. 6A,C,D). They have microvilli and their cytoplasm is ﬁlled with large vacuoles whose content is secreted into the lumen (Fig. 6A,C). Digestive cells are joined together by septate junctions and zonula adherentes. cLSM Observations Protrochophore and trochophore. No neuronal tissues could be stained in either the prototro-
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Fig. 6. Late metatrochophore of Myzostoma cirriferum (TEM). A: Parasagittal section through the larva. B,C,D: Transversal sections through the esophagus, the middle part of the digestive pouch, and the end of the digestive pouch, respectively. E: Details of a secondary chaetoblast. BL, blastocoele; CH, chaeta; DC, degenerated cells; DE, epithelium of the digestive pouch; DL, digestive lumen; EE, epithelium of the esophagus; EL, lumen of the esophagus; EP epidermis; MC, muscle cell; MI, mitochondria; MS, muscle sheath; NC, nerve cell; NU, nucleus; PL, pharyngeal lumen; SC secondary chaetoblast.
chophores or the trochophores. However, the ciliated cells of the metatroch and apical cells constituting the apical tuft showed immunoreactivity (IR) to acetylated ␣-tubulin and could therefore be distinguished from the other tissues (Fig. 7A). Young metatrochophore. In young metatrochophores (41 h, Fig. 7B), two transverse commissures show IR. The orientation of the chaetae and comparisons with the staining patterns of later stages indicate that the commissure with the thicker diameter represents the cerebral commissure and the thinner one the terminal commissure of the nervous system of the future caudal appendage. Nerves
that connect these two parts of the central nervous system could not be found. In slightly further developed stages (54 h, Fig. 7C,D) the nervous system is much more differentiated, showing characteristic features of a developing ladder-like system. The cerebral commissure expands on both sides of the pharynx and lengthens into two circumpharyngeal connectives that stretch ventrally. Behind the mouth, each circumpharyngeal connective (the right and the left) prolongs into one main ventral nerve cord and both cords run far apart from each other to the posterior body margin. A thick, postoral commissure connects the two ven-
Fig. 7. Young and intermediate metatrochophores of Myzostoma cirriferum, anti-aceytlated ␣-tubulin immunoreactivity (time of development given in the upper left corner in hours). Depth-coded images. A: Trochophore. Only the ciliated cells (cic) of the metatroch and the apical tuft (atc) show IR. B: Young metatrochophore (dorsal view) with commissures of the brain (bc) and of the caudal appendage (cac). C–I: Intermediate metatrochophore. C: Dorsal view. The dorsolateral nerves (dln) fuse and form one anterior (acdn) and one posterior (pcdn) commissure. D: Ventral view. The central nervous system consists of the brain (b), circumesophageal connectives (cc), a pair of main ventral nerves (mvn), and a postoral commissure (poc). mo, mouth opening. E: Ventral view. Paired accessory nerves (an) are added to the ventral cord. Three commissures, postoral (poc), second (c2) and terminal (tc), connect the main cords. In the hind end, ventral (vn) and dorsal (dn) nerves can be distinguished in the caudal appendage. One pair of protonephridia (pn) is located close to the chaetal follicles. ci, cilia; mn, median nerve. F: Behind the brain the dorsolateral nerves split into two ﬁbers (arrows). G: Two roots (arrowheads) form the circumesophageal connectives. H: The apical tuft (at) is in contact with the dorsolateral nerves. One apical tuft nerve (atn) projects caudally. I: The stomatogastric nerve ring (str) surrounds the mouth opening.
