Decreased pH does not alter metamorphosis but compromises juvenile calcification of the tube worm Hydroides elegans

Using CO₂ perturbation experiments, we examined the pre- and post-settlement growth responses of a dominant biofouling tubeworm (Hydroides elegans) to a range of pH. In three different experiments, embryos were reared to, or past, metamorphosis in seawater equilibrated to CO₂ values of about 480 (control), 980, 1,480, and 2,300 μatm resulting in pH values of around 8.1 (control), 7.9, 7.7, and 7.5, respectively. These three decreased pH conditions did not affect either embryo or larval development, but both larval calcification at the time of metamorphosis and early juvenile growth were adversely affected. During the 24-h settlement assay experiment, half of the metamorphosed larvae were unable to calcify tubes at pH 7.9 while almost no tubes were calcified at pH 7.7. Decreased ability to calcify at decreased pH may indicate that these calcifying tubeworms may be one of the highly threatened species in the future ocean.     

Early reproductive stages in the crustose coralline alga Phymatolithon lenormandii are strongly affected by mild ocean acidification

Coralline algae (Corallinales, Rhodophyta) are predicted to be negatively impacted by near-future ocean acidification. The effect of low pH/high pCO₂ on early life stages of Phymatolithon lenormandii (Areschoug) Adey was studied in a perturbation experiment. Several parameters including mortality, calcification (calcein staining) and development (growth and abnormalities) have been monitored for a month under experimental conditions ranging from pH_T = 8.00 (pCO₂ = 398 μatm) and pH_T = 7.55 (pCO₂ = 1,261 μatm). Our results demonstrate that survival and development of P. lenormandii early life stages can be impacted by small pH changes (ΔpH < ?0.1 pH unit). A negative impact of decreasing pH was observed including an increased mortality and a higher rate of abnormalities. Growth and calcification were still observed at the lowest pH (ΔpH = ?0.45). Growth rate was similar at all tested pH, but the maintenance of the skeleton under low pH was only possible through a persistent dynamic dissolution/calcification process, an energetically costly mechanism potentially draining resources from other vital processes.     

A morphological and structural study of the larval shell from the abalone <Emphasis Type="Italic">Haliotis tuberculata</Emphasis>

The larval shell of the marine gastropod Haliotis tuberculata was investigated by polarised light microscopy, scanning electron microscopy, Raman microspectroscopy and infra-red spectroscopy. Trochophore and veliger larval sections were used for histological examination of the growing shell and each larval stage was related to the shell development and the appearance of calcified formations. We determined the stage of initial calcification by specific staining combined with polarised light examination. The shell of 30-h-old pre-veliger larvae was found to be mineralized, confirming that calcification occurred before larval torsion. Using both infra-red and Raman spectroscopy, we showed that CaCO₃ deposition occurred at the pre-veliger stage and that the mineral phase initially deposited was essentially composed of aragonite.     

Microstructure of the paper nautilus (Argonauta nodosa) shell and the novel application of electron backscatter diffraction (EBSD) to address effects of ocean acidification

Electron backscatter diffraction (EBSD) is a powerful microscopic technique to characterise the crystallography of biomineralisation. Here, we use high-resolution EBSD to characterise one of the least studied shells in the ocean, the female argonaut brood chamber, and to examine the changes in shell microstructure in response to incubation in decreased pH conditions. The thin (225 μm) shell of Argonauta nodosa is magnesium calcite with an average magnesium content of ca. 5.1 Wt % MgCO₃. EBSD and scanning electron microscopy (SEM) revealed that calcification of the shell is bidirectional with formation of irregular crystalline grains. Following a 2 week incubation in a range of pH treatments (pH, 8.1–7.2), shell fragment weight decreased by dissolution in pH ≤ 7.8. EBSD and SEM revealed altered shell crystallography and microstructure at pH ≤ 7.4 due to preferential etching down crystallite grain boundaries and a change in crystalline orientation on both the inner and outer shell surfaces. Our study highlights the value of EBSD for the detailed examination of biogenic carbonates and its potential use in the field of ocean acidification research.     

