Experimental Biology Online - EBO

ISSN 1430-3418

Exp. Biol. Online (1999) 4:1

Vampire blood: respiratory physiology of the vampire squid (Cephalopoda: Vampyromorpha) in relation to the oxygen minimum layer

Brad A. Seibel ¹, Fabienne Chausson ², Francois H. Lallier ², Franck Zal ², and James J. Childress ²

¹Marine Science Institute and Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106, USA
²Equipe Ecophysiologie, Observatoire Océanologique de Roscoff, UPMC - CNRS-INSU, Station Biologique, BP 74, F-29682 Roscoff Cedex, France

Correspondence to: F.H. Lallier
(e-mail: lallier@sb-roscoff.fr, Tel: +33-2-98292311, Fax: +33-2-98292324)

Received: January 11 1999 / Accepted: March 17 1999

Abstract. The functional properties of the haemocyanin of Vampyroteuthis infernalis (Cephalopoda: Vampyromorpha), measured at 5°C, are reported and discussed in relation to hypoxia. The oxygen affinity of this haemocyanin ( P ₅₀=0.47-0.55 kPa) is higher than any previously measured for a cephalopod. The high cooperativity ( n ₅₀=2.20-2.23) and Bohr coefficient (-0.22) suggest a true transport function for this haemocyanin. This high-affinity haemocyanin, in conjunction with moderate gill diffusion capacity, provides a sufficient oxygen gradient from the environment to the blood to support the low routine oxygen consumption rate of V. infernalis.

Key words. Deep sea - Haemocyanin - Hypoxia - Vampyroteuthis infernalis

• Introduction
• Materials and methods
• Results
• Discussion
• Acknowledgements
• References

Introduction

Zones of minimum oxygen level are found at intermediate depths in most of the world's ocean. Although the oxygen partial pressures in some of these oxygen minimum layers are extremely low ( PO ₂<1 kPa; Schmidt, 1925; Sewell and Fage, 1948), populations of pelagic metazoans exist there ( Banse, 1964). The vampire squid, Vampyroteuthis infernalis ( Fig. 1) is the only cephalopod thought to live its entire life cycle directly in the core of the oxygen minimum layer ( Roper and Young, 1975; Hunt, 1996). Pickford (1946) coined the term "oligoaerobic" to describe V. infernalis' affinity for low oxygen. Seibel et al. (1997) demonstrated that V. infernalis is able to support its routine metabolic demands aerobically at the lowest oxygen levels that it encounters. This ability is certainly facilitated by the extremely low metabolic rate of V. infernalis ( Seibel et al., 1997, 1998). However, cephalopod species with similarly low metabo lic rates living in higher oxygen regions, such as Hawaii ( PO ₂>2.5 kPa), are unable to tolerate oxygen levels as low as those found off California ( Seibel et al., 1997). This suggests that cephalopods, such as V. infernalis, living off California possess specific physiological adaptations that enable them to survive in the extreme, persistent hypoxia of the oxygen minimum layer.

Fig. 1. A photograph of Vampyroteuthis infernalis, taken on board in a small aquarium after recovery from 700 m depth off the coast of southern California with a modified opening-closing Tucker Trawl. The specimen photographed is approximately 25-30 cm total length. Photograph taken by B. Seibel

Physiological adaptations to the oxygen minimum layer have recently been reviewed ( Childress and Seibel, 1998). The few inhabitants of the oxygen minimum layer studied in detail are able to support their routine metabolic demands aerobically via effective extraction of oxygen from the surrounding water. Adaptations of pelagic crustaceans to the oxygen minimum layer include: (1) enhanced ventilatory volume, (2) large gill surface area, (3) short diffusion distance from the water to the blood and (4) haemocyanin respiratory proteins with very high affinity for oxygen (low P ₅₀), high cooperativity of oxygen binding and a large Bohr coefficient ( >logP ₅₀/ pH).

