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Temperature Effects on Hemocyanin Oxygen Binding in an Antarctic Cephalopod.


Abstract. The functional relevance of oxygen transport by hemocyanin of the Antarctic octopod Megaleledone senoi and of the eurythermal cuttlefish Sepia officinalis was analyzed by continuous and simultaneous recordings of changes in pH and hemocyanin oxygen saturation in whole blood at various temperatures. These data were compared to literature data on other temperate and cold-water cephalopods (octopods and giant squid).

In S. officinalis, the oxygen affinity of hemocyanin changed at [delta][P.sub.50]/[degrees]C = 0.12 kPa (pH 7.4) with increasing temperatures; this is similar to observations in temperate octopods. In M. senoi, thermal sensitivity was much smaller ([less than]0.01 kPa, pH 7.2). Furthermore, M. senoi hemocyanin displayed one of the highest levels of oxygen affinity ([P.sub.50] [less than] 1 kPa, pH 7.6, 0 [degrees]C) found so far in cephalopods and a rather low cooperativity ([n.sub.50] = 1.4 at 0 [degrees]C). The pH sensitivity of oxygen binding ([delta] log [P.sub.50]/[delta] pH) increased with increasing temperature in both the cuttlefish and the Antarctic octopod. At low [Po.sub.2] (1.0 kPa) and pH (7.2), the presence of a large venous oxygen reserve (43% saturation) insensitive to pH reflects reduced pH sensitivity and high oxygen affinity in M. senoi hemocyanin at 0 [degrees]C. In S. officinalis, this reserve was 19% at pH 7.4, 20 [degrees]C, and 1.7 kPa [O.sub.2], a level still higher than in squid.

These findings suggest that the lower metabolic rate of octopods and cuttlefish compared to squid is reflected in less pH-dependent oxygen transport. Results of the hemocyanin analysis for the Antarctic octopod were similar to those reported for Vampyroteuthis--an extremely high oxygen affinity supporting a very low metabolic rate. In contrast to findings in cold-adapted giant squid, the minimized thermal sensitivity of oxygen transport in Antarctic octopods will reduce metabolic scope and thereby contribute to their stenothermality.


Cephalopods are found throughout the seas of the world, from warm tropical waters to polar oceans (Roper et al., 1984). Representatives of this group, especially squids, usually display the highest metabolic rates among marine invertebrates, even higher than those of fishes of similar size and mode of life (Webber and 0'Dor, 1985; O'Dor and Webber, 1986). Oxygen delivery via the blood is maximized to cover metabolic requirements (Portner, 1994). However, the capacity of their blood pigment, hemocyanin, for carrying oxygen is constrained by the low concentration of an extracellular pigment. This limitation is due to the unfavorable increase in colloidal osmotic pressure and blood viscosity at high pigment concentrations (Mangum, 1983, 1990). Although cephalopods, in accordance with their high rate of oxygen consumption, display the highest hemocyanin concentrations in the animal kingdom, the level of bound oxygen in squid (up to 3 mmol [1.sup.-1]; Brix et al., 1989) remains below the 4-5 mmol [1.sup.-1] of ac tive fishes (Urich, 1990). Therefore, the hearts of squids pump large volumes of blood (Wells et al., 1988; Shadwick et al., 1990) and the tissues extract most of the oxygen (Portner, 1994). Compared to that of squids, the oxygen-binding capacity of octopod blood is somewhat reduced, ranging between 0.6 and 1.6 mmol [1.sup.-1], depending on hemocyanin levels (Senozan et al., 1988; Brix et al., 1989).

In most cephalopods, cooperativity and temperature- and pH-dependent changes in affinity are the only means of modulating hemocyanin function and adjusting oxygen transport (e.g., Brix et al., 1989; Mangum, 1990; Portner, 1990). Low-molecular-weight organic substances that contribute to blood pigment function in vertebrates or crustaceans are not found in this group. In consequence, extremely large Bohr shifts ([delta] log [P.sub.50]/[delta]pH [less than] -1; Bridges, 1994) and very high levels of pH-dependent cooperativity are common (Miller, 1985; Portner, 1990). Binding of [CO.sub.2] together with [O.sub.2] in arterial blood has been suggested to support pigment function on the venous side in sepioid species (Brix et al., 1981), where both [O.sub.2] and [CO.sub.2] are released, and this [CO.sub.2] helps to exploit the large Bohr effect. In squid, supplementary oxygen uptake via the skin supports the excessive oxygen demand and provides the excess [CO.sub.2] required for the Bohr effect to function (Portne r, 1994).

