Ryan B. Shartau

Acid–base and ion balance in fishes with bimodal respiration

The evolution of air breathing during the Devonian provided early fishes with bimodal respiration with a stable O2 supply from air. This was, however, probably associated with challenges and trade-offs in terms of acid-base balance and ionoregulation due to reduced gill:water interaction and changes in gill morphology associated with air breathing. While many aspects of acid-base and ionoregulation in air-breathing fishes are similar to water breathers, the specific cellular and molecular mechanisms involved remain largely unstudied. In general, reduced ionic permeability appears to be an important adaptation in the few bimodal fishes investigated but it is not known if this is a general characteristic. The kidney appears to play an important role in minimizing ion loss to the freshwater environment in the few species investigated, and while ion uptake across the gut is probably important, it has been largely unexplored. In general, air breathing in facultative air-breathing fishes is associated with an acid-base disturbance, resulting in an increased partial pressure of arterial CO2 and a reduction in extracellular pH (pHE ); however, several fishes appear to be capable of tightly regulating tissue intracellular pH (pHI ), despite a large sustained reduction in pHE , a trait termed preferential pHI regulation. Further studies are needed to determine whether preferential pHI regulation is a general trait among bimodal fishes and if this confers reduced sensitivity to acid-base disturbances, including those induced by hypercarbia, exhaustive exercise and hypoxia or anoxia. Additionally, elucidating the cellular and molecular mechanisms may yield insight into whether preferential pHI regulation is a trait ultimately associated with the early evolution of air breathing in vertebrates.

Validation of the i-STAT system for the analysis of blood parameters in fish

The i-STAT system, a portable clinical analyzer, is increasingly being used to assess blood parameters in fish. This study validated the i-STAT system on rainbow trout blood under a broad range of conditions. Results indicate that the i-STAT is not an appropriate tool for assessing most blood parameters in rainbow trout.

Rotational behaviour of encapsulated pond snail embryos in diverse natural environments

SUMMARY Encapsulated freshwater pond snail embryos display a cilia-driven rotation behaviour that is stimulated by artificially induced hypoxia. Previous studies have suggested that the mixing effect of this behaviour causes enhanced oxygen delivery to embryos within their egg capsules. Despite extensive laboratory-based studies describing this behaviour, it is unclear how this behaviour is used to cope with changes in oxygen concentration and other environmental factors in natural water bodies. We made field measurements of embryo rotation rates in laboratory-reared Helisoma trivolvis embryos placed in ponds of different trophic levels that ranged geographically from the southern Alberta prairie to the Rocky Mountains. Abiotic factors including temperature, pH, conductivity and water oxygen concentration were measured to understand how embryonic rotation is influenced by environmental conditions. Results showed that H. trivolvis embryos exhibit differences in rotational behaviour depending on the environmental conditions. Temperature and oxygen concentration were the primary factors significantly affecting rotation rates. The effect of oxygen concentration on rotation rates was not as widespread as observed under laboratory conditions, probably because the measured oxygen concentrations were above the range that influences embryonic rotation in the laboratory. The rotational behaviour of laboratory-reared Lymnaea stagnalis provided confirmation that embryos of other encapsulated pulmonates exhibit a similar rotational response in natural environments. These results suggest that embryo rotation is influenced by a complex interplay of environmental factors.

Serotonin prolongs survival of encapsulated pond snail embryos exposed to long-term anoxia.

SUMMARY Embryos of the pond snail, Helisoma trivolvis, develop bilateral serotonergic neurons that innervate ciliary bands and stimulate cilia-driven rotation. This behaviour is postulated to increase oxygen availability during hypoxia by mixing the capsular fluid. We hypothesised that the stimulation of ciliary-driven rotation by serotonin (5-HT) enhances the survival of embryos during prolonged hypoxia. Embryo rotation and survival were monitored in different levels of oxygen for 24–48 h while in the presence or absence of 5-HT (100 μmol l−1) or a 5-HT antagonist (50 μmol l−1). Long-term hypoxia caused delayed embryonic development that appeared morphologically normal. Hypoxia also induced a transient increase in rotation rate in embryos exposed to artificial pond water (APW) or 5-HT that lasted around 3 h. 5-HT-treated embryos had an elevated rotation rate over embryos in APW throughout the long-term exposure to hypoxia. Long-term anoxia also induced a transient increase in rotation rate in embryos exposed to APW or 5-HT. Rotation ceased in embryos exposed to APW by 13 h but persisted in 5-HT-treated embryos for up to 40 h. Fifty percent mortality was reached at 9 h of anoxia in embryos in APW and at 24 h in 5-HT-treated embryos. The 5-HT antagonist mianserin partially inhibited the 5-HT enhancement of rotation but not the prolongation of survival in anoxia. The ability of 5-HT to prolong survival in anoxia reveals a 5-HT-activated metabolic pathway that liberates an alternative energy source.

