Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

The carryover effects of embryonic incubation temperature on subsequent growth and thermal tolerance in white sturgeon.

Environmental variation experienced during early periods of development can lead to persistent phenotypic alteration, known as carryover effects. Such effects increase concern for threatened or endangered species such as the white sturgeon (Acipenser transmontanus), particularly considering expected thermal changes due to climate change. We evaluated how temperature during embryonic development affects physiological parameters such as larval and early juvenile growth and thermal tolerance. Nechako River white sturgeon embryos were incubated at different environmental temperatures (Te) of 12 °C (the natural spawning temperature of this population), 15 °C (the hatchery incubation temperature), and 18 °C (representing potential increases in river temperatures given global climate change). After hatch, fish were reared at a common 15 °C for 80 days post-hatch (dph). Individuals from each temperature treatment were tested for thermal tolerance using the critical thermal maximum method (CTmax), euthanized, and measured. Fish were examined at regular intervals from 13 to 80 dph, which bridged the time from the start of exogenous feeding through the transition into early juveniles. We found carryover effects of high embryonic Te in the short term for both thermal tolerance and growth. Fish that developed at 18 °C had the lowest thermal tolerance during the start of exogenous feeding. However, differences in thermal tolerance were small for early juveniles and were unlikely to be ecologically relevant in the longer term. Fish that developed at 18 °C were smallest over the observation period, indicating a possible cost for survival from increasing environmental temperatures during embryonic development. This research represents a window into a critical period of development during which fish are particularly vulnerable to climatic variation, and shows that cooler temperatures (12 °C) during incubation are optimal for this population. The results can inform environmental managers on the best strategies to help conserve current white sturgeon populations across their range.

The origins of gas exchange and ion regulation in fish gills: evidence from structure and function.

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid-base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills.

Causes and consequences of gas bubble trauma on fish gill function.

Total dissolved gas supersaturation (TDGS) occurs when air mixes with water under pressure, which can be caused by features such as hydroelectric dams and waterfalls. Total dissolved gas supersaturation can cause harmful bubbles to grow in the tissues of aquatic animals, a condition known as gas bubble trauma (GBT). As gills are the primary gas exchange surface for most fish, it is through the gills that elevated total dissolved gases enter the blood and tissues of a fish. We describe the role of the gills in admitting TDGS into the body and discuss potential effects of bubbles in the gills on blood oxygen and carbon dioxide diffusion, blood ion and pH homeostasis, and nitrogenous waste excretion, as well as downstream effects on aerobic swimming performance.

An investigation of gill and blood carbonic anhydrase characteristics in three basal actinopterygian species: alligator gar (Atractosteus spatula), white sturgeon (Acipenser transmontanus) and Senegal bichir (Polypterus senegalus).

Many teleosts possess a unique set of respiratory characteristics allowing enhanced oxygen unloading to the tissues during stress. This system comprises three major components: highly pH sensitive haemoglobins (large Bohr and Root effects), rapid red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of membrane-bound plasma-accessible carbonic anhydrase (paCA; absence in the gills). The first two components have received considerable research effort; however, the evolutionary loss of branchial paCA has received little attention. In the current study, we investigated the availability of branchial membrane-bound CA, along with several other CA-related characteristics in species belonging to three basal actinopterygian groups: the Lepisosteiformes, Acipenseriformes and Polypteriformes to assess the earlier hypothesis that Root effect haemoglobins constrain branchial paCA availability. We present the first evidence suggesting branchial membrane-bound CA presence in a basal actinopterygian species: the Senegal bichir (Polypterus senegalus) and show that like the teleosts, white sturgeon (Acipenser transmontanus) and alligator gar (Atractosteus spatula) do not possess branchial membrane-bound CA. We discuss the varying respiratory strategies for these species and propose that branchial paCA may have been lost much earlier than previously thought, likely in relation to the changes in haemoglobin buffer capacity associated with the increasing magnitude of the Bohr effect. The findings described here represent an important advancement in our understanding of the evolution of the unique system of enhanced oxygen unloading thought to be present in most teleosts, a group that encompasses half of all vertebrates.