In some cephalopods, an increase in ambient temperature has a large effect on oxygen transport by hemocyanin; this effect is reflected by an increase in cooperativity and a fall in oxygen affinity (Brix et al., 1989, 1994; Mangum, 1990). If a rise in metabolic rate with temperature is supported by an adequate rise in [P.sub.50], the species should be able to live at a broader range of temperatures than a species in which [P.sub.50] remains constant or in which the change in [P.sub.50] is too large. For example, the high thermal sensitivity of the oxygen affinity of hemocyanin in the giant squid Architeuthis monachus suggests that arterial saturation becomes impossible at high temperatures (Brix, 1983). This question has gained general importance since comparative studies in Antarctic and temperate fish and invertebrates (including cephalopods; Portner and Zielinski, 1998) revealed that the limits of thermal tolerance are characterized by oxygen limitation, owing to the inability of circulation or ventilation to provide sufficient oxygen at extreme temperatures (for review, see Portner et al., 2000). Comparison of hemocyanin oxygen binding in cephalopods of different metabolic rates and from various latitudes should show how hemocyanin oxygen transport has adapted to different temperature regimes at various levels of metabolic activity and how blood pigment function contributes to the oxygen limitation of thermal tolerance.

These questions are especially interesting for an understanding of physiological adaptations to life in Antarctica. Here the marine environment is characterized by very stable water temperatures that are close to freezing (Clarke, 1988). Under these conditions more oxygen is physically dissolved, thereby facilitating oxygen uptake and supply to tissues. At the same time metabolic rate is reduced at lower temperatures, with the consequence that in some species blood pigments may be less important (for hemocyanin, see Mauro and Mangum, 1982b; Burnett et al., 1988). Some Antarctic fishes, the icefishes (Channichtyidae) have even lost their respiratory pigments (Ruud, 1954). The question arises as to whether the importance of blood pigment function is also reduced in Antarctic cephalopods.

Live specimens of the octopod Megaleledone senoi became available during a recent expedition to Antarctica with the RV Polarstern. This species is found in the indo-atlantic sector of the Antarctic Ocean (Taki, 1961; Kubodera and Okutani, 1986, 1994). In our study we investigated oxygen binding to the hemocyanin of this stenothermal Antarctic octopod by using a technique that allows continuous and simultaneous recordings of blood pH and oxygenation and the construction of diagrams depicting changes in oxygen saturation with pH (pH/saturation diagrams; Portner, 1990). Such an approach is most suitable for cephalopod blood owing to the extremely large pH dependence of oxygen binding (see above). It avoids the use of artificial buffers that may lead to a change in oxygen-binding properties (Portner, 1990; Brix et al., 1994). At the same time, the amount of blood required is reduced such that more sophisticated data can be collected from the very few animals accessible in remote environments like the Antarctic. The oxygen-binding properties of M. senoi hemocyanin were compared with those from other eurythermal and stenothermal cephalopods. For eurythermal octopods some literature data were available. Temperature effects on oxygen binding were studied experimentally in the cuttlefish Sepia officinalis to complement the data set available in the literature (Lykkeboe et al., 1980; Johansen et al., 1982a). To some extent, cuttlefish display a mode of life similar to that of octopods. Like octopods, they live close to the bottom of the sea (von Boletzky, 1983), but they have a larger scope for activity and metabolism, which might influence the thermal adaptation of hemocyanin function.

Materials and Methods


Antarctic octopods (Megaleledone senoi, up to 9 kg body weight) were caught in November 1996 north of Elephant Island, Antarctica, during expedition ANT XIV/2 of the RV Polarstern. The animals were collected from bottom trawls. Samples were taken immediately after capture.

Cuttlefish (Sepia officianlis, 470 to 960 g body weight) were obtained from the Marine Biomedical Institute of the University of Texas, Galveston, Texas, where this species has been bred and grown for several consecutive generations. They were kept at a salinity of 35% at temperatures of 20 to 22 [degrees]C.

