kPa CO 2 3 kPa CO 2 kPa CO 2 3 kPa CO 2

% CI 9 79.7±1.3 80.6±1.8 75.5±2.4 85.0±1.3

15 78.1±2.2 80.9±3.0 75.4±3.3 80.3±1.9

21 60.3±4.5^# 67.8±6.2 79.6±2.2 86.4±1.9

COX/OXPHOS 9 2.1±0.2 2.3±0.5 2.2±0.3 2.4±0.3

15 2.0±0.1 2.8±0.5 2.3±0.3 1.9±0.2

21 4.2±0.7 5.7±1.7^# 2.5±0.2 2.4±0.2*

COX/OXPHOS provides a comparative measure of cytochrome c oxidase capacity relative to oxidative phosphorylation. The percentage contribution of complex I (% CI) was calculated as OXPHOS CI, CII/

OXOHOS CI. It indicates the capacity of CI compared to total phosphorylation with CI and CII substrates. Both ratios were determined for permeabilised heart fibres in normocapnic (0.04 kPa CO₂) and acute hypercapnic (3 kPa CO2) respiration buffer (MiRO6) at 9, 15 and 21°C. * indicates significant differences between control and CO₂ acclimation (ANOVA, P < 0.05). ^# depicts significant differences to 9°C assay (ANOVA, P < 0.05). Means

± SEM.

P

UBLICATION

V

Metabolic capacities in relation to temperature and hypercapnia in cephalopods from various climate zones

Anneli Strobel,Hans O. Pörtner, Felix C. Mark

2013

American Journal of Physiology – Regulatory, Integrative and Comparative Physiology

(in review)

American Journal of Physiology – Regulatory, Integrative and Comparative Physiology

Metabolic capacities in relation to temperature and hypercapnia in cephalopods from various climate zones

Anneli Strobel^*, Hans O. Pörtner, Felix C. Mark

Author affiliation:

Integrative Ecophysiology, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany

Running head:

Metabolic capacities in cephalopods of various climate zones

*Corresponding author:

Anneli Strobel

Integrative Ecophysiology

Alfred Wegener Institute for Polar and Marine Research Am Handelshafen 12

D-27570 Bremerhaven Germany

phone: +49 (0) 471 4831 1716 fax: +49 (0) 471 4831 1149 e-mail: anneli.strobel@awi.de

Abstract

Coleoid cephalopods are the largest, metabolically most active invertebrates, and have adapted to habitats from the poles to the tropics. Yet, their sensitivity and acclimation capacity to ongoing ocean warming and acidification is poorly explored. This study compares Antarctic (Pareledone charcoti) and sub-tropical (Eledone moschata) octopods to metabolically more active, well-studied cuttlefish (Sepia officinalis). We acclimated E.

moschata to 16 and 21°C (0.04 kPa CO2) and S. officinalis to 0.112 kPa CO2, and measured routine metabolic rate (RMR) and mitochondrial respiration (heart) under normocapnic and hypercapnic conditions.

Mitochondria of S. officinalis were better coupled and had higher respiration rates with higher acute thermal flexibilities than the octopods. Fibre respiration in Antarctic octopods was elevated compared to the sub-tropical species, indicating cold-compensation at cellular level.

Warm-acclimated E. moschata showed uncompensated RMR and heart fibre respiration, indicating low thermal acclimation capacities. After long-term hypercapnia acclimation, the acid-base status in the haemolymph of S. officinalis was incompletely compensated with an extracellular pH remaining 0.1 pH units below control levels. Hypercapniadid not affect the RMR of S. officinalis, but led to increased mitochondrial capacities. In all three species, acute hypercapnia had no effect on the hearts’ oxidative phosphorylation capacities, but depressed COX capacities in the octopods.

Our data suggest that S. officinalis compensate elevated PCO2 by increasing mitochondrial turnover through enhanced amino acid catabolism. Higher mitochondrial COX excess capacities, which can increase mitochondrial oxygen affinity, and lower acute thermal sensitivities could render S. officinalis less vulnerable to ocean acidification and warming than octopods.

Key words

Pareledone charcoti, Eledone moschata, Sepia officinalis, ocean acidification/ warming, respiration, mitochondria

Introduction

Coleoid cephalopods are the largest, most active invertebrates, found in all marine habitats from the poles to the tropics (35). Sharing a similar lifestyle with fish, such as predation behavior and habitat distribution, a supposedly convergent evolution has led to similar performances and sophisticated respiratory and circulatory systems in cephalopods.

