SUMMARY

The Humboldt (jumbo) squid, Dosidicus gigas, is a part-time resident of the permanent oxygen minimum zone (OMZ) in the Eastern Tropical Pacific and, thereby, it encounters oxygen levels below its critical oxygen partial pressure. To better understand the ventilatory mechanisms that accompany the process of metabolic suppression in these top oceanic predators, we exposed juvenile D. gigas to the oxygen levels found in the OMZ (1% O2, 1 kPa, 10°C) and measured metabolic rate, activity cycling patterns, swimming mode, escape jet (burst) frequency, mantle contraction frequency and strength, stroke volume and oxygen extraction efficiency. In normoxia, metabolic rate varied between 14 and 29 μmol O2 g−1 wet mass h−1, depending on the level of activity. The mantle contraction frequency and strength were linearly correlated and increased significantly with activity level. Additionally, an increase in stroke volume and ventilatory volume per minute was observed, followed by a mantle hyperinflation process during high activity periods. Squid metabolic rate dropped more than 75% during exposure to hypoxia. Maximum metabolic rate was not achieved under such conditions and the metabolic scope was significantly decreased. Hypoxia changed the relationship between mantle contraction strength and frequency from linear to polynomial with increasing activity, indicating that, under hypoxic conditions, the jumbo squid primarily increases the strength of mantle contraction and does not regulate its frequency. Under hypoxia, jumbo squid also showed a larger inflation period (reduced contraction frequency) and decreased relaxed mantle diameter (shortened diffusion pathway), which optimize oxygen extraction efficiency (up to 82%/34%, without/with consideration of 60% potential skin respiration). Additionally, they breathe ‘deeply’, with more powerful contractions and enhanced stroke volume. This deep-breathing behavior allows them to display a stable ventilatory volume per minute, and explains the maintenance of the squid's cycling activity under such O2 conditions. During hypoxia, the respiratory cycles were shorter in length but increased in frequency. This was accompanied by an increase in the number of escape jets during active periods and a faster switch between swimming modes. In late hypoxia (onset ~170±10 min), all the ventilatory processes were significantly reduced and followed by a lethargic state, a behavior that seems closely associated with the process of metabolic suppression and enables the squid to extend its residence time in the OMZ.

INTRODUCTION

Dosidicus gigas (jumbo or Humboldt squid) is the largest ommastrephid squid (up to 2.5 m length and 50 kg in mass) (Nesis, 1983) and has one the highest metabolic rates of any animal in the ocean (Rosa and Seibel, 2008). It is endemic to the Eastern Pacific, and particularly abundant in the highly productive waters of the California and Peru Current systems (Nigmatullin et al., 2001) and the Costa Rica Dome (Ichii et al., 2002; Waluda and Rodhouse, 2006), where it plays a crucial role both as prey (Clarke and Paliza, 2001; Abitía-Cárdenas et al., 2002; Ruiz-Cooley et al., 2004) and predator (Markaida and Sosa-Nishizaki, 2003). In addition to its ecological role, the Humboldt squid is an economically important species and the target of the world's largest cephalopod fishing industry (Rodhouse et al., 2006).

In addition to seasonal horizontal migrations (Markaida et al., 2005), D. gigas undergoes diel vertical migrations into mesopelagic depths (around 250 m) during the daytime (Gilly et al., 2006) (Trueblood and Seibel, 2012), similar to the migration pattern of their primary prey – myctophid fishes (Markaida and Sosa-Nishizaki, 2003; Markaida et al., 2008). While at depth, D. gigas encounters the permanent oxygen minimum zone (OMZ) of the Eastern Tropical Pacific during the day, with oxygen levels often less than 5 μmol l−1 (~0.5% O2) (Kamykowski and Zentara, 1990; Morrison et al., 1999). Other top pelagic predators (e.g. tuna, swordfish, marlin and sharks) (Brill, 1994; Prince and Goodyear, 2006; Vetter et al., 2008; Nasby-Lucas et al., 2009; Stramma et al., 2012) seem to avoid these hypoxic depths, as their tolerance to hypoxic conditions is low [<150 μmol l−1 O2 (Brill, 1994; Brill, 1996); ~45 μmol l−1 O2, big-eye tuna (Lowe et al., 2000)]. One might expect that the presence of active, muscular squid in these hypoxic zones would be precluded, as, in other active squid: (i) the primary mode of locomotion, jet propulsion, is energetically inefficient (Webber et al., 2000); (ii) the oxygen-carrying capacity is limitative relative to that of fishes, requiring that cephalopods use most of the O2 in the blood on each cycle, leaving little venous oxygen reserve behind (Pörtner, 2002); (iii) the oxygen-carrying capacity is low because of viscosity-related constraints and an extracellular low-affinity respiratory protein (Pörtner, 2002); and (iv) ventilatory and locomotory systems are closely tied. Regarding the last of these, two different strategies evolved in these mollusks to deal with this restriction. Pelagic squid (like D. gigas) that routinely depend on jet propulsion optimize their locomotory apparatus by pushing a large water volume through their mantle cavity, reducing oxygen extraction efficiency (5–10%) (Pörtner, 1994; Wells et al., 1988). In contrast, nekton-benthic cephalopods (i.e. cuttlefish and octopus) developed an efficient oxygen uptake system (40–50%) (Wells and Wells, 1985) by minimizing their ventilatory volume to increase diffusion (Wells et al., 1988).

