Muscular anatomy of an entoproct creeping-type larva reveals extraordinary high complexity and potential shared characters with mollusks
© Merkel et al. 2015
Received: 6 January 2015
Accepted: 29 May 2015
Published: 3 July 2015
Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indirect development, either via a swimming- or a creeping-type larva and still debated phylogenetic position within Lophotrochozoa. Recent morphological and neuro-anatomical studies on the creeping-type larva support a close relationship of Entoprocta and Mollusca, with a number of shared apomorphies including a tetraneurous nervous system and a complex serotonin-expressing apical organ. However, many morphological traits of entoproct larvae, in particular of the putative basal creeping-type larva, remain elusive.
Applying fluorescent markers and 3D modeling, we found that this larval type has the most complex musculature hitherto described for any lophotrochozoan larva. The muscle systems identified include numerous novel and most likely creeping-type larva-specific structures such as frontal organ retractors, several other muscle fibers originating from the frontal organ, and longitudinal prototroch muscles. Interestingly, we found distinct muscle sets that are also present in several mollusks. These include paired sets of dorso-ventral muscles that intercross ventrally above the foot sole and a paired enrolling muscle that is distinct from the musculature of the body wall.
Our data add further morphological support for an entoproct-mollusk relationship (Tetraneuralia) and strongly argue for the presence of an enrolling musculature as well as seriality (but not segmentation) in the last common tetraneuralian ancestor. The evolutionary driving forces that have led to the emergence of the extraordinarily complex muscular architecture in this short-lived, non-feeding entoproct larval type remain unknown, as are the processes that give rise to the highly different and much simpler muscular bodyplan of the adult entoproct during metamorphosis.
KeywordsTetraneuralia Lacunifera Spiralia Lophotrochozoa Kamptozoa Evolution Evodevo Phylogeny Myogenesis Trochophore
Entoprocta (=Kamptozoa) are microscopic, mostly marine, sessile suspension feeders. So far, approximately 150 species have been described, which are usually divided into four subgroups: the solitary Loxosomatidae and the colonial Loxocalypodidae, Barentsiidae and Pedicellinidae [1, 2]. The typical tentacle crown which surrounds both mouth and anus is part of the calyx which houses the U-shaped gut, typically one pair of protonephridia, the reproductive organs and the cerebral ganglion. Entoprocts reproduce asexually by budding or sexually via two different larval types, the supposedly basal, lecithotrophic creeping-type and the more common planktotrophic, swimming-type larva [1, 3].
The phylogenetic relationships of Entoprocta are still highly debated and today three major concepts are considered. Classical studies have argued for a close relationship of Entoprocta and Ectoprocta (“Bryozoa”-concept), mainly based on similarities during larval metamorphosis [3, 4]. Some recent molecular studies seemingly support the traditional Bryozoa-concept [5–7], although this view has repeatedly been challenged by, e.g., embryological data that demonstrated spiral cleavage in entoprocts, and thus a significant difference to the radially cleaving ectoprocts . With the discovery of the Cycliophora, microscopic sessile organisms found on the mouthparts of lobsters , an alternative concept was established, thereby comprising cycliophorans, entoprocts and ectoprocts as a monophyletic assemblage (“Polyzoa”-concept), or - as a slight deviation from that - a sistergroup relationship solely consisting of cycliophorans and entoprocts [10, 11]. Based on a number of apomorphies shared by the entoproct creeping-type larva and larval and adult mollusks, the so-called Sinusoida- or Lacunifera-concept [12–14], which had previously suggested a monophyletic clade comprising Entoprocta and Mollusca, was recently revived, and the resulting assemblage was termed “Tetraneuralia” based on shared larval and adult neural characters [15–19]. Thereby, the creeping-type entoproct larva and the polyplacophoran larva revealed a similar architecture of the larval apical organ with numerous flask-shaped as well as peripheral cells, together with a tetraneurous condition of the longitudinal nerve cords, which so far had been considered autapomorphic for Mollusca [16, 18–21]. Even more, the creeping-type larva turned out to constitute a mosaic of larval and adult molluscan characters, featuring a distinct creeping foot with a ciliated gliding sole, pedal glands, anteriorly placed cirri and a ventrally intercrossing dorso-ventral musculature as typical traits of adult mollusks [17, 19]. All these data are particularly relevant for inferring entoproct relationships, because this complex larva is commonly considered the basal entoproct larval type and not the much simpler (and better-known) planktotrophic swimming-type larva . In order to shed further light on the morphology of the still enigmatic entoproct creeping-type larva and to contribute novel data to the discussion on entoproct evolution, we studied the myoanatomy of this larva in Loxosomella murmanica using confocal microscopy and 3D modeling.
Animals and fixation
Individuals of the solitary entoproct Loxosomella murmanica (Nilus 1909) live epizoically on the sipunculan Phascolion strombus (Montagu 1804) which inhabits empty shells of the gastropod Turritella sp. and the scaphopod Antalis sp. The shells were dredged from 30 m depth from muddy and rocky bottom at Gåsö Ränna, Gullmarsfjord, close to the Kristineberg Marine Research Station, Sweden, in June 2012. The sipunculans were removed from their shells and brooding adults of L. murmanica were detached from its host and kept in six-well dishes until larvae were released. Larvae were relaxed with 7 % MgCl2 and fixed in 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS) for 1 h at room temperature. Afterwards, the larvae were rinsed in 0.1 M PBS (3x15min) and stored at 4 °C in 0.1 M PBS containing 0.1 % sodium azide (NaN3).
Immunocytochemistry, confocal microscopy and 3D reconstruction
After storage, the larvae were rinsed (3x15min) in 0.1 M PBS and transferred into 0.1 M PBS + 0.2 % Triton X-100 (PBT) for 1 h for permeabilization. F-actin staining was carried out with a 1:20 dilution of Alexa Fluor 488 phalloidin (Invitrogen, Molecular Probes; Eugene, OR, USA) in PBT. For nucleic acid staining, 0.5 % DAPI (Invitrogen, Molecular Probes, Eugene, OR, USA) was added and the samples were incubated for 4 h in the dark. Then, larvae were rinsed in 0.1 M PBS (3x15min) and embedded on glass slides in Fluoromount G (Southern Biotech, Birmingham, AL, USA). Samples were examined using a Leica SP5 II confocal microscope. Optical sections had a step size of 0.13-0.24 μm. The confocal stacks were merged into projection images with greater focal depth. 3D reconstructions were generated with the image processing software Amira 5.4 (FEI Visualization Science Group, Hillsborow, OR, USA).
Adult and larval gross morphology of Loxosomella murmanica
The larval body is divided into an episphere and a hyposphere. The episphere comprises the region dorsal of the prototroch (pre-trochal region), including the apical organ (Fig. 2). The ventral side of the prototroch with its prominent foot sole belongs to the hyposphere (post-trochal region; see Fig. 2 and ). Compared with other spiralian larvae, the direction of moving is shifted by 90 degrees relative to the position of the apical organ and prototroch: while the “typical” trochophore is orientated with the apical organ forward, the larva of Loxosomella murmanica is oriented with the frontal organ in front during its alternating swimming and creeping movements. Therefore, the frontal organ – and not the apical organ - marks the anterior (frontal) pole of the entoproct creeping-type larva, while the opposite region is posterior (abfrontal). The gut is U-shaped and both mouth and anus reside within the hyposphere, which is framed by the prototroch and becomes the atrium in the adult entoproct (Fig. 2; see also ).
The most prominent muscle sets of adult Loxosomella murmanica are formed by the atrial ring musculature, which surrounds the tentacle crown, and the longitudinal stalk musculature (arm, lsm; Fig. 1). The latter form an outer layer of muscles which run from the pedal disc into the body, where the fibers branch and form a basket-like structure towards the dorsal side of the brood pouch. The musculature of the tentacles runs in loops to the tips of the tentacles and back to the tentacle base. The musculature of the digestive tract consists of ring muscles and longitudinal muscle fibers which seem to be twisted around the gut (Fig. 1).
