Thesis (M.E.S.)--The Evergreen State College, 2008. / Title from title screen (viewed 10/21/2009). Includes bibliographical references (leaves 62-72).

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As one of the most successful invasive species in the marine environment around the globe, the green crab Carcinus maenas possesses efficient regulatory mechanisms to quickly acclimate to environmental changes. The most important organs in this process are the nine pairs of gills that not only allow for osmoregulation, but have been shown to be involved in ammonia excretion and respiratory gas exchange. To date, however, little is known about the gills’ contribution to acid-base regulation that might become increasingly important in a “future ocean scenario” whereby surface ocean pH is predicted to drop by up to 0.5 units by the year 2100. The present thesis aims to characterize the green crab gills’ role in acid-base regulation and how it is linked to ammonia excretion. After exposure to hypercapnia (0.4 kPa pCO2 for 7 days), osmoregulating green crabs were capable of fully compensating for the resulting extracellular respiratory acidosis, while osmoconforming green crabs only partially buffered the accompanying drop in hemolymph pH after acclimation to 1% CO2 for 48 hours. Perfusion experiments on isolated green crab gills showed that different gills contributed to the excretion of H+ in an individual pattern and indicated that NH4+ is an important component of branchial acid excretion. Experiments on gill mRNA expression and pharmaceutical effects on isolated gills identified distinct epithelial transporters to play significant roles in branchial acid base regulation: Rhesus-like protein, basolateral bicarbonate transporter(s), cytoplasmic V-(H+)-ATPase, Na+/H+-exchanger, basolateral Na+/K+-ATPase, cytoplasmic and membrane bound carbonic anhydrase, and basolateral K+ channels. Regarding the latter, the present work provides the first sequence-based evidence for a potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel (CmHCN) capable of promoting NH4+ transport in the green crabs’ gill epithelium, and further demonstrates its direct involvement in branchial acid-base regulation. This highly conserved protein is a potentially important novel key-player in acid-base regulation in all animals. Interestingly, the observed principles linking acid-base to ammonia regulation in the decapod crustacean gill epithelium resemble many observations previously made in vertebrates. The data of the present thesis therefore provides valuable information for general acid-base regulation, while contributing substantially to our understanding of acid-base regulation in invertebrates. / February 2016

Since the beginning of the Industrial Revolution, carbon dioxide (CO2) has been emitted into the atmosphere at rates unprecedented to Earth’s history. Nearly 30% of the anthropogenic CO2 in the atmosphere has been absorbed in surface waters of the ocean, pushing carbonate chemistry towards increased bicarbonate ions and hydrogen protons and decreased carbonate ions. Consequently, seawater pH has decreased from pre-Industrial Revolution levels of 8.2 to current levels of 8.1, and it is expected to continue to drop to 7.8 by the year 2100 if carbon emissions continue as predicted. The combination of these effects is referred to as ocean acidification. It is at the forefront of marine research as it poses a serious threat to several marine organisms and ecosystems. Ocean acidification has the most notable direct effect on calcifying organisms with calcium carbonate skeletons and shells, because fewer carbonate ions in the water column result in reduced calcification. Coral reefs are especially vulnerable to ocean acidification since reefs are composed of complex carbonate structures. Coral reefs have a high biodiversity; thus, not only will the corals themselves be affected by ocean acidification, but so will many of the animals that dwell in them. The primary objective of this thesis was to examine the effects of ocean acidification on demersal zooplankton that reside in coral reefs. Ocean acidification research on zooplankton has primarily been single- species experiments on calcifying species or generalist copepod species. Scaling-up to experiments examining ocean acidification effects on entire zooplankton communities is logistically difficult, thus the ability to predict community changes in zooplankton due to ocean acidification has been rather limited. However, a few locations around the world have submarine volcanic CO2 seeps that can be used as natural laboratories to study ecosystem effects of ocean acidification. Two CO2 seeps located in coral reefs in Papua New Guinea were used as windows into the future to examine the effects of ocean acidification on entire