5. The Effects of Ocean Acidification in Gastropod Shell Development

Keisuke Shimizu (1), Katsunori Kimoto (2)

1 JAMSTEC, Yokosuka, Kanagawa, 237-0061, JAPAN
2 JAMSTEC, Yokosuka, Kanagawa, 237-0061, JAPAN

Background
The Molluscs constitutes one of the most diverse animal phyla, and they have evolved calcified exoskeletons called “shell”. Current high levels of CO2 leads to ocean acidification, and it affects for molluscan shell development. In fact, many previous studies have reported their phenotypic effects by ocean acidification, but the genetic response remains meagre. Thus we sought to understand not only the phenotypic effect but also the effect of gene expression pattern in high pCO2 condition using marine limpet Nipponacmea fuscoviridis.

Methods
After the artificial fertilization, embryos were cultured in different pCO2 conditions, and we checked the effects in early development.

Findings
In high pCO2 condition, we observed phenotypic and genetic effects in trochophore and veliger larvae stages.

Conclusions
Our results provide new insight of the effects for calcifying organisms in the feature high pCO2 marine condition.

4. Physiological response of a protected deep sea coral to ocean acidification

Gammon, Malindi (1), Davy, Simon (1), Tracey, Di (2)*, Cummings, Vonda (2), Marriott, Peter (2).

1 Victoria University of Wellington (VUW), Wellington 6012, New Zealand.
2 National Institute of Water and Atmospheric Research (NIWA), Wellington 6022, New Zealand.

Calcifying corals provide important habitat complexity in the deep sea and are consistently associated with a biodiversity of fish and other invertebrates. Little is understood about how deep sea calcifying corals may respond to the future predicted environmental conditions of ocean acidification (OA), but any predicted changes will have wider impacts on the ecosystem. Due to the difficulties associated with keeping deep sea corals alive in aquaria, the literature is currently limited to short-term experiments and a poor understanding as to whether there is any capacity for acclimation. Colonies of Solenosmilia variabilis, a species of deep sea protected coral found in the waters surrounding New Zealand, were collected during a cruise in March of 2014 from the Louisville Seamount chain. Over a 12-month period, coral samples were maintained in temperature controlled (~3.5°C) continuous flow-through tanks (~130 mL per minute). A control group of coral colonies were held in seawater with pH 7.88 and the treatment group in pH 7.65. These two pH levels where designed to reflect current pH conditions and end of century scenarios, respectively. In addition to investigating changes in growth for this species, measurements of respiration and intracellular pH (pHi) were taken after a mid-term (6 months for respiration; 9 months for pHi) and long-term (12 months for both respiration and pHi) exposure period. Respiration rates and pHi were independent of pH. Respiration rates ranged from 0.065 to 1.756 µm O2 (g protein)-1 hour-1. The pH manipulations used in this experiment are ecologically relevant, reflecting end of century projections for the region. This research illustrates important first-steps of understanding the sensitivity of deep sea corals to OA and the potential for acclimation. To conclude on the sensitivity of deep sea corals to OA, it is necessary that future research explores threshold responses and the role of food availability and rising temperatures.

3. Integrated impacts of temperature increase and ocean acidification on larval shell development in the American Lobster, Homerus americanus

Christine San Antonio (1)*, Michael Tlusty (2), Robyn Hannigan (3)

1 School for the Environment, University of Massachusetts Boston, Boston MA, 02125, USA
2 New England Aquarium, Central Wharf, Boston MA, 02110, USA
3 School for the Environment, University of Massachusetts Boston, Boston MA, 02125, USA

Background
To date, research on the American lobster primarily describes mechanistic consequences of OA for calcification, growth and survival rates. Here we explore the interactive effects of temperature and CO2/pH on shell development, and how these effects translate to whole animal responses to external stressor such as disease. American lobster in the Gulf of Maine provide an ideal model for studying multi-factor OA effects.