Resetting thermal limits: 10-year-old white sturgeon display pronounced but reversible thermal plasticity.

While many ectotherms improve thermal tolerance in response to prolonged thermal stress, little is known about the lasting effects of warm acclimation after returning to cooler temperatures. Furthermore, thermal stress may disproportionately impact threatened and endangered species. To address this, we repeatedly measured critical thermal maxima (CTmax; °C) and associated stress responses (hematocrit, hemoglobin concentration, plasma cortisol) of endangered subadult white sturgeon (Acipenser transmontanus) in response to control temperature (pre-acclimation; 14°C), after 1 month at either control or warm temperature (acclimation; 14°C or 20°C), and after one smonth following return to control temperature (post-acclimation; 14°C). While control fish demonstrated fairly repeatable thermal tolerance (interclass correlation coefficient = 0.479), warm-acclimated fish experienced a ∼3.1°C increase in thermal tolerance and when re-acclimated to control temperature, decreased thermal tolerance ∼1.9°C. Hematocrit, hemoglobin concentration, and final splenic somatic index (spleen mass relative to whole body mass, collected after post-acclimation CTmax) were not significantly different between control and treatment fish, suggesting no effects of warm acclimation on aerobic capacity. Plasma cortisol was significantly higher in control fish after pre-acclimation and post-acclimation CTmax trials, but importantly, acclimation temperature did not affect this response. Strikingly, final hepatosomatic index (relative liver size) was 45% lower in treatment fish, indicating warm acclimation may have lasting effects on energy usage and metabolism, even after reacclimating to control temperature. To our knowledge, these 10-year-old subadult sturgeon are the oldest sturgeon experimentally tested with regards to thermal plasticity and demonstrate incredible capacity for thermal acclimation relative to other fishes. However, more research is needed to determine whether the ability to acclimate to warm temperature may come with a persistent cost.

Heatwave resilience of juvenile white sturgeon is associated with epigenetic and transcriptional alterations.

Heatwaves are increasing in frequency and severity, posing a significant threat to organisms globally. In aquatic environments heatwaves are often associated with low environmental oxygen, which is a deadly combination for fish. However, surprisingly little is known about the capacity of fishes to withstand these interacting stressors. This issue is particularly critical for species of extreme conservation concern such as sturgeon. We assessed the tolerance of juvenile white sturgeon from an endangered population to heatwave exposure and investigated how this exposure affects tolerance to additional acute stressors. We measured whole-animal thermal and hypoxic performance and underlying epigenetic and transcriptional mechanisms. Sturgeon exposed to a simulated heatwave had increased thermal tolerance and exhibited complete compensation for the effects of acute hypoxia. These changes were associated with an increase in mRNA levels involved in thermal and hypoxic stress (hsp90a, hsp90b, hsp70 and hif1a) following these stressors. Global DNA methylation was sensitive to heatwave exposure and rapidly responded to acute thermal and hypoxia stress over the course of an hour. These data demonstrate that juvenile white sturgeon exhibit substantial resilience to heatwaves, associated with improved cross-tolerance to additional acute stressors and involving rapid responses in both epigenetic and transcriptional mechanisms.

Hydrothermal impacts of water release on early life stages of white sturgeon in the Nechako river, B.C. Canada.

Water temperature plays a crucial role in the physiology of aquatic species, particularly in their survival and development. Thus, resource programs are commonly used to manage water quality conditions for endemic species. In a river system like the Nechako River system, central British Columbia, a water management program was established in the 1980s to alter water release in the summer months to prevent water temperatures from exceeding a 20 °C threshold downstream during the spawning season of Sockeye salmon (Oncorhynchus nerka). Such a management regime could have consequences for other resident species like the white sturgeon (Acipenser transmontanus). Here, we use a hydrothermal model and white sturgeon life stage-specific experimental thermal tolerance data to evaluate water releases and potential hydrothermal impacts based on the Nechako water management plan (1980-2019). Our analysis focused mainly on the warmest five-month period of the year (May to September), which includes the water release management period (July-August). Our results show that the thermal exposure risk, an index that measures temperature impact on species physiology of Nechako white sturgeon across all early life stages (embryo, yolk-sac larvae, larvae, and juvenile) has increased substantially, especially in the 2010s relative to the management program implementations' first decade (the 1980s). The embryonic life stage was the most impacted, with a continuous increase in potential adverse thermal exposure in all months examined in the study. We also recorded major impacts of increased thermal exposure on the critical habitats necessary for Nechako white sturgeon recovery. Our study highlights the importance of a holistic management program with consideration for all species of the Nechako River system and the merit of possibly reviewing the current management plan, particularly with the current concerns about climate change impacts on the Nechako River.