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Fig. 8. Late metatrochophore of Myzostoma cirriferum, anti-aceytlated ␣-tubulin Immunoreactivity (time of development given in the upper left corner in hours). A,C: Depth-coded images. A: In late metatrochophores, two pairs of protonephridia (pn) are visible. The circumpharyngeal connectives (cc) are still split (arrowheads). b, brain; ca, caudal appendage; mn, mvn, an, median nerve, main ventral nerves, and accessory nerves of the ventral nervous chain. B: Light microscopic image. Late metatrochophore with retracted caudal appendage (ca) and four chaetae (ch) at each side. C: The dorsolateral nerves (dln) project into the dorsal commissure (dcc); the circumesophageal connectives (cc) project into the ventral (vcc) cerebral commissure. Long cilia (ci) insert at the hind end of the larva. str, stomatogastric nerve ring.
tral nerve cords at the transition between the circumpharyngeal connectives and the ventral cords (Fig. 7D). Dorsally, the nervous system consists of an anterior transversal commissure that lies directly on the ventral cerebral commissure. On each side, this dorsal commissure prolongs into one dorsolateral longitudinal nerve that runs caudally and joins just before the caudal appendage (Fig. 7C). A connection between the ventral and the dorsal parts of the nervous system could not be detected but is assumed to exist in the region of the brain. Older metatrochophores. The general arrangement of the central nervous system does not change much between intermediate and late metatrochophores. In both metatrochophores, ﬁve longitudinal nerves — one unpaired median nerve, two paired main cords, and two paired accessory nerves — compose the ventral nerve cord (Figs. 7E, 8A; see also Fig. 9). From the postoral commissure, a thin median nerve stretches along the midventral axis toward the rear end. Whether it fuses with other nerves or projects into the terminal commissure could not be clariﬁed. Approximately 7 m behind
the postoral commissure a second commissure connects the main ventral cords. Only between these two commissures are the main cords split into two branches; the inner one being thinner than the outer. Also restricted to this region is the connection of the accessory nerves with the main nerves via thin transversal nerves (Fig. 9A,B). In the hind end, each nerve cord splits into two thick branches and one thin nerve (Fig. 9C). One thick branch stretches medially and interconnects with the respective branch of the other side, forming the terminal commissure of the ventral cord. The second branch bends at a right angle dorsally and, after a few micrometers, once again at a right angle caudally. This nerve penetrates the caudal appendage and interconnects with the respective nerve of the other side to form the terminal commissure (Fig. 7E, blue coded nerves in the caudal process). Each thin nerve prolongs caudally, then bends dorsally and reaches the nerve of the contralateral side in a slight bow (Fig. 7E, green coded nerves in the caudal process). The accessory nerves emerge from the circumpharyngeal connectives and run approximately 5m
LARVAL DEVELOPMENT OF MYZOSTOMA CIRRIFERUM
stained. In younger stages this ring is more spherical (91 h, Fig. 7I), while in older ones it becomes elongated (240 h, Fig. 8C). Due to their ciliated funnels, one pair of nephridia was stained in intermediate metatrochophores of 66 h. They are located on either side, near the chaetal follicles (Fig. 7E). They are curved in a more or less right angle, with the nephridiopores pointing toward the sides. Two pairs of protonephridia show positive IR in late metatrochophores. The additional pair of excretory organs is located beside the circumpharyngeal connectives (Fig. 8A). The approximately 10 m-long ciliated funnels start dorsally and stretch ventrocaudally, where they presumably open to the outside. DISCUSSION External Morphology
Fig. 9. Myzostoma cirriferum, schematic drawing of the tubulinergic nervous system of a late metatrochophore larva. A: Ventral view, dorsal nervous system omitted. Ventral structures black, more dorsally located structures gray. Bold arrowhead, position where the main cord splits into three ﬁbers. B: Dorsal view. Dorsal neuronal structures black, ventral nervous system gray. C: Innervation of the caudal appendage. ca, caudal appendage; cc, circumesophageal connectives; ch, chaeta; ci, cilia; dcc, dorsal cerebral commissure; dln, dorsolateral longitudinal nerve; mn, mvn, an, median, main, and accessory longitudinal nerve of the ventral cord; pn, protonephridium; pcdn, posterior commissure of the dorsolateral nerves; poc, c2, tc, postoral, second and terminal commissure of the ventral cord; str, stomatogastric nerve ring; vn, dn, ventral and dorsal nerve of the caudal appendage.
