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.

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.

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 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.

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.

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.

Characterizing the hypoxic performance of a fish using a new metric: PAAS-50.

The hypoxic constraint on peak oxygen uptake (MO2,peak) was characterized in rainbow trout over a range of ambient oxygen tensions with different testing protocols and statistical models. The best-fit model was selected using both statistical criteria (R2 and AIC) and the model's prediction of three anchor points for hypoxic performance: critical PO2 (Pcrit), maximum MO2 and a new metric, the minimum PO2 that supports 50% of absolute aerobic scope (PAAS-50). The best-fitting model was curvilinear using five strategically selected PO2 values. This model predicted PAAS-50 as 70 mmHg (coefficient of variation, CV=9%) for rainbow trout. Thus, while a five-point hypoxic performance curve can characterize the limiting effects of hypoxia in fish, as envisaged by Fry over 75 years ago, PAAS-50 is a promising metric to compare hypoxic constraints on performance in a standardized manner both within and across fish species.

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.

Post-prandial respiratory gas and acid-base profiles in the gastrointestinal tract and its venous drainage in freshwater rainbow trout (Oncorhynchus mykiss) and seawater English sole (Parophrys vetulus).

The basic respiratory gas and acid-base conditions inside the lumen of the gastrointestinal tract (GIT) and blood draining the tract are largely unestablished in teleost fishes after feeding, though there have been some recent novel discoveries on freshwater rainbow trout (Oncorhynchus mykiss) and seawater English sole (Parophrys vetulus). The present study examined in greater detail the gas (PO2, PCO2, PNH3) and acid-base profiles (pH, [HCO3-], total [ammonia]) in the lumen of the stomach, the anterior, mid, and posterior intestine, as well as the venous drainage (subintestinal and/or hepatic portal vein) of the GIT in these two species 20 h post-feeding. Both species had high PCO2, PNH3, and total [ammonia], and low PO2 (virtual anoxia) in the lumens throughout all sections of the GIT, and high [HCO3-] in the intestine. Total [ammonia], PNH3, and [HCO3-] increased from anterior to posterior intestine in both species. P. vetulus had higher intestinal total [ammonia] and lower [HCO3-] than O. mykiss post feeding, but total [ammonia] was much higher in the stomach of O. mykiss. Despite the extreme conditions in the lumen, both arterial and venous blood showed relatively lower PCO2, total [ammonia] and higher PO2, implying limited equilibration between the two compartments. The higher [HCO3-] and lower total [ammonia] in the intestinal lumen of the freshwater O. mykiss than the seawater P. vetulus suggest the need for future comparative studies using conspecifics fed identical diets but acclimated to the two different salinities in order to understand the potential role of environmental salinity and associated osmoregulatory processes underlying these differences.

Continuous light (relative to a 12:12 photoperiod) has no effect on anxiety-like behaviour, boldness, and locomotion in coho salmon (Oncorhynchus kisutch) post-smolts in recirculating aquaculture systems at a salinity of either 2.5 or 10 ppt.

There is increased interest in rearing salmon in Recirculating Aquaculture Systems (RAS), where environmental conditions can be tightly controlled to optimize growth. Photoperiod and salinity are two important parameters that can be manipulated in RAS. A longer photoperiod permits more time for feeding, while intermediate salinities may reduce the energetic costs of ionoregulation, both of which may enhance growth. However, little is known about how rearing at different photoperiods and salinity affect behaviour, an understudied but important research topic for intensive fish rearing. To address this, we examined the behavioural effects of two salinities and two photoperiod regimes in coho salmon (Oncorhynchus kisutch) post-smolts reared continuously for 120 days in a RAS. Fish were reared on a photoperiod of either 12 h light:12 h dark (12:12), or 24 h light (24:0) at salinities of 2.5 and 10 ppt. To investigate behavioural differences associated with these treatments, we quantified: i) movement in an open-field test, ii) exploratory behaviour/boldness using a novel object approach test, and iii) anxiety-like behaviour with a light/dark test. The 24:0 groups displayed no differences in boldness/anxiety-like behaviour and locomotion relative to the 12:12 groups at their respective salinities. Taken together, fish reared under continuous light (24:0) show negligible behavioural alterations compared to fish reared under normal light dark conditions (12:12).

