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Abstract
Background
The shipworm
Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are
brooded, they retain the general appearance of a typical bivalve veliger-type larva.
Here, we describe myogenesis of
L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework
in order to test for homologous structures that might be part of the bivalve (larval)
muscular ground pattern.
Results
Five major muscle systems were identified: a velum retractor, foot retractor, larval
retractor, a distinct mantle musculature and an adductor system. For a short period
of larval life, an additional ventral larval retractor is present. Early in development,
a velum muscle ring and an oral velum musculature emerge. In late stages the lateral
and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor
and a pedal plexus are formed. Similar to other bivalve larvae,
L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of
them disappears in early stages of
L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval
development and are most likely incorporated into the elaborate mantle musculature
of the adult.
Conclusions
To our knowledge, this is the first account of any larval retractor system that might
contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis
shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a
ventral larval retractor are commonly found among bivalve larvae, and thus most likely
belong to the ground pattern of the bivalve larval musculature.
Electronic supplementary material
The online version of this article (doi:10.1186/s12983-014-0090-9) contains supplementary
material, which is available to authorized users.
Results and Discussion The Aculifera Hypothesis and Molluscan Ancestry The evolutionary origin of Mollusca has been a matter of long-standing debate. Thereby, reconstruction of the last common ancestor (LCA) to all mollusks, the so-called hypothetical ancestral mollusk, has been hampered by difficulties in recovering fossils of unambiguous molluscan stem species and by the lack of a general agreement concerning the relationships of the various molluscan class-level taxa to each other [2]. As a consequence, a broad consensus concerning the deep nodes in molluscan phylogeny is still lacking. Morphology-based analyses have suggested either one or the other of the sclerite-bearing but shell-less aplacophoran clades (Neomeniomorpha or Chaetodermomorpha, respectively) as the earliest molluscan offshoot [8, 9], a monophyletic Aplacophora as sister group to all remaining mollusks (the Testaria) [10], or a polyplacophoran-aplacophoran assemblage (Aculifera) as sister to all other mollusks with a primarily univalved shell (Conchifera) [11, 12]. This controversy may soon be settled, however, since two phylogenomic studies have independently confirmed the latter concept, whereby both recovered a monophyletic Aplacophora as sister group to Polyplacophora (chitons) within Aculifera [4, 5]. In the light of this phylogenetic framework, the recent description of a cylindrical (i.e., worm-shaped) sclerite- and shell-bearing Paleozoic mollusk [6] and the results of integrative molecular-paleontological studies [13, 14] have been considered as evidence for the presence of seven or eight shell plates in the LCA of crown-group aculiferans. If correct, this implies that the body plan of recent aplacophorans is the result of secondary simplification and thus a derived condition [6, 14]. Despite additional descriptions of fossils that exhibit a mixture of polyplacophoran- and aplacophoran-like features [15, 16], the morphology of the LCA of Aculifera remains elusive. This may be due to the overall paucity of well-preserved Paleozoic fossils that undoubtedly can be assigned to the aculiferan lineage, the uncertainty as to whether or not some early (Cambrian or Precambrian) fossils [17, 18] indeed represent crown- or stem-group mollusks, and the fact that solid morphological and developmental evidence from recent aplacophoran representatives is still largely lacking. The few reports of individual aplacophoran larvae and postlarvae have shown that these animals may bear six or seven rows of papillae, sclerites, or sclerite-secreting cells [19–21], but these studies found no further support by gross morphological developmental studies of two neomeniomorph representatives [22, 23]. Myogenesis Suggests that Aplacophorans Have a Secondarily Simplified Body Plan The muscular architecture of mollusks is intimately associated with the existence, number, and arrangement of shells in the respective taxa [24]. In polyplacophorans, the myoanatomy is highly complex [7, 25] (Figures 