bioRxiv preprint doi: https://doi.org/10.1101/707810; this version posted July 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Vascular Expression of Hemoglobin Alpha in Antarctic Icefish Supports 2 Iron Limitation as Novel Evolutionary Driver 3 4 Authors and Affiliations: Bruce A. Corliss1, Leon J. Delalio2, T.C. Stevenson Keller IV2,3, 5 Alexander S. Keller2, Douglas A. Keller4, Bruce H. Corliss5, Adam C Bjork6, Jody M. Beers7, 6 Shayn M. Peirce1, Brant E. Isakson2,3 * 7 8 1Biomedical Engineering Department, University of Virginia 9 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine 10 3Department of Molecular Physiology and Biophysics, University of Virginia School of 11 Medicine 12 4Sanofi, Paris, France 13 5Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island 14 6Center for Disease Control, Atlanta, Georgia 15 7Hopkins Marine Station, Stanford University, Pacific Grove, California. 16 17 18 *to whom correspondence should be addressed 19 PO Box 801394 20 Charlottesville, VA 22902 USA 21 E: [email protected] 22 P: +1 434.924.2093 23 F: +1 434.924.2828 24 25 Running title: Hemoglobin in Antarctic ice fish 26 Main text word count: 8,021 27 Figures: 6 1 bioRxiv preprint doi: https://doi.org/10.1101/707810; this version posted July 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 28 Abstract 29 Frigid temperatures of the Southern Ocean are known to be an evolutionary driver in Antarctic 30 fish. For example, many fish have reduced red blood cell (RBC) concentration to minimize 31 vascular resistance. Via the oxygen-carrying protein hemoglobin, RBCs contain the vast majority 32 of the body's iron, which is known to be a limiting nutrient in marine ecosystems. Since lower 33 RBC levels also lead to reduced iron requirements, we hypothesized that low iron availability 34 was an additional evolutionary driver of Antarctic fish speciation. Antarctic Icefish of the family 35 Channichthyidae are known to have extreme alteration of iron metabolism due to loss of two 36 iron-binding proteins, hemoglobin and myoglobin, and no RBCs. Loss of hemoglobin is 37 considered a maladaptive trait allowed by relaxation of predator selection, since extreme 38 adaptations are required to compensate for the loss of oxygen-carrying capacity. However, iron 39 dependency minimization may have driven hemoglobin loss instead of a random evolutionary 40 event. Given the variety of functions that hemoglobin serves in the endothelium, we suspected 41 the protein corresponding to the 3’ truncated Hbα fragment (Hbα-3’f) that was not genetically 42 excluded by icefish, may still be expressed as a protein. Using whole mount confocal 43 microscopy, we show that Hbα-3’f is expressed in the vascular endothelium of icefish retina, 44 suggesting this Hbα fragment may still serve an important role in the endothelium. These 45 observations support a novel hypothesis that iron minimization could have influenced icefish 46 speciation with the loss of the iron-binding portion of Hbα in Hbα-3’f, as well as hemoglobin β 47 and myoglobin. 48 49 Keywords 50 Hemoglobin alpha, Antarctic icefish, iron flux, Antarctic evolution, Antarctic Iron Flux 51 Hypothesis, Channichthyidae 2 bioRxiv preprint doi: https://doi.org/10.1101/707810; this version posted July 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 52 Introduction 53 The waters of the Southern Ocean are the coldest on Earth, with temperatures beneath the surface 54 varying between -1.9 and +1.5 Celsius (Sidell and O’Brien 2006). Such frigid conditions are 55 lethal for most mammals, in which blood plasma will quickly freeze (Verde et al. 2007), yet this 56 marine environment hosts many fish species in an ecosystem that exhibits an unexpectedly high 57 amount of biomass despite the harsh conditions (Lam and Bishop 2007; Thresher et al. 2011). 58 Species of the suborder Notothenioid make up 90 percent of the fish biomass in the seas 59 surrounding Antarctica (Sidell and O’Brien 2006), and to withstand cold conditions, they have 60 evolved adaptations such as decreased concentration of red blood cells to minimize blood 61 viscosity, with hematocrit correlating with temperature tolerance across species (Beers and Sidell 62 2011). An underappreciated consequence of reduced hematocrit is a significant decrease in 63 utilization of elemental iron, since 70 percent of red-blooded mammals’ iron is found in red 64 blood cells in the oxygen-carrying protein hemoglobin (Andrews 2000). Iron is not only 65 considered an essential nutrient for use in various iron-binding proteins (Cairo et al. 2006), but is 66 also established as a limiting nutrient in many aquatic ecosystems (Martin and Fitzwater 1988) 67 including the Southern Ocean (Thomas 2003), where addition of iron is sufficient to induce 68 transient spikes in biomass (Buesseler et al. 2004). Other limiting nutrients have been shown to 69 contribute directly to natural selection in bacteria and yeast (Lewis et al. 1986; Merchant and 70 Helmann 2012). Therefore, we propose that iron limitation could be a selection pressure 71 resulting in adaptations associated with iron binding proteins in the oceans surrounding 72 Antarctica. 