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Background: Many Antarctic notothenioid fish are considered losers of global change, due to their low thermal tolerance and lack of regulative mechanisms that enhance physiological plasticity. The Austral nototheniid congener Notothenia angustata provides an alternative model to explore the effects of ocean acidification and warming, as it inhabits cold temperate to subpolar waters. It is a eurythermal species, with greater capacities for thermal acclimation relative to Antarctic congeners, and therefore presents a useful model against which Antarctic notothenioids can be contrasted. Methods: We investigated the long-term effects of hypercarbic acclimation on whole animal and cardiac mitochondrial function for the Austral nototheniid Notothenia angustata. Fish were acclimated under hypercarbic (0.2 kPa CO2, 15 days, n=6) and normocarbic conditions (control 0.04 kPa CO2, n=10). Routine metabolic rates (RMR) were determined with acute increases in temperature (3°C/d) under normocarbic and hypercarbic conditions. Mitochondrial function was then tested within permeabilised cardiac muscle fibres, and assays conducted in normocarbic (0.04 kPa CO2) and hypercarbic (3.0 kPa CO2) media at 9, 15 and 21°C. Metabolic profiles were determined in red skeletal muscle. Findings: Whole animal critical temperature thresholds occurred below 19°C for normocarbic exposed fish, while acutely hypercarbic exposed fish maintained routine metabolic rates up to 21oC. Overall mitochondria mirrored the responses of acutely exposed whole animals, with an increased mitochondrial performance in fish acclimated to chronic hypercarbia. Chronically hypercarbic exposed animals also exhibited altered metabolomes of red muscle, but not liver with apparent increases in metabolites consistent with enhanced anaerobic metabolism and elevated contents of histidine and tryptophan that may contribute to acid-base buffering. Conclusions: Overall enhanced cardiac mitochondrial capacities coincide with increasing

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 physio

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 betwee

Oxygen equilibrium curves have been widely used to understand oxygen transport in numerous organisms. A major challenge has been to monitor oxygen binding characteristics and concomitant pH changes as they occur in vivo, in limited sample volumes. Here we report a technique allowing highly resolved and simultaneous monitoring of pH and blood pigment saturation in minute blood volumes. We equipped a gas diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and recorded changes of pigment oxygenation along PO2 and pH gradients to test the setup. Oxygen binding parameters derived from measurements in only 15 μl of haemolymph from the cephalopod Octopus vulgaris showed low instrumental error (0.93%) and good agreement with published data. Broad range spectra, each resolving 2048 data points, provided detailed insight into the complex absorbance characteristics of diverse blood types. After consideration of photobleaching and intrinsic fluorescence, pH optodes yielded accurate recordings and resolved a sigmoidal shift of 0.03 pH units in response to changing PO2 from 0-21 kPa. Highly resolved continuous recordings along pH gradients conformed to stepwise measurements at low rates of pH changes. In this study we showed that a diffusion chamber upgraded with a broad range spectrophotometer and an optical pH sensor accurately characterizes oxygen binding with minimal sample consumption and manipulation. We conclude that the modified diffusion chamber is highly suitable for experimental biologists who demand high flexibility, detailed insight into oxygen binding as well as experimental and biological accuracy combined in a single set up.

Oxygen transport measurements have been widely used to explain evolutionary adaptation and performance in fishes. Simultaneous measurements of blood pH and oxygen saturation are essential to understand oxygen transport as it occurs in vivo, but has been challenging to monitor with existing methods, particularly in minute blood volumes. We equipped a gas diffusion chamber with a broad-range fibre-optic spectrophotometer and a micro-pH optode and successfully recorded changes of pigment oxygenation and pH in only 15 µl of whole fish blood at 0°C. Highly resolved spectra ranging from 280 – 900 nm captured the complex absorbance features of Antarctic fish haemoglobin in response to changing oxygen and carbon dioxide partial pressures. After consideration of photobleaching and intrinsic fluorescence, pH optodes recorded pH shifts of 0.03 pH units. With this modified diffusion chamber experimental biologists are able to accurately characterize oxygen binding with minimal sample consumption and manipulation under diverse experimental settings.

