Student: Christian Damsgaard
University: Aarhus University, Denmark

Project description
The evolution of air-breathing represents one of the truly fascinating milestones within the vertebrate lineage by allowing aquatic animals to exploit the plentiful supply of oxygen (O2) and later to conquer land. Today we know that air-breathing arose many times amongst various groups of fish and has led to a plethora of respiratory structures spanning from buccal cavities, stomach and intestine to swimbladders and real lungs. Further, on a global scale, air-breathing fish represent one of the fastest growing sources of protein for human consumption, representing almost 10% of the current global aquaculture production (Lefevre et al., in press).

The appearance of an air-breathing organ (ABO) and transition to aerial gas exchange is typically associated with a reduction in branchial surface area, but all air-breathing fish continue the branchial exchange of ions despite the reduced gills. Thus, all air-breathing fish continue to rely on the gills for ion balance and acid-base regulation, while the kidneys assume these function in terrestrial vertebrates. Nevertheless, acid-base and ion balance have been little studied in air-breathing fish, but the existing evidence indicate that they indeed differ from water breathing fish1,2. As perfusion of the branchial surfaces of water breathers is typically driven by their respiratory demand, which unavoidably increases with temperature, it can be hypothesised that the differences between airbreathers and water-breathers become increasingly evident as temperature increases.

In tropical aquaculture, air-breathing fish are typically grown in ponds with no aeration and limited water exchange leading to an unavoidable build-up of aquatic CO2. Hypercapnia reduces growth in water-breathing fish3,4, but similar studies have not been performed in air-breathing fish. In context of the predicted global temperature increase and the rising tropospheric carbon dioxide (CO2) levels, the physiological responses and organismal consequences are of major concern. The aim of this PhD will be to study the mechanisms of acid base balance in air-breathing fish of commercial interest including Pangasionodon hypophthalmus and Monopterus albus.

Acid-base regulation during hypercapnia
The depletion of O2 by fish and microorganisms is attended by high levels of CO2 (hypercapnia), especially in aquaculture pond systems with limited water exchange. CO2 acts as an acid, and although the acidosis resulting from hypercapnia can be compensated through elevation of plasma [HCO3- ], it is clear that fluctuating CO2 levels impose metabolic costs that directly retard growth in water-breathing fish3,4. Air-breathing fish exhibit slower and reduced ability to raise plasma [HCO3-], which may reflect their reduced gills that normally account for transepithelial ion exchange. Thus, air-breathing fish may have a reduced ability for extracellular acid-base regulation during hypercapnia through HCO3- excretion, and as the organismal responses remains to be investigated in air-breathing fish, the project aims to investigate the physiological effects of environmental hypercapnia. I will study acid-base regulation in fish exposed to chronically elevated CO2 levels, applying levels that resemble those Christian Damsgaard Project description measured in aquaculture ponds, and also expose the fish to realistic diurnal variations in PCO2. CO2 excretion is likely to involve partitioning into both water and air phase, and therefore blood gasses and plasma ion concentrations will be measured in multi-catheterized fish to address the partitioning of CO2 excretion and O2 uptake between water and air. 

Regulation of ventilation
In water breathing fish, ventilation is primarily regulated by external and internal O2 sensors located on the gill arches, whereas CO2 normally plays an insignificant role due to the very high solubility of CO2 in water and hence very low levels in fish blood. In all terrestrial vertebrates the roles are reversed with central CO2 sensors providing the main stimulus for the modulation of ventilation. This shift occurred very early in the evolution of air breathing in the lungfish, which are the most terrestrial of all fish with extremely reduced gills5. The possibility exists that CO2 regulated breathing may also be found in other air-breathing fish with the report of a central CO2 sensor in the long nosed gar with much better developed gills6. I therefore seek to address if CO2 is more widely used in air-breathing fish as a factor for regulating ventilation, which could imply convergent evolution of CO2 regulated ventilation associated with air-breathing. This can be performed in set-ups where O2 and CO2 levels are manipulated in both air and water phases and both branchial and ABO ventilation frequency is studied with buccal pressure transducers. Since it has recently been demonstrated that air-breathing is associated with significant energetic costs7, this knowledge is also of immediate concern for aquaculture. 