An exploratory study with the proton microprobe of the ontogenetic distribution of 16 elements in the shell of living oysters (Crassostrea virginica)

Study of the chemical composition of shell of exoskeletonous organisms in the past has required the sacrifice of the organism. Because the beam of the proton microprobe is relatively nondestructive and analyzes the surface layer of the shell, organisms do not have to be killed. The present paper presents results of a preliminary experiment in which distribution of elements (Na to Sr) in shell of living juvenile oysters, Crassostrea virginica (Gmelin), was studied in situ with a proton microprobe at monthly intervals for four months. The relative concentration of 16 elements was measured in the newly deposited prismatic edge of the right valve of three oysters reared in controlled laboratory conditions. Na, Mg, Al, Si, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Br, and Sr were detected in concentrations as low as a few parts per million relative to the concentration of standards added to pure CaCO₃. Concentration of elements varied nominally among shells of the three individual oysters and in their successive ontogenetic stages. Fluctuations in concentration of Na, Mg, S, Cl, Ca, Mn, Fe, Cu, and Zn were generally similar in the two normally growing oysters, but differed from those in the oyster that stopped growing. Trends in concentration of Al, Si, and Sr were similar in the three oysters: those of Br were variable. Relative concentrations of Na, Cl, S, Mn, Fe, and Zn increased slightly with age of oysters, that of the other elements stayed relatively constant. Concentration of most elements was higher in shell than in seawater. Variable concentrations, especially of Na, Cl, and Si in valve edges, tend to support the hypothesis of earlier workers that separate mineral phases are present as impurities entrapped within the shell during calcification.     

Fertilisation, embryogenesis and larval development in the tropical intertidal sand dollar Arachnoides placenta in response to reduced seawater pH

We examined the response of the tropical sand dollar Arachnoides placenta to reduced seawater pH in experiments spanning ca. 50 % of the planktonic larval duration. A. placenta inhabits intertidal sandy beaches where we observed a minimum in situ pH range 0.06 pH units (pH 8.10–8.16). The responses of gametes and larvae to seawater pH were tested in vitro in ambient (pH 8.14, pCO₂ = 525.7 μatm, total alkalinity = 2,651 μmol kg soln^?1) and three reduced pH seawater treatments (7.8–7.0). Percentage fertilisation decreased significantly with decreasing pH across a range of sperm/egg ratios (4:1 up to 4,000:1). A. placenta reached the advanced pluteus stage in 4 days, and during this time, we saw no difference in survival rate of larvae between the ambient (67 %) and pH 7.79 (72 %) treatments. Four-day survival was, however, reduced to 44 and 11 % in the pH 7.65 and 7.12 treatments, respectively. Larval development and morphometrics varied among pH treatments. Embryos reared in pH 7.12 exhibited arrested development. Larvae reared at pH 7.65 showed delayed development and greater mortality compared with those reared at pH 7.79 and 8.14. When larval morphometrics are compared among larvae of the same size, differences in larval width and total arm length between pH treatments disappear. These results suggest that variation in larval size among the three highest pH treatments at a given time are likely the result of slower development and apparent shrinkage of surviving larvae and not direct changes in larval shape. There were no differences in the percentage inorganic content (a proxy for calcification) in larvae reared in either an ambient or a pH 7.7 treatment. The responses of fertilisation and development to decreased pH/increased pCO₂ in A. placenta are within the range of those reported for other intertidal and subtidal echinoid species from colder latitudes.     

Comparative effects of two antifouling paints on the oyster Crassostrea gigas

The effects of two antifouling paints, one containing organotin compounds, the other copper oxide, were studied from September 1983 to October 1984 on Crassostrea gigas (Thunberg) grown under natural field conditions in the Bay of Arcachon, France. The organotin paint reduced growth rate expressed as weight, length and width, but did not affect shell height; it drastically decreased the dry condition factor and shell density, but did not affect the viability of embryos and larvae from exposed oysters. However, some decrease in larval growth rate was observed. The copper paint had no effect on oyster growth, but lowered the condition factor compared to controls. Neither viability of embryos or larvae nor larval growth were affected by this paint.     

