Metabolic trait diversity shapes marine biogeography.

Climate and physiology shape biogeography, yet the range limits of species can rarely be ascribed to the quantitative traits of organisms1-3. Here we evaluate whether the geographical range boundaries of species coincide with ecophysiological limits to acquisition of aerobic energy4 for a global cross-section of the biodiversity of marine animals. We observe a tight correlation between the metabolic rate and the efficacy of oxygen supply, and between the temperature sensitivities of these traits, which suggests that marine animals are under strong selection for the tolerance of low O2 (hypoxia)5. The breadth of the resulting physiological tolerances of marine animals predicts a variety of geographical niches-from the tropics to high latitudes and from shallow to deep water-which better align with species distributions than do models based on either temperature or oxygen alone. For all studied species, thermal and hypoxic limits are substantially reduced by the energetic demands of ecological activity, a trait that varies similarly among marine and terrestrial taxa. Active temperature-dependent hypoxia thus links the biogeography of diverse marine species to fundamental energetic requirements that are shared across the animal kingdom.

Oxygen supply capacity breathes new life into critical oxygen partial pressure (Pcrit).

The critical oxygen partial pressure (Pcrit), typically defined as the PO2 below which an animal's metabolic rate (MR) is unsustainable, is widely interpreted as a measure of hypoxia tolerance. Here, Pcrit is defined as the PO2 at which physiological oxygen supply (α0) reaches its maximum capacity (α; µmol O2 g-1 h-1 kPa-1). α is a species- and temperature-specific constant describing the oxygen dependency of the maximum metabolic rate (MMR=PO2×α) or, equivalently, the MR dependence of Pcrit (Pcrit=MR/α). We describe the α-method, in which the MR is monitored as oxygen declines and, for each measurement period, is divided by the corresponding PO2 to provide the concurrent oxygen supply (α0=MR/PO2). The highest α0 value (or, more conservatively, the mean of the three highest values) is designated as α. The same value of α is reached at Pcrit for any MR regardless of previous or subsequent metabolic activity. The MR need not be constant (regulated), standardized or exhibit a clear breakpoint at Pcrit for accurate determination of α. The α-method has several advantages over Pcrit determination and non-linear analyses, including: (1) less ambiguity and greater accuracy, (2) fewer constraints in respirometry methodology and analysis, and (3) greater predictive power and ecological and physiological insight. Across the species evaluated here, α values are correlated with MR, but not Pcrit. Rather than an index of hypoxia tolerance, Pcrit is a reflection of α, which evolves to support maximum energy demands and aerobic scope at the prevailing temperature and oxygen level.

Oxygen supply capacity in animals evolves to meet maximum demand at the current oxygen partial pressure regardless of size or temperature.

The capacity to extract oxygen from the environment and transport it to respiring tissues in support of metabolic demand reportedly has implications for species' thermal tolerance, body size, diversity and biogeography. Here, we derived a quantifiable linkage between maximum and basal metabolic rate and their oxygen, temperature and size dependencies. We show that, regardless of size or temperature, the physiological capacity for oxygen supply precisely matches the maximum evolved demand at the highest persistently available oxygen pressure and this is the critical PO2  for the maximum metabolic rate, Pcrit-max For most terrestrial and shallow-living marine species, Pcrit-max is the current atmospheric pressure, 21 kPa. Any reduction in oxygen partial pressure from current values will result in a calculable decrement in maximum metabolic performance. However, oxygen supply capacity has evolved to match demand across temperatures and body sizes and so does not constrain thermal tolerance or cause the well-known reduction in mass-specific metabolic rate with increasing body mass. The critical oxygen pressure for resting metabolic rate, typically viewed as an indicator of hypoxia tolerance, is, instead, simply a rate-specific reflection of the oxygen supply capacity. A compensatory reduction in maintenance metabolic costs in warm-adapted species constrains factorial aerobic scope and the critical PO2  to a similar range, between ∼2 and 6, across each species' natural temperature range. The simple new relationship described here redefines many important physiological concepts and alters their ecological interpretation.

Do squid breathe through their skin?