zooplankton communities while they live naturally in their environment. Over three expeditions to two CO2 seeps, nocturnal plankton were sampled with horizontal net tows and emergence traps. Additional experiments were also conducted, and collectively this work is summarized in chapters 2-5 as outlined below. Chapter 2 reports on the observed changes in zooplankton abundance and community composition between control and high-CO2 sites. Consistent results between seep sites and expeditions showed that zooplankton abundances were reduced three-fold under high-CO2 conditions. The abundance loss was partially attributed to habitat change within the coral reef, from more structurally complex corals in the control sites to a replacement of massive bouldering corals in the high-CO2 sites. The loss of structural complexity in the reef meant there were fewer hiding spaces for the zooplankton to seek refuge in during the day. All zooplankton taxa were reduced under high-CO2 conditions but to varying levels, suggesting that each taxon reacts differently to ocean acidification. Since each taxonomic group within the zooplankton communities was reduced to varying levels under ocean acidification, the copepod genus with the largest reduction in abundance was investigated in more detail. Labidocera spp. are pontellid copepods that are generally considered surface-dwellers and are not known to inhabit coral reefs. Therefore, as a preface to the ocean acidification study, the new discovery of these copepods living in coral reefs is first described (Chapter 3). Not only were they found to be residential to the reef, but Labidocera spp. living at the control reefs preferred to reside in coral rubble, macroalgae, and turf algae. Labidocera spp. were one of the most sensitive copepods to high-CO2 conditions and were reduced by nearly 70% in abundance, prompting a more detailed investigation about the effect of ocean acidification on their physiology and habitat preference (Chapter 4). Physiological parameters, e.g. size, feeding, and oocyte development, were unaffected by ocean acidification. Unlike the zooplankton community as a whole, the main cause for the abundance loss of Labidocera spp. was not a shift in the habitat because their preferred substrata were of equal percent coverage across high-CO2 and control sites. Instead, Labidocera spp. were no longer associated with any substrata type. Multiple direct and indirect effects of ocean acidification will act on each zooplankton taxa separately, and their collective response will contribute to the community response. The effects of ocean acidification on zooplankton communities were then scaled up to potential impacts on entire ecosystems. Zooplankton are the primary food source for corals, fish, and other zooplanktivores. The impacts of ocean acidification on zooplankton communities will have cascade effects on the food chain via the pathway of zooplanktivorous organisms. A case study on the stony coral Galaxea fascicularis explored the effects of ocean acidification on the ability of corals, which had lived their entire lives under high-CO2 conditions, to feed on zooplankton (Chapter 5). Under anthropogenic changes, whether it is from bleaching, high turbidity, or ocean acidification, some corals rely more on heterotrophy and consume more zooplankton. Contrary to expectation, this study showed that when given equal quantities of food particles these corals consumed less zooplankton under ocean acidification. Corals rely on heterotrophy for essential nutrients, like nitrogen and phosphorus, which they cannot otherwise obtain from autotrophy and their symbiotic zooxanthellae. In conclusion, my thesis shows that not only is there fewer zooplankton available to consume, but the existing zooplankton is consumed with lower capture rates under high CO2 conditions. Coral reefs in future oceans will likely have reduced zooplankton abundances as an indirect effect of ocean acidification, partially caused by a change in habitat from branching corals to more massive bouldering corals. Zooplankton abundances were reduced yet the community composition was unaffected by ocean acidification. All zooplankton taxa were reduced yet present under high-CO2 conditions suggesting that the zooplankton are at least able to survive under ocean acidification. Fewer zooplankton will be available to zooplanktivores, but the fatty acid content and nutritional value of the zooplankton as a food source is expected to be similar to current food. Together this is expected to negatively impact the entire coral reef ecosystem, with some coral species unable to consume zooplankton at normal rates. In an ecosystem already highly vulnerable to ocean acidification, coral reefs may be even more threatened if the very basis of their food webs is reduced.