The closed circulatory system, driven by two branchial hearts and a powerful systemic heart, allows a high cardiac output and oxygen transport at comparable or higher rates than in comparatively active, similar-sized fish. Constraints of their molluscan heritage include for example low blood oxygen-carrying capacities and minor efficient locomotion by jet propulsion (56, 66, 68).

With rising ambient temperature, an increase in oxygen demand exceeds the capacity of oxygen supply by the circulatory and ventilatory system and causes an uncompensated decrease in oxygen levels in the body fluids (42, 45). In order to meet elevated oxygen demands, cephalopods typically increase ventilation, heart rate and stroke volume (66). The capacity of mitochondria to produce energy aerobically is one of the mechanisms supporting and constraining the performance of any tissue, as their aerobic capacities contribute to shaping the aerobic energy metabolism. In oxygen supply systems, the key tissue is the systemic heart (44). A compensatory long-term acclimation response to warmer temperatures is therefore based on changes in mitochondrial capacities. To date, little is known about mitochondrial oxidative capacities and limits to oxidative metabolism in cephalopods (but see Mommsen and Hochachka (34) and Oellermann et al. (38)).

Seibel and Walsh (57) propose an inverse relationship between vulnerability to decreasing seawater pH and metabolic rate, with less active cephalopods, such as deep-living species being more susceptible to ocean acidification than more active (e.g. shallow-living) ones. This vulnerability is founded in the ability to buffer metabolic end products, such as CO2 and protons, and the concentration of respiratory proteins buffering extracellular fluids against pH changes, which declines with metabolic rate and depth (67). In contrast, highly active animals such as squid are also extremely impacted by decreases in seawater pH (51).

This is due to the fact that blood-oxygen binding at the gills and release at the tissues in cephalopods is highly pH sensitive (5). In squids like Illex illecebrosus, who possess only little venous oxygen reserves and highly pH-sensitive oxygen transport characteristics, a blood pH change of as little as 0.25 units can be lethal (51).

As anthropogenic CO2 emissions are expected to cause a rise in atmospheric PCO2 to above 0.1 kPa by the year 2100 in realistic scenarios (‘business-as-usual’) of the Intergovernmental Panel on Climate Change (21), an efficient pHe-compensation is crucial for cephalopods to tolerate increases in seawater PCO2 (32). However, an efficient acid-base balance might occur on the expenses of ionic homeostasis by ion-transporters, which may include higher costs by ATP-dependent pumps (12, 20). If under chronically elevated seawater PCO2 acid-base imbalances in extra- and intracellular body fluids cannot be compensated, this may lead to metabolic depression (a condition expected to retard growth and reproduction), reduced activity and disruption of oxygen-transport mechanisms, and – if persistent – death (53, 58).

Furthermore, combined ocean acidification and warming might lead to additional energetic requirements, which would further reduce the thermal tolerance of marine ectotherms (41). In line with this, cephalopod sensitivity to ocean warming and acidification may vary, depending on the one hand on pH sensitivities (and concentration) of their respiratory proteins, and on the other hand on metabolic rate and mitochondrial compensation for a changing energy demand, e.g. for acid-base homeostasis.

Among cephalopods, the Octopoda have been able to adapt during their radiation to water temperatures ranging from > 30°C in tropical to -2°C in Antarctic waters (36). To date, some studies have measured respiration rates during acute thermal challenge in temperate octopods such as Octopus vulgaris (7, 23), and in the Antarctic octopod Pareledone charcoti (10), for which upper critical temperatures between 8 and 10°C have been determined (51).

Despite, long-term warm-acclimation capacities of octopods have poorly been studied so far.

Particularly in light global climate change, acclimation capacities towards chronically elevated temperatures become increasingly important (21).

In contrast to octopods, acute thermal limits (25°C, (16, 31)) and long-term warm-acclimation capacities (29) have intensively been analyzed in the cuttlefish S. officinalis.

