Ultrastructure and embryonic development of a syconoid calcareous sponge

Abstract. Recent molecular data suggest that the Porifera is paraphyletic (Calcarea+Silicea) and that the Calcarea is more closely related to the Metazoa than to other sponge groups, thereby implying that a sponge‐like animal gave rise to other metazoans. One ramification of these data is that calcareous sponges could provide clues as to what features are shared among this ancestral metazoan and higher animals. Recent studies describing detailed morphology in the Calcarea are lacking. We have used a combination of microscopy techniques to study the fine structure of Syconcoactum Urban 1905, a cosmopolitan calcareous sponge. The sponge has a distinct polarity, consisting of a single tube with an apically opening osculum. Finger‐like chambers, several hundred micrometers in length, form the sides of the tube. The inner and outer layers of the chamber wall are formed by epithelia characterized by apical–basal polarity and occluding junctions between cells. The outer layer—the pinacoderm—and atrial cavity are lined by plate‐like cells (pinacocytes), and the inner choanoderm is lined by a continuous sheet of choanocytes. Incurrent openings of the sponge are formed by porocytes, tubular cells that join the pinacoderm to the choanoderm. Between these two layers lies a collagenous mesohyl that houses sclerocytes, spicules, amoeboid cells, and a progression of embryonic stages. The morphology of choanocytes and porocytes is plastic. Ostia were closed in sponges that were vigorously shaken and in sponges left in still water for over 30 min. Choanocytes, and in particular collar microvilli, varied in size and shape, depending on their location in the choanocyte chamber. Although some of the odd shapes of choanocytes and their collars can be explained by the development of large embryos first beneath and later on top of the choanocytes, the presence of many fused collar microvilli on choanocytes may reflect peculiarities of the hydrodynamics in large syconoid choanocyte chambers. The unusual formation of a hollow blastula larva and its inversion through the choanocyte epithelium are suggestive of epithelial rather than mesenchymal cell movements. These details illustrate that calcareous sponges have characteristics that allow comparison with other metazoans—one of the reasons they have long been the focus of studies of evolution and development.     

繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。     

Comparative study of the choanosome of Porifera: 1. The Homoscleromorpha

The choanoderm and pinacoderm of representatives of the two families of Homoscleromorpha sponges, the Oscarellidae and Plakinidae, have been examined by transmission and scanning electron microscopy. Different fixative procedures have shown the dramatic influence of fixation conditions on the morphology of choanocytes. These two families of sponges have the following morphological features in common: flagellated endopinacocytes with short apical microvilli and basal pseudopods; the presence of a very thin and dense sheet of matrix material which limits the mesohyl. There are, however, only minor differences in the flagellar morphology, granule content, and anchoring system of their choanocytes. Two findings are of particular interest: (1) the presence of glycocalyx bridges between the microvilli of the choanocyte collar; and (2) the discovery of a new cell type, the apopylar cell, which has a morphology intermediate between that of pinacocytes and choanocytes. The apopylar cells limit the apopylar opening of the choanocyte chamber and indicate the transition between choanoderm and pinacoderm.     

Formation and construction of asexual buds of the freshwater spongeRadiospongilla cerebellata (Porifera,Spongillidae)

Summary The buds ofRadiospongilla cerebellata are formed asexually. Budding can be induced experimentally by injuring the sponge. The first sign of budding is a slight elevation of some surface areas, which proceed to rise rapidly so that they soon protrude conspicuously from the surface of the sponge. As a bud develops, the broad base joining it to the mother sponge narrows to a stalk, which finally breaks. The free buds drift in the water for 15–20 min and then settle, forming new sessile sponges. The buds, 1.5–2.5 mm in diameter, have an internal organization identical with that of the mother sponge. They are enclosed in a layer of pinacoderm perforated by dermal pores. Under the pinacorderm there is a shallow subdermal space, which is in communication with the incurrent canals leading to the choanocyte chambers. The water sucked into these chambers proceeds into the excurrent canal system and emerges from the sponge through the oscular tube. Spicules projecting radially from the bud bear apical tufts of microscleres. The skeletal spicules of the buds, like their choanocyte chambers, are smaller than those in the mother sponge. The chambers expand to their mature size by choanocyte mitosis. Buds and sponges are colored green by intracellular symbiotic algae of the genusChlorella.     