Methods
This laboratory-based study examined responses of larval (hatch to post-larval Stage IV; ~ 30 per tank, 3 replicate tanks per treatment) lobsters to elevated pCO2 and temperature in a 3 x 3 factoral experiment. We assessed shell morphology and chemistry using scanning electron microscopy (mineralogy), and attenuated reflectance fourier-transform infrared spectroscopy (inorganic and organic composition). We analysed the data as a nested (larval rearing container) two way MANOVA. We tested shells of bacterially exposed lobsters for evidence of disease and shell composition as well. Data from lobsters exhibiting shell disease were analyzed by a 2 way ANOVA.

Findings
Larvae reared at low pH showed loss of shell integrity with a coincident change in mineralogy from rhomboidal low-Ca calcite to prismatic low-Ca and low-Mg calcite. Temperature increases ameliorated the change in crystal morphology but did not influence growth rate which was also reduced under low pH. Bacterially challenged animals reared at lower pH and at higher temperatures showed the most significant impacts on shell chemistry and mineralogy due to the increased incidence of epizootic shell disease under the most extreme temperature and pH condition.

Conclusions
We hypothesized that ocean acidification and increased temperature would result in decreased shell growth and changes in mineral composition of larval lobster shell. We found that shell microstructure development is largely pH dependent and that OA and increased temperature alter the onset and prevalence of the shell disease.

2. Effects of ocean acidification on the photosynthetic performance, carbonic anhydrase activity and growth of the giant kelp Macrocystis pyrifera

Pamela A. Fernandez (1)*, Michael Y. Roleda (2), Catriona L. Hurd (3)

1 Department of Botany, University of Otago, Dunedin 9054, New Zealand.
2 Bioforsk Norwegian Institute for Agricultural and Environmental Research, Kudalsveien 6, Bodø 8049, Norway.
3 Institute for marine and Antarctic Studies (IMAS), University of Tasmania, TAS 7001, Australia.

Background
Temperate kelp forests play an important role in coastal environments, contributing 50% of primary production and providing food and habitat for a variety of marine invertebrates. By 2100, an increase of 192% and 14% in CO2(aq) and HCO3, respectively, is expected due to ocean acidification (OA). Most seaweeds can take up both CO2 and HCO3 as an inorganic carbon (Ci) source for photosynthesis. The projected changes in seawater carbonate chemistry may affect the ability of seaweeds to acquire Ci and, consequently, their photosynthetic performance and productivity. However, a seaweed’s response to OA will depend on their carbon physiology. This study evaluates how the relative increase in different Ci species will affect the enzyme carbonic anhydrase (CA) (external and internal), which plays an essential role in Ci acquisition, and hence photosynthesis, and growth rates of the habitat-forming giant kelp Macrocystis pyrifera.

Methods
Response variables were measured in blade discs of individuals of M. pyrifera sporophytes cultivated under two CO2(aq) concentrations (400 vs. 1,200 µatm) for 10 days in laboratory. Sampling was conducted in a time series, i.e. every 3 days, to determine regulatory and/or acclimatory processes in the carbon physiology of M. pyrifera in response to OA.

Findings
Photosynthetic and growth rates were not affected by CO2(aq). Internal CA was considerably higher than external CA under both CO2 treatments. However, external CA was reduced on day 7 of incubation in the elevated CO2(aq) treatment, but it remained active. In contrast, internal CA remained high and active across all sampling days under both CO2 treatments. Stables isotopes (Ϩ15N and Ϩ13C), carbon and nitrogen content and C:N ratio were not affected by CO2(aq).

Conclusions
The giant kelp, M. pyrifera, has two effective mechanisms for HCO3 utilization and it is not limited at current Ci concentrations. Therefore, increased CO2(aq) under an OA scenario will not affect the growth and photosynthetic performance of the giant kelp.

