Sea organisms as a source of collagen-derived polymers for skin tissue engineering applications

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin and they are usually accompanied by fibrotic reactions that result in the production of a scar. Natural biopolymers such as collagen have been a lot investigated as potential source of biomaterial for skin replacement in Tissue Engineering. Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role in connective tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches, as skin tissue engineering. In addition, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are skin and bone from bovine and porcine origin. However, these last carry high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy and immunogenic responses. On the other hand, the increase of jellyfish has led us to consider this marine organism as potential collagen source for tissue engineering applications. In the present study, novel form of acid and pepsin soluble collagen were extracted from dried Rhopilema hispidum jellyfish species in an effort to obtain an alternative and safer collagen. We studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using pepsin extraction method (34.16 mg collagen/g of tissue). The isolated collagen was characterized by SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy.

Investigations on the Chilean kelp crab Taliepus dentatus

Physiological responses of larval stages can differ from those of the adults, affecting key ecological processes. Therefore, developing a mechanistic understanding of larval responses to environmental conditions is essential vis-à-vis climate change. We studied the thermal tolerance windows, defined by lower and upper pejus (Tp) and critical temperatures (Tc), of zoea I, II, and megalopa stages of the Chilean kelp crab Taliepus dentatus. Tp limits determine the temperature range where aerobic scope is maximal and functioning of the organism is unrestrained and were estimated from direct observations of larval activity. Tc limits define the transition from aerobic to anaerobic metabolism, and were estimated from the relationship between standard metabolic rate and temperature. Zoea I showed the broadest, Zoea II an intermediate, and megalopae the narrowest tolerance window (Tp). Optimum performance in megalopae was limited to Tp between 11 and 15°C, while their Tc ranged between 7 and 19°C. Although Tc may be seldom encountered by larvae, the narrower Tp temperatures can frequently expose larvae to unfavorable conditions that can drastically constrain their performance. Temperatures beyond the Tp range of megalopae have been observed in most spring and summer months in central Chile, and can have important consequences for larval swimming performance and impair their ability to avoid predators or settle successfully. Besides the well-documented effects of temperature on development time, variability in field temperatures beyond Tp can affect performance of particular larval stages, which could drive large-scale variability in recruitment and population dynamics of T. dentatus and possibly other invertebrate species.

Phylogenetic relationships among hadal amphipods of the Superfamily Lysianassoidea : Implications for taxonomy and biogeography

Date of Acceptance: 30/8/15 Acknowledgements We thank the chief scientists, crew and company of the Japanese RV Hakuho-Maru (KH0703 and KH0803), the RV Tansei-Maru (KT-09-03), the RV Kairei (KR0716), the German FS Sonne (SO197 and SO 209) and the New Zealand RV Kaharoa (KAH0190, KAH1109, KAH1202, KAH1301 and KAH1310). This work was supported by the HADEEP projects, funded by the Nippon Foundation, Japan (2009765188), the Natural Environmental Research Council, UK (NE/E007171/1) and the Total Foundation, France. We acknowledge additional support from the Marine Alliance for Science and Technology for Scotland (MASTS) funded by the Scottish Funding Council (Ref: HR09011) and contributing institutions. We also acknowledge support from the Leverhulme Research Fellowship granted to SBP. Additional sea time was supported by NIWA’s ‘Impact of Resource Use on Vulnerable Deep-Sea Communities’ project (CO1_0906). From NIWA we thank Malcolm Clark, Ashley Rowden, Kareen Schnabel, Sadie Mills for logistical support at the NIWA Invertebrate Collection. We also thank Fredrik Søreide from Promare, USA, for supply of the Puerto-Rico samples, Marius Wenzel for helpful comments on manuscript drafts, and Dr. Tammy Horton (NOCS, UK) for identifying some of the earlier amphipod samples