apart and parallel to the main cords toward the hind end. It is assumed but unproved that they project into the terminal ventral commissure. The lateral nerves of the dorsal system are almost parallel to one another in the posterior half of the animal; they run apart from each other in the anterior half of the larval body (Figs. 7F, 8C, 9B). Approximately 10m behind the brain, each lateral nerve gives rise to two nerve branches: the inner branch bends medioventrally and projects into the circumpharyngeal connectives; the outer branch bends further toward the side, then stretches medioventrally and projects into the brain (Figs. 8C, 9B). In a few specimens it was observed that the dorsal nerve pair is in contact with the apical tuft (Fig. 7H). A thin nerve projects caudally from the cells of the apical tuft. In some metatrochophore stages (72 h), it was clearly seen that the circumpharyngeal connectives consist of two roots (Fig. 7G, arrowheads). In late metatrochophores, the dorsolateral nerves project into a dorsal cerebral commissure that is of similar size as the ventral one (Fig. 9B, dcc). From the stomatogastric nervous system, the stomatogastric ring that surrounds the mouth opening was
The term “trochophore” was ﬁrst used by Hatschek (1878) for the larva of a polychaete, Polygordius sp. That trochophore has an apical organ, preoral and postoral ciliary bands ﬂanking a groove, a complete gut, and a pair of protonephridia (Hatschek, 1878). Since this description, various definitions of trochophores have been used. Rouse (1999) recently assessed the deﬁnitions of trochophores and overall homology of ciliary bands in spiralian larvae. His results favor the conclusion that a trochophore, if deﬁned as a larva using opposed ciliary bands for feeding, should not be regarded as an ancestral type for Spiralia, or any other large taxon such as Annelida or Mollusca (Rouse, 1999). Based on the evidence that various ciliary bands could have had differing evolutionary histories, he redeﬁned the trochophore as a larval stage with a prototroch (Rouse, 1999). In that sense, Myzostoma cirriferum does not show any trochophore in its development: the three larval stages do not differentiate a prototroch but an equatorial ciliary band that develops into two ciliary tufts located below the mouth, a situation that has also been observed in M. alatum (Eeckhaut and Jangoux, 1992). These ciliary tufts are thus in location homologous to the metatroch seen in some trochophores (Rouse, 1999). The term “metatroch” is generally used with reference to feeding bands either in larvae of some polychaete annelids, echiurans, molluscs, and entoprocts, or in adult rotifers (Strathmann, 1993; Nielsen, 1995). Metatroch has certainly evolved a number of times independently (Rouse, 1999). The metatrochal tufts in M. cirriferum are not used for feeding, as individuals do not feed until postmetamorphosis, but they are used in locomotion: larvae are propelled forward mainly due to the beating of these cilia (I. Eeckhaut, pers. obs.). A locomotory function could also be attributed to the hypospheral ciliary crown that surrounds the caudal appendage and to the apical tufts, because the ultrastructure of ciliated cells of both is similar to that
