Correlating cardiac thermal performance limits with transcriptome profiles during thermal acclimation of the intertidal porcelain crab, Petrolisthes cinctipes

潮间带瓷蟹热适应过程中心脏热性能极限与转录组谱的相关性研究夏威夷大学马纳里亚分校了解温度变化的生理反应的可塑性和局限性是了解全球气候变化如何影响物种分布和丰度模式的关键组成部分。本项目旨在将功能基因组学与心脏生理学相结合，研究潮间带瓷蟹对环境相关温度变化的生理反应。海洋潮间带生物在长期（如季节性）和短期（如每日）时间尺度上经历栖息地温度的变化。本研究将研究生物对栖息地温度季节性变化的生理适应和退潮期急剧温度变化后的生理应激反应，这两者都是生物在自然栖息地热响应的重要组成部分。将解决四个具体问题：1)哪些基因参与设定心脏热性能极限？2)热驯化是否影响对急性热应激的响应？3)在适应波动温度的过程中，会发生哪些基因表达和热生理变化？4)不同季节对急性热应激的反应是否不同？为了回答第一个问题，螃蟹将在一个月的时间里适应三种温度（6摄氏度、14摄氏度和22摄氏度）。采样将包括在体内测定心脏功能的上限和下限（分别为CTmax和CTmin），并使用由13,824克隆的P. cincpes cdna组成的定制微阵列分析心脏组织转录组谱。为了回答问题2，适应这三种温度的螃蟹将暴露在急性热、冷或控制压力下，并分析从这些压力中恢复的转录组谱。为了回答问题3，螃蟹将适应一个月的热环境，包括每天短暂暴露在极热或极冷（-1C和30℃）中，之后将在四个适应周期中分析CTmax， CTmin和转录组谱。为了回答第4个问题，将在一年中的各个季节从其自然栖息地取样螃蟹，并在每个季节测量野外和应激螃蟹的CTmax， CTmin和转录组谱。使用微阵列方差分析对转录组谱进行统计分析，将揭示哪些基因在四个实验中的表达有显著变化。在这个项目中产生的数据将在斯坦福微阵列数据库中公开提供。这项研究将在动物生理学的基础领域产生新的假设，即热极限、泛温性和热适应的分子基础。这项工作的结果将直接为来自广泛生物地理区域的各种热生境的瓷蟹同属物种的温度适应比较功能基因组学研究提供信息。因此，这些数据将有助于我们了解全球温度变化如何影响物种范围分布。来自夏威夷和太平洋岛民少数族裔的本科生将通过夏威夷大学的少数族裔获得研究职业（MARC）项目被招募来参与这个项目。参与这项研究的研究生和本科生的好处包括实验室和专业技能的发展，以及在专业会议和在俄勒冈海洋生物研究所的夏季实地考察中接触“大陆”科学界。

Correlating cardiac thermal performance limits with transcriptome profiles during thermal acclimation of the intertidal porcelain crab, Petrolisthes cinctipesJonathon H. StillmanUniversity of Hawaii at ManoaUnderstanding the plasticity and limits of physiological responses to changes in temperature is a key component to understanding how global climate change will impact species distribution and abundance patterns. The aim of this project is to combine functional genomics and cardiac physiology to study the physiological responses of intertidal zone porcelain crabs to environmentally relevant temperature changes. Marine intertidal zone organisms experience changes in habitat temperature over both long (e.g., seasonal) and short (e.g., daily) time scales. This research will characterize physiological acclimatization to seasonal changes in habitat temperature and physiological stress responses following acute temperature changes during a low tide period, both of which are important components of the thermal responses of organisms in their natural habitat.Four specific questions will be addressed: 1) What genes are involved in setting cardiac thermal performance limits?, 2) Does thermal acclimation influence responses to acute thermal stress?, 3) What gene expression and thermal physiology changes occur during acclimation to fluctuating temperatures?, and 4) Does response to acute thermal stress vary across seasons? To answer question #1, crabs will be sampled during a one-month acclimation to three temperatures (6C, 14C, and 22C). Sampling will consist of in vivo determination of upper and lower thermal limits of cardiac function (CTmax and CTmin, respectively), and analysis of heart tissue transcriptome profiles using a custom microarray consisting of 13,824 cloned P. cinctipes cDNAs. To answer question #2, crabs acclimated to those three temperatures will be exposed to an acute heat, cold, or control stress and transcriptome profiles during recovery from those stresses will be analyzed. To answer question #3, crabs will be acclimated for one month to a thermal regime that includes a brief daily exposure to extreme heat or cold (-1C and 30C), after which time CTmax, CTmin and transcriptome profiles will be analyzed during four cycles of the acclimation. To answer question #4, crabs will be sampled from their natural habitat across the seasons of one year and CTmax, CTmin, and transcriptome profiles of field and stressed crabs will be measured during each season. Statistical analysis of transcriptome profiles using microarray ANOVA will reveal which genes have significant changes in expression in each of the four experiments. The data generated in this project will be made publicly available on the Stanford Microarray Database.This research will generate novel hypotheses in fundamental areas of animal physiology, namely the molecular bases of thermal limits, eurythermality, and thermal acclimation. The results of this work will directly inform comparative functional genomics studies of temperature adaptation in porcelain crab congeneric species from a range of thermal habitats across a wide range of biogeographic regions. Thus these data will contribute to our understanding of how global temperature change influences species range distributions.Undergraduate students from Hawaiian and Pacific Islander underrepresented minority groups will be recruited to work on this project through the University of Hawaii's Haumana Minority Access to Research Careers (MARC) programs. Benefits to graduate and undergraduate students involved in this research include the development of laboratory and professional skills, and exposure to the "mainland" scientific community at professional meetings and during summer field-work at the Oregon Institute of Marine Biology.