Squid are thought to obtain a large portion of their oxygen via simple diffusion across the skin in addition to uptake at the gills. Although this hypothesis has support from indirect evidence and is widely accepted, no empirical examinations have been conducted to assess the validity of this hypothesis. In this study, we examined cutaneous respiration in two squid species, Doryteuthis pealeii and Lolliguncula brevis, using a divided chamber to physically separate the mantle cavity and gills from the outer mantle surface. We measured oxygen consumption and ammonia excretion rates in the two compartments and found that, at rest, squid only obtain enough oxygen cutaneously to meet the demand of the skin tissue locally (12% of total) and excrete little ammonia across the skin. The majority of oxygen is obtained via the traditional branchial pathway. In light of these findings, we re-examine and discuss the indirect evidence that has supported the cutaneous respiration hypothesis.

Ocean acidification does not limit squid metabolism via blood oxygen supply.

Ocean acidification is hypothesized to limit the performance of squid owing to their exceptional oxygen demand and pH sensitivity of blood-oxygen binding, which may reduce oxygen supply in acidified waters. The critical oxygen partial pressure (Pcrit), the PO2  below which oxygen supply cannot match basal demand, is a commonly reported index of hypoxia tolerance. Any CO2-induced reduction in oxygen supply should be apparent as an increase in Pcrit In this study, we assessed the effects of CO2 (46-143 Pa; 455-1410 µatm) on the metabolic rate and Pcrit of two squid species - Dosidicus gigas and Doryteuthis pealeii - through manipulative experiments. We also developed a model, with inputs for hemocyanin pH sensitivity, blood PCO2  and buffering capacity, that simulates blood oxygen supply under varying seawater CO2 partial pressures. We compare model outputs with measured Pcrit in squid. Using blood-O2 parameters from the literature for model inputs, we estimated that, in the absence of blood acid-base regulation, an increase in seawater PCO2  to 100 Pa (≈1000 µatm) would result in a maximum drop in arterial hemocyanin-O2 saturation by 1.6% at normoxia and a Pcrit increase of ≈0.5 kPa. Our live-animal experiments support this supposition, as CO2 had no effect on measured metabolic rate or Pcrit in either squid species.

Synthetic capacity does not predict elasmobranchs' ability to maintain trimethylamine oxide without a dietary contribution.

Trimethylamine oxide (TMAO) is an organic osmolyte and universal protein stabilizer. Its role as a cytoprotectant is particularly important in ureosmotic elasmobranchs that accumulate high levels of urea, a macromolecular perturbant. Feeding is a key component in the turnover and maintenance of these nitrogenous compounds. However, previous studies examining TMAO regulation have been largely completed using starved individuals, when nitrogen balance is altered. Here, under fed conditions, we test the importance of dietary TMAO on long-term maintenance in three elasmobranch species with differing endogenous synthetic capacities. Smoothhounds (Mustelus canis), spiny dogfish (Squalus acanthias), and little skates (Leucoraja erinacea) exhibited species- and tissue-specific differences in their ability to conserve TMAO when fed a low TMAO diet for 56days. Smoothhounds, a species with the capacity for endogenous production, exhibited a decrease in muscle TMAO. Spiny dogfish and little skates, species with no reported ability for synthesis, exhibited decreases in plasma and liver TMAO, respectively. Our findings are contrary to previous starvation studies demonstrating constant levels of TMAO for up to 56days in elasmobranchs. Further, the previously reported synthetic capacity of these species did not correlate with their ability to conserve TMAO and cannot be used to predict a species reliance on dietary contributions for prolonged maintenance. It is possible that all species rely to a degree on absorption of TMAO from the diet or that alternate synthetic or regulatory pathways play a larger role than previously thought.

Cephalopod Susceptibility to Asphyxiation via Ocean Incalescence, Deoxygenation, and Acidification.

Squids are powerful swimmers with high metabolic rates despite constrained oxygen uptake and transport. They have evolved novel physiological strategies for survival in extreme environments that provide insight into their susceptibility to asphyxiation under anthropogenic ocean incalescence (warming), deoxygenation, and acidification. Plasticity of ecological and physiological traits, in conjunction with vertical and latitudinal mobility, may explain their evolutionary persistence and ensure their future survival.

Effects of short-term hyper- and hypo-osmotic exposure on the osmoregulatory strategy of unfed North Pacific spiny dogfish (Squalus suckleyi).