Ocean acidification and warming have been shown to affect a wide range of marine organisms and impact assemblages and ecosystems. Many of the species experiencing negative biological effects provide valuable ecosystem services, yet it is unclear how these biological effects will affect ecosystem services provision. This thesis aimed to appraise the consequences of ocean acidification and warming on important shellfish species, from physiology to provision of ecosystem services, using a multidisciplinary approach. The responses to ocean acidification and warming of two ecologically and commercially important species of oysters – the native European Flat oyster Ostrea edulis, and the non-native Pacific oyster Magallana gigas – were assessed in laboratory mesocosms following long-term exposures to a range of scenarios predicted for 2050 and 2100. Oysters provide numerous ecosystem services, including improvement of water quality, reef formation, and food provision, but are at risks from ocean acidification and other stressors due to negative impacts occurring at multiple life-stages and threatening reef maintenance and functioning (Chapter 1). The physiology of adult oysters appeared susceptible to ocean acidification and warming, with evident sub-lethal effects (Chapter 2). Magallana gigas experienced a greater degree of stress than O. edulis, displaying increased Standard Metabolic Rate, reduced Clearance Rate, and poorer Condition Indices. Reductions in Clearance Rates of M. gigas are especially concerning and may have important ecological impacts by limiting their ability to improve water quality in the future. The physiological changes experienced by individual oysters held important implications for the functioning of the reefs through changes in predation resistance. Again, M. gigas appeared to undergo more pronounced changes than O. edulis, displaying increased muscle strength but weakened shell strength. These changes are expected to alter its susceptibility to predators and influence community level interactions. Both O. edulis and M. gigas also underwent important changes to their biochemical composition with trends for impoverished nutritional quality, which holds direct implications on the provision of sea food. In particular, M. gigas contained lower lipid, carbohydrate, and protein levels, but higher contaminant concentration (copper); this change holds concerns for both future food security and future food safety. It was apparent that the physiological stress experienced (Chapter 2), led to significant energy reallocation from somatic growth to metabolism by depleting energetic reserves (Chapter 4), at the detriment of its nutritional quality. No negative effects on the eating quality of M. gigas (appearance, aroma, texture, taste, and overall acceptability) were recorded following a short-term exposure to ocean acidification and warming (Chapter 5), which was considered positive for the aquaculture sector. In order to secure future food provision and economic revenue, the UK aquaculture industry might need to reconsider its management strategy in the future, and encourage the production and consumption of O. edulis, in addition to the already popular M. gigas. It is clear that the impacts of ocean acidification and warming on oysters are multifaceted and occurring at multiple scales and levels of organisation. The risks to oysters and oyster reefs appear species-specific; in the UK, introduced M. gigas may be more vulnerable than native O. edulis. To secure benefits and minimise costs related to the management of introduced species, these findings could be integrated into the current management and conservation measures in place for these species and the reefs they can form.

Hypoxia and ocean acidification (OA) are amongst the major environmental threats to marine ecosystems worldwide. Biofilms, the signpost to guide larval settlement of many benthic invertebrates, are known to be responsive to environmental changes and thus can become the crucial factor for the response of benthic invertebrate communities. This study aimed at investigating the individual and interactive effects of hypoxia and OA on biofilms and the subsequent effects on larval settlement. Biofilms collected from two sites (clean, hypoxic) were treated with a factorial design of low dissolved oxygen and/or low pH conditions in microcosms and the bacterial cell density and viability (by LIVE/DEAD® cell viability assays) were analyzed. Larval settlement preference was tested with the marine invertebrate, Crepidula onyx. The total bacterial cell densities of biofilms of the hypoxia and hypoxia and OA combination treatment were lower than that of the control biofilms for both sites. There was generally no significant difference in cell viability among control and different treatments for both sites. While the larval settlement rate on hypoxia and hypoxia and OA combination treated biofilms was significantly lower. In conclusion, this study revealed that hypoxia and OA are likely to affect larval settlement by alteration of biofilms, and this may lead to alterations in future coastal communities.