Table 1: Station list with macrobenthos biomass results from the ocean floor

Among the Siberian shelf seas the Kara Sea is most strongly influenced by riverine runoff with nearly 1500 km fresh water discharge per year. This fresh water, discharged mainly by Ob and Yenisei, contains about 3.1 * 106 and 4.6 * 106 tons of total organic carbon per year, respectively (Gordeev et al. 1996). Little is known about the relevance of this organic material for biological communities, neither for the Kara Sea nor for the adjacent deep basins of the central Arctic Ocean. Aiming at elucidating the fate of fluvial matter transported from the rivers via estuaries into the central Arctic Ocean and the relative importance of marine organic matter being produced such information is crucial. Here we present calculations on the organic carbon demand of the Kara Sea macrozoobenthos based on measured biomass (total wet weight [ww] per 0.25 m ) from quantitative box corer samples and empirical relationships between biomass, annual production, annual respiration, and carbon remineralisation. This bottom-up approach may serve as a first estimate of the carbon remineralization potential of a given zoobenthos community (or area) as long as no data on in situ respiration rates are available. Our data basis comprises 54 stations sampled in summer seasons 1997, 1999 and 2000 in the Kara Sea at water depths between 10 and 68 m. The geographical area represented by stations analysed covers roughly 178 000 km**2, which is about one fifth of the total Kara Sea area. In this area, 290 species of invertebrate macrozoobenthos were identified with polychaeta, Crustacea, mollusca and echinodermata being the most abundant. For all stations analysed, mean biomass values ranged between 4.3 and 778.1 g ww/m**2 with organic carbon demands between 3.5 and 43.2 mg C/m**2/d. For the area of 178 000 km2 a preliminary total consumption of 1.4 * 10**6t Corg/y (equivalent to 21.5 mg C/m**2/d) was calculated for the macrozoobenthos. An extrapolation of our data would lead to an annual carbon demand of about 5-7 * 106 t for the whole Kara Sea macrozoobenthos (or 15.5-21.7 mg C/m2/d).

Seawater carbonate chemistry and morphometric coordinates and morphology of the control 8-arm pluteus of brittlestar Ophiothrix fragilis, 2008

The world's oceans are slowly becoming more acidic. In the last 150 yr, the pH of the oceans has dropped by ~0.1 units, which is equivalent to a 25% increase in acidity. Modelling predicts the pH of the oceans to fall by 0.2 to 0.4 units by the year 2100. These changes will have significant effects on marine organisms, especially those with calcareous skeletons such as echinoderms. Little is known about the possible long-term impact of predicted pH changes on marine invertebrate larval development. Here we predict the consequences of increased CO2 (corresponding to pH drops of 0.2 and 0.4 units) on the larval development of the brittlestar Ophiothrix fragilis, which is a keystone species occurring in high densities and stable populations throughout the shelf seas of northwestern Europe (eastern Atlantic). Acidification by 0.2 units induced 100% larval mortality within 8 d while control larvae showed 70% survival over the same period. Exposure to low pH also resulted in a temporal decrease in larval size as well as abnormal development and skeletogenesis (abnormalities, asymmetry, altered skeletal proportions). If oceans continue to acidify as expected, ecosystems of the Atlantic dominated by this keystone species will be seriously threatened with major changes in many key benthic and pelagic ecosystems. Thus, it may be useful to monitor O. fragilis populations and initiate conservation if needed.

Structural and functional vulnerability to elevated pCO2 in marine benthic communities

The effect of elevated pCO2/low pH on marine invertebrate benthic biodiversity, community structure and selected functional responses which underpin ecosystem services (such as community production and calcification) was tested in a medium-term (30 days) mesocosm experiment in June 2010. Standardised intertidal macrobenthic communities, collected (50.3567°N, 4.1277°W) using artificial substrate units (ASUs), were exposed to one of seven pH treatments (8.05, 7.8. 7.6, 7.4, 7.2, 6.8 and 6.0). Community net calcification/dissolution rates, as well as changes in biomass, community structure and diversity, were measured at the end of the experimental period. Communities showed significant changes in structure and reduced diversity in response to reduced pH: shifting from a community dominated by calcareous organisms to one dominated by non-calcareous organisms around either pH 7.2 (number of individuals and species) or pH 7.8 (biomass). These results were supported by a reduced total weight of CaCO3 structures in all major taxa at lowered pH and a switch from net calcification to net dissolution around pH 7.4 (Omega calc = 0.78, Omega ara = 0.5). Overall community soft tissue biomass did not change with pH and high mortality was observed only at pH 6.0, although molluscs and arthropods showed significant decreases in soft tissue. This study supports and refines previous findings on how elevated pCO2 can induce changes in marine biodiversity, underlined by differential vulnerability of different phyla. In addition, it shows significant elevated pCO2-/low pH-dependent changes in fundamental community functional responses underpinning changes in ecosystem services.