Die Aufnahme partikulärer Nahrung beiReniera sp. (Porifera)

The choanocyte chambers of the marine spongeReniera sp. protrude with their curved outer surface free into the incurrent canals. The water is sucked into the chambers by cavities between the choanocytes. Particles up to 1 µm in diameter may enter the chambers with the water current. These particles are trapped on the outer surface of the choanocyte collars and are ingested by the choanocytes and processes of the pinacocyte epithelium of the incurrent canal system, which project into the chambers. Bigger particles are retained in the incurrent canals mainly on the outer surface of the choanocyte chambers. They are ingested by pinacocytes of the canal wall and transported to cells of the mesenchyme. The present investigation shows the great importance of the pinacocyte epithelium of the incurrent canal system for suspension feeding inReniera sp.     

Microscopical aspects on symbiosis of Spongilla lacustris (Porifera,Spongillidae) and green algae

Summary When growing in the sunlight, some specimens of Spongilla lacustris are coloured green due to the presence of symbiotic unicellular chlorellae. The algae live inside most sponge cells. The chlorellae were extracted from green sponges, cultivated, added to algae-free sponges and fixed after different incubation times. In this way the uptake of the algae, their distribution and their final whereabouts in the mesenchymatic cells could be followed by in vivo microscopy, phase-contrast microscopy and electron microscopy. A few minutes after addition, the chlorellae can be found inside the choanocyte chambers. Here they are taken up by the cell bodies and collars of the choanocytes. Pinacocytes are also involved in the uptake. The distribution of algae results from a specific transmission from the donor cell to the receiver cell. The chlorellae are not released from their host vacuoles until they are extensively enclosed by the cell taking them up. Six hours after addition, all sponge cells contain algae except granulocytes, microscleroblasts, the pinacocytes of the peripheral rim region and those of the pinacoderm. The chlorellae are able to divide inside the sponge cells.Abbreviations StM Stereo-microscopical photograph - PhC Phase-contrast microscopical photograph - EM Electron microscopical photograph     

Hydrodynamics in early animal evolution

Choanoflagellates and sponges feed by filtering microscopic particles from water currents created by the flagella of microvillar collar complexes situated on the cell bodies of the solitary or colonial choanoflagellates and on the choanocytes in sponges. The filtering mechanism has been known for more than a century, but only recently has the filtering process been studied in detail and also modelled, so that a detailed picture of the water currents has been obtained. In the solitary and most of the colonial choanoflagellates, the water flows freely around the cells, but in some forms, the cells are arranged in an open meshwork through which the water can be pumped. In the sponges, the choanocytes are located in choanocyte chambers (or choanocyte areas) with separate incurrent and excurrent canals/pores located in a larger body, which enables a fixed pattern of water currents through the collar complexes. Previous theories for the origin of sponges show evolutionary stages with choanocyte chambers without any opening or with only one opening, which makes separation of incurrent and excurrent impossible, and such stages must have been unable to feed. Therefore a new theory is proposed, which shows a continuous evolutionary lineage in which all stages are able to feed by means of the collar complexes.     

Body structure of marine sponges

Summary Each choanocyte chamber of Petrosia ficiformis is formed by a slightly outpocked choanocyte epithelium and by a ring of three or four uniflagellated cone cells surrounding the apopyle. The apopyle opens into a small aphodus, which leads the water flow to larger excurrent canals. Pinacocytes of the incurrent canal system cover the basal surface of the choanocytes and separate them from the incurrent canals and the mesenchyme. The water flows into the chambers by pores in the pinacocyte cover and then through gaps between adjacent choanocytes. To our knowledge this is the first report of a leuconoid canal system in which choanocyte chambers are covered by a pinacocyte epithelium of the incurrent canal system that isolates the chambers from the mesenchyme. A future comprehensive revision of the types of canal systems in sponges seems to be necessary. Permanent affiliation: Department of Biology and Health Sciences, University of Hartford, West Hartford, CT 06117, USA     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Cell Turnover and Detritus Production in Marine Sponges from Tropical and Temperate Benthic Ecosystems