73 Out of all the Notothenioids, Antarctic Icefish of the family Channichthyidae are canonically 74 known for the most extreme alterations in iron requirement, lacking expression of hemoglobin, 75 and in half of all species, also myoglobin (Kock 2005a). In this family of icefish, oxygenation is 76 thought to occur purely through diffusion-based transport of dissolved oxygen in the blood 77 (Sidell and O’Brien 2006). The high energetic cost of circulating blood at a rate sufficient for 78 diffusion-based oxygen transport has led previous research to conclude that hemoglobin loss is a 79 net-negative or neutral trait that evolved by chance and remained due to relaxed predator 80 selection (Sidell and O’Brien 2006). However, preservation of such a deleterious trait, even 81 when paired with relaxed selection pressure, is not consistent with the extreme cardiovascular 82 adaptations found in icefish that are required in order to compensate for such inefficient 83 diffusion-based oxygen transport (Kock 2005a). Rather than the random loss of a beneficial trait 84 followed by selection for several necessary compensatory traits, it is plausible that these traits are 85 the product of directional selection resulting from an environmental factor such as limited iron 86 availability. 87 Knock-out of hemoglobin occurred with the complete genomic deletion of hemoglobin beta 88 (Hbβ), and partial ablation of hemoglobin alpha (Hbα). Given Hbα’s significant role in 89 modulating endothelial nitric oxide signaling (Straub et al. 2012a) in vertebrates, independent of 90 its function in blood oxygen transport, as well as the preservation of a 3’ fragment of hemoglobin 91 alpha (Hbα-3’f) across the genomes of all icefish (Near et al. 2006), we examine whether Hbα- 92 3’f is actively expressed in icefish tissue. While this gene fragment has been classified as an 93 inactive pseudo-gene, we present evidence that Hbα expression in the endothelium has been 94 preserved in icefish retina. This result prompts a reconsideration of whether Antarctic icefish are 3 bioRxiv preprint doi: https://doi.org/10.1101/707810; this version posted July 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 95 truly a complete hemoglobin knockout, and reveals the limitation of iron as a possible novel 96 selection pressure in aquatic Antarctic environments. Studying hemoglobin expression in 97 Antarctic icefish may yield insights into how icefish avoid pathological consequences from 98 heightened endothelial nitric oxide production seen in other species and could inform future 99 therapeutics modulating this fundamental vascular signaling pathway. 100 Materials and Methods 101 Animal Collection & Sample Preparation: Two species of Antarctic notothenioid fishes were 102 collected from the waters of the Antarctic Peninsula region during the austral autumn (April- 103 May) of 2009. Champsocephalus gunnari, an icefish species, was caught by otter trawls 104 deployed from the ARSV Laurence M. Gould at water depth of 75-150 m in Dallmann Bay 105 (64°08´S, 62°40´W). Lepidonotothen squamifrons, a red-blooded notothenioid, was collected in 106 baited traps set at a depth of 200-500 m in both Dallmann Bay and Palmer Basin (64°50´S, 107 64°04´W). Animals were transferred to the US Antarctic research base Palmer Station, where 108 they were maintained in flowing-seawater aquaria at ambient water temperatures of 0° ± 0.5°C 109 prior to sacrifice. Individuals were first anesthetized in MS-222 in seawater (1:7,500 w/v), and 110 then killed by cervical transection. Retinal tissues were excised quickly, frozen in liquid 111 nitrogen, and stored at -80°C until use. All research was in compliance with the University of 112 Alaska guidelines for work conducted on vertebrate animals (institutional approval 134774-2) 113 and endorsed by the University of Maine (UM) Institutional Animal Care and Use Committee.