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Title: Temperature sensitivity of red blood cell physiology in Atlantic cod, Gadus morhua : comparative, molecular, evolutionary and environmental aspects
Author: Barlow, S. L.
ISNI:       0000 0004 6059 8155
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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The ability of fish to withstand increased temperatures has been directly linked with the capacity of their oxygen transport system and its ability to supply the tissues of the body with sufficient oxygen to meet demand. Therefore studies on the effects of temperature on the oxygen transport systems of aquatic organisms are important for understanding their capacity to cope with climate change. An early study in Atlantic cod (Gadus morhua) suggested haemoglobin I genotype affects temperature sensitivity of red blood cell oxygen binding, with one genotype found to be temperature insensitive. However this research had many limitations and subsequent research remains inconclusive as to the significance of the haemoglobin I polymorphism. In the most comprehensive study to date, results showed statistically indistinguishable red blood cell O2 binding between any of the three haemoglobin I genotypes in wild-caught Atlantic cod. Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity, suggesting an endothermic nature to oxygen binding rarely seen in ectothermic teleosts. Reduced thermal sensitivity of oxygen binding is often attributed to the presence of increased pH sensitivity or large Bohr effect, however this is usually only considered in heterothermic fish. However, our finding of temperature insensitive oxygen binding in Atlantic cod and the presence of one of the highest Bohr effects measured suggest this mechanism may be more wide spread than previously thought. To explore this we test the effect of pH sensitivity and temperature sensitivity of oxygen binding on in six diverse fish species. A lack of thermal sensitivity of red blood cell oxygen binding was observed in two tested species and reduced thermal sensitivity was found in a further two. A strong inverse correlation was observed between the temperature sensitivity and pH sensitivity of oxygen binding in red blood cells, with a regression coefficient of 0.88. This confirms that the occurrence of thermal insensitive oxygen binding is not exclusive to heterothermic fish and as previously suggested the mechanism behind this is linked to at least one allosteric modulator. Haemoglobin polymerisation and subsequent red blood cell sickling has long been observed in fish, although little is still known about the phenomenon. Recent studies have shown haemoglobin polymerisation in fish is the result of acidosis; we confirm this in Atlantic cod and observe a strong link between the occurrence of sickling and acid-induced haemoglobin deoxygenation or the Root effect. Further, we attempt to determine the effect of temperature on the sickling in cod, as despite the ectothermic nature of fish this information is lacking and we try to determine if this too may contribute to reduction in thermal sensitivity of oxygen binding. The occurrence of sickling decreased at increased temperatures, a new finding in fish and contrary to that found in mammals. The Root effect was similarly effected by temperature, a novel observation suggesting a strong association between the two phenomena. However, sickling appeared to be exothermic in nature and as such is likely to contribute to thermal insensitive oxygen binding. Despite increasing interest in sickling in fish, the low number of observations make it difficult to determine a mechanism. Here we test a wide variety of fish to observe potential evolutionary pathways of sickling and attempt to find a genetic marker. Sickling was found to be prevalent throughout the Gadiformes, though no conclusive evidence was found in other tested species. Reconstruction of the evolution of fish RBC sickling on a composite phylogenetic tree of all studied species suggests two possible maximum parsimony evolutionary pathways and analysis of ß globin chains gives two potential markers. Finally, we determine a method to allow for DNA sequencing of the haemoglobin I polymorphism from fin clips, with the aim that this method can be used on samples taken from Atlantic cod fitted with thermal tracking tags. This will allow direct monitoring of the effect of the haemoglobin I polymorphism on temperature experience in the wild.
Supervisor: Berenbrink, M. ; Watts, P. ; Righton, D. ; Metcalfe, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral