Comparative myoanatomy of cycliophoran life cycle stages

The metazoan phylum Cycliophora includes small cryptic epibionts that live attached to the mouthparts of clawed lobsters. The life cycle is complex, with alternating sexual and asexual generations, and involves several sessile and free‐living stages. So far, the morphological and genetic characterization of cycliophorans has been unable to clarify the phylogenetic position of the phylum. In this study, we add new details on the muscular anatomy of the feeding stage, the attached Prometheus larva, the dwarf male, and the female of one of the two hitherto described species, Symbion pandora. The musculature of the feeding stage is composed of myofibers that run longitudinally in the buccal funnel (two fibers) and in the trunk (variable number of fibers). The mouth opening is lined by a myoepithelial ring musculature. A complex myoepithelial sphincter is situated proximal to the anus. In the attached Prometheus larva, three longitudinal sets of myofilaments run dorsally, laterally, and ventrally along the entire anterior‐posterior body axis. The muscular architecture of the dwarf male is complex, especially close to the penis, in the posterior part of the body. An X‐shaped muscle structure is found on the dorsal side, whereas on the ventral side, longitudinal muscles and a V‐shaped muscle structure are present. These muscles are complemented by additional dorsoventral muscles. The mesodermal muscle fibers attach to the cuticle via the epidermis in all life cycle stages studied herein. The musculature of the female is similar to that of the Pandora larva of Symbion americanus and includes dorsoventral muscles and longitudinal muscles that run in the dorsal and ventral body region. Overall, our results reveal striking similarities in the muscular arrangement of the life cycle stages of both Symbion species. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Developmental architecture of the nervous system in Themiste lageniformis (Sipuncula): New evidence from confocal laser scanning microscopy and gene expression

Sipuncula is a clade of unsegmented marine worms that are currently placed among the basal radiation of conspicuously segmented Annelida. Their new location provides a unique opportunity to reinvestigate the evolution and development of segmented body plans. Neural segmentation is clearly evident during ganglionic ventral nerve cord (VNC) formation across Sedentaria and Errantia, which includes the majority of annelids. However, recent studies show that some annelid taxa outside of Sedentaria and Errantia have a medullary cord, without ganglia, as adults. Importantly, neural development in these taxa is understudied and interpretation can vary widely. For example, reports in sipunculans range from no evidence of segmentation to vestigial segmentation as inferred from a few pairs of serially repeated neuronal cell bodies along the VNC. We investigated patterns of pan-neuronal, neuronal subtype, and axonal markers using immunohistochemistry and whole mount in situ hybridization (WMISH) during neural development in an indirect-developing sipunculan, Themiste lageniformis. Confocal imaging revealed two clusters of 5HT⁺ neurons, two pairs of FMRF⁺ neurons, and Tubulin⁺ peripheral neurites that appear to be serially positioned along the VNC, similar to other sipunculans, to other annelids, and to spiralian taxa outside of Annelida. WMISH of a synaptotagmin1 ortholog in T. lageniformis (Tl-syt1) showed expression throughout the centralized nervous system (CNS), including the VNC where it appears to correlate with mature 5HT⁺ and FMRF⁺ neurons. An ortholog of elav1 (Tl-elav1) showed expression in differentiated neurons of the CNS with continuous expression in the VNC, supporting evidence of a medullary cord, and refuting evidence of ontogenetic segmentation during formation of the nervous system. Thus, we conclude that sipunculans do not exhibit any signs of morphological segmentation during development.     

Three‐dimensional reconstruction of the musculature of various life cycle stages of the cycliophoran Symbion americanus