This study describes in vivo cell turnover (the balance between cell proliferation and cell loss) in eight marine sponge species from tropical coral reef, mangrove and temperate Mediterranean reef ecosystems. Cell proliferation was determined through the incorporation of 5-bromo-2′-deoxyuridine (BrdU) and measuring the percentage of BrdU-positive cells after 6 h of continuous labeling (10 h for Chondrosia reniformis). Apoptosis was identified using an antibody against active caspase-3. Cell loss through shedding was studied quantitatively by collecting and weighing sponge-expelled detritus and qualitatively by light microscopy of sponge tissue and detritus. All species investigated displayed substantial cell proliferation, predominantly in the choanoderm, but also in the mesohyl. The majority of coral reef species (five) showed between 16.1±15.9% and 19.0±2.0% choanocyte proliferation (mean±SD) after 6 h and the Mediterranean species, C. reniformis, showed 16.6±3.2% after 10 h BrdU-labeling. Monanchora arbuscula showed lower choanocyte proliferation (8.1±3.7%), whereas the mangrove species Mycale microsigmatosa showed relatively higher levels of choanocyte proliferation (70.5±6.6%). Choanocyte proliferation in Haliclona vansoesti was variable (2.8–73.1%). Apoptosis was negligible and not the primary mechanism of cell loss involved in cell turnover. All species investigated produced significant amounts of detritus (2.5–18% detritus bodyweight⁻¹·d⁻¹) and cell shedding was observed in seven out of eight species. The amount of shed cells observed in histological sections may be related to differences in residence time of detritus within canals. Detritus production could not be directly linked to cell shedding due to the degraded nature of expelled cellular debris. We have demonstrated that under steady-state conditions, cell turnover through cell proliferation and cell shedding are common processes to maintain tissue homeostasis in a variety of sponge species from different ecosystems. Cell turnover is hypothesized to be the main underlying mechanism producing sponge-derived detritus, a major trophic resource transferred through sponges in benthic ecosystems, such as coral reefs.     

Spicule and flagellated chamber formation in a growth zone of Aphrocallistes vastus (Porifera,Hexactinellida)

Three species of glass sponges (Class Hexactinellida) form massive deep‐water reefs by growing on the skeletons of past generations, with new growth largely vertical and away from sediment that buries the lower portions. Growth is therefore essential for reef health, but how glass sponges produce new skeleton or tissue is not known. We used fluorescence, light, and electron microscopy to study skeletal and tissue growth in the reef‐forming glass sponge Aphrocallistes vastus. The sponge consists of a single large tube (the osculum), usually with several side branches, each of which can function as an effective excurrent vent. New tissue forms at the tips of each of these extensions, but how this occurs in a syncytial animal, and how the tubes expand laterally as the sponge gets larger, are both unknown. The fluorescent dye PDMPO labeled more spicule types in the tips of the sponge than elsewhere, indicating growth that was concentrated at the edge of the osculum. New tissue production was tracked using the thymidine analog EdU. EdU‐labeled nuclei were found predominantly at the edge or lip of the osculum. In that region new flagellated chambers were formed from clusters of choanoblasts that spread out around the enlarging chamber. In cellular sponges clusters of choanocytes form flagellated chambers through several rounds of mitotic divisions, and also by immigration of mesohyl cells, to expand the chamber to full size. By contrast, chambers in glass sponges expand as choanoblasts produce enucleate collar bodies to fill them out. Growing chambers with enucleate structures may be an adaptation to life in the deep sea if chambers with cells, and therefore more nuclei, are costly to build.     

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods

Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.     

Body structure of marine sponges

Summary Specimens of Haliclona elegans (Bowerbank, 1866) are covered by a thin, double layered dermal membrane extending over large subdermal spaces. The pores in the dermal membrane are formed by single porocytes with one or sometimes several pores in the center of the cell. The subjacent tissue shows a faintly developed mesenchyme and numerous big choanocyte chambers projecting into lacunar spaces of the incurrent canal system. The outer surface of the chambers is directly covered by the pinacocyte epithelium of the incurrent canal wall, which also separates them completely from the mesenchyme. Water influx into the chambers is guaranteed by prosopylar openings in the pinacocyte cover at the outer chamber surface. The chambers are connected to the excurrent canal system in the eurypylous way by wide apopyles, each of which is surrounded by a small ring of flagellated cone cells. About 15% of the choanocyte chambers in H. elegans contain central cells, which are thought to derive from migrating pinacocytes of the canal systems.     