Enhanced oxygen unloading in two marine percomorph teleosts.

Teleost fishes are diverse and successful, comprising almost half of all extant vertebrate species. It has been suggested that their success as a group is related, in part, to their unique O2 transport system, which includes pH-sensitive hemoglobin, a red blood cell ß-adrenergic Na+/H+ exchanger (RBC ß-NHE) that protects red blood cell pH, and plasma accessible carbonic anhydrase which is absent at the gills but present in some tissues, that short-circuits the ß-NHE to enhance O2 unloading during periods of stress. However, direct support for this has only been examined in a few species of salmonids. Here, we expand the knowledge of this system to two warm-water, highly active marine percomorph fish, cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). We show evidence for RBC ß-NHE activity in both species, and characterize the Hb-O2 transport system in one of those species, cobia. We found significant RBC swelling following ß-adrenergic stimulation in both species, providing evidence for the presence of a rapid, active RBC ß-NHE in both cobia and mahi-mahi, with a time-course similar to that of salmonids. We generated oxygen equilibrium curves (OECs) for cobia blood and determined the P50, Hill, and Bohr coefficients, and used these data to model the potential for enhanced O2 unloading. We determined that there was potential for up to a 61% increase in O2 unloading associated with RBC ß-NHE short-circuiting, assuming a - 0.2 ∆pHa-v in the blood. Thus, despite phylogenetic and life history differences between cobia and the salmonids, we found few differences between their Hb-O2 transport systems, suggesting conservation of this physiological trait across diverse teleost taxa.

Innate antiviral defense demonstrates high energetic efficiency in a bony fish.

BACKGROUND: Viruses can impose energetic demands on organisms they infect, in part by hosts mounting resistance. Recognizing that oxygen uptake reliably indicates steady-state energy consumption in all vertebrates, we comprehensively evaluated oxygen uptake and select transcriptomic messaging in sockeye salmon challenged with either a virulent rhabdovirus (IHNV) or a low-virulent reovirus (PRV). We tested three hypotheses relating to the energetic costs of viral resistance and tolerance in this vertebrate system: (1) mounting resistance incurs a metabolic cost or limitation, (2) induction of the innate antiviral interferon system compromises homeostasis, and (3) antiviral defenses are weakened by acute stress. RESULTS: IHNV infections either produced mortality within 1-4 weeks or the survivors cleared infections within 1-9 weeks. Transcription of three interferon-stimulated genes (ISGs) was strongly correlated with IHNV load but not respiratory performance. Instead, early IHNV resistance was associated with a mean 19% (95% CI = 7-31%; p = 0.003) reduction in standard metabolic rate. The stress of exhaustive exercise did not increase IHNV transcript loads, but elevated host inflammatory transcriptional signaling up to sevenfold. For PRV, sockeye tolerated high-load systemic PRV blood infections. ISG transcription was transiently induced at peak PRV loads without associated morbidity, microscopic lesions, or major changes in aerobic or anaerobic respiratory performance, but some individuals with high-load blood infections experienced a transient, minor reduction in hemoglobin concentration and increased duration of excess post-exercise oxygen consumption. CONCLUSIONS: Contrary to our first hypothesis, effective resistance against life-threatening rhabdovirus infections or tolerance to high-load reovirus infections incurred minimal metabolic costs to salmon. Even robust systemic activation of the interferon system did not levy an allostatic load sufficient to compromise host homeostasis or respiratory performance, rejecting our second hypothesis that this ancient innate vertebrate antiviral defense is itself energetically expensive. Lastly, an acute stress experienced during testing did not weaken host antiviral defenses sufficiently to promote viral replication; however, a possibility for disease intensification contingent upon underlying inflammation was indicated. These data cumulatively demonstrate that fundamental innate vertebrate defense strategies against potentially life-threatening viral exposure impose limited putative costs on concurrent aerobic or energetic demands of the organism.

