The Effect of Depth on the Morphology, Bacterial Clearance, and Respiration of the Mediterranean Sponge Chondrosia reniformis (Nardo, 1847).

To support the successful application of sponges for water purification and collagen production, we evaluated the effect of depth on sponge morphology, growth, physiology, and functioning. Specimens of Eastern Mediterranean populations of the sponge Chondrosia reniformis (Nardo, 1847) (Demospongiae, Chondrosiida, Chondrosiidae) were reciprocally transplanted between 5 and 20 m depth within the Kas-Kekova Marine Reserve Area. Control sponges at 5 m had fewer but larger oscula than their conspecifics at 20 m, and a significant inverse relationship between the osculum density and size was found in C. reniformis specimens growing along a natural depth gradient. Sponges transplanted from 20 to 5 m altered their morphology to match the 5 m control sponges, producing fewer but larger oscula, whereas explants transplanted from 5 to 20 m did not show a reciprocal morphological plasticity. Despite the changes in morphology, the clearance, respiration, and growth rates were comparable among all the experimental groups. This indicates that depth-induced morphological changes do not affect the overall performance of the sponges. Hence, the potential for the growth and bioremediation of C. reniformis in mariculture is not likely to change with varying culture depth. The collagen content, however, was higher in shallow water C. reniformis compared to deeper-growing sponges, which requires further study to optimize collagen production.

Adding insult to injury: Effects of chronic oxybenzone exposure and elevated temperature on two reef-building corals.

Coral bleaching due to global warming currently is the largest threat to coral reefs, which may be exacerbated by altered water quality. Elevated levels of the UV filter oxybenzone in coastal waters as a result of sunscreen use have recently been demonstrated. We studied the effect of chronic oxybenzone exposure and elevated water temperature on coral health. Microcolonies of Stylophora pistillata and Acropora tenuis were cultured in 20 flow-through aquaria, of which 10 were exposed to oxybenzone at a field-relevant concentration of ~0.06 µg L-1 at 26 °C. After two weeks, half of the corals experienced a heat wave culminating at 33 °C. All S. pistillata colonies survived the heat wave, although heat reduced growth and zooxanthellae density, irrespective of oxybenzone. Acropora tenuis survival decreased to 0% at 32 °C, and oxybenzone accelerated mortality. Oxybenzone and heat significantly impacted photosynthetic yield in both species, causing a 5% and 22-33% decrease, respectively. In addition, combined oxybenzone and temperature stress altered the abundance of five bacterial families in the microbiome of S. pistillata. Our results suggest that oxybenzone adds insult to injury by further weakening corals in the face of global warming.

Development of an Integrated Mariculture for the Collagen-Rich Sponge Chondrosia reniformis.

In this study, novel methods were tested to culture the collagen-rich sponge Chondrosia reniformis Nardo, 1847 (Demospongiae, Chondrosiida, Chondrosiidae) in the proximity of floating fish cages. In a trial series, survival and growth of cultured explants were monitored near a polluted fish farm and a pristine control site. Attachment methods, plate materials, and plate orientation were compared. In a first trial, chicken wire-covered polyvinyl chloride (PVC) was found to be the most suitable substrate for C. reniformis (100% survival). During a second trial, survival on chicken wire-covered PVC, after six months, was 79% and 63% for polluted and pristine environments, respectively. Net growth was obtained only on culture plates that were oriented away from direct sunlight (39% increase in six months), whereas sponges decreased in size when sun-exposed. Chicken wire caused pressure on explants and it resulted in unwanted epibiont growth and was therefore considered to be unsuitable for long-term culture. In a final trial, sponges were glued to PVC plates and cultured for 13 months oriented away from direct sunlight. Both survival and growth were higher at the polluted site (86% survival and 170% growth) than at the pristine site (39% survival and 79% growth). These results represent a first successful step towards production of sponge collagen in integrated aquacultures.

Light induced intraspecific variability in response to thermal stress in the hard coral Stylophora pistillata.