However, the mitochondrial background of thermal acclimation has not been comprehensively studied in cephalopods. In fish, mitochondrial compensation to temperature includes e.g. changes in mitochondrial respiration, densities or enzyme capacities (9), or shifts from carbohydrate to fatty acid oxidation (59). In contrast, S. officinalis shows only limited temperature compensation abilities of mitochondrial capacities. Nevertheless, thermal acclimation can induce shifts in mitochondrial substrate preferences in cuttlefish (38), similar

Next to these studies on thermal sensitivities, S. officinalis is amongst the few cephalopods for which CO2 sensitivities have been previously assessed. In the highly active Humboldt squid Dosidicus gigas, a species living in association with hypercapnic oxygen minimum zones, an acute PCO2 of 0.1 kPa has been reported to depress metabolic rates by 31% (54). Similarly, acute severe hypercapnia (1 kPa CO2) reduces oxygen consumption in the temperate cephalopod S. officinalis (Schmidt et al., unpublished), but not so during acute exposure to 0.6 kPa CO2 (18). At such intermediately elevated PCO2 of 0.6 kPa, S. officinalis shows a partially compensated pHe (leading to significantly elevated extracellular bicarbonate levels) and efficient intracellular pH (pHi) regulation during acute exposure to 0.6 kPa CO2

(18), without any metabolic disturbances or depression, which probably relates to their (for invertebrates) relatively efficient acid-base regulatory system (18). However, all these studies only investigated short-term responses to elevated ambient PCO2, and no comparable studies exist for octopods so far.

To date, nobody has provided an overview over acute or long-term hypercapnia effects on mitochondrial metabolism in cephalopods. In this study, we aim to compare the susceptibility of cephalopods from different climate zones towards global warming and ocean acidification in. We investigated the difference in aerobic metabolic capacities in the benthic, Antarctic octopod Pareledone charcoti and the sub-tropical Eledone moschata, and compare these findings to those in a well-studied reference organism, the bentho-pelagic cuttlefish Sepia officinalis. In the light of ongoing global change, we measured the acclimation capacities of sub-tropical octopods towards warmer temperatures, as well as those of temperate cuttlefish towards elevated PCO2. We assessed routine oxygen consumption rates of the Antarctic octopods at their habitat temperature, of the sub-tropical octopods at their mean habitat temperature of 16°C and after five month acclimation to 21°C. Furthermore, we determined oxygen consumption of the cuttlefish at their habitat temperature of 16°C under normocapnic conditions (0.039 kPa CO2) and after five months of hypercapnia acclimation (0.112 kPa CO2; seawater PCO2 predicted for the year 2100 (21)). By measurement of their mitochondrial respiration (in heart fibres of the systemic heart) at different, rising assay temperatures in normocapnic (0.039 kPa CO2) and hypercapnic (1.6 kPa CO2) respiration medium, the thermal flexibility and potential warm/ hypercapnia acclimation capacities of cellular aerobic metabolism were investigated.

The benthic, Antarctic octopus P. charcoti is distributed on continental shelves around the Antarctic Peninsula and in the Ross Sea at temperatures between -2 and 2°C (2). The

benthic, sub-tropical (musky) octopus E. moschata, is abundant throughout the Mediterranean Sea and off the Portuguese coast (8, 39). The more active, bottom-dwelling common cuttlefish S. officinalis is one of the most abundant cephalopods and of great commercial interest. It occurs from the temperate North Atlantic to the sub-tropical Mediterranean and in Atlantic coastal waters off Senegal (3). In the Mediterranean Sea, both S. officinalis and E.

moschata face water temperatures from 10 up to 25°C (4).

Material and Methods

Animal capture, maintenance and acclimation

Specimens of adult P. charcoti were caught during the expedition ANTXXVII/3 with the German research vessel ‘RV Polarstern’ in February 2011 by means of bottom trawls off King George Island (62°18'S 58°41'W) at 350 m water depth at a local water temperature of 0.1°C and a salinity of 34.4 psu. Animals were kept in well-aerated aquaria on board of RV Polarstern with permanent seawater supply under dim light (12 h light/ dark). Logistic implications impeded any warm- or CO2- acclimations on board. Animals were not fed prior to the experiments and measurements of RMR, which were conducted one week after capture.

Adult specimens of E. moschata from the Adriatic Sea were collected in May 2010 at a water temperature of 16°C by local fisherman in Chioggia, Italy, and transported to the Alfred Wegener Institute, Bremerhaven, Germany, immediately.

Four specimens were kept under control conditions at 16°C, 0.04 kPa CO2. Another four octopods were warm-acclimated to 21°C, 0.04 kPa CO2, for a time-period of five months. The animals were acclimated in separate seawater recirculation systems in individual closed incubation boxes with a volume of 8.5 l. Each box was perfused from a header tank containing well-aerated seawater at the desired temperature (16°C/ 21°C), which was kept constant by several 250 W heating elements (Jaeger, EHEIM GmbH, Germany) controlled by a Temperature Controller TMP1380 (iSiTEC GmbH, Germany). They were fed every other day with brown shrimp (Crangon crangon).