Squid are thought to live chronically ‘on the edge of oxygen limitation’ (Pörtner, 2002) and are not well poised to adapt to low ambient O2 levels. Yet, the Humboldt squid has managed to minimize the trade-offs between high locomotory performance and hypoxia tolerance via metabolic suppression (Rosa and Seibel, 2008; Rosa and Seibel, 2010), coupled with a high-affinity respiratory protein (Seibel, 2012). Here, we exposed juvenile squid to oxygen levels found in the OMZ (1% O2, 1 kPa, 10°C, severe hypoxia) to investigate the effects on the ventilatory mechanisms that accompany the process of metabolic suppression in D. gigas, namely: (i) maximal, active, routine and inactive metabolic rates; (ii) metabolic scope; (iii) activity cycling patterns and swimming mode (with video recording); (iv) escape jet (burst) frequency; (v) mantle contraction frequency and strength; (vi) stroke volume, and ventilatory volume per minute; and (vii) oxygen extraction efficiency (with and without a potential contribution from skin respiration).

MATERIALS AND METHODS

Specimen collection

Juvenile Humboldt squid, D. gigas (d'Orbigny 1835), (5.4–13.5 g wet mass) were collected via dip net in the Gulf of California (27°N, 111°W; 28°N, 113°W), on the surface at night, in June 2011 (aboard the RV New Horizon, Scripps Institute, CA, USA) and were immediately transferred to aquaria containing 10°C seawater (environmental temperature at OMZ) on board the vessel. It is worth noting that all experiments were conducted with juvenile stages, and it is not known whether juvenile and adult jumbo squid display similar diel vertical migration behavior (i.e. whether they encounter the same minimum oxygen levels during descent).

Experimental procedure

Animals were placed in a flow-through respirometry set up (270 ml volume, Loligo Systems, Tjele, Denmark) (Rosa and Seibel, 2008; Rosa and Seibel, 2010), and allowed to acclimate for 8–12 h before measurements of oxygen consumption were started. Respirometers were immersed in a large thermostatically controlled waterbath (Lauda, Lauda-Königshofen, Germany) at 10°C, a temperature approximating that found at 250 m in the OMZ (Rosa and Seibel, 2010). Filtered (0.2 μm) and treated (50 mg l−1 streptomycin) seawater was pumped from a water-jacketed, gas-equilibration column through the respirometers at a constant flow rate (average 120 ml min−1). The water in the column was bubbled continuously to maintain incoming water at high (21% O2, 21 kPa,) or low PO2 (certified gas mixture with 1% O2, 1 kPa). Once the final 1% O2 level in the respiration chambers had been reached, the first 30 min were excluded, as they may represent an acclimatory phase after adjusting to a new oxygen concentration. Oxygen concentrations were recorded at the entrance and the exit of each chamber with two Clarke-type O2 electrodes connected to a 928 Oxygen Interface (Strathkelvin Instruments, North Lanarkshire, UK). The system was calibrated using air- and nitrogen-saturated seawater and checked for electrode drift and for microbial oxygen consumption before and after each trial. All experiments were carried out in darkness and at atmospheric pressure. Video recordings were conducted (Sony DCR-SR78, Lisbon, Portugal) during respiration runs. Afterwards, specimens were immediately weighed on a motion-compensated precision shipboard balance system (Childress and Mickel, 1980). A total of 18 specimens were investigated, with 6 replicates for the normoxic treatment (21% O2, 21 kPa) and 12 for the hypoxic one (1% O2, 1 kPa). Because of differences in squid swimming behavior (i.e. mantle contraction frequency per minute) and respiratory profile pattern (i.e. number of cycles) throughout hypoxia, this treatment was subdivided into early hypoxia (EH, hypoxia exposure time 30–160 min, N=6) and late hypoxia (LH, hypoxia exposure time >180 min, N=6). Exposure times are presented in Table 1.

Swimming behavior, metabolic rate and cycling performance

Swimming behavior

Jet propulsion was subdivided into three swimming modes, based on previous studies (Gosline et al., 1983; Bartol, 2001); namely: (1) respiratory movements, (2) steady jetting and (3) bursts/escape jets via video footage. Respiratory movements were defined by weak mantle contractions without visible thrust. Thereby, squid could be within the water column or, under LH, even lie at the bottom of the chambers. Steady jetting ranged from slow cruising (weak but visible thrusts) to vigorous jetting (powerful thrusts). Bursts or escape jets were characterized by hyperinflation (≥5% increase of relaxed mantle diameter) followed by maximum mantle contraction (Gosline and DeMont, 1985) and fast movements. The relaxed diameter was assessed under normoxia at inactive metabolic rate (IMR) using ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA), as mantle contractions at slow swimming speeds occur without hyperinflation (Gosline and DeMont, 1985). The number of escape jets per minute was quantified via video analysis.

Metabolic rate

The number of escape jets per minute and the swimming modes were linked to changes in O2 consumption during cycling periods. Based on this, inactive, routine, active and maximum active metabolic rate (IMR, RMR, AMR and MRmax, respectively) could be distinguished (see Table 2, Fig. 1) with similar results to those of a previous study (Rosa and Seibel, 2008). RMR for normoxia and hypoxia was specified as the average rate for the entirety of each recording. During periods of activity, metabolic rate was ≥15–35% (AMR) and >35–100% (MRmax) higher than the routine levels (RMR) with escape jets ≥4 bursts min−1. Thus, the AMR and MRmax were quantified by averaging the metabolic rates for all peaks exceeding ≥15% or >35%, respectively. Inactive metabolic rate was defined via swimming mode 1 (≥75% of time) and burst events <<4 min−1. These periods of apparent inactivity in the chambers correlated with metabolic rates ≤15% lower than RMR and average rates were calculated. Note that pelagic predators such as D. gigas rarely stop swimming in nature and that we did not quantify locomotion continuously for all experiments in its entirety. However, our IMR calculations seem to be a reasonable approximation to the criteria set for standard metabolic rate in mammals and other model organisms. Metabolic rate was quantified as μmol O2 g−1 wet mass h−1. The metabolic scope was calculated as the difference between maximum and minimum O2 consumption.

