Collecting and Culturing Bryozoans for Regenerative Studies.

Among marine invertebrates, bryozoans are small, not well known, and complex to identify. Nevertheless, they offer unique opportunities for whole-body generation research, because of their colonial, modular mode of growth. Here, we describe detailed methods for collection of bryozoans from a range of environments, sample preparation and identification, culture and feeding, spawning and breeding, marking colonies for growth studies, and histological preparation.

Composite branch construction by dual autozooidal budding modes in hornerids (Bryozoa: Cyclostomatida).

Horneridae (Cyclostomatida: Cancellata) is a family of marine bryozoans that forms tree-like colonies bearing functionally unilaminate branches. Colony development in this clade is not well understood. We used micro-computed tomography and scanning electron microscopy to trace zooidal budding in Hornera from the ancestrula onwards. Results show that hornerid branches are constructed by dual zooidal budding modes occurring synchronously at two separate budding sites at the growing tips. Frontal autozooids bud from a multizooidal budding lamina. Lateral autozooids bud from discrete abfrontal budding loci by "exomural budding," a previously undescribed form of frontal budding centered on hypostegal pores in interzooidal grooves on the colonial body wall. These two budding modes are integrated during primary branch morphogenesis, forming composite, developmentally bilaminate, branches. Patterns of exomural budding vary among hornerid taxa, and future studies of Cancellata taxonomy and phylogeny may benefit from morphological concepts presented here.

The epithelial layers of the body wall in hornerid bryozoans (Stenolaemata: Cyclostomatida).

Bryozoans are small colonial coelomates. They can be conceptualised as "origami-like" animals, composed of three complexly folded epithelial layers: epidermis of the zooidal/colonial body wall, gut epithelium and coelothelium. We investigated the general microanatomy and ultrastructure of the hornerid (Cyclostomatatida) body wall and polypide in four taxa, including three species of Hornera and one species belonging to an undescribed genus. We describe epithelia and their associated structures (e.g., ECM, cuticle) across all portions of the hornerid body wall, including the terminal membrane, vestibular wall, atrial sphincter, membranous sac and polypide-skeletal attachments. The classic coelomate body wall composition (epidermis-ECM-coelothelium) is only present in an unmodified form in the tentacle sheath. Deeper within a zooid it is retained exclusively in the attachment zones of the membranous sac: [skeleton]-tendon cell-ECM-coelothelium. A typical invertebrate pattern of epithelial organisation is a single, continuous sheet of polarised cells, connected by belt desmosomes and septate junctions, and resting on a collagenous extracellular matrix. Although previous studies demonstrated that polypide-specific epithelia of Horneridae follow this model, here we show that the body wall may show significant deviations. Cell layers can lose the basement membrane and/or continuity of cell cover and cell contacts. Moreover, in portions of the body wall, the cell layer appears to be missing altogether; the zooidal orifice is covered by a thin naked cuticle largely devoid of underlying cells. Since epithelium is a two-way barrier against entry and loss of materials, it is unclear how hornerids avoid substance loss, while maintaining intracolonial metabolite transport with imperfect, sometimes incomplete, cell layers along large portions of their outer body surface.

Hornera currieae n. sp. (Cyclostomatida: Horneridae): a new bathyal cyclostome bryozoan with reproductively induced skeletal plasticity.

