The silent loss of cell physiology hampers marine biosciences.

An ongoing loss of experts in marine cellular biochemistry and physiology (CBP) is stagnating the generation of knowledge upon which rapidly growing "omics" approaches rely, ultimately hampering our ability to predict organismal responses to climate change.

Transgenerational exposure to ocean acidification impacts the hepatic transcriptome of European sea bass (Dicentrarchus labrax).

Physiological effects of ocean acidification associated with elevated CO2 concentrations in seawater is the subject of numerous studies in teleost fish. While the short time within-generation impact of ocean acidification (OA) on acid-base exchange and energy metabolism is relatively well described, the effects associated with transgenerational exposure to OA are much less known. Yet, the impacts of OA can vary in time with the potential for acclimation or adaptation of a species. Previous studies in our lab demonstrated that transgenerational exposure to OA had extensive effects on the transcriptome of the olfactory epithelium of European sea bass (Dicentrarchus labrax), especially on genes related to ion balance, energy metabolism, immune system, synaptic plasticity, neuron excitability and wiring. In the present study, we complete the previous work by investigating the effect of transgenerational exposure to OA on the hepatic transcriptome of European sea bass. Differential gene expression analysis was performed by RNAseq technology on RNA extracted from the liver of two groups of 18 months F2 juveniles that had been exposed since spawning to the same AO conditions as their parents (F1) to either actual pH or end-of-century predicted pH levels (IPCC RCP8.5), respectively. Here we show that transgenerational exposure to OA significantly impacts the expression of 236 hepatic transcripts including genes mainly involved in inflammatory/immune responses but also in carbohydrate metabolism and cellular homeostasis. Even if this transcriptomic impact is relatively limited compared to what was shown in the olfactory system, this work confirmed that fish transgenerationally exposed to OA exhibit molecular regulation of processes related to metabolism and inflammation. Also, our data expand the up-regulation of a key gene involved in different physiological pathways including calcium homeostasis (i.e. pthr1), which we already observed in the olfactory epithelium, to the liver. Even if our experimental design does not allow to discriminate direct within F2 generation effects from transgenerational plasticity, these results offer the perspective of more functional analyses to determine the potential physiological impact of OA exposure on fish physiology with ecological relevance.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Windows of opportunity: Ocean warming shapes temperature-sensitive epigenetic reprogramming and gene expression across gametogenesis and embryogenesis in marine stickleback.

Rapid climate change is placing many marine species at risk of local extinction. Recent studies show that epigenetic mechanisms (e.g. DNA methylation, histone modifications) can facilitate both within and transgenerational plasticity to cope with changing environments. However, epigenetic reprogramming (erasure and re-establishment of epigenetic marks) during gamete and early embryo development may hinder transgenerational epigenetic inheritance. Most of our knowledge about reprogramming stems from mammals and model organisms, whereas the prevalence and extent of reprogramming among non-model species from wild populations is rarely investigated. Moreover, whether reprogramming dynamics are sensitive to changing environmental conditions is not well known, representing a key knowledge gap in the pursuit to identify mechanisms underlying links between parental exposure to changing climate patterns and environmentally adapted offspring phenotypes. Here, we investigated epigenetic reprogramming (DNA methylation/hydroxymethylation) and gene expression across gametogenesis and embryogenesis of marine stickleback (Gasterosteus aculeatus) under three ocean warming scenarios (ambient, +1.5 and +4°C). We found that parental acclimation to ocean warming led to dynamic and temperature-sensitive reprogramming throughout offspring development. Both global methylation/hydroxymethylation and expression of genes involved in epigenetic modifications were strongly and differentially affected by the increased warming scenarios. Comparing transcriptomic profiles from gonads, mature gametes and early embryonic stages showed sex-specific accumulation and temperature sensitivity of several epigenetic actors. DNA methyltransferase induction was primarily maternally inherited (suggesting maternal control of remethylation), whereas induction of several histone-modifying enzymes was shaped by both parents. Importantly, massive, temperature-specific changes to the epigenetic landscape occurred in blastula, a critical stage for successful embryo development, which could, thus, translate to substantial consequences for offspring phenotype resilience in warming environments. In summary, our study identified key stages during gamete and embryo development with temperature-sensitive reprogramming and epigenetic gene regulation, reflecting potential 'windows of opportunity' for adaptive epigenetic responses under future climate change.

The EFFect of dietary fat content on the recurrence of pancreaTitis (EFFORT): Protocol of a multicenter randomized controlled trial.