Table 1.

Exposure times of Dosidicus gigas to normoxic (21% O2) and hypoxic (1% O2) conditions

Exposure times of Dosidicus gigas to normoxic (21% O2) and hypoxic (1% O2) conditions
Exposure times of Dosidicus gigas to normoxic (21% O2) and hypoxic (1% O2) conditions
Table 2.

Different activity levels in Dosidicus gigas

Different activity levels in Dosidicus gigas
Different activity levels in Dosidicus gigas

Cycling performance

All control (normoxia) animals and the majority in early hypoxia (5 out of 6) animals showed a distinct periodicity in their rate of oxygen consumption (Fig. 1). These respiratory cycles are characterized by a steep increase in metabolic rate (~first 1/3 of total cycle length, Fig. 1) and video analysis confirmed that this was linked to an elevation in mantle contraction frequency. The remaining cycle length (Fig. 1) consisted of a slow transition from powerful to weaker mantle contractions. Therefore, burst frequency (number of escape jets per minute, calculated over the entire cycle length) and swimming modes were used to describe those respiratory cycles. Periodicity was further investigated by assessing the number of active and maximum active cycles per hour, including their length in minutes (via respiratory files).

Mantle contraction frequency and strength

In cephalopods, locomotion and respiration are closely tied and therefore counterproductive in their goals, even though in resting Sepia and probably to some extent in squid, ventilatory and locomotory mechanisms can be uncoupled via a collar flap system (Bone et al., 1994). However, both ventilatory and locomotory systems are highly dependent on the frequency and strength of mantle contractions, which, in turn, influence the water volume throughput. Mantle contraction frequency (ventilation rate) was quantified as the number of mantle contractions per minute (MC min−1) by video footage analysis. The strength of mantle contraction was calculated as the difference between maximum and minimum mantle diameter (in mm) using ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA).

Fig. 1.

Oscillations in oxygen levels (ΔO2) that reflect differences between the values recorded at the entrance and the exit of the chamber in normoxia (A, 10.9 g squid), early hypoxia (B, 1% O2, 5.4 g squid) and late hypoxia (C, 11.6 g squid) at 10°C. Solid lines in A represent the thresholds for inactive (IMR), routine (RMR), active (AMR) and maximum metabolic rate (MRmax) (illustrated in Table 2). Black arrows indicate the acceleration phase of MRmax and AMR cycles, and the vertical dashed lines mark the respective cycle lengths. The dashed line in C represents the onset of late hypoxia (170±10 min, N=6). Note, the 0 value on the x-axis in the hypoxia plots (B,C) does not represent the real onset of hypoxia.

Fig. 1.

Oscillations in oxygen levels (ΔO2) that reflect differences between the values recorded at the entrance and the exit of the chamber in normoxia (A, 10.9 g squid), early hypoxia (B, 1% O2, 5.4 g squid) and late hypoxia (C, 11.6 g squid) at 10°C. Solid lines in A represent the thresholds for inactive (IMR), routine (RMR), active (AMR) and maximum metabolic rate (MRmax) (illustrated in Table 2). Black arrows indicate the acceleration phase of MRmax and AMR cycles, and the vertical dashed lines mark the respective cycle lengths. The dashed line in C represents the onset of late hypoxia (170±10 min, N=6). Note, the 0 value on the x-axis in the hypoxia plots (B,C) does not represent the real onset of hypoxia.

Fig. 2.

Schematic drawing of a squid with descriptions of the measurements used to quantify ventilation stroke volume and hyperinflation in Dosidicus gigas. Arrows indicate cylinder length (Lc, 0.54 of total mantle length in D. gigas), relaxed mantle radius (rR, recorded during IMR periods in normoxia), contracted mantle radius (rC) and hyperinflated mantle radius (rH).

Fig. 2.

Schematic drawing of a squid with descriptions of the measurements used to quantify ventilation stroke volume and hyperinflation in Dosidicus gigas. Arrows indicate cylinder length (Lc, 0.54 of total mantle length in D. gigas), relaxed mantle radius (rR, recorded during IMR periods in normoxia), contracted mantle radius (rC) and hyperinflated mantle radius (rH).

Hyperinflation and stroke volume quantification

During escape jets, squid powerfully contract their radial muscles and, consequently, induce an increase in the outer mantle diameter (between 5% and 10% of relaxed mantle diameter), a process called hyperinflation (represented by the red line in Fig. 2). The relaxed mantle diameter (Fig. 2) was recorded during the IMR periods in normoxia. Changes in the relaxed diameter were determined as a percentage of the relaxed mantle diameter specified at IMR under normoxia.

Stroke volume (Vw, water throughput per mantle contraction) was quantified based on a modified O'Dor (O'Dor, 1988) equation:
formula
(1)
where, rH represents the maximum (hyperinflated) mantle radius (see also Fig. 2), rC is the contracted mantle radius, x is the mantle thickness (mm), L is the mantle length (the upper mantle is treated as a cylinder of changing diameter, while the lower region is rigid and independent of the mantle radius; in D. gigas, cylinder length Lc=0.54L, see Table 3), M is the wet mass (kg), ds is the squid density (1055 kg m−3) and 0.15 is the correction factor for visceral mass in D. gigas (see Table 3).
The ventilatory volume per minute (, ml min−1) was quantified by multiplying Vw (ml) by ventilatory frequency (MC min−1). The oxygen extraction efficiency (EO2, %) was quantified via the following equations, based on the theoretical model in I. illecebrosus (Pörtner, 1994):
formula
(2A)
formula
(2B)
formula
(2C)
formula
(2D)
without (Eqn 2A) and with a potential skin respiration contribution (20%, minimum for resting squid, Eqn 2B; 50%, maximum for resting squid, Eqn 2C; and 60%, for squid under exercise, Eqn 2D). Here, ΔO2 represents the difference between the oxygen flow in and out of the respiratory chamber (μmol l−1), is the flow rate during respiration measurement (l min−1) and is the ventilatory volume per minute (ml min−1).

