THE 2011 BULLETIN EDITORIAL COMMITTEE
Editor Dr. J.B. Claiborne Managing Editor Michael P. McKernan
Dr. J.B. Claiborne, Chair
Dr. Elizabeth Crockett
Dr. David H. Evans
Dr. Raymond Henry
Dr. Karl Karnaky
Dr. David Miller
Dr. Antonio Planchart
Dr. Robert L. Preston
Dr. Alice Villalobos
Published by the Mount Desert Island Biological Laboratory June 2011 $10.00 THE BULLETIN VOLUME 50, 2011
Mount Desert Island Biological Laboratory Salisbury Cove, Maine 04672
TABLE OF CONTENTS
Introduction ii Memorial - Raymond Rappaport, Ph.D. iv Memorial - David W. Towle, Ph.D. vii Report Titles x Reports 1-90 Officers and Trustees 92 Scientific Personnel 95 Summer Fellowship Recipients 102 Seminars, Workshops, Conferences, Courses 105 Publications 120 Author Index 122 Species Index 124 Keyword Index 125 Funding Index 126 THE MOUNT DESERT ISLAND BIOLOGICAL LABORATORY
CONNECTING SCIENCE, ENVIRONMENT, AND HEALTH
INTRODUCTION
The Mount Desert Island Biological Laboratory (MDIBL) is an independent, non-profit biological and biomedical research facility and international center for comparative physiology, environmental health sciences, marine functional genomics, and studies of regeneration. The Laboratory is located on the north shore of Mount Desert Island, overlooking the gulf of Maine about 120 miles northeast of the Portland near the mouth of the Bay of Fundy. The island, well known for Acadia National Park, provides a variety of habitats including shallow and deep saltwater, a broad intertidal zone, saltwater and freshwater marshes, freshwater lakes and streams, forests and meadows.
The Laboratory is among the oldest cold-water research facilities in the Eastern United States, and its unique site provides an outstanding environment for studying the physiology of marine and freshwater flora and fauna. During 2010, the scientific personnel included 74 doctoral level scientists (including 60 Investigators), plus 111 students, fellows, and technical staff, representing 79 institutions in 23 US states, Canada, Czech Republic, Germany, Portugal, the Netherlands, and the United Kingdom.
HISTORY AND ORGANIZATION
MDIBL was founded in 1898 at South Harpswell, Maine by J.S. Kingsley of Tufts University. The Wild Gardens of Acadia donated its present site at Salisbury Cove, and relocation was completed in 1921. The Wild Gardens of Acadia, a land-holding group headed by George B. Dorr and John D. Rockefeller, Jr., who was instrumental in the founding of Acadia National Park.
In 1914, the Laboratory was incorporated under the laws of the State of Maine as a non-profit scientific and educational institution. Founded as a teaching laboratory, MDIBL is now a center for marine research and education that attracts investigators and students from across the U.S. and around the world. Since the pioneering work of H.W. Smith, E.K. Marshall and Roy P. Forster on various aspects of renal and osmoregulatory physiology of local fauna, the Laboratory has become known worldwide as a center for investigations in electrolyte and transport physiology, developmental biology, electrophysiology and marine molecular biology.
The Mount Desert Island Biological Laboratory is owned and operated by the Board of Trustees and Members of the Corporation; at present, there are 265 members. Officers of the Corporation - Chair, Vice-Chair, Director, Secretary, Treasurer, Clerk - and an Executive Committee are elected from among the Trustees. The Chair and Executive Committee oversee and promote long-range goals of the Laboratory. The Director, with the aid of a full-time Administrative Director, staff and an external Board of Scientific Advisors is responsible for implementing the scientific, educational and public service activities of the Laboratory. APPLICATIONS AND FELLOWSHIPS
Research space is available for the entire summer season (June 1 - September 30) or for shorter research visits during this time frame. Applications for the coming summer must be submitted by early January each year. Investigators are invited to use the year-round facilities at other times of the year, but such plans should include prior consultation with the MDIBL office concerning available facilities and specimen supply.
A number of fellowships and scholarships are available to research scientists, undergraduate faculty and students, and high school students. These funds may be used to cover the cost of laboratory rent, housing and supplies. Stipends are granted with many of the student awards. Applicants for fellowships for the coming summer research period are generally due in early January.
For further information on research fellowships, please contact:
Dr. Patricia H. Hand Administrative Director Mount Desert Island Biological Laboratory P.O. Box 35 Salisbury Cove, Maine 04672 Tel. (207) 288-3605 Fax. (207) 288-2130 [email protected]
Students should contact:
Michael McKernan Director of Education and Conferences [email protected]
ACKNOWLEDGEMENTS
The Mount Desert Island Biological Laboratory is indebted to the National Institutes of Health and National Science Foundation and for substantial support. Funds for building renovations and new construction continue to permit the Laboratory to expand and upgrade its research and teaching facilities. Individual research projects served by the Laboratory are funded by private and government agencies, and all of these projects have benefited from the NSF and NIH grants to the Laboratory. For supporting our educational initiative, MDIBL acknowledges the National Science Foundation Research Experience for Undergraduates, National Institute of Environmental Health Sciences Short Term Educational Experience in Research program, Maine IDeA Network for Biomedical Research Excellence (NCRR/NIH), Cserr/Grass Foundation, Milbury Fellowship Fund, Northeast Affiliate of the American Heart Association, Cystic Fibrosis Foundation, Blum/Halsey Fellowship, Stanley Bradley Fund, Stan and Judy Fund, Adrian Hogben Fund, Bodil Schmidt-Nielsen Fellowship Fund, Maine Community Foundation, the Hearst Foundation, the Betterment Fund and many local businesses and individuals.
Ray Rappaport with his sand dollar collecting scoop at MDIBL in 2000 (left) and a first division sea urchin embryo undergoing cytokinesis (right) labeled for the microtubules of the mitotic apparatus (top) and the activated myosin (bottom) of the furrow-generating contractile ring.
Raymond Rappaport, Ph.D. 1922 – 2010
“That form ever follows function.”- Louis Sullivan.
One of the founders of modern American architecture, Louis Sullivan, was famous for emphasizing the crucial nature of the connection between form and function. Raymond Rappaport, an accomplished amateur architect in his own right, built a distinguished career employing this foundational principle in the study of cytokinesis, the final stage of cell division where the two daughter cells physically separate. Ray performed elegant experiments employing hand-fashioned tools to alter the geometry of dividing sand dollar embryos in order to gain fundamental insights into the basic relationship between form and function underpinning cytokinesis.
Ray grew up in North Bergen, New Jersey and enrolled in Bethany College in West Virginia. His education there was interrupted by service during World War II in both the Army Anti-Aircraft and Medical Corps. He finished his undergraduate studies at Columbia University and then went on to complete a masters degree at the University of Michigan in 1948. While at Michigan he met and married his life partner Barbara Nolan who was also enrolled in a masters degree program. From Michigan he went on to attend Yale University where he earned a Ph.D. in 1952. That same year he was hired as an assistant professor at Union College, a small liberal arts institution in Schenectady, New York where he spent 35 years teaching and doing research. Most summers since 1948 Ray and Barbara spent from May to September at the Mount Desert Island Biological Laboratory. In his over 60 years of affiliation with MDIBL Ray served in a number of important capacities including Director, President of the Corporation, member of the Board of Trustees, and as a full-time Senior Scientist following his retirement from Union in 1987. He also contributed to the facilities of the MDIBL through the architectural design of several MDIBL cottages and laboratory buildings as well as the cooperative dining hall. In the field of cell biology his work came to be identified with the MDIBL in the same way that in the field of nephrology the MDIBL is identified with the work of Homer Smith and E.K. Marshall.
Over a career spanning five decades Ray studied cytokinesis mainly in the large and clear embryos of the sand dollar Echinarachnius parma. Each summer season Ray and Barbara would scoop up animals from a rowboat in Emory Cove and place them in a sea water table. Ray would then sex these animals by injecting just a segment with the spawning inducer potassium chloride and in this way separate males and females into different portions of the sea water table. These animals would then be partially shed throughout the season and ultimately returned to Emory Cove in the early autumn. His operations in the lab reflected the deep sense of sustainability evident in his use of animals. It was difficult to find any plastic item in Ray’s lab as everything, from watch glasses to petri dishes to test tube to flasks, was made of glass and therefore reusable. It was also difficult to find paper towels as Ray instead had dozens of cloth towels – as he often would say it does not make sense to use paper towels in a lab in which one gets their hands wet hundreds of times a day. All of his instruments were typically the basic inexpensive models and some were customized to fit his purposes. Nothing was wasted and everything that could be recycled and reused was.
“The gift of the great microscopist is the ability to think with the eyes and see with the brain.” - Daniel Mazia
Dan Mazia was a prominent cell biologist who believed that microscopists have a different way of viewing the world. He argued that the best practitioners were those who had the ability to intuit complex meanings from simple microscopic observations. Over the years Ray’s experiments were characterized by this ability to interpret the results of his simple yet elegant experiments in a way that had profound implications for the field of cyokinesis research. Ray was interested in a fundamental question in cell biology: how does the mitotic apparatus (which mediates chromosome segregation) influence the placement and initiation of the cleavage furrow during cytokinesis? In an effort to answer this question, Ray would use needles, hooks or molds to reshape sand dollar eggs into cylinders, donuts and cones, and then observe how these alterations affected the timing or placement of the cleavage furrow. Using these deceptively simple manipulations, he was able to determine the force generated by the contractile ring, the timing and kinetics of the furrow stimulation by the mitotic apparatus, and establish that a moving mitotic apparatus could generate multiple furrows in a single cell. Overall, these studies demonstrated the crucial nature of the cross talk that exists between the microtubules making up the mitotic apparatus and the actomyosin contractile ring driving the cleavage furrow. The immunofluoresence images at the start of this article shows microtubules of the mitotic apparatus interacting with the equatorial cell cortex and activated myosin concentrated in the developing furrow of a sea urchin embryo.
Ray’s well known “torus” experiment is not only a staple of introductory cell biology and developmental biology textbooks, but has essentially framed the entire debate regarding cleavage plane determination for the last fifty years. In this experiment Ray used micromanipulation to create a torus-shaped sand dollar embryo in which the mitotic apparatus was pushed over to one side of the cell. The first cell division resulted in a horseshoe-shaped binucleate cell, and during the second cleavage, two cleavage furrows formed over the former metaphase plate as expected. However, a third cleavage furrow also formed where the astral microtubules of the two separate mitotic apparati overlapped. From these experiments, Ray proposed a mechanism for cleavage plane determination whereby the cleavage furrow was positioned by the enrichment of a “cleavage stimulus” concentrated at the cell equator by the additive influences of the two spindle poles. This equatorial stimulation model has been debated and tested to this day, and subsequent studies by other investigators have established that furrows created by the interaction of adjacent mitotic apparati have been replicated in sea urchin eggs, C. elegans embryos and mammalian tissue culture cells. So influential was Ray’s contribution that the furrows generated in this manner have now come to be referred to as “Rappaport Furrows”. One of Ray’s chief characteristics was a deep and abiding appreciation for the long history of primary scientific literature focused on cell division. His own studies combined with this respect for the literature compelled him to undertake the significant effort associated with writing a book that culminated in the publication of “Cytokinesis in Animal Cells” in 1996 by Cambridge University Press. This work is still considered the definitive text in the field and provides an excellent context for understanding past, present and future work in cell division. Ray was adamant that investigators must be aware of past experiments in order to fully understand the implications of their own work. He thought that hypotheses proposed concerning cytokinesis had to be able to explain all of the data not just the subset that the experimenter happened to discuss in his/her paper. In considering these more limited works Ray would often invoke T.H. Huxley’s famous line “the great tragedy of science is the slaying of a beautiful hypothesis by an ugly fact” or the this quote from L.V. Heilbrunn: “Usually, it is easier to invent a new theory of cell division then to test an old one”. Ray frequently cautioned that the road to understanding cytokinesis was paved with the bleached bones of many once heavily favored hypotheses and the field continues to use his crucial historical perspective in the current advances based on biochemistry, genetics, molecular biology, and new imaging technologies. One great lesson of cytokinesis research has been the prominence of mechanistic redundancy of which Ray was acutely aware. During his keynote address at an international cytokinesis meeting in 2004 Ray remarked “When I began working on cytokinesis, I thought I was tinkering with a beautifully made Swiss watch, but what I was really working on was an old Maine fishing boat engine: overbuilt, inefficient, never-failed and repaired by simple measures.” The field continues to work towards deciphering the exact molecular nature of the mitotic apparatus-based cleavage stimulus so carefully characterized by Ray’s research.
“The greatest thing a human soul ever does in this world is to see something and tell what it saw in a plain way. Hundreds of people can talk for one who can think, but thousands can think for one who can see. To see clearly is poetry, prophecy and religion, all in one.” - John Ruskin
Ray qualified as one of Ruskin’s rare “seers” in that he was a true visionary who had the ability to explain complicated concepts in a straightforward manner. In addition to being an exceptional scientist, he was an outstanding teacher who masterfully employed the now largely lost art of the “chalk talk”. In his lectures and seminars he could weave a clear and engaging story using chalk drawings as the only visual aid. His detailed and multicolor chalk diagrams of developing embryos were a legendary aspect of the embryology course that he taught for many years at Union.
On a personal level Ray was a kind, generous, humble, and community-minded man who focused on substance and paid little attention to style. He had intelligent and informed opinions on a wide spectrum of subjects and particularly enjoyed talking about the natural world and interacting with young people. He was also a progressive and cosmopolitan individual who loved music and the arts and traveled extensively and lived abroad. Ray had a real interest in Japan and fostered productive collaborations with a series of prominent Japanese cell biologists that developed into life-long friendships. One emblematic example of his thoughtfulness was the castle he constructed for the summer fund-raising raffle at the Jesup Memorial Library in Bar Harbor. He would extensively research a major European castle and then spend several months constructing a scale model in his workshop. Ever the architect, in this case Ray was building the vehicle for a child’s imagination. Ray will certainly be missed, however his legacy endures in the many scientists, students, colleagues and friends he influenced and inspired.
John H. Henson, Ph.D. C. Bradley Shuster, Ph.D. Charles A. Dana Professor of Biology Associate Professor of Biology Department of Biology Department of Biological Sciences Dickinson College New Mexico State University
David Towle 1941-2011
Songlines, used by indigenous Australians to navigate across vast distances of their land, are melodic contours and words whose sequence and rhythm describe the way. Bruce Chatwin introduced them to many of us in the 1987 book The Songlines. Anyone who knew David Towle can hopefully still hear and navigate by his songline.
In the midst of planning a June sailing trip with family and friends to the high seas and islands of the eastern Adriatic in celebration of his upcoming 70th birthday, in the midst of rebuilding his 1962 vintage 36 foot wooden cruiser Spray, in the midst of sharing with his coauthors news of his latest publication on salinity-induced changes in gene expression in the green crab Carcinus maenas, in the midst of life – David Towle died unexpectedly on January 3, 2011.
When David, until recently Mt. Desert Island Biological Laboratories Senior Investigator and Director of the Marine DNA Sequencing and Analysis Center, retired from MDIBL in the summer of 2009 to move on to further adventures, his colleagues gathered to celebrate his accomplishments. Ray Henry, longtime MDIBL summer investigator and close friend, solicited written contributions about David and wove them together into an eloquent living testimonial to the greatness of David’s person. We were so fortunate in being able to share – in the present tense - our comments in written and spoken word directly with David at his retirement fest. One cannot improve upon the word portraits expressed in MDIBL Bulletin (2009), Vol. 48, x-xviii, “Genomics With Gusto”. Please read them again, now, for an understanding of David’s attitudes and accomplishments in science and his ability to inspire students of all ages. Note particularly the multitude of comments attesting to David’s ability as a teacher – this, despite David’s oft stated declaration that he preferred research to teaching. Note also the recurrent themes of generosity, hospitality, enthusiasm, openness, and joy. The words below provide a two- dimensional timeline, in the past tense, for those who marvel at the weaving together of events that help shape a scientist
David Walter Towle was born on May 26, 1941 into a dairy-farming family in Chichester, New Hampshire. He and his two brothers and sister had ample opportunities to develop easy familiarity with machines like tractors and pickup trucks. He graduated valedictorian of his Concord High School Class, attended two years at Wesleyan University in Connecticut, and then joined the Peace Corps in the early 1960’s. He was in the first Peace Corps group sent to Nepal, where he spent two years working primarily on animal husbandry. En route back to the US, he rode his bicycle throughout Europe. He then attended the University of New Hampshire, first as an English major and eventually in Biological Sciences. After completing his Master’s degree, David earned his PhD from Dartmouth College in 1971.
David joined the faculty at the University of Richmond, Virginia, developed his research and teaching skills in comparative biochemistry and physiology, and was recognized with an Outstanding Educator Award. David began spending his entire summers at MDIBL in 1982, appreciating the opportunities to do research on live marine animals in their natural setting, to learn and share insights on ion regulation in vertebrate and invertebrate organisms with colleagues at MDIBL, and to sniff the salt air. In 1988, after 18 years at the University of Richmond, David was recruited to be Chairman of the Department of Biology at Lake Forest College, Illinois. He continued with his summer research at MDIBL, often bringing undergraduates from Lake Forest College to offer them summer research opportunities on the Maine coast. He was awarded the Foster G. McGaw Professorship of Biology in light of his administrative, research and teaching endeavors. In 1992, David and his wife, Dr. Betty Massie, purchased property in Town Hill, and David began spending summer spare time away from the lab designing and constructing their cottage. David retired from Lake Forest College in 2000 and accepted a year-round position at MDIBL. Seven years after he left Lake Forest, in recognition of his contributions to undergraduate research, the College created the David W. Towle Excellence in Research Award to be given each year to the Lake Forest senior who had undertaken the most exceptional research in the Biological Sciences.
Moving to Maine and working as a senior investigator at MDIBL, David reveled in the chance to devote his scientific energies to research. His already lengthy list of research accomplishments and publications continued to grow substantially at MDIBL. Working on the Na/K-ATPase, David was one of the first researchers to show that the activity of branchial transport enzymes was sensitive to salinity. He was also one of the first to show the Na/K-ATPase was localized to the baso-lateral membrane of the crab gill. David pioneered the preparation and use of membrane vesicles as a way to monitor ion flow and transporters in crustaceans, and he first discovered the electrogenic 2Na+H+ exchanger in crustaceans. He continued to describe transporters in crustaceans at both the functional and the molecular sequence levels, comparing them with vertebrate transporters, and demonstrating the relevance of working with what were originally called non-model invertebrates in order to understand ion regulation in animal tissues. He focused on introducing new techniques in gene expression into the laboratory, including real-time quantitative PCR, developing EST libraries, and microarray analyses to further his interests in crustacean biology. David was adamant about the obligation to share data, especially EST and sequencing data, with the scientific community. He brought many new investigators to MDIBL and generously shared his time, knowledge, space, and resources, encouraging visitors to jump into the world of molecular comparative biology by example and by direct experimentation. One of David’s most profound legacies is the number of people he trained, from undergraduates to established scientists, who now use his techniques in their research.
During his academic and scientific sojourns, David was invited to participate in numerous national and international meetings. Many in his expanding network of scientific colleagues developed into close friends and collaborators, and they exchanged research visits, postdocs and students. At the International Congress of Comparative Physiology and Biochemistry in Calgary, for example, David met Carlos Luquet and his student Griselda Genovese of Buenos Aires, Argentina, discovering their mutual interests in ion balance in marine crustaceans. This led to Carlos and Grisi coming to MDIBL to learn new techniques in David’s lab for several summers working on Maine crabs and David traveling south to work with them on Argentinian species. A research visit to Guy Charmantier’s lab in Montpelier, France, was followed by summer post-doc opportunities at MDIBL for students from Charmantier’s lab, including Celine Spanings-Pierrot. In 2005 David was awarded a Fulbright Scholarship to spend five months in Dubrovnik, Croatia, with his colleague Edo Lucu, with whom he had earlier co-organized a meeting on Transport Processes across Surfaces of Aquatic Organisms in June 1991- the day after Croatia proclaimed sovereignty and war began in Slovenia. Other meetings that David frequently attended included the APS Intersociety Meetings, Society of Experimental Biology, and European Society of Comparative Physiology and Biochemistry. Opportunities for expeditionary biology and collaborative research included a research cruise to Palau to study salt and water balance in land crabs, Baja Mexico, the Amazon River, and the Patagonian Andes. While David’s bibliography reveals a polyphyletic list of research organisms, he was a leading crustacean researcher, and he often joked that there are crabs that need to be studied all over the world.
In 2009, David decided he was ready to turn his attention away from full time science and pursue other interests. Several years ago he had purchased his first power cruiser, Summertime, and begun delving into wooden boat building, marine motors, and electronics. One of his retirement projects was to be the restoration of the wooden-hulled boat Spray (that arrived on a flatbed truck from the Midwest in his driveway the morning of his MDIBL retirement party) and a trip on the Great American Loop. He also continued with his love of music, singing with the MDI summer music chorale and redeveloping the choir at the Unitarian Universalist Church of Ellsworth, Maine, playing fiddle and accordion in the Big Moose Contra Dance Band, and traveling to musical places like Louisiana Cajun country. He leaves behind his wife, Dr. Betty Massie, three children Gail, Claire and Aaron, two grandsons Harley and Hunter, his mother Ruth, three siblings and their families, countless friends, and a strong songline.
Nora Terwilliger, Ph.D. Raymond P. Henry, Ph.D. Professor Emerita Professor and Assistant Chair University of Oregon Department of Biology Department of Biological Sciences, Oregon Institute of Marine Biology Auburn University.