Ocean acidification induces budding in larval sea urchins

Ocean acidification (OA), the reduction of ocean pH due to hydration of atmospheric CO2, is known to affect growth and survival of marine invertebrate larvae. Survival and transport of vulnerable planktonic larval stages play important roles in determining population dynamics and community structures in coastal ecosystems. Here, we show that larvae of the purple urchin, Strongylocentrotus purpuratus, underwent high-frequency budding (release of blastula-like particles) when exposed to elevated pCO2 level (>700 µatm). Budding was observed in >50 % of the population and was synchronized over short periods of time (~24 h), suggesting this phenomenon may be previously overlooked. Although budding can be a mechanism through which larval echinoids asexually reproduce, here, the released buds did not develop into viable clones. OA-induced budding and the associated reduction in larval size suggest new hypotheses regarding physiological and ecological tradeoffs between short-term benefits (e.g. metabolic savings and predation escape) and long-term costs (e.g. tissue loss and delayed development) in the face of climate change.

Sea-bed photographs (benthos) from the AWI-Hausgarten area along OFOS profile PS62/191-1

Although the use of deep-sea imagery considerably increased during the last decades, reports on nekton falls to the deep seafloor are very scarce. Whereas there are a few reports describing the finding of whale carcasses in the deep north-eastern and south-eastern Pacific, descriptions of invertebrate or vertebrate food-falls at centimetre to metre scale are extremely rare. After 4 years of extensive work at a deep-sea long-term station in northern polar regions (AWI-"Hausgarten"), including large-scale visual observations with various camera systems covering some 10 000 m2 of seafloor at water depths between 1250 and 5600 m, this paper describes the first observation of a fish carcass at about 1280 m water depth, west off Svålbard. The fish skeleton had a total length of 36 cm and an approximated biomass of 0.5 kg wet weight. On the basis of in situ experiments, we estimated a very short residence time of this particular carcass of about 7 h at the bottom. The fast response of the motile deep-sea scavenger community to such events and the rapid utilisation of this kind of organic carbon supply might partly explain the extreme rarity of such an observation.

(Table S1) Carbon and oxygen isotope values, and Mn and Sr concentrations of Early Jurassic sediments

For much of the Mesozoic record there has been an inconclusive debate on the possible global significance of isotopic proxies for environmental change and of sequence stratigraphic depositional sequences. We present a carbon and oxygen isotope and elemental record for part of the Early Jurassic based on marine benthic and nektobenthic molluscs and brachiopods from the shallow marine succession of the Cleveland Basin, UK. The invertebrate isotope record is supplemented with carbon isotope data from fossil wood, which samples atmospheric carbon. New data elucidate two major global carbon isotope events, a negative excursion of ~2 per mil at the Sinemurian-Pliensbachian boundary, and a positive excursion of ~2 per mil in the Late Pliensbachian. The Sinemurian-Pliensbachian boundary event is similar to the slightly younger Toarcian Oceanic Anoxic Event and is characterized by deposition of relatively deepwater organic-rich shale. The Late Pliensbachian strata by contrast are characterized by shallow marine deposition. Oxygen isotope data imply cooling locally for both events. However, because deeper water conditions characterize the Sinemurian-Pliensbachian boundary in the Cleveland Basin the temperature drop is likely of local significance; in contrast a cool Late Pliensbachian shallow seafloor agrees with previous inference of partial icehouse conditions. Both the large-scale, long-term and small-scale, short-duration isotopic cycles occurred in concert with relative sea level changes documented previously from sequence stratigraphy. Isotope events and the sea level cycles are concluded to reflect processes of global significance, supporting the idea of an Early Jurassic in which cyclic swings from icehouse to greenhouse and super greenhouse conditions occurred at timescales from 1 to 10 Ma.