Unlike crustaceans, ventilation and locomotion are intimately tied in cephalopods ( Wells, 1988). Thus, high ventilatory rates may not be an option for a sit-and-wait predator such as Vampyroteuthis infernalis ( Seibel et al., 1998). Limited data suggest that, while some midwater cephalopods have extremely large gill surface areas ( Eno, 1994; Madan and Wells, 1996), V. infernalis has moderate gill surface areas and diffusion distances ( Madan and Wells, 1996). Furthermore, the respiratory proteins of cephalopods generally have low affinities for oxygen (at in vivo pH and respective environmental temperatures; Bridges, 1994). Even the haemocyanins of Octopus vulgaris and Nautilus pompilius, species often discussed in the context of hypoxia tolerance ( Wells and Wells, 1983, 1985; Wells et al., 1992; Boutilier et al., 1996), have oxygen affinities ( Table 1) that are much lower than those found for Gnathophausia ingen s, a midwater crustacean living in the oxygen minimum layer off California ( Table 1) ( Belman and Childress, 1976; Sanders and Childress, 1990).

Table 1 . Metabolism ( VO ₂=ml O ₂ kg ^-1min ^-1), gill diffusion capacity (DGO ₂=ml O ₂ kg ^-1kPa ^-1 min ^-1), blood-water oxygen gradient ( P _g= VO ₂/DGO ₂; in kPa) and hemocyanin-oxygen affinity ( P ₅₀= PO ₂ in kPa at 50% hemocyanin-oxygen saturation) of Vampyroteuthis infernalis in comparison to other cephalopods. Data for the lophigastrid crustacean, Gnathophausia ingens, are also shown

^aNormalized to 5°C assuming Q ₁₀=2
^bMeasured at pH 7.4 near environmental temperature
n.a.=not available

The only midwater cephalopod for which haemocyanin oxygen binding data exist is the giant squid, Architeuthis monachus ( Brix, 1983). At its presumed habitat temperature (6.4°C) and pH 7.4, A. monachus has a P ₅₀ (1.65 kPa) lower than that of Octopus vulgaris and Nautilus pompilius, but still too high to allow aerobic survival in the oxygen minimum layer off California. However, this specimen was captured in the North Atlantic where oxygen levels are higher than those found off California (see Discussion). In order to function in the oxygen minimum layer, the haemocyanin of V. infernalis must have a P ₅₀ that is considerably lower than the ambient PO ₂ of 0.8 kPa, lower than any P ₅₀ previously measured for a cephalopod. The present study reports the first observations of the oxygen binding characteristics of the haemocyanin of Vampyroteuthis infernalis, an "oligoaerobic" cephalopod, in relation to the oxygen minimum layer off California.

Materials and methods

Specimens of Vampyroteuthis infernalis (estimated weight=250 g each) were captured in a modified opening-closing Tucker Trawl equipped with a 30 l thermally insulated cod-end off the coast of southern California (34°37'N, 122°42'W) at 700 m depth. The specimens were transferred to chilled seawater and allowed to recover for approximately 10 h prior to dissection. Blood was collected by thoroughly drying the animals and cutting the branchial veins at the gill and collecting the pooled blood. The blood was immediately frozen in liquid nitrogen and stored at -80°C until analysis (<8 weeks). Blood from only one individual was used in the present study. The remaining samples are being used for structural analysis of the haemocyanin molecule (J. Lamy, Laboratoire des Proteines Complexes, France). The protein concentration of whole blood presented below was determined at 280 nm using an absorption coefficient of 1.43, by J. Lamy (pers. comm.).

The effects of freezing on the function of haemocyanins is incompletely understood ( Morris, 1988). Long-term freezing (>1 year) may have significant effects on both the cooperativity and the affinity of crustacean haemocyanins, but the effects vary between species, both in sign and magnitude, making predictions impossible ( Lallier and Truchot, 1989; Sanders and Childress, 1990). Short-term freezing, such as that used here, seems to have a small effect on cooperativity but little or no effect on P ₅₀ ( Morris, 1988). Nothing is known of the effects of freezing on cephalopod haemocyanins.

