Hypoxia and hypercapnia often accompany seawater warming and interactively alter marine ectotherm performance, potentially threatening their populations. To detail mechanistic responses, we investigated whole-animal physiology alongside cellular homeostasis in a species expected to be relatively robust to their impacts, the oyster Ostrea edulis. Acute warming alone and combined with hypercapnia and hypoxia (deadly trio, DT) started at 18°C, increasing stepwise by 2°C per 48 h until critical temperature was reached (34°C). Death of oysters started at a lower temperature under DT than under acute warming alone, but rates equalized by 34°C. Hemolymph PO2 in DT-exposed oysters was 29% lower at 18°C, but by 34°C, was only slightly lower than that in oysters subjected to acute warming alone. In both groups, resting metabolic rate (RMR) and heart rate rose with warming. Hemolymph PO2 was stable until 26°C, whence it declined. DT elicited a higher heart rate, which began to fall after ∼32°C, whereas heart rate in oysters subjected to acute warming continued rising. Relative increases in branchial metabolite levels of alanine and fumarate, profiled via 1H-NMR spectroscopy, indicated greater contributions of anaerobic metabolism in DT-exposed oysters. Gill tissue showed higher levels of the mitochondrial stabilizer sirtuin-5 (SIRT5) alongside higher antioxidative capacity under DT compared with acute warming, before declining at temperatures beyond 30°C. Muscle intracellular pH, gill heat shock protein 70 and metabolic profiles appeared unaffected by DT compared with warming alone. Our results suggest that DT places an additional energetic burden on the oyster, lowering the critical temperature. Nevertheless, tolerance patterns indicate resilience to DT, which may require a rebalancing of passive tolerance mechanisms, especially metabolic depression.

Increasing frequencies of heatwaves threaten marine ectotherm species but not all alike. In exposed habitats, some species rely on a higher capacity for passive tolerance at higher temperatures, thereby extending time-dependent survival limits. Here, we assessed how the involvement of the cardiovascular system in extended tolerance at the margins of the thermal performance curve is dependent on warming rate. We studied organismal and heart tissue cellular responses of the European oyster, Ostrea edulis, challenged by rapid warming (+2°C per hour) and gradual warming (+2°C per 24 h). Starting at 22°C, cardiac activity was monitored as temperature was increased, tracking cardiac performance curves. Hearts were collected at discrete temperatures to determine cardiomyocyte metabolic profiles. Heart rate peaked at a lower Arrhenius breakpoint temperatures (ABT) of 30.5°C under rapid warming versus 33.9°C under gradual warming. However, oysters survived to higher temperatures under rapid than under gradual warming, with half of oysters dying (LT50) by 36.9°C versus 34.8°C, respectively. As rapid warming passed 30°C, heart rate fell and cardiomyocyte metabolic profiles suddenly changed as oysters switched to anaerobic metabolism for survival. By 36°C, severe fluctuations in Krebs cycle-related metabolites accompanied cardiac failure. In contrast, oysters exposed to gradual warming made gradual, extensive adjustments to intracellular metabolic pathways, prolonging aerobic cardiomyocyte metabolism to higher temperatures. This extended survival duration and ABT, beyond which cardiac activity decreased sharply and ceased. Our results emphasize how the rate of warming forces a trade-off between temperature maxima and survival duration, via tissue- and cellular-level impacts. European oysters possess adaptations that enable extended tolerance and survival of intertidal populations.

Unfavourable climatic conditions challenge an animal's performance and fitness. We investigated how cellular homeostasis relates to whole-animal physiology in the marine bivalve Pecten maximus under warming (W), warming plus hypercapnia (WHc) or hypoxia (WHo), and the combination of all three drivers (deadly trio, DT). Starting at 14°C, temperatures were increased stepwise by 2°C per 48 h while gill tissue was sampled from experimental exposures to test for indicators of intracellular stress, including lipid peroxidation, protein damage and degradation, apoptosis and heat shock responses. Whole-animal water filtration rate, routine metabolic rate (RMR), haemolymph PO2, pseudofaeces ejection, mantle tissue intracellular acidosis and gill tissue antioxidative capacity were measured in W and DT exposures. Filtration peaked at a lower temperature under DT, when high pseudofaeces ejection suggested that ventilation was prioritized over feeding. Warming alone doubled RMR by 22°C, whereas DT increased RMR even further, reaching higher maxima by 20°C. Haemolymph PO2 was consistently lower under DT, implying that supply was less able to meet increasing demand. Warming to 26°C stimulated a gill tissue heat-shock response, accumulated ubiquitin conjugates and apoptosis, whereas adding hypoxia or hypercapnia suppressed apoptosis. DT suppressed both heat shock and apoptotic responses, with ubiquitin conjugates and branched-chain amino acids accumulating, and gills showing visible damage. Our findings indicate that climate drivers cumulatively block protection mechanisms, increase protein damage and block protein synthesis, thereby substantially reducing passive thermal tolerance and survival under extremes. The thermal tolerance of scallops is critically reduced under DT conditions, when mechanisms defending passive tolerance are exhausted at lower temperatures.