Respiratory quotient and calcification of Nautilus macromphalus (Cephalopoda: Nautiloidea)

Respiration and calcification were investigated in the ectocochleate cephalopod Nautilus macromphalus Sowerby. Specimens were collected off New Caledonia, in October 1991, and kept at the Nouméa Aquarium until December 1991. The respiratory quotient and calcification rate of 5 individuals were measured during 14 short term incubations (63 to 363 min). Oxygen uptake was recorded with a polarographic oxygen sensor. CO₂ flux and calcification were calculated from changes in pH and alkalinity (alkalinity-anomaly technique). Several methods were used to compute the respiratory quotient (RQ); a functional regression indicated an RQ of 0.74. CaCO₃ exchange rates were linearly related to respiratory quotient, calcification occurring in individuals with a low RQ. CaCO₃ uptake from the surrounding water was noncontinuous. From the highest CaCO₃ uptake, maximum growth rate was estimated as 7.1 mg shell wt h^- (=61 g yr^-1).     

Genetic variation and covariation during larval and juvenile growth in Mercenaria mercenaria

Quantitative genetic variances and covariances were estimated for shell length of the hard clam Mercenaria mercenaria (L.) at three larval stages (prodissoconch I, 2 d and 10 d post-fertilization) in 1987 and in 1988 after ca. 9 mo of growth. At each sample interval additive genetic variance was a highly significant component of the total size variation. Narrow sense heritability estimates for shell length ranged between 0.58 (±0.10) for prodissoconch I and 1.08 (±0.29) for 2-d-old larvae. There was significant and positive genetic covariance in prodissoconch I and 2-d larval shell length which resulted in a highly significant genetic correlation (r _g=0.74) between these two traits. This covariance is not surprising since the prodissoconch I comprises the majority of the larval shell of a 2-d-old larvae. The genetic covariances between 2-d-old and 10-d-old larvae and between 10-d-old larvae and 9-mo-old juveniles were low and not significantly different from zero. These results indicate that there is substantial genetic variation for shell growth in M. mercenaria but this variation is not stable during development; the genetic variation in shell growth at one stage of development is not strongly related to the genetic variation in growth during other ontogenetic periods. In this study there were no evident constraints to natural selection for increased shell growth rate during development, which coupled with the high levels of genetic variation may suggest that in nature high rates of larval growth may not be normally subject to significant selective pressure.     

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 (Ω_calc = 0.78, Ω_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.     

Calcification in the articulated coralline alga Corallina pilulifera,with special reference to the effect of elevated CO2 concentration

Calcification in Corallina pilulifera Postels et Ruprecht displayed diurnal variations in aerated (350 ppm CO₂) culture media, with faster rates during the light than during the dark period. Addition of CO₂ (air+1250 ppm) inhibited calcification. This was attributable to the decreased pH resulting from CO₂ addition. Both photosynthesis and calcification were enhanced in seawater, with elevated dissolved inorganic carbon concentrations at a constant pH of 8.2.     

General and specific combining abilities of larval and juvenile growth and viability estimated from natural oyster populations

Diallel crosses of oysters from three geographically isolated natural populations were produced to evaluate the relative importance of genetic, maternal, and environmental effects on larval and juvenile growth and viability. Significant additive genetic effects were observed only in larval viability at Day 12 and larval shell length at Day 2. The presence of significant male and female mean square for larval viability (suggesting non-additive genetic variance) is consistent with fitness related characters. Important maternal effects were observed for the larval and juvenile shell length and viability characters. These female mean squares are probably affected by both real and spurious maternal effects and potential contributing influences are discussed. The performance of the crosses can be largely explained by two factors: parental performance and the heterotic gene effects. This is based on an apparent positive correlation of mean values between the parental populations and their crosses. The crosses' mean viability at the end of the larval phase was 14.0% lower than the pure matings and support a previous observation of lower heterozygote viability in the larval phase (Mallet and Haley, 1983b).     

