MICROMORPHOLOGY AND GENE EXPRESSION IN MUSCLE AND SHELL DEVELOPMENT OF THE MOLLUSCA

This work comprises detailed studies by scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence staining combined with confocal laser scanning microscopy (CLSM), as well as serial sectioning analyses and reconstruction techniques to elucidate the development of the larval and adult musculature of several basal representatives of the molluscan classes Polyplacophora, Bivalvia, Scaphopoda, and Gastropoda. Special reference is given to the shell musculature. In addition, aspects of the myo-anatomy of adult Solenogastres are reconsidered. A further part of this study deals with scaphopod shell morphogenesis and expression of the homeobox gene engrailed (en), in order to gain insights regarding the scaphopod-bivalve relationship. The results enable far reaching conclusions regarding the evolution and the phylogeny of the Mollusca. Solenogastres TEM analysis of adult Solenogastres revealed a mesenchymate body wall musculature which consists of outer ring, intermediate diagonal, and inner longitudinal muscles and resembles the condition of other worm-shaped taxa. The ventrally inter-crossing dorso-ventral musculature, which is diagnostic for the Mollusca, is arranged in multiple serial units along the anterior-posterior body axis. Polyplacophora During development, the chiton larva undergoes an intermediate stage in which the dorso-ventral musculature is serially arranged as in adult Solenogastres. The concentration into seven (and later eight) functional shell plate muscle units is a secondary condition which takes place after metamorphosis. Thus, assumptions of a primarily "segmented" (i.e. annelidlike) character of the polyplacophoran shell plate musculature are rejected. In addition, the anterior (i.e. pre-trochal) body region of chiton larvae shows a muscular grid which is lost at metamorphosis and resembles the body wall musculature of adult aplacophoran (Solenogastres + Caudofoveata) molluscs. Both, the multiple seriality of the dorso-ventral muscles and the apical muscle grid are regarded as ontogenetic recapitulation of the basal molluscan condition which is fully expressed in the adult body plan of Solenogastres. This infers a non-segmented, worm-shaped ancestor at the base of molluscan evolution. The existence of a larval ring-shaped muscle that underlies the prototroch cells (prototroch muscle ring) is a shared feature of polyplacophoran, gastropod, and bivalve larvae (see below) and suprataxic homology of this organ is proposed. Bivalvia Besides a rather complicated set of larval retractor muscles, the veligers of autobranchs (i.e. all Bivalvia except the Protobranchia, the latter with a test-cell larva) exhibit a distinct prototroch muscle ring similar to chitons and gastropods. Both systems are entirely larval and are resorbed during metamorphosis. Scaphopoda The general ontogeny and especially myogenesis in the dentaliid scaphopod Antalis entalis proceeds much more direct than in polyplacophorans or gastropods. Accordingly, distinct larval muscle systems are lacking. However, the paired cephalic and pedal retractors both form additional fibers which project into the region of the prototroch and are lost at metamorphosis. The existence of a distinct, paired cephalic retractor system, which is also found in the basal gastropod and cephalopod bauplan but not in the Bivalvia, suggests a clade comprising the Scaphopoda and Gastropoda + Cephalopoda. This is strengthened by expression data of the homeobox gene engrailed, which plays a significant role in molluscan shell formation. While two dorso-lateral centers of engrailed expression, which correspond to the two centers of initial shell calcification, are found in early bivalve veligers, engrailed is exclusively found in mantle margin cells surrounding the single anlage of the embryonic scaphopod shell. In contrast to bivalves, the scaphopod shell is thus formed from a single center of calcification, and a scaphopod-bivalve sistergroup relationship is therefore rejected. Gastropoda Primitive gastropods, such as the patellogastropods Patella vulgata and Patella caerulea, show one pair of asymmetrically positioned larval retractor muscles which have distinct insertion sites at the embryonic shell. Another strict larval muscle system is the prototrochal muscle ring. All these muscle are lost before, during, or shortly after metamorphosis. Parts of the adult mantle musculature as well as the muscles of the cephalic tentacles are formed prior to metamorphosis, while the buccal musculature is of entire postmetamorphic origin. The process of gastropod ontogenetic torsion is mainly caused by muscular activity of the larval retractors, while the adult shell musculature arises after the completion of torsion. Thus, ontogenetic torsion is regarded as an entirely larval process inferring that the arrangement of the adult shell musculature - which can often be reconstructed by muscle scars on fossilized shells - is not indicative for the question whether paleozoic univalved molluscs were torted or not

