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

Abstract

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.

Keywords: Cephalopod; Diffusion chamber; Eledone moschata; Haemocyanin; Hemocyanin; Octopus pallidus; Oxygen affinity; Oxygen carrying capacity; Pareledone charcoti.

Figure 1

Phylogenetic relationships of the three octopod species analysed in this study and related octopodiformes. The Bayesian phylogenetic tree was based on the mitochondrial genes cytochrome oxidase subunit I and III and the nuclear genes rhodopsin, octopine dehydrogenase and 16S rDNA. Vampyroteuthis infernalis and Argonauta nodosa served as outgroup. Posterior probabilities were shown above nodes with stars marking values of 1.0. Colours denote the climatic origin. The opening of the Drake Passage ca. 29–32 million (Ma) years ago (position marked on tree was taken from [26]), denoting the isolation of Antarctic waters from warmer waters, preceded the diversification of the Antarctic genus Pareledone. Pareledone charcoti belongs to the endemic Southern Ocean octopod family Megaleledonidae and shares ancestry with Adelieledone polymorpha. This species inhabits the northern Antarctic Peninsula and the Scotia Arc island bridge connecting shallow South American waters with the Antarctic shelf, indicating an origin from temperate shallow waters [27]. Octopus pallidus and Eledone moschata belong to distinct families of non-polar shallow water octopods [28].

Figure 2

Lowered affinity of haemocyanin for oxygen in the Antarctic Pareledone charcoti . (A) Oxygen affinity, expressed as the PO₂ of haemocyanin half-saturation, (P ₅₀), and (B) venous oxygen saturation of Pareledone charcoti were compared to two octopods originating from warmer waters, Octopus pallidus and Eledone moschata, at a comparative experimental temperature of 10°C. Calculations refer to an alpha-stat adjusted venous pH of 7.27 at 10°C and a venous PO₂ of 1 kPa. Differing letters indicate significant differences (P < 0.05) between species.

Figure 3

pH oxygen-saturation curves of haemolymph from Antarctic (A-C), South-east Australian (D-F) and Mediterranean (G-I) octopods. pH oxygen-saturation curves denote the change of oxygen saturation of haemocyanin from high to low pH at constant PO₂ (21, 13, 4, 1 kPa from left to right) and are most suitable to illustrate the high pH dependence of oxygen binding of cephalopod haemocyanin see [52]. For replicated measurements (n = 5–6), means and 95% confidence intervals (shaded area) of fitted pH oxygen-saturation curves are displayed. Replicate measurements could not be performed for Pareledone charcoti at 5°C and Eledone moschata at 15°C due to insufficient amounts of haemolymph sample. Vertical lines indicate the alpha-stat adjusted arterial (dashed) and venous pH (solid). The ten degree temperature windows cover approximate habitat temperatures for each species.

Figure 4

A) Change of arterial and venous oxygen saturation and B) venous oxygen release by octopod haemocyanin with temperature. Data refer to an arterial PO₂ of 13 kPa and to venous PO₂ for a resting (4 kPa) and exercised (1 kPa) octopus. Arterial and venous PO₂ were assumed to be constant across temperatures and not determined for the analysed octopod species and instead taken from Octopus vulgaris [50,51]. Venous pH values were alpha-stat adjusted for each temperature and arterial pH assumed to be 0.11 pH units higher than venous pH [50]. Venous oxygen release including the contribution by dissolved oxygen is indicated by dashed lines. The ten degree temperature windows cover habitat temperatures for each species except for Pareledone charcoti.

Figure 5

Total protein and haemocyanin concentrations in haemolymph of cold and warm water octopods. Haemocyanin concentrations were calculated from the haemolymph oxygen carrying capacity, based on a molecular weight of 3.5 MDa and 70 oxygen binding sites stated for octopod haemocyanin [53]. Total protein concentration was determined according to Bradford [54]. Bars depict means + 95% C.I., n = 9–13. Differing letters indicate significant differences (P < 0.05) between octopod species for total haemolymph protein (upper case) or haemocyanin concentrations (lower case). White values on bars indicate the fraction of haemocyanin relative to total haemolymph protein and asterisks significant differences between species.

Figure 6

pH at which the pH-dependent release of oxygen by haemocyanin becomes maximal. Comparison between the Antarctic Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata at an experimental temperature of 10°C. Calculations include pH oxygen-saturation curves from all analysed PO₂. Letters indicate significant differences (P < 0.05) between species. Data from different PO₂ were pooled due to similar effects by PO₂ among species.

Figure 7

Observed alpha-stat pH pattern for octopus haemolymph. The temperature dependent change of pH was determined for thawed Octopus pallidus haemolymph at 0°C, 10°C, and 20°C. Venous pH of the other species refer to freshly sampled and analysed haemolymph. pH were corrected to the free hydrogen ion scale by subtracting an experimentally determined offset of −0.136 (0.130-0.142, n = 87) pH units to account for the high ionic strength of cephalopod haemolymph [61]. Sources: Octopus pallidus, Pareledone sp., Adelieledone polymorpha (Strobel and Oellermann 2011, unpublished); Eledone moschata (Strobel and Mark 2010, unpublished); Octopus vulgaris [50,51].

Figure 8

Additional release of oxygen by haemocyanin relieves the circulation system of Pareledone charcoti at 10°C. Oxygen that remained bound to haemocyanin at 0°C (blue) was largely liberated at 10°C (red), and thereby reduces the need for increased blood circulation (i.e. expressed as number of times to circulate the whole blood volume per second, 5.2% vs. 110.4% increase in circulation) to match an increased oxygen demand at 10°C. Oxygen supply rates (O₂ release from haemocyanin between 13 and 4 kPa PO₂, solid lines) match oxygen consumption rates of Pareledone charcoti (mean MO₂ ± SD, 0.63 mmol O₂ kg⁻¹ (wet mass) h⁻¹ ± 0.12, at 0°C, vertical dashed lines, taken from [7]) at the intersections of both rates at 0°C or 10°C (values indicated on x axis). Oxygen supply comprises the oxygen transported by haemocyanin only without contributions by dissolved oxygen or oxygen absorbed via the skin. The MO₂ at 10°C was interpolated assuming a Q₁₀ of 2.12 (average Q₁₀ for Octopoda taken from [46-48]. The blood volume was assumed to be 5.2% (v/w) based on average literature values from Octopus vulgaris and Enteroctopus dofleini [78,79].