This may suggest a primary functional role of this particular group of subunits, while other subunits offer specialized or redundant functions. The P 50 of red drum non-stripped hemolysates fell within the range typically found in teleosts in both normoxia and hypoxia treatments; however, prolonged hypoxia acclimation was associated with a significantly lower non-stripped hemolysate P 50 i.
The greatest reduction was observed at lower pH; however, the interaction between P 50 and pH was not statistically significant. The Bohr coefficient for Hb was also not impacted by hypoxia acclimation for either pH range. While our data were derived from non-stripped lysates, to preserve any hypoxia driven changes in NTP content, the magnitude of observed changes between studies is comparable, with Rutjes et al. But it is important to remember that P 50 values from hemolysates are generally lower than intact red cells This is owed in part to dilution of hemoglobin, NTP and other allosteric modifiers, although the ratio of these parameters remained consistent.
Nonetheless, these data give strong evidence that hypoxia acclimation results in an increased blood P 50 in red drum, which should increase the ability of these animals to extract oxygen from the environment. A number of factors are known to impact oxygen affinity in fish. These include pH, temperature and Hb concentration, as well as allosteric modifiers such as NTPs 8 , Unfortunately, limitations on sample volume precluded measurement of NTP concentration in our experiment.
We instead chose to explore the hypothesis that differential regulation of specific Hb components may coincide with increased oxygen affinity during hypoxia acclimation. It has been previously hypothesized that species with multiple Hbs acting in concert would be better suited to tolerate environmental perturbation 1 , 30 , 31 , 32 ; however, the physiological support for this is mixed 6 , 7 , 10 , But previous work has primarily focused on temperature, with relatively less work focusing on prolonged hypoxia acclimation 3 , 5 , 33 , Here we provide evidence that specific Hb isoforms are selectively up-regulated in response to hypoxia stress, which is also correlated with a decrease in hemolysate P It is tempting to suggest that the selective up-regulation is promoting an isoHb with higher oxygen affinity, but this cannot be concluded without additional studies that involve purification and protein sequencing e.
Future work is required to more fully address this question. Nonetheless, our data provide compelling support for the hypothesis that Hb multiplicity provides a physiological benefit to environmental stress, and thus is a phenomenon of evolutionary significance. An increase in Hb oxygen affinity can play an important role in the ability of fish to maintain oxygen uptake, and aerobic metabolism, as ambient oxygen levels decline see review ref.
In normoxia, the countercurrent arrangement of blood and water flow through the gills will fully saturate blood Hb following gill transit. As ambient oxygen declines, the ability of convective processes to fully saturate blood Hb are constrained, and the degree to which oxygen uptake becomes compromised is largely a product of Hb P In fact, the minimum partial pressure of oxygen required to maintain standard metabolic rate through aerobic processes P crit has been linked to whole blood Hb-O 2 binding affinity Normoxia exposed fish showed no reduction over the same time period.
Also note that there was no effect of hypoxia acclimation on SMR, which has also been correlated to P crit in some fish species 39 , although not red drum It is important to stress that even small reductions in P crit are of substantial ecological importance. When animals are exposed to ambient oxygen levels below P crit they must augment aerobic metabolism with anaerobic metabolism.
While animals can vary substantially in their ability to survive on anaerobic metabolism, this strategy is inherently unsustainable over prolonged time scales. By lowering P crit , red drum reduce the risk associated with hypoxic environments. It would be interesting to assess whether acclimation also increases the available aerobic scope to animals in hypoxia.
Previous studies have shown that a decrease in P crit could be linked to changes in gill morphology 41 , 44 , such as an increase in gill surface area or a decrease in lamellar thickness to facilitate oxygen uptake.
In red drum, gill surface area and lamellar thickness were not different between treatments. Thus gill remodeling does not account for the altered P crit in red drum. Similarly, hematocrit was not different between normoxia and hypoxia acclimated individuals, which is a factor that has also been shown to maximize blood oxygen-carrying capacity in response to hypoxia 44 , Finally, increased heart mass as a consequence of hypoxia exposure can also impact oxygen transport properties 46 , but again no differences in relative ventricular mass were observed between treatments.
Thus it is likely that the observed changes in P crit are related specifically to the changes in Hb oxygen binding affinity.
Unfortunately, the inability to assess changes in red blood cell NTP levels make this conclusion somewhat ambiguous. The data presented here provide support for the hypothesis that marine fish endemic to hypoxia prone environments are capable of enhancing their performance through acclimation. In the case of red drum, the available evidence suggests the acclimation response was limited to the Hb system, with no changes in branchial characteristics, hematocrit or relative ventricular mass.
Differential regulation of specific Hb subunit isoforms coincident with the reduction of hemolysate P 50 is consistent with the theory that Hb multiplicity in fish provides an adaptive advantage when confronted with environmental challenges, either through the expression of functionally distinct isoHbs or the presence of distinct environmentally triggered transcriptional response elements that allow a greater level of regulation. Importantly, these gene and organ level responses also manifested at the whole animal level through a significant reduction in P crit.
All analyses were performed using the freeware MEGA version 6. Alignments were performed on putative amino acid sequences using the ClustalW function and the Gonnet protein weight matrix. Sequences were first identified using a combination of a Blast search of the NCBI database as well as a targeted selection based on literature.