Myogenesis and larval muscular anatomy in Loxosomella murmanica
The densely packed musculature of older embryonic stages already resembles the muscular condition of the hatched larva. The most prominent muscles are the ring muscles of the prototroch as well as those of the frontal organ and the apical organ (am, fm, pm; Fig. 3d-f). The prototroch longitudinal muscles and frontal organ retractor muscles are well developed (frm, plm; Fig. 3d). The openings of the paired protonephridia are laterally positioned (cf. ). The body wall ring muscles cover the entire episphere (bm; Fig. 3d-f).
Muscular architecture of the fully developed larva
Several muscles, which project straight towards the ventral side of the larva, protrude from the ring muscles of the apical organ (Fig. 5a, b, d, e). Some of these muscles seem to be attached to the apical organ ring muscles, others may be surrounded by these ring muscles (am; Figs. 4a, c, d; 5a, b, d, e). The frontal organ ring muscle surrounds several muscles which extend far within the larval body (fm; Figs. 4d, f; 5a-d). The most eye-catching muscles are formed by three prominent muscle pairs which are located laterally and in the horizontal plane of the frontal organ, herein termed “frontal organ retractors” (frm; Figs. 4a-f; 5a, b, d). Their tips, in the most frontal region of the larva, are slightly bifurcated; their abfrontal part can be branched or simple. At least one pair is attached to a lateral, thickened part of the body wall musculature close to the apical organ (Fig. 5a, b). The other pair extends even more to the lateral sides of the larva. Another pair of branched muscles is attached to the ring-shaped body wall muscles (Fig. 5a-c). A branched muscle at the apical pole of the frontal organ, the medio-dorsal frontal organ muscle, expands towards the dorsal body wall between apical and frontal organ (Fig. 5a-d). The dorso-ventral frontal organ muscle originates at the ventral pole of the frontal organ ring muscles and curves towards the dorsal side of the larva (Fig. 5a-d). The lateral short frontal organ muscle emerges from the most lateral side of the frontal organ ring muscles and projects laterally in posterior direction (Fig. 5a, b). A relative prominent muscle pair, the longitudinal frontal organ muscle, runs in parallel to the frontal organ retractor muscles, extends further, and nearly reaches the abfronto-dorsal part of the larva (Fig. 5a-d). The highly branching dorsal frontal organ muscles originate at the apical pole of the frontal organ ring muscles and curve in parallel to the dorsal body wall towards the abfrontal side of the larva (Fig. 5a-e). Some of the frontal tips of these muscles seem to be attached to the frontal organ ring muscle. Another pair of branched muscles protrudes at the ventral side of the frontal organ ring muscles and runs in parallel to the muscles that originate from the dorsal tip of the pedal dorso-ventral muscle (Fig. 5a, b, d). A bracket-shaped unpaired muscle is positioned between the region of the apical organ and frontal organ. It lies in parallel to the rib-shaped dorso-ventral muscles mentioned above and runs close to the body wall in ventral direction (Fig. 5a, c).
Comparison of the myoanatomy of entoproct larval types
Sexual reproduction in entoprocts results in the formation of one of two different larval types: the more common, but most likely derived, planktotrophic swimming-type larva or the supposedly basal, lecithotrophic, creeping-type larva [1, 3].
