Reconstructing the muscular ground pattern of phylactolaemate bryozoans: first data from gelatinous representatives.

BACKGROUND: Phylactolaemata is commonly regarded the earliest branch within Bryozoa and thus the sister group to the other bryozoan taxa, Cyclostomata and Gymnolaemata. Therefore, the taxon is important for the reconstruction of the bryozoan morphological ground pattern. In this study the myoanatomy of Pectinatella magnifica, Cristatella mucedo and Hyalinella punctata was analysed by means of histology, f-actin staining and confocal laser-scanning microscopy in order to fill gaps in knowledge concerning the myoanatomy of Phylactolaemata. RESULTS: The retractor muscles and muscles of the aperture, gut, body wall, tentacle sheath, lophophore constitute the most prominent muscular subsets in these species. The lophophore shows longitudinal muscle bands in the tentacles, lophophoral arm muscles, epistome musculature and hitherto undescribed muscles of the ring canal. In general the muscular system of the three species is very similar with differences mainly in the body wall, tentacle sheath and epistome. The body wall contains an orthogonal grid of musculature. The epistome exhibits either a muscular meshwork in the epistomal wall or muscle fibers traversing the epistomal cavity. The whole tentacle sheath possesses a regular mesh of muscles in Pectinatella and Cristatella, whereas circular muscles are limited to the tentacle sheath base in Hyalinella. CONCLUSION: This study is the first to describe muscles of the ring canal and contributes to reconstructing muscular features for the last common ancestor of all bryozoans. The data available suggest that two longitudinal muscle bands in the tentacles, as well as retractor muscles and longitudinal and circular muscles in the tentacle sheath, were present in the last common bryozoan ancestor. Comparisons among bryozoans shows that several apomorphies are present in the myoanatomy of each class- level taxon such as the epistomal musculature and musculature of the lophophoral arms in phylactolaemates, annular muscles in cyclostomes and parietal muscles in gymnolaemates.

Structural integrity and viability of Fredericella sultana statoblasts infected with Tetracapsuloides bryosalmonae (Myxozoa) under diverse treatment conditions.

Fredericella sultana is an invertebrate host of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonids. The bryozoan produces seed-like statoblasts to facilitate its persistence during unfavourable conditions. Statoblasts from infected bryozoans can harbor T. bryosalmonae and give rise to infected bryozoan colonies when conditions improve. We aimed in the present study to evaluate the integrity and viability of T. bryosalmonae-infected statoblasts after a range of harsh treatment conditions. We tested if statoblasts could survive ingestion by either brown trout or common carp. After ingestion, the fish faeces was collected at different time points. We also tested physical stressors: statoblasts collected from infected colonies were desiccated at room temperature, or frozen with and without Bryozoan Medium C (BMC). After treatments, statoblasts were assessed for physical integrity before being incubated on BMC to allow them to hatch. After 4 weeks, hatched and unhatched statoblasts were tested by PCR for the presence of the parasite. We found that statoblasts ingested by brown trout and those frozen in BMC were completely broken. In contrast, statoblasts ingested by common carp and those subjected to dry freezing were able to survive and hatch. T. bryosalmonae was detected by PCR in both hatched and unhatched infected statoblasts, but neither from broken nor uninfected statoblasts. Our results confirmed for the first time the ability of infected statoblasts to survive passage through a fish, and freezing. These findings suggest potential pathways for both persistence and spread of T. bryosalmonae-infected statoblasts in natural aquatic systems.

[FUNCTIONAL DIFFERENTIATION IN BRYOZOAN COLONY: A PROTEOMIC ANALYSIS].

Bryozoans are typical modular organisms. They consist of repetitive structural units, the zooids. Bryozoan colonies grow by zooidal budding, with the distribution pattern of the budding loci underlying the diversity of colony forms. Budding is usually restricted to the zooids at the periphery of the colony, which form a "growing edge" or local terminal growth zones. Non-budding parts of the colony can be functionally subdivided, too. In many species colonies consists of regular, often repetitive zones of feeding and non-feeding modules, associated with a periodical degeneration and regeneration of the polypide, retractile tentacle crown with a gut and the accompanying musculature. So, there is functional differentiation in bryozoan colonies but its mechanisms are unknown. Presumably, budding and/or polypide recycling in different colony parts are induced or inhibited by certain determinants of functional specialization. An effective tool of their identification is the comparison of proteomes of functionally different zones. Here we report the results of proteomic analysis of three bryozoan species from the White Sea, which have a different colony form: Flustrellidra hispida, Terminoflustra membranaceotruncata and Securiflustra securifrons. Using differential two-dimensional electrophoresis (2D-DIGE), we compared proteomes of the growing edge and the zones consisting of feeding and non-feeding zooids in these species. We estimated the overall proteome variability, revealed proteins whose relative abundance gradually changed along the proximal-distal colony axis and suggested that they might be involved in the functional differentiation of the colony.

Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages.

In all probability, natural selection began as ancient marine microorganisms were required to compete for limited resources. These pressures resulted in the evolution of diverse genetically encoded small molecules with a variety of ecological and metabolic roles. Remarkably, many of these same biologically active molecules have potential utility in modern medicine and biomedical research. The most promising of these natural products often derive from organisms richly populated by associated microorganisms (e.g., marine sponges and ascidians), and often there is great uncertainty about which organism in these assemblages is making these intriguing metabolites. To use the molecular machinery responsible for the biosynthesis of potential drug-lead natural products, new tools must be applied to delineate their genetic and enzymatic origins. The aim of this perspective is to highlight both traditional and emerging techniques for the localization of metabolic pathways within complex marine environments. Examples are given from the literature as well as recent proof-of-concept experiments from the authors' laboratories.

Showing their true colors: a practical approach to volume rendering from serial sections.

BACKGROUND: In comparison to more modern imaging methods, conventional light microscopy still offers a range of substantial advantages with regard to contrast options, accessible specimen size, and resolution. Currently, tomographic image data in particular is most commonly visualized in three dimensions using volume rendering. To date, this method has only very rarely been applied to image stacks taken from serial sections, whereas surface rendering is still the most prevalent method for presenting such data sets three-dimensionally. The aim of this study was to develop standard protocols for volume rendering of image stacks of serial sections, while retaining the benefits of light microscopy such as resolution and color information. RESULTS: Here we provide a set of protocols for acquiring high-resolution 3D images of diverse microscopic samples through volume rendering based on serial light microscopical sections using the 3D reconstruction software Amira (Visage Imaging Inc.). We overcome several technical obstacles and show that these renderings are comparable in quality and resolution to 3D visualizations using other methods. This practical approach for visualizing 3D micro-morphology in full color takes advantage of both the sub-micron resolution of light microscopy and the specificity of histological stains, by combining conventional histological sectioning techniques, digital image acquisition, three-dimensional image filtering, and 3D image manipulation and visualization technologies. CONCLUSIONS: We show that this method can yield "true"-colored high-resolution 3D views of tissues as well as cellular and sub-cellular structures and thus represents a powerful tool for morphological, developmental, and comparative investigations. We conclude that the presented approach fills an important gap in the field of micro-anatomical 3D imaging and visualization methods by combining histological resolution and differentiation of details with 3D rendering of whole tissue samples. We demonstrate the method on selected invertebrate and vertebrate specimens, and propose that reinvestigation of historical serial section material may be regarded as a special benefit.

Morphology of ctenostome bryozoans: 1. Arachnidium fibrosum.

The morphology of ctenostome bryozoans remains little investigated with only few species having been subject to more detailed studies. From all the seven main different superfamilies, only few representatives have been studied. The superfamily Arachnidioidea has particularly been neglected concerning detailed morphological and histological details. So far, not a single analysis specifically studied a representative of the family Arachnidiidae. Arachnidium-like forms have, however, often been regarded as potential cheilostome ancestors, the most successful group of bryozoans to date. The lack of any morphological data on this family called for a detailed investigation of one of its representatives. Hence, we analysed the general morphology and histology of Arachnidium fibrosum. Most striking morphological features previously unrecognized are a cardiac constrictor, previously almost unknown in the family, a single pair of apertural muscles consisting of proximal parieto-diaphragmatic and distal parieto-vestibular muscles, six pairs of duplicature bands, a lophophoral anus and retractor muscles attaching to the foregut. Although comparative data are limited, there seem to be two distinct different clades of arachnidiid ctenostomes that are characterized by their aperture and details of gut morphology. Further analysis of additional arachnidioidean species are required to confirm this.

Morphology of ctenostome bryozoans: 2. Haywardozoon pacificum, with implications of the phylogenetic position of the genus.