1. Effects of Ocean Acidification on the Growth of juvenile Mytilus edulis

Mary Margaret Stoll (1,3)*, Robert Holmberg (1), Aaron Honig (1,2), and Dr. Robyn Hannigan (1)*

1 School for the Environment, University of Massachusetts Boston, Boston, MA, 02125, USA
2 Biology Department, University of Massachusetts Boston, Boston, MA, 02125, USA
3 Chemistry and Environmental Studies Departments, Amherst College, Amherst, MA, 01002, USA

Background
Ocean acidification is the process in which surplus atmospheric carbon dioxide (CO2(g)) transfers across the ocean-atmosphere boundary and becomes CO2 (aq). This process changes the carbonate system balance leading towards increased [H+] and decreased [CO32-], ultimately causing increased acidity of the water. Furthermore, this changes the saturation state of carbonate minerals, shifting away from stability towards dissolution. As a result, carbonate biominerals such as those in bivalve shells become thermodynamically less stable and may dissolve or shift towards a more stable form.

Methods
We grew the juvenile blue mussel, Mytilus edulis, under different CO2-induced low pH conditions to explore the effect of ocean acidification on growth. We used a pH-stat CO2-dosing system designed for ocean acidification research with four replicates per treatment (n=4, control-outside room: pH=8.1, control: pH=8.1, treatment 3: pH=7.6, treatment 4: pH=7.3). We monitored carbonate chemistry parameters including pH, salinity, temperature, and total alkalinity. Juveniles were fed T-Isochrysis algae. We changed the water in each tank everyday and counted algal cells to estimate algal density.

Findings
At the end of the one-week exposure, we measured survivorship, shell length and width. We evaluated shell morphometrics using a Matlab script to determine circularity, area and perimeter. There was no difference in mussel growth between treatments (p>0.1). However, there were differences in the circularity of the mussel shells between treatments (p<0.1). Scanning electron microscopy and associated image analysis of the mussel shells indicated variations in the mineralogy and structure of the shell between treatments.

Conclusions
There was no difference in mussel growth between treatments. However, there were differences in the circularity of the shells between treatments. Analysis of the shell structure and mineralogy showed some impacts as well. Though this is a preliminary study, research is on-going to explore these impacts across life stages.

18. Vulnerability of Pteropod Shell In The Arctic Ocean: A Result of Culture Experiment Under Natural Seawater

Katsunori Kimoto (1)*, Jonaotaro Onodera (1), Naomi Harada (1), Kohei Matsuno (2), Takahito Ikenoue (3), Osamu Sasaki (4)

1 Institute of Arctic Climate and Environment Research (IACE), JAMSTEC, 2-15, Natsushima-cho, Yokosuka, 237-0061, Japan.
2 National Institute of Polar Research (NIPR), 10-3, Midori-cho, Tachikawa-shi, Tokyo 190-8518, Japan.
3 Marine Ecology Research Insitiute, 300 Iwawada, Onjuku-machi, Isumi-gun, Chiba 299-5105 Japan.
4 The Tohoku University Museum, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, 980-8578 Japan.

The pteropods (thecosome), planktic molluscs having aragonite shell is major component of marine zooplankton and it plays important roles for oceanic carbon cycles and marine ecosystems. In particular, the aragonite shell is more soluble in seawater, and could be affected by ocean acidification (OA). In this study, we performed simple culture experiment of pteropod Limacina helicina living in the Arctic Ocean and described shell degradation processes under different water mass in the natural seawater.

Culture experiment had carried out on the R/V Mirai during the Leg MR13-06 Arctic cruise in 2013. In this cruise, we stayed two weeks at fixed station (Sta. 41: 72° 45’N, 168°16’W, water depth: 52 m) from 11 Sep. to 25 Sep., 2013 and collected L. helicina by vertical plankton tow for culture experiments. During observations, we observed drastic changes of carbonate chemistry in the water column within several days. In particular, degrees of aragonite saturation (Ωara) was shown from 0.6 (around seafloor) to 2.1 (surface). We used these natural seawater with different three conditions of Ωara (0.7, 1.4, and 2.1), and cultured L. helicina in nine culture vessels (batches) during two weeks.