Electrophoretic identification and genetic analysis of bivalve larvae

Taxonomic identification and genetic analysis of larval marine invertebrates have been vexing problems. We describe a polyacrylamide mini-gel electrophoresis technique for resolving proteins from individual larval bivalves (shell length 250 to 350 m) and apply this technique to three species of laboratory-cultured larval oysters [Ostrea edulis L., 1758, Crassostrea gigas (Thunberg, 1793) and c. virginica (Gmelin, 1791)] reared during summer 1989. Electrophoretic patterns of proteins clearly discriminate among the three species and allow genetic analysis of a polymorphic allozyme locus (Pgi) in field-collected larvae and juveniles of C. virginica. This technique provides an economical tool for largescale taxonomic, ecologic, and genetic studies of meroplanktonic stages of various species.     

Combined effects of dissolved oxygen concentration and water temperature on embryonic development and larval shell secretion in the marine snail <Emphasis Type="Italic">Chorus</Emphasis><Emphasis Type="Italic">giganteus</Emphasis> (Gastropoda: Muricidae)

The present study was undertaken to determine the effects of both extracapsular oxygen concentration and temperature on embryonic development in Chorus giganteus. In normoxia increasing water temperature from 12°C to 18°C reduced by 15 days the median time required for the capsules to hatch. Hypoxia (oxygen content at 50% of air saturation) generated a low development rate and totally prevented both shell secretion and larval hatching from the egg capsule. Experimental transfer at weekly intervals, from normoxia to hypoxia and vice versa, induced a decrease and increase in the embryonic ash content, respectively, but did not affect the number of hatched larvae. Such an effect was more pronounced at 12°C than at 15°C or 18°C. The embryonic inability to produce a shell under hypoxia is likely to be a result of the low intracapsular oxygen concentration (IPO₂) generated as the combined effect of a low extracapsular oxygen concentration (environmental) added to the intracapsular embryonic oxygen demands, which lowers the IPO₂ still further. Under such conditions, a decrease in intracapsular pH is likely to take place, and, if so, embryos might divert carbonates away from shell calcification to balance such changes in pH.     

Differential timing of larval starvation effects on filtration rate and growth in juvenile Crepidula onyx

This study demonstrates that the timing of larval starvation did not only determine the larval quality (shell length, lipid content, and RNA:DNA ratio) and the juvenile performance (growth and filtration rates), but also determine how the latent effects of larval starvation were mediated in Crepidula onyx. The juveniles developed from larvae that had experienced starvation in the first two days of larval life had reduced growth and lower filtration rates than those developed from larvae that had not been starved. Lower filtration rates explained the observed latent effects of early larval starvation on reduced juvenile growth. Starvation late in larval life caused a reduction in shell length, lipid content, and RNA:DNA ratio of larvae at metamorphosis; juveniles developed from these larvae performed poorly in terms of growth in shell length and total organic carbon content because of “depletion of energy reserves” at metamorphosis. Results of this study indicate that even exposure to the same kind of larval stress (starvation) for the same period of time (2 days) can cause different juvenile responses through different mechanisms if larvae are exposed to the stress at different stages of the larval life.     