Upon thawing, the blood was centrifuged and the supernatant used for subsequent analysis. Oxygen dissociation curves for whole native blood were constructed using a step-by-step procedure ( Lykkeboe et al., 1975; Bridges et al., 1979) with a diffusion chamber ( Sick and Gersonde, 1969). Gas mixtures of known oxygen content were obtained from laboratory-grade gases (O ₂, N ₂ and CO ₂) using mass flow controllers (MKS instruments, Andover, Mass., USA). Changes in pH were induced by varying the CO ₂ tension in the gas mixtures. The in vivo blood pH of all cephalopod species studied, including the midwater Histioteuthis heteropsis ( Clarke et al., 1979), a part-time resident of the oxygen minimum layer, is believed to range from 7.2 to 7.5 ( Bridges, 1994; Pörtner, 1994). Therefore, we adjusted CO ₂ tensions aiming for this pH range for our measurements. The in vivo blood pH for Vampyroteuthis infernalis is not known. The pH was measured nea r P ₅₀ with a capillary pH electrode (Radiometer, BMS2) on a separate subsample equilibrated with the same gas mixture. The diffusion chamber and pH meter were maintained at 5°C throughout the experiment. Optical density at 365 nm was monitored continuously and used to derive Hc-O ₂ saturation as a function of PO ₂ in the gas mixture.

Sodium and potassium concentrations were determined by flame photometry (Eppendorf, Hamburg, Germany). The chloride concentration was determined by colorimetric titration (Corning 920). Calcium concentrations were measured using a colorimetric kit (Boehringer 1273574), as was magnesium (Merck 14102).

Results

The ionic composition of the blood of Vampyroteuthis infernalis is within the normal range for marine invertebrates ( Hochachka and Somero, 1984) including cephalopods ( Clarke et al., 1979). Sodium was 436.7, potassium was 11.9, magnesium was 36.7, calcium 12.2, and chloride 465.9 mmol l ^-1. The concentration of protein in the blood was determined by J. Lamy (Laboratoire des Proteines Complexes, France; pers. comm.). V. infernalis blood contained 21.5 mg protein ml ^-1.

The effects of pH on the binding of oxygen by V. infernalis haemocyanin are presented in Fig. 2. The small sample size allowed oxygen dissociation curves to be constructed at only two different pH levels. The relationship (Hill plot) between the log of fractional saturation ( S/1- S) and log of PO ₂ (kPa) is linear between about 25% and 75% oxygen saturation (log S/1- S=2.23log PO ₂+0.59; r=0.997 at pH 7.15 and log S/1- S=2.20 log PO ₂+0.71; r=0.999 at pH 7.44). Cooperativity ( n ₅₀, from the slopes of the above regressions) was high for V. infernalis ( n ₅₀=2.20 at pH 7.44 and 2.23 at pH 7.15) relative to other cephalopods at low temperatures ( Brix et al., 1989). The haemocyanin-oxygen affinity measured here is higher than any previously measured for a cephalopod ( Table 1). The effect of pH on haemocyanin oxygen affinity was significant (ANCOVA, p=0. 0001). The P ₅₀ ( PO ₂ at 50% saturation) was 0.47 kPa at pH 7.44 ( PCO ₂=0.30 kPa) and 0.55 kPa at pH 7.15 ( PCO ₂=0.8 kPa). The slope of the relationship between log P ₅₀ and pH gives a Bohr coefficient of -0.22.