Prioritization between O2 uptake and CO2 excretion at high temperature 
High temperatures impose a possible conflict between acid-base regulation and O2 transport in airbreathing ectothermic vertebrates. Thus, to maintain neutral pH as body temperature increases, animals typically decrease ventilation relative to metabolism, which causes PCO2 in the blood to increase. This relationship has not been studied in air-breathing fish. Therefore, the studies will be performed at different temperatures and will particularly address the ventilator regulation at the high temperatures, where the physiological conflict between acid-base regulation and O2 delivery is expected to be maximal. These tropical air-breathing fish are expected to live close to their thermal limits8, and with the current concept that hypercapnia is associated with lowered aerobic scope9, the physiological consequences may be extensive. This calls for an evaluation of the synergistic effects of temperature and hypercapnia on the ventilatory requirements and O2 uptake at high temperatures. This will be studied by applying pressure transducers in the buccal cavity of fish and measuring the ventilatory responses along with gas exchange at increasing temperature. 

Molecular characterization of hemoglobin isoforms and branchial ion exchangers
Air-breathing fish are likely to express several hemoglobin isoforms with distinct functional properties. At high temperatures air-breathing fish are likely to increase their reliance on aerial O2 uptake, which with time may be associated with an adaption involving altered blood O2 affinity to maintain functional Christian Damsgaard Project description efficiency. I will therefore measure blood CO2 and O2 equilibrium curves in different temperatureacclimation groups, and correlate eventual differences in O2-affinity with change in absolute and relative levels of hemoglobin isoform expression and with changes in red blood cell pH and phosphate levels. Overall contribution of the proposed research The overall project will be the first studies addressing these possible conflicts in an air-breathing fish and will address topical question regarding the how organisms prioritize different physiological functions. The studies will be performed on different acclimation groups to separate the chronic and acute effects. In addition to addressing fundamental issues for tropical aquaculture, the studies will be of importance to understand the basic physiology of air-breathing fish. 

References
1. Brauner, C. J. et al. Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis. Journal of experimental biology 207, 3381–3390 (2004). 
2. Lefevre, S. et al. Effects of nitrite exposure on functional haemoglobin levels, bimodal respiration, and swimming performance in the facultative air-breathing fish< i> Pangasianodon hypophthalmus. Aquatic Toxicology 104, 86–93 (2011). 
3. Baumann, H., Talmage, S. C. & Gobler, C. J. Reduced early life growth and survival in a fish in direct response to increased carbon dioxide. Nature Climate Change 2, 38–41 (2011). 
4. Fivelstad, S., Waagbø, R., Stefansson, S. & Olsen, A. B. Impacts of elevated water carbon dioxide partial pressure at two temperatures on Atlantic salmon (< i> Salmo salar L.) parr growth and haematology. Aquaculture 269, 241–249 (2007). 
5.Sanchez, A. et al. The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish,< i> Lepidosiren paradoxa. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 130, 677–687 (2001). 
6. Remmers, J. E. et al. Evolution of central respiratory chemoreception: a new twist on an old story. Respiration physiology 129, 211–217 (2001). 
7. Lefevre, S., Wang, T., Phuong, N. T. & Bayley, M. Partitioning of oxygen uptake and cost of surfacing during swimming in the air-breathing catfish Pangasianodon hypophthalmus. Journal of Comparative Physiology B 183, 215–221 (2013). 
8. Tewksbury, J. J., Huey, R. B. & Deutsch, C. A. Ecology - Putting the heat on tropical animals. Science 320, 1296–1297 (2008). 
9. Pörtner, H.-O. Oxygen-and capacity-limitation of thermal tolerance: a matrix for integrating climaterelated stressor effects in marine ecosystems. The Journal of experimental biology 213, 881–893 (2010).