Seawater carbonate chemistry and clownfish Amphiprion percula size and otholith development during experiments, 2011

Calcification in many invertebrate species is predicted to decline due to ocean acidification. The potential effects of elevated CO2 and reduced carbonate saturation state on other species, such as fish, are less well understood. Fish otoliths (earbones) are composed of aragonite, and thus, might be susceptible to either the reduced availability of carbonate ions in seawater at low pH, or to changes in extracellular concentrations of bicarbonate and carbonate ions caused by acid-base regulation in fish exposed to high pCO2. We reared larvae of the clownfish Amphiprion percula from hatching to settlement at three pHNBS and pCO2 levels (control: ~pH 8.15 and 404 µatm CO2; intermediate: pH 7.8 and 1050 µatm CO2; extreme: pH 7.6 and 1721 µatm CO2) to test the possible effects of ocean acidification on otolith development. There was no effect of the intermediate treatment (pH 7.8 and 1050 µatm CO2) on otolith size, shape, symmetry between left and right otoliths, or otolith elemental chemistry, compared with controls. However, in the more extreme treatment (pH 7.6 and 1721 µatm CO2) otolith area and maximum length were larger than controls, although no other traits were significantly affected. Our results support the hypothesis that pH regulation in the otolith endolymph can lead to increased precipitation of CaCO3 in otoliths of larval fish exposed to elevated CO2, as proposed by an earlier study, however, our results also show that sensitivity varies considerably among species. Importantly, our results suggest that otolith development in clownfishes is robust to even the more pessimistic changes in ocean chemistry predicted to occur by 2100.

Seawater carbonate chemistry and biological processes of Sepia officinalis during experiments, 2010

Acidification of ocean surface waters by anthropogenic carbon dioxide (CO2) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid-base physiology. Recent studies working with environmentally relevant CO2 levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO2. It is hypothesized that the differing sensitivities to elevated seawater pCO2 could be explained by taxa specific differences in acid-base regulatory capacity. In this study, we examined the acid-base regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as 31P NMR spectroscopy. During acute exposure to 0.6 kPa CO2, S. officinalis rapidly increased its blood [HCO3] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis. A minor decrease in intracellular pH (pHi) and stable intracellular phosphagen levels indicated efficient pHi regulation. We conclude that S. officinalis is not only an efficient acid-base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values, arterial O2 saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning the potentially broad range of sensitivity to changes in acid-base status amongst invertebrates, as well as to the underlying mechanistic origins. Further studies are needed to better characterize the connection between acid-base status and animal fitness in various marine species.

Seawater carbonate chemistry and biological processes during experiments with common cuttlefish Sepia officinalis, 2010

Ocean acidification and associated changes in seawater carbonate chemistry negatively influence calcification processes and depress metabolism in many calcifying marine invertebrates. We present data on the cephalopod mollusc Sepia officinalis, an invertebrate that is capable of not only maintaining calcification, but also growth rates and metabolism when exposed to elevated partial pressures of carbon dioxide (pCO2). During a 6 wk period, juvenile S. officinalis maintained calcification under ~4000 and ~6000 ppm CO2, and grew at the same rate with the same gross growth efficiency as did control animals. They gained approximately 4% body mass daily and increased the mass of their calcified cuttlebone by over 500%. We conclude that active cephalopods possess a certain level of pre-adaptation to long-term increments in carbon dioxide levels. Our general understanding of the mechanistic processes that limit calcification must improve before we can begin to predict what effects future ocean acidification will have on calcifying marine invertebrates.