Here we describe a new hornerid, Hornera currieae n. sp. (Bryozoa: Cyclostomatida) from bathyal depths across the New Zealand region. Colonies are irregular, finely branched fans attaining ~40 mm or more in height. Key characters include: (1) thick, semi-hyaline porcellanous skeleton; (2) loss or reduction of nervi (longitudinal striae) away from growing tips; (3) sparse, threadlike cancelli; and (4) small (6187 m), widely spaced autozooidal apertures. Diagnostic hornerid traits possessed by H. currieae n. sp. include vertical ancestrular tube, periancestrular budding of daughter zooids, and skeletal ultrastructure dominated by hexagonal semi-nacre grading to pseudofoliated fabric. The abfrontal incubation chamber develops from a cryptic tube arising from the frontally positioned aperture of the fertile zooid. We used SEM, micro-CT and electron backscatter diffractometry (EBSD) to investigate the ultrastructure and internal architecture of H. currieae n. sp. EBSD reveals that crystalline c-axes of laminated crystallites are perpendicular to skeletal walls. Threadlike cancelli, which traverse secondary calcification, connect autozooidal chambers to the colony-wide hypostegal cavity. Micro-CT reveals that abfrontal cancelli usually bend proximally towards the base, but turn distally towards reproductively active regions of the colony in synchrony with gonozooid development. The zone of affected cancelli extends for 47 branch internodes below the gonozooid. We assessed whether skeletal ultrastructure was similarly affected, but neither cancellus direction, nor gonozooid proximity, were predictive of the crystallite imbrication direction. We hypothesise that (1) hornerid cancelli are active conduits for colonial metabolite transport and (2) that changes in gradients of metabolites and/or reproductive morphogens within the hypostegal cavity affect cancellus morphogenesis. Potentially, H. currieae n. sp. skeletons may preserve a record of intra-colony metabolite translocation dynamics over time.

Polypide anatomy of hornerid bryozoans (Stenolaemata: Cyclostomatida).

Bryozoans are small colonial coelomates whose colonies are made of individual modules (zooids). Like most coelomate animals, bryozoans have a characteristic body wall composition, including an epidermis, an extracellular matrix (ECM) and a coelothelium, all pressed together. The order Cyclostomatida, however, presents the most striking deviation, in which the ECM and the corresponding coelothelium underlying major parts of the skeletal wall epidermis are detached to form an independent membranous sac. It forms a separate, much smaller compartment, suspended in the zooid body cavity and working as an important element of the cyclostome lophophore protrusion mechanism. The polypide anatomy and ultrastructure of this group is best known from studies of one family, the Crisiidae (Articulata). Here, we examined four species from the phylogenetically and ecologically contrasting family Horneridae (Cancellata) from New Zealand, and provide the first detailed ultrastructural description of the hornerid polypide, including tentacles, mouth region, digestive system and the funiculus. We were able to trace continuity and transitions of cell and ECM layers throughout the whole polypide. In addition, we identified that the funiculus is a lumen-free ECM cord with two associated muscles, disconnected from interzooidal pores. Except for funicular core composition, the polypide anatomy of hornerids agrees well with the general cyclostomate body plan.

Skeletal resorption in bryozoans: occurrence, function and recognition.

Skeletal resorption - the physiological removal of mineralised parts by an organism - is an important morphogenetic process in bryozoans. Reports of its occurrence and function across the phylum are patchy, however, and have not previously been synthesised. Here we show that resorption occurs routinely across a wide range of bryozoan clades, colony sizes, growth forms, ontogenetic stages, body wall types, skeletal ultrastructures and mineralogies. Beginning in the early Paleozoic, different modes and functions of resorption have evolved convergently among disparate groups, highlighting its utility as a morphogenetic mode in this phylum. Its functions include branch renovation, formation of branch articulations, excavation of reproductive chambers, part-shedding, and creation of access portals for budding beyond previously formed skeletal walls. Bryozoan skeletons can be altered by resorption at microscopic, zooidal and colony-wide scales, typically with a fine degree of control and coordination. We classified resorption patterns in bryozoans according to the morphology and function of the resorption zone (window formation, abscission or excavation), timing within the life of the skeletal element resorbed (primary or secondary), and scale of operation (zooidal or multizooidal). Skeletal resorption is probably greatly underestimated in terms of its utility and role in bryozoan life history, and its prevalence across taxa, especially in fossil forms. It is reported proportionally more frequently in stenolaemates than in gymnolaemates. Some modes of resorption potentially alter or remove the spatial-temporal record of calcification preserved within a skeleton. Consequently, knowledge that resorption has occurred can be relevant for some common applications of skeletal analysis, such as palaeoenvironmental interpretation, or growth and ageing studies. To aid recognition we provide scanning electron microscopy, backscattered electron scanning electron microscopy and transmission electron microscopy examples of skeletal ultrastuctures modified by resorption.