Sampling procedure

Animals were anesthetized by transferring them into seawater containing 2% ethanol (v/v). The animals were then removed from the seawater and the mantle was opened by a ventral incision. Blood was collected from the vena cava, the systemic heart, and the gill hearts. Blood samples from all animals were pooled, frozen, and stored for up to one year at close to -20 [degrees]C until utilized for in vitro studies of oxygen binding.

Analysis of oxygen binding

Oxygen-binding characteristics of cephalopod hemocyanin were studied using a specially constructed cuvette, built by Hellma GmbH & Co. (Mulheim, Germany; Fig. 1). The cuvette consisted of an upper and a lower compartment connected by two shafts (1.5 and 2 mm in diameter) in the left and right periphery of the cuvette, as well as a central compartment between the shafts, where blood formed a thin layer of only 0.45 mm. Stirring bars operating in the upper and lower compartments ensured continuous exchange of blood between all compartments and thus uniform mixture of the blood. Oxygen saturation was monitored continuously by using a diode array spectrophotometer with fiber optics (X-dap, IKS Optoelektronik Me[beta]gerate GmbH, Waldbronn, Germany) to measure absorbance at 345 nm through the thin layer. Blood samples were equilibrated by introducing humidified gas mixtures through a hole in the lid of the cuvette. Gas mixtures of variable P[o.sub.2] (between 1.0 and 20.0 kPa) were prepared from pure [O.sub.2], [CO.sub.2], and [N.sub.2] by gas-mixing pumps (type 2M303/a-F, Wosthoff, Bochum, Germany); complete deoxygenation occurred under pure [N.sub.2]. Blood pH was varied by changing P[co.sub.2] (between 0.09 and 1.01 kPa) or by replacing small volumes ([less than]10 [micro]1 per 2 ml of blood) of supernatant plasma after ultracentrifugation (1 h at 120,000 X g; Beckman Airfuge, Beckman Instruments, Inc., Fullerton, CA) with fixed acid (1 mol [1.sup.-1] HC1) or base (2 mol [1.sup.-1] NaOH; Morris et al., 1985; Portner, 1990). Changes in blood pH during oxygenation and deoxygenation of hemocyanin were measured continuously by using a needle pH electrode (long micro needle electrode #811, Diamond General Corp., Ann Arbor, MI) that was introduced into one of the shafts via a second hole in the lid. Total [CO.sub.2] was analyzed at 0 [degrees]C in 50-[micro]l blood samples of M. senoi; the gas chromatography method of Lenfant and Aucutt (1966) modified after Boutilier et al. (1985) was used. Measurements of oxygen-bindi ng properties were carried out at 0, 5, and 10 [degrees]C for samples of M. senoi and at 0, 10, and 20 [degrees]C for samples of S. officinalis; 10 [degrees]C was chosen since this temperature can be reached in the northern part of Sepia's distribution range (Isemer and Hasse, 1985).

Graphical analysis and calculations

Hemocyanin concentrations were measured photometrically and calculated using the extinction coefficients of Nickerson and van Holde (1971). Oxygen capacity was estimated using the molecular weights for octopods and Sepia as compiled by Miller (1994) and the assumption that there are 70 [O.sub.2]-binding sites per hemocyanin molecule in octopods and 80 in Sepia. For the evaluation of hemocyanin oxygen saturation, constant absorbance levels in the range of the highest values of P[o.sub.2] and pH were set to 100% saturation. Changes in hemocyanin oxygenation and pH were plotted in a pH/saturation diagram according to Portner (1990). The resulting oxygen-binding curves represent isobars delineating the change in oxygenation with pH at constant P[o.sub.2]. The points of intersection of the oxygen isobars with the line of half saturation quantify [P.sub.50], because it depends on pH. These [P.sub.50] and pH values were used to evaluate the Bohr coefficient, [delta] log [P.sub.50]/[delta]pH by linear regression ana lysis. For comparison, and owing to the presence of a large pH-insensitive oxygen reserve at low temperatures, the coefficient [delta] log [p.sub.80]/[delta]pH was evaluated following the same procedure. The Haldane coefficient ([delta][[HCO.sub.3].sup.-]/[delta]Hcy[O.sub.2]) was evaluated from the vertical distance between buffer lines in a pH/bicarbonate diagram (as used by Brix et al., 1981). To assess whether oxygen-linked [CO.sub.2] binding to the hemocyanin occurs (Lykkeboe et al., 1980), "measured" and calculated apparent bicarbonate levels were compared. Apparent "bicarbonate" (the sum of [[HCO.sub.3].sup.-] and [[CO.sub.3].sup.2-] levels) was calculated from measured [CO.sub.2] concentrations (C[co.sub.2]) using the applied P[co.sub.2] and the measured pH according to the formula