Cycliophora is a very recently described phylum of acoelomate metazoans with a complex life cycle and a phylogenetic position that has been under debate ever since its discovery in 1995. Symbion americanus, which lives attached to the mouthparts of the American lobster, Homarus americanus, represents the second species described for the phylum. Aiming to increase the morphological knowledge about this cryptic clade, the present study describes the muscle arrangement of the feeding stage, the attached Prometheus larva with the dwarf male inside, the free living male, the Pandora larva, and the chordoid larva of S. americanus using actin staining and confocal laser scanning microscopy. 3D reconstructions of the muscular systems are presented. In the feeding stage, circular muscles compose the buccal funnel aperture. In addition, a pair of muscles runs longitudinally in the buccal funnel. A complex sphincter was found just proximally to the anus, and six longitudinal muscles run from the trunk constriction (“neck”) in basal direction. The musculature of the larval stages and the dwarf male is very complex and includes longitudinal muscles that run dorsally and ventrally. In addition, we found dorso‐ventral muscles. The male has a complex posterior muscle apparatus in the vicinity of the penis. In this stage, X‐ and V‐shaped structures were identified on the dorsal and the ventral side, respectively. Pandora and chordoid larvae possess additional circular muscles. We discuss our findings with respect to muscle elements of other metazoan groups and the chordoid larva of Symbion pandora. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Comparative analysis of the nervous systems in presumptive progenetic dinophilid and dorvilleid polychaetes (Annelida) by immunohistochemistry and cLSM

Entire nervous systems of the dinophilids Dinophilus (two species) and Trilobodrilus (three species) and the dorvilleids Parapodrilus psammophilus and Ophryotrocha gracilis (larva) were stained with antisera directed against serotonin, Phe‐Met‐Arg‐Phe‐NH₂ (FMRFamide) and acetylated α‐tubulin and analysed by confocal laser scanning microscopy (cLSM). Adult dinophilids and the dorvilleid larva exhibit the same structure of the ventral nerve cord, with two main nerves spaced far apart, one median and two paramedian nerves. A serotonergic plexus is situated between the paramedian nerve pair, above the ventral locomotory ciliary band. These similarities between adults and larva corroborate the presumed progenetic origin of dinophilids. However, since larval nervous systems of other polychaete taxa also seem to be organized in this way, this result cannot support the view that dinophilids originate from dorvilleids. In P.psammophilus the main nerve cords are widely separated only in the last segment, indicating that this pattern may be correlated with the absence of parapodia. The unpaired median nerve of dinophilids, P. psammophilus and many other polychaetes, is considered to be part of the basic annelid body plan. The ground pattern of the ventral paired dinophilid ganglia is represented by three (anterior, main, posterior) commissures, conserved in most of the ganglia in the Dinophilus species and mostly reduced to a main commissure in the Trilobodrilus species. The dinophilid species and P. psammophilus possess six pairs of ganglia indicating six trunk segments – in contrast to former views. The two rings behind the prostomium in both the dinophilid and the dorvilleid species contain one pair of ganglia only, corroborating the presumed homology of this peristomial region in the two taxa. The Dinophilus nervous system with 12 longitudinal nerves and three perpendicular nerve rings per segment resembles orthogonal nervous structures characteristic of platyhelminths.     

Immunocytochemistry of the nervous system and the musculature of the chordoid larva of Symbion pandora (Cycliophora)

To date, the phylum Cycliophora comprises only one described extant species of acoelomate marine invertebrates, Symbion pandora. Adult specimens live commensally on the mouthparts of the Norwegian lobster, Nephrops norvegicus. Its complicated life cycle includes an asexually produced Pandora larva and a sexually produced chordoid larva. Despite detailed TEM investigations and its inclusion in recent molecular phylogenetic analyses, cycliophoran relationships still remain enigmatic. In order to increase the morphological database, I investigated the anatomy of the nervous system and the musculature of the chordoid larva by applying fluorescence-coupled antibodies against the neurotransmitters serotonin and FMRFamide, as well as FITC-coupled phalloidin to label filamentous F-actin, in combination with confocal laser scanning microscopy. The FMRFamidergic nervous system shows a bilobed anterior ganglion and one pair of ventral nerve cords, while serotonin is distributed in a scattered pattern in the anterior ganglion. In addition, there are two pairs of ventral serotonergic nerves, of which the inner pair fuses with the outer nerve cords in the posterior third of the larva. The musculature comprises an outer layer of six units of circular body wall muscles, several helicoid muscle fibers, a set of paired longitudinal muscles that span the entire anterior-posterior axis of the larva, and a few oblique muscle strands. Furthermore, an anterior muscle complex and one pair of posterior muscles are present. The chordoid organ consists of a number of distinct subunits that are each formed by a dense layer of circular muscle fibers.The overall arrangement of the oblique and longitudinal muscles as well as the body wall musculature in the chordoid larva of Symbion pandora exhibits similarities with the condition found in certain rotifers. This is congruent with some recent phylogenies based on 18S rRNA sequences but additional morphological, developmental, and molecular data are needed to clarify the phylogenetic relationships of Cycliophora.     