1A and 1C) and several components, such as a laterally positioned enrolling muscle and a dorsal rectus system that spans the longitudinal axis of the animal, have been widely considered as defining morphological features (autapomorphies) of this taxon [24]. In stark contrast to the sophisticated polyplacophoran myoanatomy, aplacophoran representatives have a much simpler muscular organization that, together with the body wall musculature, mainly comprises serially repeated dorsoventral muscles (Figures 1B and 1D). Accordingly, a scenario that suggests a shell plate-bearing aculiferan LCA with polyplacophoran-like musculature implies drastic secondary simplification of the muscular body plan of aplacophoran mollusks. Since ontogenetic data may provide important insights into the evolutionary history of a given taxon [26, 27], we investigated the development of a model neomeniomorph aplacophoran, Wirenia argentea, from hatching of the larvae until after metamorphosis. In comparing myogenesis in Wirenia with that of a polyplacophoran (Leptochiton asellus), we found striking similarities in the muscular organization of both species (see Table 1 for a summary of major larval and adult muscle systems known for the various molluscan lineages), including the presence of a rectus muscle (Figures 2A–2D), so far only known from polyplacophorans, and a laterally positioned enrolling muscle (Figures 2A–2H). Both systems are retained in adult polyplacophorans but are lost during Wirenia postlarval development (note that some neomeniomorphs do exhibit distinct enrolling muscles as adults [28, 29]). Although homology between the polyplacophoran and neomeniomorph enrolling muscles has been questioned [7], their similar position in the respective animals and, in particular, their identical mode of ontogenetic formation as independent muscle system (and not as a thickened derivative of the body wall musculature, as proposed earlier for the aplacophoran taxa [7]) argue strongly for their common evolutionary origin and thus for the presence of such a system in the LCA of both clades. Aside from these muscles, we found two additional muscular systems shared only by polyplacophorans and neomeniomorphs. These are a paired ventrolateral and a single ventromedian muscle (Figures 2E–2L). Both systems are only transiently present in advanced larvae, and neither has a counterpart in either the polyplacophoran or the neomeniomorph adult body plan. As with the rectus and the enrolling muscles, the identical positions of the ventrolateral and the ventromedian muscles in Wirenia and Leptochiton larvae, together with their identical positions relative to other muscles, clearly suggest that these respective muscles are homologous between the two species (see [30] for a recent overview on homology theory and assessment). Earlier, we had already found the ventrolateral system in larvae of another polyplacophoran, Mopalia muscosa [7], but had overlooked the ventromedian muscle. Reinvestigation of the original 3D data set, however, unambiguously revealed such a muscle also in Mopalia (data not shown; available on request). Accordingly, it appears highly likely that the ventrolateral and the ventromedian muscles together with the enrolling and the rectus muscle were all part of the muscular toolkit of the LCA of polyplacophorans and neomeniomorphs, and that the simple myoanatomy of adult Wirenia is a derived, secondary condition. Formation of the eight sets of dorsoventral muscles in polyplacophorans passes through a transitory stage of multiple individual myocytes that appear synchronously [7] (Figure 2E) and give rise to the first seven paired shell muscle units (while the eighth set forms considerably later, together with the most posterior shell plate [7]). In Wirenia, seven pairs of dorsoventral muscles develop simultaneously (Figures 3A–3E) and differentiate further in later stages (Figure 2F). A gradual numerical increase of the dorsoventral muscle sets was observed only after metamorphosis (Figure 3F). Accordingly, both neomeniomorphs and polyplacophorans exhibit a transient stage of seven-fold seriality in the arrangement of these muscles. Despite the different ontogenetic pathways that lead to this seven-fold seriality (fusion of multiple myocytes in polyplacophorans versus simultaneous formation in Wirenia), this seven-fold seriality appears to be a reoccurring pattern, at least in aculiferan mollusks (unfortunately, the ontogenetic sequence of the formation of the eight pairs of dorsoventral muscles in Monoplacophora is still unknown). This is well in line with the description of the seven-shelled fossil Kulindroplax [6], as well as with the serially arranged sclerites or papillae of some recent aplacophorans [19–21], and lends further support for an aculiferan LCA with a seven-fold seriality of epidermal hardparts and the associated musculature. This implies that the eighth set of dorsoventral muscles of recent polyplacophorans is a derived condition. The late formation of the most posterior shell plate and associated musculature in polyplacophorans [7, 31] may well be considered as ontogenetic testimony of such a scenario. The rudimentary data on myogenesis (and development in general) [21] of the second aplacophoran taxon, the Chaetodermomorpha, does not allow for definite conclusions concerning the presence of neomeniomorph- and/or polyplacophoran-like features such as the rectus or the ventromedian muscles in the LCA of this taxon. However, the ring musculature in the body wall of both aplacophoran clades, which is also rudimentarily present in the apical region of polyplacophoran larvae [7] (Figure 2), may constitute an aculiferan apomorphy uniting Polyplacophora, Neomeniomorpha, and Chaetodermomorpha (Table 1). This, together with the cylindrical shape of the aplacophorans and the polyplacophoran larva as well as the fossil Kulindroplax, argues for a worm-like body shape of the LCA of Aculifera, rendering the dorsoventrally flattened appearance of recent polyplacophorans a derived condition. The paired lateral longitudinal muscle in the larva of the chaetodermomorph Chaetoderma [21] may correspond to either the enrolling or the ventrolateral muscle of larval polyplacophorans and neomeniomorphs (Table 1), which would further support the inclusion of Chaetodermomorpha within Aculifera. The fact that Kulindroplax shares morphological features not only with polyplacophorans but also with recent chaetodermomorphs, including the absence of a pedal pit and the position of the gills [6], likewise supports such a scenario. Whether or not a monophyletic Aculifera will stand the test of future phylogenetic analyses or whether Chaetodermomorpha, despite these shared morphological characters, will be proven to have different affinities [32] remains to be seen. The transient expression of typical polyplacophoran-like muscles in the Wirenia larva, however, strongly suggests that at least neomeniomorph aplacophorans stem from an ancestor with polyplacophoran-like features that most likely also included seven shell plates.
We investigated muscle development in two chiton species, Mopalia muscosa and Chiton olivaceus, from embryo hatching until 10 days after metamorphosis. The anlagen of the dorsal longitudinal rectus muscle and a larval prototroch muscle ring are the first detectable muscle structures in the early trochophore-like larva. Slightly later, a ventrolaterally situated pair of longitudinal muscles appears, which persists through metamorphosis. In addition, the anlagen of the putative dorsoventral shell musculature and the first fibers of a muscular grid, which is restricted to the pretrochal region and consists of outer ring and inner diagonal muscle fibers, are generated. Subsequently, transversal muscle fibers form underneath each future shell plate and the ventrolateral enrolling muscle is established. At metamorphic competence, the dorsoventral shell musculature consists of numerous serially repeated, intercrossing muscle fibers. Their concentration into seven (and later eight) functional shell plate muscle bundles starts after the completion of metamorphosis. The larval prototroch ring and the pretrochal muscle grid are lost at metamorphosis. The structure of the apical grid and its atrophy during metamorphosis suggests ontogenetic repetition of (parts of) the original body-wall musculature of a proposed worm-shaped molluscan ancestor. Moreover, our data show that the "segmented" character of the polyplacophoran shell musculature is a secondary condition, thus contradicting earlier theories that regarded the Polyplacophora (and thus the entire phylum Mollusca) as primarily eumetameric (annelid-like). Instead, we propose an unsegmented trochozoan ancestor at the base of molluscan evolution. Copyright 2002 Wiley-Liss, Inc.
In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well-organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5-HT) immunocytochemical staining relative to the muscle ring differed significantly: 5-HT-immunoreactive cells were situated in the center of the striated muscle ring, while Phe-Met-Arg-Phe-NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co-expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch-contraction.
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