[[[HCO.sub.3].sup.-]] = C[co.sub.2] - [alpha]P[co.sub.2] (1)

where [alpha] is the solubility of [CO.sub.2]. For comparison, bicarbonate levels were also calculated according to equation (1) with C[co.sub.2] values derived from the Henderson-Hasselbalch equation:

C[co.sub.2] = P[co.sub.2] * ([alpha] * [10.sup.pH-pK'"] + [alpha]) (2)

[alpha] and pK'" were calculated according to Heisler (1986).

Along each isobar in the pH/saturation diagram, values of saturation S depend on pH values and the P[o.sub.2] of the isobar. The pH/saturation diagram allows comparison of S and log P[o.sub.2] with [P.sub.50] at the same pH (=p[H.sub.50]). This leads to an analysis of cooperativity at a specific pH. If this is done in the range of saturation S between 0.4 and 0.6, the analysis leads to an estimate of Hill coefficients ([n.sub.50]) according to

log(S/1 - S) = [n.sub.50](log P[o.sub.2] - log [P.sub.50]) (3)

where P[o.sub.2] is the P[o.sub.2] of the isobar, S results from P[o.sub.2] at a specific pH (p[H.sub.50]), and [P.sub.50] is the P[o.sub.2] for S = 0.5 at the same pH (Portner, 1990).


The concentration of hemocyanin in native blood (hemolymph) was 93 g [l.sup.-1] for Megaleledone senoi and 142 g [1.sup.-1] for Sepia officinalis. This is equivalent to a maximum level of hemocyanin-bound oxygen of 1.86 mmol [O.sub.2] [1.sup.-1] in the octopod and of 2.84 mmol [1.sup.-1] in Sepia. For M. senoi hemocyanin, the highest pH sensitivity of oxygen binding was found at 10 [degrees]C, as indicated by maximum slopes [delta]S/[delta]pH (Fig. 2). Lower temperatures resulted in a somewhat decreased pH sensitivity of oxygen affinity, with a maximum of [delta]S/[delta]pH = 13% per pH unit at 10 [degrees]C, compared to a maximum of 10% per pH unit at 0 [degrees]C. Saturation at 0 [degrees]C did not fall below 43% even at low pH (6.4 and 6.6) and low P[o.sub.2] (1 kPa). At 10 [degrees]C, saturation dropped to a minimum of 32% at the same P[o.sub.2]. Intermediate values of pH sensitivity and maximum unloading were found at 5 [degrees]C.

A large Bohr coefficient of -2.33 was found at 10 [degrees]C, similar to the coefficient [delta] log [P.sub.80]/[delta] pH (Table 1). The experimental evaluation of the Bohr coefficient was not possible at lower temperatures due to the fact that pH-dependent saturation did not drop below 50% at most partial pressures of oxygen. An extrapolation of binding data to very low partial pressures of oxygen revealed a Bohr coefficient of approximately -0.9 at 0 [degrees]C, below the level of [delta] log [P.sub.80]/[delta]pH. Furthermore, oxygen affinity ([P.sub.50]) at pH 7.2 changed only at [delta][P.sub.50]/[degrees]C [less than] 0.01 kPa, from 0.98 kPa at 0 [degrees]C to 1.10 kPa at 10 [degrees]C (Table 1).

