Symbion Pandora Research Articles

Transcriptome profiling of Symbion pandora (phylum Cycliophora): insights from a differential gene expression analysis

Cycliophorans are characterized by a complex life cycle that involves an asexual generation and a sexual generation. The most prominent life cycle stage in both generations is the so-called feeding stage. Using RNA-Seq, we profiled differential gene expression between feeding stages from asexual and sexual generations to study this intergenerational shift. For this, we also generated a reference transcriptome for the cycliophoran Symbion pandora. We found that a total of 2660 contigs (more than 10% of the total transcriptome) correspond to genes that are expressed differentially in the feeding stages from the asexual generation as compared to the sexual feeding stages. Among these, 1236 genes are upregulated in the asexual stages as compared to the sexual stages. Conversely, 1424 genes are upregulated in the sexual stages as compared to the asexual stages. The asexual stages express genes predominantly related to RNA processing and splicing as well as protein folding, which suggests a high degree of regulation at the transcriptional and post-transcriptional levels. In marked contrast, the sexual stages highly express genes related to signal transduction and neurotransmission. This is the first time that a large whole-transcriptome RNA-Seq expression dataset has been generated for any cycliophoran. Moreover, this study provides important information for further studies on the molecular mechanisms that are involved in the shift from asexual to sexual generations in this still enigmatic group.

Microanatomy and development of the dwarf male of Symbion pandora (Phylum Cycliophora): new insights from ultrastructural investigation based on serial section electron microscopy.

Cycliophorans have a complex life cycle that involves several sexual and asexual stages. One of the sexual stages is the 40 μm-long dwarf male, which is among the smallest free-living metazoans. Although the dwarf male has a highly complex body plan, this minute organism is composed of a very low number of somatic cells (~50). The developmental processes that give rise to this unique phenotype are largely unknown. Here we use high resolution serial block face—scanning electron microscopy to analyze the anatomy and morphogenesis of three cycliophoran dwarf males at different developmental stages ranging from internal bud to mature male. The anatomical and morphological features of the mature dwarf male stage reported here largely correspond to those reported in earlier studies. Interestingly, the organs that typically characterize the anatomy of the mature dwarf male, e.g., muscles, brain, testis and glands, are already formed in the young male. However, there are striking differences between the mature male and young male stages at the level of cellular architecture. Thus, while the young male stage, like the internal bud stage, possesses approximately 200 nucleated cells, the mature male stage comprises only around 50 nucleated cells; muscle and epidermal cells of the mature male lack nuclei. Moreover, the total body volume of the mature male is only 63% of the body of the young male implying that the maturation of the young male into a mature male involves a marked reduction of internal body volume, mainly by massive nuclei loss. Our comparative analysis of these dwarf male specimens reveals unprecedented insight into the striking morphological and developmental differences that characterize these highly miniaturized male stages both at the level of body organization and at the level of cellular ultrastructure.

External morphology of the cycliophoran dwarf male: a comparative study of Symbion pandora and S. americanus

Cycliophora is a recently described phylum to which only two species have been assigned so far, Symbion pandora and S. americanus. The cycliophoran life cycle is complex and alternates between asexual and sexual stages. Although not recognized as an entirely independent free-swimming stage when the phylum was first described, the dwarf male has a remarkably complex bodyplan albeit its very small size (approx. 30–40 μm in length). Aiming to increase the knowledge on the gross morphology of the cycliophoran dwarf male, specimens from S. pandora and S. americanus were analyzed by scanning electron microscopy. In both species, anterior and ventral ciliated fields, as well as paired lateral sensorial organs, were identified, thus confirming previous observations. However, new details are described herein such as the penial pouch that encloses the penis. We compare our findings on both Symbion species with the data currently available on other metazoan dwarf males.

