Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes

Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells-a process seen during the metamorphosis of other ciliated invertebrate larvae-not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians.

Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity

Demosponges are considered part of the most basal evolutionary lineage in the animal kingdom. Although the sponge body plan fundamentally differs from that of other metazoans, their development includes many of the hallmarks of bilaterian and eumetazoan embryogenesis, namely fertilization followed by a period of cell division yielding distinct cell populations, which through a gastrulation-like process become allocated into different cell layers and patterned within these layers. These observations suggest that the last common ancestor (LCA) to all living animals was developmentally more sophisticated than is widely appreciated and used asymmetric cell division and morphogen gradients to establish localized populations of specified cells within the embryo. Here we demonstrate that members of a range of transcription factor gene classes, many of which appear to be metazoan-specific, are expressed during the development of the demosponge Reniera, including ANTP, Pax, POU, LIM-HD, Sox, nuclear receptor, Fox (forkhead), T-box, Mef2, and Ets genes. Phylogenetic analysis of these genes suggests that not only the origin but the diversification of some of the major developmental metazoan transcription factor classes took place before sponges diverged from the rest of the Metazoa. Their expression during demosponge development suggests that, as in today's sophisticated metazoans, these genes may have functioned in the regulatory network of the metazoan LCA to control cell specification and regionalized gene expression during embryogenesis.

Cytological basis of photoresponsive behavior in a sponge larva

Ontogenetic changes in the photoresponse of larvae from the demosponge Reneira sp. were studied by analyzing the swimming paths of individual larvae exposed to diffuse white light. Larvae swam upward upon release from the adult, but were negatively phototactic until at least 12 hours after release. The larval photoreceptors are presumed to be a posterior ring of columnar monociliated epithelial cells that possess 120-mum-long cilia and pigment-filled protrusions. A sudden increase in light intensity caused these cilia to become rigidly straight. If the light intensity remained high, the cilia gradually bent over the pigmented vesicles in the adjacent cytoplasm, and thus covered one entire pole of the larva. The response was reversed upon a sudden decrease in light intensity. The ciliated cells were sensitive to changes in light intensity in larvae of all ages. This response is similar to the shadow response in tunicate larvae or the shading of the photoreceptor in Euglena and is postulated to allow the larvae to steer away from brighter light to darker areas, such as under coral rubble-the preferred site of the adult sponge on the reef flat. In the absence of a coordinating system in cellular sponges, the spatial organization and autonomous behavior of the pigmented posterior cells control the rapid responses to light shown by these larvae.

Identifizierung eines adaptiven antibakteriellen Abwehrmechanismus des marinen Schwammes Suberites domuncula

Schwämme (Porifera) sind die phylogenetisch ältesten Metazoa. Sie besitzen komplexe Abwehrsysteme, welche vor allem auf der Synthese bioaktiver niedermolekularer Sekundärmetaboliten beruhen und diese Tiere zu einer der reichhaltigsten Quellen für medizinisch nutzbare Wirkstoffe machen. Besonders der marine Einsiedler-Korkschwamm (Suberites domuncula) hat sich in den letzten Jahren zur Untersuchung der molekularen Zusammenhänge dieser Abwehrmechanismen als besonders geeignet herausgestellt. So wurden in diesem Schwamm beispielsweise zwei lyso-PAF (plättchenaktivierender Faktor) Derivate (1-O Hexadecyl-sn-glycero-3-phosphatidylcholin und 1-O-Octadecyl-sn-glycero-3-phosphatidylcholin) identifiziert und charakterisiert, sowie deren ausgeprägte antibakterielle Aktivität besonders gegenüber gramnegativen Bakterien demonstriert. Eine Behandlung mit der Modellsubstanz zur Simulation einer bakteriellen Infektion, dem Endotoxin Lipopolysaccharid (LPS), für insgesamt 72 Stunden resultierte in einem Anstieg der Expressionslevel zweier an der Biosynthese dieser bioaktiven Etherphospholipide beteiligten Proteine. Unter Anwendung der Methode des Differential Display konnte einerseits das Schlüsselenzym der Etherphospholipid Biosynthese Alkyl- Dihydroxyacetonphosphat (DHAP)-Synthase und andererseits die regulatorische Untereinheit der PAF-deacetylierenden PAF Acetylhydrolase I

Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH-terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).