For S. officinalis hemocyanin, pH sensitivity was high at 20 [degrees]C reaching a maximum [delta]S/[delta]pH of 41% per pH unit (Fig. 3). Especially in the pH range between 7.4 and 7.8, very small pH changes were sufficient to cause maximal unloading of oxygen, down to 19% saturation. The pH sensitivity at 20 [degrees]C was higher than found for M. senoi at all temperatures. As in M. senoi, lower temperatures decreased the pH sensitivity of oxygen binding with a decreased Bohr factor and level of [delta] log [P.sub.80]/[delta]pH (Table 1) and an increase in the pH-insensitive reserve at the same P[o.sub.2]. At 0 [degrees]C, [delta]S/[delta]pH reached a maximal value of only 7% per pH unit. Oxygen saturation remained above 50% at all investigated partial pressures of oxygen and values of pH. At pH 7.4, oxygen affinity fell from [P.sub.50] = 5.3 kPa at 10 [degrees]C to [P.sub.50] = = 6.5 kPa at 20 [degrees]C ([delta][P.sub.50]/[degrees]C = 0.12 kPa, Table 1).

The change in cooperativity with pH and temperature for S. officinalis is shown in Figure 4. At 20 [degrees]C the largest Hill coefficient ([n.sub.50]) of 5.9 was found at a pH (7.48) where pH sensitivity ([delta]S/[delta]pH) was also high. A decrease in temperature to 10 [degrees]C resulted in a decrease of the maximal Hill coefficient to [n.sub.50] = 4.6 (pH 7.29). The maximum was shifted to lower pH. In contrast to cuttlefish, M. sensoi had much lower Hill coefficients (Table 1). At 0 [degrees]C, [n.sub.50] was 1.4 (pH 7.43), and it varied between 1.0 (pH 6.83) and 1.4 (pH 7.31) at 10 [degrees]C. No clear maximum could be found.

Analysis of total [CO.sub.2] in M. senoi blood during variations of P[o.sub.2] and P[co.sub.2] yields the buffer lines depicted in the pH/bicarbonate diagram (Fig. 5). The position of the buffer line shifts between oxygenated and deoxygenated blood according to the quantity of [H.sup.+] bound by the pigment. The vertical distance between the buffer lines yields the Haldane coefficient ([delta][[HCO.sub.3].sup.-]/[delta]Hcy[O.sub.2]). For M. senoi hemocyanin at 0 [degrees]C the Haldane coefficient rose with falling pH (Table 1). The calculated apparent bicarbonate levels for oxygenated and deoxygenated blood diverge only slightly from the measured values, suggesting that [O.sub.2]-linked [CO.sub.2] binding does not exist (Fig. 5). The non-bicarbonate buffer value ([[beta].sub.NB]) of 4.25 mmol [1.sup.-1] pH [units.sup.-1] of M. senoi blood at 0 [degrees]C is in the same range as in the squids Illex illecebrosus and Loligo pealei (5.0 and 5.8 mmol [1.sup.-1] pH [units.sup.-1], respectively; Portner, 1990).


At the low temperatures of Antarctica icefishes rely exclusively on the transport of oxygen that is physically dissolved in the blood (see Introduction). The presence of hemocyanin-bound oxygen in Megaleledone senoi blood at levels similar to those seen in squids and temperate octopods (cf. Brix et al., 1989) suggests that oxygen transport via hemocyanin is as important in this Antarctic species as in temperate and warm-water cephalopods. In contrast to Antarctic fishes, the unchanged requirement for blood oxygen transport in Antarctic cephalopods may be related to the low level of capillarization of cephalopod musculature compared to fish muscles (Bone et al., 1981).