REPORT TITLES
Invited Review
Forrest, John N., Jr. My years as Director of MDIBL (1998-2009): Growth, Establishing a Year Round Scientific Program, and Advances in Understanding the Shark Rectal Gland ...... 1
Ionic Regulation
Kuijpers, M., Walsh, J., Katsekis, C., Cutler, C. Characterization of aquaporin 1 (AQP1) protein expression in American eel (Anguilla rostrata) ...... 10
Kratochvilova, H., Edwards, S., Claiborne, J.B. Effect of temperature and oxygen concentration on gill morphology and gill expression of sodium pump in crucian carp (Carassius carassius) ...... 11
Guerreiro, P., Bataille, A., Renfro, J.L. Parathyroid Hormone-related Protein reduces inorganic phosphate transport by PiT-like transporters in the dogfish shark (Squalus acanthias) choroid plexus...... 13
Jensen, T., Cui, L., Aleksandrov, A., Riordan, J.R. Species specific differences in CFTR strongly influence the impact of the cystic fibrosis-causing F508del mutation ...... 14
Kelley, M., Melita, A., Morris, M., Flynn, B.T., de Jonge, H., Forrest, J.N., Jr. Inhibition of type 5 Phosphodiesterase with sidenafil increases chloride secretion and augments the response to submaximal CNP in the rectal gland of the spiny dogfish, Squalus acanthias ...... 16
Comparative Biochemistry
Littlechild, S., Brummer, G., Conrad, G. The use of fibrinogen, riboflavin and UVA to immobilize the LASIK flap in corneas of the spiny dogfish shark (Squalus acanthias)...... 18
Kelley, K., Crockett, E. Production of reactive oxygen species in mitochondria from cold- and warm-acclimated striped bass, Morone saxatilis...... 19
Rabeneck, B., Diamanduros, A., Claiborne, J.B. Molecular and immunohistochemical identification of a sodium hydrogen exchanger-2c (NHE2c) paralog in the gills of marine longhorn sculpin, (Myoxocephalus octodecemspinosus) ...... 21
Kinne, R., Spokes, K., Silva, A., Silva, P. The effect of KCN on the secretion of chloride by the rectal gland of Squalus acanthias...... 24
Kinne, R., Spokes, K., Silva, P. Glycogen measurement in the rectal gland of Squalus acanthias...... 26
Silva, P., Spokes, K., Kinne, R. Complete nucleotide sequence and translated protein sequence of a sodium-glucose cotransporter in the rectal gland of the spiny dogfish (Squalus acanthias)...... 28
Silva, P., Spokes, K., Kinne, R. Molecular identification of glycogen synthase in the rectal gland of Squalus acanthias...... 30 Silva, P., Spokes, K., Kinne, R. Failure to detect a urea transporter in the rectal gland of Squalus acanthias...... 32
Comparative Physiology
Currie, S., Kolhatkar, A., Walker, N., Gamperl, K. Chemical and molecular chaperones and their importance to dogfish (Squalus acanthias) hemoglobin-oxygen affinity following temperature and osmotic stress...... 34
Wheeler, M., Tapley, D., Crockett, E. Analysis of non-heme iron levels from select tissues from cold- and warm-acclimated striped bass (Morone saxatilis) ...... 36
Preston, R., Petit, M., Willis, J., Marshall, A., Chuaypanang, S. Factors contributing to desiccation tolerance by killifish embryos (Fundulus heteroclitus)...... 38
Cai, S.Y., Yeh, C.Y., Lionarons, D., Smith, V., Li, W., Boyer, J. Adult sea lamprey (Petromyzon marinus) tolerates biliary atresia through renal excretion of bile salts and organic solutes...... 40
Brunk, E., Perrone, S., Cyr, J., Swanberg, C., Preziosi, C., Staggs, L., Marquis, H., Ashworth, S. The actin binding protein, Cofilin 1-like, regulates zebrafish (Danio rerio) development...... 42
Orcine, M., Hartline, D. Histaminergic neurons of two copepods, Centropages hamatus and Calanus finmarchicus ...... 44
Vasquez, M., Crombie, T., Julian, J. Lysosome number and size do not vary during a tidal cycle in erythrocytes of the bloodworm Glycera dibranchiata ...... 45
Marshall, A., Preston, R. Predation of killifish embryos by adult killifish (Fundulus heteroclitus)..... 48
Schnettler, E., Wray, C., Kieffer, A., Danner, R. Identification of Parathyroid Hormone Related Peptide (PTHrP) in brook trout (Salvelinus fontinalus) ...... 49
Young, R., Guerreiro, P., Renfro, J.L., Perry, D., Taylor, R., Villalobos, A. Accumulation of zinc by choroid plexus of spiny dogfish shark (Squalus acanthias) ...... 51
Molecular Toxicology
Masereeuw, R., Prevoo, B., Miller, D.S. Glucocorticoids signal through tyrosine kinase to regulate Multidrug resistance-associated protein 2 (Mrp2) in killifish (Fundulus heteroclitus) renal proximal tubules...... 53
Mahringer, A., Miller, D., Kläs, J., Reichel, V., Fricker, G. Signaling mechanisms of aryl hydrocarbon receptor (AhR) – mediated regulation of ABC transporters in killifish (Fundulus heteroclitus) kidney tubules...... 55
Lionarons, D., DeGroote, M., Boyer, J., Cai, S.Y. The apical sodium-dependent bile salt transporter (Asbt ) maintains the enterohepatic circulation of bile salts in the little skate, Leucoraja erinacea ...... 57 Chorover, J., Wickramasekara, S., Chorover, N., Hernandez-Ruiz, S., Amistadi, M.K., Abrell, L. Endocrine disrupting compounds and pharmaceuticals in water containing natural organic matter ..... 59
Cell Biology
Gianakas, A., Morris, R., Henson, J. The rocketing motility of cytoplasmic ridges present in spreading sea urchin (Strongylocentrotus droebachiensis) coelomocytes is driven by Arp2/3 complex-facilitated actin polymerization ...... 62
Saunders, D., Ertl, R., Coffman, J. Defining the ectopic expression pattern of nodal induced by nickel, zinc, and Dynasore in embryos of the sea urchin Strongylocentrotus purpuratus ...... 63
Davis, B., Coffman, J. Developmental plasticity of sea urchin (Strongylocentrotus purpuratus) larvae in response to varying diet ...... 66
Vo, N., Mikhaeil, M., Lee, L. Development and partial characterization of two cell lines derived from pituitaries of adult Atlantic salmon, Salmo salar...... 69
Ecology
Kidder, G., Norden, W. Surface seawater temperature at MDIBL dock - 2009 and 2010...... 73
Brann, D., Conrad, G. First step in establishing a new marine model organism: Induced spawning, rearing, and surface reactivity of Mytilus edulis embryos...... 74
Quinby, D., Petersen, C. Variable spawning periodicity in Fundulus heteroclitus within in a New England salt marsh...... 75
Van Dyke, R., Hess, H., Petersen, C. Patterns of trematode parasite prevalence in Littorina spp...... 77
Disney, J., Kidder, G., Balkaran, K., Brestle, C., Brestle, G. Blue mussel (Mytilus edulis) settlement on restored eelgrass (Zostera marina) is not related to proximity of eelgrass beds to a bottom mussel aquaculture lease site in Frenchman Bay ...... 80
Correa, E., Petersen, C. Wray, C. Population genetics of estuarine fish of Acadia National Park: connectivity among estuaries in Downeast Maine ...... 83
Petersen, C., Wray, C. Preliminary population genetic analysis of eelgrass and Fundulus at the Callahan Mine Superfund Site...... 85
Rabeneck, B., Claiborne, J.B. Second-year study of water quality parameters along a north to south transect in Frenchman Bay, ME...... 87
Computational Biology and Bioinformatics
Congdon, C.B., Nolan, D., Olson, I., Shea, C., Christie, A. Toward a new online computational mining tool for peptide precursor prediction...... 89
Immunology
Ho, E., Buckley, K., Messier, C., Rast, J. A screen for bacteria that induce a gut-associated immune response in the larval sea urchin...... 90
INVITED REVIEW
My years as Director of MDIBL (1998-2009): Growth, Establishing a Year Round Scientific Program, and Advances in Understanding the Shark Rectal Gland
John N. Forrest, Jr., M.D. Department of Medicine, Yale University School of Medicine, New Haven, CT 06510
The editor of the MDIBL Bulletin, Dr. J.B. Wray) and were admirably led by Patricia Hand, our Claiborne, asked me to write a history of my years as Administrative Director. DAC met weekly during director of MDIBL (1998-2009). In this account I summers and monthly during the rest of the year. will emphasize the growth of the lab during these These meetings always had practically everyone in transforming 11 years, our expanding educational attendance, which still astounds me. DAC meetings programs, the maintenance of the special ambience of were open debates on the goals of the lab, new grant the lab, the establishing of a year-round scientific opportunities, ideas for educational programs, needed program and the accomplishments of these recruited improvements in scientific resources and facilities, scientists, and conclude with a summary of the work including cottages and dorms, recruiting summer PIs, on the shark rectal gland done in my lab during this maintaining the informality of the lab, and the period. In preparing for this task, I reviewed the establishment of a permanent year-round scientific histories written by previous officers, including program. These committed individuals had an Burger, Marshall, Smith and Forster, the historical indefatigable passion for the lab and inspired us to account of MDIBL, 1898-1963, by Thomas Maren, achieve lofty goals. and the centennial book published in 1998 “A Laboratory by the Sea" by Franklin H. Epstein 21. Development of Long-term Goals. If we were to succeed in continuing to attract world-class Personal Beginnings. I first came to MDIBL in summer PI’s, and develop a year-round program, we 1970, at the invitation of Dr. Epstein, my mentor at needed outstanding resources and facilities. In Yale, who invited me to spend the first months of my anticipation of expanding the year-round scientific renal fellowship in Salisbury Cove. I returned to the program, in 2004, with partial funding from the lab each summer for the next 41 years and was Maren Foundation, we insulated and expanded the greatly influenced by the renal investigators here, co-op Dining Hall, originally constructed by Ray particularly Epstein, Roy Forster, Arnost Kleinzeller, Rappaport We set about the task of renovating all of Bodil Schmidt-Nielson, and John Boylan. Without our lab buildings. Only one, the first building on question, MDIBL has had a profound impact on campus, the old Neal laboratory, had been renovated many of the important biological advances of the last in 1989; the rest were built many decades ago and in century 21,22. The lab has also been the keystone and need of upgrading. With grant support from the NIH source of renewal for my scientific and personal life. and NSF and energized fundraising, we were able to When the chance came to assume the directorship in renovate and equip 11 of 12 laboratory buildings, 1998, I seized the opportunity with some confidence, build two new residence halls (Spruce and Birch as I knew the workings of the lab in depth, and Halls), and in July of 2008 dedicate a new year-round understood how to choose advisers who were laboratory building, with three floors of laboratory committed to MDIBL 6. and core space. This was the first new lab building at MDIBL in 35 years. This structure, a state-of-the-art The Roles of DAC and the Board. Key to our 15,000 square foot “green” research building successes was the work of the summer PIs on the currently houses several cores, the Wistar and Martha Director’s Advisory Committee (DAC), (Drs. Ed Morris Center for the Environment and Human Benz, Jim Boyer, J.B. Claiborne, David Evans, Ray Health, the John and Jean Boylan Center for Cellular Frizzell, Larry Renfro, Bruce Stanton and David and Molecular Physiology, a teaching laboratory, and Barnes) and our extraordinary Board Chair Terence the Katherine Davis Center for Regenerative Biology Boylan. Our senior staff worked to make each and Medicine. It received a Gold LEED certification successive initiative a sparkling reality (Jeri Bowers, from the U.S. Green Building Council and was the Steve Bryant, Mark Hanscome, George Kidder, first green laboratory building in the state of Maine. Claudine Lurvey, Mike McKernan, and Charles Terrence Boylan, whose father, Dr. John Boylan, medical students, residents, fellows and was a prominent summer investigator invited to undergraduates, are now the leading courses in the MDIBL by Homer Smith, became the first layperson country for training in research techniques. to chair the Board of Trustees and offered sound advice at each step of the new building. Boylan Financial support. To reach the long held organized two charrettes that brought together goal of establishing a year-round scientific program, architects, environmentalists and Salisbury Cove MDIBL required financial growth. In 1998, our residents. With its vistas overlooking Frenchman’s external grant support consisted of one Toxicology Bay, it has been called aboratory building in the Center grant from the NIEHS for $500,000. In the United States. A NIH-funded 10,000 square foot ensuing years we were successful in progressively addition to this building is now under construction raising support from the NIH, the NSF, state of and is scheduled to open in 2012. A congressional Maine bond funds through the Maine Biomedical appropriation helped establish the Center for Research Coalition, US Congressional funds, and Regenerative Medicine. The appropriation was the private philanthropy, so that our budget in 2009 had result of many visits to Washington and the risen to over $11 million dollars of external grant realization of our Congressional delegation that funding per year (Figure 2). understanding the regenerative capacity of marine organisms could lead to human medical therapies.
Educational Programs. In 1998, MDIBL had two symposia and no short courses. For the Centennial year, under Director David Dawson and Board Chair Jim Boyer, we built a new building, the Maren auditorium, seating 150 persons. Recognizing that the new auditorium and renovated lab space were ideal for hands-on courses and symposia, we started to organize and recruit faculty leaders and students to both. By 2008 we had 18 short courses and symposia with participation of 122 faculty members from medical schools and universities, 73 medical Figure 2. Growth of External Grant Funding to MDIBL residents, 58 graduate students including 44 medical Year-Round Scientific Program by Year students, and 96 undergraduates, all receiving hands- on research training at MDIBL (Figure 1). Several of The NIH-NCRR supported Biomedical Research theseMDIBL courses, forCourses example, and quantitative Conferences fluorescence! Institutional Network (BRIN) and the IDeA microscopy, and intensive pedagogical courses for Networks of Biomedical Research Excellence grants awarded to MDIBL, both under the extraordinary leadership of Dr. Patricia Hand, brought more than $44 million to the lab over the past decade. Through these grants MDIBL became the lead institution for 12 other research and academic organizations in the state of Maine. Dr. Hand’s leadership resulted in the Maine INBRE’s recognition as the number one INBRE in the U.S. Our financial success over the years enabled us to build Scientific Program Funds and Second Century Funds totaling $5.25 million for the use of the next Director, Dr. Kevin Strange.
Maintaining the special ambience and informality of MDIBL. The special features and traditions that attracted scientists and their families to Figure 1. Growth of Educational Programs at MDIBL the lab for decades were continued while we were by Year expanding. These elements included; 1) the outdoor mail shed, where PIs gather to exchange scientific ideas while picking up mail; 2) a Co-op dining hall Recruitment of year round scientists and their open 24 hours a day for students and PIs; 3) financial accomplishments at MDIBL. The first three year- support from the administration for fish procurement round investigators came from the summer program: and the animal care program, keeping costs for Drs. Hermann Haller, David Towle, and George specimens reasonable; 4) Monday morning seminars Kidder. Haller was the first, in 2000. I had known throughout the summer on the point (without slides); him from Yale where he was a talented postdoctoral 5) loaning of lab equipment to summer PIs; 6) Friday fellow in endocrinology with Howard Rasmussen. noon “brown bag” seminars where students and PIs He is a physician scientist who now heads the largest learn what other groups are doing: 7) free Friday transplant program in Europe (in Hannover, evening pasta at the Co-op for families; 8) the Fourth Germany) and is deeply committed to renal of July and August picnics on the lab beach to investigation using marine models. He aided me welcome families; 9) softball games, and “coffee greatly in establishing our first Marine DNA gatherings” for spouses, and finally 10) no charge for sequencing facility at MDIBL. Dr. Haller began our parking! These traditions add so much to the foray into regenerative medicine by showing for the ambience for which MDIBL is known. They may be first time that a regenerative zone in skate and shark challenged or changed in the future, as we become a kidney is able to respond to renal insufficiency by more “formal and structured” institution. I hope that generating entire new nephrons, glomeruli and future generations will realize that these are tubules, in adult animals 20. Since 2006, his group distinguishing features of MDIBL that set us apart established the zebrafish facility at MDIBL and used from other places. this animal model to investigate the molecular mechanisms of proteinuria. He developed a novel Establishing a year-round scientific Program. zebrafish model of proteinuria 30 and has identified Why did MDIBL have the goal of establishing a genes, including the actin binding protein, cofilin-1, year-round scientific program? First and foremost, whose inactivation leads to proteinuria in zebrafish, we believed that the pace of scientific discovery at mice and humans 3. the lab using marine and other model organisms was too important not to be pursued year-round. Of the Dr. David Towle had been a summer investigator many (some say more than 20) marine laboratories at MDIBL for many years. In July 2001 I asked him along the East Coast in the first years of the 20th to join us as the second year-round investigator and century, only three (Woods Hole, Cold Spring the first Director of our Marine DNA Sequencing and Harbor, and MDIBL) reached their Centennial year. Analysis Center. It was a productive appointment as The first two of these established year-round Towle was extremely generous and productive in scientific programs along the way and both were near teaching many NSF funded summer scientists the large metropolitan areas (Boston and New York) and techniques of cloning, microarrays and use of small major universities. MDIBL had neither of these inhibitory RNAs. His favorite marine tissues were advantages, but had a history of outstanding gills of the euryhaline green shore crab Carcinus scientists, (Warren and Margaret Lewis, Dahlgren, maenas where he used microarrays to examine the Marshall, Shannon, Pitts, Berliner, Forster, Maren, interaction between environmental salinity and Boylan, Bradley, Kinter, Schmidt-Nielson, and transport gene expression 53. With many others at the Epstein) and a splendid location on Mount Desert lab, including Christie, Lenz, Hartline, Henry, Lucu, Island near Acadia National Park. Second, for the lab Lovett, Dickenson, Luquet, Genovese, and Coblenz, to continue to grow in scientific accomplishments, it he identified new transport and regulatory genes in needed to develop year-round programs in emerging the American lobster Homarus americanus, the blue disciplines: molecular biology, functional genomics, crab, Callinectes sapidus, and the euryhaline green regenerative biology and medicine, and crab Chasmagnathus granulatus 8,11,18,26,29,40-42. David neurophysiology, to complement our historic viewed science as “high adventure”; 69 articles in strengths in physiology and toxicology. Third, the peer reviewed journals with collaborators are lab was a visible national and international center for attributed to his scientific capacity and generosity. scientific scholarship and education, able to attract David died suddenly last winter. A wonderful tribute excellent scientists from around the world to its to Dr. Towle organized by Raymond Henry of seasonal summer programs. Why not capitalize on Auburn University, was published in the 2009 this stature to expand to year round? Bulletin and can be viewed at http://www.mdibl.org/ press_releases/David_Towle_PhD_1941-2011/298. Dr. George Kidder is a long-term summer PI who defense pathways in zebrafish embryos and the in July 2001 was asked to join the scientific staff and effects of dioxin derivatives (tetrachlorodibenzo-p- served effectively for many years as our Instrument dioxin or TCDD) to up-regulate the FoxQ1b gene Officer and Head of our Animal Care Program. With tetrachlorodibenzo-p-dioxin (TCDD). Their results Dr. Robert Preston, Kidder studied the energetics of suggest that FoxQ1b may play a role in craniofacial osmoregulation, including oxygen consumption and abnormalities induced during development by water flux using the killifish, Fundulus heteroclitus exposure to TCDD 44,46. 34,35. He later received EPA and foundation grants for MDIBL’s well-known eel grass project in I met Dr. David Barnes at a symposium in Texas Frenchman’s Bay 19,36. and a year later in July 2002 recruited him from ATCC to MDIBL, first as a summer investigator, Dr. Carolyn Mattingly was recruited in October then as a year round scientist. Dr. Barnes served as 2001 and is now the Director of Bioinformatics at the Associate Director of our Center for Marine MDIBL. With a background in laboratory Functional Genomic Studies and led the way for the toxicology, Dr. Mattingly expanded and promoted sequencing of the little skate (Leucoraja erinacea) the Comparative Toxicogenomics Database (CTD) genome, a joint project of MDIBL and the that Jim Boyer and I had begun a few years earlier Universities of Delaware and Vermont. (See http: under the NIEHS Marine and Freshwater Toxicology //www.mdibl.org/mdibl_press_releases/MDIBL_Ann initiative 43. CTD is a public resource that promotes ounces_Cooperative_Project_to_Sequence_the_Little understanding about the interaction of environmental _Skate_Genome/252/). Dr. Barnes and his senior chemicals with gene products, and their effects on associate Angela Parton, are experts in cell culture human health. In the beginning, CTD received early and derived the first multipass, continuously helpful advice from leaders of the mouse genome proliferating cell line from a cartilaginous fish using database at the Jackson Lab5, the first scientific embryos of the spiny dogfish shark 45. Using this cell collaboration between these institutions in many line they identified lengthy, highly conserved gene- years. specific nucleotide sequences in the non-coding 3' UTRs of eight genes involved in the regulation of cell For gene sequence and EST data, CTD first used growth and proliferation 24. Their results indicate that MDIBL’s Marine DNA Sequencing and Analysis highly conserved gene sequences dating from the Center as we sequenced ESTs from shark and skate appearance of jawed vertebrates can be identified tissues2. Under Dr. Mattingly’s guidance, biocurators through the use of cartilaginous fish. Thus, this cell now curate manually a triad of chemical-gene, line may be useful to test hypotheses on the role of chemical-disease and gene-disease relationships from these ancient conserved sequences in comparative the literature. These data are integrated into cell biology. chemical-gene-disease networks to predict novel relationships that can be examined in experiments. Dr. Denry Sato came to MDIBL with Dr. Barnes Since 2009, the content of CTD has expanded in 2002 and described a growth factor defined feeder- dramatically to 1.4 million chemical-gene-disease free culture medium that maintained mouse data points, with statistical analyses and analytical embryonic stem (ES) cells in a pluripotent state for tools, including GeneComps and ChemComps, to over 100 passages. In the process he observed that identify related genes sets and chemicals that share leukemia inhibitory factor (LIF), commonly used to toxicogenomic profiles. Venn diagrams help the maintain pluripotency, was also an anti-apoptotic investigator discover unique attributes of any set of mitogen for mouse ES cells 25,28. Sato, in chemicals, genes or diseases. This wealth of collaboration with Stanton and Frizzell, also showed expanded chemical-gene-disease data allows users that human serum- and glucocorticoid-inducible around the world to generate testable hypotheses kinase 1 (SGK1) stimulated CFTR-mediated Cl- about molecular mechanisms of environmental currents by increasing CFTR levels in the plasma diseases15. CTD is available free of charge at membrane when the two proteins were co-expressed http://ctd.mdibl.org and is a major accomplishment of in Xenopus oocytes 47. The Stanton lab at MDIBL MDIBL. continues to work on elucidating the mechanism of this interaction to determine whether SGK1 might be In their labs Drs Mattingly and Planchart have a useful therapeutic target in cystic fibrosis. studied the effects of low-dose arsenic to perturb 14!
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Figure 3. Recruitment of Year-Round Scientific Investigators at MDIBL by Year
Dr. Antonio Planchart was recruited in discovering that mitochondrial hydrogen peroxide is September 2004 from the Faculty of Bates College to a signal required for activation of nodal expression, teach undergraduate courses at the College of the and hence for specification of the oral-aboral axis of Atlantic and establish a year-round laboratory at the sea urchin embryo 12. MDIBL and is now an Assistant Professor. His work with Dr. Mattingly on arsenic and dioxin derivates Dr. Andrew Christie is a Maine native who has been cited previously 44,46 and his work in the sea joined MDIBL as an INBRE investigator in June urchin embryo with Dr. Coffman is described 2008 and is the Director of our Imaging Core Facility below12. and a member of the Program in Neuroscience in the John W. and Jean C. Boylan Center for Cellular and Dr. James Coffman, an Associate Professor at Molecular Physiology. In 2008 he used molecular MDIBL, was recruited in 2005 and one year later techniques to identify the cardiotropic actions of published in Science the major findings from the brain/gut-derived tachykinin-related peptides (TRPs) annotated sequence of the sea urchin, from the American lobster Homarus americanus 7 Strongylocentrotus purpuratus 49. In 2008 with Tony and also identified A-type allatostatins possessing- Robertson he showed that expression of Runt-1 in the YXFGI/Vamide carboxy-termini from the nervous sea urchin embryo is required for blastula stage cell system of the copepod crustacean Calanus proliferation as well as embryonic expression of finmarchicus 9. He recently identified a calcitonin- several wnt genes and cyclin D, shedding light on the like diuretic hormone that functions as a modulator of oncogenic potential of Runx family of transcription the cardiac neuromuscular system in the American factors. In 2009, he collaborated with Tony lobster, Homarus americanus 10. Planchart in studies of the sea urchin embryo, Dr. Randy Dahn was recruited MDIBL in 2008 In 1990 during a sabbatical with Sydney Brenner after training at the University of Chicago where he at the MRC in Cambridge, England, I learned the published major papers on sonic hedgehog function techniques of molecular biology. PCR was in its in chondrichthyan fins 13 and shared developmental infancy. No genes had yet been cloned in the dogfish mechanisms in the vertebrate gill arch and paired fin shark. With Steve Jones and Paul Schofield at skeletons 27. MDIBL we used a PCR product to probe a shark heart cDNA library, and cloned the first gene in the Dr. Jane Disney was hired in July 2009 as the dogfish shark, the cardiac peptide C–type natriuretic Director of MDIBL’s Community Environmental peptide (CNP). It was the first of many genes to be Health Lab and has worked with Dr. Kidder in eel cloned at MDIBL. This was a surprise, as it was the grass restoration 19. first description of CNP occurring outside of the central nervous system 48. We provided evidence that Dr. Kevin Strange was recruited to MDIBL as CNP is the dominant cardiac peptide that stimulates our first on-site Director in January 2010, moving salt secretion via the CFTR chloride channel on the from a tenured Professorship in the Department of apical surface of the rectal gland, when the shark is Anesthesiology at Vanderbilt University. He was volume expanded by ingestion of seawater 48. CNP accompanied by his scientist wife Becky Morrison has a unique receptor (NPR-B), a single and holds three NIH RO1 grants dealing with the transmembrane receptor that is not activated by atrial genetic pathways by which animal cells sense and natriuretic peptides. If CNP, acting through NPR-B maintain salt and water balance, using the model was an activator of secretion, then this receptor roundworm C. elegans. His recent work at MDIBL should be present in the rectal gland. Using PCR and focuses on regulation of ortholog-mediated inhibition genomic walking, Steve Aller in our group cloned of ClC anion channel activity by WNK-independent this receptor from the gland and showed that CNP ERK kinase signaling in C. elegans 14,23. Dr. Strange (but not ANP) generated cyclic GMP (cGMP) and recently wrote that our institution has evolved activated chloride secretion in both rectal gland tissue rapidly, and that working at MDIBL, either full time and Xenopus oocytes expressing shark NPR-B and or as a visiting scientist, provides freedom from CFTR 1. Dr. Aller began working in my lab as a traditional academic boundaries and bureaucracies, in local high school student. After graduating from an inspiring and collegial atmosphere. He concludes college he supervised my labs at Yale and MDIBL that MDIBL’s second century will be an exciting one for almost a decade, received a PhD from Yale in “as the institution grows and as its core values serve structural biology (Molecular Biophysics and as an important driving force for the evolution of its Biochemistry) and is now an Assistant Professor in research efforts” 50. the Department of Department of Pharmacology & I would add that the greatest challenge to MDIBL Toxicology at the University of Alabama. The in the future will be to maintain the exceptional high cellular mechanism by which CNP acting through quality of the seasonal scientific program while cGMP activates CFTR remained unknown, until building a “Rockefeller University-like” year-round recent work with Dr. Hugo de Jonge of Erasmus program. There are many obstacles to overcome University in Rotterdam, cited below. before this widely held goal can be achieved. A post-doc fellow, Rudiger Lehrich, now an Advances in Understanding the Shark Rectal Assistant Professor of Medicine at Duke University Gland. From my earliest days at MDIBL, the carried out three studies with us on rectal gland digitform rectal gland of the dogfish shark held a physiology. We showed that the drug genestein was capable of activating chloride secretion by a cyclic special fascination for me. It is an ideal model tissue, 38 composed of a single cell type and with a single AMP independent mechanism . We then known function, the excretion of a salt load that demonstrated that protein kinase C zeta (PKC ζ) is cannot be handled by the shark kidney alone. During involved in mitogenic signal transduction. Using years as Director, I was fortunate to have a immunocytochemistry, we found that PKC ζ co- substantial number of undergraduates, medical localizes with microtubules in both interphase and students, and postdoctoral fellows who pursued metaphase cells of the shark rectal gland in primary questions of cell physiology using the rectal gland in culture. During mitosis, PKC ζ co-localizes with our beautiful waterfront laboratory in Neal 2. beta-tubulin in spindle microtubules, while entirely sparing astral microtubules. These findings provided the first evidence that PKC ζ is associated with the inhibitors of this PDE-3 isoform (amrinone, mitotic apparatus and indicated a functional role for milrinone, and cilostamide) alone cause comparable this kinase isoform in cell division39. Using chloride secretion in the gland and are not additive to immunofluorescence, perfusion studies of the gland CNP, strongly indicating that they have identical and oocyte expression, we provided the first evidence inhibitory effects on PDE-3 33. that chloride secretagogues (vasoactive intestinal peptide, forskolin, and genistein) increase apical It was a great privilege and a labor of love to CFTR trafficking in an intact epithelium 37. direct MDIBL through these eleven transforming years. Our accomplishments together were the We next turned our attention to cloning and highlights of my professional life. MDIBL continues expressing the VIP receptor (sVIP-R) from the gland, to occupy a special place in my heart. the oldest cloned VIP receptor to date 4. The cloned sVIP-R from shark rectal gland is only 61% identical Dr. Forrest’s research at MDIBL was supported by to the human VIP-R1. The agonist affinity for NIH grants DK 34208, NIEHS P30-ES 3828, NCRR activating chloride conductance by the cloned INBRE grant P20RR016463, NSF grant DBI- receptor when expressed with CFTR in oocytes was 0139190 and American Heart Association grant VIP > GHRH = PHI > PACAP > secretin, a profile 92011310. mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a very low affinity for PACAP. 