Counts of benthos communities in Kongsfjorden in 2009

In order to reveal the structure of the sparsely known deeper sublittoral hard bottom communities of glacial Kongsfjorden, the macroepibenthos from six depth zones (30-200 m) was analysed. A total of 180 still images derived from 6-h video recorded at the Kongsfjordneset remotely operated vehicle station were assessed quantitatively. Overall 27 mainly suspension-feeding species/taxa were observed. Of these, two-thirds have an arcto-boreal distribution, while the remainder are cosmopolitan. The overall mean epibenthos abundance was 33 ind./m**2 with maximum values at 150 m depth (97.9 ind./m**2). The majority of the taxa inhabited the entire depth range. Encrusting red algae, an unidentified sponge and the sea anemone Urticina eques, characterized the assemblage of the shallow zone. The sea anemones Hormathia spp. were important below 30 m, the Serpulid polychaete Protula tubularia was characteristic for the community below 50 m and the demosponge Haliclona sp. was a key taxon between 100 and 200 m depth. Cluster analysis and non-metrical multidimensional scaling based on abundance data showed differences between the assemblages along the bathymetric gradient, but only in the shallower depths in relation to the substratum surface incline. As surface and tidal current impacts attenuate with increasing depth, there is a gradual trend from robust key species towards more fragile ones (i.e. P. tubularia), in line with the 'Physical control hypothesis'.

Distribution of sponge spicule types, Goniothecium odontella and Diploneis cf. bomboides in selected spicule clusters from ODP Leg 112 holes (Table 1)

Clusters of sponge spicules found in Quaternary deep-water sediments at Sites 685 and 688 off Peru represent single individuals of small sponges or fragments of larger sponges. The spicule assemblages constituting these clusters probably represent a few demosponge species of the subclass Tetractinomorpha and order Astrophorida, because triaenes and microscleric euasters, as well as abundant monaxons, are present. As proved by incorporated Neogene diatoms, these spicule clusters are allochthonous. The sponge individuals probably inhabited deeper neritic environments during late Neogene time.

Effects of ocean acidification on sponge communities

The effects of ocean acidification on lower invertebrates such as sponges may be pronounced because of their low capacity for acid-base regulation. However, so far, most studies have focused on calcifiers. We present the first study of the effects of ocean acidification on the Porifera. Sponge species composition and cover along pH gradients at CO2 vents off Ischia (Tyrrhenian Sea, Italy) was measured at sites with normal pH (8.1-8.2), lowered pH (mean 7.8-7.9, min 7.4-7.5) and extremely low pH (6.6). There was a strong correlation between pH and both sponge cover and species composition. Crambe crambe was the only species present in any abundance in the areas with mean pH 6.6, seven species were present at mean pH 7.8-7.9 and four species (Phorbas tenacior, Petrosia ficiformis, Chondrilla nucula and Hemimycale columella) were restricted to sites with normal pH. Sponge percentage cover decreased significantly from normal to acidified sites. No significant effect of increasing CO2 levels and decreasing pH was found on spicule form in Crambe crambe. This study indicates that increasing CO2 concentrations will likely affect sponge community composition as some demosponge species appear to be more vulnerable than others. Further research into the mechanisms by which acidification affects sponges would be useful in predicting likely effects on sessile marine communities.

Sea-floor videos and photographs (benthos) along 6 shelf profiles from the eastern Weddell Sea, Antarctica taken with remote operated vehicel CHEROKEE during Polarstern cruise ANT-XXI/2

The ROV operations had three objectives: (1) to check, whether the "Cherokee" system is suited for advanced benthological work in the high latitude Antarctic shelf areas; (2) to support the disturbance experiment, providing immediate visual Information; (3) to continue ecological work that started in 1989 at the hilltop situated at the northern margin of the Norsel Bank off the 4-Seasons Inlet (Weddell Sea). The "Cherokee" is was equipped with 3 video cameras, 2 of which support the operation. A high resolution Tritech Typhoon camera is used for scientific observations to be recorded. In addition, the ROV has a manipulator, a still camera, lights and strobe, compass, 2 lasers, a Posidonia transponder and an obstacle avoidance Sonar. The size of the vehicle is 160 X 90 X 90cm. In the present configuration without TMS (tether management system) the deployment has to start with paying out the full cable length, lay it in loops on deck and connect the glass fibres at the tether's spool winch. After a final technical check the vehicle is deployed into the water, actively driven perpendicular to the ship's axis and floatings are fixed to the tether. At a cable length of approx. 50 m, the tether is tightened to the depressor by several cable ties and both components are lowered towards the sea floor, the vehicle by the thruster's propulsion and the depressor by the ship's winch. At 5 m intervals the tether has to be tied to the single conductor cable. In good weather conditions the instruments supporting the navigation of the ROV, especially the Posidonia system, allow an operation mode to follow the ship's course if the ship's speed is slow. Together with the lasers which act as a scale in the images they also allow a reproducible scientific analysis since the transect can be plotted in a GIS system. Consequently, the area observed can be easily calculated. An operation as a predominantly drifting system, especially in areas with bottom near currents, is also possible, however, the connection of the tether at the rear of the vehicle is unsuitable for such conditions. The recovery of the system corresponds to that of the deployment. Most important is to reach the surface of the sea at a safe distance perpendicular to the ship's axis in order not to interfere with the ship's propellers. During this phase the Posidonia transponder system is of high relevance although it has to be switched off at a water depth of approx. 40 m. The minimum personal needed is 4 persons to handle the tether on deck, one person to operate the ship's winch, one pilot and one additional technician for the ROV's operation itself, one scientist, and one person on the ship's bridge in addition to one on deck for whale watching when the Posidonia system is in use. The time for the deployment of the ROV until it reaches the sea floor depends on the water depth and consequently on the length of the cable to be paid out beforehand and to be tightened to the single conductor cable. Deployment and recovery at intermediate water depths can last up to 2 hours each. A reasonable time for benthological observations close to the sea floor is 1 to 3 hours but can be extended if scientifically justified. Preliminary results: after a first test station, the ROV was deployed 3 times for observations related to the disturbance experiment. A first attempt to Cross the hilltop at the northern margin of the Norsel Bank close to the 4- Seasons Inlet was successful only for the first hundreds of metres transect length. The benthic community was dominated in biomass by the demosponge Cinachyra barbata. Due to the strong current of approx. 1 nm/h, the design of the system, and an expected more difficult current regime between grounded icebergs and the top of the hilltop the operation was stopped before the hilltop was reached. In a second attempt the hilltop was successfully crossed because the current and wind situation was much more suitable. In contrast to earlier expeditions with the "sprint" ROV it was the first time that both slopes, the smoother in the northeast and the steeper in the southwest were continuously observed during one cast. A coarse classification of the hilltop fauna shows patches dominated by single taxa: cnidarians, hydrozoans, holothurians, sea urchins and stalked sponges. Approximately 20 % of the north-eastern slope was devastated by grounding icebergs. Here the sediments consisted of large boulders, gravel or blocks of finer sediment looking like an irregularly ploughed field. On the Norsel Bank the Cinachyra concentrations were locally associated with high abundances of sea anemones. Total observation time amounted to 11.5 hours corresponding to almost 6-9 km transect length.