In cephalopods, pH and temperature are most important factors in the regulation of hemocyanin oxygen transport (Brix et al., 1989; Bridges, 1994; Portner, 1994). The temperature dependence of [P.sub.50] varies greatly between species. For example, the oxygen affinity of the hemocyanin of giant squid (Architeuthis monachus) decreases at [delta][P.sub.50]/[delta]T = 1.89 kPa per degree Celsius (pH 7.4), while a value of only 0.20 kPa/[degrees]C (pH 7.4) was found for the octopod Octopus vulgaris (calculated after Brix et al., 1989). A lower value of [delta][P.sub.50]/[delta]T = 0.10 kPa/[degrees]C (pH 7.4) was found for the octopod Eledone cirrhosa (calculated after Bridges, 1994); this was similar to the value of 0.12 kPa/[degrees]C (pH 7.4) calculated for Sepia officinalis hemocyanin in the present study. In eurythermal cephalopods like Sepia officinalis, Octopus vulgaris, or Eledone cirrhosa and in some squids, a moderate rise in [P.sub.50] with temperature occurs (cf. Brix et al., 1994). In this way capill ary P[o.sub.2] is maintained ("buffered") at progressively higher levels, which are required for elevated diffusive oxygen flux to mitochondria during increased rates of oxygen consumption. Such changes in [P.sub.50] with temperature allow S. officinalis to be distributed over a wide range, from the Mediterranean to the North Sea (von Boletzky, 1983). In contrast, the extreme thermal sensitivity of [P.sub.50] seen in giant squid may eliminate oxygen transport by hemocyanin and contribute to the heat intolerance of these animals (Brix, 1983). The very small adjustments of [P.sub.50] found in M. senoi hemocyanin ([delta][P.sub.50]/[delta]T = 0.001 kPa/[degrees]C, pH 7.2) may also be detrimental, because when temperature rises, blood P[o.sub.2] will be held at levels too low to adequately support an increase in metabolic rate. In consequence, Antarctic octopods like M. senoi are very stenothermal (Portner and Zielinski, 1998).

For S. officinalis the greatest pH sensitivity (Fig. 3) at 20 [degrees]C was found in the range of in vivo blood pH (7.4-7.8 at 17-19 [degrees]C; Johansen et al;., 1982a). The same phenomenon occurs in squid (Portner, 1990). The fact that the oxygen transport system responds to even small changes in extracellular acid-base status is consistent with the pH-dependent P[o.sub.2] buffer function of the hemocyanin (Portner, 1994). This is not surprising, because cephalopods regulate primarily extracellular, not intracellular, acid-base balance (Portner, 1994).

The blood pH of the Antarctic octopod is not known. Assuming that blood pH follows [alpha]-stat predictions (Reeves, 1972) and is high in the cold, as seen in Loligo pealei blood in vitro (Howell and Gilbert, 1976), the in vivo pH range for M. senoi is between pH 7.7 and 7.9 at 0 [degrees]C. With the highest pH sensitivity in this pH range, oxygen unloading would occur at very low oxygen tensions ([less than]1 kPa), supporting only very low metabolic rates (Fig. 2). A [P.sub.50] below i kPa (pH 7.6; 0 [degrees]C reflects one of the highest oxygen affinities reported so far for cephalopods. This value is close to the [P.sub.50] of 0.47 to 0.55 kPa evaluated for the cold-water vampire squid Vampyroteuthis infernalis (5 [degrees]C; Seibel et al., 1999). These findings suggest that M. senoi displays a low metabolic rate similar to that of the Antarctic octopod Pareledone charcoti (0.3 [micro]mol [g.sup.-1] [h.sup.-1] at 0 [degrees]C and about 50 g body weight; H. O. Portner, T. Hirse, V. Wegewitz, unpubl. data, 15 times lower than similar sized S. officinalis at 17 [degrees]C, 4.4 [micro]mol [g.sup.-1] [h.sup.-1], Johansen et al., 1982b) or even lower and close to the 0.1 [micro]mol [g.sup.-1] [h.sup.-1] measured at 5 [degrees]C in the deep-sea squid Vampyroteuthis infernalis (Seibel et al, 1997).

The Bohr coefficient evaluated for both investigated cephalopod species dropped when temperature decreased. This result is similar to findings in the crustaceans Cancer magister and Cancer anthonyi (Burnett et al., 1988) and in the octopod Eledone cirrhosa (Bridges, 1994). In S. officinalis, [delta] log [P.sub.50]/[delta]pH decreased moderately, from -1.33 at 20 [degrees]C to -0.99 at 10 [degrees]C (Table 1). In M. senoi, the Bohr coefficient fell drastically, from an extremely high value of -2.33 at 10 [degrees]C to a much smaller value evaluated by extrapolation to be -0.9 at 0 [degrees]C (Table 1). The Bohr factor in the vampire squid was found to be even lower (-0.22; Seibel et al., 1999). These results indicate that the Bohr effect becomes less important at low temperature and low metabolic rate.