1. Aller SG, Lombardo ID, Bhanot S, and Forrest JN, Jr. Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland. Am With a medical student, Connor Telles (now an J Physiol 276: C442-449, 1999. orthopedic surgeon at Yale) and a team of 2. Aller SG, Smith CM, Towle DW, and Forrest JN, Jr. undergraduates, we next set out to determine which Analysis of 172 expressed sequence tags from the shark of the 90 known potassium channels was the (Squalus acanthias) rectal gland Bulletin of the Mt Desert Island Biol Lab 39: 123-125, 2000. dominant potassium channel in the rectal gland that 3. Ashworth S, Teng B, Kaufeld J, Miller E, Tossidou I, functions to set the resting membrane potential and Englert C, Bollig F, Staggs L, Roberts IS, Park JK, thus the driving force for chloride exit through CFTR Haller H, and Schiffer M. The actin binding protein, in this tissue. In an extensive series of perfusion cofilin-1, plays a significant role in the regulation of actin + dynamics, we examined its role in podocytes to determine studies using type specific K inhibitors, molecular the impact of cofilin-1 dysfunction on glomerular filtration. cloning, and oocyte expression studies, we PLoS One 5: e12626, 2010. determined that the TASK-1 leak potassium channel 4. Bewley MS, Pena JT, Plesch FN, Decker SE, Weber GJ, was the dominant basolateral K+ responsible for this and Forrest JN, Jr. Shark rectal gland vasoactive intestinal function in the gland 16,31,51. This channel was later peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels. Am J Physiol Regul found to be coupled to CFTR in mammalian epithelia Integr Comp Physiol 291: R1157-1164, 2006. and is under investigation as a modifier gene in 5. Blake JA, Eppig JT, Bult CJ, Kadin JA, and Richardson patients with cystic fibrosis 17. JE. The Mouse Genome Database (MGD): updates and enhancements. Nucleic Acids Res 34: D562-567, 2006. 6. Boyer JL, and Forrest JN, Jr. The J.I.M. interview. James Returning to the longstanding puzzle of how L. Boyer, MD and John N. Forrest, Jr, MD. J Investig Med CNP activates CFTR in the rectal gland, we knew 46: 338-341, 1998. that CNP through its membrane receptor NBR-B 7. Christie AE, Cashman CR, Stevens JS, Smith CM, Beale markedly stimulates tissue cGMP 1. A paradox that KM, Stemmler EA, Greenwood SJ, Towle DW, and puzzled us for years was that the cell permeant Dickinson PS. Identification and cardiotropic actions of 32 brain/gut-derived tachykinin-related peptides (TRPs) from analog, dibutyryl cGMP, had no effect on secretion . the American lobster Homarus americanus. Peptides 29: Through a series of perfusion experiments, molecular 1909-1918, 2008. techniques, and short circuit current experiments in 8. Christie AE, Rus S, Goiney CC, Smith CM, Towle DW, primary tubular cell cultures of the gland, performed and Dickinson PS. Identification and characterization of a cDNA encoding a crustin-like, putative antibacterial protein over the past three years with Hugo DeJonge, we from the American lobster Homarus americanus. Mol established that endogenous cGMP (but not the Immunol 44: 3333-3337, 2007. dibutyryl derivative) is a phosphodiesterase-3 (PDE- 9. Christie AE, Sousa GL, Rus S, Smith CM, Towle DW, 3) inhibitor that impairs the breakdown of cAMP and Hartline DK, and Dickinson PS. Identification of A-type thus increases its tissue content 32,52. We have now allatostatins possessing -YXFGI/Vamide carboxy-termini from the nervous system of the copepod crustacean Calanus identified PDE-3 activity in the gland. Specific finmarchicus. Gen Comp Endocrinol 155: 526-533, 2008. 10. Christie AE, Stevens JS, Bowers MR, Chapline MC, kinase signaling. Am J Physiol Cell Physiol 300: C624-635, Jensen DA, Schegg KM, Goldwaser J, Kwiatkowski MA, 2011. Pleasant TK, Jr., Shoenfeld L, Tempest LK, Williams 24. Forest D, Nishikawa R, Kobayashi H, Parton A, Bayne CR, Wiwatpanit T, Smith CM, Beale KM, Towle DW, CJ, and Barnes DW. RNA expression in a cartilaginous Schooley DA, and Dickinson PS. Identification of a fish cell line reveals ancient 3' noncoding regions highly calcitonin-like diuretic hormone that functions as an conserved in vertebrates. Proc Natl Acad Sci U S A 104: intrinsic modulator of the American lobster, Homarus 1224-1229, 2007. americanus, cardiac neuromuscular system. J Exp Biol 213: 25. Furue M, Okamoto T, Hayashi Y, Okochi H, Fujimoto 118-127, 2010. M, Myoishi Y, Abe T, Ohnuma K, Sato GH, Asashima 11. Coblentz FE, Towle DW, and Shafer TH. Expressed M, and Sato JD. Leukemia inhibitory factor as an anti- sequence tags from normalized cDNA libraries prepared apoptotic mitogen for pluripotent mouse embryonic stem from gill and hypodermal tissues of the blue crab, cells in a serum-free medium without feeder cells. In Vitro Callinectes sapidus. Comp Biochem Physiol Part D Cell Dev Biol Anim 41: 19-28, 2005. Genomics Proteomics 1: 200-208, 2006. 26. Genovese G, Senek M, Ortiz N, Regueira M, Towle DW, 12. Coffman JA, Coluccio A, Planchart A, and Robertson Tresguerres M, and Luquet CM. Dopaminergic regulation AJ. Oral-aboral axis specification in the sea urchin embryo of ion transport in gills of the euryhaline semiterrestrial crab III. Role of mitochondrial redox signaling via H2O2. Dev Chasmagnathus granulatus: interaction between D1- and Biol 330: 123-130, 2009. D2-like receptors. J Exp Biol 209: 2785-2793, 2006. 13. Dahn RD, Davis MC, Pappano WN, and Shubin NH. 27. Gillis JA, Dahn RD, and Shubin NH. Shared Sonic hedgehog function in chondrichthyan fins and the developmental mechanisms pattern the vertebrate gill arch evolution of appendage patterning. Nature 445: 311-314, and paired fin skeletons. Proc Natl Acad Sci U S A 106: 2007. 5720-5724, 2009. 14. Dave S, Sheehan JH, Meiler J, and Strange K. Unique 28. Hayashi Y, Furue MK, Okamoto T, Ohnuma K, Myoishi gating properties of C. elegans ClC anion channel splice Y, Fukuhara Y, Abe T, Sato JD, Hata R, and Asashima variants are determined by altered CBS domain M. Integrins regulate mouse embryonic stem cell self- conformation and the R-helix linker. Channels (Austin) 4: renewal. Stem Cells 25: 3005-3015, 2007. 289-301, 2010. 29. Henry RP, Thomason KL, and Towle DW. Quantitative 15. Davis AP, King BL, Mockus S, Murphy CG, Saraceni- changes in branchial carbonic anhydrase activity and Richards C, Rosenstein M, Wiegers T, and Mattingly CJ. expression in the euryhaline green crab, Carcinus maenas, in The Comparative Toxicogenomics Database: update 2011. response to low salinity exposure. J Exp Zool A Comp Exp Nucleic Acids Res 39: D1067-1072, 2011. Biol 305: 842-850, 2006. 16. Decker SE, Kelley CA, Beltz E, Telles CJ, Ratner M, 30. Hentschel DM, Mengel M, Boehme L, Liebsch F, Burks K, Epstein M, Peters A, Motley W, and Forrest Albertin C, Bonventre JV, Haller H, and Schiffer M. JN, Jr. Inhibitors of 4TM-2P potassium channels inhibit Rapid screening of glomerular slit diaphragm integrity in chloride secretion in the perfused rectal gland of the spiny larval zebrafish. Am J Physiol Renal Physiol 293: F1746- dogfish, Squalus acanthias. Bulletin of the Mt Desert Island 1750, 2007. Biol Lab 44: 15-17, 2005. 31. Kelley CA, Decker SE, Cohen AD, Epstein M, Kelley P, 17. Decker SE, Poyan Mehr A, Telles CJ, Butterworth MB, and Forrest JNJ. TASK-1 but not TASK-3 potassium Hallows K, Frizzell RA, and Forrest JN, Jr. Coupling of channel inhibitors block chloride secretion in the rectal K2P TASK potassium channels and CFTR in marine and gland of the shark (Squalus acanthias). Bulletin of the Mt mammalian chloride secreting epithelia. Jounal of the Desert Island Biol Lab 46: 36-37, 2007. American Society of Nephrology 17: 47, 2006. 32. Kelley CA, Kufner A, Epstein W, Melita A, Hart M, B.C. 18. Dickinson PS, Stevens JS, Rus S, Brennan HR, Goiney T, de Jonge HR, and Forrest JN, Jr. Stimulation of CC, Smith CM, Li L, Towle DW, and Christie AE. chloride secretion by CNP is mediated by Cyclic GMP Identification and cardiotropic actions of sulfakinin peptides inhibition of phosphodiesterase III in the rectal gland of the in the American lobster Homarus americanus. J Exp Biol spiny dogfish, Squalus acanthias: Evidence from in vitro 210: 2278-2289, 2007. perfusion studies. Bull. Mt. Desert Isl. Bio. Lab 48:31-34, 19. Disney JE, and Kidder GW. Community-Based Eelgrass 2009. Bulletin of the Mt Desert Island Biol Lab 48: 31-34, (Zostera marina) Restoration in Frenchman Bay 50 50: 108- 2009. 109, 2010. 33. Kelley MH, Melita A, Edelstein H, Kelley CA, Epstein 20. Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, WS, Vosburgh B, A.E. K, Burks KL, de Jonge HR, and Luft FC, and Haller H. Nephrogenesis is induced by Forrest J.N. J. Effects of type specific phosphodiesterase partial nephrectomy in the elasmobranch Leucoraja inhibitors on chloride secretion in the perfused rectal gland erinacea. J Am Soc Nephrol 14: 1506-1518, 2003. of the dogfish shark (Squalus acanthias). 48: 21-22, 2010. 21. Epstein FH. A Laboratory by the Sea. The Mount Desert 34. Kidder GW, 3rd, Petersen CW, and Preston RL. Island Biological Laboratory. Rhinebeck, N.Y.: The River Energetics of osmoregulation: I. Oxygen consumption by Press, 1998. Fundulus heteroclitus. J Exp Zool A Comp Exp Biol 305: 22. Evans DH. A brief history of the study of fish 309-317, 2006. osmoregulation: The central role of the Mt. Desert Island 35. Kidder GW, 3rd, Petersen CW, and Preston RL. Biological Laboratory. 1, 1-10. . Front Physiol 1: 1-10, Energetics of osmoregulation: II. Water flux and 2010. osmoregulatory work in the euryhaline fish, Fundulus 23. Falin RA, Miyazaki H, and Strange K. C. elegans heteroclitus. J Exp Zool A Comp Exp Biol 305: 318-327, STK39/SPAK ortholog-mediated inhibition of ClC anion 2006. channel activity is regulated by WNK-independent ERK 36. Kidder GW, and Miller M. Drift buoys monitor surface III mediates C-type natriuretic peptide (CNP) stimulation of currents driving dispersal of eelgrass (Zostera marina) seeds. chloride secretion in the rectal gland of the spiny dog fish Bulletin of the Mt Desert Island Biol Lab 110-113, 2010. (Squalus acanthias) . Bulletin of the Mt Desert Island Biol 37. Lehrich RW, Aller SG, Webster P, Marino CR, and Lab 48: 27-30, 2009. Forrest JN, Jr. Vasoactive intestinal peptide, forskolin, and 53. Towle DW, Henry RP, and Terwilliger NB. Microarray- genistein increase apical CFTR trafficking in the rectal detected changes in gene expression in gills of green crabs gland of the spiny dogfish, Squalus acanthias. Acute (Carcinus maenas) upon dilution of environmental salinity. regulation of CFTR trafficking in an intact epithelium. J Comp Biochem Physiol Part D Genomics Proteomics 2010. Clin Invest 101: 737-745, 1998. 38. Lehrich RW, and Forrest JN, Jr. Protein kinase C zeta is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland. J Biol Chem 269: 32446-32450, 1994. 39. Lehrich RW, and Forrest JN, Jr. Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland. Am J Physiol 269: F594-600, 1995. 40. Lovett DL, Verzi MP, Burgents JE, Tanner CA, Glomski K, Lee JJ, and Towle DW. Expression profiles of Na+,K+- ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus. Biol Bull 211: 58-65, 2006. 41. Lucu C, and Towle DW. Characterization of ion transport in the isolated epipodite of the lobster Homarus americanus. J Exp Biol 213: 418-425, 2010. 42. Luquet CM, Weihrauch D, Senek M, and Towle DW. Induction of branchial ion transporter mRNA expression during acclimation to salinity change in the euryhaline crab Chasmagnathus granulatus. J Exp Biol 208: 3627-3636, 2005. 43. Mattingly CJ, Colby GT, Forrest JN, and Boyer JL. The Comparative Toxicogenomics Database (CTD). Environ Health Perspect 111: 793-795, 2003. 44. Mattingly CJ, Hampton TH, Brothers KM, Griffin NE, and Planchart A. Perturbation of defense pathways by low- dose arsenic exposure in zebrafish embryos. Environ Health Perspect 117: 981-987, 2009. 45. Parton A, Forest D, Kobayashi H, Dowell L, Bayne C, and Barnes D. Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias. Comp Biochem Physiol C Toxicol Pharmacol 145: 111-119, 2007. 46. Planchart A, and Mattingly CJ. 2,3,7,8- Tetrachlorodibenzo-p-dioxin upregulates FoxQ1b in zebrafish jaw primordium. Chem Res Toxicol 23: 480-487, 2010. 47. Sato JD, Chapline MC, Thibodeau R, Frizzell RA, and Stanton BA. Regulation of human cystic fibrosis transmembrane conductance regulator (CFTR) by serum- and glucocorticoid-inducible kinase (SGK1). Cell Physiol Biochem 20: 91-98, 2007. 48. Schofield JP, Jones DS, and Forrest JN, Jr. Identification of C-type natriuretic peptide in heart of spiny dogfish shark (Squalus acanthias). Am J Physiol 261: F734-739, 1991. 49. Sodergren E, and Consortium. The genome of the sea urchin Strongylocentrotus purpuratus. Science 314: 941-952, 2006. 50. Strange K. Cell physiology at the Mount Desert Island Biological Laboratory: a brief look back and forward. Am J Physiol Cell Physiol 300: C1-5, 2011. 51. Telles CJ, Motley W, Decker SE, Beltz E, and Forrest JNJ. Cloning and molecular identification of a TASK-1 potassium channel cDNA and protein in the rectal gland of the spiny dogfish, Squalus acanthias. Bulletin of the Mt Desert Island Biol Lab 44: 51-53, 2005. 52. Tilly BC, Hogema BM, Kelley CA, Forrest JN, Jr., and de Jonge HR. Cyclic GMP inhibition of phosphodiesterase Characterization of aquaporin 1 (AQP1) protein expression in American eel (Anguilla rostrata)
Marcela V. Kuijpers, Jonathon D. Walsh, Christopher J. Katsekis and Christopher P. Cutler Department of Biology, Georgia Southern University, Statesboro, GA 30460
This study was carried out to test a new eel AQP1 antibody that was generated and to test the feasibility of knocking-down (reducing) eel intestinal AQP1 protein expression, as prelude to testing the role of AQP1 proteins in the process of marine eel intestinal water absorption.
Studies in eels have shown previously that the aquaporin water channel protein isoform, aquaporin 1 (AQP1) is expressed in the intestine and its mRNA and protein expression increase markedly when eels are acclimated from freshwater (FW) to seawater (SW)1. The protein-coding region of the AQP1 gene sequence and the derived amino acid sequence of AQP1 were obtained by PCR, cloning and sequencing (data not shown). A new affinity-purified polyclonal antibody was raised commercially (Genscript) against the C-terminal 19 amino acids of the protein. Characterization of the AQP1 antibody involved determining that bands on Western blots were around the correct molecular weight (27.5 kDa), and that there was an increase in expression between the intestinal epithelia of FW- and SW- acclimated eels. The AQP1 antibody produced staining in the region above and below the 25 kDa molecular weight marker (Fig 1). The level of apparent AQP1 staining increased dramatically between FW- and SW- acclimated fish rectum as had been shown in European eel1. One major reason for raising the antibody was to perform morpholino gene knock- down experiments to demonstrate whether AQP1 plays a major role in water absorption in the intestine, particularly in SW eels. As evidence from the manufacturer of the eel AQP1-specific vivo-morpholinos (Genetools LLC) suggested that vivo-morpholinos usually work best when given to animals using intra venus (i.v.) injections, a study was undertaken to knock-down AQP1 protein expression in anesthetized small adult American eels using i.v. injections into the caudal artery. FW fish (approx. 100 g wet wt) were injected 1 day prior to SW-acclimation and for 3 days subsequent to it. A group of control fish were also sham-injected with vehicle (phosphate buffered saline) alone. Fish were then sacrificed and epithelial scrapes (using a glass slides) were removed from the lumen of the intestine and rectum and homogenized in the presence of buffer containing protease inhibitor cocktail (Research Product International). Homogenates were measured for protein content using a spectrophotometric Bradford’s assay (Boston Bioproducts). The level of AQP1 protein expression was compared between morpholino- and sham- injected eels using Western blotting (Figure 2). Although AQP1 protein expression was variable between different animals (probably due to variable timing of the switch from freshwater to seawater expression levels), there appeared to be no significant difference between morpholino- injected and control animals. This was probably due to the difficulty of being sure of the location of the needle in the caudal artery during injections into such small eels. Future studies will be performed using intraperitoneal (i.p.) injections, which should prove more reliable. J. Walsh was supported by an NSF REU supplement grant, M. Kuijpers by a Georgia Southern University Chandler Scholarship, and C. Katsekis and C.P. Cutler by an NSF IOS 0844818 grant.
1. Martinez A.-S., Cutler C. P., Wilson G., Phillips C., Hazon N. and Cramb G. Regulation of expression of two aquaporin homologues in the intestine of the European eel: Effects of seawater acclimation and cortisol treatment. Am. J. Physiol. 288: R1733-43, 2005. Effect of temperature and oxygen concentration on gill morphology and gill expression of sodium pump in crucian carp (Carassius carassius).
Hana Kratochvilova1,4, Susan L. Edwards2,4 James B. Claiborne3,4 1Faculty of Science, University of South Bohemia, 37005 Ceské Budejovice, 2Appalachian State University, Boone NC 28608, 3Georgia Southern University, Statesboro GA 30458, 4Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
The crucian carp is known for its great ability to survive low oxygen conditions for a very long time in ice-covered forest pools. We found that it is also capable of changing the cell structure of the gills to decrease surface area and prevent the loss of minerals to the water when in normal water, but uncover additional gill cells to take up more oxygen to live in low oxygen environments. Both water oxygen concentrations and temperature can also affect the level of specific proteins synthesized by the gill cells that help the animal to regulate salts within the body under varying environmental conditions.
The teleost fish gill serves not only as a site of gas exchange but also as the main osmoregulatory and acid/base balancing organ2. Freshwater fish tend to lose ions by diffusion or by renal excretion and have to counteract this loss by absorbing the ions against the concentration gradient through the branchial epithelium6. One of the most important ion pumps in the epithelium is the Na+/K+-ATPase3.
Crucian carp (C. carassius) is a freshwater cyprinid fish that has a great ability to survive long anoxic conditions at low ambient temperatures (over five months at 4°C)1. It has a large hepatic glycogen reserve (up to 15% of the body weight before over wintering)5 which is the main source of substrate for anaerobic glycolysis. To conserve this limited source, carp reduce energy expenditure during hypoxia. One way to do so is to lower the surface area of the gill and therefore reduce ion loss to the water. It is well described that the crucian carp is able to change the morphology of its gill depending on the changing environmental conditions (oxygen level and temperature)7. An additional strategy could be the “channel arrest” – a reduced consumption of ATP by various pumps by lowering the number of the pumps and/or decreasing their activity4.
Hypoxia exposure experiments on crucian 1,8 carp were conducted at the University of South 1,6 % covered of ILS/100 Bohemia, Czech Republic. The fish were kept nr. of IPCL 1,4 in an outdoor pool with the normal light cycle and temperature. When the water reached the 1,2 desired temperature (25°C, 15°C, 5°C), the fish 1 abundance were placed into an indoor water tank. The 0,8 concentration of dissolved oxygen was kept at relative 0,6 normoxic level or was gradually decreased to hypoxia and afterwards to anoxia by aerating 0,4 with nitrogen. The experimental fish (n=3 for 0,2 each group) were sacrificed after 7 days of 0 acclimation and their gills were removed, fixed 25N 15N 5N 5H 5A categories of experimental fish with 4% PFA (paraformaldehyde) followed by dehydration and embedding in paraffin wax. Fig. 1 Percentage coverage of the interlamellar space (ILS; white columns). Number of immunopositive cells per one lamella (IPLC) Further processing and microscopic + + stained for Na /K -ATPase (black columns). Values are group visualization were conducted at the MDIBL. means ±SD. Numbers in the categories refer to the acclimation Wide-field fluorescence microscope (Zeiss temperature in °C, N=normoxia, H=hypoxia, A=anoxia. Axiovert 200) was used for observation of the gill morphology in hematoxilin and eosin stained sections and confocal microscope (Zeiss 510 Meta) for immunohistochemically processed sections stained for sodium pump.
Figure 1 shows the coverage of the interlamellar space (ILS) in the gills of crucian carp. The data agree with the hypothesis that carp prevent ion loss to the water by lowering the surface area of the gill when there is enough oxygen in the ambient environment6. At 15°C normoxia (15N), we observed the highest coverage of the ILS (80%). With decreasing temperature (5°C) and oxygen concentration (hypoxia; 5H), the gill coverage decreased to a half (40%) of the 15N levels and may indicate that the animal is trying to compensate for the hypoxic conditions by increasing the surface area. Interestingly, at 5°C anoxia (5A), the cell mass returned to cover the ILS almost to the original extent (76%). We speculate that it is adaptive for the animal to maintain ILS coverage to reduce ion loss (and energy conservation) when there is no ambient oxygen available. These results correspond with the observation that the number of immunopositive cells per lamella (IPCL) at 5A is the lowest of all groups and may indicate a second energy conservation strategy via channel arrest as fewer cells express Na+-K+-ATPase during these extreme conditions.
Funded in part by NSF IOB-061687 to JBC and MDIBL New Investigators Award to SLE.
1. Blažka, P. O biologii karasia obyknobennogo (Carassius carassius L. morpha humilis heckel). Zoologičeskij žurnál XXXIX, 1958. vyp. 9: p. 1384-1389. 2. Evans, DH, PM Piermarini, and KP Choe. The Multifunctional Fish Gill: Dominant Site of Gas Exchange, Osmoregulation, Acid-Base Regulation, and Excretion of Nitrogenous Waste. Physiol. Rev., 2005. 85(1): p. 97-177. 3. Gibbs, A and GN Somero. Na+-K+-adenosine triphosphatase activities in gills of marine teleost fishes: Changes with depth, size and locomotory activity level. Marine Biology, 1990. 106(3): p. 315-321. 4. Hochachka, PW and PL Lutz. Mechanism, origin, and evolution of anoxia tolerance in animals. Comparative Biochemistry and Physiology, Part B, 2001. 130: p. 435-459. 5. Nilsson, GE. Long-term anoxia in crucian carp: changes in the levels of amino acid and monoamine neurotransmitters in the brain, catecholamines in chromaffin tissue, and liver glycogen. J Exp Biol, 1990. 150(1): p. 295-320. 6. Perry, SF. The Chloride Cell:Structure and Function in the Gills of Freshwater Fishes. Annual Review of Physiology, 1997. 59(1): p. 325-347. 7. Sollid, J, P De Angelis, K Gundersen and GE Nilsson. Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills. J Exp Biol, 2003. 206(20): p. 3667-3673.
Parathyroid Hormone-related Protein reduces inorganic phosphate transport by PiT-like transporters in the dogfish shark (Squalus acanthias) choroid plexus
Pedro M. Guerreiro1, Amy M. Bataille2, and J. Larry Renfro2 1 Center of Marine Sciences, University of Algarve, Faro, Portugal 2Physiology and Neurobiology, Univ. Connecticut, Storrs, CT 06269
The choroid plexus is responsible for the composition of cerebrospinal fluid (CSF) which nourishes and offers a stable envi- ronment for neurons. We have established that specific inorganic phosphate (Pi) transporters (PiT, Slc20), localized in its apical membrane, mediate phosphate concentration in CSF and that these transporters may be regulated, in part, by para- thyroid hormone-related protein (PTHrP).
Phosphate is critical for numerous vital functions, and its regulation in body fluids is of paramount impor- tance. In the CSF of intact animals changes in Pi can produce oscillations in pH, Ca2+, etc. with high impact on cellular health, ventilation rate and renal Pi excretion. In both humans and sharks, Pi in plasma is about twice that in CSF1. The choroid plexus (CP) has a significant role in control of CSF, and the33P-labelled Pi unidirec- tional fluxes across dogfish shark CP revealed a high flux ratio in the CSF-to-blood direction1. This transport was inhibited by arsenate and has characteristics consistent with a PiT-type, Na+-dependent transporter, specifi- cally, its insensitivity to phosphonoformic acid (1 mM) and the ability to use Li+ as a substrate2,3. We localized this PiT2-like transporter in the apical region of the CP cells by immunohistochemistry2.
PTHrP is a pleiotropic factor in which the N-terminus has high similarity to that of parathyroid hormone and interacts with the same receptor, evoking similar actions on calcium and phosphate metabolism4. Our previous studies demonstrated that this is a normal factor in fish, increasing whole-body calcium uptake in teleost larvae4 and increased renal Ca2+ reabsorption together with increased Pi secretion and reduced Pi reabsorption by floun- der renal proximal tubule epithelium 5. PTHrP is present in the shark genome, and its occurrence, together with that of the receptor, has been demonstrated in the elasmobranch CP4.
Following the characterization of the transporter type involved in Pi transport, we tested the effects of PTHrP on Pi flux across dogfish IVth CP. Flux measurements were taken after consecutive 60 min periods in normal elasmobranch physiological saline with or without 50 nM PTHrP. After correction for passive Pi flux (leak), PTHrP exposure induced a significant (P = 0.039; Student's paired t-test, n =9) reduction in CSF-to-blood Pi active transepithelial transport which averaged about 40% less than that of the control period (28.7 ± 6.56 SEM vs. 15.2 ± 2.34 SEM nmol x cm-2 x h-1). There was no effect on transepithelial electrical resistance.
In conclusion, the active removal of Pi from the CSF-side of the dogfish shark CP by a PiT-type, Na+- dependent transporter is inhibited to PTHrP. This, together with its effect on renal Pi excretion may be part of an endocrine mechanism responsible for controlling CSF Pi/Ca2+ composition. Supported by NSF.
1. Guerreiro, PM, Villalobos, AR, and Renfro, JL. Active transepithelial transport of inorganic phosphate by choroid plexus of spiny dogfish shark (Squalus acanthias). Bulletin of the Mt. Desert Island Biol. Lab. 47: p. 28, 2008. 2. Guerreiro, PM, Bataille, AM, and Renfro, JL. PiT-like transporters are associated with inorganic phosphate trans- port by choroid plexus of spiny dogfish shark (Squalus acanthias). Bulletin of the Mt. Desert Island Biol. Lab. 49: p. 5, 2010. 3. Virkki, LV, Biber, J, Murer, H, and Forster, IC. Phosphate transporters: a tale of two solute carrier families. Am J Physiol Renal Physiol. 293(3): p. F643-654, 2007. 4. Guerreiro, PM, Renfro, JL, Power, DM, and Canario, AVM. The parathyroid hormone family of peptides: struc- ture, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish. Am J Phy- siol Regul Integr Comp Physiol. 292(2): p. R679-696, 2007. 5. Guerreiro, PM, Canario, AVM, Power, DM and Renfro, JL. Piscine PTHrP regulation of calcium and phosphate transport in winter flounder renal proximal tubule primary cultures. Am J Physiol Regul Integr Comp Physiol. 299(2): p. R603-611, 2010.
Species specific differences in CFTR strongly influence the impact of the cystic fibrosis-causing F508del mutation
Tim Jensen, Liying Cui, Andrei A. Aleksandrov and John R. Riordan Department of Biochemistry and Biophysics and Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC 27599
A single mutation is responsible for most cystic fibrosis. At the cellular level the F508del lesion causes the protein to be recognized as abnormal by quality control systems and degraded, resulting in loss of its function to control epithelial ion and fluid homeostasis. At the protein level the primary impact of the absence of F508 is thermodynamic instability. This property is expected to be context dependent, and we postulated that the impact might be different in CFTRs of different species. We have confirmed this hypothesis and been able to mimic features of the protein, in species less influenced by the mutation, to stabilize the human mutant.
Most patients with cystic fibrosis possess the F508del mutation in the CFTR gene, resulting in defective ion and fluid transport in epithelial cells of respiratory, intestinal and other tissues. A great deal has been learned about the impact of the deletion of the single PHE508 amino acid over the past twenty years.
At the cellular level the mutant protein is recognized as abnormal by quality control systems in both the early and late secretory pathways and degraded. The small amount that may reach the cell surface plasma membrane is partially functional at temperatures below ~ 30oC, but activity is rapidly lost at physiological temperature1. Thus, although manipulations of constituents of the cellular quality control systems can result in traffic of the mutant protein to the plasma membrane and retention there, the molecule remains thermodynamically unstable and dysfunctional.
At the protein level, studies of the NBD1 domain3,4 in which PHE508 is located, and with the full-length molecule1 have demonstrated that the thermal instability is the basic defect to be overcome. As one means of identifying features of the protein that influence its stability, we have compared the influence of the F508del mutation on CFTRs from various species. Ostedgaard et al.2 initiated this approach and found that both murine and porcine F508del CFTR matured at least partially at 37oC and were functional at least at 25oC.
We now have examined wild-type and F508del versions of CFTR from several other species heterologously expressed in human cells and observed that, in several other mammalian species tested, the mutation had generally similar effects on maturation and stability as in human. In contrast, the F508del mutants of several non-mammalian species including amphibian, elasmobranch and avian matured nearly as well as their wild-type counterparts at 37oC (Fig 1). Most striking was the result with avian CFTR which not only matured similarly whether PHE508 was present or not but also was not thermally destabilized by the mutation. Thus, strong channel activity persisted at temperatures of 40oC and above where human F508 CFTR was entirely inactive. While the half-life in cells of bird F508del CFTR was somewhat shortened relative to the wild-type, it remained greater than 10 h, more than adequate to maintain a steady-state level comparable with that of the wild-type human protein.
Figure 1: Western blot showing varying degrees of maturation of DF508 CFTR in various species. CFTR cDNAs (wild-type and DF508) from each species indicated were expressed in HEK-293 cells grown at 37oC or 27oC. All constructs except the human had GFP fused at their C-termini accounting for their larger sizes. The more slowly migrating diffuse bands represent the mature forms with complex oligosaccharide chains, the sharper more rapidly migrating bands, the immature core-glycosylated forms.
As human and avian CFTR are ~80% identical in sequence, differences exist at >200 residue positions. We focused on ones in NBD1 that might be expected to influence structural stability and dynamics. In addition to the I539T substitution, present in nearly all species except human and which promotes ER export but not thermostability of F508del, we found a strong correlation between the presence of proline residues at specific positions and both maturation and thermostability of the F508del protein among different species. Introduction of proline residues at these positions into human F508del CFTR provided it with a similar level of thermodynamic stability. We believe that these strategically placed prolines are part of an allosteric network connecting dynamic elements of the mutant NBD1 enabling overall domain stability similar to the wild-type and hypothesize that the binding of specific ligands to the mutant protein may have similar influence. We plan to test both small and large molecule binders for this ability as the basis of a therapeutic strategy. (Supported by the NIH and CFF).