Statistics

Two-way ANOVA were performed to evaluate significant differences between oxygen treatments (21% and 1% O2), metabolic rates, mantle contraction frequencies and strengths, % of relaxed diameter and stroke volumes. Subsequently, Tukey HSD post hoc tests were conducted. In some ventilatory measurements, no data for the late hypoxia treatment were recorded and, as result, independent Student's t-tests were applied. Linear and polynomial regressions were performed to assess the correlations between mantle contraction frequency, contraction strength and metabolic rate at different activity levels under control and hypoxic conditions. ANCOVA were conducted to detect significant differences in the relationship between mantle contraction strength/frequency and metabolic rate. For all statistical analysis, STATISTICA (Tulsa, OK, USA) version 10.0 was used.

RESULTS

The effect of hypoxia on D. gigas metabolic rate is shown in Fig. 3A. In control animals, activity level only showed significant differences (P<0.05) between IMR and MRmax, increasing from 13.7±6.1 to 28.4±12.0 μmol O2 g−1 wet mass h−1 (Fig. 3A; supplementary material Table S1). Exposure to severe hypoxia (1% O2, 1 kPa) led to a significant decrease in O2 consumption (≤25% of control values, Fig. 3A). AMR and RMR were significantly reduced in EH (exposure time 30–160 min) and IMR at LH (exposure time >180 min; two-way ANOVA, P<0.05; supplementary material Table S1). MRmax was only achieved under normoxic conditions and, overall, activity levels (AMR and MRmax) declined with hypoxia exposure from 36% to 15% in EH to 0% in LH (Fig. 3A, Fig. 4). RMR dominated in EH treatments, increasing from 47% (normoxia) to 77%, but was not present in LH (100% IMR; Fig. 4). The metabolic scope also changed dramatically with hypoxia (Fig. 3B), decreasing from 15.0±5.9 μmol O2 g−1 wet mass h−1 in normoxia to 1.7±0.7 μmol O2 g−1 wet mass h−1 in EH, reaching almost 0 (0.3±0.2 μmol O2 g−1 wet mass h−1) in LH (one-way ANOVA, F=34.06, P<<0.0001). The number of escape jets per minute increased significantly during AMR from normoxia (11.2 jets min−1) to EH (14.3 jets min−1), but no jets were recorded at LH (Fig. 5A; supplementary material Table S1). Overall (disregarding the activity level), there was a significant decrease in total bursts per hour from normoxia to EH (Fig. 5B, t-test, t=5.55, P<0.05).

Table 3.

Species-specific correction factors for visceral mass and cylinder mantle length to determine stroke volume in D. gigas

Species-specific correction factors for visceral mass and cylinder mantle length to determine stroke volume in D. gigas
Species-specific correction factors for visceral mass and cylinder mantle length to determine stroke volume in D. gigas
Fig. 3.

Effects of hypoxia (1% O2) on (A) metabolic rate (MR) and (B) metabolic scope (maximum MR–minimum MR) of D. gigas. Values are expressed as means and s.d. (N=6). EH, early hypoxia; LH, late hypoxia. Significant differences between the level of activity (capital letters) and between treatments (lowercase letters) are indicated (P<0.05, see supplementary material Table S1).

Fig. 3.

Effects of hypoxia (1% O2) on (A) metabolic rate (MR) and (B) metabolic scope (maximum MR–minimum MR) of D. gigas. Values are expressed as means and s.d. (N=6). EH, early hypoxia; LH, late hypoxia. Significant differences between the level of activity (capital letters) and between treatments (lowercase letters) are indicated (P<0.05, see supplementary material Table S1).

All control animals showed a distinct periodicity in oxygen consumption rate (Fig. 1A) and video analysis confirmed that these cycles were correlated with burst frequency and level of activity. Cycles of AMR and MRmax differed significantly in length (18±4 and 52±8 min, respectively; Fig. 6A) and frequency (1.5 and 0.2 cycles h−1, respectively; t-test, P<<0.001; Fig. 6B). In early hypoxia, periodicity was also observed (in 5 out of 6 animals, illustrated in Fig. 1B), but with a significantly shorter cycle length (5±1 min; Fig. 6A) and higher frequency (4±1 cycles h−1; Fig. 6B; t-tests, P<0.001).

The effect of hypoxia (1% O2, 1 kPa) on ventilation rate, a function of mantle contraction frequency and strength, is presented in Fig. 7. Contraction frequency significantly increased with activity, from 45±7 MC min−1 in IMR to 74±3 MC min−1 in MRmax in normoxia, and from 32±6 MC min−1 in IMR to 50±5 MC min−1 in AMR during EH (Fig. 7A; supplementary material Table S1). During LH, the contraction frequency decreased to 6±3 MC min−1. Contraction strength (Δ mantle diameter; Fig. 7C) increased significantly with metabolic rate in normoxia and EH (supplementary material Table S1). At IMR, mantle contraction strength was more or less constant (around 1.5 mm) during EH, but then decreased dramatically with duration of exposure (LH) down to ≤0.4 mm (two-way ANOVA, P<0.05; supplementary material Table S1). The correlations between contraction frequency/strength and metabolic rate are given in Fig. 7B,D. In both cases, the relationship is linear for both normoxic (Fig. 7B: R2=0.977, F=130.3, P=0.001; Fig. 7D: R2=0.982, F=167.6, P<0.001) and hypoxic treatments (Fig. 7B: R2=0.850, F=17, P<0.05; Fig. 7D: R2=0.855, F=17.7, P<0.05). Interestingly, the slope for hypoxia is significantly steeper (F=12.8, P<0.01, frequency; F=5.2, P<0.05, strength) than that for normoxia, indicating that much higher ventilation rates (frequency and strength) are required to achieve even modest metabolic rates during exposure to hypoxia. There was also a significant correlation between mantle contraction frequency and strength (Fig. 8). Under normoxic conditions, squid showed a linear relationship between strength and frequency (R2=0.993, P<<0.001), whereas under hypoxia (1% O2, 1 kPa) the relationship was polynomial (R2=0.988, P=0.01), indicating reduced relative ventilation frequency.

Fig. 4.

Changes in IMR, RMR, AMR and MRmax of D. gigas, under control conditions (21% O2), and in EH and LH (1% O2).

Fig. 4.

Changes in IMR, RMR, AMR and MRmax of D. gigas, under control conditions (21% O2), and in EH and LH (1% O2).

There was a significant increase in the percentage relaxed mantle diameter in AMR and MRmax (i.e. hyperinflation) under normoxia (Fig. 9; supplementary material Table S1). However, the opposite trend was observed in EH and LH.

The effect of hypoxia on ventilation stroke volume Vw is shown in Fig. 10A. The highest value was observed during maximum activity (MRmax) under normoxia (4.8 ml). During EH, there was a significant increase in Vw, with activity ranging from 2.7 ml (IMR) to 3.6 ml (RMR) to 4.3 ml (AMR; P<0.05). In LH, there was a significant drop (42%) during the inactive state (IMR). By multiplying the Vw (Fig. 10A) by ventilation (contraction) frequency (Fig. 7A), we obtained the ventilatory volume per minute (Fig. 10B). As a result, it became evident that in EH the increase in Vw (at AMR and RMR; Fig. 10A) was followed by a decrease in contraction frequency (Fig. 7A), leading to similar values of in normoxia and EH (Fig. 10B). In LH, this ventilatory parameter was significantly lower (Fig. 10B, P<0.05; supplementary material Table S1). Oxygen extraction efficiency EO2 under normoxia ranged between 6.4% and 13.4% (2.6% and 5.4%, taking into account 60% potential skin respiration) from IMR to MRmax and was significantly elevated as a result of exposure to hypoxia, with a minimum of 43.7% (15.2% including 60% cutaneous uptake) in IMR during LH, and a maximum of 81.5% (33.6%) in AMR during EH (Fig. 10C,D, P <0.05; supplementary material Table S1). In Table 4, EO2 without, and with 20%, 50% and 60% potential skin respiration is presented.

Fig. 5.

Effect of hypoxia (1% O2) on burst frequency in D. gigas. (A) Number of escape jets per minute during IMR, RMR, AMR and MRmax, and (B) total number of burst events per hour. Values are expressed as means ± s.d. (N=6). Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Fig. 5.

Effect of hypoxia (1% O2) on burst frequency in D. gigas. (A) Number of escape jets per minute during IMR, RMR, AMR and MRmax, and (B) total number of burst events per hour. Values are expressed as means ± s.d. (N=6). Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

DISCUSSION

Effect of hypoxia on metabolic rate and metabolic scope

Dosidicus gigas metabolic rate varied between 14 and 29 μmol O2 g−1 wet mass h−1, depending on the level of activity. These values were in agreement with those from our previous studies (Rosa and Seibel, 2008; Rosa and Seibel, 2010), which also indicated a wide metabolic scope in well-oxygenated water. Dosidicus gigas is able to maintain these high metabolic rates down to an ambient O2 level of ~20 μmol l−1 (~2 kPa, ~200 m depth) (Gilly et al., 2006), below which they dramatically drop. In the present experiments, with the temperature and oxygen conditions found in the OMZ, squid metabolic rate dropped more than 75% under exposure to early hypoxia with no further significant reduction with exposure time. Moreover, MRmax was not recorded under such conditions and the metabolic scope was significantly decreased (Fig. 1B, Fig. 3B, Fig. 4). In general, organisms suppress their metabolism by 50–95% by shutting down expensive processes (i.e. protein synthesis, muscle activity) (Guppy and Withers, 1999) and supplement the remaining energy demand using a combination of available O2 and anaerobic metabolic pathways (Seibel, 2011). Cephalopods that routinely depend on jet propulsion extract only a small proportion (5–10%) of the available O2 from the ventilatory stream, but are able to alter the O2 extraction efficiency (≤25%) when necessary, including cutaneous respiration, for example (Wells et al., 1988; Wells and O'Dor, 1991; Pörtner, 1994). Yet, the most common strategy among powerful predators that managed to minimize the trade-offs between high performance and hypoxia tolerance is a high-affinity respiratory protein (Lowe et al., 2000; Gilly et al., 2006; Seibel, 2011). Hemocyanin of cephalopods is usually characterized by a low O2 affinity with high pH sensitivity, properties that facilitate O2 release to demanding tissues, but presumably interfere with O2 extraction from hypoxic or CO2-rich seawater (Pörtner, 1994; Pörtner, 2002; Melzner et al., 2007). Yet, it has been reported (Seibel, 2012) that the respiratory protein in D. gigas is both highly temperature and pH sensitive, but, in addition, is characterized by a high O2-binding affinity. These properties facilitate O2 uptake in cold, deep waters where O2 is scarce and enhances O2 release in warmer surface waters where O2 and O2 demand are elevated. Furthermore, Seibel (Seibel, 2012) could link the hypoxia tolerance down to Pcrit (1.6 kPa, 10°C) (Trueblood and Seibel, 2012) to the optimized hemocyanin function in D. gigas, and suggests that below Pcrit (~170 m, Gulf of California) the onset of metabolic suppression kicks in.