Recent research suggests that prior exposure of several months to elevated irradiance induces enhanced thermal tolerance in scleractinian corals. While this tolerance has been reported at the species level, individual coral colonies may react differently due to individual variability in thermal tolerance. As thermal anomalies are predicted to become common in the upcoming future, intraspecific variation may be key to the survival of coral populations. In order to study light-history based thermal stress responses on individual colonies, we developed a preliminary microcosm experiment where three randomly chosen, aquacultured colonies of the model coral Stylophora pistillata were exposed to two irradiance treatments (200 and 400 µmol photons m-2 s-1) for 31 days, followed by artificially induced heat stress (∼33.4 °C). We found different responses to occur at both the intraspecific and the intracolonial levels, as indicated by either equal, less severe, delayed, and/or even non-necrotic responses of corals previously exposed to the irradiance of 400 compared to 200 µmol photons m-2 s-1. In addition, all individual colonies revealed light-enhanced calcification. Finally, elevated irradiance resulted in a lower chlorophyll a concentration in one colony compared to the control treatment, and the same colony displayed more rapid bleaching compared to the other ones. Taken together, this study highlights the potential importance of intra-individual variability in physiological responses of scleractinian corals and provides recommendations for improving methodological designs for future studies.

Interactive effects of oxygen, carbon dioxide and flow on photosynthesis and respiration in the scleractinian coral Galaxea fascicularis.

Rates of dark respiration and net photosynthesis were measured for six replicate clonal fragments of the stony coral Galaxea fascicularis (Linnaeus 1767), which were incubated under 12 different combinations of dissolved oxygen (20%, 100% and 150% saturation), dissolved carbon dioxide (9.5 and 19.1 µmol l-1) and water flow (1-1.6 versus 4-13 cm s-1) in a repeated measures design. Dark respiration was enhanced by increased flow and increased oxygen saturation in an interactive way, which relates to improved oxygen influx into the coral tissue. Oxygen saturation did not influence net photosynthesis: neither hypoxia nor hyperoxia affected net photosynthesis, irrespective of flow and pH, which suggests that hyperoxia does not induce high rates of photorespiration in this coral. Flow and pH had a synergistic effect on net photosynthesis: at high flow, a decrease in pH stimulated net photosynthesis by 14%. These results indicate that for this individual of G. fascicularis, increased uptake of carbon dioxide rather than increased efflux of oxygen explains the beneficial effect of water flow on photosynthesis. Rates of net photosynthesis measured in this study are among the highest ever recorded for scleractinian corals and confirm a strong scope for growth.

Evidence for water-mediated mechanisms in coral-algal interactions.

Although many coral reefs have shifted from coral-to-algal dominance, the consequence of such a transition for coral-algal interactions and their underlying mechanisms remain poorly understood. At the microscale, it is unclear how diffusive boundary layers (DBLs) and surface oxygen concentrations at the coral-algal interface vary with algal competitors and competitiveness. Using field observations and microsensor measurements in a flow chamber, we show that coral (massive Porites) interfaces with thick turf algae, macroalgae, and cyanobacteria, which are successful competitors against coral in the field, are characterized by a thick DBL and hypoxia at night. In contrast, coral interfaces with crustose coralline algae, conspecifics, and thin turf algae, which are poorer competitors, have a thin DBL and low hypoxia at night. Furthermore, DBL thickness and hypoxia at the interface with turf decreased with increasing flow speed, but not when thick turf was upstream. Our results support the importance of water-mediated transport mechanisms in coral-algal interactions. Shifts towards algal dominance, particularly dense assemblages, may lead to thicker DBLs, higher hypoxia, and higher concentrations of harmful metabolites and pathogens along coral borders, which in turn may facilitate algal overgrowth of live corals. These effects may be mediated by flow speed and orientation.

Cell kinetics during regeneration in the sponge Halisarca caerulea: how local is the response to tissue damage?

Sponges have a remarkable capacity to rapidly regenerate in response to wound infliction. In addition, sponges rapidly renew their filter systems (choanocytes) to maintain a healthy population of cells. This study describes the cell kinetics of choanocytes in the encrusting reef sponge Halisarca caerulea during early regeneration (0-8 h) following experimental wound infliction. Subsequently, we investigated the spatial relationship between regeneration and cell proliferation over a six-day period directly adjacent to the wound, 1 cm, and 3 cm from the wound. Cell proliferation was determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h). The percentage of proliferative choanocytes increased over time in all areas and after six days of regeneration choanocyte proliferation rates were comparable to steady-state tissue. Tissue areas farther from the wound had higher rates of choanocyte proliferation than areas closer to the wound, indicating that more resources are demanded from tissue in the immediate vicinity of the wound. There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges. Our data suggest that the production of collagen-rich wound tissue is a key process in tissue regeneration for H. caerulea, and helps to rapidly occupy the bare substratum exposed by the wound. Regeneration and choanocyte renewal are competing and negatively correlated life-history traits, both essential to the survival of sponges. The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

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.

Marine microorganism-invertebrate assemblages: perspectives to solve the "supply problem" in the initial steps of drug discovery.