Fig. 2. Haemocyanin-oxygen fractional saturation for Vampyroteuthis infernalis as a function of oxygen partial pressure (kPa) at pH 7.44 ( triangles) and pH 7.15 ( inverted triangles). The circled region of the curve indicates the environmental PO 2 value within the oxygen minimum layer at 700 m depth off California. Also shown ( inset) is the haemocyanin oxygen binding expressed as a Hill plot (log fractional saturation as a function of log PO 2). The P 50 ( PO 2 at half saturation, or log S/1- S=0) is 0.47 kPa at pH 7.44 and 0.55 kPa at pH 7.15

Discussion

The extreme hypoxia characterizing the oxygen minimum layer requires effective extraction of oxygen from the ambient water. The oxygen gradient between the water and blood ( P _g) required to support the oxygen demand can be calculated from the rate of oxygen consumption and the morphometrics of the gills ( Krogh, 1941). The morphometrics of the gills of Vampyroteuthis infernalis have been determined for a single specimen (11 g) captured in the Atlantic. The oxygen concentration at minimum layer depths in the Atlantic is considerably higher than that at comparable depths in the Pacific. Gill size, and presumably diffusion capacity, are known to increase for cephalopods, including V. infernalis, in areas of low oxygen concentration ( Roper, 1969; Young, 1972). Therefore, we view the following calculations as conservative estimates.

With a metabolic rate of 0.04 ml kg ^-1 min ^-1 (normalized to 10 g wet mass; Seibel et al., 1997) and a gill diffusion capacity of 0.31 ml O ₂ kg ^-1mmHg ^-1min ^-1 ( Madan and Wells, 1996) we calculate a P _g of only 0.02 kPa O ₂ for V. infernalis. A PO ₂ difference of only 0.02 kPa is required between ambient seawater at 0.8 kPa and the blood to provide sufficient oxygen diffusion to support the routine metabolic rate. This indicates that while the gill diffusion capacity of V. infernalis is only moderately high among cephalopods ( Eno, 1994), it is extremely high in relation to its metabolic rate ( Table 1). This is a much smaller gradient than that required by the midwater crustacean, Gnathophausia ingens (0.15 kPa; Belman and Childress, 1976). The extremely high affinity ( P ₅₀=0.19 kPa) found for G. ingens haemocyanin ( Sanders and Childress , 1990) is necessary to create this gradient because of the considerably higher metabolic rate of this species. The haemocyanin oxygen affinity ( P ₅₀) of 0.47-0.55 kPa ( Fig. 2; Table 1) measured here for V. infernalis, although the highest ever measured for a cephalopod, is sufficient for oxygen extraction only in conjunction with an extremely low metabolic rate ( Seibel et al., 1997) and moderate gill diffusion capacity ( Madan and Wells, 1996). As in octopods ( Wells and Wells, 1982), extraction efficiency may be increased somewhat by the counter-current blood flow in the gills of V. infernalis ( Young, 1964). The measured cooperativity and affinity should result in just over 70% haemocyanin-oxygen saturation at the ambient PO ₂ of 0.8 kPa ( Fig. 2). The relatively low haemocyanin (protein) concentration found for V. infernalis (21.5 mg ml ^-1) is similar to that found for G. ingens (24 mg ml ^-1; Childress and Seibel, 1998) and provides an oxygen-carrying capacity far greater than would dissolved oxygen in plasma.

A number of factors are known to influence oxygen binding of respiratory proteins. Among the most important for cephalopods are temperature ( Brix et al., 1989) and pH ( Bridges, 1994; Pörtner, 1994). The temperature regime of Vampyroteuthis infernalis is narrow (5±1°C) and extremely stable in space and time. Therefore, temperature effects were not measured in the present study. Protons generated during anaerobic metabolism cause pH shifts that affect haemocyanin-oxygen binding. However, the low tissue-buffering capacity of the mantle ( Seibel et al., 1997) and low glycolytic enzymatic activities ( Seibel et al., 1998) suggest that protons generated during anaerobic bursts of swimming are of little importance for V. infernalis. The lactate produced during anaerobic glycolysis can be an important moderator of respiratory protein function in vertebrates and crustaceans ( Truchot and Lallier, 1992). Octopine, the equivalent of lactate in cepha lopods, is probably metabolized in the muscle tissues of V. infernalis as in other cephalopods ( Pörtner, 1994) rather than excreted into the blood. Like anaerobic proton generation, octopine production is probably low in V. infernalis. Furthermore, organic effectors have not been evidenced in molluscan haemocyanins. Therefore, acidification by respiratory CO ₂ production is probably the most important moderator of oxygen binding in V. infernalis.

