Vascular Expression of Hemoglobin Alpha in Antarctic Icefish Supports Iron Limitation as Novel Evolutionary Driver

Frigid temperatures of the Southern Ocean are known to be an evolutionary driver in Antarctic fish. For example, many fish have reduced red blood cell (RBC) concentration to minimize vascular resistance. Via the oxygen-carrying protein hemoglobin, RBCs contain the vast majority of the body’s iron, which is known to be a limiting nutrient in marine ecosystems. Since lower RBC levels also lead to reduced iron requirements, we hypothesize that low iron availability was an additional evolutionary driver of Antarctic fish speciation. Antarctic Icefish of the family Channichthyidae are known to have an extreme alteration of iron metabolism due to loss of RBCs and two iron-binding proteins, hemoglobin and myoglobin. Loss of hemoglobin is considered a maladaptive trait allowed by relaxation of predator selection since extreme adaptations are required to compensate for the loss of oxygen-carrying capacity. However, iron dependency minimization may have driven hemoglobin loss instead of a random evolutionary event. Given the variety of functions that hemoglobin serves in the endothelium, we suspected the protein corresponding to the 3’ truncated Hbα fragment (Hbα-3’f) that was not genetically excluded by icefish may still be expressed as a protein. Using whole mount confocal microscopy, we show that Hbα-3’f is expressed in the vascular endothelium of icefish retina, suggesting this Hbα fragment may still serve an important role in the endothelium. These observations support a novel hypothesis that iron minimization could have influenced icefish speciation with the loss of the iron-binding portion of Hbα in Hbα-3’f, as well as hemoglobin β and myoglobin

The Impact of Hemoglobin Expression on Cardiovascular Physiology and Thermal Tolerance of Antarctic Notothenioid Fishes

Antarctic fishes from the suborder Notothenioidei provide excellent opportunities to investigate evolutionary adaptations to chronically cold body temperature. One family within the group, Channichthyidae, has the unique feature of lacking completely the oxygen-binding protein, hemoglobin (Hb). These animals have compensated for absence of an oxygen carrier with a suite of cardiovascular modifications: large hearts with numerous mitochondria, large blood volumes pumped through large diameter vessels, and high cardiac outputs. This study examines a number of questions related to the cardiovascular physiology and biochemistry of channichthyids, or \u27icefishes,\u27 using a variety of methodologies that include techniques from those used on the whole animal to others at the molecular level. I examined mechanistic underpinnings driving formation of one of the unusual traits we see in icefishes today, a remarkably dense vasculature within their eyes compared to those of red-blooded notothenioids. Results indicate that a biochemical pathway, nitric oxide-mediated angiogenesis, is patent in notothenioid fishes and may have led to the remarkable pattern of blood vessels in eyes of icefishes. I also found that mitochondrial populations within eyes of icefishes are particularly dense compared to those in Hb-expressing notothenioids. High densities of mitochondria provide a lipid-rich membranous network that aids diffusive flux of oxygen in species having reduced oxygen-carrying capacity because of the absence of a circulating oxygen carrier in their blood. Finally, organismal temperature sensitivities of notothenioid fishes reveal that the pattern of Hb expression in notothenioids influences thermal tolerance limits. Icefishes, in particular, are susceptible to acute elevations of temperature and my results support the hypothesis that oxygen may be the limiting factor in setting their maximum thermal limits. Thus, hemoglobinless icefishes may be a sentinel taxon for climatic warming in the Antarctic region. This research provides insights into several different physiological/biochemical processes that are fundamental to vertebrate animals and may have important applications to both biomedicine and problems associated with global climate change