The chemical diversity associated with marine natural products (MNP) is unanimously acknowledged as the "blue gold" in the urgent quest for new drugs. Consequently, a significant increase in the discovery of MNP published in the literature has been observed in the past decades, particularly from marine invertebrates. However, it remains unclear whether target metabolites originate from the marine invertebrates themselves or from their microbial symbionts. This issue underlines critical challenges associated with the lack of biomass required to supply the early stages of the drug discovery pipeline. The present review discusses potential solutions for such challenges, with particular emphasis on innovative approaches to culture invertebrate holobionts (microorganism-invertebrate assemblages) through in toto aquaculture, together with methods for the discovery and initial production of bioactive compounds from these microbial symbionts.

Coral calcification under daily oxygen saturation and pH dynamics reveals the important role of oxygen.

Coral reefs are essential to many nations, and are currently in global decline. Although climate models predict decreases in seawater pH (∼0.3 units) and oxygen saturation (∼5 percentage points), these are exceeded by the current daily pH and oxygen fluctuations on many reefs (pH 7.8-8.7 and 27-241% O2 saturation). We investigated the effect of oxygen and pH fluctuations on coral calcification in the laboratory using the model species Acropora millepora. Light calcification rates were greatly enhanced (+178%) by increased seawater pH, but only at normoxia; hyperoxia completely negated this positive effect. Dark calcification rates were significantly inhibited (51-75%) at hypoxia, whereas pH had no effect. Our preliminary results suggest that within the current oxygen and pH range, oxygen has substantial control over coral growth, whereas the role of pH is limited. This has implications for reef formation in this era of rapid climate change, which is accompanied by a decrease in seawater oxygen saturation owing to higher water temperatures and coastal eutrophication.

Red light represses the photophysiology of the scleractinian coral Stylophora pistillata.

Light spectrum plays a key role in the biology of symbiotic corals, with blue light resulting in higher coral growth, zooxanthellae density, chlorophyll a content and photosynthesis rates as compared to red light. However, it is still unclear whether these physiological processes are blue-enhanced or red-repressed. This study investigated the individual and combined effects of blue and red light on the health, zooxanthellae density, photophysiology and colouration of the scleractinian coral Stylophora pistillata over 6 weeks. Coral fragments were exposed to blue, red, and combined 50/50% blue red light, at two irradiance levels (128 and 256 µmol m(-2) s(-1)). Light spectrum affected the health/survival, zooxanthellae density, and NDVI (a proxy for chlorophyll a content) of S. pistillata. Blue light resulted in highest survival rates, whereas red light resulted in low survival at 256 µmol m(-2) s(-1). Blue light also resulted in higher zooxanthellae densities compared to red light at 256 µmol m(-2) s(-1), and a higher NDVI compared to red and combined blue red light. Overall, our results suggest that red light negatively affects the health, survival, symbiont density and NDVI of S. pistillata, with a dominance of red over blue light for NDVI.

Surviving in a marine desert: the sponge loop retains resources within coral reefs.

Ever since Darwin's early descriptions of coral reefs, scientists have debated how one of the world's most productive and diverse ecosystems can thrive in the marine equivalent of a desert. It is an enigma how the flux of dissolved organic matter (DOM), the largest resource produced on reefs, is transferred to higher trophic levels. Here we show that sponges make DOM available to fauna by rapidly expelling filter cells as detritus that is subsequently consumed by reef fauna. This "sponge loop" was confirmed in aquarium and in situ food web experiments, using (13)C- and (15)N-enriched DOM. The DOM-sponge-fauna pathway explains why biological hot spots such as coral reefs persist in oligotrophic seas--the reef's paradox--and has implications for reef ecosystem functioning and conservation strategies.

Coral aquaculture to support drug discovery.

Marine natural products (NP) are unanimously acknowledged as the 'blue gold' in the urgent quest for new pharmaceuticals. Although corals are among the marine organisms with the greatest diversity of secondary metabolites, growing evidence suggest that their symbiotic bacteria produce most of these bioactive metabolites. The ex hospite culture of coral symbiotic microbiota is extremely challenging and only limited examples of successful culture exist today. By contrast, in toto aquaculture of corals is a commonly applied technology to produce corals for aquaria. Here, we suggest that coral aquaculture could as well be a viable and economically feasible option to produce the biomass required to execute the first steps of the NP-based drug discovery pipeline.

Principles of biofouling protection in marine sponges: a model for the design of novel biomimetic and bio-inspired coatings in the marine environment?