Fig. 6.

Effect of hypoxia (1% O2) on the periodicity of activity cycles in D. gigas. (A) Average cycle length for MRmax and AMR, and (B) cycle frequency. Values are expressed as means and s.d. (N=5; 1 of the 6 was not cycling). Significant differences between AMR and MRmax cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Fig. 6.

Effect of hypoxia (1% O2) on the periodicity of activity cycles in D. gigas. (A) Average cycle length for MRmax and AMR, and (B) cycle frequency. Values are expressed as means and s.d. (N=5; 1 of the 6 was not cycling). Significant differences between AMR and MRmax cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

However, metabolic suppression is time limited, as anaerobic glycolysis results in the accumulation of protons (H+) and organic compounds such as lactate and octopine (Hochachka and Somero, 2002; Rosa and Seibel, 2010) with harmful consequences on acid–base status (Hochachka and Mommsen, 1983). Recent biochemical analysis revealed that under EH ~60% of the depressed metabolism is still achieved aerobically but this drops to ~45% at LH (K.T., unpublished). No significant further drop in metabolic rate between EH and LH could be quantified, indicating a limitation in the potential to suppress metabolism and a possible switch in energy expenditure from muscle activity to acid–base and ion regulation processes.

Fig. 7.

Effect of hypoxia (1% O2) on ventilation rates of D. gigas. (A) Mantle contraction (MC) frequency, and (C) mantle contraction strength (change in mantle diameter). Significant differences between activity levels (capital letters) and between treatments (lowercase letters) are indicated (P<0.05, see supplementary material Table S1). Values are expressed as means and s.d. (N=6). (B,D) Ventilation frequency and strength, plotted against oxygen consumption. Diamonds, normoxia; circles, EH; triangles, LH. Yellow, IMR; red, RMR; bright blue, AMR; dark blue, MRmax.

Fig. 7.

Effect of hypoxia (1% O2) on ventilation rates of D. gigas. (A) Mantle contraction (MC) frequency, and (C) mantle contraction strength (change in mantle diameter). Significant differences between activity levels (capital letters) and between treatments (lowercase letters) are indicated (P<0.05, see supplementary material Table S1). Values are expressed as means and s.d. (N=6). (B,D) Ventilation frequency and strength, plotted against oxygen consumption. Diamonds, normoxia; circles, EH; triangles, LH. Yellow, IMR; red, RMR; bright blue, AMR; dark blue, MRmax.

Vertical migrations and respiratory cycling behavior

Jumbo squid are diel vertical migrators that spend their nights in shallower, well-oxygenated waters (~70 m) and dive into the OMZ during the day (peak depth range 200–300 m; 2–0.2 kPa) (Gilly et al., 2006) (Trueblood and Seibel, 2012). Thereby, jumbo squid spend more than 70% of the day below 200 m (≤2% O2, 2 kPa) (Gilly et al., 2006) following their main prey (myctophid fishes) (Markaida and Sosa-Nishizaki, 2003; Markaida et al., 2008) and in this way also escape from: (i) unfavorable warmer sea surface temperatures, and (ii) elevated predation pressure and resource (food) competition, as most active fish predators are excluded from the OMZ (Prince and Goodyear, 2006).

Nonetheless, acoustic data revealed short-term cyclic vertical movements (~15–90 min), during day and night time, that have been interpreted as active forages (Gilly et al., 2006). In a previous study, we (Rosa and Seibel, 2010) discovered a similar periodicity in the rate of metabolism in the confines of a respiration chamber (cycle length ~20 min), which was also observed in the present experiments (20/60 min for active/maximum active cycles; Fig. 1A, Fig. 6A). As the frequency and duration of brief vertical movements are similar to the periodicity reported here, we argue that the activity cycles (MRmax and AMR) at normoxia may reflect the capacity to perform migratory forays.

During early hypoxia, the respiratory cycles were shorter in length but increased in frequency (Fig. 1B, Fig. 6). This was accompanied by an increase in the number of escape jets in active periods (see AMR in Fig. 5A) and a faster switch between swimming modes (data not shown). As high muscular activity involves the use of anaerobic ATP production, cycling between aerobic and anaerobic swimming phases over short time intervals may reduce the cost of transport and permit a long-term use of anaerobic resources, a strategy that in the long term may imply a maximized use of ambient O2 (Finke et al., 1996). Finke and colleagues assumed that this oscillation between periods of high and low muscular activity may have been related to the hypoxia tolerance of Lolliguncula brevis in coastal waters. It is plausible that D. gigas uses the same strategy. Furthermore, it displayed an enhanced usage of its lateral fins under progressive hypoxia, with a peak at the transition from EH to LH (data not shown). This also might contribute to minimize the costs of transport, as lateral fins can push a large mass of water especially at low velocities (O'Dor et al., 1988; Wells and O'Dor, 1991). At LH (onset ~170±10 min), jumbo squid moved into a lethargic state and ventilatory cycles stopped (Fig. 1C, Fig. 6). This lethargic behavior has been associated with metabolic suppression (Rosa and Seibel, 2010). Gilly and colleagues (Gilly et al., 2006) claimed that a substantial number of excursions lasted for many hours (maximum duration of 400/800 min below 300/200 m), during which time rhythmic diving occurred that appeared to be as robust as that observed at well-oxygenated, shallower depths. The majority of these extended dives at 300 m (~1% O2, 1 kPa) peaked between 140 and 240 min, which is in close agreement with the onset of late hypoxia (~170±10 min) in the present study. Yet, the switch to severe hypoxia within the respiration chambers could have been faster than during average, natural descents [see fig. 11 of Gilly (Gilly et al., 2006)], even though fast migrations (i.e. hunting, escape) in jumbo squid are known, which might have elevated stress. Also the lack of food within the chambers might have accelerated the depletion of anaerobic energy reserves, resulting in the onset of lethargy.