Eggs of sub-tropical S. officinalis were collected by local fisherman in the Venice Lagoon, Chioggia, Italy, in May 2009 at a local temperature of 16°C. They were transported to and raised in a closed, recirculating aquarium system at the Alfred Wegener Institute, Bremerhaven, Germany, on a diet of mysids (Neomysis integer) and brown shrimp (C.

Adult cuttlefish were either kept under control conditions (16°C, 0.04 kPa CO2) in the general aquaria facilities of the Alfred Wegener Institute (30-32 psu), or acclimated to 16°C and 0.112 kPa CO2 for five months, respectively (N = 8). Cuttlefish were kept in a recirculating seawater system in individual closed incubation boxes (volume 8.5 l), fed by a header tank containing well-aerated, hypercarbic seawater. To adjust seawater PCO2, the water was injected with gas mixed from compressed air and CO2 using a mass flow controlling gas mixing system (HTK Hamburg GmbH, Germany). Cuttlefish were fed every other day with live shrimp (Palaemon sp. or C. crangon).

The pH was monitored daily with a NIST (National Institute of Standards and Technology) buffer calibrated WTW 3310 pH meter (WTW, Germany) and a glass electrode (InLab Routine Pt1000®, Mettler Toledo GmbH, Germany). Dissolved inorganic carbon (DIC) was measured by gas chromatography (Agilent 6980 N, Agilent Technologies, Germany), following a protocol modified from Lenfant and Aucutt (27), and Pörtner et al.

(40). Seawater carbonate chemistry was calculated with the CO2SYS software (28), using the measured pH and dissolved inorganic carbon values.

Routine metabolic rate

Routine metabolic rate of E. moschata (control/ after long-term warm-acclimation), P.

charcoti and S. officinalis (control/ after long-term acclimation to 0.112 kPa) was measured via intermittent-flow respirometry. Cephalopods were not fed for five days prior to the respiration experiments to ensure complete digestion of the last meal (17). RMR in this study is defined as the oxygen consumption during rest in unfed (not starving) animals, including spontaneous activity. The respiration chambers were covered with black cloth in order to minimize external disturbance and spontaneous activity of the animal. Each animal was placed in a 870 ml cylindrical respirometer under acclimation conditions (E. moschata: 16°C, 0.04 kPa, N=4 54-76g; 21°C, 0.04 kPa N=4, 25-31g. P. charcoti 0°C 0.04 kPa, N=4, 43.6–

91.5g. S. officinalis 16°C, 0.04 kPa, N=6, 88-146g; 16°C, 0.112 kPa, N=7, 33-46g).

Individuals were allowed to recover within the respiration chambers for 24 hours, an appropriate period to overcome the effect of any handling stress (10, 69). Constant seawater-mixing within the respirometer was generated by an aquarium pump. In the intermittent-flow system, water exchange between chamber and ambient water was interrupted every 15 min for 15 min to measure oxygen depletion (max. 10% O2 depletion) by the animal within the chamber, then oxygen concentration was replenished to 100% by flush pumps. Oxygen

concentration within the chamber was detected once per minute using a fluoroptic sensor connected to a FiBox2 (PreSens – Precision Sensing GmbH, Germany) oxygen meter.

Oxygen consumption of the animal was calculated by using the linear declining rate of oxygen content within the respiration chamber for each 15 min measurement interval. The oxygen meter was calibrated before each measurement in well-aerated seawater at the respective acclimation-temperature, calibration at zero oxygen was conducted in nitrogen-bubbled seawater.

Blank measurements of bacterial respiration in the respirometer were carried out for all species and each acclimation group, values for RMR were corrected accordingly.

Dissection and heart fibre preparation

After anaesthetization of the animals in 2.5% ethanol, the mantle cavity was opened and samples of blood were taken from the anterior vena cava for further experiments. The animals were then sacrificed by decapitation. Branchial hearts were excised, immediately freeze-clamped and stored in liquid nitrogen. After dissection, a piece of systemic heart tissue was transferred into ice-cold modified biopsy medium (in mmol*l^-1: 2.77 Ca2EGTA, 7.23 K2EGTA, 14.46 KOH, 5.77 Na2ATP, 6.56 MgCl2*6H2O, 20 taurine, 20 imidazole, 0.5 dithiothreitol (DTT), 50 MES, 588 Sucrose and 252 glycine, 1000 mOsm, pH 7.4 at 24°C).