Subarctic fjord systems are facing a continuous temperature increase as well as more frequent and more intense marine heatwaves (MHWs). MHWs are periods of exceptionally high temperatures above the long-term average. In the Porsangerfjord (Northern Norway), the average water temperature ranges from 2.5 degrees C in March to 10 degrees C in August. MHWs frequently exceed the summer maximum by up to 6 degrees C with so far unknown ecological effects. The green sea urchin, Strongylocentrotus droebachiensis, is a key grazer on habitat-forming kelp in Subarctic fjord systems. At high abundances, sea urchins transform productive kelp beds through pronounced grazing into depleted sea urchin barrens. In a laboratory experiment, we investigated the influence of acute temperature change on the grazing activity and the metabolic performance of the green sea urchin. Grazing rates increased continuously from 2 to 10 degrees C but decreased again at 14 degrees C. At 22 degrees C, rapid decay of sea urchins were associated with changes of the metabolic energy state from aerobiosis to anaerobiosis. We propose two scenarios: Moderate warming might lead to the proliferation of the sea urchin population and increased grazing pressure on kelp forests, while extreme warming, as predicted by the end of the century, as well as the occurrence of extreme MHWs, may severely impair the sea urchin population and, in turn, might favour the proliferation of adaptable kelp species.

Current climate change, particularly ocean warming, will induce shifts in marine species distribution and composition, affecting the marine food web and, thus, trophic interactions. Analyses of the stable isotopes 13C and 15N are commonly used to detect trophic markers for food web analyses. With the current standard methods used in food web ecology, it is still challenging to identify potential changes in the uptake and utilization of trophic markers. In this work, we present a 13C-enrichment analysis by NMR spectroscopy to track the uptake and utilization of dietary carbon in a simple laboratory experiment of a primary producer and its consumer (algae and bivalve). In particular, we tested the hypothesis of a temperature-dependent use of dietary carbon by tracing the incorporation of 13C-atoms. Unicellular phytoplankton, Phaeodactilum tricornutum, was reared in a medium containing 13C-labeled bicarbonate. The accompanying 13C-NMR spectra of labeled P. tricornutum showed a specific profile of 13C-labeled compounds, including typical trophic markers such as the polyunsaturated omega-3 fatty acid eicosapentaenoic acid (EPA). Afterwards, 13C-labeled P. tricornutum was fed to King scallops, Pecten maximus, kept at two different temperatures (15°C and 20°C). Tissue-specific NMR spectra of P. maximus revealed elevated 13C-NMR signals, particularly of the fatty acid EPA in the digestive gland, which was not evident in muscle tissue. The comparison between the two temperatures indicated a change in trophic markers. At the higher temperature, less unsaturated fatty acids were detected in the digested gland, but increased 13C-labels in sugars were detected in the adductor muscle. This might indicate a change in the uptake and utilization of the trophic marker EPA in P. maximus due to a shift in energy conversion from favored beta-oxidation at colder temperatures to conversion from carbohydrates in the warmth. Our approach indicates that besides the accumulation of trophic markers, their incorporation and conversion are additional important factors for the reliable interpretation of trophic linkages under climate change.

Recently, we could show that scallops show limitations of muscular performance like a reduced force under ocean warming and acidification. However, the underlying mechanisms at the cellular level are not completely understood. Metabolomics has become a valuable tool to evaluate the responses of marine organisms to various stressors. In the present study we therefore used a semi-targeted, multi tissue NMR based metabolomic approach to analyze metabolite patterns in the Atlantic king scallop, Pecten maximus, that were long-term acclimated to different end of century conditions of ocean warming (OW), ocean acidification (OA) and their combination (OWA). We investigated tissue specific metabolic profiles and metabolite concentrations in frozen tissues from gills, mantle and phasic and tonic adductor muscle of P. maximus under present conditions using 1H-HR-MAS NMR spectroscopy. A set of 33 metabolites revealed a clear tissue-specific pattern which can be attributed to the individual functions of the respective tissue type. We then evaluated the impact of OW, OA and OWA on the metabolic profiles of the different tissues. OW was the main driver of the changes in metabolites. In particular, energy-related metabolites seem to play an important role in the physiological response of scallops to OW and OWA. In combination with pathway analysis and network exploration we propose a possible correlation between metabolic changes in the adductor muscle and limited swimming performance of P. maximus under future climate. While the metabolic response of the phasic muscle seems to mainly depend on net consumption of energy related metabolites such as ATP and phospho-L-arginine, the tonic muscle seems to rely on metabolizing specific amino acids and beta-oxidation to account for the elevated energetic requirements under ocean warming and acidification.