1. Aleksandrov AA, Kota P, Aleksandrov LA, He L, Jensen T, Cui L, Gentzsch M, Dokholyan NV, Riordan JR Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR. J Mol Biol. 401(2):194- 210 2010. 2. Ostedgaard LS, Rogers CS, Dong Q, Randak CO, Vermeer DW, Rokhlina T, Karp PH, Welsh MJ. Processing and function of CFTR-DeltaF508 are species-dependent. Proc Natl Acad Sci U S A. 104(39):15370-5 2007. 3. Protasevich I, Yang Z, Wang C, Atwell S, Zhao X, Emtage S, Wetmore D, Hunt JF, Brouillette CG. Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide- binding domain 1. Protein Sci. 19(10):1917-31 2010. 4. Wang C, Protasevich I, Yang Z, Seehausen D, Skalak T, Zhao X, Atwell S, Spencer Emtage J, Wetmore DR, Brouillette CG, Hunt JF. Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis. Protein Sci. 19(10):1932-47 2010.
Inhibition of type 5 Phosphodiesterase with sidenafil increases chloride secretion and augments the response to submaximal CNP in the rectal gland of the spiny dogfish, Squalus acanthias
Megan H. Kelley1,2,5, August M. Melita1,2,5, Montana Morris1,5, Bob Tom Flynn,3,5 Hugo R. de Jonge4,5 and John N. Forrest Jr1,5. 1Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510 2University of Vermont, Burlington, VT 0540 3John Bapst Memorial High School, Bangor ME, 05501 4Department of Biochemistry, Erasmus University Medical Center, 3000CA Rotterdam, The Netherlands 5Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
At least 11 isoforms of phosphodiesterase (PDE) enzymes metabolize the second messenger cyclic nucleotides Cyclic AMP and /or cyclic GMP in mammalian cells. To determine the roles of specific PDEs in the shark rectal gland, we carried out both perfusion of the intact gland and electrophysiological studies in primary cultures of tubular cells using the specific PDE- 5 inhibitor sidenafil. Our results indicate that inhibition of PDE-5 with sidenafil alone stimulates chloride secretion modestly, but is highly synergistic when added to submaximal concentrations of CNP, providing indirect evidence that PDE types 3 and 5 are present in the gland and contribute to the effects of CNP.
C-type natriuretic peptide (CNP) is the cardiac peptide in the shark heart that stimulates chloride secretion by the shark rectal gland (SRG)5. Our laboratory has cloned both this peptide and its receptor NPR-B from shark rectal gland and shown that CNP activates Cl- conductance when CFTR and shark NPR-B are co-expressed in oocytes1,5. CNP markedly elevates endogenous cyclic GMP (cGMP) in the rectal gland and recent evidence from our laboratories indicates that this increased level of cGMP activates Cl- secretion in the rectal gland by inhibiting type III phosphodiesterase (PDE-3), raising cAMP levels2.4.6. PDE-3 is the only phosphodiesterase enzyme that is inhibited by cGMP. In previous studies from our laboratory only inhibitors of type 3 phosphodiesterase (amrinone, milrione and cilostamide) stimulated chloride secretion in this model4. The only substrate of PDE-5 is the second messenger, cGMP.
To determine if PDE-5 is present in the gland and plays a role in chloride secretion, we used the specific inhibitor of type 5 PDE, sidenafil (Viagra). Glands were perfused as previously described3. As shown in Figure 1, at 30 min of basal secretion, sidenafil (20 µM) was added to the perfusate (30-45 min) and an increase in chloride secretion was observed, rising from 160 ± 53 µEq/h/g to a maximum of 661 ± 145.
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Figure 1. In each group, glands were perfused for 30 min to reach basal secretion. X indicate basal secretion for 60 min without the addition of agonists (n=5). ♦ indicates the addition of sidenafil 20 µM from 30-45 min (n=5). Closed triangles indicate the addition of submaximal (2 nM) CNP from 30-45 min followed by perfusion with 20 µM sidenafil + 2nM CNP from 45-60 min (n=7). Sidenafil alone moderately stimulates chloride secretion but markedly enhances the response to submaximal CNP. Chloride secretion was measured as µEq Cl/h/g. When glands were perfused with a submaximal concentration of CNP (2 nM from 30-45 min), chloride secretion also rose only slightly, from 187 ± 58 µEq/h/g to a maximum of 462 ± 148. However in this group the addition of sidenafil to the perfusate containing 2 nM CNP (45-60 min) caused a dramatic rise in chloride secretion to a maximum of 1632 ± 120 µEq/h/g. Thus, inhibition of PDE-5 with sidenafil alone causes a modest stimulation, but the addition of sidenafil to a submaximal concentration of CNP causes a clear synergism in the rate of chloride secretion.
Similar results were seen in electrophysiological studies in primary cultured tubular epithelial cells of the gland (Figure 2). Cultures were obtained as previously described7.
Figure 2. Similar to the perfusion studies, a low concentration of CNP has only a modest effect but the addition of Sidenafil (20 µM) again increases chloride secretion (measured as Isc) and augments the response to CNP, reaching a maximal Isc that is only modestly increased by the addition of forskolin (10 µM).
We conclude that both PDE-3 and PDE-5 are present in the shark rectal gland and interact to elicit a maximal chloride secretory response to CNP.
This work was supported by NIH grants DK 34208, NIEHS 5 P30 ES03828 (Center for Membrane Toxicity Studies) to J.N.F., an NSF grant DBI-0139190 (REU site at MDIBL), a STERR award (R25- ES)16254 to B.T.F and a MDIBL New Investigator Award to H. d. J.
1. Aller, SG, Lombardo, ID, Bhanot S. and Forrest JN Jr. Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland. Am. J. Physiol. 276:C442-9, 1999. 2. Kelley, CA, Kufner, A, Epstein, W, Melita, A, Hart, M, Tilly, BC, de Jonge, HR, and Forrest, JN, Jr. Stimulation of chloride secretion by CNP is mediated by Cyclic GMP inhibition of phosphodiesterase III in the rectal gland of the spiny dogfish, Squalus acanthias: Evidence from in vitro perfusion studies. Bull. Mt. Desert Isl. Bio. Lab 48:31-34, 2009. 3. Kelley, GG, Poeschla EM, Barron HV, and Forrest JN Jr. A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP. J. Clin. Invest. 85 (5): 1629-36,1990. 4. Kelley, MH, Melita A, Edelstein H, Kelley, CA, Epstein WS, Vosburgh B, Kufner AE, Burks KL, de Jonge HR, Forrest, JN Jr. Effects of type specific phosphodiesterase inhibitors on chloride secretion in the perfused rectal gland of the dogfish shark (Squalus acanthias) ). Bull. Mt. Desert Isl. Bio. Lab. 48: 21-22, 2010. 5. Schofield, JP, Jones, DS, and Forrest JN Jr. Identification of C-type natriuretic peptide in heart of spiny dogfish shark (Squalus acanthias). Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F734-F739, 1991. 6. Tilly BC, Hogema BM, Kelley CA, Forrest JN Jr, and De Jonge HR. Cyclic GMP inhibition of phosphodiesterase III mediates C-type natriuretic peptide (CNP) stimulation of chloride secretion in the rectal gland of the spiny dog fish (Squalus acanthias). Bull. Mt. Desert Isl. Bio. Lab. 48: 27-30, 2009. 7. Valentich JD, and Forrest JN Jr. Cl- secretion by cultured shark rectal gland cells. I. Transepithelial transport. Am. J. Physiol. 260: C813-823, 1991.
The use of fibrinogen, riboflavin and UVA to immobilize the LASIK flap in corneas of the spiny dogfish shark (Squalus acanthias)
Stacy Littlechild, Gage Brummer and Gary W. Conrad Division of Biology, Kansas State University, Manhattan, KS 66502
In human corneas that have undergone LASIK, the flap normally lays very smoothly on the surface of the laser-modified stroma, but it never actually attaches to it, even several years after the surgery, thus constituting a hazard in case of physical trauma to the eye, such as sudden contact with an automobile air bag. Work performed on shark corneas suggests that such flaps might be glued firmly to the stroma if a solution of fibrinogen were applied to the interface between the flap and the stroma at any time following the surgery.
Laser-Assisted In Situ Keratomileus (LASIK) is a common procedure used to correct eye conditions such as nearsightedness, farsightedness and astigmatism. One liability that results from this procedure is that the permanent flap that results from cutting into and exposing the middle layer of the cornea (the stroma), forever remains non-attached to the underlying laser-modified stroma3. Such a potentially loose layer represents a medical risk. To decrease the risk of re-exposure of the stroma and immobilize this LASIK flap, a protocol using fibrinogen, riboflavin and UVA light (RF+FIB+UVA)1 was tested for its ability to adhere the layers of the stroma resulting from LASIK surgery.
To represent the LASIK flap, a model flap was created in the isolated corneas of the Spiny dogfish shark (Squalus acanthias). Then, experimental and control solutions were applied between the stromal flap and underlying stroma. Protocol controls included varying the solution applied between the stromal layers to contain either riboflavin only (RF), fibrinogen only (FIB) or both (RF+FIB). Concentration of RF in RF-containing solutions was 0.1%, and concentration of FIB in FIB-containing solutions was 18%. Experimental corneas received RF+FIB and long wavelength (365nm) ultraviolet light (UVA). To quantitatively measure the adhesion strength, corneas were clipped to a digital force gauge and constant force was applied. The data produced by the force gauge was interpreted by computer software to record the peak tension as the stromal flap was pulled from the underlying stroma surface at a constant rate.
The experimental RF+FIB+UVA protocol generated adhesion that reached an average peak tension of 2.09 Newtons (N) while controls, such as RF only, reached an average peak tension of only 0.76N. Similarly, the current LASIK protocol, which uses no RF, FIB or UVA to seal the LASIK flap, produced an average peak tension of only 0.39N. From the data collected, the RF+FIB+UVA protocol generates an average of a 5-fold increase in adhesion. Further data are being collected from other controls using the albino rabbit cornea (Oryctolagus cuniculus) ex vivo as a mammalian model.
These results suggest that some molecules currently in clinical trials for treating keratoconus2, may also be used to immobilize the LASIK flap onto its laser-modified stroma, thus reducing risk of flap dislodgement. Thank you to all who supported and funded this work: NIH EY0000952; K-INBRE (P20-RR16475); Terry C. Johnson Cancer Center, Kansas State University; and NCRR ME-INBRE (2-P20-RR016463).
1. Khadem J, Truong T, and Ernest JT, Photodynamic Biologic Tissue Glue. Cornea. 1994;13(5):406-410. 2. McCall AS, Kraft S, Edelhauser HF, Kidder GW, Lundquist RR, Bradshaw HE, Dedeic Z, Dionne MJ, Clement EM, and Conrad GW, Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long wavelength ultraviolet radiation (UVA). Invest Ophthalmol Vis Sci. 2010 Jan;51(1):129-138. 3. Zhang Y, Schmack I, Dawson DG, Grossniklaus HE, Conrad AH, Kariya Y, Suzuki K, Edelhauser HF, and Conrad GW, Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas. Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2390-2396
Production of reactive oxygen species in mitochondria from cold- and warm-acclimated striped bass, Morone saxatilis
Kathleen M. Kelley1 and E.L. Crockett2 1University of Maine Farmington, Farmington, Maine 04938 2Department of Biological Sciences, Ohio University, Athens, Ohio 45701
Many cold-blooded animals, including invertebrates and fishes, are able to withstand a range of environmental (body) temperatures. For the animals to persist in varying thermal environments, a number of physiological and biochemical modifications are necessary. Common responses to low body temperatures include an increase in polyunsaturated fatty acid contents in (in order to keep cellular membranes fluid at cold temperatures), and an increase in the content of mitochondria, the primary source of reactive oxygen species including free radicals. As part of our ongoing work to elucidate the consequences of these physiological changes on the susceptibility to oxidative stress, this study investigates the effect of acclimation temperature on the production of reactive oxygen species by mitochondria from striped bass.
Eurythermal organisms regularly experience seasonal or diurnal swings in temperatures and have evolved physiological and biochemical adaptations that enable them to survive in varying thermal environments. One such adaptation is the adjustment in composition and fluidity of biological membranes, which can be altered with even a modest change in temperature4. Many studies have shown that cellular membranes of ectothermic animals acclimated to cold temperatures display an increase in polyunsaturated fatty acids to maintain membrane fluidity4. These alterations, however, should make these membranes more prone to oxidative damage initiated by reactive oxygen species (ROS) 1. At the same time, animals at cold body temperatures also display elevated contents of mitochondria2, the primary source of ROS, which could potentially lead to high rates of production of damaging ROS within the cells of animals living at cold body temperatures. Our research investigates the effects of acclimation temperature on the production of ROS in mitochondria prepared from skeletal muscle of the eurythermal striped bass, Morone saxatilis.
Fingerling M. saxatilis were obtained from Delmarva Aquatics (Smyrna, DE) and held at MDIBL for one week in recirculating 18˚C brackish water (8 ppt) to provide a similar thermal history in all individuals. Temperatures were then increased (or decreased) incrementally from 18˚C at 2.5˚C/day until the desired acclimation temperatures of 22˚C or 9.5˚C were reached. Animals were held at final acclimation temperatures for a minimum of one week before sacrifice. Glycolytic (white) muscle was obtained from the dorsal axial region, and mitochondria were prepared using differential centrifugation according to Moyes et al.5. Activity of the electron transport complex, cytochrome c oxidase (CCO) was measured using a method from Hansen and Sidell3. Amplex UltraRed (AUR, Invitrogen, Carlsbad, CA) was used to quantify production of hydrogen 6 peroxide (H2O2) (according to Treberg et al. ) with mitochondria prepared from temperature acclimated fish. Horseradish peroxidase (20U/ml) and superoxide dismutase
(100 U/ml) were added to the reaction mixture to couple H2O2 and superoxide production to the oxidation of AUR. Preliminary work indicated that the substrate succinate (5 mM) yielded the highest rates of ROS production, and so was used in all reactions. ROS production was monitored over time (3 hours) at a temperature intermediate (15˚C) to the acclimation temperatures. A standard curve of H2O2 was performed alongside the rate assays.
Mitochondria from M. saxatilis acclimated to cold temperatures (9.5˚C) show no statistically significant Figure 1: ROS production in mitochondria from differences in rates of ROS production compared with temperature acclimated M. saxatilis as normalized mitochondria prepared from animals acclimated to a warmer to CCO activity (n = 6). Error bars = SEM. p = temperature (22˚C) when rates of ROS production are either 0.18. normalized to mitochondrial protein (not shown) or CCO
activity (Figure 1). These results indicate that any changes in polyunsaturated fatty acids that occur after one week of temperature acclimation do not impact rates of ROS production in mitochondria.
This research was supported by NSF IOS 0842624. Kathleen Kelley was supported by Maine IDeA Network of Biomedical Research Excellence (2-P20-RR016463).
1. Crockett, EL. The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation, a review. J. Comp. Physiol. B 178: 795-809, 2008. 2. Egginton S, and Sidell, BD. Thermal acclimation induces adaptive changes in subcellular structure of fish skeletal muscle. Am. J. of Physiol. 256: R1-R9, 1989. 3. Hansen, CA, and Sidell, BD. Atlantic hagfish cardiac muscle: metabolic basis of tolerance to anoxia. Am.J. Physiol. 244: R356-R362, 1983. 4. Hazel JR, and Williams, EE. The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog in Lipid Res. 29: 167–227, 1990. 5. Moyes, CD, Buck, LT, Hochachka P, and Suarez, RK. Oxidative properties of carp red and white muscle. J.Exp. Biol. 143, 321-331, 1989. 6. Treberg JR, Quinlan, CL, and Brand, MD. Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production-a correction using glutathione depletion. FEBS J. 277: 2766-2778, 2010.
Molecular and immunohistochemical identification of a sodium hydrogen exchanger-2c (NHE2c) paralog in the gills of marine longhorn sculpin, (Myoxocephalus octodecemspinosus)
Brett Rabeneck, Andrew Diamanduros, and James Claiborne Department of Biology, Georgia Southern University, Statesboro, GA 30460
Sodium hydrogen exchanger proteins are present in all animals for fluid, ion, and acid-base regulation essential for life. Many more of these proteins exist in teleost fish than in mammals as a result of gene duplication that occurred after the divergence of teleosts from the tetrapod lineage. Here we investigate one of the paralogs from the NHE2 subfamily in the marine longhorn sculpin.
Gill sodium/hydrogen antiporter (NHE) proteins are thought to be involved in osmoregulation, cell volume control and acid-base adjustments. NHE’s have evolved into multiple paralogs in the teleost linage following a putative whole genome duplication that did not occur in mammals 4, 6, 7, 9. Here, we have begun to characterize a new NHE isoform found in the gills of marine longhorn sculpin, Myoxocephalus octodecemspinosus which we have designated NHE2c.
Sculpin were caught by fishermen from Frenchman Bay, ME. Total RNA from gill tissue was isolated from longhorn sculpin using the Tri-Reagent RT method of extraction (Molecular Research Center, Inc.; Cincinnati, OH). First-strand cDNA was synthesized with GeneRacer oligo-dT using SuperScript III RT according to manufacture’s protocol (Invitrogen; Carlsbad, CA). The 3’ and subsequently the 5’ ends were obtained with Rapid Amplification of cDNA Ends (RACE) after ligating a Marathon Adaptor (Clontech; Mountain View, CA) to double-stranded, template cDNA, then PCR amplifying with Marathon Adaptor Primers and cross- compatible, Gasterosteus aculeatus, gene-specific primers 8.
A cloned insert (pCR®4-TOPO®plasmid, TOPO TA Cloning Kit for Sequencing; Invitrogen) of the 3’ PCR product was sequenced at the MDIBL. Sequence was verified in three different fish and PCR amplified from 5’ to 3’ ends to gain the entire NHE2c coding sequence. Sequence trace files were aligned with MacVector (MacVector, Inc.; Cary, NC). Once the specific 3’ end was acquired, polyclonal IgG antibodies were synthesized against NHE2c epitopes within the open reading frame (ORF) with the highest antigenicity values (ProSci;
Poway, CA). At the time of excision, gills were also taken for immunohistochemistry (IHC). Tissue was fixed in 4% paraformaldehyde in PBS at pH
7.4 for 24 hours, rinsed in pH 7.4 PBS before being dehydrated in a series of increasing EtOH solutions Figure 1: Phylogenetic tree of selected NHE1-3 sequences for 45 minutes each. Fixed tissue was embedded in using Clustal W from the EMBL-EBI. See Edwards for paraffin wax at 60°C and 5µm sections were sliced Ensembl and NCBI accession numbers5. M. parallel to the lateral plane of the gill arch and octodecemspinosus amino acid sequence NHE2c is not yet published to GenBank. mounted onto Superfrost®Plus microscope slides (Fisher; Pittsburgh, PA).
The putative NHE2c paralog is 3,933 nucleotides in length with 46.13% GC content. The 5’ untranslated region is within the 1,374 bps sequenced up to the KOZAC site. Protein-coding sequence is a 2,388 bp open reading frame (ORF), followed by a 292 bp 3’ UTR region. The ORF codes for a peptide that is 795 amino acids (AA) in length with a molecular weight of 89.2 kDa. The sculpin NHE2c amino acid sequence is 51% identical to the rabbit and rat NHE2 and shows 50% identity with NHE2 in humans. The sculpin NHE2c nucleotide sequence aligns with sculpin NHE2b from 1363 to 3122 with 78% identity in this region. The sNHE2c sequence aligns with the F. heteroclitus NHE2b sequence from 1830-2479 with 79% identity. In Figure 1, the M. octodecemspinosus NHE2c amino acid sequence grouped with G. aculeatus NHE2c amino acid sequence, the only other NHE2c sequence in the multiple alignments and the sequence that was used to make cross- compatible primers.
The species specific NHE2c polyclonal antibody stained thin, elongate structures along the apical edge of the interlamellae regions of the Na+/K+-ATPase-expressing cells (Figure 2, Image A, Na+/K+-ATPase not shown). This staining pattern was seen in the same cells with sculpin NHE2b (not shown), but the latter protein has a more punctate, less apical orientation1. Peptide-competition for IHC, shown in Figure 2 Image B, was incubated in a 3:1 ratio (peptide:antibody) for 4 hours at room temperature at a peptide concentration of 1mg/mL, which is approximately a 50-fold excess of peptide to antibody and showed little or no edge staining. The apical staining of NHE2c noted here is similar to the general location within Na+/K+-ATPase–rich gill cells of NHE2 in the dogfish (Squalus acanthias), and NHE3 in the gills of Atlantic stingray (Dasyatis sabina) and the sand flathead (Platycephalus bassensis), albeit covering a larger area in these latter species 2, 3. Image A Image B
Figure 2: Image A on the left is M. octodecemspinosus gill tissue labeled with a rabbit polyclonal antibody against the putative NHE2c epitope sequenced from marine longhorn sculpin. Image B on the right is gill labeled the peptide- competition incubation in which the rabbit NHE2c antibody that was incubated with its corresponding NHE2c peptide.
NHE2 studies in mammals and fish to date have mostly described one isoform of the subfamily, but the finding of NHE2c paralog in marine longhorn sculpin, unique from NHE2b, lends credence to a new phylogenetic arrangement of the NHE2 subfamily initially proposed by Edwards 5. The functional purpose of the NHE2c is unknown but its location suggests a contribution to apical Na+/H+ exchange in a fashion similar to NHE3.
Funded by NSF IOB-061687 to JBC and GSU GSPDF Research Award.
1. Catches JS, Burns JM, Edwards SL, and Claiborne JB. Na+/H+ antiporter, V-H+-ATPase and Na+/K+-ATPase immunolocalization in a marine teleost (Myoxocephalus octodecemspinosus). J Exp Biol 209: 3440-3447, 2006. 2. Choe KP, Kato A, Hirose S, Plata C, Sindic A, Romero MF, Claiborne JB, and Evans DH. NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 289: R1520-R1534, 2005. 3. Claiborne JB, Choe KP, Morrison-Shetlar AI, Weakley JC, Havird J, Freiji A, Evans DH, and Edwards SL. Molecular detection and immunological localization of gill Na+/H+ exchanger in the dogfish (Squalus acanthias). American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 294: R1092-R1102, 2008. 4. Claiborne JB, Edwards SL, and Morrison-Shetlar AI. Acid-base regulation in fishes: cellular and molecular mechanisms. Journal of Experimental Zoology 293: 302-319, 2002. 5. Edwards SL, Weakley JC, Diamanduros AW, and Claiborne JB. Molecular identification of Na+–H+ exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus. Journal of Fish Biology 76: 415-426, 2010. 6. Grinstein S, Woodside M, Sardet C, Pouyssegur J, and Rotin D. Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism. Journal of Biological Chemistry 267: 23823-23828, 1992. 7. Jaillon O., et al (61 authors). Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431: 946-957, 2004. 8. LaRue K, Tarley, M., Wilbur, B., Diamanduros, A., Claiborne, J. Tissue distribution of NHE isoform transcripts in the longhorn sculpin, Myoxocephalus octodecemspinosus The Bulletin, MDI Biological Laboratory 48: 48-49, 2009. 9. Scott GR, Claiborne JB, Edwards SL, Schulte PM, and Wood CM. Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. Journal of Experimental Biology 208: 2719-2729, 2005.
The effect of KCN on the secretion of chloride by the rectal gland of Squalus acanthias
Rolf Kinne,1 Katherine C. Spokes,2 Anya Silva,3 and Patricio Silva.4 1Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany 2Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 3Grinnell High School, Grinnell IA 50112 4Department of Medicine Temple University School of Medicine, Philadelphia, PA 19140
All cells require fuel to perform their work. This fuel generally comes from outside the cells. This report shows that the cells of the rectal gland of the shark can use fuel stored within the cells to accomplish their work.
Isolated rectal glands perfused without glucose continue to secrete chloride albeit at a rate about 40% of that attained in the presence of glucose.1 In preliminary experiments that were uncontrolled we found that the addition of KCN reduced that rate even further, indicating that the residual secretion of chloride was sustained by endogenous metabolism of an additional source of energy.1 In the present series of experiments we examined the effect of KCN on chloride secretion in the presence and absence of glucose in a more systematic fashion.
Isolated rectal glands of S. acanthias were perfused through their single artery by gravity at 16°C and 40 mm Hg pressure with oxygenated shark Ringer’s solution containing 5 x 10-4M theophylline, in a single pass perfusion. Venous effluent and duct fluid were collected separately from PE-90 catheters placed in the vein and duct of the gland. Collections were made every ten minutes. Glucose at a concentration of 5 mM was used during the initial thirty minutes of all experiments. After thirty minutes of perfusion, the perfusate was changed to an experimental perfusate and collections continued at ten-minute intervals. Reagents were purchased from Sigma-Aldrich. Chloride was measured using a Buchler-Cotlove chlorhidometer. Chloride secretion was calculated from the chloride concentration in the duct fluid, the volume of the fluid, the collection time, and the weight of the gland and expressed as µEq per gram of gland per hour. Statistical analysis was done using Student’s “t” test, or “paired t” as applicable.
The addition of 10-3M KCN to the perfusate of gland perfused with glucose resulted in a rapid decrease in the secretion of chloride to about 15% in all experiments. The results are shown in Figure 1. When the perfusate was changed to a solution that did not contain glucose, the secretion of chloride fell to about 40% of the baseline secretion. The further addition of 10-3M KCN reduced the secretion of chloride to the same level as that observed in the presence of glucose. The results are also shown in Figure 1.
Figure 1. Effect of KCN on the secretion of chloride in the presence and absence of glucose. Control experiments are shown in the open symbols. Squares represent KCN in the presence of glucose. Closed circles depict the effect of KCN in the absence of glucose. Data is shown as percentage of the rate of chloride secretion at time 30 minutes. The arrows indicate the time of KCN addition. The rate of chloride secretion fell significantly (p < 0.01) immediately after the addition of KCN in the presence or absence of glucose. Symbols are mean ± SEM.
To fully support the secretion of chloride stimulated by theophylline or other stimulatory agents, rectal glands require the supply of exogenous fuels. Glucose alone appears to be sufficient to support stimulated secretion of chloride even at high rates of secretion. The previous observation that a significant proportion of the secretion of chloride remained after the removal of glucose suggested that the rectal gland cells contain an endogenous fuel supply capable of supporting at least in part that secretion. The observation that KCN reduces the secretion of chloride in the presence or absence of glucose further supports the notion that there is an endogenous source of fuel. The additional observation that KCN does not completely eliminate the secretion of chloride suggests that a portion of the secretion of chloride is independent of oxidative metabolism, as measured by the sensitivity to KCN, and is probably anaerobic glycolysis.
1. Kinne, R, Spokes, KC, Silva, P. Secretion of chloride and mechanism of transport of glucose in the rectal gland of Squalus acanthias. Bull. Mt. Desert Isl. Bio. Lab. 49: 44-46, 2010.
Glycogen measurement in the rectal gland of Squalus acanthias
Rolf Kinne,1 Katherine C. Spokes,2 and Patricio Silva.3 1Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany 2Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 3Department of Medicine Temple University School of Medicine, Philadelphia, PA19140
Although most cells rely on fuel provided from an external source to perform their work, some cells store reserves of fuel within them. This report shows that the rectal gland cells store glucose in the form of its storage molecule glycogen.
The most likely endogenous energy source for the secretion of chloride in the rectal gland is glycogen. Glycogen granules have been identified in the rectal gland of the round sting-ray Urolophus jamaicensis1 and it is likely that other elasmobranchs share this trait. Glycogen has not been reported in the rectal gland of S. acanthias. In these series of experiments we assayed the rectal gland for evidence of glycogen.