The North Pacific spiny dogfish (Squalus suckleyi) is a partially euryhaline species of elasmobranch that often enter estuaries where they experience relatively large fluctuations in environmental salinity that can affect plasma osmolality. Previous studies have investigated the effects of altered salinity on elasmobranchs over the long term, but fewer studies have conducted time courses to investigate how rapidly they can adapt to such changes. In this study, we exposed unfed (no exogenous source of nitrogen or TMAO) spiny dogfish to hyper- and hypo-osmotic conditions and measured plasma and tissue osmolytes, nitrogen excretion, and changes in enzyme activity and mRNA levels in the rectal gland over 24h. It was shown that plasma osmolality changes to approximately match the ambient seawater within 18-24h. In the hypersaline environment, significant increases in urea, sodium, and chloride were observed, whereas in the hyposaline environment, only significant decreases in TMAO and sodium were observed. Both urea and ammonia excretion increased at low salinities suggesting a reduction in urea retention and possibly urea production. qPCR and enzyme activity data for Na(+)/K(+)-ATPase did not support the idea of rectal gland activation following exposure to increased salinities. Therefore, we suggest that the rectal gland may not be a quantitatively important aspect of the dogfish osmoregulatory strategy during changes in environmental salinity, or it may be active only in the very early stages (i.e., less than 6h) of responses to altered salinity.

The thermal stress response to diel vertical migration in the hyperiid amphipod Phronima sedentaria.

The hyperiid amphipod Phronima sedentaria experiences a temperature change of 15 °C during diel migration in the Eastern Tropical North Pacific (ETNP) from 8-10 °C at depth to 25-27 °C at night in the surface waters. The aim of this study was to determine if the natural temperature gradient experienced by P. sedentaria results in a thermal stress response. Individuals were initially exposed to their night time temperatures (23 °C) and subsequently subjected to temperatures within and above the range they typically experience. In the Eastern Tropical North Pacific P. sedentaria tolerates its normal night-time temperature (~23 °C), but only for the duration of its stay there (~9 h). Longer exposures (24 h) result in elevated heat shock protein (hsp) expression. 29 °C results in hsp expression, increased lactate production and 50% mortality at all exposure durations. This represents an upper critical temperature. Understanding the adaptations of pelagic amphipods to their current environment will help predict the physiological impacts of global warming for amphipods and their predators.

Slow swimming, fast strikes: effects of feeding behavior on scaling of anaerobic metabolism in epipelagic squid.

Many pelagic fishes engage prey at high speeds supported by high metabolic rates and anaerobic metabolic capacity. Epipelagic squids are reported to have among the highest metabolic rates in the oceans as a result of demanding foraging strategies and the use of jet propulsion, which is inherently inefficient. This study examined enzymatic proxies of anaerobic metabolism in two species of pelagic squid, Dosidicus gigas and Doryteuthis pealeii (Lesueur 1821), over a size range of six orders of magnitude. We hypothesized that activity of the anaerobically poised enzymes would be high and increase with size as in ecologically similar fishes. In contrast, we demonstrate that anaerobic metabolic capacity in these organisms scales negatively with body mass. We explored several cephalopod-specific traits, such as the use of tentacles to capture prey, body morphology and reduced relative prey size of adult squids, that may create a diminished reliance on anaerobically fueled burst activity during prey capture in large animals.

Metabolic suppression during protracted exposure to hypoxia in the jumbo squid, Dosidicus gigas, living in an oxygen minimum zone.

The jumbo squid, Dosidicus gigas, can survive extended forays into the oxygen minimum zone (OMZ) of the Eastern Pacific Ocean. Previous studies have demonstrated reduced oxygen consumption and a limited anaerobic contribution to ATP production, suggesting the capacity for substantial metabolic suppression during hypoxic exposure. Here, we provide a more complete description of energy metabolism and explore the expression of proteins indicative of transcriptional and translational arrest that may contribute to metabolic suppression. We demonstrate a suppression of total ATP demand under hypoxic conditions (1% oxygen, PO2 =0.8 kPa) in both juveniles (52%) and adults (35%) of the jumbo squid. Oxygen consumption rates are reduced to 20% under hypoxia relative to air-saturated controls. Concentrations of arginine phosphate (Arg-P) and ATP declined initially, reaching a new steady state (~30% of controls) after the first hour of hypoxic exposure. Octopine began accumulating after the first hour of hypoxic exposure, once Arg-P breakdown resulted in sufficient free arginine for substrate. Octopine reached levels near 30 mmol g(-1) after 3.4 h of hypoxic exposure. Succinate did increase through hypoxia but contributed minimally to total ATP production. Glycogenolysis in mantle muscle presumably serves to maintain muscle functionality and balance energetics during hypoxia. We provide evidence that post-translational modifications on histone proteins and translation factors serve as a primary means of energy conservation and that select components of the stress response are altered in hypoxic squids. Reduced ATP consumption under hypoxia serves to maintain ATP levels, prolong fuel store use and minimize the accumulation of acidic intermediates of anaerobic ATP-generating pathways during prolonged diel forays into the OMZ. Metabolic suppression likely limits active, daytime foraging at depth in the core of the OMZ, but confers an energetic advantage over competitors that must remain in warm, oxygenated surface waters. Moreover, the capacity for metabolic suppression provides habitat flexibility as OMZs expand as a result of climate change.