BACKGROUND: Around 20% of patients with acute pancreatitis (AP) will develop acute recurrent pancreatitis (ARP) and 10% will progress to chronic pancreatitis. While interventions to avoid recurrences exist for the two most common causes - abstinence for alcoholic and cholecystectomy for biliary pancreatitis - the are no known preventive measures in idiopathic ARP. Though it is not included in any of the guidelines, a low-fat diet is often recommended. Our aim is to test dietary fat reduction's effect on AP recurrence in a randomized controlled setting, in order to provide high-quality evidence for the validity of such an intervention. METHODS, DESIGN: Participants with at least 2 episodes of AP in the preceding 2 years of which the last episode was idiopathic will be randomized to one of two diets with different fat contents: a 'reduced fat diet' (15% fat, 65% carbohydrate, 20% protein) and a 'standard healthy diet' (30% fat, 50% carbohydrate, 20% protein; based on WHO recommendations). Participants will be followed-up for 2 years (visits will be scheduled for months 3, 6, 12, 18 and 24) during which they will receive a repeated session of nutritional guidance, complete food frequency questionnaires and data on relapse, mortality, BMI, cardiovascular parameters and serum lipid values will be collected. DISCUSSION: This study will determine the effect of modifying the dietary fat content on AP recurrence, mortality, serum lipids and weight loss in idiopathic cases.

Effects of ocean acidification over successive generations decrease resilience of larval European sea bass to ocean acidification and warming but juveniles could benefit from higher temperatures in the NE Atlantic.

European sea bass (Dicentrarchus labrax) is a large, economically important fish species with a long generation time whose long-term resilience to ocean acidification (OA) and warming (OW) is not clear. We incubated sea bass from Brittany (France) for two generations (>5 years in total) under ambient and predicted OA conditions (PCO2: 650 and 1700 µatm) crossed with ambient and predicted OW conditions in F1 (temperature: 15-18°C and 20-23°C) to investigate the effects of climate change on larval and juvenile growth and metabolic rate. We found that in F1, OA as a single stressor at ambient temperature did not affect larval or juvenile growth and OW increased developmental time and growth rate, but OAW decreased larval size at metamorphosis. Larval routine and juvenile standard metabolic rate were significantly lower in cold compared with warm conditioned fish and also lower in F0 compared with F1 fish. We did not find any effect of OA as a single stressor on metabolic rate. Juvenile PO2,crit was not affected by OA or OAW in both generations. We discuss the potential underlying mechanisms resulting in the resilience of F0 and F1 larvae and juveniles to OA and in the beneficial effects of OW on F1 larval growth and metabolic rate, but contrastingly in the vulnerability of F1, but not F0 larvae to OAW. With regard to the ecological perspective, we conclude that recruitment of larvae and early juveniles to nursery areas might decrease under OAW conditions but individuals reaching juvenile phase might benefit from increased performance at higher temperatures.

Early occurrence of pseudocysts in acute pancreatitis - A multicenter international cohort analysis of 2275 cases.

BACKGROUND: Pseudocysts being the most frequent local complications of acute pancreatitis (AP) have substantial effect on the disease course, hospitalization and quality of life of the patient. Our study aimed to understand the effects of pre-existing (OLD-P) and newly developed (NEW-P) pseudocysts on AP. METHODS: Data were extracted from the Acute Pancreatitis Registry organized by the Hungarian Pancreatic Study Group (HPSG). 2275 of 2461 patients had uploaded information concerning pancreatic morphology assessed by imaging technique. Patients were divided into "no pseudocyst" (NO-P) group, "old pseudocyst" (OLD-P) group, or "newly developed pseudocyst" (NEW-P) groups. RESULTS: The median time of new pseudocyst development was nine days from hospital admission and eleven days from the beginning of the abdominal pain. More NEW-P cases were severe (15.9% vs 4.7% in the NO-P group p < 0.001), with longer length of hospitalization (LoH) (median: 14 days versus 8 days, p < 0.001), and were associated with several changed laboratory parameters. OLD-P was associated with male gender (72.2% vs. 56.1%, p = 0.0014), alcoholic etiology (35.2% vs. 19.8% in the NO-P group), longer hospitalization (median: 10 days, p < 0.001), a previous episode of AP (p < 0.001), pre-existing diagnosis of chronic pancreatitis (CP) (p < 0.001), current smoking (p < 0.001), and increased alcohol consumption (unit/week) (p = 0.014). CONCLUSION: Most of the new pseudocysts develop within two weeks. Newly developing pseudocysts are associated with a more severe disease course and increased length of hospitalization. Pre-existing pseudocysts are associated with higher alcohol consumption and smoking. Because CP is more frequently associated with a pre-existing pseudocyst, these patients need closer attention after AP.