Serotonin immunoreactivity in the nervous system of the Pandora larva, the Prometheus larva, and the dwarf male of Symbion americanus (Cycliophora)

Cycliophora is a recently described phylum of enigmatic metazoans with a very complex life cycle that includes several sexual and asexual stages. Symbion pandora and Symbion americanus are the only two cycliophoran species hitherto described, of which morphological and genetic knowledge is still deficient to clarify the phylogenetic position of the phylum. Aiming to increase the database on the cycliophoran neural architecture, we investigated serotonin immunoreactivity in the free swimming Pandora larva, the Prometheus larva, and the adult dwarf male of S. americanus. In the larval forms, serotonin is mainly expressed in a ring-shaped pattern at the periphery of the antero-dorsal cerebral ganglion. Additionally, several serotonergic perikarya emerge from both sides of the cerebral ganglion. Thin neurites project anteriorly from the cerebral ganglion, while a pair of ventral longitudinal neurites emerges laterally and runs along the anterior-posterior body axis. Posteriorly, the ventral neurites fuse and extend as a posterior projection. In the dwarf male, serotonin is found mainly in the commissural neuropil of the large anterior cerebral ganglion. In addition, serotonin immunoreactivity is present in the most anterior region of the ventral neurites. Comparative analysis of spiralian nervous systems demonstrates that the neuroanatomy of the cycliophoran larval stages resembles much more the situation of adult rather than larval spiralians, which may be explained by secondary loss of larval structures and heterochronic shift of adult components into the nervous system of the Pandora and the Prometheus larva, respectively .

Expression of synapsin and co‐localization with serotonin and RFamide‐like immunoreactivity in the nervous system of the chordoid larva of Symbion pandora (Cycliophora)

Abstract. Cycliophora is one of the most recently described metazoan phyla and hitherto includes only two species: Symbion pandora and Symbion americanus. With a very complex life cycle, cycliophorans are regarded as an enigmatic group with an uncertain phylogenetic position, although they are commonly considered lophotrochozoan protostomes. In order to extend the database concerning the distribution of immunoreactive substances in the free‐swimming chordoid larva of S. pandora, we investigated synapsin immunoreactivity using fluorescence‐coupled antibodies in combination with confocal laserscanning microscopy. Moreover, we analyzed the co‐localization patterns of synapsin, serotonin, and RFamide‐like immunoreactivity in the chordoid larva by 3D imaging technology based on the confocal microscopy image stacks. Synapsin is expressed in large parts of the bilobed anterior cerebral ganglion including anterior and dorsal projections. Two pairs of ventral neurites run longitudinally into the larval body of which the inner pair shows only weak, scattered synapsin immunoreactivity. In addition, a lateral synapsin immunoreactive projection emerges posteriorly from each ventral longitudinal axon. Double immunostaining shows co‐localization of synapsin and serotonin in the cerebral ganglion, the outer and the inner ventral neurites, and the anterior projections. Synapsin and RFamide‐like immunoreactivity co‐occur in the cerebral ganglion, the outer ventral neurites, and the dorsal projections. Accordingly, the cerebral ganglion and the outer ventral neurites are the only neural structures that co‐express the two neurotransmitters and synapsin. The overall neuroanatomical condition of the cycliophoran chordoid larva resembles much more the situation of adult rather than larval life cycle stages of a number of spiralian taxa.

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.

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.

A new species of Cycliophora from the mouthparts of the American lobster, Homarus americanus (Nephropidae, Decapoda)

The present study describes a new species of Cycliophora with the aid of light- and electron microscopy. The animals live attached or free-living on the mouth appendages of the American lobster, Homarus americanus. Individuals occur in dense clusters of up to several thousand individuals. The new species is named Symbion americanus sp. nov. according to the name of its host; it can be distinguished from the previously described species Symbion pandora by the presence of a posterior pair of retractable tubular appendages or toes in the Prometheus larva. Morphological variation among cycliophorans on H. americanus collected in different localities seems to be high. In several sexual populations of S. americanus, older feeding individuals with a female typically have 5–13 rings of cuticular scars and a thicker cuticle. Moreover, attached Prometheus larvae frequently contain three males, chordoid cysts possess a distal appendix, and chordoid larval morphology varies among localities. These differences in morphology might indicate the existence of cryptic species. The presence of toes in the Prometheus larva could support a cycliophoran relationship with rotiferan taxa, although additional ultrastructural studies are needed. Considering that the genus Homarus is at least 60 My years old, and with regard to its history of speciation, it seems possible that the two Symbion species separated during the Pleistocene.