Slow heating rates increase thermal tolerance and alter mRNA HSP expression in juvenile white sturgeon (Acipenser transmontanus).

Freshwater fish such as white sturgeon (Acipenser transmontanus) are particularly vulnerable to the effects of anthropogenically induced global warming. Critical thermal maximum tests (CTmax) are often conducted to provide insight into the impacts of changing temperatures; however, little is known about how the rate of temperature increase in these assays affects thermal tolerance. To assess the effect of heating rate (0.3 °C/min, 0.03 °C/min, 0.003 °C/min) we measured thermal tolerance, somatic indices, and gill Hsp mRNA expression. Contrary to what has been observed in most other fish species, white sturgeon thermal tolerance was highest at the slowest heating rate of 0.003 °C/min (34.2 °C, and CTmax of 31.3 and 29.2 °C, for rates 0.03 and 0.3 °C/min, respectively) suggesting an ability to rapidly acclimate to slowly increasing temperatures. Hepatosomatic index decreased in all heating rates relative to control fish, indicative of the metabolic costs of thermal stress. At the transcriptional level, slower heating rates resulted in higher gill mRNA expression of Hsp90a, Hsp90b, and Hsp70. Hsp70 mRNA expression was increased in all heating rates relative to controls, whereas expression of Hsp90a and Hsp90b mRNA only increased in the two slower trials. Together these data indicate that white sturgeon have a very plastic thermal response, which is likely energetically costly to induce. Acute temperature changes may be more detrimental to sturgeon as they struggle to acclimate to rapid changes in their environment, however under slower warming rates they demonstrate strong thermal plasticity to warming.

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; α2ß/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (ß/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrates that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas ß/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, ß/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; ζ2ε2 and HbF; α2γ2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (γ38Thr→Ile and γ101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the ß/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

In 1741, shipwrecked naturalist Georg Wilhelm Steller made detailed observations of large marine mammals grazing on seaweed in the shallow waters surrounding a remote island in the North Pacific Ocean. Within thirty years, these 'Steller's sea cows' had been hunted to extinction. Unlike their remaining tropical relatives ­ dugongs and manatees ­ Steller's sea cows were specialized to cold, sub-Arctic environments. Measuring up to 10 meters long, they were much larger than other sea cow species. This, along with having very thick skin, helped them to reduce heat loss. Previous work showed that the hemoglobin protein ­ which binds to and carries oxygen around mammalian bodies ­ of Steller's sea cows had a decreased affinity for oxygen, resulting in greater delivery of oxygen to organs and tissues. It was thought that this could be an adaptation to fuel heightened metabolic heat production in cold conditions. Studies of ancient DNA also identified the substitution of a single building block in the Steller's sea cow hemoglobin protein that is not present in other mammals and was suspected to underlie this modification. To determine how this unique substitution affects Steller's sea cow hemoglobin function ­ and whether it contributed to their ability to live in cold environments ­ Signore et al. generated hemoglobin proteins of Steller's sea cows, dugongs and Florida manatees. Testing their biochemical properties showed that this single exchange profoundly alters multiple aspects of how the Steller's sea cow hemoglobin works. Alongside reducing hemoglobin's oxygen affinity, the Steller's sea cow substitution also makes the protein more soluble, potentially increasing the level of hemoglobin within red blood cells. Additionally, it eliminates hemoglobin sensitivity to a molecule involved in oxygen binding ­ known as DPG ­ saving energy by no longer requiring production of this molecule. Furthermore, the same substitution makes hemoglobin less sensitive to changes in temperature, which would have helped to safeguard the delivery of oxygen to cool limbs and other extremities, reducing costly heat loss. Together, these changes in hemoglobin would have helped the Steller's sea cow to more efficiently transport oxygen around the body. Importantly, generating and testing Steller's sea cow pre-natal hemoglobins suggested this substitution may have also helped to enhance the fetal growth rate of these immense marine mammals by improving gas exchange between the mother and fetus. Signore et al. have revealed how a mutated form of hemoglobin allowed an extinct mammal to adapt to an extreme environment. Similar methods could be used to understand the physiological attributes of other extinct animals. In the future, this increased understanding of hemoglobin mutations could aid the development of human hemoglobin substitutes for therapeutic uses.