Isolated rectal glands of S. acanthias were perfused through their single artery by gravity at 16°C and 40mm Hg pressure with oxygenated shark Ringer’s solution containing 5 x 10-4M theophylline, in a single pass perfusion. Venous effluent and duct fluid were collected separately from PE-90 catheters placed in the vein and duct of the gland. Collections were made every ten minutes. There was no glucose in the perfusate at any time during the experiments. After the initial thirty minutes of perfusion, a portion of the gland, labeled “PRE,” was tied off and cut coronally and processed for the assay of glycogen. After ninety minutes of perfusion the experiment was terminated, the glands were cut coronally, flash frozen in liquid nitrogen, weighed and a weighed piece, labeled “POST,” transferred into an ice-cold mortar and crushed into small pieces; the tissue pieces were placed into 1:10 volume (weight/volume) of cold 0.03 N HCl, and homogenized at 0° C using a Tekmar homogenizer. The homogenate was boiled for 5 minutes, centrifuged, and a sample of the supernatant was separated and stored at 4°C for the measurement of glucose. The remaining homogenate was then diluted 1:10 in 1 N HCl, boiled for three hours to hydrolyze the glycogen, centrifuged, and the supernatant saved for the glucose determination. The acid extracts of rectal gland tissue were neutralized with equal volumes of 0.03 N or 1 N NaOH. Glucose was then measured using a glucose oxidase assay kit purchased from Sigma. The assay kit was adapted for small volumes for use with a spectrophotometer plate reader, and read at 540 nm. The glycogen content was determined by subtracting the amount of glucose measured in the initial homogenate from the level found after the acid hydrolysis, values are expressed as micromoles per gram of tissue. Values are reported as mean±SD.
Table 1 Glucose content in rectal glands 29-32 in μmoles/g tissue Gland # Free glucose Total glucose after hydrolysis Glycogen derived glucose 29 PRE 5.4 45.9 40.5 29 POST 5.1 44.3 39.2 30 PRE 12.7 40.3 27.6 30 POST 6.1 23.9 17.8 31 PRE 19.1 36.4 17.3 31 POST 7.8 28.6 20.8 32 PRE 6.3 30.7 24.4 32 POST 5.34 28.7 23.4
Mean PRE 10.9±6.4 38.3±6.4 27.5±9.7 Mean POST 6.1±1.2 31.4±8.9 25.3±9.5
The results are shown in Table 1. In all glands investigated glucose content after acid hydrolysis was significantly higher. The average amount of glucose in the cells was 8.4 μM/g, and the average amount of glucose derived from glycogen was 26.4 μM/g. Thus, rectal gland cells of S. acanthias contain significant amounts of glycogen. Interestingly there is also free glucose in glands perfused without glucose, suggesting breakdown of glycogen under these experimental conditions. However, due to the large scatter of the results no statistical significant difference could be detected either in content of free glucose, total glucose after hydrolysis, or glucose derived from glycogen between samples taken at the beginning or after a period of chloride secretion.
These experiments indicate that rectal gland cells contain a substantial amount of glycogen to provide glucose to the cells. The glycogen content did not change significantly after an hour of perfusion further suggesting that there is enough glycogen stored for the metabolic needs of the cells even at a high rate of chloride secretion. A rough calculation of the amount of glycogen expected to be used for active chloride secretion during the perfusion period suggests that only a small percentage would be hydrolyzed, well within the experimental error of the glycogen determination. Of note, there is sufficient free glucose generated intracellularly to support the secretion of chloride even when there is no glucose in the perfusate. In fact the amount of glucose present in the cells is similar if not larger than the concentration of glucose in the perfusate; such intracellular accumulation would be expected from the operation of SGLT1 in the plasma membrane.2
1. Doyle, WL. Tubule cells of the rectal gland of Urolophus. Am. J. Anat. 111: 223-237, 1962. 2. Silva, P, Spokes, KC, Kinne, R. Molecular identification of a sodium-glucose cotransporter in the rectal gland of S. acanthias. Bull. Mt. Desert Isl. Bio. Lab. 49: 47-50, 2010. Complete nucleotide sequence and translated protein sequence of a sodium-glucose cotransporter in the rectal gland of the spiny dogfish (Squalus acanthias)
Patricio Silva, 1 Katherine C. Spokes,2 and Rolf Kinne.3 1Department of Medicine Temple University School of Medicine, Philadelphia, PA 19140 2Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 3Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany
Glucose needs to enter the cells to be useful as a fuel, but it cannot cross the cell membrane. This report identifies a transporter that moves glucose into the cells of the rectal gland of the spiny dogfish, Squalus acanthias.
We previously reported the partial identification of a sodium glucose transporter present in the rectal gland of S. acanthias.1 In the present series of experiments we extended the sequence beyond that previously reported.
A rectal gland and a piece of spiral valve from a single dogfish were homogenized separately in lysis buffer from Qiagen using a Tekmar tissue homogenizer. The homogenate was passed through a Qiagen shredder column. mRNA was prepared using Qiagen Rneasy minikit, and treated with DNase. Single strand cDNA was prepared using an Invitrogen First-Strand synthesis kit. PCR amplification was done using RedTaq ready mix from Sigma and the primers shown in Table I. Primers for Na-K-ATPase, also shown in Table I, were used as controls. The amplified products were separated in a 2% agarose gel in TAE. The products were eluted using MinElute Gel extraction kit from Qiagen, purified and sequenced in the MDIBL DNA Sequencing Core.
Table I Primer sequence Predicted # bases Starting base Sodium/Glucose cotransporter 1 Left tcagtcaggacctctgctca 436 24 Right cgcctcttcaggtactcagg 459 2 Left cagagctggagttgtgacca 439 418 Right ccggaacaagtggaatgagt 856 3 Left aagtgttacacgcccagacc 669 815 Right aaagatggccagcaagaaga 1483 4 Left tcttcttgctggccatcttt 704 1464 Right gttccgtgtttcgatctggt 2167 5 Left acgcggggagtgagcctcc 459 1 Right cgcctcttcaggtactcagg 459 6 Left tcttcttgctggccatcttt 764 1464 Right tcaaggcctgaaaacagaaa 2226 Na-K-ATPase Left gacagctctttggtggcttc 679 373 Right gcttcaagccagctgtatcc 1051
Figure 1. Agarose gel showing the products of six PCR reactions using six different pairs of primers for six different portions of the SGLT1 cDNA. The primers used and labeled 1 through 6 are shown in Table I. All primers yielded products of the expected number of bases in both the rectal gland and the intestine. Primers for Na-K- ATPase shown as N were used as control and also resulted in a product of the expected number of bases. The results are shown in Figure 1. All the primers resulted in products of the predicted size both in the rectal gland and in the intestine. The bands were cut out eluted and submitted for sequencing to the MDIBL DNA Sequencing Core using the same primers. The resulting sequences were spliced together and the combined sequence is shown in Figure 2.
Figure 2. This figure shows the complete sequence of a sodium- glucose cotransporter present in both the rectal gland and the intestine of S. acanthias.
Figure 3. Aminoacid sequence translated from the nucleotide sequence shown in Figure 2.
The sodium-glucose cotransporter identified in the rectal gland and intestine of S. acanthias is identical to that previously described in the intestine of the same species (AM184079.1). This sodium-glucose cotransporter is a member of the superfamily of sodium dependent solute transporters.2, This co-transporter has significant homology to all known sodium dependent glucose transporters. Although there may be other glucose transporters in the rectal gland cells, based on the observation that phlorizin inhibits chloride secretion3 this may be the transporter that mediates the entry of glucose from the blood into rectal gland cells.
1. Silva, P, Spokes, KC, and Kinne, R. Molecular identification of a sodium-glucose cotransporter in the rectal gland of S.acanthias. Bull. Mt. Desert Is. Bio. Lab. 49: 47-50, 2010. 2. Saier, MH, Jr, Beatty, JT, Goffeau, A, Harley, KT, Heijne, WH, Huang, SC, Jack, DL, Jähn, PS, Lew, K, Liu, J, Pao, SS, Paulsen, IT, Tseng, TT, and Virk, PS. The major facilitator superfamily. J Mol Microbiol Biotechnol. 1: 257-279, 1999. 3. Kinne,R, Spokes, KC, and Silva, P. Secretion of chloride and mechanism of transport of glucose in the rectal gland of Squalus acanthias. Bull. Mt Desert Is. Bio. Lab. 49: 44-46, 2010.
Molecular identification of glycogen synthase in the rectal gland of Squalus acanthias
Patricio Silva,1 Katherine C. Spokes, 2 and Rolf Kinne.3 1Department of Medicine Temple University School of Medicine, Philadelphia, PA 19140 2Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 3Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany
Many cells store glucose, the most common exogenous fuel, to maintain a fuel source necessary for their work. Glucose is stored in the form of glycogen. Glycogen is built up when there is enough glucose and taken down when it is scarce. This report shows that rectal gland cells have glycogen synthase, the enzyme that builds up glycogen from glucose, and can therefore make glycogen.
When the rectal gland of the shark is perfused in vitro without glucose it continues to secrete chloride albeit at a much reduced rate.1 This observation suggests that the rectal gland cells contain an endogenous source of energy. Electron microscopic studies have shown that the rectal gland cells contain granules with the morphology of glycogen granules.2 To determine whether rectal gland cells have the capacity to synthesize glycogen we designed experiments using rtPCR to ascertain whether these cells have glycogen synthase. A sequence for glycogen synthase from the shark has not yet been published.
A rectal gland and brain from a single dogfish were homogenized in lysis buffer from Qiagen using a Tekmar tissue homogenizer. The homogenate was passed through a Qiagen shredder column, messenger RNA was prepared using Qiagen RNAeasy minikit, and treated with DNase. Single strand cDNA was then prepared using an Invitrogen First-Strand synthesis kit. PCR amplification was done using RedTaq ready mix from Sigma and the degenerate primers shown in Table I. The amplified products were separated using 2% agarose gel in TAE. The products were eluted from the gel using MinElute Gel extraction kit from Qiagen, purified and sequenced at the MDIBL DNA Sequencing Core.
Table I Primer sequence Predicted # bases Starting base Glycogen synthase Left 5’-garttycaraayytncaygc-3’ 676 1086 Right 5’-ccntggggntayacnccngc-3’ 1762 Left 5’-garttccagaayytgcaykc-3’ 676 1086 Right 5’-grnggsggctcycaccagtg-3’ 1762 Left 5’-garttccagaaiitgcaikc-3’ 676 1086 Right 5’-grscgsggctgycacctykg-3’ 1762 SGLT1 Left 5’-cagagctggagttgtgacca-3’ 439 418 Right 5’-ccggaacaagtggaatgagt-3’ 856
Figure 1. rtPCR amplification of glycogen synthase from S. acanthias rectal gland and brain. The first primer pair yielded a product of the expected size. The other two did not result in any amplification. The control SGLT1 resulted in a product of the expected size.
The primers were designed using the consensus sequence of the known sequences for glycogen synthase from zebra fish D. rerio, zebra finch T. guttata, and P acific oyster C. pacifica. A primer for sodium dependent glucose transporter 1, known to work in shark tissues was used as a control. Three different degenerate primer pairs for the same region of glycogen synthase were used, and the results are shown in Figure 1. Only the first primer pairs yielded results of the expected size. The control primer pair for sodium dependent glucose transporter 1 (SGLT1) resulted in a product of the expected number of bases.
The amplified product yielded the sequence shown in Figure 2. The sequence is 68% to 75% similar to that of glycogen synthase 1 and 2 in a variety of tissues in species ranging from invertebrates to vertebrates. The amino acid sequence translated from the base sequence is shown in Figure 3.
The results reported here show that the cells of the rectal gland of S. acanthias express glycogen synthase and are therefore capable of synthesizing glycogen. Thus, glycogen stored in the gland can serve as endogenous fuel when the exogenous sources of energy are not available.
CACATCCAGGAGTTTGTCCGAGGGCACTTCTACGGATGTCTG
AACTTTGACCTAGATAAGACCCTGTTCATTTTCATCGCGGGAA
GATACGAGTTCTCAAACAAGGGTGCTGACGTATTTCTAGAAG
CTCTGGCTCGTCTAAATTATCTGCTCAAGGCGAACCGAAGTG
AAGTAACCGTGGTTGCGTTTTTCATCATGCCTGCGAAGACCA
ATAACTTCAACGTAGAAACTCTCAAGGGACAGGCAGTACGGA
AACAGCTTTGGGACACCGCCAATGTGGTCAAAGAAAAGTTTG Figure 2. Partial nucleotide sequence of GCAAAAAGCTTTACGAGTCACTCTTAGAAGGTGAGATGCCTG glycogen synthase from the rectal gland of S. ATATAAACTCAATTCTGGATCGCGAGGACTTCACTATGATGA acanthias. The sequence contains 596 bases. AAAGAGCCATATATGCTACTCAGCGCCGTTCGCTGCCTCCTAT
CTGCACGCATAACATGCTGGATGATTCCAATGATCTCATTCTC AAGGCAGTTCGAAGGATTGGATTGTTTAATAGCACGAGTGAC AGAGTCAAGGTCATCTTTCACCCAGAGTTCCTGTCGTCCACCA GCCCATTGCTCCCTTTGGATTATGAGGAGTTTGTGCGTGGCTG CCA
IQEFVRGHFYGCLNFDLDKTLFIFIAGRYEFSNKGADVFLEALA RLNYLLKANRSEVTVVAFFIMPAKTNNFNVETLKGQAVRKQL Figure 3. Amino acid sequence translated WDTANVVKEKFGKKLYESLLEGEMPDINSILDREDFTMMKRAI from the base sequence shown in Figure 2. YATQRRSLPPICTHNMLDDSNDLILKAVRRIGLFNSTSDRVKVIF HPEFLSSTSPLLPLDYEEFVRGC
1. Kinne, R. Spokes, KC, and Silva, P. Secretion of chloride and mechanism of transport of glucose in the rectal gland of Squalus acanthias. Bull. Mt. Desert Isl. Bio. Lab. 49: 44-46, 2010. 2. Doyle, WL. Tubule cells of the rectal gland of Urolophus. Am. J. Anat. 111: 223-237, 1962. Failure to identify a urea transporter in the rectal gland of Squalus acanthias
Patricio Silva,1 Katherine C. Spokes,2 Alice R. Villalobos,3 and Rolf Kinne.4 1Department of Medicine Temple University School of Medicine, Philadelphia, PA 19140 2Department of Medicine Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 3Department of Nutrition and Food Science, Texas A&M University, College Station, TX 77853 4Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany
Sharks need to get rid of the salt that they take in when they eat. The organ that performs this is the rectal gland. The rectal gland secretes a solution of salt back into the ocean that is more concentrated than the salt in the blood of the animal. This increase in concentration is due to the absence of urea in the secretion, and this report explains that this absence is due to the lack of urea transporters in the cells of the rectal gland.
The concentration of salt in the fluid secreted by the rectal gland averages 450 mM, sixty percent greater than the concentration of salt in the dogfish blood or in the Ringer’s solution used to perfuse the isolated rectal gland; the osmolality of the blood, perfusate, and secreted fluid being the same. We, and others, have assumed that the difference in the concentration of salt is due to the fact that the concentration of urea in the blood or perfusate is 350 mM while there is little or no urea in the fluid secreted by the gland. In fact, such an assumption seemed warranted by experiments that showed that decreasing the concentration of urea in the perfusate of isolated glands decreased the concentration of salt in the secreted fluid.1 Conversely, increasing the concentration of urea in the perfusate increased the concentration of salt in the fluid.1 The absence of urea from the secretion of the gland indicates that the rectal gland cells represent a barrier for the diffusion of urea across the rectal gland epithelia. The site for this barrier whether apical or basolateral is not known. There are no reports on the concentration of urea in the rectal gland cells. We decided to look into this issue by ascertaining whether the rectal gland of Squalus acanthias expresses a urea transporter, necessary for urea to get into the cells.
A piece of rectal gland, intestine, kidney, the brain and the choroid plexus from a single dogfish and a rectal gland from a skate were homogenized in lysis buffer from Qiagen using a Tekmar tissue homogenizer. The homogenate was then passed through a Qiagen shredder column. Messenger RNA was then prepared using Qiagen RNAeasy minikit, and treated with DNase. Single strand cDNA was then prepared using an Invitrogen First-Strand synthesis kit. PCR amplification was then done using RedTaq ready mix from Sigma and the primers shown in Table I. The amplified products were separated using 2% agarose gel in TAE.
Table I Primer sequence Preicted # Starting bases base Urea transporter Left 5’-ctatccctgtgggagttgga-3’ 191 754 Right 5’-atgcaggcaaggacagagtt-3’ 945 Left 5’-gcagggctttatgggtacaa-3’ 387 435 Right 5’-aacaaggaaaatgccaccag-3’ 821 NaKATPase Left 5’-gacagctctttggtggcttc-3’ 680 371 Right 5’-gcttcaagccagctgtatcc-3’ 1051 NKCCT Left 5’-cgagatctgcttgtggaaca-3’ 200 1527 Right 5’-cattggctgggatgaaagtt-3’ 1728 SGLT1 Left 5’-aagtgttacacgcccagacc-3’ 669 815 Right 5’-aaagatggccagcaagaaga-3’ 1483
The primers for urea transporter were designed using the reported sequence for a S. acanthias urea transporter.2 Primers for NKCCT, NaKATPase, and sodium dependent glucose transporter 1 (SGLT1) that have previously been reported3 were used as controls. The results are shown in Figure 1. Primers for NaKATPase resulted in a product of the expected number of bases in the kidney. SGLT1 primers produced results of the expected size in rectal gland, intestine, kidney, brain and choroid plexus. The primers for NKCCT resulted in a product of the predicted size in the rectal gland of the skate (data not shown). The primers for urea transporter did not produce any results in rectal gland of S. acanthias or R. erinacea, or in S. acanthias intestine. These primers resulted in the appropriate sized products in kidney, brain and choroid plexus samples.
In accordance with previous reports,2 urea transporter mRNA, as demonstrated by rtPCR, is present in kidney, brain, and choroid plexus of S. acanthias. No signals were obtained when using mRNA from either intestine of S. acanthias or rectal gland of either S. acanthias or L. erinacea. It is certainly possible that there is a different urea transporter in the rectal gland and intestine of S. acanthias that was not identified by these experiments. However, the apparent absence of urea transporter from the rectal gland does agree with the physiological observation of rectal gland function. There is little or no urea in the secretion of the rectal gland. The osmolality of the secretion of the rectal gland varies in proportion with the concentration of urea in the perfusate indicating that urea does not cross the rectal gland epithelia.1 The absence of a urea transporter in the gland suggests that the barrier for urea is at the basolateral cell membrane. In fact, very low urea permeability has been demonstrated in both apical and basolateral membranes of rectal gland cells.4 The absence of urea transporter from the intestine is in keeping with the rectal gland being an appendix of the gastrointestinal tract.
Figure 1. rtPCR amplification of urea transporter from S. acanthias rectal gland, kidney, intestine, brain and choroid plexus. Primer pairs for NakATPase and SGLT1 yielded a products of the expected size in all the tissues examined. The urea transporter was only identified in kidney, brain and choroid plexus.
1. Silva, P, Stoff, JS, Solomon, RJ, Rosa, R, Stevens, A, and Epstein, J. Oxygen cost of chloride transport in perfused rectal gland of Squalus acanthias. Journal of Membrane Biology. 53: 215-221, 1980. 2. Smith, CP, and Wright, PA. Molecular characterization of an elasmobranch urea transporter. Am. J. Physiol. 276: R622-R626, 1999. 3. Silva, P, Spokes, KC, and Kinne, R. Molecular identification of a sodium-glucose cotransporter in the rectal gland of S. acanthias. Bull. Mt. Desert Isl. Bio. Lab. 49: 47-50, 2010. 4. Zeidel, JD, Mathai, JC, Campbell, JD, Ruiz, WG, Apodaca, GL, Riordan, J, and Zeidel ML. Selective permeability barrier to urea in shark rectal gland. Am. J. Physiol. 289: F83-89, 2005. Chemical and molecular chaperones and their importance to dogfish (Squalus acanthias) hemoglobin-oxygen affinity following temperature and osmotic stress
Suzanne Currie1, Ashra Kolhatkar1, Nathan S.B. Walker1 and A. Kurt Gamperl2 1Department of Biology, Mount Allison University, Sackville, NB, Canada E4L 1G7 2Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NF, Canada A1C 5S7
In most cells, proteins are protected from cellular stress by both chemical and molecular chaperones – organic compounds that stabilize protein folding. Using the dogfish red blood cell as a model, our goal was to determine if these chaperones are required to preserve cell function in the face of high temperature or osmotic stress. We show that hemoglobin-oxygen affinity, a measure of red blood cell function, is not dependent on the presence of chemical or molecular chaperones.
Elasmobranchs are marine osmoconformers, and use the organic osmolytes trimethylamine oxide (TMAO) and urea to maintain the osmotic concentration of their internal fluids similar to that of their environment. TMAO is also known to rescue proteins experiencing temperature-induced folding defects, and thus functions as a chemical chaperone5. The heat shock proteins (HSPs) are molecular chaperones that also prevent protein denaturation. Recently, we demonstrated that acute heat stress does not induce HSP70 expression in red blood cells of the spiny dogfish shark if physiological levels of TMAO are present2, but that hypo-osmotic stress only results in an induction of HSP70 in cells with TMAO. Furthermore, we showed that cells experiencing hypo- osmotic stress have significantly more oxidative damage despite the protection afforded by both TMAO and HSPs. Thus, the goal of this work was to determine if cellular function is compromised following: 1) heat stress when cells are protected by TMAO but not HSPs, and 2) hypo-osmotic stress when cells have both TMAO and HSPs but appear to incur some degree of cellular damage. To this end and given that the main function of vertebrate red blood cells is to carry and deliver oxygen, we used hemoglobin-oxygen affinity as an indicator of cell function in dogfish red blood cells.
We sampled whole blood from pithed spiny dogfish in a procedure approved by the MDIBL IACUC. Blood was washed in elasmobranch saline, resuspended at a hematocrit of 15% in saline with and without TMAO and stored at 4°C for 24-48 h1. Red blood cells were then placed in a shaking water bath at 13°C for 1 h, and then subjected to either a 1 h heat shock at 24°C or a hypo-osmotic shock (at 50% NaCl concentration). We constructed oxygen dissociation curves3 using the Tucker method4 and a Cameron OM 200 oxygen meter prior to the stress at 13°C (control) and 2 and 20 h following the initiation of heat or hypo-osmotic stress. Using Hill plots, we calculated the PO2 at which hemoglobin was 50% saturated with oxygen (P50) to determine if hemoglobin-oxygen affinity is affected by the stress (heat or hypo-osmotic) and TMAO.
A. Heat Shock B. Hypo-osmotic shock
Figure 1. P50 values (mean ± SEM) for dogfish red blood cells incubated with (dark bars) and without (light bars) TMAO, before (control) and 2 and 20 h following a 1 h heat shock (A) or 2 and 20 h into a hypo-osmotic stress (B). N = 6 for both experiments. Two-way repeated measures ANOVAs revealed no significant differences between groups in both the heat shock (time: p = 0.881; TMAO: p = 0.645) and hypo-osmotic (time: p = 0.984; TMAO: p = 0.933) experiments. Our results to date indicate that neither heat nor hypo-osmotic stress significantly affect how tightly oxygen is bound to hemoglobin in dogfish red blood cells (Figure 1). Furthermore, the presence or absence of exogenous TMAO in these cells does not impact P50. These data suggest that hemoglobin - oxygen affinity is preserved in the face of potentially damaging stressors regardless of the presence of chemical or molecular chaperones.
This research was supported by an MDIBL Blum Halsey Fellowship and an NSERC Discovery grant to SC, and an NSERC Undergraduate Research Award to NSBW.
1. Caldwell, S., Rummer, J.L. and Brauner, C.J. Blood sampling techniques and storage duration: Effects on the presence and magnitude of the red blood cell β-adrenergic response in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. A 144: 188-195, 2006. 2. Currie, S and Robertson, C. Cellular and molecular chaperones in the red blood cells of the spiny dogfish, Squalus acanthias. Mount Desert Island Biological Laboratory Bulletin 49: 58-59, 2010. 3. Gollock, M. J., Currie, S., Petersen, L.H. and Gamperl, A.K. Cardiovascular and haematological responses of Atlantic cod (Gadus morhua) to acute temperature increase. J. Exp. Biol. 209: 2961-2970, 2006. 4. Tucker, V.A. Method for oxygen content and dissociation curves on microlitre blood samples. J. Appl. Physiol. 23: 410-414, 1967. 5. Welch, W.J. and Brown, C.R. Influence of chemical and molecular chaperones on protein folding. Cell Stress Chap. 1: 109-115, 1996.
Analysis of non-heme iron levels from select tissues from cold- and warm-acclimated striped bass (Morone saxatilis)
Mark S. Wheeler1, David W. Tapley1 and E.L. Crockett2 1Department of Biology, Salem State University, Salem, Massachusetts 01970 2Department of Biological Sciences, Ohio University, Athens, Ohio 45701
Ectotherms, such as striped bass (Morone saxatilis), living in cold water face several oxidative stresses, including higher levels of oxygen, and polyunsaturated fatty acids, and an abundance of mitochondria when compared to animals in warmer water. We investigated non-heme iron, which may cause oxidative stress, to see whether concentration varied with acclimation temperature. Elevated levels of non-heme iron have long been associated with oxidative stress. We wished to see if any particular tissues were at a greater risk of such damage and to determine if a short acclimation at elevated or lowered temperatures would in fact cause a significant change in non-heme iron content in either acclimation group.
Eurythermal animals, such as striped bass (Morone saxatilis), regularly experience swings in temperatures and have evolved physiological and biochemical adaptations that enable them to survive these extreme conditions. Many studies have shown that cellular membranes of ectothermic animals acclimated to cold temperatures display an increase in polyunsaturated fatty acids to maintain membrane fluidity2. These alterations, however, should make these membranes more prone to oxidative damage initiated by reactive oxygen species (ROS) 1, particularly those generated by greater numbers of mitochondria. Increased ROS may lead to elevated superoxide activity, which in turn may lead to elevated levels of free-iron which induces oxidative damage to DNA3. We investigated concentrations of non-heme (free) iron from two acclimation groups of striped bass, one group acclimated at warmer temperatures and the other acclimated at lower temperatures.
Fingerling M. saxatilis were obtained from Delmarva Aquatics of Delaware and held at MDIBL for one week in recirculating, brackish water (8 ppt) at the local ambient sea water temperature (18˚C) to provide a common thermal history in all individuals. Temperatures were then altered at 2˚C/day or 1˚C/day on alternating days from 18˚C until final acclimation temperatures of 22˚C or 9.5˚C were reached. Animals were held at final acclimation temperatures for a minimum of one week prior to sacrifice. Glycolytic (white) muscle was obtained from the dorsal axial region, as was oxidative (red) muscle. The hearts (excluding the bulbus arteriosis), whole spleens, and the right distal lobes of the livers were collected, avoiding bile ducts. Non-heme iron was analyzed using a micro-scale assay described by Figure 1. Comparison of non-heme iron Rebouche, Wilcox, and Widness4, but modified to utilize a concentrations in samples of homogenized cardiac tissue. (Warm n=6, Cold n=7) NanoDrop ND-1000™ UV/VIS spectrophotometer to allow for smaller samples.
Non-heme iron concentrations from M. saxatilis showed no statistically significant differences between acclimation groups, except in cardiac tissues, in which cold acclimated fish showed a 52% increase in iron concentrations when compared to warm acclimated fish, representing a P-value of 0.027 (Figure 1). This could indicate that cardiac tissue in cold acclimated fish may be more prone to oxidative damage.