The texture of shell surface of L. helicina showed remarkable changes under stereomicroscope within 48 hours from starting experiment: All shells of L. helicina kept hyaline under 2.1 and 1.4 of Ωara conditions. On the other hand, cloudy and whitish (damaged) shells were appeared under 0.7 Ωara condition. Seven days after, shell surface became whitish moderately in 1.4 Ωara conditions. It had never appeared whitish colored shells under 2.1 Ωara condition through the period of culture experiment. Onshore laboratory, all cultured shell density of L. helicina had analyzed to detect aragonite shell density by the Microfocus X-ray CT (MXCT). We will discuss about shell density changes during the culture experiment.

39. Adaptive capacity of the sea urchin Heliocidaris erythrogramma to ocean change: responses from fertilisation to the juvenile

Shawna A. Foo (1), Symon A. Dworjanyn (2), Alistair Poore (3), Januar Harianto (1), Maria Byrne (4)*

1 School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia
2 National Marine Science Centre, Southern Cross University, Coffs Harbour, New South Wales, Australia
3 Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, New South Wales, Australia
4 Schools of Medical and Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia

Background
To accurately predict impacts of ocean acidification and warming on the responses of marine populations, it is important to determine an organism’s capacity for phenotypic plasticity and the potential of species for genetic adaptation.

Methods
We determined the effects of near-future acidification and warming across the life cycle of Heliocidaris erythrogramma from fertilisation to metamorphosis in the progeny of 16 sire-dam crosses. Sources of variation in tolerance to warming (+3 °C) and acidification (-0.3-0.5 pH units) were investigated for fertilisation, larval success and juvenile metamorphosis.

Findings
Across all life stages, maternal legacy was important, with dam identity significantly interacting with stressors. Across the genotypes tested, fertilisation was negatively affected by increased temperature, but not pH. Larval development was compromised in low pH, but not temperature. By the settled juvenile stage, no impact of warming or acidification was evident and this was likely due to selective mortality of sensitive individuals. Across all environments tested, the juveniles exhibited a similar ability to calcify.

Conclusions
The impact of warming and acidification on development after fertilisation was influenced by parental identity, with the offspring of some dam-sire pairs more sensitive than others. That the progeny of some sire-dam pairs showed high stress tolerance indicates the potential for selection of resistant genotypes and adaptation that could facilitate the persistence of H. erythrogramma populations. Performance of progeny at one stage could not predict the performance later in development and shows the importance of assessing impacts of ocean change across the life cycle of marine invertebrates.

17. Two-current choice flumes for fish chemosensory behaviour – method validation and limited effects of high pCO2

Fredrik Jutfelt (1), Josefin Sundin (2), Graham D Raby (3), Timothy D Clark (4)

1 Norwegian University of Science and Technology, Trondheim, 7491, Norway
2 Uppsala University, Uppsala, 751 24, Sweden
3 Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, N9C1A2, Canada
4 University of Tasmania and CSIRO Agriculture Flagship, Hobart, 7000, Australia

Background
Ocean acidification has been suggested to disturb fish behavioural responses to chemosensory cues. Most experiments have investigated coral reef species, while the effects on species from other parts of the world are largely unknown. The methods used to quantify chemosensory behaviour in fish are variable and in need of standardisation.

Methods
Two-current choice flumes were constructed in a range of sizes to accommodate fish from 3 to 200 mm length. The flumes carry two parallel laminar water flows through an arena where the experimental animal can choose between the two flows, and the two flows can be manipulated (e.g. hypoxia, hypercapnia, prey/predatory cues). The methodology was validated using a range of flow, dye and pilot testing. To eliminate bias in behavioural observation we used automated video analysis.

Findings
Stable laminar flow can be difficult to maintain and can be disrupted by deviations in flow. Flow controllers, baffles and multiple layers of honeycomb collimators are vital components. Long observation times are needed to quantify side preference. Subconscious biases make objective observation difficult and blinded observation or automated video analysis is therefore needed.

Using the flumes we show that predator avoidance was high in both control and high CO2 (1000 μatm) exposed Atlantic cod. Furthermore, Atlantic cod strongly avoided high CO2 water, even after one month of acclimation to high CO2. In goldsinny wrasse, the predator avoidance was slightly reduced by high CO2 exposure compared to control.

