General description of larvae stages
To study the development of muscles in Siboglinum fiordicum, we reconstructed the muscular architecture and anatomy in four successive stages: trochophora, early metatrochophora, late metatrochophora, and competent larva (Figs. 1–5).
The size and shape of the trochophores are comparable to those of the eggs. The widest part of the larva is in the middle of the body (Figs. 1A-C). The trochophore larva has a small convex episphere (esp) (without an apical tuft of cilia) and an unusually long hyposphere (hsp). The earliest trochophore stage have a thin, irregular prototroch (p). In late trochophores, an irregular strip of mesotroch (m) appears (Fig. 1C), and a wide prototroch is formed by large multicilated trochoblast cells (mbl) arranged in 2–3 rows (Figs. 1B-C, 2A-B, 3A).
The early metatrochophora has a wide prototroch, represented by first short (Fig. 2A), then longer cilia (Fig. 2B) of similar length (there are no opposed beating cilia). The mesotroch becomes a regular annular stripe (Fig. 2A) with cilia soon extending length and density (Fig. 2B), and a neurotroch and a dorsal ciliary spot (cp) appear (Figs. 2A, B). The neurotroch is a wide ventral ciliary field, consisting of two zones, a small anterior (na) and a larger posterior (np) (Fig. 2A). At this stage, the chaetae of the annula (ca) (middle part of “trunk” or 3rd segment) and the chaetae of the first segment of the opisthosoma (co) (i.e., the 4th body segment) are laid under the epidermis (Figs. 2C-H). The chaetae are not visible externally, but scanning electron micrographs show depressions of the future chaetae of the opisthosoma (Fig. 2B).
Late metatrochophores of S. fiordicum are noticeably elongated. In the cone-shaped prostomium (pr) of the larva, a frontal fold (aff) appears on the ventral side (Fig. 3A). The peristomium (pe), located behind the prototroch, carries a tentacle rudiment (tb), which is laid on the dorsal side to the left of the ciliary spot (Fig. 3B). S. fiordicum does not have a mouth opening, but a gap in the musculature (Fig. 3F’). The second segment bears a neurotroch, comprising an anterior and a posterior zone. The third segment of the larva (the trunk of adult pogonophorans) is a narrow part of the body, located posterior to the middle fold of the body and stretching to the first septum which borders the anterior segment of the opisthosoma (Fig. 3A). In the first segment of the opisthosoma, large chaetae are clearly visible and a thin mesotroch posterior to the chaetae (Fig. 3a).
In the competent larva, the prototroch almost disappears. In the peristomium, there is an anlage of tentacle and a dorsal ciliary spot. In the second segment (“forepart segment” in adult frenulates), the frenulum (f), or bridle, appears. The third segment (trunk segment of adult frenulates) is delineated anteriorly and posteriorly by septa (s1), and in the middle part, chaetae of the annula, extend externally (Fig. 4A). The first segment of the opisthosoma, between the first septum and the mesotroch, is relatively long. In the relaxed larvae, this segment exceeds the length of all subsequent segments of the opisthosoma (Figs. 4A-E).
Phalloidin staining results. General description. The somatic musculature is formed at the early metatrochophore stage and is represented by layers of longitudinal and circular muscles (Figs. 2–5). The longitudinal musculature comprise four separate strands located ventrally (mv), dorsally (md), and ventro-laterally (mvl). The circular muscles (mc) are evenly distributed as complete wide bands along the body of the larvae from the apical end to the annular chaetae. From the annular chaetae to the first septum, there is a body region (future trunk segment) devoid of the circular muscles and longitudinal muscles constricted into four bundles. The bundles of longitudinal muscles form the first septum separating the trunk segment from the first segment of the opisthosoma (Fig. 6). In the opisthosoma, posterior to the first septum, the circular muscles are organized in distinctly separate bundles (Figs. 2–6). In the larvae, the musculature of the future tubiparous glands is laid, as well as the muscular apparatus that controls the movement of the opisthosomal chaetae (Figs. 2E-H, 7, 8).
Longitudinal musculature. Four main longitudinal muscle strands extend from the prostomium to the posterior end of the opisthosome in the larvae: one ventral (mv), one dorsal (md), and two ventro-lateral (mvl).
The dorsal (md) and ventral (mv) longitudinal strands are unpaired for the most of their length. The ventral strand is the narrowest. The widest strands are the ventro-lateral ones, which have two longitudinal components at all studied larval stages: ventral (mvvl) and lateral (mlvl). The ventral component (mvvl) originate at the anterior prototroch and insert at the posteriormost body, while the lateral component (mlvl) originate at the anterior prostomium and insert at the first septum.