Teleost red blood cells actively enhance the passive diffusion of oxygen that was discovered by August Krogh.

In the early 20th century, August and Marie Krogh settled one of the most controversial questions in physiology, showing through elegant experiments that oxygen (O2) uptake at the lung is driven by passive diffusion alone. Krogh's later work, on the regulation of local blood flow and capillary recruitment at the tissues, was awarded with the Nobel Prize in 1920. A century later it is still undisputed that O2 moves across tissues by diffusion, however, animals use active mechanisms to regulate and facilitate the passive process. Teleost fishes have evolved a mechanism by which adrenergic sodium-proton-exchangers (ß-NHEs) on the red blood cell (RBC) membrane actively create H+ gradients that are short-circuited in the presence of plasma-accessible carbonic anhydrase (CA) at the tissue capillaries. The rapid acidification of the RBC reduces the O2 affinity of pH-sensitive haemoglobin, which increases the O2 diffusion gradient to the tissues. When RBCs leave the site of plasma-accessible CA, ß-NHE activity recovers RBC pH during venous transit, to promote renewed O2 loading at the gills. This mechanism allows teleosts to unload more O2 at their tissues without compromising O2 diffusion gradients and therefore, to use the available O2 carrying capacity of the blood to a greater degree. In Atlantic salmon, ß-NHE short-circuiting reduces the requirements on the heart by up to 30% during moderate exercise and even at rest, with important ecological implications. Thus, in some teleosts, the RBCs participate in regulating the systemic O2 flux by actively altering the passive diffusion of O2 that Krogh discovered.

Thermal tolerance and hypoxia tolerance are associated in blacktip reef shark (Carcharhinus melanopterus) neonates.

Thermal dependence of growth and metabolism can influence thermal preference and tolerance in marine ectotherms, including threatened and data-deficient species. Here, we quantified the thermal dependence of physiological performance in neonates of a tropical shark species (blacktip reef shark, Carcharhinus melanopterus) from shallow, nearshore habitats. We measured minimum and maximum oxygen uptake rates (MO2 ), calculated aerobic scope, excess post-exercise oxygen consumption and recovery from exercise, and measured critical thermal maxima (CTmax), thermal safety margins, hypoxia tolerance, specific growth rates, body condition and food conversion efficiencies at two ecologically relevant acclimation temperatures (28 and 31°C). Owing to high post-exercise mortality, a third acclimation temperature (33°C) was not investigated further. Acclimation temperature did not affect MO2  or growth, but CTmax and hypoxia tolerance were greatest at 31°C and positively associated. We also quantified in vitro temperature (25, 30 and 35°C) and pH effects on haemoglobin-oxygen (Hb-O2) affinity of wild-caught, non-acclimated sharks. As expected, Hb-O2 affinity decreased with increasing temperatures, but pH effects observed at 30°C were absent at 25 and 35°C. Finally, we logged body temperatures of free-ranging sharks and determined that C. melanopterus neonates avoided 31°C in situ We conclude that C. melanopterus neonates demonstrate minimal thermal dependence of whole-organism physiological performance across a seasonal temperature range and may use behaviour to avoid unfavourable environmental temperatures. The association between thermal tolerance and hypoxia tolerance suggests a common mechanism warranting further investigation. Future research should explore the consequences of ocean warming, especially in nearshore, tropical species.

The gaseous gastrointestinal tract of a seawater teleost, the English sole (Parophrys vetulus).