Conclusions
We present a reliable flume methodology for measurements of chemosensory behaviour that provides stable laminar flow and unbiased behavioural quantification. We show that high CO2 appears to have limited effects on temperate fishes. We encourage the use of these approaches in all future studies to enable a comprehensive and robust understanding of any CO2 effects on the chemosensory behaviour of fish.

16. The impact of angel’s wing on pteropod population in the Gulf of Naples (Western Mediterranean)

Sergio d’Albero (1,2,3)*, Paola Rumolo (2), Angelo Bonanno (2), Enrico Dinelli (3), Clara Manno (1)

1 BAS (British Antarctic Survey), Cambridge, Cambridgeshire, CB30ET, UK
2 IAMC-CNR, Napoli, Campania, Calata Porta di Massa, 80133, Italy
3 University of Bologna, Dep. Of Marine Biology, Emilia Romagna, 48123, Italy

Background
Thecosomate Pteropods are particularly vulnerable to Ocean Acidification since their shells are made by of aragonite, a highly soluble form of biogenic carbonate in sea water. Marine areas with naturally high levels of carbon dioxide in the water provide a natural laboratory to study the adaptation strategy of calcifying organisms exposed to chronic low pH level. The Naples of Bay (Western Mediterranean) is a notable case where the presence of CO2 vents was already documented by local fishermans in the 70’s when they described the phenomenon of bubbles coming out from the sea surface as “the Wing of the Angel”. Despite several studies investigated the impact of the CO2 vents on calcifying benthic compartment, the effect on pelagic calcifying community, such as pteropods, is still poor understood.

Methods
Thecosomate Pteropods were collected during a cruise in two regions characterised by similar hydrological and biological regime but different carbonate chemistry regime (with and without the presence of CO2 vents). To understand the impact of high pCO2 -low pH levels on the pteropod population, the variability in biodiversity, abundance and shell quality of pteropods between the two regions were investigated. Shell morphology, dissolution and repair were examined using scanning electron microscopy.

Findings
Pteropod abundance and biodiversity were higher in the control than the CO2 vents stations. In all the stations the dominant pteropod was C. conica. We observed a difference in shell degradation of C. conica where in CO2 vent stations this pteropod presented higher shell dissolution and lower shell weight than those collected in the control stations. Moreover C. conica found at CO2 vent stations were smaller than those found in control conditions.

Conclusions
We suggest that C. conica could develop physiological and morphological change to counteract the higher energy demand due to the presence of chronic low pH exposition.

37. Toward improved modelling of CaCO3 dissolution in seawater

Jim E. Greenwood (1)*, Victor W. Truesdale (2)

1 CSIRO Oceans and Atmosphere, Perth, WA, 6014, Australia.
2 School of Life Sciences, Oxford-Brookes University, Oxford, OX3OBP, UK.

Background
Accurate representation of the dissolution kinetics of calcium carbonate in seawater is needed to reduce uncertainty in ocean carbon models. The dissolution mechanism is known to be complex, and despite having been studied over several decades, substantial disagreements remain as to the correct form, and accompanying parameter values, of the necessary rate equation. The use of a number of different experimental approaches to control the chemical background of the dissolving solution, and the application of several different models, has added to the confusion.

Methods
We apply a commonly used model of calcium carbonate dissolution to the Hawaiian Ocean Time-series site in the North Pacific, and test the sensitivity of calcite export to the suggested parameter values. We also present some new laboratory results for the dissolution of calcite and compare them against previous studies.

Findings
We find that large uncertainties in the export of CaCO3 can arise from the use of the most commonly used model of CaCO3 dissolution in the ocean, depending on the choice of parameter values reported by different workers. Meanwhile, our laboratory experiments confirm that the dissolution of calcite is non-ideal with respect to existing models. A new model that takes account of changes in surface reactivity is tested.

Conclusions
The activity of determining empirical rates for fieldwork purposes from arbitrary curve fittings to laboratory plots of rate versus concentration data has limited value. Instead, much more detailed experimentation is needed. We recommend that this follows strict guidelines recommended for chemical kinetics study.

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