I. EECKHAUT ET AL.
of ciliated cells of the metatrochal tufts. Only a sensory function can be attributed to the cilia located at the level of the mouth and at the apex of the caudal appendage, as they are associated with sensory processes. Such ciliated sensory processes were not observed in the apical tuft, although cLSM observations showed that it is well supplied by nerves. Chaetae Even if Myzostoma cirriferum larvae cannot be categorized within trochophores sensu Rouse (1999), they differentiate chaeta as some trochophores do. The pair of chaetal follicles in M. cirriferum is intimately associated with the epidermis and appears in young metatrochophores. Larval chaetae regress totally during metamorphosis and ﬁve pairs of deﬁnitive chaetae develop only later in adults (Ja¨gersten, 1936; Eeckhaut and Jangoux, 1993a). The chaetae of adult myzostomids are located in parapodia and function in locomotion or attachment to the host. In M. cirriferum larvae, chaetae probably favor the suspension of individuals in the water column and enable larvae to hang onto the echinoderm hosts, where they complete their metamorphosis (Eeckhaut and Jangoux, 1993a). Chaetae and chaetal follicles in M. cirriferum are both quite similar to those found in annelids, pogonophorans, and echiurans, the last two taxa having been recently considered as belonging to polychaetes (McHugh, 1997; McHugh, 2000): in all these taxa, each chaetal follicle includes chaetoblasts that secrete the chaetae and lateral cells that enclose the chaetae until they penetrate the larval body (see above and Specht, 1988; Gardiner, 1992, for reviews about polychaete chaetae). The holes in chaetae are usually considered as being due to the presence of chaetoblast microvilli: the granular material that forms the core of the chaetae would be secreted into the lumens of chaetal follicles around the microvilli (Gustus and Cloney, 1972; Orrhage, 1973). Chaetigerous structures, although not named chaetae, are also found in other taxa, e.g., the Ko¨lliker’s organs of incirrate octopods (Budelmann et al., 1997) and the hair-like structures or bristles found on the mantle lobe of brachiopods (Gustus and Cloney, 1972; James, 1997). Gustus and Cloney (1972), particularly, pointed out the remarkable resemblance between the bristles of brachiopod larvae and the chaetae of polychaetes. This means either that chaetae are plesiomorphic for these two taxa, or that they evolved independently several times. Orrhage (1973) pointed out that, whichever of the two interpretations will be the ﬁnal one, it is difﬁcult to interpret the presence of chaetae in a phylogenetic context. Integument The integument of the late metatrochophore, that is, the competent larva, is made of an epidermis and
a parenchyma that ﬁlls the blastocoele between the internal organs. It differs from the integument of postmetamorphic individuals studied by Eeckhaut and Jangoux (1993b) in some respects: 1) larval epidermal microvilli do not end in bulges, 2) myoepithelial cells are absent, 3) gland cells are not observed (although covering cells participate in the formation of the cuticle), and 4) there is not a continuous muscle layer below the epidermis. We did not observe any closed internal cavity lined by a mesothelium that would have suggested the existence of a coelom in larvae. The presence of a coelom in myzostomids (in larvae and adults) has been a subject of controversy since their discovery and is still much debated (Haszprunar, 1996; Fauchald and Rouse, 1997; Zrzarvy et al., 1998). In adult myzostomids, the female genital tract has been considered to be homologous to the coelom of polychaetes because, in some myzostomids, a pair of ducts (unfortunately named metanephridia) connects the genital tract to the digestive system (see Grygier, 2000, for review). However, since the discovery of pairs of protonephridia in Myzostoma cirriferum (Pietsch and Westheide, 1987), these ducts are no longer considered as having an excretory function and the coelomic nature of the female genital tract has become extremely dubious. Here we show that no coelomic cavity appears during larval development, thus strongly suggesting that the female genital cavity of adults does not differentiate from such a cavity and cannot therefore be homologous to a coelom, or at least not to the coelom of polychaetes. The acoelomate condition of myzostomids can be considered as an apomorphic feature, as is also the case in some interstitial polychaetes (Fransen, 1980, 1987; Smith et al., 1986). According to Smith et al. (1986), secondary reduction of the coelomic cavity of polychaetes may arise either by the obliteration of the formed coelom by mesodermally derived cells or by the total absence of a coelom during ontogeny, which is the case in M. cirriferum (present study). Digestive System There are a few differences between the larval digestive system of Myzostoma cirriferum and that of the polychaete trochophore. In the former, the pharyngeal and esophageal epithelia are glabrous, while, according to Heimler (1988), they are always ciliated in the latter. Moreover, in the only extensive study made on the ontogenesis of a polychaete stomodeum, Åkesson (1962) observed that the pharynx ﬁrst appears as two hollow rudiments that fuse together before the appearance of the mouth. Such a process is very different from what we observed in M. cirriferum. Indeed, the formation of the pharynx in the latter includes the following steps: 1) the appearance of the mouth, i.e., the blastopore; 2) the formation of the pharyngeal epithelium and lumen
LARVAL DEVELOPMENT OF MYZOSTOMA CIRRIFERUM
by the invagination of the epidermis consisting exclusively of covering cells; 3) the differentiation of the resting cells that surround the pharyngeal epithelium into muscle cells forming the pharyngeal muscle sheath; and 4) the innervation of the pharynx by nerve processes. The midgut forms almost at the same time as the pharynx in a way similar to that observed in some polychaete trochophores ˚ kesson, 1962; Heimler, 1988): it differentiates (A within the hyposphere from resting cells as a hollow rudiment that will connect afterwards to the pharynx through a short esophagus. Digestive caeca and the anus of M. cirriferum form later, after metamorphosis (Eeckhaut and Jangoux, 1993a).