The body wall musculature of the swimming-type larva of Loxosomella atkinsae consists of ring muscles which surround the entire larval body . The apical ring muscles and the ring muscles of the prototroch form the most prominent muscle sets of this larval type. Furthermore, about 40 prototroch longitudinal muscles originate from the most ventral prototroch ring muscle (“main prototroch constrictor” in ) and extend in direction of the body wall above the most dorsal prototroch ring muscle . In the swimming-type larva, the apical organ ring muscle is connected to several longitudinal muscles: one pair of main inner longitudinal retractor muscles, one abfrontal longitudinal muscle and a pair of median muscles . A pair of longitudinal muscles splits below the apical organ ring muscle, at the level of the frontal ganglion, and extends laterally to the digestive tract and establishes contact with the prototroch ring muscles. On the abfrontal side of the larva, a pair of diagonal muscles originates from the prototroch ring muscles and inserts below the apical organ ring muscles . An additional short abfronto-ventral pair of muscles arises close to the abfrontal diagonal muscle but seems to terminate already at the most ventral set of the episphere body wall ring muscles. A pair of frontal diagonal muscles inserts at the prototroch constrictor muscle and terminates below the apical organ ring muscle .
While this brief summary shows that a number of distinct larval muscle systems are present in this relative small and simple-looking entoproct swimming-type larva, the muscular architecture of the creeping-type larva appears far more complex. Similar to the swimming-type larva, the episphere of the larval body contains numerous outer ring muscles (Fig. 4b-d). Both larval types share a prominent apical organ muscle system, consisting of several densely packed ring muscles (cf.  and Figs. 4d, f; 5c herein).
Differences between the swimming- and creeping-type larva are found concerning the number of the prototroch longitudinal muscles, which is considerably higher in the swimming-type larva (40 instead of 28; cf.  and Figs. 4; 5a, d, e herein). Probably due to a much simpler architecture of the frontal organ in the swimming-type larva of Loxosomella atkinsae, the number of muscle bundles originating from the frontal organ is very low compared to the creeping-type larva of L. murmanica. Only one pair of muscles, the paired lateral longitudinal muscle, protrudes from the level of the frontal organ of the swimming larva and splits ventrally to form contact with the prototroch ring muscles . A similar muscle pair can be found in the creeping-type larva (the lateral longitudinal muscle), whereby small muscle fibers branch off to come in contact with the prototroch longitudinal muscles rather than with the prototroch ring muscles, as in the swimming larva (Fig. 5a, d, e). The dorsal end of this muscle seems to terminate not far from the frontal organ ring muscle, right below the prominent ring muscle set between apical and frontal organ (Fig. 5a). The position of the paired abfrontal diagonal muscles of the swimming-type larva between apical organ and prototroch is similar to the position of the abfrontal dorso-ventral muscles of the creeping-type larva (Fig. 5e). However, since the latter muscle pair does not contain fibers that intercross ventrally, homology of these two muscles between both larval types appears at least questionable.
Probably one of the most interesting muscle sets of the creeping-type larva, the horseshoe-shaped, posteriorly open enrolling muscle, is most likely a homolog of the main prototroch constrictor of the swimming-type larva (cf. ). We suggest the enrolling muscle/main prototroch constrictor muscle to be independent of the prototroch ring muscle system because (i) the position of this muscle changes by contraction of the prototroch longitudinal muscles from a ventral to dorsal position relative to the prototroch ring muscles and (ii) the shape of the enrolling muscle/main prototroch constrictor muscle is horseshoe-shaped (i.e., posteriorly open) in contrast to the prototroch muscle which forms a closed ring. The entoproct enrolling muscle may have a central function during metamorphosis, when the larva has already settled. During this process the hyposphere retracts and is enclosed through the contraction of a ring of cells above the prototroch [3, 4]. We suggest this ring of cells to be part of the enrolling muscle due to its position in the contracted larva.
Comparison of the musculature of the entoproct creeping-type larva with that of other lophotrochozoans
Due to the highly unique overall morphology of the entoproct creeping-type larva and the high complexity of its musculature, which undoubtedly contains numerous apomorphies, comparisons to other lophotrochozoans is somewhat difficult. To our surprise, however, we found some muscle systems that bear important differences as well as similarities to that of other taxa and we focus on these in the following.
