Environmental, Organismal and Evolutionary Physiology of Freeze Avoidance in Antarctic Notothenioid Fishes

南极诺氏类鱼进化出了抗冻(AF)蛋白质，可以防止进入体液的冰晶生长，从而避免在结冰的栖息地结冰。然而，即使在极端寒冷的南极海洋环境中，也发现了严重的地区性梯度。与鱼类环境严重程度相关的生物学因素是内源性冰负荷，这可能决定了诺氏藻类栖息地的环境严重程度的可容忍限度。内源性冰负荷是由环境中的冰晶通过身体表面进入并以某种方式定位到脾脏而产生的。表面组织有多容易被冰进入，冰是如何到达脾的，以及脾冰的命运是什么，需要澄清。脾隔离冰提出了一个假设，即巨噬细胞可能在房颤结合的冰晶的移位和消除中发挥作用。抗冻糖蛋白(AFGP)与最近发现的第二种抗冻剂一起作用，称为抗冻增强蛋白(AFPP)，因此有必要评估AFPP对避免冻结的贡献。最近的研究表明，外分泌的胰腺和前胃，而不是肝脏，合成AFGP并将其分泌到肠道，从那里它们可能返回血液。对于一种主要的血浆蛋白来说，胃肠道到血液的运输是一种非常非常规的途径，也回避了这样的问题：血液AFPP的来源是什么？为什么需要两种不同的房颤蛋白？它们的进化时间是什么？DNA中的哪些基因组变化与抗冻特性的发展或丧失有关？这项提案中描述的实验解决了环境、生物和进化生理学的这些相互关联的问题，并将进一步加深我们对脊椎动物的新生理学、环境适应的局限性和气候驱动的基因组变化的理解。拟议的研究将(1)确定在大麦克默多海湾地区和帕尔默站较温和的亚瑟港逐渐严重的鱼类栖息地中，环境温度和冰冷的时空异质性。居住在这些地点的鱼类的脾冰负荷将被确定为与环境严重程度和宜居性相关。(2)评估完整鱼类和分离组织中冰进入的表层组织位置及其相对屏障性能(3)评估免疫细胞在内源性冰命运中的作用，(4)确定血液AFGP是否来自肠道/直肠摄取，(5)检查AFPP对血液总AF活性的贡献(6)通过对大插入DNA BAC克隆的分析，评估AFGP基因座跨Notothenioidei在不同热环境调控下的基因组变化的进展。还将通过分析包含AFPP基因组位点的BAC克隆来研究AFPP的起源和进化。拟议研究的更广泛影响包括对研究生和本科生进行从物理场测量到尖端基因组学等各种研究方法的培训。本科生的研究项目已经促成了共同撰写的出版物，并将继续这样做。外联活动包括建立关于南极鱼类生物学和避免冻结主题的维基网站，向旧金山科学探索馆提供咨询服务，以及向感兴趣的极地生物学家提供BAC图书馆。这一研究主题多次受到国内和国际科学新闻的报道，并将继续以这种方式向公众传播。

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.