A total descontaminação do sistema de canais radiculares e da massa dentinária é uma constante preocupação clínica. Diante disso, esta pesquisa teve o objetivo de avaliar o nível de descontaminação dentinária alcançada após irrigação com soluções de hipoclorito de sódio em diferentes concentrações valores de pH. Oitenta dentes bovinos unirradiculados foram divididos em 9 grupos experimentais diferentes. As coroas foram seccionadas e despregadas das raízes. Foram obtidos segmentos de 12mm e os canais instrumentados até a lima K120, preenchidos com EDTA 17%, durante 10 minutos. As raízes foram impermeabilizadas externamente com duas camadas de esmalte. Suspensões de Enterococcus faecalis (ATCC 29212) foram padronizadas em espectrofotômetro (3x108 UFC/mL) e depositadas em microtubos com caldo BHI e um espécime. O protocolo de contaminação seguiu a metodologia de MA et al. (2011) com adaptações. Após 5 dias, os espécimes foram fixados em um dispositivo de apoio esterilizado e irrigados durante 5 minutos com as soluções-teste estabilizadas com tampões: (G1) NaOCl 1% - pH5; (G2) NaOCl 1% - pH7; (G3) NaOCl 1% - pH10; (G4) NaOCl 2,5% - pH5; (G5) NaOCl 2,5% - pH 7; (G6) NaOCl 2,5% - pH10; (G7) NaOCl 5% - pH5; (G8) NaOCl 5% - pH7; (G9) NaOCl 5% - pH10, contendo 8 espécimes cada, além dos grupos controles. Após o tratamento com as soluções irrigadoras, metade dos espécimes é avaliada por cultura microbiológica (contagem de unidades formadoras de colônias UFC/mL das raspas de dentina retiradas das paredes dos canais) e a outra metade do mesmo grupo avaliada por microscopia confocal de varredura a laser (MCVL) e corante Live & Dead. Houve diferença estatística entre diversos grupos, analisados pelos testes de Kruskal-Wallis e Dunn. (p<0,05) Concluiu-se que a solução de hipoclorito de sódio acidificada provocou uma redução significante no número de bactérias. Essa redução foi ainda maior quando a concentração da solução era elevada. / The complete decontamination of root canal system and dentinal mass is a constant clinical concern. Therefore, this study aimed to evaluate the level of dentin decontamination achieved after irrigation with sodium hypochlorite solutions at different concentrations and pH values. Eighty single rooted bovine teeth were divided into 9 different groups. The crowns were sectioned and separated from the roots. Then, there was obtained segments of 12mm and the root canals were instrumented until size #120 K-file and filled with 17% EDTA during 10 minutes. The roots were externally sealed with two layers of nail polish. Suspensions of Enteroccus faecalis (ATCC 29212) were standardized using a spectrophotometer (3x108 CFU/mL) and placed in microtubes containing BHI broth and one sample. The protocol of contamination followed the methodology of MA et al. (2011) with some adjustments. After five days, the samples were fixed in a sterilized device and irrigated during 5 minutes with the tested solutions stabilized by buffers substances: (G1) 1% NaOCl - pH5; (G2) 1% NaOCl - pH7; (G3) NaOCl 1% - pH10; (G4) 2.5% NaOCl - pH5, (G5) 2.5% NaOCl - pH 7, (G6) 2.5% NaOCl - pH10; (G7) 5% NaOCl - pH5; (G8 ) NaOCl 5% - pH 7 (G9) 5% NaOCl - pH10, containing 8 specimens each, and the control groups. After the treatment with the irrigating solutions, half of the specimens were evaluated by microbiological cultures (counting colony forming units CFU/mL of dentine chips removed from the wall of root canals) and the other half of the same group were evaluated by confocal laser scanning microscopy (CLSM) with fluorescent Live & Dead stain. There was statistical difference among various groups (p<0.05) by Kruskal-Wallis and Dunns test. It was concluded that the acidified sodium hypochlorite solution resulted in a significant reduction in the number of bacteria.