Background Haemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance. Results The cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight functional units and an internal duplicated functional unit d. Our molecular phylogenetic analyses reveal the third isoform representing a potentially ancestral haemocyanin isoform, and the analyses of the expression of haemocyanin type 3 reveal that haemocyanin type 3 only can be observed within eggs and during early development. Isoforms 1 and 2 are absent at these stages. After hatching, isoform 3 is downregulated, and isoform 1 and 2 are upregulated. Conclusions Our study clearly shows an embryonic relevance of the third isoform, which will be further discussed in the light of the changes in the physiological function of haemocyanin during ontogeny. Taken together with the fact that it could also be the isoform closest related to the common ancestor of cuttlefish haemocyanin, the phylogeny of cuttlefish haemocyanin may be recapitulated during its ontogeny.

In the eurythermal cuttlefish Sepia officinalis, performance depends on hearts that ensure systemic oxygen supply over a broad range of temperatures. We therefore aimed to identify adjustments in energetic cardiac capacity and underlying mitochondrial function supportin g therma l acclimati on th at cou ld be criti cal for the cuttlef ish’s comp etitive success in variable environments. Two genetically distinct cuttlefish populations were acclimated to 11°C, 16°C and 21°C, respectively. Subsequently, skinned and permeabilised heart fibres were used to assess mitochondrial functioning by means of high-resolution respirometry and a substrate-inhibitor protocol, followed by measurements of cardiac citrate synthase and cytosolic enzyme activities. In cuttlefish hearts, thermal sensitivity of mitochondrial substrate oxidation was high for proline and pyruvate but low for succinate. Oxygen efficiency of catabolism rose from 11°C to 21°C via shifts to oxygen-conserving oxidation of proline and pyruvate as well as via reduced relative proton leak. Acclimation to 21°C decreased mitochondrial complex I activity in Adriatic cuttlefish and increased complex IV activity in English Channel cuttlefish. However, compensation of mitochondrial capacities did not occur during cold acclimation to 11°C. Moreover, temperate English Channel cuttlefish had larger hearts with lower mitochondrial capacities than subtropical Adriatic cuttlefish. The changes observed for substrate oxidation, mitochondrial complexes, relative proton leak or heart weights improve energetic efficiency and essentially seem to extend tolerance to high temperatures and reduce associated tissue hypoxia. We conclude that cuttlefish sustain cardiac performance and thus, systemic oxygen delivery over short and long-term changes of temperature and environmental conditions by multiple adjustments in cellular and mitochondrial energetics.

The efficiency and adaptive plasticity of mitochondrial metabolism is one of the key factors shaping organismal tolerance towards environmental change.We thus investigated the adaptive capacities of mitochondrial metabolism in Antarctic & Austral notothenioids (Nothothenia rossii, N. coriiceps & N. angustata) and temperate Sea Bass (Dicentrarchus labrax) with respect to some important factors that will influence the survival of marine life in the near future: global warming (elevated sea temperatures), ocean acidification (elevated CO2 levels) and hypoxia tolerance.We namely analysed the function and contribution of the single respiratory complexes to total mitochondrial metabolism, as well as membrane potential and proton leak. In all experiments, mitochondrial measurements were made under acute thermal challenges, compairing liver and heart mitochondria from either differently acclimated individuals (thermal acclimation, CO2 acclimation the combination thereof) or from individuals selected for a specific phenotypic trait (hypoxia tolerance in D. labrax).The implications of mitochondrial plasticity for long-term adaptation in a changing marine environment will be discussed.