Preferential intracellular pH regulation: hypotheses and perspectives

ABSTRACT The regulation of vertebrate acid–base balance during acute episodes of elevated internal PCO2 is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as ‘coupled pH regulation’. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe. Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid–base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid–base homeostasis during severe acute acid–base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid–base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land. Summary: Preferential intracellular pH regulation confers exceptional tolerance to a severe acute respiratory acidosis. This trait may represent a basal pattern of acid–base regulation used by developing vertebrates that is lost or retained in adults.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid–base challenges

ABSTRACT The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2 and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid–base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid–base homeostasis during development of amniotes, and vertebrates in general. Summary: Embryonic turtles preferentially regulate tissue pH in the absence of blood pH regulation during acid–base disturbances. This pattern of acid–base regulation has never been observed before in amniotes.

Characterization of Na+ transport to gain insight into the mechanism of acid-base and ion regulation in white sturgeon (Acipenser transmontanus)

Freshwater fish actively take up ions via specific transporters to counter diffusive losses to their hypotonic environment. While much is known about the specific mechanisms employed by teleosts, almost nothing is known about the basal fishes, such as white sturgeon (Acipenser transmontanus) which may offer insight into the evolution of osmo- and ionoregulation in fishes. We investigated Na+ uptake in juvenile white sturgeon in the presence and absence of transporter inhibitors. We found that sturgeon acclimated to 100μmoll-1 Na+ have Na+ uptake kinetics typical of teleosts and that a Na+/H+ exchanger (NHE) is the predominant transporter for Na+ uptake. White sturgeon are tolerant to hypercarbia-induced respiratory acidoses and recover blood pH (pHe) at 1.5kPa PCO2 but not at higher PCO2 (6kPa PCO2) where they preferentially regulate intracellular pH (pHi). It was hypothesized that during exposure to hypercarbia Na+ uptake would increase at CO2 tensions at which fish were capable of pHe regulation but decrease at higher tensions when they were preferentially regulating pHi. We found that Na+ uptake did not increase at 1.5kPa PCO2, but at 6kPa PCO2 Na+ uptake was reduced by 95% while low water pH equivalent to 6kPa PCO2 reduced Na+ uptake by 71%. Lastly, we measured net acid flux during hypercarbia, which indicates that net acid flux is not associated with Na+ uptake. These findings indicate Na+ uptake in sturgeon is not different from freshwater teleosts but is sensitive to hypercarbia and is not associated with pHe compensation during hypercarbia.

White sturgeon (Acipenser transmontanus) acid–base regulation differs in response to different types of acidoses

White sturgeon (Acipenser transmontanus) completely protect intracellular tissue pH (pHi) despite large reductions in extracellular (blood) pH (pHe), termed preferential pHi regulation, in response to elevated environmental PCO2 (hypercarbia) and in general appear to be relatively resilient to stressors. Preferential pHi regulation is thought to be associated with hypercarbia tolerance in general, but has also recently been observed to protect pHi against metabolic acidoses induced by exhaustive exercise and anoxia in a tropical air breathing catfish. We hypothesized that preferential pHi regulation may also be a general strategy of acid–base regulation in sturgeon. To address this hypothesis, severe acidoses were imposed to reduce pHe, and the presence or absence of preferential pHi regulation was assessed in red blood cells (RBC), heart, brain, liver and white muscle. A respiratory acidosis was imposed using hyperoxia, while metabolic acidoses were induced by exhaustive exercise, anoxia or air exposure. Reductions in pHe occurred following hyperoxia (0.15 units), exhaustive exercise (0.30 units), anoxia (0.10 units) and air exposure (0.35 units); all acidoses reduced RBC pHi. Following hyperoxia, heart, brain and liver pHi were preferentially regulated against the reduction in pHe, similar to hypercarbia exposure. Following all metabolic acidoses heart pHi was protected and brain pHi remained unchanged following exhaustive exercise and air exposure, however, brain pHi was reduced following anoxia. Liver and white muscle pHi were reduced following all metabolic acidoses. These results suggest preferential pHi regulation may be a general strategy during respiratory acidoses but during metabolic acidoses, the response differs between source of acidoses and tissues.

Water pH limits extracellular but not intracellular pH compensation in the CO2 tolerant freshwater fish, Pangasianodon hypophthalmus

ABSTRACT Preferentially regulating intracellular pH (pHi) confers exceptional CO2 tolerance on fish, but is often associated with reductions in extracellular pH (pHe) compensation. It is unknown whether these reductions are due to intrinsically lower capacities for pHe compensation, hypercarbia-induced reductions in water pH or other factors. To test how water pH affects capacities and strategies for pH compensation, we exposed the CO2-tolerant fish Pangasianodon hypophthalmus to 3 kPa PCO2 for 20 h at an ecologically relevant water pH of 4.5 or 5.8. Brain, heart and liver pHi was preferentially regulated in both treatments. However, blood pHe compensation was severely reduced at water pH 4.5 but not 5.8. This suggests that low water pH limits acute pHe but not pHi compensation in fishes preferentially regulating pHi. Hypercarbia-induced reductions in water pH might therefore underlie the unexplained reductions to pHe compensation in fishes preferentially regulating pHi, and may increase selection for preferential pHi regulation. Summary: Low water pH limits extracellular pH compensation in a CO2-tolerant fish. This may increase selection for a more robust CO2 defence strategy where intracellular pH is preferentially regulated.