The number of bundles in each muscle strand remains unchanged from the prostomium to the posterior end of the opisthosoma. But the number of fine fibers that make up the bundles varies. For example, the dorsal muscle strand (md) is formed as a single wide strand, which bears about 8–9 bundles in all stages (Figs. 2–5). The number of bundles within a strand does not change with age, but the thickness of each bundle changes due to the increasing number of muscle fibers that make up the individual bundles.
At the early stage of formation, the fibers of the longitudinal muscles are grouped in separate bundles (Figs. 2C-E), while at the later stages of development, the fibers form a continuous muscle layer (Figs. 3C, D, 4D, E). For example, in the trunk segment (3rd segment), the longitudinal fibers of all four muscle strands run widely so that they almost form a continuous layer of longitudinal muscles (here the longitudinal strands are interconnected by short lateral circular muscle fibers (mcc) (Figs. 3C, D, 4C, D). In the posterior trunk, the longitudinal muscle fibers are grouped into dense bundles. The muscle fibers that are grouped into bundles in the posterior part of the trunk segment have a similar pattern in all studied samples: the dorsal strand is represented by two bundles (md1-2), the ventral one - by one bundle, and the ventro-lateral muscle strand is represented by three (mvvl1-3) and two (mlvl1-2).
At the stage of late metatrochophora, in the area of the potential temporary mouth anlagen, the bundles of the ventral longitudinal muscle strand split (Fig. 3F’). At an earlier and later stage, this hole is not visible.
Circular musculature. The circular musculature forms a muscular dense corset in the anterior part of the larva (Figs. 2, 3, 4, 5). From the apical end till the anterior trunk segment, the circular muscle fibres are distributed as wide bands, surrounding the larval body. Each band includes about eight complete circular muscle bundles. Under the prototroch, the bundles are semi-circular and overlap mid-dorsally and mid-ventrally (Figs. 2C, D’, 3F’, 4E). On the dorsal side posterior to the prototroch, the circular muscles form two slightly asymmetrical holes (Fig. 2C). The left hole corresponds to the future unpaired tentacle (tm). In the later stages (competent larva), a muscle ring appears at the base of the tentacular anlage (Figs. 4C, D, 5I).
In the trunk segment of the early metatrochophore, the circular bands are sparsely located (Figs. 2C, 3B, 4C, 5G-I). Later, the trunk segment lengthens and forms two parts. In the anterior part (until to the annular chaetae), 4–5 circular bundles are visible, and in the posterior part of the body (behind the annular chaetae), there are no circular muscles at all.
Septal musculature and sequence of their formation. The first septum (sI) divides the body of the early metatrochophora into two parts: an anterior segment and a posterior opisthosomal segment (Figs. 4, 5). The formation of the first septum completes already in the late metatrochophora, which encompass the closure of the ecm layer and the formation of a continuous layer of longitudinal muscles (Figs. 6, 11). The formation of specialized longitudinal musculature along the lateral sides of the first opisthosomal segment accompanies the development of the first septum between the trunk segment and the first segment of the opisthosoma. The bundles of this musculature are laid down at the stage of the early metatrochophore and gradually grow in thickness at later stages. The first septum is based on a layer of extracellular matrix, which gradually deepens from the body wall into the body of the larva. In early metatrochophora, the septum is incomplete, there is a lumen in the middle of the septum, through which the larval intestine runs. At later stage, the edges of the extracellular matrix of the septum join, and the septum completely separates the trunk segment and the opisthosoma. Following this, the longitudinal muscle bundles of the septum also transverse deep into the body. In the late stages, the longitudinal musculature extends from the first septum till the second septum of the opisthosoma. There are ca 24 pairs of longitudinal bundles building the first septum. With the larval development the bundles significantly increase in length (Figs. 2F, 6). Ca 12 large myoepithelial cells with large nuclei (mcn) form this septal muscle (Figs. 6D, 9A).