Anthropogenic carbon dioxide (CO2) emissions are increasing the atmospheric CO2 concentration and the oceans are absorbing around 1/3 them. The CO2 hydrolysis increases the H+ concentration, decreasing the pH, while the proportions of the HCO3- and CO32- ions are also affected. This process already led to a decrease of 0.1 pH units in surface seawater. According to "business-as-usual" models, provided by the Intergovernmental Panel on Climate Change (IPCC), the pH is expected to decrease 0.3-0.5 units by 2100 and 0.7-0.8 by 2300. As a result the surface ocean carbonates chemistry will also change: with increasing pCO2, dissolved inorganic carbon will increase and the equilibrium of the carbonate system will shift to higher CO2 and HCO3– levels, while CO32– concentration will decrease. Surface seawaters will progressively become less saturated towards calcite and aragonite saturation state and some particular polar and cold water regions could even become completely undersaturated within the next 50 years. <p>Responses of marine organisms to environmental hypercapnia, i.e. to an excess of CO2 in the aquatic environment, can be extremely variable and the degree of sensitivity varies between species and life stages. Sea urchins are key stone species in many marine ecosystems. They are considered to be particularly vulnerable to ocean acidification effects not only due to the nature of their skeleton (magnesium calcite) whose solubility is similar or higher than that of aragonite, but also because they lack an efficient ion regulatory machinery, being therefore considered poor acid-base regulators. Populations from polar regions are expected to be at an even higher risk since the carbonate chemical changes in surface ocean waters are happening there at a faster rate. <p>The goal of this work was to study the effects of low seawater pH exposure of different life stages of sea urchins, in order to better understand how species from different environments and/or geographic origins would respond and if there would be scope for possible adaptation and/or acclimatization.<p>In a first stage we investigated the effects of ocean acidification on the early stages of an intertidal species from temperate regions, the Atlantic Paracentrotus lividus sea urchin, and of a sub-Antarctic species, Arbacia dufresnei. The fertilization, larval development and larval growth were studied on specimens submitted through different pH experimental treatments. The fertilization rate of P. lividus gametes whose progenitors came from a tide pool with high pH decrease was significantly higher, indicating a possible acclimatization or adaptation of gametes to pH stress. Larval size in both species decreased significantly in low pH treatments. However, smaller A. dufresnei echinoplutei were isometric to those of control treatments, showing that size reduction was most likely due to a slower growth rate. In the pH 7.4 (predicted for 2300) treatment, P. lividus presented significantly more abnormal forms than control ones, but A. dufresnei did not. The latter does not seem to be more vulnerable than temperate species, most likely due to acclimatization/adaptation to lower pH seasonal fluctuations experienced by individuals of this population during spring time.<p>In a second stage, adult physiological responses of P. lividus and A. dufresnei to low pH seawaters were studied. Intertidal field P. lividus specimens can experience pH fluctuations of 0.4 units during low tidal cycles, but their coelomic fluid pH will not change. During experimental exposure to low pH, the coelomic fluid (extracellular) pH of both species decreased after weeks of exposure to low seawater pH. However, it owned a certain buffer capacity (higher than that of seawater) which did not seem to be related to passive skeleton dissolution. In laboratory studies, the feeding rate of P. lividus, the RNA/DNA ratio (proxy for protein synthesis and thus metabolism) of both the gonads and the body wall of the studied species and the carbonic anhydrase activity in the body wall (an enzyme involved in calcification and respiratory processes) of A. dufresnei did not differ according to seawater pH. The same was true for spine regeneration (a proxy for calcification) of both species. This shows that both P. lividus and A. dufresnei are able to cope when exposed to mild hypercapnia (lowest investigated pH 7.4) for a mid-term period of time (weeks). In a different set of experiments, pH effects were tested on P. lividus individuals together with two temperatures (10ºC and 16ºC). The pH decrease of the coelomic fluid did not vary between temperatures, neither did its buffer response. The oxygen uptake rates of P. lividus (as a proxy for global metabolic state of the whole organism) increased in lower pH treatments (7.7 and 7.4) in organisms exposed to lower temperatures (10ºC), showing that this was upregulated and that organisms experienced a higher energetic demand to maintain normal physiological functions. For instance, gonad production (given by the RNA/DNA ratio) was not affected neither by temperature, nor pH.<p>Finally, possible morphological and chemical adaptations of cidaroid (“naked”) spines, which are not covered by epidermis, to low magnesium calcite saturation states were investigated. Deep sea field specimens from the Weddell Sea (Antarctica), Ctenocidaris speciosa were studied. Cidaroid spines have an exterior skeleton layer with a polycrystalline constitution that apparently protects the interior part of the monocrystaline skeleton, the stereom (tridimensional magnesium calcite lattice). The cortex of C. speciosa was by its turn divided into two layers. From these, it presented a thicker inner cortex layer and a lower Mg content in specimens collected below the aragonite saturation horizon. The naked cortex seems able to resist to low calcium carbonate saturation state. We suggest that this could be linked to the important organic matrix that surrounds the crystallites of the cortex.