Characterization and role of carbonic anhydrase in the calcification process of the azooxanthellate coral <Emphasis Type="Italic">Tubastrea aurea</Emphasis>

In zooxanthellate corals, the photosynthetic fixation of carbon dioxide and the precipitation of CaCO₃ are intimately linked both spatially and temporally making it difficult to study carbon transport mechanisms involved in each pathway. When studying Tubastrea aurea, a coral devoid of zooxanthellae, we can focus on carbon transport mechanisms involved only in the calcification process. We performed this study to characterize T. aurea carbonic anhydrase and to determine its role in the calcification process. We have shown that inhibition of tissular carbonic anhydrase activity affects the calcification rate. We have measured the activity of this enzyme both in the tissues and in the organix matrix extracted from the skeleton. Our results indicate that organic matrix proteins, which are synthesized by the calcifying tissues, are not only structural proteins, but they also play a crucial catalytic role by eliminating the kinetic barrier to interconversion of inorganic carbon at the calcification site. By immunochemistry we have demonstrated the presence of a protein both in the tissues and in the organic matrix, which shares common features with prokaryotic carbonic anhydrases.     

Latitudinal patterns of shell thickness and metabolism in the eastern oyster Crassostrea virginica along the east coast of North America

The goal of this study was to quantify growth and metabolic responses of oysters to increased temperatures like those that will occur due to global warming. Impact of temperature on eastern oyster (Crassostrea virginica) shell growth and metabolism was investigated by sampling 24 sites along the eastern North American seaboard ranging from New Brunswick, Canada, to Florida, USA, in March and August 2013. There was a positive correlation between oyster shell thickness and site temperature. At southern sites, shells were up to 65 % thicker than at the northernmost site, likely due to higher precipitation of CaCO₃ in warmer water. This was supported by laboratory experiments showing that thicker shells were produced in response to temperatures 2, 4, and 6 °C above ambient seawater temperatures (8–14 °C) in Connecticut, USA. Field experiments with oyster respiration were conducted during winter and summer at 13 sites to compare responses to thermal stress with latitude. Respiration rates were much higher during summer than winter, but the combination of summer and winter data fell along the same exponential curve with respect to temperature. At all sites, temperature-specific metabolic rates at elevated temperatures were lower than predicted, indicating significant seasonal acclimatization by C. virginica.     

Response of sea urchin pluteus larvae (Echinodermata: Echinoidea) to reduced seawater pH: a comparison among a tropical, temperate, and a polar species

Ocean acidification, as a result of increased atmospheric CO₂, is predicted to lower the pH of seawater to between pH 7.6 and 7.8 over the next 100 years. The greatest changes are expected in polar waters. Our research aimed to examine how echinoid larvae are affected by lower pH, and if effects are more pronounced in polar species. We examined the effects of lowered pH on larvae from tropical (Tripneustes gratilla), temperate (Pseudechinus huttoni, Evechinus chloroticus), and a polar species (Sterechinus neumayeri) in a series of laboratory experiments. Larvae were reared in a range of lower pH seawater (pH 6.0, 6.5, 7.0, 7.5, 7.7, 7.8 and ambient), adjusted by bubbling CO₂ gas. The effect of pH on somatic and skeletal growth, calcification index, development and survival were quantified, while SEM examination of the larval skeleton provided information on the effects of seawater pH on the fine-scale skeletal morphology. Lowering pH resulted in a decrease in survival in all species, but only below pH 7.0. The size of larvae were reduced at lowered pH, but the external morphology (shape) was unaffected. Calcification of the larval skeleton was significantly reduced (13.8–36.9% lower) under lowered pH, with the exception of the Antarctic species, which showed no significant difference. SEM examination revealed a degradation of the larval skeletons of Pseudechinus and Evechinus when grown in reduced pH. Sterechinus and Tripneustes showed no apparent difference in the skeletal fine structure under lowered pH. The study confirms the need to look beyond mortality as a single endpoint when considering the effects of ocean acidification that may occur through the 21st century, and instead, look for a suite of more subtle changes, which may indirectly affect the functioning of larval stages.     

Ocean acidification and temperature rise: effects on calcification during early development of the cuttlefish Sepia officinalis

This study investigated the effects of seawater pH (i.e., 8.10, 7.85 and 7.60) and temperature (16 and 19 °C) on (a) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid), (b) growth, development and (c) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO₂ conditions in the perivitelline fluid (>3-fold higher pCO₂ than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas ⁴⁵Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.