A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

Temperature-dependent RNA editing in octopus extensively recodes the neural proteome.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

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.

Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata.

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNaV1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNaV1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.

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.

Experimental determination of the spatial scale of a prey patch from the predator's perspective.

Foraging theory predicts that predators should prefer foraging in habitat patches with higher prey densities. However, density depends on the spatial scale at which a "patch" is defined by an observer. Ecologists strive to measure prey densities at the same scale that predators do, but many natural landscapes lack obvious, well-defined prey patches. Thus one must determine the scale at which predators define patches of prey. We estimated the scale at which guppies, Poecilia reticulata, selected patches of zooplankton prey using a behavioral assay. Guppies could choose between two prey arrays, each manipulated to have a density that depended on the spatial scale at which density was calculated. We estimated the scale of guppy foraging by comparing guppy preferences across a series of trials in which we systematically varied the scale associated with "high" prey density. This approach enables the application of foraging theory to non-discrete habitats and prey landscapes.