Taxa with a so-called three-layered body wall musculature, consisting of an outer circular, inner longitudinal and intermediate oblique muscle layer, can be found in nearly all worm-shaped lophotrochozoan phyla, e.g., in various annelids including sipunculans and hirudineans [26–28], nemertines [29, 30], aculiferan molluscs [31–33] and in polyclad and rhabditophoran Platyhelminthes [34–36]. However, individual elements of the three-layered body wall pattern are often absent in the body wall of lophotrochozoan worms as, e.g., diagonal muscle fibers in many polychaetes [37–39]. The loss of specific muscle layers is therefore likely to be the result of secondary simplification.
Our data show that the episphere of the creeping-type larva is more or less covered with ring muscles (Figs. 3; 4), while longitudinal and oblique elements are completely missing. The adult muscle system of Loxosomella murmanica consists, among others, of longitudinal stalk musculature and the atrial ring muscles (Fig. 1). Muscle sets covering the complete adult body are lacking. In contrast, adult Loxosomella vivipara and L. parguerensis possess a fine outer ring muscle layer, covering parts of the body, as well as longitudinal and oblique muscle elements in the foot, stalk and calyx . A true body wall musculature could not be observed in any of the investigated entoproct species, and we suggest that a lophotrochozoan-like body wall is absent in entoprocts due to secondary loss. Whether or not the adult muscles develop from larval muscle sets is still uncertain, but rather than a complete loss of the complex larval musculature, incorporation of some larval elements into the adult muscular bodyplan by remodeling seems probable. A metamorphosing creeping-type larva already shows striking similarities to an adult entoproct: the larva attaches to the substratum with the contracted frontal organ, while a ring of cells above the prototroch contracts below the retracted hyposphere [3, 4]. The larval gut rotates by 90°, larval ciliary bands disintegrate and adult cilia are formed on tentacle buds, which are exposed when the atrium reopens [3, 4]. A transformation of the frontal organ ring musculature as foot musculature and/or of the prototroch ring muscles as atrial ring musculature (Fig. 2) appears highly likely, especially when considering the relative position of these prominent muscle sets in a settled larva. Further muscle units, such as the frontal organ retractors, may contribute to the formation of the longitudinal stalk musculature, but since details on the emergence of the adult entoproct body plan from the larval one are entirely lacking, these issues require reassessment of entoproct metamorphosis using modern methods.
Dorso-ventral muscle fibers are part of many lophotrochozoan body plans, e.g., polychaetes , platyhelminths  and mollusks [33, 41]. Thereby, diagnostic for mollusks and entoprocts alone is the medioventral intercrossing of parts of the dorso-ventral musculature [15–17]. Recent studies have shown that the larvae of polyplacophorans and the neomeniomorph Wirenia argentea share a transitory seven-fold seriality of dorso-ventral muscles in their ontogeny . The entoproct creeping-type larva possesses different sets of dorso-ventral muscle fibers (Figs. 5a, e, f; 6a, b). The most prominent set, the abfrontal dorso-ventral musculature, is located in the abfrontal part of the larvae, originating around the anus and running close to the hindgut, straight to the dorsal body wall (Figs. 5e; 6a, b).
Six pairs of rib-shaped muscles present another set of dorso-ventral musculature. These muscle fibers originate ventrally, surrounded by the prototroch longitudinal muscles, and bend laterally, probably inserting at the dorsal body wall (Figs. 5a; 6a, b). Additional dorso-ventral muscle pairs are found in the medio-frontal part of the larva and run towards the apical organ (Figs. 5a, b; 6a, b; frontal dorso-ventral muscles) and into the foot (Figs. 5a, d, f; 6a, b; pedal dorso-ventral muscles). These pedal dorso-ventral muscles are very strong muscle fibers, shaped like an inverted U, and seem to intercross ventrally in the posterior-third part of the animal, right above the pedal sole (Figs. 4b, f; 5a, d, f; 6a, b). These muscles are most likely the intercrossing dorso-ventral muscles previously recognized by transmission electron microscopy (see Fig. 6c in ).