Identification of upper thermal thresholds during development in the endangered Nechako white sturgeon with management implications for a regulated river.

Climate change-induced warming effects are already evident in river ecosystems, and projected increases in temperature will continue to amplify stress on fish communities. In addition, many rivers globally are impacted by dams, which have many negative effects on fishes by altering flow, blocking fish passage, and changing sediment composition. However, in some systems, dams present an opportunity to manage river temperature through regulated releases of cooler water. For example, there is a government mandate for Kenney dam operators in the Nechako river, British Columbia, Canada, to maintain river temperature <20°C in July and August to protect migrating sockeye salmon (Oncorhynchus nerka). However, there is another endangered fish species inhabiting the same river, Nechako white sturgeon (Acipenser transmontanus), and it is unclear if these current temperature regulations, or timing of the regulations, are suitable for spawning and developing sturgeon. In this study, we aimed to identify upper thermal thresholds in white sturgeon embryos and larvae to investigate if exposure to current river temperatures are playing a role in recruitment failure. We incubated embryos and yolk-sac larvae in three environmentally relevant temperatures (14, 18 and 21°C) throughout development to identify thermal thresholds across different levels of biological organization. Our results demonstrate upper thermal thresholds at 21°C across physiological measurements in embryo and yolk-sac larvae white sturgeon. Before hatch, both embryo survival and metabolic rate were reduced at 21°C. After hatch, sublethal consequences continued at 21°C because larval sturgeon had decreased thermal plasticity and a dampened transcriptional response during development. In recent years, the Nechako river has reached 21°C by the end of June, and at this temperature, a decrease in sturgeon performance is evident in most of the traits measured. As such, the thermal thresholds identified here suggest current temperature regulations may not be suitable for developing white sturgeon and future recruitment.

An atlas of plasma-accessible carbonic anhydrase availability in the model teleost, the rainbow trout.

The unique teleost oxygenation system that permits enhanced oxygen unloading during stress comprises three main characteristics: pH-sensitive haemoglobin, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma-accessible carbonic anhydrase (paCA). A heterogeneous distribution of paCA is essential; its presence permits enhanced oxygen unloading during stress, while its absence at the gills maintains conditions for oxygen uptake by pH-sensitive haemoglobins. We hypothesised that paCA would be absent in all four gill arches, as has been previously indicated for arch two, and that paCA would be present in all other tissues. Through a suite of biochemical and molecular methods, we confirmed the absence of paCA from all four arches. We also found evidence for paCA in nine other tissues and a lack of paCA availability in the stomach. Expression was highly variable between tissues and suggests these differences may be associated with their respective metabolic activities. Additionally, we analysed the specific CA-IV isoform expressed within each tissue and showed almost complete separation of expression between tissues; CA-IVa was detected in the heart, brain, anterior intestine, and liver, whereas CA-IVb was detected in all intestine sections, pyloric caeca, kidney, and white muscle. This adds to a growing collection of work suggesting CA-IVa and b play divergent roles in gas exchange and ion/acid-base balance, respectively. The current study represents the first comprehensive atlas of paCA availability within the circulatory system of the model teleost, rainbow trout, and fills important gaps in our knowledge of this unique oxygenation system.

Do extreme postprandial levels of oxygen, carbon dioxide, and ammonia in the digestive tract equilibrate with the bloodstream in the freshwater rainbow trout (Oncorhynchus mykiss)?