Fibre preparation followed the description by Kuznetsov et al. (25). The heart tissue was mechanically dissected in ice-cold medium by scissors and forceps in small fibre bundles and stored on ice in respiration medium. For each respiration experiment, a subsample of the heart fibre bundles was permeabilized for 30 min with 50 g/ml saponin by gentle mixing on ice.

Afterwards, the fibres were washed three times for 10 min in 2 ml ice-cold, modified assay medium (mitochondrial respiration medium, MiRO5), modified from (25, 34)) containing (in mmol*l^-1) 50 Na⁺Hepes, 25 KH2PO4, 50 KCl, 50 NaCl, 350 sucrose, 0.5 EGTA, 10 MgCl2*6 H2O, 20 taurine, 50 lactobionate, 150 glycine and 10g*l^-1 freshly added BSA (fatty acid free) (1000 mOsm, pH 7.4 at 24°C). Then, the subsample was blotted dry, divided in two and weighed, and immediately transferred into 2 ml air saturated assay medium (MiRO5) + 300U/ml catalase (for reoxygenation with hydrogen-peroxide) in glass-chambers of an Oroboros Oxygraph-2k^TM respirometer (Oroboros Instruments, Austria) at the respective assay temperature for respirometric analysis.

Heart fibre respiration assays were measured in the standard, normocapnic respiration

CO2. Considering a maximum gradient of about 0.4 kPa between the intracellular and extracellular space (46), 1.6 kPa would be the corresponding intracellular PCO2 to the extracellular PCO2 in S. officinalis long-term acclimated to 0.112 kPa CO2. Although we did not measure intracellular acid-base parameters in systemic heart due to sample shortage, the calculated PCO2 of 1.6 is well within a range that can be expected for cuttlefish heart, as intracellular PCO2 values of 0.4 kPa was measured in control S. officinalis (Häfker 2012, unpublished) and 0.7 kPa in Lolliguncula brevis (47) mantle tissue.

Heart fibre respiration assays

Respiration of each subsample was measured at 0, 6 and 12°C (P. charcoti) or 16, 21 and 26°C (E. moschata and S. officinalis) in randomized order. The heart fibre respiration was converted to nmol O2 min^-1 mgfresh weight (fw)^-1. For measurement of heart mitochondrial capacities, the concentration and choice of substrates for the combined substrate-inhibitor protocol was made in accordance to previous experiments on systemic heart fibre respiration of S. officinalis by Oellermann et al. (38). Resting respiration (state II) was measured with complex I (CI) substrates, 5mM proline, 5mM malate, and 5mM pyruvate. State III respiration (maximum coupled oxidative phosphorylation) of complex I (OXPHOS CI) was induced by 1mM ADP, state III respiration of complex I and complex II (CII) by adding 5mM succinate (OXPHOS CI, CII). Integrity of the mitochondrial membranes was tested wit 0.01mM cytochrome c. Leak capacity (state IV⁺) was evaluated by adding 4 g/ml oligomycin, followed by uncoupling of the respiration induced by titration of up to 2.5 M carbonylcyanide-p-(trifluoromethyl) phenylhydrazone (FCCP). After inhibition of complex I with 0.005mM rotenone (state IIIu of complex II), non-mitochondrial respiration (ROX) was detected by adding 0.0025mM antimycin A, followed by addition of the artificial substrates for complex IV (cytochrome C oxidase, COX), 2mM ascorbate and 0.5mM N,N,N’,N’-tetramethyl-p-phenylendiamine dihydrochloride (TMPD). To avoid low oxygen tensions within the chambers during the respiration experiments, oxygen concentrations were restored to air saturation when reaching 100nmol O2 ml^-1 by re-oxygenation with 1% hydrogen-peroxide.

Extracellular acid-base parameters.

Hemolymph pH (extracellular pH, pHe) from S. officinalis was measured immediately after sampling at the acclimation temperature with a pH meter (WTW 340i, WTW, Germany.

Electrode: In Lab^® Viscous, Mettler Toledo GmbH, Germany). The pH meter was calibrated daily with NIST buffers (WTW, Germany). Measurements were carried out in a closed microcentrifuge tube (0.5 ml) to minimize contact with environmental air. Plasma total CO2

(CCO2)was measured after centrifugation by means of a Gas Chromatography (Agilent 6980 N, Agilent Technologies, Germany). Blood carbonate chemistry was calculated using the following, modified Henderson-Hasselbalch equation:

(1)

(2)

[HCO3-] represents bicarbonate concentration. Values for the CO2-solubility coefficient and pK’’’ (first apparent dissociation constant of carbonic acid) were calculated for 16°C and 32 psu from values for Carcinus maenas hemolymph (64), which possesses a similar extracellular hemocyanin concentration and ionic composition (18).