Climate change challenges marine organisms by constraining their temperature-dependent scope for performance, fitness, and survival. According to the concept of Oxygen and Capacity Limited Thermal Tolerance (OCLTT), the overall thermal performance curve relates to an organism’s aerobic power budget, its overall aerobic scope for growth, exercise, reproduction, and other performances. We hypothesize that physiological principles shaping tolerance in extant ecosystems have also been operative during climatic changes in the distant past. To compare response patterns in extant fauna and their palaeo-relatives, we started here by studying the metabolic background of performance in the European flat oyster Ostrea edulis at organismic and cellular levels, focusing on the acute thermal window and the metabolic changes towards upper lethal temperatures. We investigated the response of the oysters (pre-acclimated at 12 °C) to a short-term warming protocol (by 2 °C every 48 h) from 14 to 36 °C which we identified as the lethal temperature. At the organismic level, heart and filtration rates were recorded. Gill metabolites were studied by 1 H NMR spectroscopy to address thermal responses at the cellular level. Feeding activity by O. edulis (assessed by the filtration rates) was highest between 18 and 24 °C when overall energy expenditure (indicated by heart rate as a proxy for routine metabolic rate) was moderate. We conclude that this range reflects the thermal optimum of this species. Beyond 26 °C, the gill tissue of O. edulis became partly anaerobic, and cardiac dysfunction (arrhythmia) developed at 28 °C followed by an Arrhenius break point (30 °C). This mirrors performance constraints and indicates a wide temperature range of passive tolerance which may be a long-standing characteristic of ostreids supporting survival in extreme environments as well as during past and present climate oscillations.

In the ocean the main climate drivers affecting marine organisms are warming, hypercapnia, and hypoxia. We investigated the acute effects of warming (W), warming plus hypercapnia (WHc, ~1800 μatm CO2), warming plus hypoxia (WHo, ~12.1 kPa O2), and a combined exposure of all three drivers (Deadly Trio, DT) on king scallops (Pecten maximus). All exposures started at 14 °C and temperature was increased by 2 °C once every 48 h until the lethal temperature was reached (28 °C). Gill samples were taken at 14 °C, 18 °C, 22 °C, and 26 °C and analyzed for their metabolic response by 1H-nuclear magnetic resonance (NMR) spectroscopy. Scallops were most tolerant to WHc and most susceptible to oxygen reduction (WHo and DT). In particular under DT, scallops' mitochondrial energy metabolism was affected. Changes became apparent at 22 °C and 26 °C involving significant accumulation of glycogenic amino acids (e.g. glycine and valine) and anaerobic end-products (e.g. acetic acid and succinate). In line with these observations the LT50 was lower under the exposure to DT (22.5 °C) than to W alone (~ 25 °C) indicating a narrowing of the thermal niche due to an imbalance between oxygen demand and supply.

Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsin-like (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Cas-like to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L−1. The K M values ranged from 0.072 mmol L−1 for the Try-like activity of P. vannamei to 0.309 mmol L−1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing ~80–90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves-Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4-5 weeks to clean seawater (control) and to either 50 μg L(-1) Cu or 50 μg L(-1) Cd at one of three partial pressures of CO2 ( [Formula: see text] ∼ 395, ∼ 800 and ∼ 1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated [Formula: see text] enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated [Formula: see text] , but [Formula: see text] modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated [Formula: see text] . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure [Formula: see text] . The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated [Formula: see text] ) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated [Formula: see text] . Cellular energy status of oysters was maintained at all metal and CO2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia (∼ 800 μatm [Formula: see text] ) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO2 levels can modulate accumulation and physiological effects of metals in bivalves in a species-specific manner which can affect their fitness and survival during the global change in estuaries.

The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg2+, Zn2+, Cu2+, and Cd2+ on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg2+ and Cu2+ while Zn2+ had only a moderate effect and Cd2+ caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus.

Fluorophores have become a ubiquitous and powerful tool for life science researchers. They may be used as reporter groups of fluorogenic substrates needed for the determination of enzyme activities. These substrates are nonfluorescent in their initial “structure” and only through enzymatic cleavage have their fluorescent properties become apparent. Thereby, they are very sensitive and suitable to detect even very low enzyme activities. If fluorogenic substrates are designed for very specific research purpose, they are often very expensive or limited in quantity.

The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.

NanoDrop spectrophotometry and NanoDrop fluorospectrometry are used almost exclusively to determine the concentrations of nucleic acids and proteins. We propose that NanoDrop fluorospectrometry can also be applied for measuring enzyme activities using fluorogenic substrates such as the proteolytic activities of the 26S proteasome. Because the NanoDrop ND-3300 device requires only 2μl of sample, the amount of sample extract, substrate, and cofactors used for an enzyme assay can be significantly reduced. In this report, we present exemplary microassays for proteasomal activities (chymotrypsin-, trypsin-, and PGPH [peptidyl-glutamyl peptide hydrolase]-like sites) in extracts of isolated hemocytes from a marine crab, Cancer pagurus (Crustaceae).

Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40–75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg- and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1–3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates during larval development.