The gastrointestinal tract (GIT) lumen of teleosts harbors extreme conditions, especially after feeding: high PCO2 (20-115 Torr), total ammonia (415-3710 µM), PNH3 (79-1760 µTorr in the intestine), and virtual anoxia (PO2 < 1 Torr). These levels could be dangerous if they were to equilibrate with the bloodstream. Thus, we investigated the potential equilibration of O2, CO2, and ammonia across the GIT epithelia in freshwater rainbow trout by monitoring postprandial arterial and venous blood gases in vivo and in situ. In vivo blood was sampled from the indwelling catheters in the dorsal aorta (DA) and subintestinal vein (SIV) draining the posterior intestine in the fasting state and at 4 to 48 h following catheter-feeding. To investigate possible ammonia absorption in the anterior part of the GIT, blood was sampled from the DA, SIV and hepatic portal vein (HPV) from anaesthetized fish in situ following voluntary feeding. We found minimal equilibration of all three gases between the GIT lumen and the SIV blood, with the latter maintaining pre-feeding levels (PO2 = 25-49 Torr, PCO2 = 6-8 Torr, and total ammonia = 117-134 µM and PNH3 = 13-30 µTorr at 48 h post-feeding). In contrast to the SIV, we found that the HPV total ammonia more than doubled 24 h after feeding (128 to 297 µM), indicative of absorption in the anterior GIT. Overall, the GIT epithelia of trout, although specialized for absorption, prevent dangerous levels of PO2, PCO2 and ammonia from equilibrating with the blood circulation.

Ammonia transport is independent of PNH3 gradients across the gastrointestinal epithelia of the rainbow trout: A role for the stomach.

Although the gastrointestinal tract (GIT) is an important site for nitrogen metabolism in teleosts, the mechanisms of ammonia absorption and transport remain to be elucidated. Both protein catabolism in the lumen and the metabolism of the GIT tissues produce ammonia which, in part, enters the portal blood through the anterior region of the GIT. The present study examined the possible roles of different GIT sections of rainbow trout (Oncorhynchus mykiss) in transporting ammonia in its unionized gas form-NH3 -by changing the PNH3 gradient across GIT epithelia using in vitro gut sac preparations. We also surveyed messenger RNA expression patterns of three of the identified Rh proteins (Rhbg, Rhcg1, and  Rhcg2) as potential NH3 transporters and NKCC as a potential ammonium ion (NH4 + ) transporter along the GIT of rainbow trout. We found that ammonia absorption is not dependent on the PNH3 gradient despite expression of Rhbg and Rhcg2 in the intestinal tissues, and Rhcg2 in the stomach. We detected no expression of Rhbg in the stomach and no expression of Rhcg1 in any GIT tissues. There was also a lack of correlation between ammonia transport and [NH4 + ] gradient despite NKCC expression in all GIT tissues. Regardless of PNH3 gradients, the stomach showed the greatest absorption and net tissue consumption of ammonia. Overall, our findings suggest nitrogen metabolism zonation of GIT, with stomach serving as an important site for the absorption, handling and transport of ammonia that is independent of the PNH3 gradient.

The effect of temperature on haemoglobin-oxygen binding affinity in regionally endothermic and ectothermic sharks.

Haemoglobin (Hb)-O2 binding affinity typically decreases with increasing temperature, but several species of ectothermic and regionally endothermic fishes exhibit reduced Hb thermal sensitivity. Regionally endothermic sharks, including the common thresher shark (Alopias vulpinus) and lamnid sharks such as the shortfin mako shark (Isurus oxyrinchus), can maintain select tissues and organs warmer than ambient temperature by retaining metabolic heat with vascular heat exchangers. In the ectothermic bigeye thresher shark (Alopias superciliosus), diurnal movements above and below the thermocline subject the tissues, including the blood, to a wide range of operating temperatures. Therefore, blood-O2 transport must occur across internal temperature gradients in regionally endothermic species, and over the range of environmental temperatures encountered by the ectothermic bigeye thresher shark. While previous studies have shown temperature-independent Hb-O2 affinity in lamnid sharks, including shortfin mako, the Hb-O2 affinity of the common and bigeye thresher sharks is unknown. Therefore, we examined the effect of temperature on whole-blood Hb-O2 affinity in common thresher shark and bigeye thresher shark. For comparison, analyses were also conducted on the shortfin mako shark and two ectothermic species, blue shark (Prionace glauca) and spiny dogfish (Squalus acanthias). Blood-O2 binding affinity was temperature independent for common thresher shark and shortfin mako shark, which should prevent internal temperature gradients from negatively affecting blood-O2 transport. Blue shark and spiny dogfish blood-O2 affinity decreased with increasing temperature, as expected, but bigeye thresher shark blood exhibited both a reduced temperature dependence and a high Hb-O2 affinity, which likely prevents large changes in environment temperature and low environmental oxygen from affecting O2 uptake.

Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

Temperature independence of haemoglobin-oxygen affinity in smalleye Pacific opah (Lampris incognitus) and swordfish (Xiphias gladius).

Smalleye Pacific opah and swordfish can conserve metabolic heat and maintain specific body regions warmer than ambient water temperature (i.e. regional heterothermy). Consequently, blood O2 uptake at the gills occurs at the environmental temperature at which the individual is found, but O2 offloading will occur at different temperatures in different tissues. While several regionally heterothermic fishes (e.g. billfishes, tunas and sharks) show a reduced temperature effect on haemoglobin (Hb)-O2 affinity, the temperature dependence of Hb-O2 affinity in opah and swordfish is unknown. We hypothesized that the Hb of opah and swordfish would also show a reduced temperature dependence. Opah whole-blood-O2 affinity exhibited a reverse temperature dependence above 50% Hb-O2 saturation (10-20°C, pH 7.2-8.0), while the temperature dependence of swordfish blood-O2 affinity (10-25°C) was saturation and pH dependent, becoming temperature independent below 50% Hb-O2 saturation and pH 7.4. Experiments on stripped haemolysates showed that adding ATP ([ATP]/[Hb]=30) decreased the temperature sensitivity of Hb-O2 affinity, changing the overall oxygenation enthalpy (ΔH') values of opah (10-20°C) and swordfish (10-25°C) Hbs at pH 7.4 from -15 and -42 kJ mol-1 O2, respectively, to +84 and -9 kJ mol-1 O2. Swordfish blood-O2 affinity was high compared with that of other large, pelagic, marine teleosts, which may be the result of unusually low ATP/Hb levels, but might also enable swordfish to forage in the potentially low-oxygenated water of the upper reaches of the oxygen minimum layer. The existence of Hbs with reduced temperature sensitivity in regionally heterothermic fishes may prevent marked changes in Hb-O2 affinity between the cold and warm tissues.

Air exposure moderates ocean acidification effects during embryonic development of intertidally spawning fish.

Ocean acidification can negatively impact the early life-stages of marine fish, due to energetic costs incurred by the maintenance of acid-base homeostasis, leaving less energy available for growth and development. The embryos of intertidally spawning fishes, such as Pacific herring, are often air exposed for hours. We hypothesized that air exposure would be beneficial to the developing embryo due to a higher oxygen availability (and thus reduced metabolic costs to secure adequate oxygen) and permitting excess CO2 associated with ocean acidification to be off-gassed during emersion. To investigate this, we reared Pacific herring (Clupea pallasii) embryos under three tidal regimes (subtidal: fully immersed, low intertidal: 2 × 2 h air exposure, and high intertidal: 5 + 9 h air exposure) fully crossed with three aquatic CO2 levels (400, 1500 and 3200 µatm) at a water temperature of 9.5 °C and naturally fluctuating air temperature during air exposure. We measured the effects on embryonic development and hatch, as well as carry-over effects on larval development and survival. Air exposure during embryonic development had significant positive effects on growth, condition and survival in larval Pacific herring, with some interactive effects with CO2. Interestingly, CO2 by itself in the fully immersed treatment had no effect, but had significant interactions with air exposure. Our research suggests that air exposure during low tide can be highly beneficial to intertidally spawning fishes and needs to be taken into account in climate change studies and modeling.