At the late metatrochophore stage, three more separate circular bands are added at the posterior end of the opisthosoma (Figs. 3C, F). Subsequent septa (sII) form posterior to the first septum, splitting the opisthosoma into additional segments. There is an outstripping development of circular muscle bands, which seem to outline the boundaries of the opisthosomal segments, followed by the development of muscle-less inner septal divisions (Figs. 2F-H, D, 5I, 11). In the early metatrochophora, three circular bands are formed (Figs. 2C, F): the first at the level of the first pair of chaetae, the second beneath the mesotroch. The third is located posterior to the second one. The early metatrochophora has these three opisthosomal circular muscle bands but no internal septa yet (Figs. 2E-H), while the late metatrochophora already has six opisthosomal circular muscle bands and six septa (Figs. 3C, F).
At the stage of the competent larva, a distinct circular muscle bands (mf) is visible in the middle of the anterior body segment. We assume these muscles correlate to the position of the anterior septum (sIII) which divides the anterior body segment into two segments (forepart and trunk) (Fig. 4A, C, E). These two segments (following the peristomium or 1st segment) grow without further divisions into the adult stage. The anterior segment is termed the forepart (following Southward (43)), which is equivalent to the "fused protosome and mesosome”, according to the now abandoned deuterostomic concept of Ivanov (44). The posterior segment is termed the trunk (following Southward (43)) and is equivalent to the "metasome", according to the now abandoned deuterostomic concept of Ivanov (44)). In this second body segment (forepart) there is a frenulum (or bridle, a characteristic structure found in all the currently described frenulates (see, for example, Ivanov (26); Southward et al. (45)), and in the third segment (trunk) there are annular chaetae that detach the preannular (genital) and postannular (trophosomal) parts. The following circular bands and septa are formed at the very end of the opisthosoma one by one; For example, at the stage of the competent larva, a seventh circular band forms (Fig. 4H). Later in the post-larval stages a new circle band and a new segment form at the very posterior end of the juvenile. Adults of S. fiordicum have up to 21 muscle septa in the opisthosoma (unpublished data) Within the circular opisthosomal bands, number of fibers increases with the age of larva. For example, from 7 to 12 fibers within the bundle located under the mesotroch (Figs. 2F, 4H).
Musculature of chaetae. The movement of the chaetae of the first segment of the opisthosoma is controlled by both specialized chaetal muscles and somatic muscle bundles. The latter include the longitudinal septal musculature (mls) and the circular septal musculature (moc1) (Fig. 7, 8). The chaetal muscles include separate circular and longitudinal muscle bundles. In total, there are 3 circular muscle bundles (mcch1-3) and about 10 longitudinal muscle bundles (mlch1-10) for each pair of chaetae (Figs. 2F-H, 7, 8). The circular muscles of the chaetal apparatus lie in the ventro-lateral and dorso-lateral directions (mcch1-3). Among the circular muscles, the first one (mcch1) stands out, one end reaching the ventral cord of the longitudinal muscles (mv), and the other attaching the body wall at the level of the septal muscles (mls) (Figs. 7A, 8). The first circular muscle lies at the base of the chaetae in such a way that the ends of the chaetae "abut against it". This circular muscle serves also as an anchor for almost all longitudinal muscles of the chaetae apparatus (mlch1-4, 6–10) (Figs. 7A-D). The rest of the circular chaetal muscles originate at the ventral longitudinal cord and supply the distal ends of the chaetae.
The longitudinal muscles of the chaetal apparatus attach with their distal end to the body wall either at the level of the first segment (mlch1-6) or at the level of the second segment (mlch7-10). The proximal end of the longitudinal chaetal muscles anchors either on the chaetae (mlch5) or on the first circular muscle or in the immediate vicinity of it (mlch1-4, 6–10) (Figs. 7A-D).
The annular chaetae (ca) at the stage of the competent larva are not yet fully formed; therefore, we assume that the musculature is not yet fully formed. At the studied stages, we see that the four pairs of annular chaetae are controlled by their own muscles, which includes a pair of circular bundles (mca) and longitudinal bundles that extend from the ventrolateral longitudinal muscle strands (mvl) (Figs. 2G, 3B, 4G).
Musculature of tubiparous glands. Phalloidin stained the muscular sheaths of the sacs and ducts (mtg) of tubiparous glands in larvae, from the early metatrochophorа to the competent larva (Figs. 2E, 3F, 4D-E). At all stages, they are located in the posterior half of the anterior part, throughout the entire trunk segment, and in the first segment of the opisthosoma (Figs. 2E, 3C, 4G). Moreover, the tubiparous glands are associated with radial muscle bundles (mrtg) extended from the somatic longitudinal strands to the distal portion on the gland (Figs. 2E, 4C).
