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Vampire blood:respiratory physiology of the
vampire squid (Cephalopoda:Vampyromorpha)
in relation to the oxygen minimum layer
1
Brad A. Seibel1,Fabienne Chausson2,Francois H.Lallier2, Franck Zal2,
and James J.Childress2
1Equipe Ecophysiologie,Observatoire Océanologique de Roscoff,
UPMC – CNRS-INSU,Station Biologique, BP 74, F-29682 Roscoff Cedex, France
2Marine Science Institute and Department of Ecology, Evolution
and Marine Biology, University of California, Santa Barbara, CA 93106, USA
Received: January 11 1999 / Accepted: March 17 1999 / Published: March 24 1999
Abstract. The functional properties of the haemocyanin of Va mpy roteu thi s in-
fernalis (Cephalopoda: Vampyromorpha), measured at 5°C, are reported and
discussed in relation to hypoxia. The oxygen affinity of this haemocyanin
(P50=0.47–0.55 kPa) is higher than any previously measured for a cephalopod.
The high cooperativity (n50=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. i n f e r n a l i s .
Key words. Deep sea – Haemocyanin – Hypoxia
Vampyroteuthis infernalis
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 ox-
ygen minimum layers are extremely low (PO2<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. i n f e r n a l i s ’ affinity for low oxygen. Seibel et al.
(1997) demonstrated that V. i n f e r n a l i s is able to support its routine metabolic
demands aerobically at the lowest oxygen levels that it encounters. This abili-
F.H. Lallier (e-mail: lallier@sb-roscoff.fr,Tel: +33-2-98292311, Fax: +33-2-98292324)
2
ty is certainly facilitated by the extremely low metabolic rate of V. i n f e r n a l i s
(Seibel et al., 1997, 1998). However,cephalopod species with similarly low met-
abolic rates living in higher oxygen regions, such as Hawaii (PO2>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. i n f e r n a l i s , living off Cali-
fornia possess specific physiological adaptations that enable them to survive
in the extreme, persistent hypoxia of the oxygen minimum layer.
Physiological adaptations to the oxygen minimum layer have recently been
reviewed (Childress and Seibel, 1998).The few inhabitants of the oxygen min-
imum layer studied in detail are able to support their routine metabolic de-
mands aerobically via effective extraction of oxygen from the surrounding
water. Adaptations of pelagic crustaceans to the oxygen minimum layer in-
clude: (1) enhanced ventilatory volume,(2) large gill surface area,(3) short dif-
fusion distance from the water to the blood and (4) haemocyanin respiratory
proteins with very high affinity for oxygen (low P50), high cooperativity of ox-
ygen binding and a large Bohr coefficient (∆logP50/∆pH).
Unlike crustaceans,ventilation and locomotion are intimately tied in ceph-
alopods (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 af-
finities 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 affin-
ities (Table 1) that are much lower than those found for Gnathophausia ingens,
a midwater crustacean living in the oxygen minimum layer off California (Ta-
ble 1) (Belman and Childress, 1976; Sanders and Childress, 1990).
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 Califor-
nia with a modified open-
ing-closing Tucker Trawl.
The specimen photo-
graphed is approximately
25–30 cm total length. Pho-
tograph taken by B.Seibel
The only midwater cephalopod for which haemocyanin oxygen binding
data exist is the giant squid, Architeuthis monachus (Brix, 1983).At its pre-
sumed habitat temperature (6.4°C) and pH 7.4, A. monachus has a P50
(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 lev-
els are higher than those found off California (see Discussion). In order to
function in the oxygen minimum layer,the haemocyanin of V. i n f e r n a l i s must
have a P50 that is considerably lower than the ambient PO2of 0.8 kPa, lower
than any P50 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.
3
Tabl e 1 . Metabolism (VO2=ml O2kg–1min–1), gill diffusion capacity (DGO2=ml O2kg–1kPa–1
min–1), blood-water oxygen gradient (∆Pg=VO2/DGO2; in kPa) and hemocyanin-oxygen
affinity (P50=PO2in kPa at 50% hemocyanin-oxygen saturation) of Vam py ro te ut hi s i nf er n -
alis in comparison to other cephalopods. Data for the lophigastrid crustacean, Gnathop-
hausia ingens, are also shown
Species VO2aDGO2DDPgP50bReferences
Va mp y r o te u t h i s 0.04 2.32 0.02 0.47 Madan and Wells,
infernalis 1996; Seibel et al.,
1997
Nautilus 0.28 0.38 0.74 2.3 Brix et al., 1989;
pompilius Wel l s et al., 1992;
Eno, 1994
Octopus 0.35 0.45 0.77 2.45 Wells and Wells,
vulgaris 1983; Bridges,
1994; Eno, 1994
Architeuthis n.a. n.a. n.a. 1.65 Brix et al., 1989
monachis
Gnathophausia 0.56 3.73 0.15 0.19 Belman and
ingens Childress, 1976;
Sanders and
Childress, 1990
aNormalized to 5°C assuming Q10=2
bMeasured at pH 7.4 near environmental temperature
n.a. = not available
4
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 be-
ing 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 pre-
dictions 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 P50 (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 (O2,N
2and
CO2) using mass flow controllers (MKS instruments, Andover, Mass., USA).
Changes in pH were induced by varying the CO2tension in the gas mixtures.
The in vivo blood pH of all cephalopod species studied, including the midwa-
ter Histioteuthis heteropsis (Clarke et al., 1979), a part-time resident of the ox-
ygen minimum layer, is believed to range from 7.2 to 7.5 (Bridges, 1994;
Pörtner, 1994). Therefore, we adjusted CO2tensions aiming for this pH range
for our measurements. The in vivo blood pH for Vampyroteuthis infernalis is
not known. The pH was measured near P50 with a capillary pH electrode (Ra-
diometer, BMS2) on a separate subsample equilibrated with the same gas mix-
ture. 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-O2saturation as a function of PO2in the gas mixture.