Ventilation rates and activity levels of juvenile jumbo squid under metabolic suppression in the oxygen minimum zone.

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% O(2), 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 O(2) 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 O(2) 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.

Temperature compensation of aerobic capacity and performance in the Antarctic pteropod, Clione antarctica, compared with its northern congener, C. limacina.

In ectotherms living in cold waters, locomotory performance is constrained by a slower generation of the ATP that is needed to fuel muscle contraction. Both polar and temperate pteropods of the genus Clione, however, are able to swim continuously by flapping their parapodia (wings) at comparable frequencies at their respective habitat temperatures. Therefore, we expected polar species to have increased aerobic capacities in their wing muscles when measured at common temperatures. We investigated muscle and mitochondrial ultrastructure of Clione antarctica from the Southern Ocean (-1.8°C) and populations of a sister species, Clione limacina, from the Arctic (-0.5 to 3°C) and from the North Atlantic (10°C). We also measured oxygen consumption and the activity of the mitochondrial enzyme citrate synthase (CS) in isolated wings of the two species. The Antarctic species showed a substantial up-regulation of the density of oxidative muscle fibers, but at the expense of fast-twitch muscle fibers. Mitochondrial capacity was also substantially increased in the Antarctic species, with the cristae surface density (58.2±1.3µm(2)µm(-3)) more than twice that found in temperate species (34.3±0.8µm(2)µm(-3)). Arctic C. limacina was intermediate between these two populations (43.7±0.5µm(2)µm(-3)). The values for cold-adapted populations are on par with those found in high-performance vertebrates. As a result of oxidative muscle proliferation, CS activity was 4-fold greater in C. antarctica wings than in temperate C. limacina when measured at a common temperature (20°C). Oxygen consumption of isolated wing preparations was comparable in the two species when measured at their respective habitat temperatures. These findings indicate complete compensation of ATP generation in wing muscles across a 10°C temperature range, which supports similar wing-beat frequencies during locomotion at each species' respective temperature. The elevated capacity in the wing muscles is reflected in the partial compensation of whole-animal oxygen consumption and feeding rates.

Trade-off between aerobic capacity and locomotor capability in an Antarctic pteropod.

At -1.8 degrees C, the waters of Antarctica pose a formidable physiological barrier for most ectotherms. The few taxa that inhabit this zone have presumably made specific adjustments to their neuromuscular function and have enhanced their metabolic capacity. However, support for this assertion is equivocal and the details of specific compensations are largely unknown. This can generally be attributed to the fact that most Antarctic organisms are either too distantly related to their temperate relatives to permit direct comparisons (e.g., notothenioid fishes) or because they are not amenable to neuromuscular recording. Here, as a comparative model, we take advantage of 2 pelagic molluscs in the genus Clione to conduct a broadly integrative investigation on neuromuscular adaptation to the extreme cold. We find that for the Antarctic congener aerobic capacity is enhanced, but at a cost. To support a striking proliferation of mitochondria, the Antarctic species has shed a 2-gear swim system and the associated specialized neuromuscular components, resulting in greatly reduced scope for locomotor activity. These results suggest that polar animals have undergone substantial tissue-level reorganizations to accommodate their environment, which may reduce their capacity to acclimate to a changing climate.

Critical oxygen levels and metabolic suppression in oceanic oxygen minimum zones.