Glucose levels show independent and dose-dependent association with worsening acute pancreatitis outcomes: Post-hoc analysis of a prospective, international cohort of 2250 acute pancreatitis cases.

BACKGROUND: Metabolic risk factors, such as obesity, hypertension, and hyperlipidemia are independent risk factors for the development of various complications in acute pancreatitis (AP). Hypertriglyceridemia dose-dependently elicits pancreatotoxicity and worsens the outcomes of AP. The role of hyperglycemia, as a toxic metabolic factor in the clinical course of AP, has not been examined yet. METHODS: We analyzed a prospective, international cohort of 2250 AP patients, examining associations between (1) glycosylated hemoglobin (HbA1c), (2) on-admission glucose, (3) peak in-hospital glucose and clinically important outcomes (mortality, severity, complications, length of hospitalization (LOH), maximal C-reactive protein (CRP)). We conducted a binary logistic regression accounting for age, gender, etiology, diabetes, and our examined variables. Receiver Operating Characteristic Curve (ROC) was applied to detect the diagnostic accuracy of the three variables. RESULTS: Both on-admission and peak serum glucose are independently associated with AP severity and mortality, accounting for age, gender, known diabetes and AP etiology. They show a dose-dependent association with severity (p < 0.001 in both), mortality (p < 0.001), LOH (p < 0.001), maximal CRP (p < 0.001), systemic (p < 0.001) and local complications (p < 0.001). Patients with peak glucose >7 mmol/l had a 15 times higher odds for severe AP and a five times higher odds for mortality. We found a trend of increasing HbA1c with increasing LOH (p < 0.001), severity and local complications. CONCLUSIONS: On-admission and peak in-hospital glucose are independently and dose-dependently associated with increasing AP severity and mortality. In-hospital laboratory control of glucose and adequate treatment of hyperglycemia are crucial in the management of AP.

Early changes in laboratory parameters are predictors of mortality and ICU admission in patients with COVID-19: a systematic review and meta-analysis.

Despite the growing knowledge of the clinicopathological features of COVID-19, the correlation between early changes in the laboratory parameters and the clinical outcomes of patients is not entirely understood. In this study, we aimed to assess the prognostic value of early laboratory parameters in COVID-19. We conducted a systematic review and meta-analysis based on the available literature in five databases. The last search was on July 26, 2020, with key terms related to COVID-19. Eligible studies contained original data of at least ten infected patients and reported on baseline laboratory parameters of patients. We calculated weighted mean differences (WMDs) for continuous outcomes and odds ratios (ORs) with 95% confidence intervals. 93 and 78 studies were included in quantitative and qualitative syntheses, respectively. Higher baseline total white blood cell count (WBC), C-reactive protein (CRP), lactate-dehydrogenase (LDH), creatine kinase (CK), D-dimer and lower absolute lymphocyte count (ALC) (WMDALC = - 0.35 × 109/L [CI - 0.43, - 0.27], p < 0.001, I2 = 94.2%; < 0.8 × 109/L, ORALC = 3.74 [CI 1.77, 7.92], p = 0.001, I2 = 65.5%) were all associated with higher mortality rate. On admission WBC, ALC, D-dimer, CRP, LDH, and CK changes could serve as alarming prognostic factors. The correct interpretation of laboratory abnormalities can guide therapeutic decisions, especially in early identification of potentially critical cases. This meta-analysis should help to allocate resources and save lives by enabling timely intervention.

High gene flow in polar cod (Boreogadus saida) from West-Svalbard and the Eurasian Basin.

The current and projected environmental change of the Arctic Ocean contrasts sharply with the limited knowledge of its genetic biodiversity. Polar cod Boreogadus saida (Lepechin, 1774) is an abundant circumpolar marine fish and ecological key species. The central role of polar cod in the Arctic marine food web warrants a better understanding of its population structure and connectivity. In this study, the genetic population structure of 171 juveniles, collected from several fjords off West-Svalbard (Billefjorden, Hornsund and Kongsfjorden), the northern Sophia Basin and the Eurasian Basin of the Arctic Ocean, was analysed using nine DNA microsatellite loci. Genetic analyses indicated moderate to high genetic diversity, but absence of spatial population structure and isolation-by-distance, suggesting ongoing gene flow between the studied sampling regions. High levels of connectivity may be key for polar cod to maintain populations across wide spatial scales. The adaptive capacity of the species will be increasingly important to face challenges such as habitat fragmentation, ocean warming and changes in prey composition. In view of a limited understanding of the population dynamics and evolution of polar cod, a valuable next step to predict future developments should be an integrated biological evaluation, including population genomics, a life-history approach, and habitat and biophysical dispersal modelling.