The microhabitat of Symbion pandora (Cycliophora) on the mouthparts of its host Nephrops norvegicus (Decapoda: Nephropidae)

The microhabitat of Symbion pandora (Cycliophora) was described by recording the prevalence and distribution of two life cycle stages, namely feeding individuals and chordoid cysts on the mouth appendages of 65 Norway lobsters. The commensals infested lobsters with a carapace length >35 mm, while the intensity of the commensals increased with host size up to more than 1,100 feeding individuals and 173 chordoid cysts. Feeding individuals and chordoid cysts were found on all six pairs of mouth appendages, but were rare on Mxp3. A Kruskal–Wallis analysis of variance showed that the distribution of the two stages over individual segments deviated significantly from random expectations (P<0.001). Feeding individuals densely aggregated on a few medial segments, generally those that are pervaded by suspended food particles during host feeding. Chordoid cysts, which arise from non-feeding females occurred in low numbers and were more evenly distributed over the segments, often aggregating on the lateral parts and in the articulations of the segments. Based on these findings we suggest that the feeding activity of S. pandora is synchronized with that of the host.

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.

Occurrence of the phylum Cycliophora in the Mediterranean

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 277:297-299 (2004) - doi:10.3354/meps277297 Occurrence of the phylum Cycliophora in the Mediterranean Oldrich Nedved* Faculty of Biological Sciences, University of South Bohemia, and Institute of Entomology, Academy of Sciences, Branisovská 31, 37005 Ceské Budejovice, Czech Republic *Email: nedved@bf.jcu.cz ABSTRACT: Symbion pandora is the single described species of the animal phylum Cycliophora, and was discovered in 1995 on the coast of northern Europe. Specimens have recently been found in the Adriatic Sea, at the Istria Peninsula on the Croatian coast, on the mouthparts of lobster Homarus gammarus. KEY WORDS: Symbion pandora · Lobster · Homarus · Croatia · Adriatic Sea Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 277. Online publication date: August 16, 2004 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2004 Inter-Research.

Dwarf male of Symbion pandora (cycliophora).

This study clarifies the identity and development of the male in the life cycle of Symbion pandora. The male is not produced directly by the feeding stage, as previously thought, but arises as a distinct individual from budding cells inside an intermediate stage named the Prometheus larva. The morphology and the development of the two distinct stages are described with light and electron microscopy. Furthermore, the following terminology is suggested to clearly distinguish between the different individuals: 1) the Prometheus larva, which is the free-swimming individual being produced inside the feeding stage; 2) the attached Prometheus larva on the feeding stage, which mostly degenerates following settlement, except for the internal budding cells; and 3) the dwarf male, which is the ciliated, sexually mature stage. The budding cells inside the attached Prometheus larva usually develop two internal dwarf males. Each dwarf male is heavily ciliated and has a well-developed nervous system with a relatively large brain, numerous gland and muscle cells, testis with bundles of sperm, and one penial structure. The male lacks a gut, as in the other free stages in the life cycle of Symbion pandora. This study also indicates that the dwarf male is freed from the attached Prometheus larva. Copulation, which has not been observed yet, probably takes place between a free-swimming male and the female, either while the female is released or afterwards.

Performance of 18S rDNA helix E23 for phylogenetic relationships within and between the Rotifera–Acanthocephala clades

The species diversity of the phylum Rotifera has been largely studied on the basis of morphological characters. However, cladistic relationships within this group are poorly resolved due to extensive homoplasy in morphological traits, substantial phenotypic plasticity and a poor fossil record. We undertook this study to determine if a phylogeny based on partial 18S rDNA, which included the helix E23 of 18S rDNA sequence, was concordant with established taxonomic relationships within the order Ploimida (class: Monogononta). We also estimated the level of polymorphism within clones and populations of Ploimida ‘species’. Finally, we included the Cycliophora Symbion pandora as outgroup and the variable helix E23 region to examine the influence of their signal on the evolutionary relationships among Acanthocephala, Bdelloidea and Ploimida. Phylogenetic reconstruction was performed using maximum parsimony, neighbour joining and maximum likelihood methods. We found 1) that morphologically similar Ploimida ‘species’ show vastly different 18S E23 rDNA sequences; 2) inclusion of the helix E23 of 18S rDNA and its secondary structure analysis results in better resolution of family level relationships within the Ploimida; 3) an impact of Symbion pandora as an outgroup with inclusion of the helix E23 on the relationships between the Rotifera and the Acanthocephala; and 4) partial incongruence and differential substitution rate between conserved region and helix E23 region of the 18S rDNA gene depending on the taxomic group studied.