There has been considerable recent progress in understanding the respiratory physiology of the gastrointestinal tract (GIT) in teleosts, but the respiratory conditions inside the GIT remain largely unknown, particularly the luminal PCO2 and PO2 levels. The GIT of seawater teleosts is of special interest due to its additional function of water absorption linked to HCO3- secretion, a process that may raise luminal PCO2 levels. Direct measurements of GIT PCO2 and PO2 using micro-optodes in the English sole (Parophrys vetulus; anaesthetized, artificially ventilated, 10-12 °C) revealed extreme luminal gas levels. Luminal PCO2 was 14-17 mmHg in the stomach and intestinal segments of fasted sole, considerably higher than arterial blood levels of 5 mmHg. Moreover, feeding, which raised intestinal HCO3- concentration, also raised luminal PCO2 to 34-50 mmHg. All these values were higher than comparable measurements in freshwater teleosts, and also greater than environmental CO2 levels of concern in aquaculture or global change scenarios. The PCO2 values in subintestinal vein blood draining the GIT of fed fish (28 mmHg) suggested some degree of equilibration with high luminal PCO2, whereas subintestinal vein PO2 levels were relatively low (9 mmHg). All luminal sections of the GIT were virtually anoxic (PO2 ≤ 0.3 mmHg), in both fasted and fed animals, a novel finding in teleosts.

Ionome and elemental transport kinetics shaped by parallel evolution in threespine stickleback.

Evidence that organisms evolve rapidly enough to alter ecological dynamics necessitates investigation of the reciprocal links between ecology and evolution. Data that link genotype to phenotype to ecology are needed to understand both the process and ecological consequences of rapid evolution. Here, we quantified the suite of elements in individuals (i.e., ionome) and differences in the fluxes of key nutrients across populations of threespine stickleback. We find that allelic variation associated with freshwater adaptation that controls bony plating is associated with changes in the ionome and nutrient recycling. More broadly, we find that adaptation of marine stickleback to freshwater conditions shifts the ionomes of natural populations and populations raised in common gardens. In both cases ionomic divergence between populations was primarily driven by differences in trace elements rather than elements typically associated with bone. These findings demonstrate the utility of ecological stoichiometry and the importance of ionome-wide data in understanding eco-evolutionary dynamics.

Limits and patterns of acid-base regulation during elevated environmental CO2 in fish.

Aquatic CO2 tensions may exceed 30-60 Torr (ca. 30,000-79,000 µatm, respectively; hypercarbia) in some environments inducing severe acid-base challenges in fish. Typically, during exposure to hypercarbia blood pH (pHe) is initially reduced and then compensated in association with an increase in plasma HCO3- in exchange for Cl-. Typically, intracellular pH (pHi) is reduced and recovery is to some degree coupled to pHe recovery (coupled pH regulation). However, during acute hypercarbia, pHe recovery has been proposed to be limited by an "apparent upper bicarbonate threshold", restricting complete pHe recovery to below 15 Torr PCO2. At PCO2 values beyond that which fish can compensate pHe, some fish are able to fully protect pHi despite large sustained reductions in pHe (preferential pHi regulation) and can tolerate PCO2 > 45 Torr. This review discusses pHe and pHi regulation during exposure to hypercarbia starting with modeling the capacity and theoretical limit to pHe compensation in 19 studies. Next, we discuss how fish compensate severe acute hypercarbia exposures beyond the putative limit of pHe compensation using preferential pHi regulation which has recently been observed to be common among fish subjected to severe hypercarbia. Finally, we consider the evolution of pH regulatory strategies in vertebrates, including how the presence of preferential pHi regulation in embryonic reptiles may indicate that it is an embryonic trait that is either lost or retained in adult vertebrates and may have served as an exaptation for key evolutionary transitions during vertebrate evolution.

The effect of temperature acclimation on thermal tolerance, hypoxia tolerance and aerobic scope in two subspecies of sheepshead minnow; Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi.