Data analysis and statistics

Using the heart fibre respiration data of all species, the following indicators for mitochondrial capacities were calculated:

1) Capacity of complex I relative to total coupled oxidative phosphorylation (OXPHOS), calculated as OXPHOS CI, CII/ OXPHOS CI (%).

2) Percentage proton leak fraction of OXPHOS I, II (% Leak): State IV⁺/ OXPHOS CI, CII, and respiratory control ratio (RCR⁺) as State IV⁺/ OXPHOS CI, CII

3) Capacity of cytochrome c oxidase (COX) relative to OXPHOS CI, CII (COX/ OXPHOS CI, CII)

4) Temperature coefficient Q10 was calculated for RMR and heart fibre respiration (only for complex IV/ COX) according to the formula:

(3)

R denotes the respiratory rate (of the heart fibres/ RMR) at a higher (T2) or lower (T1) temperature. Differences in heart fibre oxygen consumption at the assay temperatures 0, 6 and 12, or 16, 21 and 26°C, and between the different acclimation groups were tested using unpaired, two-tailed t-test and one-way analysis of variance (ANOVA, with Tukey post-hoc test). A p0.05 was considered the significance threshold. All values were tested for normality (Kolmogorov-Smirnov) and homogeneity of variance and are given in means ± SEM.

Results Cuttlefish

Whole-animal oxygen consumption. The respiration rate of hypercapnia-acclimated S.

officinalis (16°C, 0.112 kPa CO2; 0.10±0.0 mol O2 g^-1 min^-1) was not different to the RMR of control S. officinalis (0.04 kPa CO2; 0.11±0.0 mol O2 g^-1 min^-1) at 16°C. Both were similar compared to the RMR of E. moschata at 16°C (0.09±0.1 mol O2 g^-1 min^-1).

Permeabilized heart fibre respiration. After five months of acclimation to 0.112 kPa CO2, the heart fibres of S. officinalis showed a higher state III respiration than the control group at the warmest assay temperature of 26°C in the normocapnic respiration buffer. In the same 26°C assay, state III respiration of the hypercapnia acclimated cuttlefish measured in the hypercapnic (1.6 kPa CO2) mitochondrial respiration medium was significantly lower than in the normocapnic medium (Figure 3).

State IV⁺ respiration rose significantly with assay temperature only in normocapnic mitochondrial respiration medium in the control cuttlefish. The percentage contribution of state IV⁺ to state III respiration (% Leak) was lower in the cuttlefish acclimated to elevated PCO2, while their heart fibres exposed to acute hypercapnia had an elevated COX/ OXPHOS CI, CII ratio.

Extracellular acid-base parameters. Control extracellular pH (pHe) of S. officinalis was 7.49±0.03, HCO3- 5.3±1.6 mM and PCO2 0.7±0.2kPa. After long-term hypercapnia acclimation, a new extracellular steady-state HCO3- of 9.9±1.7 mM and PCO2 of 1.1±0.3 kPa was calculated at a significantly lower pHe of 7.40±0.01.

Octopods

Whole-animal oxygen consumption. Respiration rates were mass corrected (gramm wet weight) for all species using the weight exponent 0.75 (37). As respiration rate of P.

charcoti was only measured at 0°C (0.03±0.0 mol O2 g^-1 min^-1), a combined Q10 value was calculated using the respiration rates of P. charcoti at 0°C and E. moschata at 16°C (0.09±0.1 mol O2 g^-1 min^-1), a procedure already used for a comparison between Antarctic and temperate octopus species (10). The resulting Q10 of 2.2 is also depicted in Figure 1, and demonstrates an uncompensated shift from P. charcoti respiration rates at its habitat temperature to the respiration rates of E. moschata at their habitat temperature of 16°C.

RMR of the warm-acclimated E. moschata (21°C, 0.04 kPa CO2) measured at 21°C (0.13±0.0 mol O2 g^-1 min^-1) was only non-significantly higher than the RMR of control E.

moschata (16°C, 0.04 kPa CO2) measured at 16°C, with a Q10 of 2.7 between the two groups (Figure 1).