The Stylonematophyceae is an early diverging red algalclass that contains organisms in which sexual reproductionhas not been reported. Previous studies havehypothesized a single copy of the actin gene in thisclass. Amplification of the actin gene in members of theStylonematophyceae, including several isolates of Stylonemaalsidii, reveals a more complex evolutionaryhistory. The data support independent gene duplicationsin Goniotrichopsis reniformis and Stylonemaalsidii. Three of the seven isolates of S. alsidii had threecopies of actin. Analyses indicate that all copies arefunctional and not pseudogenes. Due to our lack ofknowledge of the ploidy level in Stylonema and theasexual nature of these organisms, an ancient changein ploidy level, producing diploid or even triploid organisms,cannot be ruled out to explain two copies of actinwithin these organisms. Within Stylonema alsidii two ofthe actin copies (Groups 1 and 2) show similar levels ofvariation; p-distances and the number of synonymousand non-synonymous sites are roughly the same. Yet thechanges are distributed differently. One group showedlittle shared variation among the samples, mutationstherefore represent autoapomorphies, while variation inthe other group is usually shared among samples(parsimony-informative). Variation in the other group isusually shared among samples (parsimony-informative).A third group of actin genes also observed in S. alsidiiare highly divergent from the other two copies, yet theymaintain all of the signatures of functional proteins.Our data reveal a complex evolutionary history of theactin gene in these species and highlight the lack ofknowledge into the basic biology (ploidy level, life cyclecharacteristics) of this class.

Cuttlefish (Sepia officinalis) display diurnal changes in activity levels that are linked to the gas loading of the cuttlebone, which regulates buoyancy (Denton and Gilpin-Brown, 1961). To investigate diurnal changes of standard and active metabolic rates, we analysed activity patterns of S. officinalis using new techniques of video tracking. We monitored S. officinalis activity by video continuously for 5 days. In a parallel experiment we measured their rates of oxygen consumption in order to explore whether metabolic rate changes in parallel with activity levels. Both measurements of activity and oxygen consumption indicate a strong diurnal pattern with a maximum physical activity shortly after midnight and a relatively constant minimum value during daytime. In order to determine whether patterns of metabolic regulation go hand in hand with the diurnal rhythm of activity-dependent metabolic slowing (daytime) and stimulation (nighttime), we analysed the activities of enzymes involved in standard and digestive metabolism in mantle and hepatopancreas tissues to complete the picture. Moreover, we could show that different feeding schemes strongly affect the distinctiveness of the diurnal activity pattern.

The Southern Ocean hosts a rich and diverse fauna that required specialist adaptations to colonize and persist at temperatures close to freezing. While much has been revealed about key adaptations in Antarctic fishes little is known about evolutionary strategies of other Antarctic ectotherms, particularly the abundant benthic incirrate octopods. Their oxygen demand is largely fuelled by the blue oxygen transporter haemocyanin that however, due to its increasing functional failure towards colder temperatures, poses a prime target for cold-adaptive adjustment. While haemocyanin structure has been well understood it remains unclear which molecular features and evolutionary trajectories explain functional properties of octopod haemocyanin. This thesis thus aimed to unravel cold-adaptive features of octopod haemocyanin and the underlying molecular features that evolved to sustain oxygen supply at sub-zero temperatures. Haemocyanin function is best assessed by oxygen binding experiments, which however was challenged due to the minute haemolymph volumes non-model organisms like Antarctic octopods, yield. I thus upgraded an oxygen diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and tested the setup for Octopus vulgaris, the Antarctic eelpout and a Baikal amphipod. This technical advancement enabled simultaneous recordings of pH and oxygen dependent pigment absorbance in only 15 microliter of sample. Results were highly reproducible and accurate and provided detailed insight to the complex and dynamic spectral features of three diverse blood-types. To identify cold-adaptive functional traits of blood oxygen transport this study compared haemocyanin oxygen binding properties, oxygen carrying capacities and haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the temperate Octopus pallidus and the subtropical Eledone moschata. Compared to octopods from warmer climates, Pareledone charcoti showed inco