The genus Haywardozoon represent a little known genus of ctenostome bryozoans that has only been found in the deep-sea. It forms small, mostly uniserial colonies lacking polymorphs. Zooids have a conspicuous apertural closure mechanism consisting of a cuticular lower lip that closes the aperture. The systematic placement of the genus remains uncertain, detailed morphological studies that include soft-body morphological traits are missing. Consequently, this is the first study analyzing H. pacificum by means of histological serial sections and 3d-reconstruction. Zooids are ovoid and in some cases solitary, that is, showing no interconnected zooids. Most prominent is the large vestibular wall that can be more than half of the total length of the zooid. Its vestibular wall is particularly lined by a complex, multilayered and branched cuticle. A single pair of lateral parieto-diaphragmatic muscles is present. The polypide is small and comprises about 17 tentacles. The digestive tract is short, has an elongated cardia, a vestigial caecum and a vestibular anus. An ovipositor/intertentacular organ and several oligolecithal oocytes were detected. Several aspects of zooidal morphology, including the structure of the bilateral aperture, parieto-diaphragmatic muscles, general structure of the gut and the thick cuticle, clearly indicate an association to the ctenostome superfamily Alcyonidioidea. Therefore, we reject the previous placement into Hislopioidea and suggest a possible association to pherusellid ctenostomes. New reproductive characters show that H. pacificum is a broadcaster contrary to some other deep-sea forms that are brooding. RESEARCH HIGHLIGHT: Morphology of ctenostome bryozoans remain little investigated. This contribution is the second of a series of detailed morphological analyses of this understudied clade of bryozoans. The morphological investigation of Haywardozoon pacificum revealed numerous characters that show a closer relationship to Flustrellididrae rather than Hislopiidae as previously assumed.

Ganglion ultrastructure in phylactolaemate Bryozoa: evidence for a neuroepithelium.

In contrast to other Bryozoa, members of the subtaxon Phylactolaemata bear a subepithelial cerebral ganglion that resembles a hollow vesicle rather than being compact. In older studies this ganglion was said to originate by an invagination of the pharyngeal epithelium. Unfortunately, documentation for this is fragmentary. In chordates the central nervous system also arises by an invagination-like process, but this mode is uncommon among invertebrate phyla. As a first attempt to gather more data about this phenomenon, cerebral ganglia in two phylactolaemate species, Fredericella sultana and Plumatella emarginata, were examined at the ultrastructural level. In both species the ganglion bears a small central lumen. The ganglionic cells are organized in the form of a neuroepithelium. They are polarized and interconnected by adherens junctions on their apical sides and reside on a basal lamina. The nerve cell somata are directed towards the central lumen, whereas the majority of nervous processes are distributed basally. Orientation of the neuroepithelial cells can be best explained by the possibility that they develop by invagination. A comparison with potential outgroups reveals that a neuroepithelial ganglion is at least derived. Since, however, a reliable phylogenetic system of the Bryozoa is missing, a decision on whether such a ganglion is apomorphic for Bryozoa or evolved within this taxon can hardly be made.

The morphology and evolutionary significance of the ciliary fields and musculature among marine bryozoan larvae.

Despite the embryological and anatomical disparities present among lophotrochozoan phyla, there are morphological similarities in the cellular arrangements of ciliated cells used for propulsion among the nonfeeding larval forms of kamptozoans, nemerteans, annelids, mollusks, and bryozoans. Evaluating whether these similarities are the result of convergent selective pressures or a shared (deep) evolutionary history is hindered by the paucity of detailed cellular information from multiple systematic groups from lesser-known, and perhaps, basal evolutionary phyla such as the Bryozoa. Here, I compare the ciliary fields and musculature among the major morphological grades of marine bryozoan larvae using light microscopy, SEM, and confocal imaging techniques. Sampling effort focused on six species from systematic groups with few published accounts, but an additional four well-known species were also reevaluated. Review of the main larval types among species of bryozoans and these new data show that, within select systematic groups of marine bryozoans, there is some conservation of the cellular arrangement of ciliary fields and larval musculature. However, there is much more morphological diversity in these structures than previously documented, especially among nonfeeding ctenostome larval types. This structural and functional diversification reflects species differences in the orientation of the apical disc during swimming and crawling behaviors, modification of the presumptive juvenile tissues, elongation of larval forms in the aboral-oral axis, maximizing the surface area of cell types with propulsive cilia, and the simplification of ciliary fields and musculature within particular lineages due to evolutionary loss. Considering the embryological origins and functional plasticity of ciliated cells within bryozoan larvae, it is probable that the morphological similarities shared between the coronal cells of bryozoan larvae and the prototrochal cells of trochozoans are the result of convergent functional solutions to swimming in the plankton. However, this does not rule out cell specification pathways shared by more closely related spiralian phyla. Overall, among the morphological grades of larval bryozoans, the structural variation and arrangement of the main cell groups responsible for ciliary propulsion have been evolutionarily decoupled from the more divergent modifications of larval musculature. The structure of larval ciliary fields reflects the functional demands of swimming and substrate exploration behaviors before metamorphosis, but this is in contrast to the morphology of larval musculature and presumptive juvenile tissues that are linked to macroevolutionary differences in morphogenetic movements during metamorphosis.