The process of biofouling of marine structures and substrates, such as platforms or ship hulls, proceeds in multiple steps. Soon after the formation of an initial conditioning film, formed via the adsorption of organic particles to natural or man-made substrates, a population of different bacterial taxa associates under the formation of a biofilm. These microorganisms communicate through a complex quorum sensing network. Macro-foulers, e.g., barnacles, then settle and form a fouling layer on the marine surfaces, a process that globally has severe impacts both on the economy and on the environment. Since the ban of tributyltin, an efficient replacement of this antifouling compound by next-generation antifouling coatings that are environmentally more acceptable and also showing longer half-lives has not yet been developed. The sponges, as sessile filter-feeder animals, have evolved antifouling strategies to protect themselves against micro- and subsequent macro-biofouling processes. Experimental data are summarized and suggest that coating of the sponge surface with bio-silica contributes to the inhibition of the formation of a conditioning film. A direct adsorption of the surfaces by microorganisms can be impaired through poisoning the organisms with direct-acting secondary metabolites or toxic peptides. In addition, first, compounds from sponges have been identified that interfere with the anti-quorum sensing network. Sponge secondary metabolites acting selectively on diatom colonization have not yet been identified. Finally, it is outlined that direct-acting secondary metabolites inhibiting the growth of macro-fouling animals and those that poison the multidrug resistance pump are available. It is concluded that rational screening programs for inhibitors of the complex and dynamic problem of biofilm production, based on multidisciplinary studies and using sponges as a model, are required in the future.

Epizoic acoelomorph flatworms impair zooplankton feeding by the scleractinian coral Galaxea fascicularis.

Many scleractinian coral species host epizoic acoelomorph flatworms, both in aquaculture and in situ. These symbiotic flatworms may impair coral growth and health through light-shading, mucus removal and disruption of heterotrophic feeding. To quantify the effect of epizoic flatworms on zooplankton feeding, we conducted video analyses of single polyps of Galaxea fascicularis (Linnaeus 1767) grazing on Artemia nauplii in the presence and absence of symbiotic flatworms. 18S DNA analysis revealed that flatworms inhabiting G. fascicularis belonged to the genus Waminoa (Convolutidae), which were hosted at a density of 3.6±0.4 individuals polyp(-1). Polyps hosting flatworms exhibited prey capture rates of 2.2±2.5, 3.4±4.5 and 2.7±3.4 nauplii polyp(-1) 30 min(-1) at prey concentrations of 250, 500 and 1,000 nauplii L(-1), respectively. Polyps that had their flatworms removed displayed prey capture rates of 2.7±1.6, 4.8±4.1 and 16.9±10.3 nauplii polyp(-1) 30 min(-1). Significant main and interactive effects of flatworm presence and ambient prey concentration were found, reflected by the fact that flatworms significantly impaired host feeding rates at the highest prey density of 1,000 nauplii L(-1). In addition, flatworms displayed kleptoparasitism, removing between 0.1±0.3 and 0.6±1.1 nauplii 30 min(-1) from the oral disc of their host, or 5.3±3.3 to 50.0±2.1% of prey acquired by the coral. We suggest classifying the coral-associated Waminoa sp. as an epizoic parasite, as its presence may negatively affect growth and health of the host.

Cultivation of sponges, sponge cells and symbionts: achievements and future prospects.

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential. Since biological production is one option to supply materials for early drug development, the main challenge is to establish generic techniques for small-scale production of marine organisms. We analysed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts. To date, cultivation of whole sponges has been most successful in situ; however, optimal conditions are species specific. The establishment of sponge cell lines has been limited by the inability to obtain an axenic inoculum as well as the lack of knowledge on nutritional requirements in vitro. Approaches to overcome these bottlenecks, including transformation of sponge cells and using media based on yolk, are elaborated. Although a number of bioactive metabolite-producing microorganisms have been isolated from sponges, and it has been suggested that the source of most sponge-derived bioactive compounds is microbial symbionts, cultivation of sponge-specific microorganisms has had limited success. The current genomics revolution provides novel approaches to cultivate these microorganisms.

Oxygen and heterotrophy affect calcification of the scleractinian coral Galaxea fascicularis.