Sodium and potassium concentrations were determined by flame photo-
metry (Eppendorf, Hamburg, Germany). The chloride concentration was de-
termined by colorimetric titration (Corning 920). Calcium concentrations
were measured using a colorimetric kit (Boehringer 1273574), as was magne-
sium (Merck 14102).
Results
The ionic composition of the blood of Vampyroteuthis infernalis is within the
normal range for marine invertebrates (Hochachka and Somero,1984) includ-
ing 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 concentra-
tion of protein in the blood was determined by J. Lamy (Laboratoire des Pro-
teines Complexes, France; pers. comm.). V. i n f e r n a l i s 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 PO2(kPa) is
linear between about 25% and 75% oxygen saturation (logS/1-S=2.23
logPO2+0.59; r=0.997 at pH 7.15 and logS/1-S=2.20logPO2+0.71; r=0.999 at
pH 7.44). Cooperativity (n50, from the slopes of the above regressions) was
high for V. i n f e r n a l i s (n50=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 cephalo-
pod (Table 1). The effect of pH on haemocyanin oxygen affinity was signifi-
cant (ANCOVA, p=0.0001). The P50 (PO2at 50% saturation) was 0.47 kPa at
pH 7.44 (PCO2=0.30 kPa) and 0.55 kPa at pH 7.15 (PCO2=0.8 kPa).The slope of
the relationship between log P50 and pH gives a Bohr coefficient of -0.22.
5
Fig. 2. Haemocyanin-oxy-
gen fractional saturation
for Vamp yr ot eu thi s i nf er n-
alis 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 environ-
mental PO2value within the
oxygen minimum layer at
700 m depth off California.
Also shown (inset) is the
haemocyanin oxygen bind-
ing expressed as a Hill plot
(log fractional saturation as
a function of log PO2). The
P50 (PO2at half saturation,
or logS/1–S=0) is 0.47 kPa
at pH 7.44 and 0.55 kPa at
pH 7.15
6
Discussion
The extreme hypoxia characterizing the oxygen minimum layer requires ef-
fective extraction of oxygen from the ambient water.The oxygen gradient be-
tween the water and blood (∆Pg) 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 Va m py roteut h i s i n -
fernalis have been determined for a single specimen (11 g) captured in the At-
lantic. 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,in-
cluding V. i n f e r n a l i s , 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 O2kg-1mmHg-1min-1
(Madan and Wells, 1996) we calculate a ∆Pgof only 0.02 kPa O2for V. i n f e r n -
alis.APO2difference 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. i n f e r n a l i s is only moderately high among cephalopods (Eno, 1994), it is ex-
tremely high in relation to its metabolic rate (Table 1). This is a much smaller
gradient than that required by the midwater crustacean, Gnathophausia in-
gens (0.15 kPa; Belman and Childress, 1976). The extremely high affinity
(P50=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 (P50) of
0.47–0.55 kPa (Fig. 2; Table 1) measured here for V. i n f e r n a l i s , 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. i n fe r n a l i s (Young, 1964). The
measured cooperativity and affinity should result in just over 70%
haemocyanin-oxygen saturation at the ambient PO2of 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 swim-
ming are of little importance for V. i n f e r n a l i s . 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 cephalopods, is probably metabolized in the mus-
cle tissues of V. i n f e r n a l i s as in other cephalopods (Pörtner, 1994) rather than
excreted into the blood. Like anaerobic proton generation, octopine produc-
tion is probably low in V. i n f e r n a l i s . Furthermore, organic effectors have not
been evidenced in molluscan haemocyanins. Therefore,acidification by respi-
ratory CO2production is probably the most important moderator of oxygen
binding in V. i n f e r n a l i s .
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 in-
creased 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 CO2binding,as proposed by Lykkeboe
et al. (1980). In any case, the relatively low oxygen affinities of most cephalo-
pod haemocyanins will allow sufficient release of oxygen at the tissues.In con-
trast, oxygen unloading at the tissues may be problematic forV. i n f e r n a l i s due
to the high-affinity haemocyanin reported here. The high cooperativity (rela-
tive to other cephalopods at low temperatures; Brix et al., 1989) of this
haemocyanin allows release of most of its bound oxygen with a relatively
small drop in PO2.The magnitude of the Bohr effect in V. i n f e r n a l i s (-0.22) will
further facilitate oxygen unloading at the tissues during respiratory CO2re-
lease and subsequent acidosis. Given that arterial PO2is very near ambient
PO2, any release of oxygen at the tissues will occur at PO2levels within the co-
operative region of the oxygen dissociation curve (see Fig. 2). This suggests
that the haemocyanin does indeed play a transport function in V. i n f e r n a l i s .
Cephalopods have received considerable attention in the context of hy-
poxia tolerance. Hypoxia is believed to have played a large role in cephalopod
evolution.However,those species reported to be hypoxia tolerant are general-
ly 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 af-
finities relative to V. i n f e r n a l i s (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 PO2(Wells and Wells, 1982; Wells et al., 1992). Instead, Octopus spp.
(Seibel, 1998) and Nautilus spp. (Boutilier et al., 1996) have considerable ca-
7
8
pacities for metabolic suppression and/or anaerobic metabolism to wait out
periods of intolerably low oxygen. They can survive complete anoxia for sev-
eral 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 suffi-
cient 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 Malacolo-
gists 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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