Enrolling muscles are independent lateral muscle systems that occur in some larval and adult aculiferan mollusks [33, 41]. In both entoproct larval types, a muscle system is present which bears striking similarities to the enrolling muscle of polyplacophoran and larval neomeniomorph aplacophoran mollusks: in both phyla, the enrolling muscle is independent of the body wall and extends along the entire length of the pedal sole on the ventro-lateral side (Figs. 5f; 6a, b cf. [25, 33]). In the larva of the neomeniomorph Wirenia as well as in the swimming- and creeping-type entoproct larvae, this enrolling muscle is horseshoe-shaped, i.e., posteriorly open (Figs. 5f; 6a, b [25, 33]). Similar to (adult) polyplacophoran mollusks, the creeping-type larva is able to contract across the entire length of the body. Due to its position above the prototroch in the contracted creeping-type larva, the entoproct enrolling muscle might have an important role during metamorphosis. While this muscle system persists in adult polyplacophorans and is remodeled to become a part of the longitudinal body wall musculature in the neomeniomorph Wirenia, its postmetamorphic fate in entoprocts remains unknown.
Compared to other spiralian trochophore-like larvae, the muscular system of the creeping-type larva of the solitary entoproct Loxosomella murmanica is highly complex. Despite some muscle sets, which can be homologized between the two entoproct larval types (e.g., the prototroch longitudinal muscles, which most probably are an apomorphic character of Entoprocta), other muscle types are unique to the creeping-type larva. These are, e.g., muscles which are associated with the frontal organ, such as the frontal organ ring muscles and the frontal organ retractors. Recent studies on the myogenesis of aculiferan mollusks (neomeniomorphs and polyplacophorans) propose a common evolutionary origin of the enrolling muscle as well as a seven-fold seriality of the dorso-ventral muscles in the last common ancestor of polyplacophorans and aplacophorans . The data presented herein likewise demonstrate a distinct enrolling muscle and, among others, a set of six pairs of serially arranged and one pair of ventrally intercrossing dorso-ventral muscle fibers in the entoproct creeping-type larva. In light of the Tetraneuralia hypothesis, which suggests entoproct-mollusk monophyly based on numerous morphological characters [16, 19], this strongly argues for the existence of a distinct set of enrolling muscles as well as (muscular) seriality (albeit not annelid-like segmentation) in the ur-tetraneuralian. Whether this last common ancestor of mollusks and entoprocts bore (seven) shell plates (as is assumed for the ur-aculiferan), or whether such dorsal armature constitutes an invention of Mollusca alone, remains unknown and first requires reconstruction of the last common ancestor to all mollusks before further assessments can be made.
Apical ring muscles
Atrial ring muscles
Body wall musculature
Frontal organ ring muscles
Frontal organ retractor muscles
Intestinal longitudinal muscles
Intestinal ring muscles
Longitudinal stalk muscles
- MgCl2 :
- NaN3 :
Phosphate buffered saline
Phosphate buffered saline with Triton X-100
Pedal dorso-ventral muscles
Paired lateral longitudinal muscle
Prototroch longitudinal muscles
Prototroch ring muscles
We thank the staff of the Sven Lovén Centre for Marine Sciences, Kristineberg, Sweden, in particular Ola Björlin, for their hospitality and support of our field work; Thomas Schwaha, Alen Kristof and Sabrina Kaul-Strehlow (University of Vienna) for their advice on confocal microscopy and Amira. This work was supported by the “Equal opportunities office” of the Faculty of Biology, University of Mainz, to JM and a grant of the European Community through an Assemble grant (no. 227799) to Ricardo Neves (main applicant), University of Basel, and JM. AW is grateful for financial support from the Austrian Science Fund (FWF, grant number P24276-B22) for a project on the evolution and development of basal mollusks, with which this work was tightly connected, and to generous support by the Faculty of Life Sciences, University of Vienna. BL kindly acknowledges grants from the German Science Foundation (Li998/9-1) and the Inneruniversitäre Forschungsförderung (University of Mainz).
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