The larval and postmetamorphic protonephridia in Myzostoma cirriferum both consist of four cells. However, larval protonephridia consist of one ciliated terminal cell and three duct cells while, in adults, they include three terminal cells and one duct cell. It is probable that two of the larval duct cells acquire cilia during metamorphosis. Larval protonephridia in M. cirriferum are probably nonfunctional because the cilia lie within a blind protonephridial lumen that is not in contact with the blastocoel. Such a situation has been observed in the polychaete Lanice conchilega, where no microvillous weir apparatus occurs between the terminal cell and duct cells (Heimler, 1987). A pair of protonephridia, also called head kidneys, is characteristically situated within the blastocoel of planktotrophic polychaete trochophores (Anderson, 1966; Smith, 1992) and in some larvae of Platyhelminthes (Ruppert, 1978), of Entoprocta (Nielsen, 1995), of Mollusca (Bartolomaeus, 1989), and of Echiura (Newby, 1940). Nephridia have also been reported in two species of lecithotrophic polychaete trochophores (Heimler, 1988; Smith, 1992; Bartolomaeus, 1998). According to Bartolomaeus (1998), a protonephridium consisting of three cell types (i.e., monociliated terminal cell, duct cell, and epidermal nephridiopore cell) is the plesiomorphic stage of larval nephridia in all these taxa. In M. cirriferum larvae, we observed the tricellular organization of the protonephridia, although terminal cells are multiciliated. The late metatochophores of M. cirriferum differ from all trochophores described previously in having two pairs of protonephridia. Based on their location, the pair of protonephridia in the episphere and that in the hyposphere probably corresponds to the ﬁrst and fourth pair of postmetamorphic protonephridia, respectively.
larvae (e.g., Ophryotrocha gracilis) and progenetic adult dinophilids (Mu¨ller and Westheide, 1997, 2002; Mu¨ller, 1999). In contrast to these polychaete nervous systems, however, in M. cirriferum the accessory nerves are not located between the median nerve and the main cords, but beside the latter ones. In adult Myzostoma, the accessory nerves are missing. They could either be degenerated or, more probably, fused with the main cords, as is the case in some polychaetes. It also seems to be unlikely that the accessory nerves transform into the serotonergic ﬁbers that enclose the central nerve mass in adults (Mu¨ller and Westheide, 2000). A median nerve has been found in many polychaete (Hanstro¨m, 1968; Mu¨ller, 1999) and hirudinean species (Bristol, 1898; Bullock and Horridge, 1965), and is regarded by some authors as being part of the articulate body plan (Mu¨ller, 1999; Mu¨ller and Westheide 2000, 2002). In platyhelminthes, with their even-numbered longitudinal nerves from two up to ten, on the other hand, a median nerve rarely exists (Bullock and Horridge, 1965; Reuter et al., 1998). Whereas the larval dorsal nerves of Myzostoma run straight, they show iterated bulges and arborizations, innervating the marginal body part in adults (Mu¨ller and Westheide, 2000). Neither the projection of these nerves into the brain nor their posterior commissure, demonstrated here for the larvae, could be seen in adults. Paired dorsal nerves also occur in platyhelminthes and in polychaetes; in the latter they are regarded by Mu¨ller (1999) as belonging to the basic polychaete body plan. Other striking features of the larval nervous system of Myzostoma cirriferum are the paired circumpharyngeal connectives. Splitting of the circumpharyngeal connectives into a dorsal and a ventral root, each forming two commissures within the brain, is assumed to be the ancestral condition of the polychaete cephalic nervous system (Orrhage, 1995). Furthermore, in regeneration studies it could be demonstrated that the polychaete circumesophageal connectives are paired structures that during ongoing differentiation fuse to a different degree, leaving longer or shorter dorsal roots behind (Mu¨ller and Henning, unpubl. data). Therefore, it is possible that the single-stranded connectives in adult M. cirriferum were built by fusion of the paired larval structures as well. No structures of the prominent nervous system of the larval caudal appendage can be identiﬁed in adults, indicating that it gets completely reduced together with the appendage during metamorphosis.