Sacculogenesis of Buddenbrockia plumatellae (Myxozoa) within the invertebrate host Plumatella repens (Bryozoa) with comments on the evolutionary relationships of the Myxozoa.

Members of the phylum Myxozoa are obligate parasites, primarily of aquatic organisms. Their phylogeny has remained problematic, with studies placing them within either the Bilateria or Cnidaria. The discovery that the enigmatic Buddenbrockia plumatellae is a myxozoan that possesses distinct bilaterian features appeared to have finally resolved the debate. B. plumatellae is described as a triploblastic 'worm-like' organism, within which typical myxozoan malacospores form. Using EM we examined the early development of the B. plumatellae 'worms' within the bryozoan host Plumatella repens. The initial development involved numerous unicellular, amoeboid pre-saccular stages that were present within the basal lamina of the host's body wall. These stages migrate immediately beneath the peritoneum where a significant host tissue reaction occurs. The stages aggregate, initiating the formation of a 'worm'. The base of a developing 'worm' forms a pseudosyncytium which resolves into an ectoderm surrounding a mesendoderm. The pseudosyncytium is directly anchored into neighbouring host cells via masses of striated fibres. The replication of the ectodermal and mesendodermal cells extends the developing 'worm' into the coelom of the host. The mesendoderm resolves to form a mesoderm and an endoderm. Myogenesis appears to be initiated from the anchored end of the 'worm' and develops along the mesoderm. The aggregation and differentiation of amoeboid pre-saccular stages to initiate the 'worm' draws analogies to the sacculogenesis observed for Tetracapsuloides bryosalmonae, B. plumatellae's sister taxon within the class Malacosporea. The development of a multicellular, spore forming organism, from single cells does not correlate to any bilaterian or cnidarian species. Current phylogenies indicate the Myxozoa are basal bilaterians along with the Acoela and Mesozoa. Comparison with these other basal groups may help to resolve the placement of Myxozoa within the tree of life.

Rearing gymnolaemate bryozoan larvae for cellular and molecular analyses.

Gymnolaemates represent the largest group of extant bryozoans, having more than 3,000 described species. Gymnolaemates display a diverse array of reproductive and developmental patterns including planktotrophy, lecithotrophy, and matrotrophy. The larvae of gymnolaemates have been broadly grouped into three types, cyphonautes (shelled, feeding), pseudocyphonautes (shelled, nonfeeding), and coronate (unshelled, nonfeeding), although each group is heterogeneous and probably includes various morphologies that are largely undescribed. Here, methods for rearing bryozoan colonies and larvae are presented.

Nine new species of Bugula Oken (Bryozoa: Cheilostomata) in Brazilian shallow waters.