Acid suppression therapy, gastrointestinal bleeding and infection in acute pancreatitis - An international cohort study.

BACKGROUND: Acid suppressing drugs (ASD) are generally used in acute pancreatitis (AP); however, large cohorts are not available to understand their efficiency and safety. Therefore, our aims were to evaluate the association between the administration of ASDs, the outcome of AP, the frequency of gastrointestinal (GI) bleeding and GI infection in patients with AP. METHODS: We initiated an international survey and performed retrospective data analysis on AP patients hospitalized between January 2013 and December 2018. RESULTS: Data of 17,422 adult patients with AP were collected from 59 centers of 23 countries. We found that 23.3% of patients received ASDs before and 86.6% during the course of AP. ASDs were prescribed to 57.6% of patients at discharge. ASD administration was associated with more severe AP and higher mortality. GI bleeding was reported in 4.7% of patients, and it was associated with pancreatitis severity, mortality and ASD therapy. Stool culture test was performed in 6.3% of the patients with 28.4% positive results. Clostridium difficile was the cause of GI infection in 60.5% of cases. Among the patients with GI infections, 28.9% received ASDs, whereas 24.1% were without any acid suppression treatment. GI infection was associated with more severe pancreatitis and higher mortality. CONCLUSIONS: Although ASD therapy is widely used, it is unlikely to have beneficial effects either on the outcome of AP or on the prevention of GI bleeding during AP. Therefore, ASD therapy should be substantially decreased in the therapeutic management of AP.

Future ocean warming may prove beneficial for the northern population of European seabass, but ocean acidification will not.

The world's oceans are acidifying and warming as a result of increasing atmospheric CO2 concentrations. The thermal tolerance of fish greatly depends on the cardiovascular ability to supply the tissues with oxygen. The highly oxygen-dependent heart mitochondria thus might play a key role in shaping an organism's tolerance to temperature. The present study aimed to investigate the effects of acute and chronic warming on the respiratory capacity of European sea bass (Dicentrarchus labrax L.) heart mitochondria. We hypothesized that acute warming would impair mitochondrial respiratory capacity, but be compensated for by life-time conditioning. Increasing PCO2  may additionally cause shifts in metabolic pathways by inhibiting several enzymes of the cellular energy metabolism. Among other shifts in metabolic pathways, acute warming of heart mitochondria of cold life-conditioned fish increased leak respiration rate, suggesting a lower aerobic capacity to synthesize ATP with acute warming. However, thermal conditioning increased mitochondrial functionality, e.g. higher respiratory control ratios in heart mitochondria of warm life-conditioned compared with cold life-conditioned fish. Exposure to high PCO2  synergistically amplified the effects of acute and long-term warming, but did not result in changes by itself. This high ability to maintain mitochondrial function under ocean acidification can be explained by the fact that seabass are generally able to acclimate to a variety of environmental conditions. Improved mitochondrial energy metabolism after warm conditioning could be due to the origin of this species in the warm waters of the Mediterranean. Our results also indicate that seabass are not yet fully adapted to the colder temperatures in their northern distribution range and might benefit from warmer temperatures in these latitudes.

Aerobic capacities and swimming performance of polar cod (Boreogadus saida) under ocean acidification and warming conditions.