Permeabilized heart fibre respiration. In E. moschata, thermal acclimation did not affect complex I or II activity (state III respiration; data of complex I respiration not shown), nor net state IV⁺ respiration (proton leak capacities). Only complex IV (COX) assayed at 21°C displayed increased activity following warm-acclimation to 21°C (Figure 2). A shift was also visible in the Q10 for COX activity over the whole thermal range: Q10 of control E.

moschata was 1.8, and 2.2 in warm-acclimated E. moschata (Table 2). Furthermore, the COX/state III ratio measured in the 26°C assay was higher in heart fibres of the warm-acclimated octopus compared to their control.

In P. charcoti, state III respiration (CI, CII) rose from 0.44±0.0 mol O2 g^-1 min^-1 at 0°C, to 0.53±0.0 mol O2 g^-1 min^-1 at 6°C and was significantly higher at 12°C compared to the 0°C assay with values of 0.73±0.1 mol O2 g^-1 min^-1 in normocapnic buffer. The respiration rates of all other respiratory states investigated also rose acutely with temperature, and were significantly elevated at the warmest assay temperature of 12°C (data not shown).

In both octopus species, P. charcoti and E. moschata, heart fibre respiration measured in the hypercapnic (1.6 kPa CO2) respiration medium was not significantly affected when compared to the respiration rates in normocapnic mitochondrial respiration medium (MiRO5).

Only state IV⁺ respiration in the warm-acclimated E. moschata assayed at 16°C was lower in the hypercapnic respiration medium compared to the normocapnic respiration medium

ratios was visible at the highest assay temperatures in the hypercapnic vs. normocapnic respiration medium in E. moschata and P. charcoti (Table 2).

Comparison between species

State III respiration of P. charcoti at 0°C was 0.44±0.0 mol O2 g^-1 min^-1, of E.

moschata at 16°C 0.79±0.2 mol O2 g^-1 min^-1 and 1.11±0.2 mol O2 g^-1 min^-1 in the warm-acclimated E. moschata at 21°C. In control S. officinalis, state III respiration at 16°C was 1.40±0.5 mol O2 g^-1 min^-1 and 2.66±0.4 mol O2 g^-1 min^-1 in the hypercapnia-acclimated group (Figure 4). The values of E. moschata and S. officinalis were not significantly different, only the fibre respiration of P. charcoti at 0°C was significantly lower than the one of E.

moschata and S. officinalis at 16°C.

The respiratory control ratio (RCR⁺) and percentage of OXPHOS CI to total OXPHOS flux of S. officinalis (control and hypercapnia acclimated) was significantly higher than in both octopus species. Consequently, control S. officinalis showed a lower contribution of state IV⁺ to OXPHOS CI, CII (% Leak) than P. charcoti, and hypercapnia acclimated S. officinalis had the lowest state IV⁺ contribution (% Leak) of all species investigated (Table 2).

Discussion

Hypercapnia sensitivity of S. officinalis

A number of studies report reduced metabolic rates (i.e. metabolic depression) during acute and long-term severe hypercapnia, e.g. in mussels or sipunculid worms (33, 49), usually accompanied by an uncompensated decrease in pHe, independent of reduced or restored pHi

levels. Several studies show that pHe can be already altered at low to intermediate hypercapnia levels in invertebrates, however, they are not necessarily accompanied by depressed metabolic rates (e.g. burrowing shrimp (11) or spider crabs (71)).

In our study, the cuttlefish S. officinalis also showed no sign of metabolic depression in their oxygen consumption rates after five months of exposure to 0.112 kPa CO2 (RMR: 0.1 mol O2 g^-1 min^-1), and also another study demonstrated that S. officinalis is able to maintain stable RMRs around 0.09 mol O2 g^-1 min^-1 during acute exposure to an intermediate PCO2

of 0.6 kPa(18). In contrast, the Humboldt squid D. gigas showed considerably reduced RMRs

during acute exposure to 0.1 kPa CO2 (54), which supports the findings on a relatively lower hypercapnia sensitivity of S. officinalis compared to other cephalopods.

When S. officinalis was exposed to acute hypercapnia in another study, it displayed rapid bicarbonate accumulation, which nevertheless was insufficient for complete compensation of pHe (18). Similarly, pHe of the long-term hypercapnia-acclimated cuttlefish remained significantly below control levels (0.09 pH units), paralleled by slightly elevated HCO3- and PCO2 levels. However, it is postulated that such incomplete compensation (up to 0.2 pH units below control) is within the tolerance limit to maintain proper functioning of the respiratory protein, hemocyanin, due to very large Bohr factors below -1 in this species (18).