Seawater carbonate chemistry and biological processes during experiments with common cuttlefish Sepia officinalis, 2008

Ocean acidification and associated changes in seawater carbonate chemistry negatively influence calcification processes and depress metabolism in many calcifying marine invertebrates. We present data on the cephalopod mollusc Sepia officinalis, an invertebrate that is capable of not only maintaining calcification, but also growth rates and metabolism when exposed to elevated partial pressures of carbon dioxide (pCO2). During a 6 wk period, juvenile S. officinalis maintained calcification under ~4000 and ~6000 ppm CO2, and grew at the same rate with the same gross growth efficiency as did control animals. They gained approximately 4% body mass daily and increased the mass of their calcified cuttlebone by over 500%. We conclude that active cephalopods possess a certain level of pre-adaptation to long-term increments in carbon dioxide levels. Our general understanding of the mechanistic processes that limit calcification must improve before we can begin to predict what effects future ocean acidification will have on calcifying marine invertebrates.

Provenance of Holocene Calcareous Aeolianites, Eastern South Australia

The southeastern coast of South Australia contains a spectacular and world-renown suite of Quaternary calcareous aeolianites. This study is focused on the provenance of components in the Holocene sector of this carbonate breach-dune succession. Research was carried out along seven transects from ~30 meters water depth offshore across the beach and into the dunes. Offshore sediments were acquired via grab sampling and SCUBA. Results indicate that dunes of the southern Lacepede and Otway coasts in particular are mostly composed of modern invertebrate and calcareous algal allochems. The most numerous grains are from molluscs, benthic foraminifera, coralline algae, echinoids, and bryozoans. These particles originate in carbonate factories such as macroalgal forests, rocky reefs, seagrass meadows, and low-relief seafloor rockgrounds. The incorporation of carbonate skeletons into coastal dunes, however, depends on a combination of; 1) the infauna within intertidal and nearshore environments, 2) the physical characteristics of different allochems and their ability to withstand fragmentation and abrasion, 3) the wave and swell climate, and 4) the nature of aeolian transport. Most aeolian dune sediment is derived from nearshore and intertidal carbonate factories. This is particularly well illustrated by the abundance of robust infaunal bivalves that inhabit the nearshore sands and virtual absence of bryozoans that are common as sediment particles in water depths > 10mwd. Thus, the calcareous aeolianites in this cool-water carbonate region are not a reflection of the offshore marine shelf factories, but more a product of shallow nearshore-intertidal biomes.

Fossil associations from the middle and upper Eocene strata of the Pamplona Basin and surrounding areas (Navarre, western Pyrenees)

Fossil associations from the middle and upper Eocene (Bartonian and Priabonian) sedimentary succession of the Pamplona Basin are described. This succession was accumulated in the western part of the South Pyrenean peripheral foreland basin and extends from deep-marine turbiditic (Ezkaba Sandstone Formation) to deltaic (Pamplona Marl, Ardanatz Sandstone and Ilundain Marl formations) and marginal marine deposits (Gendulain Formation). The micropalaeontological content is high. It is dominated by foraminifera, and common ostracods and other microfossils are also present. The fossil ichnoasssemblages include at least 23 ichnogenera and 28 ichnospecies indicative of Nereites, Cruziana, Glossifungites and ?Scoyenia-Mermia ichnofacies. Body macrofossils of 78 taxa corresponding to macroforaminifera, sponges, corals, bryozoans, brachiopods, annelids, molluscs, arthropods, echinoderms and vertebrates have been identified. Both the number of ichnotaxa and of species (e. g. bryozoans, molluscs and condrichthyans) may be considerably higher. Body fossil assemblages are comparable to those from the Eocene of the Nord Pyrenean area (Basque Coast), and also to those from the Eocene of the west-central and eastern part of South Pyrenean area (Aragon and Catalonia). At the European scale, the molluscs assemblages seem endemic from the Pyrenean area, although several Tethyan (Italy and Alps) and Northern elements (Paris basin and Normandy) have been recorded. Palaeontological data of studied sedimentary units fit well with the shallowing process that throughout the middle and late Eocene occurs in the area, according to the sedimentological and stratigraphical data.