Locomotion and contraction in an asconoid calcareous sponge

Various large‐scale behaviors (e.g., locomotion, shape changes, contractions) have been documented numerous times in intact sponges of the class Demospongiae. However, little is known about such motile events in calcareous sponges (Class Calcarea). Here, we report on whole‐sponge behaviors of the calcareous asconoid sponge Leucosolenia botryoides, as revealed by time‐lapse videos. These behaviors included locomotion and contraction. Locomotion in these sponges appeared as an outward movement (25–130 μm h^?1) of the asconoid tubes away from the sponge's center; such translocations were always accompanied by extensive movements of protruding spicules, which appear to act as anchoring hooks for the sponge's translocations. This is the first report of whole‐sponge locomotion in the Calcarea. Contractile waves also were propagated in these sponges at speeds of 50–150 μm h^?1, and they involved systemic contraction, then re‐extension of the asconoid tubes. The observations suggest that, like the more complex demosponges, these simple calcareous sponges are capable of adaptive whole‐animal behaviors (changes in flow, shape, and location), which occur in response to environmental stimuli such as crawling intruders.     

Choanoflagellates, choanocytes, and animal multicellularity

Abstract. It is widely accepted that multicellular animals (metazoans) constitute a monophyletic unit, deriving from ancestral choanoflagellate‐like protists that gave rise to simple choanocyte‐bearing metazoans. However, a re‐assessment of molecular and histological evidence on choanoflagellates, sponge choanocytes, and other metazoan cells reveals that the status of choanocytes as a fundamental cell type in metazoan evolution is unrealistic. Rather, choanocytes are specialized cells that develop from non‐collared ciliated cells during sponge embryogenesis. Although choanocytes of adult sponges have no obvious homologue among metazoans, larval cells transdifferentiating into choanocytes at metamorphosis do have such homologues. The evidence reviewed here also indicates that sponge larvae are architecturally closer than adult sponges to the remaining metazoans. This may mean that the basic multicellular organismal architecture from which diploblasts evolved, that is, the putative planktonic archimetazoan, was more similar to a modern poriferan larva lacking choanocytes than to an adult sponge. Alternatively, it may mean that other metazoans evolved from a neotenous larva of ancient sponges. Indeed, the Porifera possess some features of intriguing evolutionary significance: (1) widespread occurrence of internal fertilization and a notable diversity of gastrulation modes, (2) dispersal through architecturally complex lecithotrophic larvae, in which an ephemeral archenteron (in dispherula larvae) and multiciliated and syncytial cells (in trichimella larvae) occur, (3) acquisition of direct development by some groups, and (4) replacement of choanocyte‐based filter‐feeding by carnivory in some sponges. Together, these features strongly suggest that the Porifera may have a longer and more complicated evolutionary history than traditionally assumed, and also that the simple anatomy of modern adult sponges may have resulted from a secondary simplification. This makes the idea of a neotenous evolution less likely than that of a larva‐like choanocyte‐lacking archimetazoan. From this perspective, the view that choanoflagellates may be simplified sponge‐derived metazoans, rather than protists, emerges as a viable alternative hypothesis. This idea neither conflicts with the available evidence nor can be disproved by it, and must be specifically re‐examined by further approaches combining morphological and molecular information. Interestingly, several microbial lin°Cages lacking choanocyte‐like morphology, such as Corallochytrea, Cristidiscoidea, Ministeriida, and Mesomycetozoea, have recently been placed at the boundary between fungi and animals, becoming a promising source of information in addition to the choanoflagellates in the search for the unicellular origin of animal multicellularity.     