<p>Some echinoid species present adaptive features that enable them to deal with low pH stresses. This seems to be related to the environmental conditions to which populations are submitted to. Therefore, organisms already submitted to pH daily or seasonal fluctuations or living in environments undersaturated in calcium carbonate seem to be able to cope with environmental conditions expected in an acidified ocean. Under the realistic scenario of a decrease of ca. 0.4 units of pH by 2100, sea urchins, and echinoderms in general, appear to be robust for most studied processes. Even thought, this general response can depend on different parameters such as exposure time, pH level tested, the process and the life stage considered, our results show that there is scope for echinoids to cope with ocean acidification.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Ocean acidification may cause biodiversity loss, alter ecosystems and impact food security, yet uncertainty over ecological responses to ocean acidification remains considerable. Most work on the impact of ocean acidification on foraminifera has been short-term laboratory experiments on single species. To expand this, benthic foraminiferal assemblages were examined across shallow water CO2 gradients in the Gulf of California, off the islands of Ischia and Vulcano in Italy and off Papua New Guinea. Living assemblages from the Gulf of California did not appear to show a response across a pH range of 7.55 – 7.88, although the species assemblage was impoverished in all locations and the dead assemblage was less diverse at the lowest pH sites where there was evidence of post mortem dissolution. At Vulcano, the small macroalga, Padina pavonica, did not protect calcareous foraminifera from the adverse effects of ocean acidification. Calcareous taxa disappeared from the assemblage and were replaced by agglutinated foraminifera as mean pH reduced from 8.19 to 7.71. Settlement of benthic foraminifera onto artificial collectors off Vulcano was adversely affected in the acidified water, with few species as pCO2 increased and evidence of post-mortem dissolution. The foraminiferal tests, collected off Papua New Guinea, had lower δ11B as mean pH decreased from 7.99 – 7.82 for small (250 – 500 µm) Amphistegina lessonii, but not for A. lessonii or Calcarina spengleri >500 µm. In the larger foraminifera, photosynthetic activity by symbionts may begin to dominate the boron isotopic signature. Overall, the responses of foraminiferal assemblages to ocean acidification are complex, but there was an overall reduction in species diversity in infaunal, epifaunal and epiphytic assemblages as pCO2 increased. This raises serious concerns for the survival of shallow water calcareous benthic foraminifera as the oceans continue to acidify, with implications for benthic ecosystems and inorganic carbon cycling.

Elevated atmospheric concentrations of carbon dioxide (CO₂), partly driven by anthropogenic activity, are decreasing the pH of the oceans. This thesis aimed to assess the biological response of foraminifera to ocean acidification. Foraminifera are single-celled organisms that form the dominant component of many marine communities. A series of laboratory experiments were carried out on benthic intertidal foraminifera from the Eden and Ythan estuaries, NE Scotland, to assess the impacts of ocean acidification. The responses of two dominant intertidal species of foraminifera (Haynesina germanica and Elphidium williamsoni) to ocean acidification were initially investigated in a short-term (6 week) experiment. Multiple species and multiple stressors (seasonal temperature regime and elevated CO₂) were then incorporated in a long-term (18 month) mesocosm study to investigate the physiological consequences (e.g. survival, growth) of ocean acidification. Survival of both Haynesina germanica and Elphidium williamsoni was significantly reduced under low pH conditions. Live specimens of both these calcareous species were however recorded at low pH, in reduced numbers. Following long-term exposure to ocean acidification, foraminiferal populations were still dominated by calcareous forms. Agglutinated foraminifera were recorded throughout the long-term incubations but their numbers were not high enough in the initial sediment collections to allow them to contribute significantly to the populations. Overall, survival of all foraminifera was greatly reduced in elevated CO₂ treatments. Temperature effects were observed on foraminiferal survival and diversity with the largest CO₂ effects recorded under the seasonally varying temperature regime. Foraminiferal test damage for all live species was also highest under elevated CO₂ conditions. Test dissolution was particularly evident in Haynesina germanica with important morphological features, such as functional ornamentation, becoming reduced or completely absent under elevated CO₂ conditions. A reduction in functionally important ornamentation could lead to a reduction in feeding efficiency with consequent impacts on this organism's survival and fitness. In addition, changes in the relative abundance and activities of these important species could affect biological interactions (e.g. food web function) and habitat quality.