Sponge bioerosion accelerated by ocean acidification across species and latitudes?

In many marine biogeographic realms, bioeroding sponges dominate the internal bioerosion of calcareous substrates such as mollusc beds and coral reef framework. They biochemically dissolve part of the carbonate and liberate so-called sponge chips, a process that is expected to be facilitated and accelerated in a more acidic environment inherent to the present global change. The bioerosion capacity of the demosponge Cliona celata Grant, 1826 in subfossil oyster shells was assessed via alkalinity anomaly technique based on 4 days of experimental exposure to three different levels of carbon dioxide partial pressure (pCO2) at ambient temperature in the cold-temperate waters of Helgoland Island, North Sea. The rate of chemical bioerosion at present-day pCO2 was quantified with 0.08-0.1 kg/m**2/year. Chemical bioerosion was positively correlated with increasing pCO2, with rates more than doubling at carbon dioxide levels predicted for the end of the twenty-first century, clearly confirming that C. celata bioerosion can be expected to be enhanced with progressing ocean acidification (OA). Together with previously published experimental evidence, the present results suggest that OA accelerates sponge bioerosion (1) across latitudes and biogeographic areas, (2) independent of sponge growth form, and (3) for species with or without photosymbionts alike. A general increase in sponge bioerosion with advancing OA can be expected to have a significant impact on global carbonate (re)cycling and may result in widespread negative effects, e.g. on the stability of wild and farmed shellfish populations, as well as calcareous framework builders in tropical and cold-water coral reef ecosystems.

The origin of the pelagobenthic metazoan life cycle: what's sex got to do with it?

The biphasic (pelagobenthic) life cycle is found throughout the animal kingdom, and includes gametogenesis, embryogenesis, and metamorphosis. From a tangled web of hypotheses on the origin and evolution of the metazoan pelagobenthic life cycle, current opinion appears to favor a simple, larval-like holopelagic ancestor that independently settled multiple times to incorporate a benthic phase into the life cycle. This hypothesis derives originally from Haeckel's (1874) Gastraea theory of ontogeny recapitulating phylogeny, in which the gastrula is viewed as the recapitulation of a gastracan ancestor that evolved via selection on a simple, planktonic hollow ball of cells to develop the capacity to feed. Here, we propose an equally plausible hypothesis that the origin of the metazoan pelagobenthic life cycle was a direct consequence of sexual reproduction in a likely holobenthic ancestor. In doing so, we take into account new insights from poriferan development and from molecular phylogenies. In this scenario, the gastrula does not represent a recapitulation, but simply an embryological stage that is an outcome of sexual reproduction. The embryo can itself be considered as the precursor to a biphasic lifestyle, with the embryo representing one phase and the adult another phase. This hypothesis is more parsimonious because it precludes the need for multiple, independent origins of the benthic form. It is then reasonable to consider that multilayered, ciliated embryos ultimately released into the water column are subject to natural selection for dispersal/longevity/feeding that sets them on the evolutionary trajectory towards the crown metazoan planktonic larvae. These new insights from poriferan development thus clearly support the intercalation hypothesis of bilaterian larval evolution, which we now believe should be extended to discussions of the origin of biphasy in the metazoan last common ancestor.