Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes

We investigated whether fatigue from sustained aerobic swimming provides a sub-lethal endpoint to define tolerance of acute warming in fishes, as an alternative to loss of equilibrium (LOE) during a critical thermal maximum (CTmax) protocol. Two species were studied, Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus). Each fish underwent an incremental swim test to determine gait transition speed (UGT), where it first engaged the unsteady anaerobic swimming mode that preceded fatigue. After suitable recovery, each fish was exercised at 85% of their own UGT and warmed 1°C every 30 min, to identify the temperature at which they fatigued, denoted as CTswim. Fish were also submitted to a standard CTmax, warming at the same rate as CTswim, under static conditions until LOE. All individuals fatigued in CTswim, at a mean temperature approximately 2°C lower than their CTmax. Therefore, if exposed to acute warming in the wild, the ability to perform aerobic metabolic work would be constrained at temperatures significantly below those that directly threatened survival. The collapse in performance at CTswim was preceded by a gait transition qualitatively indistinguishable from that during the incremental swim test. This suggests that fatigue in CTswim was linked to an inability to meet the tissue oxygen demands of exercise plus warming. This is consistent with the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, regarding the mechanism underlying tolerance of warming in fishes. Overall, fatigue at CTswim provides an ecologically relevant sub-lethal threshold that is more sensitive to extreme events than LOE at CTmax

Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus

Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy

Renal plasticity in response to feeding in the Burmese python, Python molurus bivittatus

Burmese pythons are sit-and-wait predators that are well adapted to go long periods without food, yet subsequently consume and digest single meals that can exceed their body weight. These large feeding events result in a dramatic alkaline tide that is compensated by a hypoventilatory response that normalizes plasma pH; however, little is known regarding how plasma HCO3− is lowered in the days post-feeding. The current study demonstrated that Burmese pythons contain the cellular machinery for renal acid–base compensation and actively remodel the kidney to limit HCO3− reabsorption in the post-feeding period. After being fed a 25% body weight meal plasma total CO2 was elevated by 1.5-fold after 1day, but returned to control concentrations by 4days post-feeding (dpf). Gene expression analysis was used to verify the presence of carbonic anhydrase (CA) II, IV and XIII, Na+ H+ exchanger 3 (NHE3), the Na+ HCO3− co-transporter (NBC) and V-type ATPase. CA IV expression was significantly down-regulated at 3dpf versus fasted controls. This was supported by activity analysis that showed a significant decrease in the amount of GPI-linked CA activity in isolated kidney membranes at 3dpf versus fasted controls. In addition, V-type ATPase activity was significantly up-regulated at 3dpf; no change in gene expression was observed. Both CA II and NHE3 expression was up-regulated at 3dpf, which may be related to post-prandial ion balance. These results suggest that Burmese pythons actively remodel their kidney after feeding, which would in part benefit renal HCO3− clearance

Multi-linear regression models predict the effects of water chemistry on acute lead toxicity to Ceriodaphnia dubia and Pimephales promelas

The current study examined the acute toxicity of lead (Pb) to Ceriodaphnia dubia and Pimephales promelas in a variety of natural waters. The natural waters were selected to range in pertinent water chemistry parameters such as calcium, pH, total CO2 and dissolved organic carbon (DOC). Acute toxicity was determined for C. dubia and P. promelas using standard 48h and 96h protocols, respectively. For both organisms acute toxicity varied markedly according to water chemistry, with C. dubia LC50s ranging from 29 to 180μg/L and P. promelas LC50s ranging from 41 to 3598μg/L. Additionally, no Pb toxicity was observed for P. promelas in three alkaline natural waters. With respect to water chemistry parameters, DOC had the strongest protective impact for both organisms. A multi-linear regression (MLR) approach combining previous lab data and the current data was used to identify the relative importance of individual water chemistry components in predicting acute Pb toxicity for both species. As anticipated, the P. promelas best-fit MLR model combined DOC, calcium and pH. Unexpectedly, in the C. dubiaMLR model the importance of pH, TCO2 and calcium was minimal while DOC and ionic strength were the controlling water quality variables. Adjusted R2 values of 0.82 and 0.64 for the P. promelas and C. dubia models, respectively, are comparable to previously developed biotic ligand models for other metals

Implications of pH manipulation methods for metal toxicity: Not all acidic environments are created equal

The toxicity of many metals is impacted by environmental pH, through both competition and complexation by hydroxide and carbonate ions. To establish safe environmental regulation it is important to properly define the relationship between pH and metal toxicity, a process that involves manipulating the pH of test water in the lab. The current study compares the effects of the three most common pH manipulation methods (carbon dioxide, acid–base addition, and chemical buffers) on acute Pb toxicity of a model fish species, Pimephales promelas. Acidification of test water revealed that the Pb and Pb2+ LC50 values were impacted by the pH manipulation method, with the following order of effects: HCl<CO2<MOPS. Conversely no differences in toxicity were observed when test pH was alkalinized using MOPS or NaOH. The different impacts of pH manipulation methods on Pb toxicity are likely due to different physiological stresses resulting from the respective methods; the physiological implications of each method are discussed. The results suggest that when studying the impacts of pH on metal toxicity it is important to properly replicate the ambient conditions of interest as artificial buffering using CO2 environments or organic buffers significantly affects the physiology of the test organisms above and beyond what is expected from pH alone. Thus, using CO2 and organic buffers overestimates the impact of acid pH on Pb toxicity

Mechanisms of acid-base regulation in the African lungfish Protopterus annectens

10.1242/jeb.02776Journal of Experimental Biology210111944-1959JEBI