Ultrastructure of the female pedal gonad in Phoxichilidium femoratum (Chelicerata, Pycnogonida).

Phoxichilidium femoratum is a common species of sea spiders - a small and unique group of chelicerates with unusual adult anatomy. In particular, substantial parts of the reproductive system in pycnogonids (unlike euchelicerates) are located in the appendages. Existing studies of pycnogonid gonads are often limited to light-microscopic level, cover a small range of species, and focus on the contents of the gonad diverticula. Ultrastructural data are rare and contradictory, and the organisation of the gonad wall and the gonoducts is unknown. Here we present a detailed light and transmission electron microscopy-based examination of the pedal portion of the adult female reproductive system in Phoxichilidium femoratum Rathke, 1799. We describe its gross anatomy and the ultrastructure of the gonad diverticulum, oviduct and gonopore, as well as development of the oocytes. Each gonad diverticulum is enclosed in the extracellular matrix of the horizontal septum and bears some internal cellular lining. However, neither the gonad lining, nor the septum sheath cells, ever form a continuous epithelial layer. Oocytes, which undergo maturation in the diverticulum, remain, until very late in the process, attached to the gonad wall though specialised stalk cells. Interestingly, stalk cells do not participate in egg envelope or yolk formation: both are synthesized endogenously in the oocytes. The oviduct is supplied with musculature, which assists in egg transport to the gonopore, whereas the gonopore itself is surrounded by specialised glands.

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.

Early lecithotrophic stages of Nymphon grossipes, and the role of larval appendages and glands in different larval types of pycnogonids.

Nymphon grossipes is a common subtidal species belonging to a small and unique group of chelicerates, that is, the sea spiders. These animals have an anamorphic phase during post-embryonic development and often hatch as small, oligomeric and exotrophic larvae (protonymphons) with four postocular segments, cheliphores, and two pairs of larval legs. A common alternative to protonymphons is a large lecithotrophic larval type, where animals hatch at more advanced stages and have a foreshortened anamorphic development. Based on external morphology, N. grossipes was believed to be an intriguing intermediate between these two conditions and its hatchlings were called "lecithotrophic protonymphons." Here, we examine the anatomy and ultrastructure of instars I and II and review the variety of roles of larval appendages and associated glands in other sea spiders in order to correctly place the larva of this species among pycnogonid larval types. Compared to "typical protonymphons," N. grossipes young hatch with an advanced segmental and appendage composition: six postocular segments instead of four, buds of walking legs 1 and hidden buds of walking legs 2. This state corresponds to the instars II/III (rather than larvae) of Nymphon brevirostre and Pycnogonum litorale. Modifications of the larval appendages, chelar, and spinning glands are aligned with ecological needs of different larval types along a few typical dimensions: locomotion and feeding, dispersal, and attachment to the parent. Although the main challenge for N. grossipes young is secure attachment to the egg package while they growth, there are some discrepancies in their anatomy: N. grossipes retains an oyster basket, but an otherwise nonfunctional digestive system, and a strong silken thread for attachment, but no corresponding reduction of the larval legs. Thus, it is likely that the switch to lecithotrophy happened in the recent evolutionary history of this species.

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.

Anatomical changes in postembryonic development of Pycnogonum litorale.

Sea spiders (Pycnogonida) are a small group of arthropods, sister to other chelicerates. They have an unusual adult bauplan, oligosegmented larvae, and a protracted postembryonic development. Pycnogonum litorale (Strøm, 1762) is an uncommonly long-lived sea spider with a distinctive protonymphon and adult anatomy. Although it was described ~250 years ago, little is known about its internal organization and development. We examined the anamorphic and early epimorphic development of this species using histology, light microscopy, and SEM, and provide the first comprehensive anatomical study of its many instars. Postembryonic development of P. litorale includes transformations typical of pycnogonids: reorganization of the larval organs (digestive, nervous, secretory), formation of the abdomen, trunk segments (+ appendages), primary body cavity and reproductive system. Specific traits include the accelerated articulation of the walking legs, formation of the subesophageal and posterior synganglia, and the system of twin midgut diverticula. In addition, P. litorale simultaneously lose the spinning apparatus and all larval appendages. We found that developmental changes occur in synchrony with changes in ecology and food sources. The transition from the anamorphic to the epimorphic period in particular is marked by considerable anatomical and lifestyle shifts. HIGHLIGHTS: Postembryonic development of P. litorale includes numerous anamorphic and epimorphic stages. The instars acquire abdomen, trunk segments, body cavity, and gonads, while losing all larval appendages. Developmental changes are synchronized with changes in lifestyle and food sources.