Fig. 8.

Relationship between mantle contraction (MC) strength (change in mantle diameter) and frequency (ventilation rate) in D. gigas under normoxia and hypoxia (1% O2). The black straight line and diamonds represent normoxia, the dashed line represents hypoxia, with circles for EH and triangles for LH. Yellow, IMR; red, RMR; bright blue, AMR; dark blue, MRmax. Values are expressed as means (N=6).

Fig. 8.

Relationship between mantle contraction (MC) strength (change in mantle diameter) and frequency (ventilation rate) in D. gigas under normoxia and hypoxia (1% O2). The black straight line and diamonds represent normoxia, the dashed line represents hypoxia, with circles for EH and triangles for LH. Yellow, IMR; red, RMR; bright blue, AMR; dark blue, MRmax. Values are expressed as means (N=6).

Fig. 9.

Changes in the relaxed mantle diameter of IMR, RMR, AMR and MRmax in D. gigas, under control conditions (21% O2), EH and LH (1% O2). Percentage relaxed mantle diameter was calculated based on the relaxed diameter under IMR normoxia. Values are expressed as means and s.d. (N =6). Red arrows indicate a hyperinflation mechanism. Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Fig. 9.

Changes in the relaxed mantle diameter of IMR, RMR, AMR and MRmax in D. gigas, under control conditions (21% O2), EH and LH (1% O2). Percentage relaxed mantle diameter was calculated based on the relaxed diameter under IMR normoxia. Values are expressed as means and s.d. (N =6). Red arrows indicate a hyperinflation mechanism. Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Ventilatory mechanisms and oxygen extraction efficiency during hypoxia

In normoxia, the D. gigas mantle contraction frequency and strength were linearly correlated (Fig. 8) and increased significantly with activity level (Figs 7, 8). Additionally, an increased stroke volume and ventilatory volume per minute (Fig. 10) were observed, followed by a hyperinflation process during high activity periods (red arrows in Fig. 9). The squid Illex illecebrosus and L. opalescens, in contrast, do not vary their ventilation via frequency with vigorous jetting, but via powerful contractions of their circular fibres (Wells et al., 1988; Webber and O'Dor, 1985). A linear increase in ventilation frequency with speed (<0.6 m s−1) was found in I. illecebrosus, but at speeds >0.6 m s−1 the correlation was linear with cavity pressure (Webber and O'Dor, 1986). As a result, I. illecebrosus and L. opalescens seem to be restricted to regulating ventilation strength and frequency at the same time, while D. gigas seems to have developed an optimized fine-tuning between locomotion and ventilation, and seems to have the potential to regulate mantle contraction frequency, strength and stroke volume according to activity.

Yet, EH changed the relationship between ventilation strength and frequency from linear to polynomial with increasing activity (Fig. 8). This indicates that D. gigas, under hypoxic conditions, primarily increases the power of mantle contraction and does not regulate its frequency, as was observed for I. illecebrosus and L. opalescens under normoxia (Wells et al., 1988).

Fig. 10.

Changes in (A) stroke volume Vw, (B) ventilatory volume per minute VV, (C) O2 extraction efficiency EO2 and (D) O2 extraction efficiency with potential skin respiration contribution (EO2,60%) in IMR, RMR, AMR and MRmax of D. gigas, under control conditions (21% O2), EH and LH (1% O2). Values are expressed as means and s.d. (N=6). Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Fig. 10.

Changes in (A) stroke volume Vw, (B) ventilatory volume per minute VV, (C) O2 extraction efficiency EO2 and (D) O2 extraction efficiency with potential skin respiration contribution (EO2,60%) in IMR, RMR, AMR and MRmax of D. gigas, under control conditions (21% O2), EH and LH (1% O2). Values are expressed as means and s.d. (N=6). Significant differences between active and maximum active cycles (capital letters) and between oxygen treatments (lowercase letters) are indicated (P<0.05).

Table 4.

Oxygen extraction efficiencies without (0%) and with (20%, 50% and 60%) consideration of potential skin respiration

Oxygen extraction efficiencies without (0%) and with (20%, 50% and 60%) consideration of potential skin respiration
Oxygen extraction efficiencies without (0%) and with (20%, 50% and 60%) consideration of potential skin respiration

In EH, jumbo squid showed a larger inflation period leading to reduced contraction frequency and decreased relaxed mantle diameter, which optimize O2 uptake (i.e. shorter diffusion ways) via gills and skin (see Fig. 10C). Additionally, they seem to breathe ‘deeply’, with more powerful contractions (see different slopes in Fig. 7D) and enhanced stroke volume (Fig. 10A). This deep-breathing behavior allows the squid to have the same amount of water passing through the gills per period of time, i.e. a stable ventilatory volume per minute, and explains the maintenance of the squid's cycling activity under such O2 conditions.

