Since the advent of Antarctic continental glaciation, the opening of the Drake Passage between South America and the Antarctic Peninsula, and the onset of cooling of the Southern Ocean ~40-25 million years ago, evolution of the Antarctic marine biota has been driven by the development of extreme cold temperatures. The biochemical and physiological challenges facing ectotherms living in the Southern Ocean include the reduction of reaction rates and metabolic fluxes and a pervasive weakening of macromolecular interactions. Yet, Southern Ocean ectotherms are now threatened by warming over periods measured in centuries or less. The proposed research seeks to understand the molecular mechanisms of cold adaptation in Antarctic marine fishes and to assess the physiological capacity of these organisms to resist or compensate for rapid oceanic warming. The P.I. will characterize two important and interacting protein systems, the tubulins that form microtubules and the chaperonin CCT (cytoplasmic chaperonin-containing TCP-1, a family of proteins that assists the folding of the tubulins). Higher-level, integrative responses to global temperature change will be analyzed by studying the thermal dependence of cleavage in Antarctic fish embryos (a microtubule-dependent process). The objectives are (1) to determine the contributions of five novel amino acid substitutions found in Antarctic fish beta-tubulins to microtubule assembly at cold temperature. (2) to compare the functional properties of CCT from testis tissues of Antarctic fishes and mammals. (3) to evaluate the effects of increased temperature on embryogenesis in Antarctic fishes. The research will introduce graduate and REU undergraduate students to state-of-the-art biochemical, cellular, and molecular-biological research relevant to ecological and environmental issues of the Antarctic marine ecosystem. The proposed work also will benefit society by developing a cold-functioning chaperonin protein folding system, of great value to the biopharmaceutical and biotechnological industries for use in folding insoluble proteins.
The long-term goals of this three-year award were to understand the molecular basis of cold adaptation in Antarctic marine fishes and to assess the physiological capacity of these fishes to resist or compensate for the rapid warming of the Southern Ocean. Our specific objectives were 1) to evaluate the molecular features of the chaperonin CCT of Antarctic fishes that enable it to provide folding assistance to client proteins at the extremely low body temperatures (-2 to +2 deg C) of these vertebrates; and 2) to assess the effects of above normal temperatures on the development of embryos of Antarctic fish species. Intellectual Merit The successful adaptive evolution of the major group of Antarctic fishes, the notothenioids, to the cold Southern Ocean involved constraints and trade-offs at many levels of biological organization: molecular, cellular, organismal, and ecological. At the molecular and cellular levels, we have shown that CCT and its important client proteins, tubulin and actin, have evolved greater structural flexibility so that they are able to productively interact to yield properly folded proteins in an energy-poor environment. Furthermore, our results indicate that the CCT-client protein folding system can function at temperatures 5-10 deg C above the normal range, thus indicating that the assisted protein folding system is unlikely to provide a molecular bottleneck to cellular function in the notothenioid fishes as seawater temperatures rise. We have evaluated higher-level, integrative responses to oceanic warming by comparing the development of Antarctic fish embryos at normal (0 deg C) and above-normal (5, 10 deg C) temperatures. To support this work, we have developed in vitro fertilization systems for three species of notothenioids: the rockcod Notothenia coriiceps and two icefish species, Chaenocephalus aceratus and Champsocephalus gunnari. Our results show that N. coriiceps embryos acclimated to 5 deg C develop normally, but 3X faster, than embryos reared at -2 deg C. We suggest that Antarctic fish embryos developing at atypically "warm" temperatures may hatch in winter, before the return of the sun in spring increases their prey (phytoplankton, zooplankton) abundance. Thus, Southern Ocean warming may impact negatively an important, intermediate component (fish) of the Antarctic food web. To support the research on embyonic development, we have generated high quality reference transcriptomes (i.e., the totality of gene transcripts) for N. coriiceps and for C. aceratus. Furthermore, we have nearly completed the genome sequence of N. coriiceps and are well underway with the genome of C. aceratus. The transcriptome and genome sequences will enable us to examine the impacts of climate change on Antarctic fishes at the level of gene expression and will provide important resources for analysis of genome evolution in the cold-adapted Antarctic fishes. Our transcriptome and genome sequences are among the first to be generated for the Antarctic notothenioids. Broader Impacts I have disseminated my group's results by publishing numerous articles in the scientific literature and through presentations to national and international scientific meetings. I have trained numerous undergraduates, graduate students, and postdoctoral fellows in advanced molecular, cellular, and developmental-biology techniques under the auspices of this award. Many of these individuals have deployed to Antactica (Palmer Station), where they have learned first hand how to manage complex, expeditionary biological research in a remote field location. I have reached out to diverse communities through lecture presentations (Science for the Public: Contemporary Science, Northeastern University Alumni, etc.). My SftPublic presentation, "Antarctic Fishes: Models for Climate Change and Human Disease," was videotaped and is available on the WGBH Forum Network, on the SftPublic website (www.scienceforthepublic.org/archive/sftpublic-programs-2013/), and on many web and Boston-area tv channels. I have conducted Skype videoconferences with the InSciEd Out program of the Lincoln K-8 Choice School (Rochester, MN). I discussed climate change with 4-5th graders and evolutionary adaptation of Antarcic fishes with 6-8th graders. I coordinated Skype videoconferences with Ms. Ashley Nelson (Palmer Station, Antarctica) at the Marine Science Center Open House in 2013. Ms. Nelson conducted a tour of Palmer Station and described my research program for several hundred attendees of the Open House. I filmed an educational/ scientific video, "The Making of the Fittest: The Birth and Death of Genes," with Dr. Sean Carroll (Director of the Department of Science Education for the Howard Hughes Medical Institute). This 12-min video can be downloaded at http://media.hhmi.org/fittest/birth_death_genes.html. As of early 2013, HHMI had shipped over 37,000 DVDs of this program. Based on DVD user survey data alone, over 1 million students are viewing this video each year (an average of 87 students per instructor, > 15,000 instructors). This is just the DVD audience - more than 200,000 copies of the video have been downloaded from the HHMI website.