The Development of the Brachiopod Crania (Neocrania) anomala (O. F. Müller)and its Phylogenetic Significance

The freely spawned eggs of Crania go through radial cleavage, embolic gastrulation, and the posteroventral part of the archenteron forms mesoderm through modified enterocoely. The blastopore closes in the posterior end of the larva. The ciliated, lecithotrophic larva has four pairs of coelomic pouches and three pairs of dorsal setal bundles. At metamorphosis, the larva curls ventrally by contraction of a pair of midventral muscles, which are extensions of the first pair of coelomic sacs; the larva attaches by the epithelium just behind the closed blastopore. The brachial valve is secreted by the middle part of the dorsal epithelium and the pedicle valve is secreted by the attachment epithelium. The second pair of coelomic sacs develop small attachment areas at the edge of the dorsal valve and become the lophophore coelom (mesocoel); the third pair of coelomic sacs become the body coelom (metacoel) with the adductor muscles. The posterior position of the closing blastopore is characteristic of deuterostomes. The ventral curving of the settling larva and the formation of both valves from dorsal epithelial areas indicate that the brachiopods have a very short ventral side as opposed to the phoronids. It is concluded that both groups have originated from a creeping ancestor with a straight gut.     

The pregenital abdominal musculature in phasmids and its implications for the basal phylogeny of Phasmatodea (Insecta: Polyneoptera)

Recently several conflicting hypotheses concerning the basal phylogenetic relationships within the Phasmatodea (stick and leaf insects) have emerged. In previous studies, musculature of the abdomen proved to be quite informative for identifying basal taxa among Phasmatodea and led to conclusions regarding the basal splitting events within the group. However, this character complex was not studied thoroughly for a representative number of species, and usually muscle innervation was omitted. In the present study the musculature and nerve topography of mid-abdominal segments in both sexes of seven phasmid species are described and compared in detail for the first time including all putative basal taxa, e.g. members of Timema, Agathemera, Phylliinae, Aschiphasmatinae and Heteropteryginae. The ground pattern of the muscle and nerve arrangement of mid-abdominal segments, i.e. of those not modified due to association with the thorax or genitalia, is reconstructed. In Timema, the inner ventral longitudinal muscles are present, whereas they are lost in all remaining Phasmatodea (Euphasmatodea). The ventral longitudinal muscles in the abdomen of Agathemera, which span the whole length of each segment, do not represent the plesiomorphic condition as previously assumed, but might be a result of secondary elongation of the external ventral longitudinal muscles. Sexual dimorphism, common within the Phasmatodea, also applies to the muscle arrangement in the abdomen of some species. Only in the females of Haaniella dehaanii (Heteropteryginae) and Phyllium celebicum (Phylliinae) the ventral external longitudinal muscles are elongated and span the length of the whole segment, possibly as a result of convergent evolution.     

Description and innervation of the lateral line system in two gobioids, <Emphasis Type="Italic">Odontobutis obscura</Emphasis> and <Emphasis Type="Italic">Pterogobius elapoides</Emphasis> (Teleostei: Perciformes)

Components of the lateral line system and their innervation were studied in Odontobutis obscura (Odontobutidae) and Pterogobius elapoides (Gobiidae), which are benthic and pelagic species, respectively. Innervation of the superficial neuromasts constituting the trunk lateral line system by way of three continuous longitudinal series (dorsal, middle, and ventral series: ld, lm, and lv series, respectively) became apparent for the first time. Innervation patterns indicated that the ld and lv series represented a mixture of displaced rows (from lm series) and new additional rows. In O. obscura, the ld and lv series were poorly developed, whereas both series were well developed in the pelagic P. elapoides, possibly as an adaptation to receive stimuli from above and below. Two extremely elongated nerve branches derived from the lateral ramus of the posterior lateral line nerve innervated the ld and lv series, respectively, in P. elapoides. Homologies of the neuromast rows on the head and body were discussed on the basis of their innervation patterns.     

Caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth

In order to address the question of the conservation of posterior growth mechanisms in bilaterians, we have studied the expression patterns of the orthologues of the genes caudal, even-skipped, and brachyury in the annelid Platynereis dumerilii. Annelids belong to the still poorly studied third large branch of the bilaterians, the lophotrochozoans, and have anatomic and developmental characteristics, such as a segmented body plan, indirect development through a microscopic ciliated larva, and building of the trunk through posterior addition, which are all hypothesized by some authors (including us) to be present already in Urbilateria, the last common ancestor of bilaterians. All three genes are shown to be likely involved in the building of the anteroposterior axis around the slit-like amphistomous blastopore as well as in the patterning of the terminal anus-bearing piece of the body (the pygidium). In addition, caudal and even-skipped are likely involved in the posterior addition of segments. Together with the emerging results on the conservation of segmentation genes, these results reinforce the hypothesis that Urbilateria had a segmented trunk developing through posterior addition.     

Anatomy of the serotonergic nervous system of an entoproct creeping-type larva and its phylogenetic implications

Abstract. Although the internal phyletic relationships of Spiralia (and Lophotrochozoa) remain unresolved, recent progress has been made due to molecular phylogenetic analyses as well as developmental studies of crucial taxa such as Mollusca, Sipuncula, or Annelida. Despite this progress, the phylogenetic position of a number of phyla, such as Entoprocta, remains problematic, mainly due to their unique morphology, their aberrant mode of development, and their exclusion in most large-scale phylogenetic analyses. In order to extend the morphological dataset of this enigmatic taxon, we herein describe the anatomy of the serotonergic nervous system of the creeping-type larva of Loxosomella murmanica . The apical organ is very complex and comprises six to eight centrally positioned flask cells and eight bipolar peripheral cells. In addition, a prototroch nerve ring, an anterior nerve loop, a paired buccal nerve, and an oral nerve ring are found. Moreover, the larva of L. murmanica has one pair of pedal and one pair of lateral longitudinal nerve cords and thus expresses a tetraneurous condition. Several paired serotonergic perikarya, which form contact with the pedal nerve cords but not with the lateral ones, are found along the anterior–posterior axis. The combination of a complex larval serotonergic apical organ and (adult) tetraneury, comprising one pair of ventral and one pair of more dorsally situated lateral longitudinal nerve cords without ganglia, has so far only been reported for basal molluscs and may be diagnostic for a mollusc–entoproct clade. In addition, the larva of Loxosomella expresses a mosaic of certain neural features that are also found in other larval or adult Spiralia, e.g., a prototroch nerve ring, an anterior nerve loop, and a buccal nervous system.     

The delineation of the fourth walking leg segment is temporally linked to posterior segmentation in the mite Archegozetes longisetosus (Acari: Oribatida,Trhypochthoniidae)

Acari (mites and ticks) lack external segmentation, with the only indication of segmentation being the appendages of the prosoma (chelicerae, pedipalps, and four pairs of walking legs). Acari also have a mode of development in which the formation of the fourth walking leg is suppressed until the nymphal stages, following a hexapodal larva. To determine the number of segments in the posterior body region (opisthosoma) of mites, and to also determine when the fourth walking leg segment is delineated during embryogenesis, we followed the development of segmentation in the oribatid mite Archegozetes longisetosus using time‐lapse and scanning electron microscopy, as well as in situ hybridizations of the A. longisetosus orthologues of the segmentation genes engrailed and hedgehog. Our data show that A. longisetosus patterns only two opisthosomal segments, indicating a large degree of segmental fusion or loss. Also, we show that the formation of the fourth walking leg segment is temporally tied to opisthosomal segmentation, the first such observation in any arachnid.     