A mechanism of oxygen-linked [CO.sub.2] binding has been proposed for Sepia hemocyanin, which transports both [O.sub.2] and [CO.sub.2] to the tissues. The [CO/.sub.2] produced in metabolism and the [CO.sub.2] released during deoxygenation would elicit a drop in pH, as required for the large Bohr effect ([less than] -1.0) to function normally (Lykkeboe et al., 1980; Brix et al., 1981). No oxygen-linked [CO.sub.2] transport was found in M. senoi (Fig. 5). At 0 [degrees]C, the estimated Bohr coefficient of [delta] log [P.sub.50]/[delta]pH [approximate] -0.9 would reflect normal function of the Bohr effect, whereas the extremely high Bohr coefficient at 10 [degrees]C would be counterproductive for oxygen transport, a finding consistent with the stenothermality of Antarctic animals.

A reduced pH sensitivity of hemocyanin oxygen binding in M. senci and other octopods compared to S. officinalis and squids is reflected in the magnitude of the pH-independent venous reserve, which rises as temperature falls (Figs. 2, 3). This reserve represents the amount of oxygen that remains bound to the respiratory pigment at constant P[o.sub.2], even when pH falls to very low values. Comparison of this venous reserve for several cephalopod species at normal environmental temperatures and low [Po.sub.2] shows that it is below 5% (at 1.7 kPa) for the squid Illex illecebrosus (15 [degrees]C; Portner, 1990), 19% (at 1.7 kPa) for S. officinalis (20 [degrees]C; this study), and 43% (at 1 kPa) for the Antarctic octopod M senoi (0 [degrees]C; this study). A value of below 10% results for the hemocyanin of the octopod Octopus dofleini (at 1.7 kPa and 20 [degrees]C; Portner, 1990; calculated after Miller and Mangum, 1988); however, the in vivo value may be higher for this species because it lives at lower tempera tures. The high pH sensitivity of squid hemocyanins maximizes the release of oxygen in the tissues and supports their high metabolic rate (Portner, 1990, 1994). Sepioids and, even more so, octopods display a less active life style with lower metabolic rates (for example: Houlihan et al., 1982; Webber and O'Dor, 1985, 1986; Finke et al., 1996; Seibel et al., 1997). A low-activity mode of life may eliminate the necessity to maximize pH-dependent oxygen transport to the extent seen in squids.

With falling pH, the pH-independent venous reserve increased and was reached at higher P[o.sub.2] (88% at pH 6.8, 20 kPa [O.sub.2], and 0 [degrees]C in M. senoi, or 40% at pH 6.8, 20 kPa [O.sub.2], and 20 [degrees]C in S. officinalis). At normoxic P[o.sub.2] (20 kPa [O.sub.2]) and low pH, this resembles a Root effect (Bridges, 1994) but at the same time, further pH sensitivity (the Bohr effect) is eliminated and deoxygenation depends exclusively on P[o.sub.2] (Figs. 2 and 3).

The cooperativity of respiratory pigments is characterized by the Hill coefficient ([n.sub.50]). In S. officinalis at 20 [degrees]C (Fig. 4) and in the squids Illex illecebrosus and Loligo vulgaris, the highest cooperativity correlates with the highest pH sensitivity of oxygen binding ([delta]S/[delta]pH) in the range of in vivo pH (Portner, 1990). Here maximal deoxygenation occurs at minimal pH change (Portner, 1990, 1994). A decrease in temperature caused the maximal Hill coefficient of S. officinalis hemocyanin to drop from [n.sub.50] = 5.9 at 20 [degrees]C to [n.sub.50] = 4.6 at 10 [degrees]C. At the same time, maximum cooperativity was shifted to lower pH values, when in vivo pH should rise according to [alpha]-stat predictions (Reeves, 1972). A similar temperature dependence of the Hill coefficient was found for several crustaceans (Mauro and Mangum, 1982a,b). As with the Bohr effect, the progressive mismatch between the pH range of maximum cooperativity and the actual blood pH suggests that cooperativ ity becomes less important in oxygen transport at lower temperatures.

Accordingly, a low cooperativity of [n.sub.50] = 1.4 was found for M. senoi, at 0 [degrees]C (Table 1) and of [n.sub.50] = 2.2 for the vampire squid (Seibel et al., 1999). Surprisingly, cooperativity did not increase with temperature in M. senoi hemocyanin.