Ultrastructure. In general, the somatic musculature is formed by myoepithelial cells of the coelomic lining. These are large cells with a prominent apical part and large basal projections (Figs. 9A, 10A). Cells connect via adherence junctions (aj) which are located at the border between the apical part of cell and the basal projections and also on the membranes of muscular projections (Figs. 9A, 10A). The apical part of the cell bears large irregular-shaped nucleus containing a few amounts of condensed chromatin and large nucleolus (Figs. 9A, 10A). Cytoplasm around nucleus is filled with numerous mitochondria of small diameter and electron dense matrix, canals of rough endoplasmic reticulum (re), and inclusions of different types. Basal projections of myoepithelial cells abut on the thin layer of extracellular matrix (ecm), which is highly electron dense (Fig. 9B). In some places, which are probably able to stretch extensively, the basal membrane of the muscular projections forms numerous finger-like outgrowths (flo), which repeat the folds of extracellular matrix and epithelium of body wall (Fisg. 9A, 10A). The cytoplasm of muscular projections is filled with numerous myofilaments, which mostly extend in longitudinal direction (Fig. 9B) but can also extend at the angle to the body wall (Fig. 9A, 10B). Myofilaments connect the basal membrane to cell via electron dense hemidesmosomes (he) (Fig. 10C). Myofilaments are organized as in cross-striated musculature: there are sarcomeres (sa) and Z-bodies (Zn) (Fig. 10B). Cytoplasm of muscular projections contains mitochondria of small diameter and a few electron dense inclusions (Fig. 10A).
In the somatic longitudinal muscle strands, several myoepithelial cells connect and form a pseudo-multilayer (Fig. 10A). This construction forms because muscular projections of cells overlay each other. However, all cells contact the layer of extracellular matrix and, hereby, myoepithelial cells form the monolayer (Fig. 10A). In some places where the myoepithelial cells and their projections contact each other, the cell membranes form numerous finger-like protrusions, which all together work as interdigitate cell junction (Fig. 10D).
In septa, the myoepithelial cells have large apical part that extend into coelom lumen of the 1st opisthosomal segment and contains large nucleus in basal parts (Figs. 11A, B). These cells connect not only via adherence junctions, but also by septate junction (Figs. 11B, B’). Muscular projections of cells extend in radial direction, i.e., from body wall to the center of the septum, at a right angle to anterior-posterior body axis (Figs. 11A-C).
According to CLSM, the tubiparous glands are associated with radial muscle bundles (mrtg), which extend between dorsal and ventrolateral longitudinal muscles strands and distal portion on the gland (Fig. 2E, 4C). Cells of the radial muscles directly abut on the cells of gland lumen (Fig. 11D). The contraction of radial muscles promotes the extrusion of the secret from the lumen of the gland to environment.
The complicated musculature of the chaetal sacs is organized by the myoepithelial cells (Fig. 12A). All cells are connected via adherence junctions and have large muscular projections (Fig. 12B). Some of myoepithelial cells form the circular muscle lining, which envelopes the follicle cells and chaetae (co) (Fig. 12B). Other cells form the longitudinal muscles of chaetal sac (Fig. 12C). These cells extend between the layers of extracellular matrix of the chaetal sac and the epithelium of the body wall. On both sides, the myofilaments adhere to the extracellular matrix via hemidesmosomes. Myofilaments are mostly passed in a longitudinal direction. Interestingly, myofilaments are attached peripherally to the extracellular matrix exactly where the thick bundles of electron dense tonofilaments (tf) of epithelial cells are attached on the opposite side of the ecm basal membrane. These tonofilaments extend into tips of microvilli and attach to the cuticle (Fig. 12C). Thus, the myofilaments of the longitudinal muscles of the chaetal sac indirectly attach to the cuticle via thick tonofilaments of the epidermal cells.
During observations of the larvae, we have recorded videos on the characteristic movement of the larvae at each of the stages of development described in the work. Examples of detailed movements can be seen in the videofile, Additional file 1. Trochophores float due to the beating of cilia, their body does not bend. They actively swim out of the maternal tube and can spin and swim on the bottom of the dishes. Metatrochophores still actively swim using cilia, but they bend the body, so that their swimming can be accompanied by rotational movements. Late metatrochophores stop actively swimming, they often lie at the bottom of the glass dish or sediment but continue to bend the body. Chaetae are often anchored in the sediment particles. Competent larvae burrow into the sediment head downward, their rotating posterior end of the body is visible outside the sediment.