The whole-body shortening reflex of the medicinal leech: motor pattern,sensory basis,and interneuronal pathways

The leech whole-body shortening reflex consists of a rapid contraction of the body elicited by a mechanical stimulus to the anterior of the animal. We used a variety of reduced preparations — semi-intact, body wall, and isolated nerve cord — to begin to elucidate the neural basis of this reflex in the medicinal leech Hirudo medicinalis. The motor pattern of the reflex involved an activation of excitatory motor neurons innervating dorsal and ventral longitudinal muscles (dorsal excitors and ventral excitors respectively), as well as the L cell, a motor neuron innervating both dorsal and ventral longitudinal muscles. The sensory input for the reflex was provided primarily by the T (touch) and P (pressure) types of identified mechanosensory neuron. The S cell network, a set of electrically-coupled interneurons which makes up a fast conducting pathway in the leech nerve cord, was active during shortening and accounted for the shortest-latency excitation of the L cells. Other, parallel, interneuronal pathways contributed to shortening as well. The whole-body shortening reflex was shown to be distinct from the previously described local shortening behavior of the leech in its sensory threshold, motor pattern, and (at least partially) in its interneuronal basis.Abbreviations conn connective - DE dorsal excitor motor neuron - DI dorsal inhibitor motor neuron - DP dorsal posterior nerve - DP:B1 dorsal posterior nerve branch 1 - DP:B2 dorsal posterior nerve branch 2 - MG midbody ganglion - VE ventral excitor motor neuron - VI ventral inhibitor motor neuron     

And Lophotrochozoa makes three: Notch/Hes signaling in annelid segmentation

Segmentation is unquestionably a major factor in the evolution of complex body plans, but how this trait itself evolved is unknown. Approaching this problem requires comparing the molecular mechanisms of segmentation in diverse segmented and unsegmented taxa. Notch/Hes signaling is involved in segmentation in sequentially segmenting vertebrates and arthropods, as judged by patterns of expression of one or more genes in this network and by the disruption of segmental patterning when Notch/Hes signaling is disrupted. We have previously shown that Notch and Hes homologs are expressed in the posterior progress zone (PPZ), from which segments arise, in the leech Helobdella robusta, a sequentially segmenting lophotrochozoan (phylum Annelida). Here, we show that disrupting Notch/Hes signaling disrupts segmentation in this species as well. Thus, Notch/Hes functions in either the maintenance of the PPZ and/or the patterning processes of segmentation in representatives of all three superphyla of bilaterally symmetric animals. These results are consistent with two evolutionary scenarios. In one, segmentation was already present in the ancestor of all three superphyla. In the other, Notch/Hes signaling functioned in axial growth by terminal addition in an unsegmented bilaterian ancestor, and was subsequently exapted to function in segmentation as that process evolved independently in two or more taxa.     

Early development of the aplacophoran mollusc Chaetoderma

The early development of the trochophore larva of the aplacophoran Chaetoderma nitidulum (Mollusca: Caudofoveata = Chaetodermomorpha) is described using scanning and transmission electron microscopy and using fluorescence staining and confocal laser scanning microscopy of the muscle system. The most important features include the presence of a prototroch and a telotroch, serially arranged glandular cells (spiculoblasts) at the dorsal mantle, the occurrence of a ventral suture as a possible remnant of a former foot sole, and the presence of a pair of protonephridia. The development of the early anlagen of the circular body wall muscles does not show the anterior–posterior mode of formation that is typical for annelids, thus strengthening the hypothesis of a non‐segmented ancestry of Mollusca.     

Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

Some aspects of spiralian development

Nielsen, C. 2010. Some aspects of spiralian development. —Acta Zoologica (Stockholm) 91 : 20–28 Spiralian development is not only a characteristic early cleavage pattern, with shifting orientations of the cleavage planes, but also highly conserved cell lineages, where the origin of several organs can be traced back to identifiable cells in the lineage. These patterns are well documented in annelids, molluscs, nemertines, and platyhelminths and are considered ancestral of a bilaterian clade including these phyla. Spiral cleavage has not been documented in ecdysozoans, and no trace of the spiral development pattern is seen in phoronids and brachiopods. Origin of the spatial organization in spiralian embryos is puzzling, but much of the information appears to be encoded in the developing oocyte. Fertilization and “pseudofertilization” apparently provides the information defining the secondary, anterior‐posterior body axis in many species. The central nervous system consists of three components: an apical organ, derived from the apical blastomeres 1a¹¹¹‐1d¹¹¹, which degenerates before or at metamorphosis; the cerebral ganglia derived from other blastomeres of the first micromere quartet and retained in the adult as a preoral part of the brain; and the originally circumblastoporal nerve cord, which has become differentiated into a perioral part of the brain, the paired or secondarily fused ventral nerve cords, and a small perianal nerve ring.