Comparative larval myogenesis and adult myoanatomy of the rhynchonelliform (articulate) brachiopods Argyrotheca cordata, A. cistellula, and Terebratalia transversa

<p>Abstract</p> <p>Background</p> <p>Despite significant methodological progress, Brachiopoda remains one of the lophotrochozoan phyla for which no recent ontogenetic data employing modern methodologies such as fluorescence labelling and confocal microscopy are available. This is particularly astonishing given the ongoing controversy concerning its phylogenetic position. In order to contribute new morphogenetic data for phylogenetic and evolutionary inferences, we describe herein the ontogeny and myoanatomy of larvae and adults of the rhynchonelliform brachiopods <it>Argyrotheca cordata, A. cistellula</it>, and <it>Terebratalia transversa </it>using fluorescence F-actin labelling combined with confocal laserscanning microscopy.</p> <p>Results</p> <p>Fully grown larvae of <it>A. cordata </it>and <it>T. transversa </it>consist of three distinct body regions, namely an apical lobe, a mantle lobe with four bundles of setae, and a pedicle lobe. Myogenesis is very similar in these two species. The first anlagen of the musculature develop in the pedicle lobe, followed by setae muscles and the mantle lobe musculature. Late-stage larvae show a network of strong pedicle muscles, central mantle muscles, longitudinal muscles running from the mantle to the pedicle lobe, setae pouch muscles, setae muscles, a U-shaped muscle, serial mantle muscles, and apical longitudinal as well as apical transversal muscles. Fully developed <it>A. cistellula </it>larvae differ from the former species in that they have only two visible body lobes and lack setae. Nevertheless, we found corresponding muscle systems to all muscles present in the former two species, except for the musculature associated with the setae, in larvae of <it>A. cistellula</it>. With our survey of the adult myoanatomy of <it>A. cordata </it>and <it>A. cistellula </it>and the juvenile muscular architecture of <it>T. transversa </it>we confirm the presence of adductors, diductors, dorsal and ventral pedicle adjustors, mantle margin muscles, a distinct musculature of the intestine, and striated muscle fibres in the tentacles for all three species.</p> <p>Conclusion</p> <p>Our data indicate that larvae of rhynchonelliform brachiopods share a common muscular bodyplan and are thus derived from a common ancestral larval type. Comparison of the muscular phenotype of rhynchonelliform larvae to that of the other two lophophorate phyla, Phoronida and Ectoprocta, does not indicate homology of individual larval muscles. This may be due to an early evolutionary split of the ontogenetic pathways of Brachiopoda, Phoronida, and Ectoprocta that gave rise to the morphological diversity of these phyla.</p

Capitellid connections: contributions from neuromuscular development of the maldanid polychaete Axiothella rubrocincta (Annelida)

