Antarctic notothenioid fish evolved antifreeze (AF) proteins that prevent ice crystals that enter their body fluids from growing, and thereby avoid freezing in their icy habitats. However, even in the extreme cold Antarctic marine environment, regional gradations of severity are found. The biological correlate for environmental severity in fish is the endogenous ice load, which likely determines the tolerable limit of environmental severity for notothenioid habitation. The endogenous ice load develops from environmental ice crystals entering through body surfaces and somehow localizing to the spleen. How prone the surface tissues are to ice entry, how ice reaches the spleen, and what the fate of splenic ice is, requires elucidation. Spleen sequestration of ice raises the hypothesis that macrophages may play a role in the translocation and perhaps elimination of AF-bound ice crystals. Antifreeze glycoproteins (AFGP) act in concert with a second, recently discovered antifreeze called antifreeze potentiating protein (AFPP), necessitating an assessment of the contribution of AFPP to freezing avoidance. Recent research suggests that the exocrine pancreas and the anterior stomach, not the liver, synthesize AFGPs and secrete them into the intestine, from where they may be returned to the blood. A GI-to-blood transport is a highly unconventional path for a major plasma protein and also begs the questions, What is the source of blood AFPP?. Why are two distinct AF proteins needed and what is the chronology of their evolution? What genomic changes in the DNA are associated with the development or loss of the antifreeze trait? Experiments described in this proposal address these interrelated questions of environmental, organismal, and evolutionary physiology, and will further our understanding of novel vertebrate physiologies, the limits of environmental adaptation, and climatically driven changes in the genome. The proposed research will (1) determine the temporal and spatial heterogeneity of environmental temperature and iciness in progressively more severe fish habitats in the greater McMurdo Sound area, and in the milder Arthur Harbor at Palmer Station. The splenic ice load in fishes inhabiting these sites will be determined to correlate to environmental severity and habitability. (2) Assess the surface tissue site of ice entry and their relative barrier properties in intact fish and isolated tissues preparations (3) Assess the role of immune cells in the fate of endogenous ice, (4) determine whether the blood AFGPs are from intestinal/rectal uptake, (5) examine the contribution of AFPP to the total blood AF activity (6) evaluate the progression of genomic changes in the AFGP locus across Notothenioidei as modulated by disparate thermal environments, in four selected species through the analyses of large insert DNA BAC clones. The origin and evolution of AFPP will be examined also by analyzing BAC clones encompassing the AFPP genomic locus. The broader impacts of the proposed research include training of graduate and undergraduate students in research approaches ranging from physical field measurements to cutting edge genomics. Undergraduate research projects have lead to co-authored publications and will continue to do so. Outreach includes establishing Wiki websites on topics of Antarctic fish biology and freeze avoidance, providing advisory services to the San Francisco Science Exploratorium, and making BAC libraries available to interested polar biologists. This research theme has repeatedly received national and international science news coverage and will continue to be disseminated to the public in that manner.