Polar cod (Boreogadus saida) is an important prey species in the Arctic ecosystem, yet its habitat is changing rapidly: climate change, through rising seawater temperatures and CO2 concentrations, is projected to be most pronounced in Arctic waters. This study aimed to investigate the influence of ocean acidification and warming on maximum performance parameters of B. saida as indicators for the species' acclimation capacities under environmental conditions projected for the end of this century. After 4 months at four acclimation temperatures (0, 3, 6, 8°C) each combined with two PCO2  levels (390 and 1170 µatm), aerobic capacities and swimming performance of B. saida were recorded following a Ucrit protocol. At both CO2 levels, standard metabolic rate (SMR) was elevated at the highest acclimation temperature indicating thermal limitations. Maximum metabolic rate (MMR) increased continuously with temperature, suggesting an optimum temperature for aerobic scope for exercise (ASex) at 6°C. Aerobic swimming performance (Ugait) increased with acclimation temperature irrespective of CO2 levels, while critical swimming speed (Ucrit) did not reveal any clear trend with temperature. Hypercapnia evoked an increase in MMR (and thereby ASex). However, swimming performance (both Ugait and Ucrit) was impaired under elevated near-future PCO2  conditions, indicating reduced efficiencies of oxygen turnover. The contribution of anaerobic metabolism to swimming performance was very low overall, and further reduced under hypercapnia. Our results revealed high sensitivities of maximum performance parameters (MMR, Ugait, Ucrit) of B. saida to ocean acidification. Impaired swimming capacity under ocean acidification may reflect reduced future competitive strength of B. saida.

Effects of ocean acidification increase embryonic sensitivity to thermal extremes in Atlantic cod, Gadus morhua.

Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO2 -driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO2 conditions (400 µatm vs. 1100 µatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO2 ) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid-base-relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3-6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO2 caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO2 and mitochondrial capacities. Elevated PCO2 stimulated MO2 at cold and intermediate temperatures, but exacerbated warming-induced constraints on MO2 , indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO2 . Increased MO2 in response to elevated PCO2 was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO2 treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO2 conditions and suggest that acclimation to elevated PCO2 requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains the thermal performance window of embryos, which has important implication for the susceptibility of cod to projected climate change.

Mitochondrial acclimation potential to ocean acidification and warming of Polar cod (Boreogadus saida) and Atlantic cod (Gadus morhua).

BACKGROUND: Ocean acidification and warming are happening fast in the Arctic but little is known about the effects of ocean acidification and warming on the physiological performance and survival of Arctic fish. RESULTS: In this study we investigated the metabolic background of performance through analyses of cardiac mitochondrial function in response to control and elevated water temperatures and PCO2 of two gadoid fish species, Polar cod (Boreogadus saida), an endemic Arctic species, and Atlantic cod (Gadus morhua), which is a temperate to cold eurytherm and currently expanding into Arctic waters in the wake of ocean warming. We studied their responses to the above-mentioned drivers and their acclimation potential through analysing the cardiac mitochondrial function in permeabilised cardiac muscle fibres after 4 months of incubation at different temperatures (Polar cod: 0, 3, 6, 8 °C and Atlantic cod: 3, 8, 12, 16 °C), combined with exposure to present (400µatm) and year 2100 (1170µatm) levels of CO2. OXPHOS, proton leak and ATP production efficiency in Polar cod were similar in the groups acclimated at 400µatm and 1170µatm of CO2, while incubation at 8 °C evoked increased proton leak resulting in decreased ATP production efficiency and decreased Complex IV capacity. In contrast, OXPHOS of Atlantic cod increased with temperature without compromising the ATP production efficiency, whereas the combination of high temperature and high PCO2 depressed OXPHOS and ATP production efficiency. CONCLUSIONS: Polar cod mitochondrial efficiency decreased at 8 °C while Atlantic cod mitochondria were more resilient to elevated temperature; however, this resilience was constrained by high PCO2. In line with its lower habitat temperature and higher degree of stenothermy, Polar cod has a lower acclimation potential to warming than Atlantic cod.

Oxygen- and capacity-limited thermal tolerance: bridging ecology and physiology.

Observations of climate impacts on ecosystems highlight the need for an understanding of organismal thermal ranges and their implications at the ecosystem level. Where changes in aquatic animal populations have been observed, the integrative concept of oxygen- and capacity-limited thermal tolerance (OCLTT) has successfully characterised the onset of thermal limits to performance and field abundance. The OCLTT concept addresses the molecular to whole-animal mechanisms that define thermal constraints on the capacity for oxygen supply to the organism in relation to oxygen demand. The resulting 'total excess aerobic power budget' supports an animal's performance (e.g. comprising motor activity, reproduction and growth) within an individual's thermal range. The aerobic power budget is often approximated through measurements of aerobic scope for activity (i.e. the maximum difference between resting and the highest exercise-induced rate of oxygen consumption), whereas most animals in the field rely on lower (i.e. routine) modes of activity. At thermal limits, OCLTT also integrates protective mechanisms that extend time-limited tolerance to temperature extremes - mechanisms such as chaperones, anaerobic metabolism and antioxidative defence. Here, we briefly summarise the OCLTT concept and update it by addressing the role of routine metabolism. We highlight potential pitfalls in applying the concept and discuss the variables measured that led to the development of OCLTT. We propose that OCLTT explains why thermal vulnerability is highest at the whole-animal level and lowest at the molecular level. We also discuss how OCLTT captures the thermal constraints on the evolution of aquatic animal life and supports an understanding of the benefits of transitioning from water to land.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

BACKGROUND: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. RESULTS: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. CONCLUSIONS: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Blue blood on ice: modulated blood oxygen transport facilitates cold compensation and eurythermy in an Antarctic octopod.