Preliminary analyses of all identified sequences were performed using the neighbor joining method, after which the sequences used for analysis were pruned to limit species specific duplication events excluding red drum. This was performed to simplify analysis and graphical display to emphasize deeper phylogenetic trends.
Similarly, targeted Blast search efforts were made to ensure proper coverage of the Acanthopterygii, Protacanthopterygii, and Otocephala superorders across the various Hb subunit isoforms. Phylogenies were tested using bootstrap methods with iterations. All sequence identifiers within the final analysis are included in Fig.
Unless specified all chemicals were obtained from Fisher Scientific. The recirculation system was equipped with a common biological filter tank to control ammonia levels. Fish were fed daily with commercial fish pellets Aquafeed, Cargill, USA and tanks were siphoned daily to remove debris. All fish were acclimated to the laboratory setting for at least two weeks prior to experimentation.
Standard metabolic rate SMR of fish was measured in advance of P crit measurement. Background tests were conducted for 0.
Hypoxia was maintained using an automated nitrogen bubbling system, which consisted of a fiber optic oxygen electrode and associated meter PreSens interfaced to a gas flow solenoid, which was controlled using AutoResp software Loligo Systems. Fish were sub-dermally tagged with VI alpha tags Northwest Marine Technology, USA , according to the manufacturer guidelines, 1 week prior to experimentation.
Individuals were then randomly assigned to either hypoxia or normoxia treatments, with 7 fish per treatment. P crit of all experimental fish was measured before subjecting them to their respective treatments and at the end of the 2-week acclimation period. Fish were fed ad libitum.
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After the final P crit measurement, fish were placed back into their respective treatments for 1 week to recover from the P crit test, after which individuals were euthanized and sampled as described below. The heart was excised and the ventricle was separated and weighed. The first gill arch on the left side of the fish was sampled for gill surface area and gas diffusion distance analysis. Primer sets were designed using Primer3 48 , 49 and specificity of each set was verified using standard PCR and gel electrophoresis procedures.
Successful DNase treatment was verified using a no reverse transcriptase control for each treatment. Methemoglobin concentration was assessed spectrophotometrically and used to correct total Hb concentration to ensure a functional Hb 4 concentration of 0. There was no significant difference in methemoglobin levels between control and hypoxia acclimated fish. Hb-O 2 equilibria were constructed using a previously described custom built gas equilibration chamber 52 placed within a plate spectrophotometer Molecular Devices. Assays consisted of equilibration at 0, 0.
Each plot was assessed manually to verify proper dose response relationships with respect to absorbance and O 2 level. The pH of each diluted hemolysate was assessed using a microelectrode system, which was used for calculation of Bohr coefficients. Measurements of gill surface area were carried out according to methods outlined by Hughes Briefly, the total number of filaments on the first gill arch was counted and the length of every tenth filament was measured under a dissecting microscope.
The linear spacing between lamellae along the filament was measured along at least 8 lamellae at X magnification under a light microscope Nikon Eclipse TEU at the base, midsection and tip of 5 filaments evenly distributed along the gill arch using digital image capture software QED Capture.
An image of a stage micrometer was also digitally captured to calibrate the measurements. At least 5 sections were mounted onto each slide. Slides were stained with hematoxylin solution, gill no. Digital images of 20 lamellae were captured for each fish to quantify lamellar area. All lamellae were selected from the midsection of the filament and no more than 4 lamellae were chosen from a single filament. Images were assigned random numbers before surface areas were scored to minimize bias. Surface area was standardized to the mass of the fish for comparisons between different fish.
Fifteen digital images of lamellae were then taken for each fish under X magnification and 10 measurements of lamellar blood-to-water diffusion distance were taken to estimate the lamellar blood-to-water diffusion distance of each fish. Similar to surface area measurements, images were also assigned random numbers before diffusion distances were scored. Gene expression levels and morphometric data collected after the acclimation experiment were analyzed using unpaired t-test to identify differences between the normoxia and hypoxia group.
Note that a significant interaction between factors was not observed, thus treatment effects at a specific pH could not be assessed.
P crit from hypoxia acclimation experiment was analyzed using one-tailed paired t-test, under the a priori assumption that dynamic Hb expression and reduced hemolysate P 50 should reduce P crit. Borza, T. Atlantic cod Gadus morhua hemoglobin genes: multiplicity and polymorphism. BMC genetics 10 , 51 Brix, O. An ecophysiological interpretation of hemoglobin multiplicity in three herbivorous marine teleost species from New Zealand.
Frey, B. The haemoglobin system of the mudfish, Labeo capensis: adaptations to temperature and hypoxia. Fyhn, U. Hemoglobin heterogeneity in amazonian fishes. Comparative Biochemistry and Physiology a-Physiology 62 , 39—66 Rutjes, H.
Multiple strategies of Lake Victoria cichlids to cope with lifelong hypoxia include hemoglobin switching. Olianas, A. Striped mullet Mugil cephalus hemoglobin system: multiplicity and functional properties. Shimada, T. The multiple hemoglobins of the japanese eel, Anguilla japonica - molecular basis for hemoglobin multiplicity and the subunit interactions.