BACKGROUND: Bugula is a speciose genus of marine bryozoans, represented by both endemic and cosmopolitan species distributed in tropical and temperate waters and important to marine biologists because of the occurrence of many species in harbor and fouling communities, therefore as potential invaders. The southeastern Brazilian coast in the southern Atlantic hosts the highest known diversity of the genus, a status intimately associated with the intensity of collecting efforts. METHODOLOGY: Morphological data based on the examination of living specimens, scanning electron and light microscopic images, and morphometric analyses were used to assess the diversity of Bugula along the coastal areas of southern, northeastern, and southeastern Brazil. In this study, morphological species boundaries were based mainly on avicularian characters. For two morphologically very similar species, boundaries are partially supported by 16 S rDNA molecular data. RESULTS: Nine species are newly described from Brazil, as follows: Bugula bowiei n. sp. ( = Bugula turrita sensu Marcus, 1937) from the southern, northeastern, and southeastern coasts; Bugula foliolata n. sp. ( = Bugula flabellata sensu Marcus, 1938), Bugula guara n. sp., Bugula biota n. sp. and Bugula ingens n. sp from the southeastern coast; Bugula gnoma n. sp. and Bugula alba n. sp. from the northeastern coast; Bugula rochae n. sp. ( = Bugula uniserialis sensu Marcus, 1937) from the southern coast; and Bugula migottoi n. sp., from the southeastern and southern coasts. CONCLUSION: The results contribute to the morphological characterization and the knowledge of the species richness of the genus in the southwestern Atlantic (i.e., Brazil), through the description of new species in poorly sampled areas and also on the southeastern coast of that country. Additionally, the taxonomic status of the Brazilian specimens attributed to B. flabellata, B. turrita and B. uniserialis are clarified by detailed studies on zooidal and avicularia morphology.

From cells to colonies: at what levels of body organization does the 'temperature-size rule' apply?

An inverse relationship between temperature during ontogeny and final body size is widespread in ectotherms, but poorly understood. Evidence suggests that within organs, this "temperature-size rule" (TSR) may also apply to cell size with no change in numbers. So how closely do reductions in size and number of cells and other repeated structures correlate with size reduction at higher levels of organization? We examine this in the context of a proposal that size and/or number changes at various organizational levels are adaptive responses to temperature- and size-dependent oxygen supply. We subjected two clones of the modular colonial bryozoan, Celleporella hyalina, to orthogonal combinations of two temperatures and two oxygen concentrations during ontogeny, observing effects on sizes of colonies and larvae, and sizes and numbers of cells, tentacles, and modules (autozooids). We found that the size:number responses varied among cell types and among structures at different levels of organization, with the inverse temperature-size relationship applying only to larval parenchymal cells and colony modules. Using our findings and other evidence we propose a unifying adaptive hypothesis that predicts how temperature affects the sizes of mitochondria, cells, organs, modules and organisms, and their relationships with processes that determine the functional capacity of aerobic metabolism.

The complete mitochondrial genome of Flustrellidra hispida and the phylogenetic position of Bryozoa among the Metazoa.

The complete mitochondrial genome of Flustrellidra hispida (Bryozoa, Ctenostomata, Flustrellidridae) was sequenced using a transposon-mediated approach. All but one of the 36 genes were identified (trnS2). The genome is 13,026 bp long, being one of the smallest metazoan mitochondrial genomes sequenced to date with a unique gene order when compared to other Metazoa. The genome has an overall AT richness of 59.4%. We found seven regions of overlaps between tRNAs and protein-coding genes ranging from 2 to 11 nt, and seven regions of overlap between tRNAs, ranging from 1 to 8 nt, resulting in a total number of 46 overlapping nucleotides. Genes nad4, cox2, atp8, and nad3 are terminated by the abbreviated stop codon T and cytb is suggested to terminate on (ACT)AA; we postulate that mRNA editing is required to remove AC for TAA to be functional in terminating translation. Phylogenetic analysis of nucleotide and amino acid data place Flustrellidra in the Lophotrochozoa. DNA for this study originated from two populations resulting in a contig consisting of multiple haplotypes. Twenty-seven SNP sites were detected, the majority occurring in cox1 and nad5. With cox1 already established as a marker in bryozoan studies, we advocate the further testing of nad5.

[Systematic value of the larval structure and details of postlarval morphogenesis in Bryozoa gymnolaemates]. / Valeur systématique de la structure larvaire et des particularités de la morphogenèse post-larvaire chez les Bryozoaires Gymnolaemates.

Among the various species of Bryozoa Gymnolaemata, the larvae and their development were studied, comparing the larval structure and the evolution of their cellular categories during the post-larval morphogenesis the existence of nine well-defined larval types could be revealed. Cases of insufficiently described larvae are discussed. The present systematic of Bryozoa Gymnolaemata is compared with the classification of various larval types. For the major part of cases, each systematic family is marked by a precise type of larva; however there are some exceptions, especially in the ordre Ctenostomata. These discordances may suggest some rearrangements of the classification utilized at the present time.