Magdalena A. Gutowska

Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid–base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H+-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pHe and pHi) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO2 conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO2. Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pHe whenever seawater pH changes. However, measurements of pHi demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na+ and HCO3−, suggesting a bicarbonate buffer mechanism involving secondary active Na+-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pHi enables calcification to proceed despite decreased pHe. However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation.

BackgroundBiogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient.ResultsThe combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes.ConclusionsBeneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.

Adaptation of a globally important coccolithophore to ocean warming and acidification

Although ocean warming and acidification are recognized as two major anthropogenic perturbations of today’s oceans we know very little about how marine phytoplankton may respond via evolutionary change. We tested for adaptation to ocean warming in combination with ocean acidification in the globally important phytoplankton species Emiliania huxleyi. Temperature adaptation occurred independently of ocean acidification levels. Growth rates were up to 16% higher in populations adapted for one year to warming when assayed at their upper thermal tolerance limit. Particulate inorganic (PIC) and organic (POC) carbon production was restored to values under present-day ocean conditions, owing to adaptive evolution, and were 101% and 55% higher under combined warming and acidification, respectively, than in non-adapted controls. Cells also evolved to a smaller size while they recovered their initial PIC:POC ratio even under elevated CO2. The observed changes in coccolithophore growth, calcite and biomass production, cell size and elemental composition demonstrate the importance of evolutionary processes for phytoplankton performance in a future ocean.

Elevated seawater pco2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immunohistochemical methods, we demonstrate that Na(+)/K(+)-ATPase (soNKA), a V-type H(+)-ATPase (soV-HA), and Na(+)/HCO(3)(-) cotransporter (soNBC) are colocalized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater Pco(2) (0.16 and 0.35 kPa) over a time course of 6 wk in different ontogenetic stages. The applied CO(2) concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late-stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII, and COX. In contrast, no hypercapnia-induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However, a transiently increased ion regulatory demand was evident during the initial acclimation reaction to elevated seawater Pco(2). Gill Na(+)/K(+)-ATPase activity and protein concentration were increased by ~15% during short (2-11 days) but not long-term (42-days) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the downregulation of ion regulatory and metabolic genes in late-stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater Pco(2).

Intragel oxygen promotes hypoxia tolerance of scyphomedusae.

SUMMARY Populations of jellyfish are known to thrive in many low oxygen environments, however, the physiological mechanisms that permit these organisms to live in hypoxia remain unknown. The oxyregulatory abilities of four species of scyphomedusae were investigated, and it was found that Aurelia labiata, Phacellophora camtschatica, Cyanea capillata and Chrysaora quinquecirrha maintain steady oxygen consumption to below 20 hPa oxygen (<10% air saturation). Oxygen content of the mesoglea of A. labiata was measured using a fibre optic oxygen optode, and oxygen profiles through the gel are characterised by a gradient that decreases from just below normoxia at the aboral subsurface to ∼85% air saturation near the subumbrellar musculature. This gradient sustains oxyregulation by scyphomedusae, and it is demonstrated that A. labiata must be using intragel oxygen to meet its metabolic needs. Gel can also be used as an oxygen reservoir when A. labiata moves into hypoxia. Gel oxygen is depleted after about 2 h in anoxia and recovers to 70% of normal after 2.5 h in normoxia. Behaviour experiments in the laboratory showed that Aurelia labiata behaves similarly in normoxia and hypoxia (30% and 18% air saturation). The acute threshold for provoking behavioural changes in A. labiata is somewhere near its critical partial pressure, and oxygen stratification stimulates swimming back and forth across the oxycline. Intragel oxygen dynamics are recognised as a fundamental component of medusan physiology.

Influence of temperature, hypercapnia, and development on the relative expression of different hemocyanin isoforms in the common cuttlefish Sepia officinalis.

The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and acclimation to elevated seawater pCO(2) and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juveniles, and adults), under different temperatures and elevated seawater pCO(2). Our results indicate moderately altered hemocyanin expression in all embryonic stages acclimated to higher pCO(2), while hemocyanin expression in hatchlings and juveniles remained unaffected. During the course of development, total hemocyanin expression increased independently of pCO(2) or thermal acclimation status. Expression of isoform 3 is reported for the first time in a cephalopod in this study and was found to be generally low but highest in the embryonic stages (0.2% of total expression). Despite variable hemocyanin expression, hemolymph total protein concentrations remained constant in the experimental groups. Our data provide first evidence that ontogeny has a stronger influence on hemocyanin isoform expression than the environmental conditions chosen, and they suggest that hemocyanin protein abundance in response to thermal acclimation is regulated by post-transcriptional/translational rather than by transcriptional modifications.

A shell regeneration assay to identify biomineralization candidate genes in mytilid mussels.

Biomineralization processes in bivalve molluscs are still poorly understood. Here we provide an analysis of specifically expressed sequences from a mantle transcriptome of the blue mussel, Mytilus edulis. We then developed a novel, integrative shell injury assay to test, whether biomineralization candidate genes highly expressed in marginal and pallial mantle could be induced in central mantle tissue underlying the damaged shell areas. This experimental approach makes it possible to identify gene products that control the chemical micro-environment during calcification as well as organic matrix components. This is unlike existing methodological approaches that work retroactively to characterize calcification relevant molecules and are just able to examine organic matrix components that are present in completed shells. In our assay an orthogonal array of nine 1mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36days to regenerate. Structural observations using stereo-microscopy, SEM and Raman spectroscopy revealed organic sheet synthesis (day 20) as the first step of shell-repair followed by the deposition of calcite crystals (days 20 and 29) and aragonite tablets (day 36). The regeneration period was characterized by time-dependent shifts in gene expression in left central mantle tissue underlying the injured shell, (i) increased expression of two tyrosinase isoforms (TYR3: 29-fold and TYR6: 5-fold) at day 20 with a decline thereafter, (ii) an increase in expression of a gene encoding a nacrein-like protein (max. 100-fold) on day 29. The expression of an acidic Asp-Ser-rich protein was enhanced during the entire regeneration process. This proof-of-principle study demonstrates that genes that are specifically expressed in pallial and marginal mantle tissue can be induced (4 out of 10 genes) in central mantle following experimental injury of the overlying shell. Our findings suggest that regeneration assays can be used systematically to better characterize gene products that are essential for distinct phases of the shell formation process, particularly those that are not incorporated into the organic shell matrix.

A sea urchin Na+K+2Cl− cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na(+)K(+)2Cl(-) cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50=6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4 μM) and furosemide (IC50=315.4 μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.