The O2 extraction efficiency reached ~82% in EH at active metabolic rates (Fig. 10C) without consideration of cutaneous uptake. Other cephalopods, such as nautilus and squid (L. brevis), also alter the effectiveness of oxygen extraction (i.e. cutaneous uptake) from the ventilatory stream as a result of falling ambient O2 levels or exercise when necessary, but at around 15–20% (once 43% in nautilus) (Wells et al., 1988; Wells, 1990; Pörtner, 1994). Maximum O2 extraction efficiency (AMR at EH), considering 60% potential skin respiration in squid (theoretical model, I. illecebrosus under exercise) (Pörtner, 1994), was still found to be 34% in our study (Fig. 10D). The organization of squid mantle muscle structure (mitochondria-rich inner and outer layer) supports the importance of skin respiration in cephalopods (Gosline and DeMont, 1985). Under hypoxia, PO2 gradients drastically decline and therefore do not favor diffusion processes (O2 uptake via skin and gills). Under LH, when jumbo squid cease swimming, cutaneous uptake might even decrease as a result of a lack of convection. Deep-breathing behavior observed at EH, instead, might favour cutaneous uptake (inner and outer mantle), but here we were not able to distinguish between O2 uptake via skin and gills.

In cuttlefish and octopus, organisms that are less dependent on jet propulsion, the fraction of oxygen extraction from the ventilatory stream is higher [50–90% in cuttlefish (Melzner et al., 2006); 45–75% in octopus (Wells and Wells, 1985; Wells, 1990)], and their O2 uptake can only be enhanced by an elevation in water throughput and increased diffusion gradients at the water–blood threshold (Melzner et al., 2006). Other OMZ residents in the Gulf of California, like Gnathophausia ingens, can regulate oxygen uptake mainly via an increase in ventilation volume (5–6 times, maximum 8) at constant (or slightly increasing) O2 extraction efficiencies that can reach 48–60%, with a maximum ~95% (Childress, 1971). However, these large changes in ventilation volume are not possible for squid because of the constraints imposed by the massive collagenous tissues in the mantle walls of muscular squid (Gosline and Shadwick, 1983; Wells et al., 1988).

In contrast, at LH, all the ventilatory processes were significantly reduced, except for O2 extraction efficiency followed by a lethargic state. Also, Boutilier and colleagues (Boutilier et al., 1996) reported that Nautilus cease ventilatory movements, cardiac output and heartbeat to survive prolonged periods of hypoxia. In the majority of investigated squid, ventilation was completely shut down, but we cannot rule out the possibility that there were further very weak contractions that could not be assessed by the method of analysis used. Another explanation could be that D. gigas has the potential to uncouple its ventilatory and locomotory mechanisms via a collar flap system, as is known for resting sepia and to some extent for squid (Bone et al., 1994). This original respiratory design of cephalopods (Wells, 1988) drives expiration via an active inward movement of the collar flaps (and probably via stored elastic energy during inhalation) (Gosline and Shadwick, 1983) without the activity of circular muscle fibers (Bone et al., 1994).

Unfortunately, the experiments were interrupted a maximum of 20 min after the jumbo squid stopped moving. Nonetheless, recent data suggest that juvenile (R.R., unpublished) and adult jumbo squid (B.A.S., unpublished) can sustain severe hypoxia (1% O2) for at least 6–12 h and therefore might use lethargy as a strategy to extend the residence time in the OMZ. It is noteworthy that our experiments were conducted with juveniles and therefore our results might not apply in adults, even though metabolic rates in juvenile and adult squid should not differ, as muscular squid display an unusual scaling relationship (tendency to isometry). This might be a consequence of: (1) tubular geometry (mantle diameter increases faster than thickness with growth), exchange surfaces (surface area1/2:volume1/3 increases with size) (O'Dor and Hoar, 2000) and cutaneous respiration (~60%) (Pörtner, 1994; Pörtner, 2002); (2) ontogenetic differences in locomotory expenditure, because the squid cost of transport may not decrease as much with size as that in other animals (e.g. mammals, birds and fish), where adults are more energy efficient; and (3) energetic requirements during all stages of the squid's short life cycle (namely, for growth and reproduction). High sustained production costs during all ages may result in a B value (scaling coefficient) that does not decrease with increasing body mass (Seibel, 2007; Rosa et al., 2009). However, it remains unclear whether adults will adjust their ventilatory mechanisms to low oxygen levels in a similar way to juveniles, especially as little is known about the potential of oxygen uptake via the skin under hypoxia.

Acknowledgements

We would like to thank Unai Markaida for his assistance with collection of the specimens.

FOOTNOTES

FUNDING

This work was supported by research grants to R.R. from the Portugal–US Research Networks Program (FLAD/US National Science Foundation Research Grant) and The Portuguese Foundation for Science and Technology (FCT, Ciência 2007 Program) and to B.A.S. from the National Science Foundation [grant OCE-0851043].

LIST OF SYMBOLS AND ABBREVIATIONS

     
  • AMR

    active metabolic rate

  •  
  • ds

    squid density

  •  
  • EO2

    O2 extraction efficiency

  •  
  • IMR

    inactive metabolic rate

  •  
  • L

    mantle length

  •  
  • Lc

    cylinder length

  •  
  • M

    weight mass (kg)

  •  
  • MC

    mantle contractions

  •  
  • MR

    metabolic rate

  •  
  • MRmax

    maximum active metabolic rate

  •  
  • OMZ

    oxygen minimum zone

  •  
  • rC

    contracted mantle radius

  •  
  • rH

    maximum (hyperinflated) mantle radius

  •  
  • RMR

    routine metabolic rate

  •  
  • flow rate

  •  
  • ventilatory volume per minute

  •  
  • Vw

    stroke volume

  •  
  • x

    mantle thickness (mm)

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