With its ﬁve longitudinal cords, the ventral nervous system of Myzostoma cirriferum larvae greatly resembles the nervous system of some polychaete
The ﬁrst phylogenetic analyses of morphological characters including myzostomids placed them either basal to the polychaetes or as a derived group
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within a phyllodocidan polychaete clade, depending on the character scoring scheme used (Rouse and Fauchald, 1997). More recently, phylogenetic analyses of two independent genes (18S rDNA, EF-1 alpha; Eeckhaut et al., 2000) or based on both molecular (18S rDNA and 28S rDNA) and morphological characters (Zrzavy et al., 2000) provide strong support for the placement of myzostomids within the Spiralia but outside the Annelida clade. In the light of the present work, it is unquestionable that Myzostoma cirriferum larvae share similarities with some polychaete trochophores: chaetae of both taxa are homologous structures and the nervous system of M. cirriferum larvae displays features that are shared with some polychaete annelids (e.g., the ﬁve ventral longitudinal cords). However, it is interesting to note that the myzostomid trochophore does not ﬁt the restricted deﬁnition of Rouse (1999), i.e., they do not have a prototroch, only a metatroch. The absence of a coelom in the larval stages of myzostomids is also intriguing; these early developmental stages show a derived condition that is often viewed as a characteristic of aberrant adult morphology associated with unusual adult lifestyles of metazoans, which in the case of the myzostomids is the close association with echinoderms. Given the undisputed spiralian afﬁnities of myzostomids, the present work coupled with recent phylogenetic analyses strongly suggests that a trochophore is a plesiomorphic spiralian condition and that the myzostomid larva evolved from a trochophore in which the prototroch has been lost. The evolution to a myzostomid trochophore also implied the total disappearance of the coelom and the development of a parenchyma. Rieger (1986), in analyzing various interstitial polychaete body plans, postulated that acoelomate taxa could be derived by progenesis from coelomates that exhibit an acoelomate larva. In this way, an origin of acoelomates from coelomates is possible without the reduction of the coelom (Rieger, 1986). Recently, Mu¨ller and Westheide (2000) suggested that the ancestor of myzostomids was a coelomate worm with six segments. Considering the recent phylogenetic position of myzostomids within Platyzoa (Eeckhaut et al., 2000; Zrzavy et al., 2000), the numerous homologies put in evidence between myzostomids and polychaetes by ultrastructural works strongly support the view that a trochophore larva and segmentation are two features that appeared early during the evolution of Spiralia.
ACKNOWLEDGMENTS The authors thank Professor Andre´ Toulmond, Director of the Observatoire Oce´anologique de Roscoff, for the research facilities provided. We also thank the members of the station for their help in collecting some of the Antedon by scuba diving. We thank Dr. Damnhait McHugh and two anonymous
reviewers for correcting the manuscript and for helpful comments.
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