The freshwater teleost Cyprinodon variegatus hubbsi (Cvh) diverged from its euryhaline relative Cyprinodon variegatus variegatus (Cvv) ~150 kya and these subspecies are physiologically distinct in their osmoregulatory capabilities. Cvv inhabits intertidal estuaries and saltwater marshes along the Gulf of Mexico and Atlantic coast, where they experience a broad temperature range from -1.9 to 43 °C and frequent bouts of hypoxia. In contrast, Cvh lives in several lakes in central Florida, where temperature is more stable (12-31 °C) and hypoxia is uncommon. To assess whether relaxed selective pressure on Cvh has resulted in reduced temperature and hypoxia tolerance, a comparative study on the effects of acclimation to 25, 30 and 35 °C on critical thermal tolerance (CTMax), hypoxia tolerance, and aerobic scope was performed. The CTMax was similar between subspecies and positively correlated with acclimation temperature. Neither subspecies, however, could survive at 38 °C for a prolonged period of time. In general, Cvv displayed greater hypoxia tolerance and aerobic scope relative to Cvh over the range of acclimation temperatures. Routine metabolic rate was significantly lower while maximum metabolic rate and aerobic scope were significantly higher in Cvv, but only in fish acclimated to 30 °C. Overall, the different responses of Cvh to relaxed selective pressure suggest these traits are weakly linked physiologically in these fishes.

The effects of salinity and photoperiod on aerobic scope, hypoxia tolerance and swimming performance of coho salmon (Oncorhynchus kisutch) reared in recirculating aquaculture systems.

Land-based recirculating aquaculture systems (RAS) have been used to rear salmon from smolt to market-sized adults, but high operating costs have limited their wide spread adoption. One clear advantage of using RAS for salmon aquaculture over open net pens is that fish can be reared under optimal conditions in an attempt to maximize growth and physiological performance and reduce overall production costs. However, few studies have attempted to define the optimal conditions for the long-term rearing of salmon. Thus, the goal of this study is to determine the effects of salinity and photoperiod, two factors that can be easily manipulated in RAS, on the physiological performance of coho salmon (Oncorhynchus kisutch) during long-term rearing. To address this goal, post-smolt coho salmon were reared for 150 days in replicate RAS at 2.5, 5, 10 and 30 ppt under either 12:12 and 24:0 (light:dark) photoperiods. Routine metabolic rate, maximum metabolic rate, aerobic scope and hypoxia tolerance were measured at 60 and 120 days of rearing, while swimming performance was assessed at 60 and 150 days of rearing. There were no effects of salinity or photoperiod on metabolic rate measurements, hypoxia tolerance or swimming performance at any sampling time. There were, however, significant effects of salinity and photoperiod on post-swimming hematology. The results suggest that physiological disturbances continue to manifest due to different environmental conditions, despite acclimation, but do not hinder the animal's ability to cope with physiological stressors. Overall, rearing salinity and photoperiod had very few measurable effects on the physiology and performance of coho salmon except the ionoregulatory disturbances following swimming at salinities of 2.5 and 30 ppt.

The effect of salinity and photoperiod on thermal tolerance of Atlantic and coho salmon reared from smolt to adult in recirculating aquaculture systems.

Land-based, closed containment salmon aquaculture involves rearing salmon from smolt to adult in recirculating aquaculture systems (RAS). Unlike in open-net pen aquaculture, rearing conditions can be specified in RAS in order to optimize growth and physiological stress tolerance. The environmental conditions that yield optimal stress tolerance in salmon are, however, unknown. To address this knowledge gap, we reared Atlantic (Salmo salar) and coho (Oncorhynchus kisutch) salmon in 7 separate RASs for 400 days post-smoltification under 2 photoperiods (24:0 or 12:12, light:dark) and 4 salinities (2.5, 5, 10 or 30 ppt.) and assessed the effects of these conditions on thermal tolerance. We found that over the first 120 days post-smoltification, rearing coho under a 24:0 photoperiod resulted in a ~2 °C lower critical thermal maxima (CTmax) than in coho reared under a 12:12 photoperiod. This photoperiod effect did not persist at 200 and 400 days, which was coincident with an overall decrease in CTmax in coho. Finally, Atlantic salmon had a higher CTmax (~28 °C) compared to coho (~26 °C) at 400 days post-smoltification. Overall, these findings are important for the future implications of RAS and for the aquaculture industry to help identify physiologically sensitive time stages.