This research was supported by NSF IOS 0842624.
1. Crockett, EL. The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation, a review. J. Comp. Physiol. B 178: 795-809, 2008. 2. Hazel JR and Williams EE. The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog in Lipid Res. 29: 167–227, 1990. 3. Keyer J and Imlay JA. Superoxide accelerates DNA damage by elevating free-iron levels. PNAS 93: 13635-13640, 1996. 4. Rebouche CJ, Wilcox CL, and Widness JA. Microanalysis of non-heme iron in animal tissues. J. Biochem. Biophys. Methods 58: 239-251, 2004.
Factors contributing to desiccation tolerance by killifish embryos (Fundulus heteroclitus)
Robert L. Preston1,5, Michaela K. Petit2, Jennifer L. Willis3, Arhea V. Marshall4 and Sirilak Chuaypanang1 1School of Biological Sciences, Illinois State University, Normal, IL 61790, 2 Arcadia University, Glenside, PA 19038 3 Southern Maine Community College, South Portland, ME 04106 4 High School for Math, Science and Engineering at City College of New York, NY 10031 5Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
Killifish spawn at the margins of estuaries and stranded embryos can develop normally in air in about 14 days. Hatching is triggered by immersion in seawater at spring tides. Aerially developing embryos may be severely desiccated but under some conditions can develop and hatch normally. We found that these embryos resist desiccation by increasing proteins that stabilize enzymes (heat shock proteins). In addition, desiccation damage is likely to be ameliorated by high concentrations of free amino acids that function as compatible solutes to stabilize proteins in tissues.
During late May through July the northern killifish subspecies, Fundulus heteroclitus macrolepidotus, spawns during high tides and spring tides at the edges of estuaries in brackish water (about 10 ppt)3. Some embryos remain immersed and some are stranded in vegetation and on the rocky shoreline by the ebbing tide and therefore are exposed to air and desiccation stress for extended periods2,3. Flooding the aerially incubated embryos with seawater at spring tides after 14 days triggers hatching. In the laboratory, aerially incubated embryos have greater viability and hatching success2. During mid-stages of development (5-9 days, stages 28- 33)1 killifish embryos can resist severe desiccation for short periods (2-6 hrs)6 and this results in increased hatching success compared with early stage embryos (0-2 days, stages 1-20)1 and late stage embryos (12-14 days, stages 36-39)1,6. Mid-stage embryos can tolerate severe desiccation conditions, 22% relative humidity (RH) for 2 hours, that always kill early and late stage embryos6.
We proposed that several physiological factors may play a role in enhanced desiccation tolerance by mid- stage embryos including: down regulation of aquaporins (AQPs) in mid-stage embryos7, constitutive and/or induced expression of stress proteins (heat shock proteins, HSPs)6 especially HSP 90; and the presence of high levels of compatible solutes that may help stabilize cellular native protein structure during water loss5. The preliminary data presented here focus on two of these factors, HSP mRNA expression as a function of embryo developmental age and the presence of glucose as a possible compatible solute in embryo tissues6,7. We previously measured total free amino acid concentrations of about 40mM - 60mM in killifish embryos at all developmental stages and concluded that at these concentrations they are likely to be a major component of possible compatible solutes5.
Killifish were collected from Northeast Creek, Mount Desert Island, ME, and held in aquaria with running natural SW (about 30 ppt). Eggs and milt were expressed manually into a beaker containing 10 ppt artificial seawater (Instant Ocean, Mentor, OH; ASW). After a 30 minute incubation, the embryos were placed on filter paper moistened with 10 ppt ASW for aerial incubation at 20o C in a closed chamber whose vapor phase was in equilibrium with 10 ppt ASW. The embryos developed normally over 14 days and at maturity hatched after flooding with 10 ppt ASW. For HSP90 experiments, primer pairs were designed from the alignment of fish HSP90 sequences in Genbank. Total RNA was isolated from the embryos preserved in RNAlater (Qiagen, Valencia, CA) using RNeasy Plus Midi Kit (Qiagen, Valencia, CA). RNA concentration was determined spectrophotometrically (NanoDrop, Wilmington, DE). cDNA was synthesized using the Quantitech reverse transcriptase (RT) Kit (Qiagen, Valencia, CA). Quantitative PCR was performed on a Stratagene (Stratagene, La Jolla, CA) real-time PCR machine (for more detail7).
The relative expression of HSP90 mRNA in control developing embryos (2, 7 and 14 days old) and desiccated embryos was determined using F. heteroclitus ornithine decarboxylase as a internal reference which has been shown by other workers as an adequate internal reference in this species8 (Fig 1). Control embryos were incubated in air in equilibrium with 10ppt SW. Desiccated embryos were exposed to 22.5% RH for 2 hours using an incubation chamber containing saturated K acetate solution4 and then returned to control incubation conditions. The relative expression of HSP90 mRNA increased in as a function of developmental stage in both 1600 control and desiccated embryos. We predicted that there may be a peak in HSP90 mRNA expression in the 7 day old embryos, but these data show a 1200 consistent increase with development. Earlier experiments showed variability in these patterns, 800 but in general the increase HSP90 mRNA expression was observed. This consistent increase Fold change seemed reasonable since aerially incubated embryos 400 are exposed to desiccation stress throughout all stages. These data were also consistent with 0 preliminary Western blots (data not shown) that 2D 7D 14D show expression of HSP90 at all stages.
Figure 1. Relative expression of HSP90 mRNA compared In the glucose experiments 7 day old embryos with ornithine decarboxlase in Fundulus embryos at three treated (as described above) in control and developmental ages. The white bars are controls and the dark desiccated conditions were extracted by boiling in bars embryos exposed to 22% relative humidity (RH) for 2 water for 5 min and centrifuged. In control hrs and then returned to normal conditions (see text). The values are ratios of the means ± SE for triplicate experiments, this treatment denatures proteins but measurements. The values are significantly different for all does affect detection of glucose. The supernatant comparisons at p ≤ 0.05 or better (Student’s t-test). was analyzed for glucose using a colorimetric coupled enzyme microplate assay (EnzyChrom Glucose Assay Kit; BioAssay Systems, Hayward, CA) and a standard addition protocol that corrects for endogenous inhibitors of the assay. A minimum of three replicate experiments were done. The data showed that the difference between control and desiccated embryos was not significant (p = 0.1) using the standard criterion (p ≤ 0.05). Additional repeat measurements may nudge the data into statistical significance. The apparent concentration corrected for embryo water content and estimated tissue content8 ranged from 95.6 ± 57.9 µM in control embryos to 289.7 ± 77.1 µM in desiccated embryos which had 25% less water content after desiccation. These values were much lower than those determined previously using a glucometer for human blood recalibrated for killifish extracts (4-9 mM)7 which suggests that the glucometer method may be inappropriate for animal extracts, or must be calibrated in a way that corrects for interfering substances. The conclusion is that glucose concentrations in embryos measured by either method are not likely to substantially contribute to the pool of compatible solutes (predominately free amino acids) that stabilize cell proteins.
Support: M. Petit, NSF- REU (DBI-0453391); J. Willis, Maine IDeA Network of Biomedical Research Excellence (2-P20-RR016463); A. Marshall, SETH program (Science Education Through Health).
1. Armstrong, PB and Child, JS. Stages in the normal development of Fundulus heteroclitus. Bio Bull. 128: 143-168, 1965. 2. Baldwin, JL, Petersen, CW, Preston, RL and Kidder, GW. Aerobic and submerged development of embryos of Fundulus heteroclitus. Bull. Mt. Desert Isl. Biol. Lab. 45: 45-46, 2006. 3. Petersen, C.W., S. Salinas, R. L. Preston, and G. W. Kidder III (2010) Spawning periodicity and reproductive behavior of Fundulus heteroclitus in a New England salt marsh. Copeia : 203-210, 2010. 4. Preston, RL, Flowers, AE, Lahey, BC, McBride, SR, Petersen, CW and Kidder, GW. Measurement of the desiccation of Fundulus heteroclitus embryos in controlled humidities. Bull. Mt. Desert Isl. Biol. Lab. 45: 101-103, 2006. 5. Preston, RL, Baumhardt, PE, Petit, ET, Ruensirikul, S, Kidder, GW. Measure of apparent free amino acid and protein content in single Fundulus heteroclitus embryo during development. Bull. Mt. Desert Isl. Biol. Lab. 46: 133- 135, 2007. 6. Preston, RL, Edwards, BR, Baumhardt, PE, Lantigua, J, Ruensirikul, S and Kidder, GW. Desiccation resistance by mid-stage Fundulus heteroclitus embryos. Bull. Mt. Desert Isl. Biol. Lab. 47: 94-96, 2008. 7. Preston, RL, Petit, MK, Fontaine, EP, Clement, EM, Ruensirikul, S. Factors contributing to desiccation tolerance by Fundulus heteroclitus embryos. Bull. Mt. Desert Isl. Biol. Lab. 49: 53-55, 2010. 8. Tingaud-Sequeira A, Zapater C, Chauvigné F, Otero D, Cerdà J. Adaptive plasticity of killifish (Fundulus heteroclitus) embryos: dehydration-stimulated development and differential aquaporin-3 expression. Am J Physiol Regulat. Integr. Comp Physiol. 296: 1041-1052, 2009. Adult sea lamprey (Petromyzon marinus) tolerates biliary atresia through renal excretion of bile salts and organic solutes
Shi-Ying Cai1, Chu-Yin Yeh2, Daniel Lionarons1, Victoria Smith3, Weiming Li2, and James Boyer1,3 1 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 2 Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824 3 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672
The adult sea lamprey is a unique cholestatic animal model of biliary atresia which maintains normal plasma bile salt homeostasis despite loss of its biliary system. Here we found that adult lamprey maintains normal plasma bile salt and biliverdin levels mainly by excreting these substrates through its kidneys although small amount of these compounds can also be eliminated through the intestine. Understanding these mechanisms at the molecular level may lead to new medical treatment for cholestasis.
Sea lamprey (Petromyzon marinus, here after lamprey) larvae lose their bile ducts and gallbladder during metamorphosis/transformation to the adult form and thus resemble biliary atresia in humans5. In contrast to children with this disease who develop progressive liver failure due to the inability to excrete bile, the post- metamorphosis lamprey grows normally to adult size. Our previous studies indicate that the adult lamprey’s liver is cholestatic, containing high levels of bile salts and biliverdin similar to levels seen in the liver of cholestatic patients and rodents models but without liver injury2. Paradoxically plasma levels of bile salt and biliverdin are normal in contrast to elevations of these substances in cholestatic animals and man. It is not known how the adult lamprey maintains normal plasma levels of bile salts and bilirubin in the face of cholestatic levels of these substances in the liver. In this report, we examine the roles of kidney and intestine in the excretion of bile salts and other organic solutes.
Upstream migratory sea lamprey were caught in the Kenebunck River in southern Maine and maintained at MDIBL in 11 °C fresh water tanks with 12 hours light-dark cycle. Males and females were kept separated. All animals were anesthetized in 0.1g/L Tricaine prior to intravenous injections, blood sampling and all surgical procedures, including ureter and/or intestinal cloacae cannulations with balloon attachments, intestinal ligation or sacrifice. To assess the clearance of organic anions into intestine and kidney, BSP (10 mg/kg body weight) was injected intravenously via the caudal tail vein, and cannulated or intestinal ligated fish were immediately returned to their fresh water tanks for 48 hrs. For clearance of bile acids, 3H-TCA (~ 30 μCi) was injected into animals that were then maintained in isolated plexiglass holding chambers as previously described4 for ~ 12 hours. BSP concentration was assayed as we have previously reported1.
Previous observations by us and others have detected biliverdin, 3H-TCA and BSP in washes of adult lamprey intestine, suggesting that organic solutes may be excreted across intestinal epithelium1,3. However, it is also possible that these organic solutes might enter the intestine from unidentified remnants of the biliary system since the proximal intestine is closely adherent to the liver in the lamprey. To test this possibility, the mid intestine was ligated and BSP was injected intravenously. Both segments were washed independently. BSP was detected in both the proximal and distal intestines with higher concentrations found in the proximal segments. However, detection of BSP in the distal intestine segment indicates that organic anions can cross the intestinal epithelium from plasma into the intestinal lumen. To further assess the magnitude of this process, cannulas were secured in the intestinal cloacae and intestinal secretions were collected for 72 hrs after i.v. BSP. However, less than 0.2% of the total injected BSP was recovered in this experiment indicating that the intestine may not be the major route for organic solute excretion.
While working on this project, one lamprey was serendipitously discovered to have an obstructed ureter where high concentrations of biliverdin (200 μM) and bile salts were detected. Prior studies of ureter washings had failed to detect significant amounts of these solutes after their i.v. injection. Therefore, we cannulated the lamprey ureters and repeated the organic anion clearance studies. Urine flow rates in these migrating adults averaged ~ 10 ml/hr/kg body weight. Biliverdin’s green color could be seen in the collected urine. Biochemical analysis showed that lamprey excretes biliverdin at a rate of ~ 5 μg/hr/kg body weight. When 3H-TCA or BSP were injected i.v., ~ 15% 3H-TCA was collected in the urine over a 12 hr period while ~ 35% of the injected BSP was excreted into urine over 72 hr period confirming that the kidney is the major route for organic solute excretion in the adult migrating lamprey.
In summary, our current studies indicate that the adult lamprey can excrete organic solutes via both kidney and intestine. However, the kidney is the major route of excretion for these compounds. These findings provide an explanation for how adult lamprey maintains normal plasma levels of bile salts and biliverdin when there is no intestinal secretion of bile. Future studies will need to determine the specific transport systems that the lamprey uses for excretion of organic anions in the kidney and intestine.
These studies were supported by National Institutes of Health Grants DK34989, and DK25636 and an NIEHS new investigator award (ES03828) from MDIBL to W.L.
1. Cai, SY, Li, W, Yeh, CY, Hagey, L, Soroka, CS, Mennone, A, Smith, V, Han, K, and Boyer, JL. Sea lamprey (Petromyzon marinus) - a unique cholestatic animal model. Bull. Mt. Desert Isl. Biol. Lab. 49:61-63, 2010. 2. Cai, SY, Yeh, CY, Hagey, L, Soroka, CS, Mennone, A, Li, W, and Boyer, JL. Sea lamprey (Petromyzon marinus) - a unique cholestatic animal model that tolerates biliary atresia. Hepatology, 52(S4):665A, 2010. 3. Makos, BK and Youson, JH. Tissue levels of bilirubin and biliverdin in the sea lamprey, Petromyzon marinus L., before and after biliary atresia. Comp. Biochem. Physiol. 91A:701-710, 1988. 4. Siefkes, MJ, Scott, AP, Zielinski, B, Yun, S-S and Li, W. Male Sea Lampreys, Petromyzon marinus L. excret a sex pheromone from gill epithelia. Biology of Reproduction. 69:125-132, 2003. 5. Youson, JH, and Sidon, EW. Lamprey biliary atresia: First model system for the human condition? Experientia. 34:1084-6, 1978. The actin binding protein, Cofilin 1-like, regulates zebrafish (Danio rerio) development
Erin Brunk1, Sharon Perrone3, Justine Cyr4, Caleb Swanberg1, Christopher Preziosi1, Lynne Staggs2, Hannah Marquis1, and Sharon Ashworth1 1Department of Molecular and Biomedical Sciences and the School of Biology and Ecology, University of Maine, Orono, ME 04469 2Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672 3Dickinson College, Carlisle, PA 17013 4University of Maine-Presque Isle, ME 04769
The actin depolymerizing factor/cofilin (AC) family of actin binding proteins plays a critical role in maintaining the structural and metabolic integrity of eukaryotic cells. The zebrafish (Danio rerio) has three cofilin isoforms: cofilin 1, cofilin 1-like and cofilin 2. Loss of expression of these cofilin isoforms leads to severe and often fatal developmental abnormalities.
Regulation of the actin cytoskeleton is essential for numerous cellular processes including cell movement, phagocytosis, cytokinesis, intracellular trafficking and cellular structural integrity. The proteins that bind actin primarily dictate actin dynamics and the ability of actin to respond to the needs of the cell. One family of proteins, the actin depolymerizing factor/cofilin (AC) family, regulates actin dynamics by binding actin filaments, altering the filament twist and severing the filaments. This results in an increase in the globular actin pool2. Although in vitro biochemistry studies and cell culture models have contributed to our current understanding of the role the AC protein family plays in actin regulation, little information about this protein family has been documented in studies of whole organisms.
The research in our laboratory focuses on the role in development of the three zebrafish cofilin isoforms: non-muscle cofilin 1, cofilin 1-like and muscle cofilin 2. Zebrafish cofilin 1 shares 74-77% identity with rat, mouse and human cofilin 1 and zebrafish cofilin 2. Zebrafish cofilin 1-like shares only 49.7% identity with zebrafish cofilin 1, but critical amino acid residues for cofilin activation and actin binding are highly conserved. These include the MASGV cofilin phosphorylation site containing Ser-3 and the actin binding motifs WAPECAPLKSKM and DAIKKK located within the actin binding domain2. To study the role of the zebrafish cofilin isoforms in development, we have used cofilin expression knockout and knockdown approaches using a cofilin 1 mutant and morpholino gene silencing (Gene Tools, LLC). The morpholino induced cofilin 1 morphants mimicked the severe and fatal phenotype observed in hi3736aTg proviral insertional cofilin 1 mutants1,3,4. The cofilin 1 mutant larvae had severe edema, abnormal craniofacial features including a gaping mouth, an abnormal heart with a slower heart rate, an uninflated swim bladder, an arched back and compromised kidney glomerular filtration1,3,4. Preliminary studies of morpholino induced knockdown of cofilin 2 expression suggested cofilin 2 was essential for zebrafish to survive to 48 hours post fertilization (hpf) and complete embryogenesis. Studying the absence of cofilin 1-like is the focus of this report.
B C A
Figure 1. One to two cell zebrafish embryos injected with morpholinos designed to knockdown cofilin 1-like protein expression developed severe abnormalities such as pericardial edema (B, arrow) and stunted anterior-posterior axis (C) compared to wildtype embryos (A). Images were taken at 120 hpf. Bar - 100µm
To determine how loss of cofilin 1-like expression affects zebrafish development, we utilized a morpholino designed to knockdown cofilin 1-like expression. One to two cell zebrafish embryos were injected with 4.6 nL of 100 µM cofilin 1-like morpholino. These embryos were observed for 120 hpf and compared to control injected and wildtype embryos. Sixty-nine percent of the cofilin 1-like morphants survived until 96 (hpf) compared to 83% of the control embryos. At 48 hpf pericardial edema was observed around the yolk, heart and occasionally around the head. At 96 hpf 74% of cofilin 1-like morphants exhibited an abnormal phenotype compared to 7.7% of control zebrafish. These abnormalities ranged from severe pericardial edema (Figure 1B) to stunted anterior-posterior axis (Figure 1C) to a curved spine. The majority of cofilin 1- A B like morphants had bent and twisted pectoral fins (Figure 2B). These studies suggested cofilin 1-like was crucial for normal zebrafish development.
Future studies will investigate the localization of actin and cofilin 1-like in developing wildtype and cofilin 1-like Figure 2. Cofilin 1-like morphants developed malformed pectoral morphant embryos using immunofluorescence fins (B, arrow) compared to wildtype pectoral fins (A, arrow). techniques. Images were taken at 72 hpf. Bar – 100µm.
A special thank you to Mark Nilan at the University of Maine zebrafish facility and to Dennis Moran at the MDIBL zebrafish facility for their support of this project.
This work was funded by MDIBL New Investigator Awards, University of Maine Start-up funds and Research Faculty Awards to Sharon Ashworth, INBRE – Maine IDeA Network of Biomedical Research Excellence (2-P20-RR016463) to Erin Brunk, Caleb Swanberg and Justine Cyr and NSF REU (MDIBL) (NSF DBI 0453391) to Sharon Perrone.
1. Ashworth S, Teng B, Kaufeld J, Miller E, Tossidou I, Englert C, Bollig F, Staggs L, Roberts ISD, Park J, Haller H, Schiffer M. Cofilin-1 inactivation leads to proteinuria – studies in zebrafish, mice and humans. PLoSONE: 5(9): e12626, 2010. 2. Bamburg JR. Proteins of the ADF/Cofilin Family: Essential Regulators of Actin Dynamics. Annu Rev Cell Dev Biol 15:185-230, 1999. 3. Marquis H, Bell EP, Miller EE, Gilman MS, Bond SK, Grimaldi R, Ashworth SL. Analysis of the Danio rerio cofilin mutant. MDIBL Bulletin: 48, 52-53, 2009. 4. Preziosi C, Swanberg C, Marquis H, Morse C, Miller E, Brunk E, Ashworth S. Cofilin 1 is essential for zebrafish (Danio rerio) survival. MDIBL Bulletin: 49, 33-34, 2010. Histaminergic neurons of two copepods, Centropages hamatus and Calanus finmarchicus
Monica I. Orcine and Daniel K. Hartline Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, HI 967822
Many invertebrate nerve cells can be individually recognized by their location, size, contacts and the chemical “transmitter” they use for communication with other cells. These characters change slowly with evolution and so can help establish relatedness (“phylogeny”) among different groups. We identified the same pair of histamine-containing neurons in two different copepod species, a first step in establishing the rate of evolution of histamine transmitters in copepods.
Calanoid copepods are minute planktonic crustaceans making up much of the oceanic biomass. Immunohistochemistry was used to label neurons containing histamine against a contrastingly- labeled nervous system in Centropages hamatus and Calanus finmarchicus with a goal of identifying homologous neurons.
Calanus finmarchicus were collected using vertical net tows in the Gulf of Maine and Centropages hamatus with sub-surface horizontal tows.in Frenchman Bay The dorsal cuticle was removed and specimens were given three sequential tratements interspersed with rinses: 1) fixation at 5°C for <24 hours in 4% 1- ethyl-3-(3-dimethyl-amino-propyl) carbodiimide (Sigma, Cat. #E7750) in 0.1 M phosphate buffer (PB) (Electron Microscopy Sciences, Cat. #11600-10); 2) incubation overnight at 4°C in primary antibodies for histamine (Panula et al., 1988; ImmunoStar Inc., Cat. #22939; 1:500) and acetylated a-tubulin (Santa Cruz Fig. 1. MU2HA neurons in C. hamatus (arrow) homologous to pair in C. finmarchicus. Biotechnology Cat. # sc-23950; 1:50) PB with 0.3% Triton X-100 (PBTX) and 10% normal donkey serum (NDS); 3) incubation overnight at 5°C in fluorescently-tagged secondary antibodies (1:300 each, Alexafluor 488 antimouse and Alexafluor 594 antirabbit) in PBTX and NDS. Rinses following treatments 1 and 2 were in PBTX and following 3 in PB, for ≥15 minutes per change with 5 changes at 22-25°C. Following the final rinse, specimens were mounted in Vectashield with DAPI to label nuclei. Imaged with an Olympus Fluoview 1000 confocal microscope and analyzed with ImageJ (Rasband).
Using a map of the histaminergic cells in C. finmarchicus1 as a reference, the cell body pair designated
“MU2HA” was observed in three additional C. finmarchicus. A pair with very similar form and location was identified in two Centropages hamatus, and we believe it to be homologous (ig. 1 arrow). It was commonly observed that the two cell bodies appeared dorsally with short axons that could be traced ventrally to two distinct dots. From there, axons projected in a lateral anterior curvature (a U shape). Additional support for the identification of this soma pair was its location in the maxillular region. However, the one displayed in Figure 1 showed a more enlarged pair of cell bodies displaced posteriorly.
We hope to identify additional neurons needed for further insight into the neurophylogenetic relationships among these different copepods species. In addition, we are attempting to identify the differences among the histaminergic systems in different copepod species: what features are modified or seem to be evolutionarily lost. Based on the morphology of this histaminergic system, which tends to be conserved in the animal kingdom, this study may also assist in a better classification of the Calanoida.
We thank Dr. A. Christie for help with immunohistochemical procedures and Capt. A. Peterson for animal collections. Supported by NSF IOB-0923692, to D.K.H., and NSF REU site at MDIBL DBI 0453391.
1. Hartline, D.K. and A.E. Christie (2010) Immunohistochemical mapping of histamine, dopamine and serotonin in the central nervous system of the copepod Calanus finmarchicus (Crustacea; Maxillopoda; Copepoda) Cell Tissue Res 341(1):49-71.
Lysosome number and size do not vary during a tidal cycle in erythrocytes of the bloodworm Glycera dibranchiata
M. Christina Vasquez¹, Timothy A. Crombie² and David Julian² ¹ Department of Animal Science, ² Department of Biology, University of Florida, Gainesville, FL 32611
In intertidal marine organisms, exposure to tidal cycles has been proposed to damage cellular components by causing an increase in the production of free radicals. In animals, damaged cellular components are usually removed and recycled by an intracellular process called autophagy. In this study, we investigated whether cells from the intertidal bloodworm Glycera dibranchiata showed changes during a tidal cycle that are characteristic of increased autophagy. We found no evidence of biologically significant changes in autophagy associated with a tidal cycle, suggesting that either exposure to tidal cycles does not increase free radical production, or that adaptations in G. dibranchiata minimize any resulting cellular damage.
Intertidal marine organisms experience a variety of abiotic stressors during tidal emersion, including exposure to hypoxia, anoxia and hydrogen sulfide (H₂S). However, the return of oxygenated water during tidal immersion may also be a stressor. Biomedical research on mammalian systems has shown that a rapid return to normoxia from hypoxia or anoxia (termed ischemia-reperfusion) may cause cellular damage from increased reactive oxygen species (ROS) production, which can damage intracellular proteins and organelles8. To remove these damaged components, cells utilize lysosome-mediated autophagy, in which specialized vacuoles termed autophagosomes isolate the damaged proteins and organelles and then bind to lysosomes, creating autophagolysosomes3. These then allow the degradation and recycling of the damaged components3. If immersion after low tide increases ROS production, similar to reperfusion after ischemia, then tidal immersion may activate lysosome-mediated autophagy. Previous studies investigating whether tidal cycles increase ROS production have not produced consistent results1, 7. To our knowledge, no studies have investigated whether tidal cycles are associated with changes in lysosomes, although it has previously been suggested that exposure to H₂S increases autophagic responses11.
The bloodworm Glycera dibranchiata may be an ideal organism for studying autophagic responses to tidal cycles. G. dibranchiata inhabits burrows in hypoxic mudflats along the northeastern US coast, including extensive populations in Maine9. Its coelomic cavity contains abundant erythrocytes which, unlike mammalian erythrocytes, contain cellular organelles, including lysosomes6. Previous studies using in vitro exposures and laboratory exposures in vivo have shown that conditions similar to those that occur during low tide can cause increased oxidative stress and oxidative damage in G. dibranchiata4, 5. In this study we investigated whether erythrocytes from a natural population of G. dibranchiata exhibit lysosomal changes characteristic of increased autophagy during a tidal cycle.
This study was completed in June 2010 at Salsbury Cove, ME, which is adjacent to the Mount Desert Island Biological Laboratory. To provide a preliminary assessment of the population distribution within the cove, we laid an 80 m transect during a maximal low tide of -0.15 m. This transect extended from the center of the cove to the beach. We then counted the number of worms present in 0.25 m2 quadrats spaced 5 m apart along the transect. The average density of bloodworms in the cove was 5.2 ± 0.82 (SD) worms per m2. Worms ranged in length from 5 to 23 cm with an average of 12.7 cm. At 45 m away from the center of the cove, worm density dropped to 0 and this coincided with reduced mud depth and the lower distribution range of the blue mussel Mytilus edulis. To determine the effect of tidal emersion and immersion on lysosomes, we selected a location within the cove approximately 15 m from the center of the cove, where the distribution data predicted that G. dibranchiata could be reliably collected. When the mean lower low water level was 0 m, the G. dibranchiata burrows at this location were still Figure 1: Matched brightfield (A) and immersed. Just as the tide began to recede from the sample fluorescent (B) images of live G. dibranchiata location, we collected six worms in their surrounding erythrocytes labeled with LysoSensor sediment and returned these immediately to MDIBL. We refer Yellow/Blue.