Other studies on the very active squids I. illecebrosus and L. pealei suggest an even tighter regulation of blood parameters (i.e. pHe) in order to optimize hemocyanin function when blood PCO2 rises, as a drop in arterial pH by only 0.15 pH units could hamper oxygen saturation of their pigment (43, 50). Furthermore, active cephalopods like squid (50) or S.

officinalis probably possess quite efficient intracellular acid-base compensatory mechanisms.

Acute exposure to 0.6 kPa CO2 only caused a very minor decrease in pHi by 0.03 units in Sepia mantle tissue, without involving anaerobic metabolic pathways in order to provide extra energy for acid-base regulation (18).

In line with these findings, Hu et al. (20) report expression patterns of ion-transporters compensated back to control levels after 42 days exposure to 0.3 kPa CO2. As a consequence, metabolic costs largely remain constant during hypercapnia acclimation in S. officinalis, which is also reflected in our RMR data of hypercapnia vs. control cuttlefish.

Nevertheless, the study by Hu et al. (20) revealed an increase in ATP-synthase and COX mRNA expression during 42 days exposure of juvenile S. officinalis to 0.3 kPa CO2, which gives a first hint of slight alterations in mitochondrial energy metabolism.

Indeed, state III respiration of the control group measured in normocapnic respiration buffer showed the same pattern as the heart fibres of the hypercapnia acclimated group measured in the hypercapnic buffer, which can be taken as a first indicator for compensatory mechanisms in mitochondrial metabolism towards chronic hypercapnia. Following a hypothesis for mammalian and fish mitochondria, acutely elevated bicarbonate concentrations can competitively inhibit citrate synthase and succinate-dehydrogenase within the TCA-cycle (60, 65), (Strobel et al. 2013, under review). This acute effect might then be compensated by shifts in altered activities and/ or quantities of mitochondrial complexes and shifts in metabolic pathways, such as enhanced glutamate oxidation feeding into the TCA-cycle via

2-Furthermore, the intra-mitochondrial soluble adenylyl cyclase (sAC) is being discussed to be directly stimulated by bicarbonate, and then produces the second messengers cyclic adenosine monophosphate (cAMP) in mammals. In the course of this, it activates protein kinase A (PKA), which in the end phosphorylates Complex IV of the electron transfer system (ETS), and possibly Complex I as well (63). As a long-term response, the sAC and cAMP signaling cascade may directly influence the expression or activity of metabolic enzymes and mitochondrial complexes via transcription factors and post-translational modifications, and thereby elicit constantly elevated levels of e.g. COX (70). In line with these hypotheses, transcriptional or post-translational regulation of slightly elevated ATP synthase and COX expression patterns has been demonstrated in hypercapnia exposed, juvenile S. officinalis (20). In consequence, elevated intracellular bicarbonate is postulated to increase OXPHOS efficiency and ATP synthesis due to a high flux through the ETS (1, 6, 63).

In fact, mitochondrial state III respiration of the hypercapnia acclimated S. officinalis in this study was significantly elevated when assayed at 26°C in normocapnic respiration buffer (Figure 3), which indicates a shift towards compensated mitochondrial capacities compared to the control group via an increase in ATP synthesis capacities to overcome the inhibitory effect of elevated PCO2. Due to acclimation to new intracellular conditions, i.e.

slightly lower pHi and higher bicarbonate levels, mitochondrial capacities of the hypercapnia acclimated S. officinalis then show similar respiration patterns (state III) in the hypercapnic buffer, as the control group in the normocapnic buffer (Figure 3). This corresponds to their

‘new’ intracellular environment with increased bicarbonate levels. Accordingly, shifts towards enhanced mitochondrial complex and enzyme activities, induced by sAC/ cAMP, seem to bring mitochondrial capacities back to normal levels.

When the heart fibres of the hypercapnia acclimated cuttlefish are measured in normocapnic buffer, their excessive capacities (i.e. higher enzyme/ ETS activities) become visible (Figure 3, lower panel). Thus, compensation of mitochondrial capacities as a response to bicarbonate induced mitochondrial inhibition seems likely.

In the control animals, the relative proton leak increased with assay temperature, similar to findings in Antarctic bivalves (Laternula elliptica) (48) and in the lugworm Arenicola marina (61). In the hypercapnia acclimated cuttlefish, state IV⁺ respiration did not increase with acute assay temperature. Reduced proton leak capacities can be mediated by structural changes of mitochondrial membranes or by an increase in ATP synthase activity relative to electron flux through the ETS, thereby reducing mitochondrial membrane potential

Im Dokument Mitochondrial plasticity in response to changing abiotic factors in Antarctic fish and cephalopods (Seite 181-200)