<p>Abstract</p> <p>Background</p> <p>Numerous phylogenetic analyses on polychaete annelids suggest a taxon Capitellida that comprises the three families Maldanidae, Arenicolidae and Capitellidae. Recent molecular studies support the position of the Echiura, traditionally ranked as a separate phylum, within the capitellids. In order to test the robustness of this molecular-based hypothesis we take a different approach using comparative analyses of nervous and muscle system development in the maldanid <it>Axiothella rubrocincta</it>. Employing immunocytochemistry in combination with confocal laserscanning microscopy, we broaden the database on capitellid organogenesis, thereby incorporating classical histological data in our analysis. Besides assessing possible shared features with the echiurans, we also discuss the variability of neural and muscular characters within the Capitellida.</p> <p>Results</p> <p>The scaffold of the adult central nervous system, which is already established in early developmental stages of <it>Axiothella</it>, consists of cerebral commissures that give rise to simple circumesophageal connectives with fused ventral and dorsal roots and a single ventral neurite bundle. From the latter arise segmental neurites that innervate the peripheral bodywall. Since there is no observable regular pattern, and individual neurites are lost during ontogeny, their exact arrangement remains elusive. The pharynx is encircled by a prominent stomatogastric nerve ring, with a pair of anterior and lateral proboscis neurites directly connecting it to the central nervous system. One pair of ventral and one pair of dorsal longitudinal muscles form the earliest rudiments of the bodywall musculature in late larval stages, while a continuous layer of circular muscles is lacking throughout ontogeny.</p> <p>Conclusions</p> <p>Comparative neurodevelopmental analysis of capitellid and echiuran species reveals several common characters, including simple circumesophageal connectives, a single fused ventral nerve strand, and a stomatogastric ring nerve, that support a close relationship of both taxa, thus corroborating recent molecular phylogenetic analyses. The data on myogenesis show that four longitudinal muscle bands most likely represent an ancestral character not only for the Capitellida, but for the Annelida in general. Whether or not circular muscles are part of the annelid groundpattern remains uncertain.</p

Of tests, trochs, shells, and spicules: Development of the basal mollusk Wirenia argentea (Solenogastres) and its bearing on the evolution of trochozoan larval key features

<p>Abstract</p> <p>Background</p> <p>The phylogenetic status of the aplacophoran mollusk taxon Solenogastres (Neomeniomorpha) is controversially discussed. Some authors propose the clade to represent the most basal branch within Mollusca, while others claim aplacophoran mollusks (Solenogastres and Caudofoveata) to be derived. Larval characters are central in these discussions, specifically the larval test (calymma, apical cap), the ontogeny of the epidermal scleritome, and the proposed absence of larval protonephridia. To date, developmental data are available for five solenogaster species, but most reports are incomplete and need confirmation.</p> <p>Results</p> <p><it>Wirenia argentea </it>deposit small batches of uncleaved embryos that are tightly enclosed by a smooth and transparent egg hull. Cleavage is spiral and unequal. The ciliated larvae hatch about 45 hours after deposition and swim actively in the water column. Within 48-60 hours after hatching they become mushroom-shaped with a pronounced apical cap partly enclosing a posterior trunk. The cells covering the apical cap are large and cleavage arrested. On the apical cap there is a prominent prototrochal band of compound cilia and an apical ciliary tuft and the trunk bears a terminal ciliary band (telotroch). Obscured by the apical cap, a ciliary band originates in the stomodaeal pore and surrounds the trunk. As development is proceeding, the trunk elongates and becomes covered by cuticle with the exception of a ventral ciliary band, the future foot. The larvae have a pair of protonephridia. At 5 days after hatching they begin to settle and within the following 7-9 days the apical cap is gradually reduced. Scattered epidermal sclerites form under the cuticle. <it>Wirenia argentea </it>lack iterated groups of sclerites at any developmental stage. At 40 days after hatching, the postlarvae have a fully developed foregut, but the midgut and hindgut are not yet interconnected.</p> <p>Conclusions</p> <p>Solenogastres develop via a trochophore-like lecitotrophic larva with a preoral apical cap that at least partly represents an enlarged prototrochal area. Homology of this larval type (pericalymma larva) to test cell larvae of other spiralian clades is doubtful. The ontogeny of <it>W. argentea </it>does not provide any evidence for a derived status of Solenogastres within Mollusca.</p