The question arises as to why thermal sensitivity is so low in M. senoi hemocyanin but so high in the blood pigment of cold-adapted giant squid (cf. Brix, 1983). A high value of [delta][P.sub.50] [degrees][C.sup.-1] reflects a high heat of oxygenation (cf. Brix et al., 1994) or Arrhenius activation energy. Giant squid probably display higher metabolic rates and thus must maintain [P.sub.50] levels higher than those of M. senoi. A high heat of oxygenation may be required for setting [P.sub.50] values high at low temperature as in giant squid. In that respect the low thermal sensitivity of hemocyanin in the Antarctic octopod is again in accordance with the low metabolic rate of this group.

In summary, the pH sensitivity of oxygen binding in cephalopod hemocyanins is adjusted to metabolic rate. The pH-insensitive oxygen reserve in hemocyanin was largest in M. senoi and intermediate in S. officinalis, if compared to squids (this study and Portner, 1990). Furthermore, the Bohr effect is reduced and the pH-insensitive oxygen reserve rises during cooling, suggesting that pH sensitivity falls in the cold. The temperature dependence of the Bohr factor is less pronounced in the eurythermal S. officinalis, which would, together with an appropriate change in [P.sub.50], ensure a supply of oxygen at changing temperatures. In M. senoi, a high oxygen affinity of hemocyanin, a moderately high Bohr coefficient, and a low cooperativity at 0 [degrees]C cause blood [Po.sub.2] to be maintained ("buffered") at low values matching a low rate of oxygen consumption. In this species the low thermal sensitivity of oxygen affinity prevents an upward shift of the buffered [Po.sub.2] at higher temperatures, suggesting th at oxygen transfer to tissues may become limiting when oxygen demand rises. This observation is in contrast to the findings in giant squid, where arterial oxygen uptake is hampered by an excessive drop in oxygen affinity, thereby limiting heat tolerance (Brix, 1983). Accordingly, hemocyanin function probably contributes to an oxygen limitation of heat tolerance that sets in early and characterizes thermal tolerance in Antarctic octopods (Portner and Zieliaski, 1998) and probably also in giant squid.


The authors thank Iris Hardewig and Boris Klein for sampling hemolymph from Megaleledone senoi during the expedition with RV Polarstern. The technical and logistical help by the staff of the Marine Biomedical Institute is gratefully acknowledged. Supported by grants of the Deutsche Forschungsgemeinschaft to H.O. Portner (Po 278).

(*.) To whom correspondence should be addressed. E-mail:

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                    Oxygen affinity ([P.sub.50]), Bohr,
                 Haldane, and Hill coefficients evaluated
                   for the hemocyanins of the Antarctic
                   octopod Megaleledone senoi and of the
                      cuttlefish Sepia officinalis at
                          different temperatures
                                            Bohr coefficient
Temperature ([degrees]C) [P.sub.50] (kPa)     ([delta] log
Megaleledone senoi
0                         0.98 (pH 7.2)    [approximate] -0.9
5                                                  ND
10                        1.10 (pH 7.2)           -2.33
Sepia officinalis
10                         5.3 (pH 7.4)           -0.99
20                         6.5 (pH 7.4)           -1.33
                                               Haldane coefficient
Temperature ([degrees]C)     ([delta] log      ([delta][HCO.sub.3]-/
                         [P.sub.80]/[delta]pH) [delta][HcyO.sub.2]
Megaleledone senoi
0                                -1.51         0.66 (pH 6.2)
                                               0.50 (pH 6.8)
                                               0.39 (pH 7.2)
5                                -1.46         ND
10                               -2.13         ND
Sepia officinalis
10                               -1.44
20                               -1.94
                         Hill coefficient
Temperature ([degrees]C) ([n.sub.50])
Megaleledone senoi
0                        1.4 (pH 7.43)
5                        1.5 (pH 7.24)
                         1.0 (pH 7.31)
10                       1.2 [+ or -] 0.1
                           (pH 6.83-7.31;
                            n = 6)
Sepia officinalis
10                       4.6 (pH 7.29)
20                       5.9 (pH 7.48)
[P.sub.50], Haldane, and Hill
coefficients are valid for the pH
values given in brackets. ND, not
determined; [HcyO.sub.2], concentration
of oxygenated hemocyanin.
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Publication:The Biological Bulletin
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Date:Feb 1, 2001
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