1
to this sample time as “pre- Parameter Pre-emersion Emersion Immersion emersion”. As the worms were Time 3:50 a.m. 5:35 a.m. 6:35 a.m. collected, we also measured the Temperature (°C) 12.9 12.5 12.5 sediment temperature and obtained interstitial water samples for H₂S (µM), 5 cm depth 0 - 49 4 - 94 0 analysis of H₂S and salinity11 (we H₂S (µM), 10 cm depth 0 - 267 3 - 43 0 were unable to measure dissolved Salinity (ppt) 26* 22* 36 oxygen due to equipment failure). No. of worms sampled 6 6 6 Upon their arrival at MDIBL less Worm length (cm) 13.0 ± 2.2 9.6 ± 3.5 11.2 ± 4.4 than 15 minutes later, the worms Table 1: Changes in salinity (ppt), temperature (°C), and range of hydrogen were immediately measured for sulfide (µM) in the environment of G. dibranchiata burrows during a tidal length and the erythrocytes were cycle. Worm length (cm) reported ± SD. *Freshwater runoff from a rainstorm collected, washed4, and stained for reduced salinity over the mudflat prior to immersion lysosomes using a fluorescent, pH- dependent marker (LysoSensor Yellow/Blue, Invitrogen, Carlsbad, CA) that typically labels lysosomes and autophagolysosomes2. Fluorescent images of the erythrocytes were acquired as z-stacks and analyzed using ImageJ (http://rsb.info.nih.gov/ij). For 10-15 cells per worm, we determined the number of lysosomes within each cell, the cross-sectional area of each lysosome (from the z-slice estimated to be closest to center of that lysosome), and the total lysosome area per cell (the sum of all of the lysosome cross-sectional areas within each cell). The worm collection and lysosome labeling procedure was repeated at the same approximate location after 1.5 h of continuous tidal emersion and 45 min after tidal immersion, which we refer to as “emersion” and “immersion” respectively. In all, 3,934 lysosomes were measured from a total of 258 cells from 18 worms (6 at each time point). The results for lysosome count and area were not normally distributed, so we used Kruskal- Wallis one-way ANOVA on ranks to test for a significant effect of tidal cycle. Total lysosome area per cell was grouped by worm and was normally distributed, so standard one-way ANOVA was used to test for a significant effect of tidal cycle.
At the collection site, H₂S was variable and patchy, and a light rain during the collection significantly reduced the salinity of surficial water (Table 1). The lysosomal dye identified lysosomes, presumably including autophagolysosomes (Fig. 1). We found statistically significant effects of tidal cycle on the number of lysosomes present per cell (df=2; H=10.7; p=0.005) and on the cross-sectional area of the lysosomes per cell (df=2; H=24.5; p<0.001). Overall, the number of lysosomes and the lysosome cross-sectional area decreased from pre-emersion to immersion (Fig. 2A and 2B, respectively), but the magnitudes of these changes were very small. We found no significant differences between time points for the total lysosome area (Fig. 2C; F2, 17 =2.199; p=0.215).
) 2
40 ) 0.4 10 m 2
ABCm 8 30 0.3 6 20 0.2 4 10 0.1 2 Lysosomes per cell per Lysosomes
0 Area perlysosome ( 0.0 0
n n n n Lysosome area percell ( n o o o o o si rsi sion si er ersion er er m m mersi me m e E -emersi Emersion em Emersion Im e Im - Im e- re Pr Pr P Figure 2: Changes in lysosome characteristics in G. dibranchiata erythrocytes during a tidal cycle. A) Number of lysosomes present per cell ± 1 SD. B) Mean cross-sectional area of the lysosomes measured per cell ± 1 SD. C) Total lysosome area per cell ± 1 SD. Box plots show the median plus the 5th, 10th, 25th, 75th, 90th and 95th percentiles. Refer to text for results of statistical tests. 2
We expected to observe lysosomal changes consistent with activation of lysosome-mediated autophagy during a tidal cycle, and we hypothesized that reoxygenation (i.e., the immersion sample) would be associated with the greatest increase in oxidative stress and subsequent cellular damage, and therefore that the increased autophagy would results in a higher number of large lysosomes in the immersion sample. However, we instead measured a slight decrease in the number and size (cross-sectional area) of lysosomes during emersion and then immersion. It is important to point out that these conclusions are based on a limited sample size. Furthermore, the observed effect is quite small, so even if it is valid, it may be of relatively little biological significance. Based on these limited data, we failed to find support for the hypothesis that re-immersion during a tidal cycle is associated with increased lysosome-mediated autophagy. This suggests that either cycles of tidal emersion and immersion do not increase ROS production, or that protective mechanisms in G. dibranchiata minimize cellular damage. A more conclusive determination would require a greater sample sizes and increased resolution in sampling intervals.
We are grateful to Robert Tilden for generously sharing his extensive knowledge and experience on G. dibranchiata life history and collection techniques. We thank Lauren Gravois, Breanna Sipley, Maria Duarte and Stephanie Wohlgemuth for help with collections. This project was supported by an MDIBL Graduate Student Research Award to MCV and TAC, and an MDIBL New Investigator Award to DJ.
1. Abele, D & Puntarulo, S. Formation of reactive species and induction of antioxidant defense systems in polar and temperate marine invertebrates and fish. Comp. Biochem. Physiol. 138A: 405-415, 2004. 2. Bains, M & Heidenreich, KA. Live-cell imaging of autophagy induction and autophagosome-lysosome fusion in primary cultured neurons. Meth. Enzymol. 453: 145-58, 2009. 3. Dunn Jr., WA. Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J. Cell. Biol. 110: 1923-1933, 1990. 4. Hance, JM, Andrzejewski, JE, Predmore, BL, Dunlap, KJ, Misiak, KL and Julian, D. Cytotoxicity from sulfide exposure in a sulfide-adapted marine invertebrate. J. Exp. Mar. Biol. Ecol. 359: 102-109, 2008. 5. Joyner-Matos, J, Predmore, BL, Stein, JR, Leeuwenburgh, C and Julian, D. Hydrogen sulfide induces oxidative damage to RNA and DNA in a sulfide-tolerant marine invertebrate. Physiol. Biochem. Zool. 83: 356-65, 2010. 6. Mangum, CP, Colacino, JM and Vandergon TL. Oxygen binding of single red blood-cells of the annelid bloodworm Glycera dibranchiata. J. Exp. Zool. 249: 144-149, 1989. 7. Pannunzio, TM and Storey, KB. Antioxidant defenses and lipid peroxidation during anoxia stress and aerobic recovery in the marine gastropod Littorina littorea. J. Exp. Mar. Biol. Ecol. 221: 277-292, 1998. 8. Turer, AT, and Hill, JA. Pathogenesis of myocardial ischemia-reperfusion injury and rationale for therapy. Am. J. Cardiol. 106: 360-8, 2010. 9. Wilson, W and Ruff, R. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (North Atlantic) – sandworms and bloodworms. U.S. Fish. Wildl. Serv. Biol. Rep. 82(11.80). US Army Corps of Engineers, TR EL-82-4, 1988, p. 23. 10. Yorimitsu, T and Klionsky, DJ. Autophagy: molecular machinery for self-eating. Cell Death Differ. 12: 1542-1552, 2005. 11. Wohlgemuth, SE, Arp, AJ, Bergquist, DC and Julian, D. Rapid induction and disappearance of electron-dense organelles following sulfide exposure in the marine annelid Branchioasychis americana. Invertebr. Biol. 126: 163-172, 2007.
3 Predation of killifish embryos by adult killifish (Fundulus heteroclitus)
Arhea V. Marshall1 and Robert L. Preston2,3 1 High School for Math, Science and Engineering at City College of New York, NY 10031 2School of Biological Sciences, Illinois State University, Normal, IL 61790 3Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
Killifish spawn in early summer at the margins of estuaries. Because of the wide tidal range in coastal Maine, not all embryos may remain immersed in seawater but some are exposed to air and survive for long periods. Immersed embryos are exposed to a suite of possible aquatic predators (including adult killifish). We determined that adult killifish readily eat freshly fertilized killifish embryos within a few minutes of exposure to adult fish. These data suggest that the unusual ability of embryos to survive in air provides a selective advantage under the potentially intense predation pressure of adult killifish.
Adult northern killifish, Fundulus heteroclitus macrolepitodus, spawn in estuaries during peak high tides3. The eggs are deposited at the margins of the streams and some embryos are stranded in air as the tide recedes. Since the low to high tidal range may vary from 6-12 feet on a daily basis, and are highest at spring tides, some embryos may be exposed to air for extended periods of time. Investigations in this and other labs have shown that F. heteroclitus embryos can resist desiccation stress, and develop normally to hatching in ~14 days1,4. Flooding of the aerially incubated mature embryos with SW has been shown to trigger hatching1,4. Experiments have shown that aerial incubation favors embryo survival and the production of larger hatchlings compared with immersed embryos1. Immersed killifish embryos would be subjected to a different suite of predators than those embryos in air. Immersed embryos can be ingested by larger aquatic organisms, including fishes and possibly by killifish adults2. We conducted preliminary experiments to test the hypothesis that F. heteroclitus adults may be important predators of killifish embryos.
Fish were caught in minnow traps at Northeast Creek, Mount Desert Island, ME and kept in aquaria with natural running seawater (SW; ~30 ppt). Approximately 150 eggs were fertilized with milt from five fish, in a beaker with 25 ml of 10 ppt artificial seawater (ASW; Instant Ocean, Mentor, OH). After sitting for 30 min, embryos were rinsed three times with 10 ppt SW and held for another 30 min. Opaque four liter containers were used containing three liters of SW diluted with deionized water to 10 ppt. Two female and two male fish were placed in each of three containers (triplicate conditions) and covered with filter paper. The fish were acclimated in the containers for 20 min. Thirty embryos were then gently added to each container and the number of embryos remaining counted after 5, 15 or 60 min exposure to the adult fish. At the 5 min and 60 min times, each triplicate experiment was repeated. As controls for embryo viability under these conditions, 30 embryos were placed in a dark container in 10 ppt SW, without fish present. In another control embryos were placed beneath a nylon screen in a container where adult fish were present (60 min time period). In both conditions, all embryos survived (triplicate measurements).
In 15 min and 60 min conditions all embryos were eaten. In the 5 min condition survival ranged from 0 to 29 embryos in six replicate containers (mean 9.3 ± 4.7; n = 6; ~31%). Adult fish are very sensitive to disturbance and it is likely that after 5 min some fish had not acclimated fully to the disturbance caused by adding embryos. These data strongly support the hypothesis that adult killifish may be a major predator of killifish embryos. The data also suggest that from an evolutionary standpoint selection for the physiological mechanisms that permit survival of killifish embryos in an aerial environment may remove the embryos from the potentially serious threat of predation by adult killifish as well as other aquatic predators. (A. Marshall was supported by the SETH program, Science Education Through Health).
1. Baldwin, JL, Petersen, CW, Preston, RL and Kidder, GW. Aerobic and submerged development of embryos of Fundulus heteroclitus. Bull. Mt. Desert Isl. Biol. Lab. 45: 45-46, 2006. 2. Kneib, RT. Size-specific patterns in the reproductive cycle of the killifish, Fundulus heteroclitus (Pisces: Fundulidae) from Sapelo Island, Georgia. Copeia, 1986: 342-351, 1986. 3. Petersen, CW, Salinas, S, Preston, RL and Kidder III, GW. (2010) Spawning periodicity and reproductive behavior of Fundulus heteroclitus in a New England salt marsh. Copeia 2010: 203-210. 4. Preston, RL, Edwards, BR, Baumhardt, PE, Lantigua, J, Ruensirikul, S and Kidder, GW. Desiccation resistance by mid-stage Fundulus heteroclitus embryos. Bull. Mt. Desert Isl. Biol. Lab. 47: 94-96, 2008. Identification of Parathyroid Hormone Related Peptide (PTHrP) in brook trout (Salvelinus fontinalus)
Erin Schnettler1, Charles Wray2, Alison Kieffer3 and Russell Danner1 1Department of Biology, Colby College, Waterville, Maine 04901 2Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672 3University of Maine, Presque Isle, Presque Isle, Maine 04769
Brook trout reared in hatcheries in Maine exhibit symptoms of calcium-related metabolic diseases. To investigate genes responsible for calcium regulation the parathyroid hormone-related protein gene was partially sequenced.
Brook trout occupying waters with low dissolved calcium concentrations (< 2.5mg/L) have been shown to exhibit symptoms of nutritional hyperparathyroidism. This disease causes the development of rubbery deformed jaws (Figure 1), renal failure, and reproductive failure and is consequently a costly burden to the fish hatcheries raising brook trout in locations deplete of this divalent ion3. One of the objectives of an ongoing study investigating the causes, diagnosis and treatment of nutritional hyperparathyroidism in brook trout is to examine the underlying cell physiology responsible for calcium (Ca2+) uptake in salmonid gills. Previous studies examining the functional genomics of calcium deficiency in brook trout sequenced and quantified the expression of the epithelial calcium channel gene (ECaC) as well as the calcium sensing receptor (CaSR) in brook trout5. While the highest average gene expression levels were found in samples from low Ca2+ conditions, the difference in expression level was not Figure 1. Brook trout broodfish with statistically significant. affected mandibles. Clinically brook trout with this condition make an effort The parathyroid hormone-related protein gene (PTHrP, to eat but are unable to close their mouth around food pellets. Phillips Hatchery, human PTHLH) was identified as a promising candidate to photo courtesy of GR Danner, DVM. investigate Ca2+ deficiency in brook trout1,2,4. PTHrP has been shown to be one of the hypercalcemic factors that are upregulated in low Ca2+ conditions to restore and maintain healthy Ca2+ concentrations within the body. Thus, this study pursued the identification of the PTHrP gene from the brook trout genome and sought to investigate whether fish taken from low Ca2+ conditions would exhibit elevated gene expression levels of PTHrP. In addition, samples from five different tissues; gill, kidney and corpuscle of Stannius, intestine, skin and brain were taken in order to establish anatomical locations where PTHrP is expressed in brook trout.
Previous attempts to isolate PTHrP from brook trout using degenerate PCR had been unsuccessful. However, new PTHrP sequences from several species of fish became available and 12 new pairs of degenerate primers based on these sequences were designed. PCR, using different combinations of degenerate primers and sequencing, revealed a 245 bp DNA sequence with high homology to known piscine PTHrP sequences and thus, is believed to be the PTHrP gene in brook trout (Figure 2). Using this sequence, species-specific primers were synthesized and Q-PCR studies analyzing average expression levels among five tissue samples from both low and high Ca2+ conditions are underway. Furthermore, future studies will be designed to address whether PTHrP expression levels are affected when fish are exposed to short-term or long-term Ca2+ deficiency2.
*** * **** * **** *** Brook Trout THKGRSLHEFKRRHWIQELLDQVHTSDSERAPAP------QSRTNANECHS------TFSGSALSPPK Pufferfish HDKGRALQDFKRRMWLQELLDEVHTADIRELPVR------TTSTGGGDSRGVGLGQ-AVVSPSSTSSTLHPK Tetraodon HDKGRTLQDFKRRMWLQELLDDVHTADIRELAA------GGPSGGP------QPR Stickleback HDKGRTLQDFKRRMWLQELLDEVHTAAVRDLGG------GGGSSVGLPGG--TAVNPSTTGSTLHSK Zebrafish HDKGRTLQDFKRRMWLQELLHEVHTAEVREAQQPRGGVSISSGAGGGVGAGVSITLPAGVGVSTGAGTAHPK Sea Bream HDKGRSLQEFKRRMWLHELLEEVHTADDRP--VQSRT------QSQTFSGN------ALHEK Eur. Flounder HDKGRSLQEFKRRMWLQELLEEVHTADEQAPHVQSRT------PNQTFSGN------ALPQK * * **** Brook Trout PSGGTKNLPLSFRLRGEGS----NLPQETHKFRYI- Pufferfish PPGGTKNLPETFKLEDEEG---TNLPQETNKSTSHK Tetraodon APAGAKDAALAFQPEDQEG---TKLPQESHQSTGHK Stickleback PPGATKHLPAGFGLEEEVGGGGTNLPQETNKSQAYK Zebrafish PAGGTKNLPIGFGLEDEEG---TNLPQETHKSQNYK SeaBream PPGATKNIPDRFRLDREGP----NLPQETNKALAYK Eur. Flounder PPGATKNLPDRFRLDKEVT----NLPQETNKELAYK
Figure 2: Sequence comparison of piscine PTHrP AA sequences: Brook Trout (Salvelinus fontinalus) Pufferfish (Takifugu rubripes and Tetraodon nigroviridis), Stickleback (Gasterosteus aculeatus), Zebrafish (Danio rerio), Sea Bream (Sparus auratus), and European Flounder (Platichthys flesus). All published sequences are available in Genbank; sequences were aligned using the ClustalW. Fully conserved amino acid residues are marked by *.
This work was supported by a Morris Animal Foundation Grant (D07Z0-105) to G. Russell Danner, DVM.
1. Abbink, W, Bevelander, GS, Rotlant, J, Canario, AVM and Flik, G. Calcium handling in Sparus auratus: effects of water and dietary calcium levels on mineral composition, cortisol and PTHrP levels. Jour. Ex. Biol. 207: 4077-4084, 2004. 2. Abbink, W, Bevelander, Hang, X, Lu, W, Guerreiro, PM, Spannings, T, Canario, AVM and Flik, G. PTHrP regulation and calcium balance in sea bream (Sparus auratus) under calcium constraint, Jour. Ex. Biol. 209: 3550- 3557, 2006. 3. Danner GR. Investigating the Causes, Diagnosis and Treatment of Brook Trout Nutritional Hyperparathyroidism (Phase II). Morris Animal Foundation. Grant D07Z0-105, Maine Department of Inland Fisheries & Wildlife, Augusta, ME. 4. Guerreiro, PM, Renfro, JL, Power, DM and Canario, AVM, .The parathyroid family of peptides: structure, tissue distribution, and potential functional roles in calcium and phosphate balance in fish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: R679-696, 2006. 5. Wray, CG and Kieffer, A. unpublished DNA sequence data, available upon request.
Accumulation of zinc by choroid plexus of spiny dogfish shark (Squalus acanthias)
Robin K. Young1, Pedro M. Guerreiro2, J. Larry Renfro3, Deborah Perry4, Robert J. Taylor4 and Alice R.A. Villalobos1 1Nutrition and Food Science, Texas A&M University, College Station, TX 77843 2Center of Marine Sciences, University of Algarve, Faro, Portugal 3Physiology and Neurobiology, Univ. Connecticut, Storrs, CT 06269 4Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843
Zinc is a nutrient mineral that is essential to nearly every aspect of the cell’s biology. We have studied how the choroid plexus, the tissue that forms the barrier between blood and cerebrospinal fluid (CSF), accumulates Zn in response to changes in the availability of this nutrient mineral. We determined that choroid plexus accumulates more Zn as more is made available and possibly increases expression of the Zn-binding protein metallothionein-1. However, under conditions of decreased Zn availability, the choroid plexus continues to accumulate Zn but decreases expression of metallothionein. These adaptations may help ensure adequate intracellular supply of this essential nutrient for this brain barrier.
The choroid plexus forms the blood-cerebrospinal fluid (CSF) barrier and actively secretes CSF and transports inorganic and organic solutes into and out of CSF. Therefore, this barrier epithelium regulates fluid/electrolyte balance, nutrient availability, and accumulation of metabolites and xenobiotics in the brain. The epithelium also transports and accumulates the essential nutritive mineral zinc (Zn). The unique roles of Zn in the biology and physiology of choroid plexus have not been elucidated. However, Zn is the essential cofactor for carbonic anhydrase, an enzyme that facilitates formation of CSF1, and is a pro-antioxidant that protects transport function under conditions of oxidative stress4. Thus, intracellular Zn accumulation in choroid plexus may represent the intracellular step in transepithelial transport of Zn and ensure sufficient availability of this essential nutrient to support basic biology and unique physiological functions of choroid plexus. The broader research objective is to elucidate the mechanisms that facilitate and regulate transport and accumulation of Zn in choroid plexus under control versus Zn deficient conditions. Transport and accumulation of Zn in choroid plexus has been studied in a limited number of mammalian species and no non-mammalian species. Thus, isolated intact lateral and IVth choroid plexus of dogfish shark, an established model for characterization of solute transport in choroid plexus2,3 was used to initiate investigation of regulation of Zn accumulation elicited in response to changes in Zn availability.
To establish that dogfish choroid plexus accumulates Zn, concentrations of endogenous Zn were measured and compared among lateral choroid plexus, IVth choroid plexus, plasma and CSF collected from individual sharks by inductively coupled plasma mass spectrometry (ICP-MS; Trace Elemental Research Laboratory, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University). Samples of cerebral cortex adjacent to the lateral ventricle and cerebellum adjacent to the IVth ventricle were collected for Zn analysis. Kidney and liver were analyzed for Zn; these peripheral epithelial tissues accumulate essential minerals and are target tissues of heavy metals. Skeletal muscle served as a general reference tissue. The choroid plexus has been described as a ‘sink’ for both essential minerals and contaminant heavy metals. However, this is based on experimental dosing of laboratory-reared and short-lived animals, predominantly mammalian species, e.g., rat, rabbit, and has not been established for longer-lived vertebrates reared in less controlled environments. Thus, as is possible by ICP-MS the same tissue samples were also analyzed for the essential minerals, iron (Fe), copper (Cu), and selenium (Se) and contaminant heavy metals arsenic (As), cadmium (Cd), and nickel (Ni). Concentration of each metal was expressed as parts per million (ppm) based on wet tissue weight (± 0.1 mg) and reported as mean ± SE; tissues were collected from 4-5 animals. Zn concentrations in plasma and CSF were 0.22 ± 0.06 ppm and 0.18 ± 0.02 ppm; whereas concentrations in lateral choroid plexus and IVth choroid plexus were 14.16 ± 0.06 ppm and 10.92 ± 1.78 ppm. Thus, total Zn concentration in either plexus exceeded that in plasma by roughly 40-60-fold and that in CSF by 50-75-fold. Choroid plexus tissues accumulated Zn to levels comparable to those in kidney (13.26 ± 0.50 ppm) and liver (11.70 ± 1.16 ppm), as well as brain cortex (10.92 ± 0.89 ppm) and cerebellum (9.79 ± 0.19 ppm). Choroid plexus tissue also accumulated Fe and Cu. Concentrations of Fe in plasma and CSF were 0.73 ± 0.09 ppm and 0.25 ± 0.04 ppm. Tissue:plasma Fe concentration ratios for lateral choroid plexus were 40-80 and for IVth choroid plexus were 27-45; these values were similar to those for kidney, but tissue:plasma ratios for liver exceeded 100. Concentrations for Cu in plasma and CSF were 0.13 ± 0.01 ppm and 0.07 ± 0.01 ppm; tissue:plasma Cu concentration ratios for lateral choroid plexus were 8-12 and for IVth choroid plexus were 7-10, values similar to those for both kidney and liver. However, although plasma Se concentration (0.17 ± 0.01 ppm) were similar to plasma Cu concentrations, tissue:plasma Se concentration for lateral and IVth choroid plexus and liver were 3-5, but were as great as 16 for kidney. Both lateral and IVth choroid plexus tissues accumulated heavy metals. Ni concentration in plasma was 0.003 ± 0.001 ppm, whereas concentrations in lateral choroid plexus and IVth choroid plexus were 0.067 ± 0.009 ppm and 0.025 ± 0.001 ppm; however, Ni concentration in muscle was 0.011 ± 0.004 ppm. Cd concentration in plasma was 0.0003 ± 0.0001 ppm; however, concentrations in lateral and IVth choroid plexus tissues were 1.22 ± 0.19 ppm and 0.66 ± 0.16 ppm and in kidney and liver were 2.02 ± 0.17 ppm and 0.87 ± 0.10 ppm. Tissue accumulation of As was markedly different, with most tissues accumulating As to levels 8- 10X that of plasma. Plasma As concentration was 0.28 ± 0.04 ppm. However, whereas As concentrations in lateral and IVth choroid plexus were 3.07 ± 0.18 ppm and 2.22 ± 0.31 ppm, 4.53 ± 0.34 ppm in kidney, 3.95 ± 0.48 ppm skeletal muscle, and 6.66 ± 0.48 ppm in liver.
To determine whether changes in extracellular Zn concentration might elicit changes in Zn accumulation, isolated segments of lateral and IVth choroid plexus were incubated for 24 or 48 hours in elasmobranch- modified Leibovitz’s L-15 medium with different concentrations of Zn (ZnCl2) or the Zn chelator N,N,N’,N’- tetrakis-2 pyridylmethyl-ethylenediamine (TPEN). This laboratory had demonstrated that isolated shark choroid plexus incubated in modified L-15 medium for up to 72 h maintains bioelectrical properties and capacity to transport organic anions comparable to freshly harvested tissues3. After treatment tissues were analyzed for Zn content by ICP-MS and for gene expression of the Zn-binding protein metallothionein-I (MT-1) by quantitative real-time PCR with SYBR green detection and normalization to actin and GAPDH gene expression. In isolated IVth choroid plexus incubated for 24 h and 48 h with 10 µM Zn, MT-1 gene expression in Zn-treated tissue increased by 425-fold and 700-fold as compared to untreated tissues. At 24 h, total Zn concentration was 9 ppm in control tissue and 20 ppm in Zn-treated tissues (per wet weight; ± 0.01 mg). A separate set of tissues were incubated for 24 h with 10 µM Zn2 or 2 µM TPEN. Zn accumulation in TPEN-treated tissues (7 ppm) was comparable to that in Zn-supplemented tissues (6 ppm). However, TPEN treatment decreased MT-1 gene expression by 80% as compared to Zn-supplemented tissue.
Based on data for intact dogfish shark, both lateral choroid plexus and IVth choroid plexus accumulate Zn to concentrations some 50X and 70X those in plasma and CSF. Accumulation of Zn in choroid plexus was comparable to that in kidney and liver. Choroid plexus also accumulated the essential minerals, Fe, Cu and Se, and the contaminant heavy metals, As, Cd and Ni. Preliminary data for isolated choroid plexus suggest that the choroid plexus adapts to changes in Zn availability. Increases in extracellular Zn elicited an increase in Zn accumulation and an increase in MT-1 gene expression. Thus, although additional Zn is transported into the cell, increased expression of MT-1 would serve to bind Zn and regulate levels of free Zn. However, experimental Zn depletion did not result in a marked decrease in Zn accumulation, but did elicit decreased MT-1 gene expression. The sustained capacity to accumulate Zn might also involve altered expression of Zn transporters; this will be pursued in future studies. This work was supported in part by funds from the MDIBL Forrest Fund awarded to ARAV.
1. Brown PD, Davies SL, Speake T, Millar ID. Molecular mechanisms of cerebrospinal fluid production. Neuroscience 129: 957-970, 2004. 2. Villalobos AR, Miller DS, Renfro JL. Transepithelial organic anion transport by shark choroid plexus. Am J Physiol Regul Integr Comp Physiol 282: R1308-1316, 2002. 3. Villalobos AR, Renfro JL. Trimethylamine oxide suppresses stress-induced alteration of organic anion transport in choroid plexus. J Exp Biol 210: 541-552, 2007. 4. Villalobos AR, Young RK. Regulation of apical choline transport in cultured choroid plexus by oxidative stress. FASEB J. 2010 24:1024.15. Glucocorticoids signal through tyrosine kinase to regulate Multidrug resistance-associated protein 2 (Mrp2) in killifish (Fundulus heteroclitus) renal proximal tubules
Rosalinde Masereeuw1, Brigitte Prevoo1 and David S. Miller2 1Dept. Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre / Nijmegen Centre for Molecular Life Sciences, The Netherlands 2Laboratory of Toxicology and Pharmacology, NIH/NIEHS, Research Triangle Park, NC, USA
Multidrug resistance protein 2 (Mrp2) in kidney tubules transports foreign chemicals into forming urine. Here we show that the anti-inflammatory drug, dexamethasone, rapidly increases Mrp2 activity by acting through a non-glucocorticoid receptor, and a protein phosphorylation cascade.