Asexual and puzzling sexual reproduction of the Mediterranean sponge Haliclona fulva (Demospongiae): life cycle and cytological structures

Despite the common assumption that most Haplosclerida are viviparous sponges, this study of the reproductive cycle of Haliclona fulva demonstrates that this species is actually oviparous and gonochoric. Intriguingly, not a single male was recorded in 15 months of sampling. Oogenesis is synchronous, starting in late April and terminating in September. Asexual reproduction is represented by cyclic budding, which occurs from late November to early March. During the season of asexual reproduction, the reproductive effort represents from 0.21% to 1.49% of the parental tissue, with the highest values being recorded in winter. During the season of sexual reproduction, the female reproductive effort ranges 0.05–1.15%, with the highest effort appearing in early summer. However, no significant correlation between reproductive efforts and seawater temperature fluctuations could be detected. We describe the ultrastructural morphogenesis of the buds for the first time in this species. This process is asynchronous, with buds of variable size being attached to the maternal apical surface via a short stalk. Young buds lack any particular anatomical organization, whereas bud maturity is characterized by the development of mesohyl and by the appearance of an increasing number and volume of lacunae in the central part of each bud. At this stage, buds harbor numerous small choanocyte chambers scattered throughout the inner region, and all cell types known from the mesohyl of parental sponges: microgranular cells, granular cells, archaeocytes, endopinacocytes and exopinacocytes, central cells, and sclerocytes.     

Viviparous development in the Antarctic sponge Stylocordyla borealis Loven, 1868

The complete larval development of the deep-sea sponge Stylocordyla borealis (from eggs to young sponges) was followed in sponges from the Antarctic waters of Terra Nova Bay. S. borealis shows a viviparous strategy which leads to young complete sponges incubated in the mother body, with cortex, spicules and choanocyte chambers. This development can be considered a K-strategy, which is usually employed by deep-sea organisms and cold-water benthic invertebrates.     

Evidence for the existence of microtubule protein in the extracellular space of marine sponges

The medulla region of sponges (= mesohyl) is only rarely dispersed with cells (approximately 25% of the total tissue volume). In vivo studies with a series of anti-cytoskeletal drugs revealed that taxol caused a significant contraction of intact specimens. Therefore, we investigated the possibility that microtubules are one component of the dynamic extracellular network in sponges, especially in the mesohyl. Using the sponge Geodia cydonium we found that most cells are not intracellulary stained in indirect immunofluorescence microscopy. However, all cells were brightly stained by the monoclonal antibody (mAb) at their plasma membranes. After incubating cryostat sections with a buffer, containing ATP, large (length up to 75 mum) aster- to filamentous-like structures became visible in the extracellular space of the mesohyl region. By three cycles of assembly and disassembly microtubules could be isolated from the extracellular material which were composed of alpha- and beta-tubulin (M(r): 55,000), several polypeptides within the M(r) range 55,000-82,000 (presumably tau proteins) and one protein of an M(r) higher than 100,000. Electron microscopical analysis revealed morphologically intact microtubules with typical side projections (very probably composed of microtubule-associated protein). Some aspects of the possible involvement of microtubules in the extracellularly localized displacement and motion network are discussed.     

First observations on egg release in the oviparous sponge Chondrilla nucula (Demospongiae, Chondrosida, Chondrillidae) in the Mediterranean Sea

Abstract. The reproduction of the demosponge Chondrilla nucula in Portofino (Ligurian Sea, Italy) was studied during August 2001. Eighteen individuals were sampled and examined with light microscopy for the presence of gametes, and 5 individuals carrying oocytes were found. In addition to microscopic observations, reproductive individuals could be easily identified as female even at the macroscopic level because of the presence of a grayish layer in the mesohyl where oocytes were concentrated. Oogenesis resulted in modifications of the external sponge morphology and of the aquiferous system. Approximately one‐third of the sponge body was filled with oocytes with the consequent disappearance of choanocyte chambers in the reproductive portion of the sponge. Under laboratory conditions, we obtained fertilized eggs from females and observed the first stages of embryonic development. Our observations suggest that fertilization in specimens of Chondrilla nucula occurs internally and not in the water. During the 2 years following these observations, no reproductive specimens were found among the same population during the reported reproductive period.