Heterotrophy is known to stimulate calcification of scleractinian corals, possibly through enhanced organic matrix synthesis and photosynthesis, and increased supply of metabolic DIC. In contrast to the positive long-term effects of heterotrophy, inhibition of calcification has been observed during feeding, which may be explained by a temporal oxygen limitation in coral tissue. To test this hypothesis, we measured the short-term effects of zooplankton feeding on light and dark calcification rates of the scleractinian coral Galaxea fascicularis (n=4) at oxygen saturation levels ranging from 13 to 280%. Significant main and interactive effects of oxygen, heterotrophy and light on calcification rates were found (three-way factorial repeated measures ANOVA, p<0.05). Light and dark calcification rates of unfed corals were severely affected by hypoxia and hyperoxia, with optimal rates at 110% saturation. Light calcification rates of fed corals exhibited a similar trend, with highest rates at 150% saturation. In contrast, dark calcification rates of fed corals were close to zero under all oxygen saturations. We conclude that oxygen exerts a strong control over light and dark calcification rates of corals, and propose that in situ calcification rates are highly dynamic. Nevertheless, the inhibitory effect of heterotrophy on dark calcification appears to be oxygen-independent. We hypothesize that dark calcification is impaired during zooplankton feeding by a temporal decrease of the pH and aragonite saturation state of the calcifying medium, caused by increased respiration rates. This may invoke a transient reallocation of metabolic energy to soft tissue growth and organic matrix synthesis. These insights enhance our understanding of how oxygen and heterotrophy affect coral calcification, both in situ as well as in aquaculture.

Extracoelenteric zooplankton feeding is a key mechanism of nutrient acquisition for the scleractinian coral Galaxea fascicularis.

Internal and external feeding on zooplankton may provide scleractinian corals with important nutrients. However, the latter process has never been properly quantified. To quantify the dynamics of zooplankton capture, digestion and release for a scleractinian coral, we performed detailed video analyses of Galaxea fascicularis feeding on Artemia nauplii. A highly dynamic process of prey capture, digestion and release was observed. A single G. fascicularis polyp (N=3) captured 558±67 and released 383±75 Artemia nauplii over a 6 h interval. On average, 98.6% of prey captured was not ingested. Instead, prey items were clustered into aggregates that were digested externally by mesenterial filaments. In addition, we employed carbon, nitrogen and phosphorus analysis of zooplankton before and after digestion by G. fascicularis colonies (N=6). For total organic carbon, 43.1% (0.298±0.148 µg Artemia(-1)) was lost after 6 h of digestion. For total organic nitrogen, total organic phosphorus and orthophosphate (PO(4)(3-)), these values were 51.3% (0.059±0.028 µg Artemia(-1)), 50.9% (0.009±0.004 µg Artemia(-1)) and 84.6% (0.0019±0.0008 µg Artemia(-1)), respectively. For extracoelenteric zooplankton feeding alone, total estimated nutrient inputs for G. fascicularis colonies were 76.5±0.0 µg organic carbon, 15.2±0.0 µg organic nitrogen, 2.3±0.2 µg organic phosphorus and 0.5±0.8 µg inorganic phosphorus per cm(2) coral tissue per day. These values exceed calculations based on intracoelenteric feeding by up to two orders of magnitude. Our results demonstrate that extracoelenteric zooplankton feeding is a key mechanism of nutrient acquisition for a scleractinian coral. These results are of importance to coral aquaculture and our understanding of benthic-pelagic coupling on coral reefs.

The biology and economics of coral growth.

To protect natural coral reefs, it is of utmost importance to understand how the growth of the main reef-building organisms-the zooxanthellate scleractinian corals-is controlled. Understanding coral growth is also relevant for coral aquaculture, which is a rapidly developing business. This review paper provides a comprehensive overview of factors that can influence the growth of zooxanthellate scleractinian corals, with particular emphasis on interactions between these factors. Furthermore, the kinetic principles underlying coral growth are discussed. The reviewed information is put into an economic perspective by making an estimation of the costs of coral aquaculture.

Hypothesized kinetic models for describing the growth of globular and encrusting demosponges.

The marine sponges Dysidea avara and Chondrosia reniformis (globular forms) were cultured in the laboratory on a diet of viable Phaeodactylum tricornutum cells and dissolved nutrients (algae and fish powders). Our growth data were combined with literature data for Pseudosuberites andrewsi (a globular sponge) and for the encrusting sponges Oscarella lobularis, Hemimycale columella, and Crambe crambe. The suitability of three growth models-linear, exponential, and radial accretive-for describing the growth of globular and encrusting sponges was assessed. Radial accretive growth was determined to be the best model to describe growth of both encrusting and globular sponges. Average growth rates of 0.051+/-0.016 and 0.019+/-0.003 mm/day (calculated as the increase of the radius of the sponge per day) were obtained experimentally for D. avara and C. reniformis, respectively.