The role of the large negative Bohr coefficients found for most cephalopods (<-1.0) is still actively debated (see Bridges, 1994; Pörtner, 1994 for review). In some cases a large Bohr coefficient may improve oxygen loading with increased ventilation during temporary hypoxia ( Lykkeboe and Johannsen, 1982; Brix et al., 1989). It may also be related to cutaneous oxygen uptake ( Pörtner, 1994). Alternatively, it may serve a more traditional oxygen transport role in conjunction with oxygen-linked CO ₂ binding, as proposed by Lykkeboe et al. (1980). In any case, the relatively low oxygen affinities of most cephalopod haemocyanins will allow sufficient release of oxygen at the tissues. In contrast, oxygen unloading at the tissues may be problematic for V. infernalis due to the high-affinity haemocyanin reported here. The high cooperativity (relative to other cephalopods at low temperatures; Brix et al., 1989) of this haemocyanin allows release of mos t of its bound oxygen with a relatively small drop in PO ₂. The magnitude of the Bohr effect in V. infernalis (-0.22) will further facilitate oxygen unloading at the tissues during respiratory CO ₂ release and subsequent acidosis. Given that arterial PO ₂ is very near ambient PO ₂, any release of oxygen at the tissues will occur at PO ₂ levels within the cooperative region of the oxygen dissociation curve (see Fig. 2). This suggests that the haemocyanin does indeed play a transport function in V. infernalis.

Cephalopods have received considerable attention in the context of hypoxia tolerance. Hypoxia is believed to have played a large role in cephalopod evolution. However, those species reported to be hypoxia tolerant are generally from unstable oxygen environments. Animals in these environments, tide-pools and burrows, and those with shells experience oxygen regimes that vary from near air saturation to complete anoxia. Octopus spp. and Nautilus spp. have high critical oxygen partial pressures and low haemocyanin oxygen affinities relative to V. infernalis ( Table 1). While they have clearly adjusted their physiology for enhancement of oxygen extraction relative to active squids ( Wells, 1988), they can not regulate their oxygen consumption much below 2.6 kPa PO ₂ ( Wells and Wells, 1982; Wells et al., 1992). Instead, Octopus spp. ( Seibel, 1998) and Nautilus spp. ( Boutilier et al., 1996) have considerable capacities for me tabolic suppression and/or anaerobic metabolism to wait out periods of intolerably low oxygen. They can survive complete anoxia for several hours. Inhabitants of oxygen minimum layers must rely on their abilities to extract oxygen from the ambient water to support their routine metabolic rates and generally have very limited abilities to survive complete anoxia ( Childress and Seibel, 1998). The high oxygen affinity haemocyanin reported here, in conjunction with a moderate gill diffusion capacity, provides a sufficient oxygen gradient between the environment and the blood to support the low routine oxygen consumption rate of Vampyroteuthis infernalis.

Acknowledgements. This research was supported in part by a University of California Graduate Division Fellowship and a Western Society of Malacologists Student Grant to B.A.S., National Science Foundation grant (OCE-9415543) to J.J.C., and Ifremer-URM 7 to F.H.L. and F.C. We thank the Monterey Bay Aquarium for allowing participation on research and collection cruises and we thank the Captain and Crew of the R/V Point Sur for their assistance at sea. We thank Joan Company and Shana K. Goffredi for critically reviewing this manuscript.

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Online publication: March 24, 1999
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