The survival of oceanic organisms in oxygen minimum zones (OMZs) depends on their total oxygen demand and the capacities for oxygen extraction and transport, anaerobic ATP production and metabolic suppression. Anaerobic metabolism and metabolic suppression are required for daytime forays into the most extreme OMZs. Critical oxygen partial pressures are, within a range, evolved to match the minimum oxygen level to which a species is exposed. This fact demands that low oxygen habitats be defined by the biological response to low oxygen rather than by some arbitrary oxygen concentration. A broad comparative analysis of oxygen tolerance facilitates the identification of two oxygen thresholds that may prove useful for policy makers as OMZs expand due to climate change. Between these thresholds, specific physiological adaptations to low oxygen are required of virtually all species. The lower threshold represents a limit to evolved oxygen extraction capacity. Climate change that pushes oxygen concentrations below the lower threshold (~0.8 kPa) will certainly result in a transition from an ecosystem dominated by a diverse midwater fauna to one dominated by diel migrant biota that must return to surface waters at night. Animal physiology and, in particular, the response of animals to expanding hypoxia, is a critical, but understudied, component of biogeochemical cycles and oceanic ecology. Here, I discuss the definition of hypoxia and critical oxygen levels, review adaptations of animals to OMZs and discuss the capacity for, and prevalence of, metabolic suppression as a response to temporary residence in OMZs and the possible consequences of climate change on OMZ ecology.

Post-spawning egg care by a squid.

Gonatus onyx is one of the most abundant cephalopods in the Pacific and Atlantic Oceans and is an important prey species for a variety of vertebrate predators, but a full understanding of its life history has been hampered because spawning occurs at great depths, where observation is difficult. Here we describe post-spawning egg care, or brooding, in this deep-sea squid. Our finding is unexpected because this behaviour differs from the reproductive habits of all other known squid species.

Synergistic effects of climate-related variables suggest future physiological impairment in a top oceanic predator.

By the end of this century, anthropogenic carbon dioxide (CO(2)) emissions are expected to decrease the surface ocean pH by as much as 0.3 unit. At the same time, the ocean is expected to warm with an associated expansion of the oxygen minimum layer (OML). Thus, there is a growing demand to understand the response of the marine biota to these global changes. We show that ocean acidification will substantially depress metabolic rates (31%) and activity levels (45%) in the jumbo squid, Dosidicus gigas, a top predator in the Eastern Pacific. This effect is exacerbated by high temperature. Reduced aerobic and locomotory scope in warm, high-CO(2) surface waters will presumably impair predator-prey interactions with cascading consequences for growth, reproduction, and survival. Moreover, as the OML shoals, squids will have to retreat to these shallower, less hospitable, waters at night to feed and repay any oxygen debt that accumulates during their diel vertical migration into the OML. Thus, we demonstrate that, in the absence of adaptation or horizontal migration, the synergism between ocean acidification, global warming, and expanding hypoxia will compress the habitable depth range of the species. These interactions may ultimately define the long-term fate of this commercially and ecologically important predator.

Hypoxic Jumbo Squid Activate Neuronal Apoptosis but Not MAPK or Antioxidant Enzymes during Oxidative Stress.

AbstractThe limitations that hypoxia imparts on mitochondrial oxygen supply are circumvented by the activation of anaerobic metabolism and prosurvival mechanisms in hypoxia-tolerant animals. To deal with the hypoxia that jumbo squid (Dosidicus gigas) experience in the ocean's depth, they depress their metabolic rate by up to 52% relative to normoxic conditions. This is coupled with molecular reorganization to facilitate their daily descents into the ocean's oxygen minimum zone, where they face not only low oxygen levels but also higher pressures and colder frigid waters. Our current study explores the tissue-specific hypoxia responses of three central processes: (1) antioxidant enzymes responsible for defending against oxidative stress, (2) early apoptotic machinery that signals the activation of cell death, and (3) mitogen-activated protein kinases (MAPKs) that act as central regulators of numerous cellular processes. Luminex xMAP technology was used to assess protein levels and phosphorylation states under normoxic and hypoxic conditions in brains, branchial hearts, and mantle muscles. Hypoxic brains were found to activate apoptosis via upregulation of phospho-p38, phospho-p53, activated caspase 8, and activated caspase 9, whereas branchial hearts were the only tissue to show an increase in antioxidant enzyme levels. Hypoxic muscles seemed the least affected by hypoxia. Our results suggest that hypoxic squid do not undergo large dynamic changes in the phosphorylation states of key apoptotic and central MAPK factors, except for brains, suggesting that these mechanisms are involved in squid hypometabolic responses.