INTRODUCTION: The Antarctic Ocean hosts a rich and diverse fauna despite inhospitable temperatures close to freezing, which require specialist adaptations to sustain animal activity and various underlying body functions. While oxygen transport has been suggested to be key in setting thermal tolerance in warmer climates, this constraint is relaxed in Antarctic fishes and crustaceans, due to high levels of dissolved oxygen. Less is known about how other Antarctic ectotherms cope with temperatures near zero, particularly the more active invertebrates like the abundant octopods. A continued reliance on the highly specialised blood oxygen transport system of cephalopods may concur with functional constraints at cold temperatures. We therefore analysed the octopod's central oxygen transport component, the blue blood pigment haemocyanin, to unravel strategies that sustain oxygen supply at cold temperatures. RESULTS: To identify adaptive compensation of blood oxygen transport in octopods from different climatic regions, we compared haemocyanin oxygen binding properties, oxygen carrying capacities as well as haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata. In the Antarctic Pareledone charcoti at 0°C, oxygen unloading by haemocyanin was poor but supported by high levels of dissolved oxygen. However, lower oxygen affinity and higher oxygen carrying capacity compared to warm water octopods, still enabled significant contribution of haemocyanin to oxygen transport at 0°C. At warmer temperatures, haemocyanin of Pareledone charcoti releases most of the bound oxygen, supporting oxygen supply at 10°C. In warm water octopods, increasing oxygen affinities reduce the ability to release oxygen from haemocyanin at colder temperatures. Though, unlike Eledone moschata, Octopus pallidus attenuated this increase below 15°C. CONCLUSIONS: Adjustments of haemocyanin physiological function and haemocyanin concentrations but also high dissolved oxygen concentrations support oxygen supply in the Antarctic octopus Pareledone charcoti at near freezing temperatures. Increased oxygen supply by haemocyanin at warmer temperatures supports extended warm tolerance and thus eurythermy of Pareledone charcoti. Limited haemocyanin function towards colder temperatures in Antarctic and warm water octopods highlights the general role of haemocyanin oxygen transport in constraining cold tolerance in octopods.

Gene expression profiling in gills of the great spider crab Hyas araneus in response to ocean acidification and warming.

BACKGROUND: Hypercapnia and elevated temperatures resulting from climate change may have adverse consequences for many marine organisms. While diverse physiological and ecological effects have been identified, changes in those molecular mechanisms, which shape the physiological phenotype of a species and limit its capacity to compensate, remain poorly understood. Here, we use global gene expression profiling through RNA-Sequencing to study the transcriptional responses to ocean acidification and warming in gills of the boreal spider crab Hyas araneus exposed medium-term (10 weeks) to intermediate (1,120 µatm) and high (1,960 µatm) PCO2 at different temperatures (5°C and 10°C). RESULTS: The analyses reveal shifts in steady state gene expression from control to intermediate and from intermediate to high CO2 exposures. At 5°C acid-base, energy metabolism and stress response related genes were upregulated at intermediate PCO2, whereas high PCO2 induced a relative reduction in expression to levels closer to controls. A similar pattern was found at elevated temperature (10°C). There was a strong coordination between acid-base, metabolic and stress-related processes. Hemolymph parameters at intermediate PCO2 indicate enhanced capacity in acid-base compensation potentially supported by upregulation of a V-ATPase. The likely enhanced energy demand might be met by the upregulation of the electron transport system (ETS), but may lead to increased oxidative stress reflected in upregulated antioxidant defense transcripts. These mechanisms were attenuated by high PCO2, possibly as a result of limited acid-base compensation and metabolic down-regulation. CONCLUSION: Our findings indicate a PCO2 dependent threshold beyond which compensation by acclimation fails progressively. They also indicate a limited ability of this stenoecious crustacean to compensate for the effects of ocean acidification with and without concomitant warming.