Neurogenesis suggests independent evolution of opercula in serpulid polychaetes

<p>Abstract</p> <p>Background</p> <p>The internal phylogenetic relationships of Annelida, one of the key lophotrochozoan lineages, are still heavily debated. Recent molecular analyses suggest that morphologically distinct groups, such as the polychaetes, are paraphyletic assemblages, thus questioning the homology of a number of polychaete morphological characters. Serpulid polychaetes are typically recognized by having fused anterior ends bearing a tentacular crown and an operculum. The latter is commonly viewed as a modified tentacle (= radiole) and is often used as an important diagnostic character in serpulid systematics.</p> <p>Results</p> <p>By reconstructing the developmental neuroanatomy of the serpulid polychaete <it>Spirorbis </it>cf. <it>spirorbis </it>(Spirorbinae), we found striking differences in the overall neural architecture, the innervation pattern, and the ontogenetic establishment of the nervous supply of the operculum and the radioles in this species. Accordingly, the spirorbin operculum might not be homologous to the radioles or to the opercula of other serpulid taxa such as <it>Serpula </it>and <it>Pomatoceros </it>and is thus probably not a part of the tentacular crown.</p> <p>Conclusion</p> <p>We demonstrate that common morphological traits such as the prostomial appendages may have evolved independently in respective serpulid sublineages and therefore require reassessment before being used in phylogenetic analyses. Our findings corroborate recent molecular studies that argue for a revision of serpulid systematics. In addition, our data on <it>Spirorbis </it>neurogenesis provide a novel set of characters that highlight the developmental plasticity of the segmented annelid nervous system.</p

Myoanatomy and serotonergic nervous system of the ctenostome Hislopia malayensis: evolutionary trends in bodyplan patterning of ectoprocta

<p>Abstract</p> <p>Background</p> <p>Ectoprocta is a large lophotrochozoan clade of colonial suspension feeders comprising over 5.000 extant species. Their phylogenetic position within the Lophotrochzoa remains controversially discussed, but also the internal relationships of the major ectoproct subclades -Phylactolaemata, Stenolaemata, and Gymnolaemata - remains elusive. To gain more insight into the basic configuration of ectoproct muscle systems for phylogenetic considerations, we analysed the adult myoanatomy and the serotonergic nervous system as well as myogenesis in budding stages of the ctenostome <it>Hislopia malayensis</it>.</p> <p>Results</p> <p>In adults, the serotonergic nervous system is restricted to the lophophoral base with a high concentration in the cerebral ganglion and serotonergic perikarya between each pair of tentacles. Prominent smooth apertural muscles extend from the basal cystid wall to each lateral side of the vestibular wall. The musculature of the tentacle sheath consists of regular strands of smooth longitudinal muscles. Each tentacle is supplied with two bands of longitudinal muscles that show irregular striation. At the lophophoral base several muscles are present: (i) Short muscle fibres that proximally diverge from a single point from where they split distally into two separate strands. (ii) Proximally of the first group are smooth, longitudinal fibres that extend to the proximal-most side of the lophophoral base. (iii) Smooth muscle fibres, the buccal dilatators, traverse obliquely towards the pharynx, and (iv) a circular ring of smooth muscle fibres situated distally of the buccal dilatators. Retractor muscles are mainly smooth with short distal striated parts. The foregut consists mainly of striated ring musculature with only few longitudinal muscle fibres in the esophagus, while the remaining parts of the digestive tract solely exhibit smooth musculature. During budding, apertural and retractor muscles are first to appear, while the parietal muscles appear at a later stage.</p> <p>Conclusions</p> <p>The apertural muscles show high similarity within Ectoprocta and always consist of two sets of muscles. Gymnolaemates and Phylactolaemates show clear differences within their digestive tract musculature, the former showing smooth and longitudinal muscles to a much greater extent than the latter. The complex musculature at the lophophoral base appears promising for inferring phylogenetic relationships, but sufficient comparative data are currently lacking.</p