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.

The (not very) typical protonymphons of Pycnogonum litorale.

Sea spiders are unique and poorly known marine chelicerates. Their larvae are even less studied, especially at the ultrastructural level. Here, we examined the hatchlings of Pycnogonum litorale (Strøm, 1,762) using histology, SEM and TEM. Existing classifications place these larvae among "typical" protonymphons, together with Nymphon brevirostre. Our results, however, revealed major differences between the two species. Hatchlings of P. litorale are endotrophic for 1-2 weeks, with yolk deposits in the body wall and a reduced secretory apparatus. They lack a body cavity, demonstrate an unusual modification of the midgut sheath cells and a complex subesophageal ganglion, which includes neuromeres of the prospective walking legs 1. These larvae also possess well-developed glia and complex sensory structures: eyes, V-shaped mechanoreceptive bristles, integrated chemo- and mechanoreceptors, and three types of concealed mechanoreceptors embedded into the body wall and only seen on the sections. In this paper we also propose a new interpretation of the pycnogonid larval types: we present a set of traits useful for diagnosis and a preliminary classification. Finally, we discuss the complexity of glial types in sea spiders and other arthropods.

Body cavities in bryozoans: Functional and phylogenetic implications.

Based on morphological evidence, Bryozoa together with Phoronida and Brachiopoda are traditionally combined in the group Lophophorata, although this view has been recently challenged by molecular studies. The core of the concept lies in the presence of the lophophore as well as the nature and arrangement of the body cavities. Bryozoa are the least known in this respect. Here, we focused on the fine structure of the body cavity in 12 bryozoan species: 6 gymnolaemates, 3 stenolaemates and 3 phylactolaemates. In gymnolaemates, the complete epithelial lining of the body cavity is restricted to the lophophore, gut walls, and tentacle sheath. By contrast, the cystid walls are composed only of the ectocyst-producing epidermis without a coelothelium, or an underlying extracellular matrix; only the storage cells and cells of the funicular system contact the epidermis. The nature of the main body cavity in gymnolaemates is unique and may be considered as a secondarily modified coelom. In cyclostomes, both the lophophoral and endosaccal cavities are completely lined with coelothelium, while the exosaccal cavity only has the epidermis along the cystid wall. In gymnolaemates, the lophophore and trunk cavities are divided by an incomplete septum and communicate through two pores. In cyclostomes, the septum has a similar location, but no openings. In Phylactolaemata, the body cavity is undivided: the lophophore and trunk coeloms merge at the bases of the lophophore arms, the epistome cavity joins the trunk, and the forked canal opens into the arm coelom. The coelomic lining of the body is complete except for the epistome, lophophoral arms, and the basal portions of the tentacles, where the cells do not interlock perfectly (this design probably facilitates the ammonia excretion). The observed partitioning of the body cavity in bryozoans differs from that in phoronids and brachiopods, and contradicts the Lophophorata concept.

Postembryonic development of pycnogonids: A deeper look inside.