Relations of the new phylum Cycliophora

The Cycliophora is the most recently described animal phylum and is based on a single species, Symbion pandora, which was discovered on the mouthparts of the Norway lobster Nephrops norvegicus1. Because only a few morphological data1,2,3 are available for Symbion, the precise nature of its phylogenetic relationships is highly controversial2,4,5. Here we present a phylogenetic analysis of 18S ribosomal RNA sequence data, including a new Symbion sequence, which places Symbion in a lophophorate-aschelminth-protostome clade and which suggests a sister-group relationship between Cycliophora and a Rotifera-Acanthocephala clade.

The chordoid larva of Symbion pandora (Cycliophora) is a modified trochophore.

Developmental and free-living stages of the chordoid larva of the cycliophoran species, Symbion pandora Funch and Kristensen 1995, were studied using light and electron microscopy. In the free-living stage of the larva, about 200 μm long, four ciliated areas are found: two anterior bands, a ventral ciliated field, and a posterior unit on the ventral side of the foot. The nervous system consists of a dorsal brain and a pair of ventral longitudinal nerves. A gut is absent. A pair of protonephridia, each with a single multiciliated terminal cell and at least one duct cell, is present. Nephridiopores are not localized. A pair of corsal ciliated organs is posterior to the brain. The homology between these and the apical organ of a trochophore larva is discussed. A distinctive longitudinal rod, the chordoid organ, consists of vacuolized cells with circular myofilaments. The organ is comparable to a similar structure in gastrotrichs. In the discussion of the phylogenetic position of Cycliophora among protostomians, important morphological observations that are described in the present study indicate that, despite some dissimilarities, the chordoid larva is a modified trochophore. © 1996 Wiley-Liss, Inc.

The chordoid larva of Symbion pandora (Cycliophora) is a modified trochophore

Developmental and free-living stages of the chordoid larva of the cycliophoran species, Symbion pandora Funch and Kristensen 1995, were studied using light and electron microscopy. In the free-living stage of the larva, about 200 μm long, four ciliated areas are found: two anterior bands, a ventral ciliated field, and a posterior unit on the ventral side of the foot. The nervous system consists of a dorsal brain and a pair of ventral longitudinal nerves. A gut is absent. A pair of protonephridia, each with a single multiciliated terminal cell and at least one duct cell, is present. Nephridiopores are not localized. A pair of corsal ciliated organs is posterior to the brain. The homology between these and the apical organ of a trochophore larva is discussed. A distinctive longitudinal rod, the chordoid organ, consists of vacuolized cells with circular myofilaments. The organ is comparable to a similar structure in gastrotrichs. In the discussion of the phylogenetic position of Cycliophora among protostomians, important morphological observations that are described in the present study indicate that, despite some dissimilarities, the chordoid larva is a modified trochophore. © 1996 Wiley-Liss, Inc.

Cycliophora is a new phylum with affinities to Entoprocta and Ectoprocta

THE mouthparts of the Norway lobster Nephrops are colonized by an acoelomate metazoan, Symbion pandora gen. et sp. nov. Sessile stages continually produce inner buds replacing feeding structures. They also produce one of three motile stages: (1) larvae containing new feeding stages, (2) dwarf males, which settle on feeding stages, or (3) females, which settle onto lobster mouthparts, and eventually degenerate, giving rise to dispersive larvae. All motile stages are short-lived, and do not feed. The structure and function of the cilia suggest a phylogenetic position in Protostomia, while some aspects of inner budding and brooding of larvae are similar to those of Entoprocta and Ectoprocta. The dispersive larva possesses a mesodermal supporting chordoid structure, otherwise absent in protostomian larvae. We believe that all the above features of this previously undescribed species warrant the recognition of a new phylum with affinities to Ectoprocta and Entoprocta.