Sea spiders form a small, enigmatic group of recent chelicerates, with an unusual bodyplan, oligosegmented larvae and a postembryonic development that is punctuated by many moults. To date, only a few papers examined the anatomical and ultrastructural modifications of the larvae and various instars. Here we traced both internal and external events of the whole postembryonic development in Nymphon brevirostre HODGE 1863 using histology, SEM, TEM and confocal microscopy. During postembryonic development, larvae of this species undergo massive reorganization: spinning apparatus and chelar glands disappear; larval legs redifferentiate; three new segments and the abdomen are formed with their corresponding internal organs and appendages; circulatory and reproductive systems develop anew and the digestive and the nervous systems change dramatically. The body cavity remains schizocoelic throughout development, and no traces of even transitory coeloms were found in any instar. In Nymphon brevirostre, just like in Artemia salina LINNAEUS 1758 the heart arises through differentiation of the already existing schizocoel, and thus the circulatory systems of arthropods and annelids are not homologous. We found that classical chelicerate tagmata, prosoma and opisthosoma, are inapplicable to adult pycnogonids, with the most striking difference being the fate and structure of the seventh appendage-bearing segment.

Oligomeric larvae of the pycnogonids revisited.

Organization and ultrastructure of the protonymphon larva were never adequately described, despite it being the common larval type of the enigmatic sea spiders and the only example of oligosegmented life stage among recent chelicerates. We have made a comprehensive examination of the newly hatched free-living protonymphons of Nymphon brevirostre using SEM, TEM, light, and confocal microscopy. Although fairly typical in their broad characters, protonymphon larvae have a number of unique and unexpected traits. Body cavity, already present at this stage, is lined with extracellular matrix and thus is conclusively identified as primary body cavity. Central nervous system includes four postocular neuromeres arranged in three ganglia: supraesophageal, subesophageal, and the first ganglion of the ventral nerve cord. Examination of the sensory organs revealed unusually organized eyes, mechanoreceptors, and chemoreceptors. We have uncovered a mixed sensory-secretory nature of chelar glands and proposed possible modalities of its receptory part. We gave first descriptions of the complex ultrastructure of three secretory organs (spinning glands, slit-like organs, proboscis glands) and hypothesized on their mode of functioning. Comparisons with another oligomeric larva, for example, nauplius, revealed discrepancies in the segmentation of these animals. Although both larvae are externally unsegmented and bear three pairs of homologous appendages, the protonymphon body includes a fourth segment of the prospective walking legs which is absent in nauplius.

Tentacle structure in freshwater bryozoans.

Tentacles are the main food-gathering organs of bryozoans. The most common design is a hollow tube of extracellular matrix (ECM), covered with ten columns of epithelial cells on the outside, and a coelothelium on the inside. Nerves follow the ECM, going between the bases of some epidermal cells. The tentacle musculature includes two bundles formed by myoepithelial cells of the coelothelium. The tentacles of freshwater (phylactolaemate) bryozoans, however, differ somewhat in structure from those of marine bryozoans. Here, we describe the tentacles of three species of phylactolaemates, comparing them to gymnolaemates and stenolaemates. Phylactolaemate tentacles tend to be longer, and with more voluminous coeloms. The composition of the frontal cell row and the number of frontal nerves is variable in freshwater bryozoans, but constant in marine groups. Abfrontal cells form a continuous row in Phylactolaemata, but occur intermittently in other two classes. Phylactolaemata lack the microvillar cuticle reported in Gymnolaemata. Abfrontal sensory tufts are always composed of pairs of mono- and/or biciliated cells. This arrangement differs from individual abfrontal ciliary cells of other bryozoans: monociliated in Stenolaemata and monociliated and multiciliated ones in Gymnolaemata. In all three groups, however, ciliated abfrontal cells probably serve as mechanoreceptors. We confirm previously described phylactolemate traits: an unusual arrangement of two-layered coelothelium lining the lateral sides of the tentacle and oral slits in the intertentacular membrane. As previously reported, tentacle movements involved in feeding differ between bryozoan groups, with phylactolaemates tending to have slower movements than both gymnolaemates and stenolaemates, and a narrower behavioral repertoire than gymnolaemates. The morphological and ultrastructural differences between the freshwater species we studied and marine bryozoans may be related to these functional differences. Muscle organization, tentacle and coelom size, and degree of confluence between tentacle and lophophore coeloms probably account for much of the observed behavioral variability.