Mrp2 (Abcc2) is expressed at the luminal membrane of vertebrate renal proximal tubule, where it drives ATP-dependent efflux of anionic xenobiotics and metabolic wastes into the urine. We recently used isolated killifish (Fundulus heteroclitus) renal tubules to show that dexamethasone rapidly activates Mrp2 transport through a nongenomic mechanism involving glucocorticoid receptor (GR)1. Here, we define the intracellular signaling pathway downstream of GR.
For transport experiments, freshly isolated killifish renal tubules were exposed to dexamethasone without and with inhibitors of signaling. After a predetermined exposure time, 2 μM fluorescein-methotrexate (FL- MTX) was added to the medium and FL-MTX transport into the tubules lumen was measured by confocal microscopy and quantitative image analysis as before1,2. Previous experiments have established this assay as a measure of Mrp2 transport activity2.
We previously showed that dexamethasone and triamcinolone acetonide, potent synthetic glucocorticoids, and cortisol, the natural fish glucocorticoid, increased Mrp2-mediated transport of FL-MTX through a glucocorticoids receptor agonist-sensitive process; the mineralocorticoid receptor antagonist, spironolactone, was without effect 1. These results established the glucocorticoid receptor (GR) through which these glucocorticoids activate Mrp2. Second, dexamethasone stimulation of Mrp2 transport activity was rapid and was not altered when transcription or translation was inhibited; immunostaining showed no increase in Mrp2 protein expression. These observations argue for a nongenomic mechanism of GR regulation of Mrp2 function. Down- stream GR, the tyrosine kinase c-Met receptor was identified. The natural ligand for c-Met is hepatocyte growth factor (HGF). Exposing killifish tubules to human recombinant HGF produced a rapid and potent increase in FL-MTX transport; significant stimulation of luminal FL-MTX was evident within 5 min of exposure (Fig. 1). This HGF-mediated effect was blocked by pretreatment with PHA-665752, but not by RU486, importantly indicating that HGF signaling is downstream of GR (data not shown).
Figure 1. HGF stimulates Mrp2-mediated transport. A; Stimulation by HGF is highest at 25 nM. Tubules were incubated without (control) or with HGF for 1 h. B; Rapid stimulation of FL-MTX transport by HGF. Tubules were incubated with FL-MTX for 30 min until steady state after which 25 nM HGF was added. Mean values ± S.E.M. are shown for 11-18 (A), 12-30 (B) tubules. Significantly different from control, ***p<0.001.
Activation of c-Met can initiate intracellular signaling through further downstream phosphorylation cascades. We have found evidence for the involvement of the MEK/ERK pathway in GR/cMet signaling to Mrp2. When tubules were exposed to U0126, which blocks a MAPK pathway by inhibiting MEK1/2 31, the effects of dexamethasone and HGF on FL-MTX transport were abolished (Fig. 2A). No such effects were seen with LY294002, which inhibits the PI-3kinase/Akt pathway 32 (Fig. 2B), nor with SB203580, which inhibits Akt and p38 MAP kinase 33 (Fig. 2C). These findings are consistent with dexamethasone acting through GR, cMet and MEK/ERK 1/2 to increase Mrp2 transport activity.
Figure 2. Dexamethasone and HGF stimulate Mrp2 through MEK1/2 signaling. A; Tubules were incubated without (control) or with 1 μM dexamethasone (dex) or 25 nM HGF and the MEK1/2 inhibitor U0126 (10 µM) for 1 h. B; Co-treatment with the PI3K inhibitor LY294002 (10 µM) appears to be ineffective in reversal of the dex or HGF induced increase in FL-MTX secretion. C; Also up to 10 µM of SB203580 (SB) was not efficient in abolishing the dexamethasone-induced effect, indicating absence of p-Akt signaling. The fluorescence intensities in lumen and cell compartments are depicted as percentage of the fluorescence in control lumen. Mean values ± S.E.M. are shown for 20-39 (A), 10-19 (B) and 11-21 (C) tubules. Significantly different from control, *p<0.05, ***p<0.001. Significantly different from dexamethasone or HGF, ### p<0.001.
In conclusion, our results disclose a novel mechanism by which glucocorticoids acting through GR, c-Met and MEK1/2 cause rapid, nongenomic stimulation of Mrp2-mediated transport in renal proximal tubules. This upregulation may enhance efflux of metabolic wastes during cell and tissue stress.
This work was supported by a Dutch Society of Pharmacology-Schering-Plough award to Dr. R. Masereeuw, a Blum Halsey Fund of MDIBL for a collaborative program between Dr. D.S. Miller, Dr. R. Masereeuw and Prof. dr. G. Fricker and the Intramural Research Program of NIEHS/NIH.
1. B. Prevoo, R. Masereeuw, G. Flik and D.S. Miller. Rapid, non-genomic regulation of multidrug resistance protein 2 (Mrp2) by glucocorticoids in killifish (Fundulus heteroclitus) renal proximal tubules. Bull. Mt Desert Isl. Biol. Lab 49:79-80, 2010 2. Terlouw SA, Masereeuw R, Russel FG and Miller DS. Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux. Mol. Pharmacol. 59: 1433-1440, 2001.
Signaling mechanisms of aryl hydrocarbon receptor (AhR) – mediated regulation of ABC transporters in killifish (Fundulus heteroclitus) kidney tubules
Anne Mahringer1,3, David S. Miller2,3, Juliane Kläs1,3, Valeska Reichel1,3 and Gert Fricker1,3 1Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany 2Laboratory of Pharmacology, NIH/NIEHS, Research Triangle Park, NC 27709 3Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
Environmental toxicants can stimulate detoxifying genes by the aryl hydrocarbon receptor, a ligand-activated transcription factor, that binds to the promoter region of target genes. However, contaminants like dioxin are also able to activate protein kinases that modulate transcription factor function1. Here, we show that tyrosine kinase, p38 MAP kinase and protein kinase C are involved in the dioxin-mediated activation of P-glycoprotein efflux function in renal killifish tubules.
We recently have reported that ABC transporter expression and function are increased upon AhR stimulation by the agonist β-napthoflavone (BNF) or the highly toxic compound dioxin (TCDD, 2,3,7,8-tetra- chlorodibenzodioxine)2. Transport function and protein expression of P-glycoprotein (P-gp), Breast cancer resistance protein (BCRP) and Multidrug resistance-associated protein 2 (Mrp2) but not of organic anion transporters (oats) have been upregulated by AhR agonists in renal killifish tubules after 3h. This mechanism affected both transcriptional and translational steps as was shown by reversal with specific inhibitors of mRNA (actinomycin D) and protein synthesis (cycloheximide). Hence, activation of AhR by exposure to environmental contaminants leads to the induction of cytochromes, phase II conjugating enzymes and ABC efflux transporters potentiating metabolic processes3. In parallel, AhR activity is also dependent on phosphorylation steps that can modulate shuttling of the transcription factor in and out of the nucleus or its binding to promoter regions4,5,6.
To determine the possible cross-talk between AhR and protein kinases, isolated tubules from killifish were incubated with 1 nM TCDD and inhibitors of tyrosine kinase (genistein, 10, 25 µM), p38 MAP kinase (SB203580, 5, 10 µM) and MAP kinase kinase (MEK1, MEK2) (PD98059, 1, 5, 10 µM) for 3h. Additionally, inhibitors of protein kinase C (PKC) (BIM (bisindolylmaleimide), 100 nM), PI3 kinase (wortmannin, 50, 250, 500 nM) and protein kinase A (PKA) (H89, 0.1, 1, 10 µM) were tested for their modulatory effect on TCDD induced AhR activation. P-gp efflux activity in killifish tubules was determined using confocal microscopy to measure specific accumulation of the fluorescent substrate NBD-CSA in lumens of intact killifish renal tubules7,8. In this study, we showed that inhibition of tyrosine kinase, p38 MAPK and PKC caused a significant decrease in TCDD mediated induction of P-gp efflux activity. Incubation of kidney tubules with PD98059 (MEK1, MEK2), wortmannin (PI3K) or H89 (PKA) did not have any effect. These data suggest that tyrosine kinase as well as p38 MAPK and PKC are additional targets through which TCDD modulates the function of AhR. Whether those kinases affect the transcriptional response of AhR (stabilization, nuclear translocation) or whether they mediate AhR-independent, non-genomic effects of TCDD remains to be determined. Another target of AhR signaling includes Nrf2 (NF-E2-related factor 2), a transcription factor that binds to ARE (antioxidant response element) in phase II enzymes9. Nrf2 mediates antioxidative stress responses that could be evoked by toxic pollutants. Incubation of renal tubules with the Nrf2 agonist sulforaphane resulted in an increased transport activity of P-glycoprotein indicating that the ABC transporter is responsive to Nrf2.
The present study shows for the first time the involvement of tyrosine kinase, p38 MAPK and PKC in the AhR-mediated induction of P-gp activity by TCDD in killifish tubules. Besides, we determined P-gp efflux as a target of the antioxidative transcription factor Nrf2. Future investigations will focus on the cross-talk between kinases and AhR as well as on a linkage between Nrf2 and AhR considering ABC efflux transporter. Funded by: DFG grants GF1211/12-1 and GF1211/13-1, NIH grant MDIBL-CMTS (ES03838) and the NIEHD Division of Intramural Research.
1. Puga A, Ma C and Marlowe JL. The arylhydrocarbon receptor cross-talks with multiple signal transduction pathways. Biochem Pharmacol. 77:713-22, 2009. 2. Mahringer A, Miller DS and Fricker G. Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules (submitted to Am J Physiol Regul Integr Comp Physiol). 3. Köhle C and Bock KW. Coordinate regulation of human drug-metabolizing enzymes, and conjugate transporters by the Ah receptor, pregnane X receptor and constitutive androstane receptor. Biochem Pharmacol. 77:689-99, 2009. 4. Kasai S and Kikuchi H. The inhibitory mechanisms of the tyrosine kinase inhibitors herbimycin a, genistein, and tyrphostin B48 with regard to the function of the aryl hydrocarbon receptor in Caco-2 cells. Biosci Biotechnol Biochem. 74:36-43, 2010. 5. Chen YH and Tukey SH. Protein kinase C modulates regulation of the CYP1A1 gene by the aryl hydrocarbon receptor. J Biol Chem. 271:26261-26266, 1996. 6. Tan Z, Chang X, Puga A and Xia Y. Activation of mitogen-activated protein kinases (MAPKs) by aromatic hydrocarbons: role in the regulation of aryl hydrocarbon receptor (AHR) function. Biochem Pharmacol. 64:771-80, 2002. 7. Gutmann H, Miller DS, Droulle A, Drewe J, Fahr A and Fricker G. P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule. Br J Pharmacol. 129:251-6, 2000. 8. Terlouw SA, Graeff C, Smeets PH, Fricker G, Russel FG, Masereeuw R and Miller DS. Short-and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule. J Pharmacol Exp Ther. 301:578-85, 2002. 9. Miao W, Hu L, Srivens PJ and Batist G. Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway: direct cross-talk between phase I and II drug-metabolizing enzymes. J Biol Chem. 280:20340-8, 2008. 10. Osburn WO and Kensler TW. Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults. Mutat Res. 659:31-9, 2008.
The apical sodium-dependent bile salt transporter (Asbt) maintains the enterohepatic circulation of bile salts in the little skate, Leucoraja erinacea
Daniël A. Lionarons1, Maya de Groote2, James L. Boyer1 and Shi-Ying Cai1 1Liver Center, Yale University School of Medicine, New Haven, CT 06520 2Boothbay Region High School, Boothbay Harbor, ME 04538
ASBT facilitates absorption of bile salts in the enterohepatic circulation in mammals. Here we characterized the bile salt transport system in the intestine of the little skate (Leucoraja erinacea), an evolutionarily primitive vertebrate. Our results indicate that the sodium-dependent bile salt transport system is conserved in skate, and an ortholog of Asbt functions for this activity.
In vertebrates, the liver excretes bile salts into the intestine, where they facilitate absorption of dietary lipids. In the terminal ileum, bile salts are re-absorbed and transported back to liver to maintain an enterohepatic circulation4. The apical sodium-dependent bile acid transporter (ASBT/SLC10A2) is the functional determinant of this activity in the ileum of humans and rodents3. However, little is known about this transport system in non-mammalian vertebrates. Our previous studies indicate that bile salts can be re-absorbed in the intestine of the little skate (Leucoraja erinacea)1, an evolutionarily primitive vertebrate. But it is not known whether this function is sodium-dependent, or whether an Asbt ortholog is responsible for this function.
To address this question, the little skates were captured off the coast of Southwest Harbor, Maine, and maintained in sea-water tanks at MDIBL. After anesthesia, the intestine was removed proximally at the common bile duct and distally at the start of the rectum and divided in the middle into two everted gut sacs, prepared as described by Wilson and Wiseman5. Both sacs were incubated in 16 ºC elasmobrach ringers solution (with or without sodium) supplemented with 50µM 3H-taurocholic acid (3H-TCA, 2 mCi/mmol), and incubated for 30, 60 or 120 minutes with gentle agitation. The sacs were then washed in ice-cold medium, and homogenized. The concentration of 3H-TCA in the lysate was measured in a liquid scintillation counter.
To identify the full-length cDNA of skate Asbt, we first cloned a fragment from skate intestine using degenerate primers, followed by 5´ and 3´ RACE PCR. To functionally characterize skate Asbt, the coding region was cloned into pcDNA3 vector and transiently transfected into COS-7 cells. 3H-TCA uptake assay was performed as described previously2, while human ASBT served as a positive control. To test the sodium-dependency of [3H]-TCA uptake, sodium in the medium was substituted with choline. Real-time PCR was applied to determine tissue distribution of skate Asbt. Data were analyzed using Student’s independent t-test.
As illustrated in Figure 1A, uptake of 3H-TCA was significantly higher in the distal intestine than in the proximal intestine. In addition, uptake of 3H-TCA increased in a time-dependent manner in the distal intestine suggestive of active transport. In contrast, transport was abolished when sodium was substituted with choline, demonstrating sodium-dependency. Further molecular cloning identified an ortholog of ASBT in skate intestine. DNA sequencing indicates that the full-length skate Asbt cDNA is 2.3kb, encodes 393 amino acids and has 189 bp at the 5´-UTR and 918 bp at the 3´-UTR. Protein sequence alignment showed that skate Asbt shares 67% identity to human ASBT and has an extra 38 amino acid at its C-terminal end. Phylogenetic analysis indicates this gene is a member of SLC10A2 subfamily. Real-time PCR indicates that skate Asbt mRNA is most abundant in the distal segment of intestine followed by kidney and gallbladder, whereas the levels in other tissues are relatively low, including brain, liver, muscle, pancreas and spleen (data not shown). This tissue distribution of skate Asbt is very similar to its mammalian orthologs.
To test if skate Asbt can transport bile salts in a sodium-dependent manner, we transfected a skate Asbt expression construct into COS-7 cells. As demonstrated in Figure 1B, skate Asbt transports 3H-TCA only when sodium is present in the uptake solution. The transport activity of skate Asbt was also confirmed in a gene reporter assay where scymnol sulfate, the major endogenous bile salt in skate, was transported into the cells. When compared to human ASBT transport activity, skate Asbt was less efficient at 10 µM 3H-TCA solution (Fig. 1B). However, this difference disappeared at concentrations higher than 100 µM (data not shown),
indicating that human ASBT may have higher affinity for TCA than skate Asbt.
Figure 1 A, 3H-TCA uptake in the proximal and distal intestine of skate, in sodium (Na+) or choline (Ch) medium. B, 3H-TCA uptake by skate Asbt and human ASBT expressed in COS-7 cells. Cells were incubated with solution containing 10µM 3H-TCA (0.2 Ci/mmol) at 37°C for 10 minutes. All values are expressed as mean ± SD and represent 3-4 independent experiments. #P<0.05 distal intestine (Na+) versus each other group at the same time point, *P<0.05 as indicated.
In summary, we demonstrate for the first time that the distal but not proximal intestine of the skate absorbs bile salts in a sodium-dependent manner. Molecular cloning experiments indicate that Asbt is the functional component for this transport activity in skate. These findings signify that intestinal sodium-dependent bile salt transport systems are highly conserved throughout vertebrate evolution. Future studies will determine whether skate Asbt can transport substrates other than bile salts.
This work was supported by National Institutes of Health Grants DK34989, and DK25636 (J.L.B.) and a student grant from the Dutch Digestive Foundation (D.A.L).
1. Fricker, G, Wossner, R, Drewe, J, Fricker, R, and Boyer, JL. Enterohepatic circulation of scymnol sulfate in an elasmobranch, the little skate (Raja erinacea). Am.J.Physiol 273:G1023-G1030, 1997. 2. Liang, D, Hagenbuch, B, Stieger, B, Meier, PJ. Parallel decrease of Na1-taurocholate otransport and its encoding mRNAin primary cultures of rat hepatocytes. Hepatology 18:1162–1166, 1993. 3. Oelkers, P, Kirby, LC, Heubi, JE, Dawson, PA. Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2). J Clin Invest. 99:1880–1887, 1997. 4. Trauner, M and Boyer, JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 83(2):633-71, 2003. 5. Wilson, TH and Wiseman, G. The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J Physiol 123(1):116-25, 1954.
Endocrine disrupting compounds and pharmaceuticals in water containing natural organic matter
Jon Chorover1,2, Samanthi Wickramasekara1, Nathan Chorover2, Selene Hernandez-Ruiz1, Mary Kay Amistadi1 and Leif Abrell1
1Arizona Laboratory for Emerging Contaminants, University of Arizona, Tucson, AZ 85721 2Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
A wide range of trace organic contaminants is emerging in water sources as a result of expanding human usage of pharmaceuticals, hormones and personal care products (EDC/PPCPs) that are incompletely destroyed during wastewater treatment. Aquatic natural organic matter that is produced during decay of vegetation, biomass and anthropogenic waste is postulated to affect the environmental transport and fate of these contaminants. This project combines solid-phase-extraction methods with tandem mass spectrometry to quantify EDC/PPCP concentrations in wastewater effluent affected regions of the Charles River Watershed (MA) and in source water intakes and wastewater outfalls on Mount Desert Island (ME).
A wide range of “emerging contaminants” including pharmaceuticals and personal care products (PPCPs) and endocrine disrupting compounds (EDCs) have been measured in water sources throughout the US1. The presence of PPCPs and EDCs in streams has been attributed to widespread domestic use of these compounds, their eventual appearance in municipal wastewater, and wastewater treatment that is only partially effective in their removal prior to effluent discharge into surface waters. Thousands of different pharmaceutical substances are produced and used daily including painkillers, antibiotics, antidiabetics, β-blockers, contraceptives, lipid regulators, antidepressants, and impotence drugs. Since many of these compounds exert biological effects even at very low concentrations2, an understanding of their environmental transport behavior is needed to assess risks to human health. Effluent and biosolids from wastewater treatment plants are primary entryways for PPCPs and EDCs into the environment. Given that EDC/PPCPs may form soluble aqueous complexes with natural dissolved organic matter (DOM) potentially affecting their transport behavior and detection3, we are conducting an assessment of relations between DOM concentration/structure and EDC/PPCP profiles in watersheds of several participating municipalities including Austin (TX), Bar Harbor (ME), Boston (MA), Phoenix, and Tucson (AZ). Trace inorganic constituents also have potential use as surrogates for medical waste. For example, rare earth element (REE) data can be used to screen for gadolinium (Gd) anomalies, which provide an indication of hospital waste-derived contamination.4 The results reported herein pertain to samples collected in New England during July 2010.
Water samples were collected for trace organic and inorganic chemistry analyses from ten New England locations during July 2010 (see Table 1, Figure 1). For analysis of trace organic compounds (PPCPs and EDCs), four replicate water samples were collected in 1 L amber, glass bottles (I-CHEM certified to meet or exceed US EPA contaminent-free specifications; from VWR, cat. # IR249-1000). Water collections were acquired as grab samples or 24 hour drip composites. For trace inorganic solution chemistry analysis, water samples were collected in duplicate using 60 mL HDPE bottles, and then filtered through 0.45 μm nylon membrane filters prior to ICP-MS analyses. All water samples were held at 4 C until processing.
Water samples collected for trace organic compounds analysis were filtered through PALL 0.7 m glass fiber filters (VWR, cat. # 28149-456) before solid phase extraction (SPE) using either a hydrophobic lipid balance (Oasis HLB, 6 ml, 150 mg Waters Corp, cat. # 186003365) or an octadecyl reverse phase matrix (Strata C18-U, 6 ml, 500 mg, Phenomenex Inc., cat. # 8B-S002-HCH). All SPE cartridges were conditioned with 7 mL acetonitrile, 5 mL methanol, and 5 mL water. Filtered water samples were loaded under vacuum at ca. 10 mL min-1 followed by air drying under vacuum for ca. 20 min. Cartridges were eluted with a series including methanol, acetonitrile and MTBE. Eluents were subsequently evaporated to dryness and re-dissolved in 1 mL 50% aqueous methanol for LCMSMS injection. All solvents were liquid chromatography grade. Liquid chromatography was performed with 5 uL sample injections on a Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (1.7 µm, 2.1 x 50 mm ) and a gradient mobile phase of water and acetonitrile for 30 min. Ionization and detection of each indicator compound has been optimized resulting in cone voltage, capillary voltage, and drying gas flow settings optimized for electrospray ionization (ESI), and collision energy and collision gas pressure optimized for multiple reaction monitoring (MRM) by tandem mass spectrometry. Calibration curves consisting of at least seven points were developed for each analyte. Deuterated forms of target analytes were employed as internal standards.
Figure 1. Emerging contaminant concentrations measured in surface water samples collected during summer of 2010 in Charles River Watershed (MA, sites 1-9) and Mount Desert Island locations (ME, sites 10-13): (a) tonalide, fragrance, (b) triclosan, antibiotic, (c) nonylphenol, surfactant, (d) estrone, estrogenic hormone, (e) bisphenol A, plasticizer, (f) carbemazapine, anti-epileptic drug, (g) DEET, insect repellent, (h) ibuprofen, analgesic, (i) TCEP, flame retardant, (j) sulfamethoxazole, antibiotic, (k) iopromide, contrasting agent, (l) PFOS, surfactant, (m) ciprofloxacin, antibiotic, (n) salicylic acid, analgesic measured in water samples from nine different New England locations (Table 1) using two different extraction methods (HLB and C18). Data pertaining to the concentrations of EDC/PPCP compounds sampled from sites in MA and ME are shown in Figure 1. Data for 14 compounds are plotted in two columns in order of increasing water solubility (decreasing compound hydrophobicity or octanol-water partition coefficient). In addition, the graphs show a comparison of compound recoveries made using a hydrophobic (C18) resin versus a hydrophilic-lipophilic balance (HLB) resin. In general, HLB resins appear to exhibit greater recoveries with our current methods than do the C18 resins, and so we are using this method of SPE in our related research. Sites 1 through 9 in the Charles River Watershed are sampled with progressive distance down gradient in the stream. Site 1, a river stem site that is up-gradient from all wastewater treatment plants (WWTPs), but is impacted by septic tank leachate, shows relatively low concentrations of most target compounds. Sites 2, 4, 5 and 6 are WWTP outfalls that exhibit the highest concentrations of analytes, and the concentrations of these compounds are attenuated with increasing distance down river (Sites 7-9) from the lowest gradient WWTP (site 6). Very high concentrations of carbemazepine were observed in WWTP outfalls from the Wrentham DC, a hospital for mentally disabled patients. In contrast, relatively high concentrations of nonylphenol, estrone, DEET, and ibuprofen were observed in discharges from the Bar Harbor WWTP.
As indicated above, an issue of particular concern relates to the extent to which EDC/PPCP compounds form soluble complexes with natural DOM and, if so, whether that affects our ability to detect and quantify trace organic contaminants in natural waters. To answer that question, we employ spectroscopic (fluorescence quenching) experiments on solutions containing wastewater DOM at concentrations typical for the region to determine DOM-EDC/PPCP and while also assessing target analyte recoveries by LC-MSMS.
Figure 2. Fluorescence spectroscopy / LC-MSMS experiment to effects of EDC/PPCP-DOM bonding interactions on detection and quantification of target analytes. (DOM concentrations of 8 mg L-1 are used, which is characteristic of Charles River and Mount Desert Island Example here is for bisphenol A, ibuprofen and carbamazepine. Top fluorescence excitation-emission matrix is for wastewater DOM. Bottom matrices show intensity of DOM fluorescence quenching that results from complexation with target analytes. A large quenching of fluorescence occurs in the case of ibuprofen, concurrent with only 51% recovery of spiked analyte via LC-MSMS. More complete recovery of BPA and carbamazepine is consistent with lower fluorescence quenching.3
Funding for this research was provided in part by the U.S. National Science Foundation (Major Research Instrumentation Program Grant no. 0722579). University of Arizona gratefully acknowledges that the Water Research Foundation is the joint owner of certain technical information upon which this report is based. University of Arizona thanks the Foundation for their financial, technical, and administrative assistance in funding the project through which this information was discovered. The comments and views detailed herein may not necessarily reflect the views of the Water Research Foundation, its officers, directors, affiliates, or agents. Copyright 2009, Water Research Foundation and the University of Arizona, ALL RIGHTS RESERVED.
1. Kolpin, DW, Furlong, ET, Meyer, MT, Thurman, EM, Zaugg, SD, Barber, LB, Buxton, HT. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: A national reconnaissance. Environ. Sci. Technol. 36: 1202-1211, 2002. 2. Diamanti-Kandarakis, E, Bourguignon, JP, Giudice, LC, Hauser, R, Prins, GS, Soto, AM, Zoeller, RT, Gore, AC. Endrocrine-disrupting chemicals: An Endocrine Society scientific statement. Endocrine Rev. 30: 293-342, 2009. 3. Hernandez-Ruiz, S, Abrell, L, Wickramasekara, S, Chorover, J. Evaluation of chemical interactions between dissolved organic matter and model EDC/PPCPs. Abstracts of the American Chemical Society National Meeting, Fall 2009. 4. Kulaksiz, S, Bau, M. Contrasting behaviour of anthropogenic gadolinium and natural rare earth elements in estuaries and the gadolinium input into the North Sea. Earth Planet. Sci. Let. 260: 361-371.
The rocketing motility of cytoplasmic ridges present in spreading sea urchin (Strongylocentrotus droebachiensis) coelomocytes is driven by Arp2/3 complex-facilitated actin polymerization
Anastasia Gianakas1, Robert L. Morris2, and John H. Henson1 1Department of Biology, Dickinson College, Carlisle, PA 17013 2Department of Biology, Wheaton College, Norton, MA 02766
Cell motility is critical for aspects of development, immune defense and tissue function and is based on the dynamics of the protein filaments that constitute the cellular skeleton. The growth of actin filaments can be facilitated by a number of other proteins including the Arp2/3 complex. In this study we demonstrate that the motility of cytoplasmic ridges in spreading sea urchin blood cells (coelomocytes) is mediated by Arp2/3 complex-driven actin filament growth.
We have used sea urchin coelomocytes as a model experimental system for studying actin-based motility. As these disc-shaped cells spread onto glass substrates they exhibit extensive actin-based centripetal flow towards the cell center 1 and at the same time phase dense linear processes can be observed rocketing around the periphery of the cell, often in a centrifugal direction. We hypothesized that the movement of these ridge-like processes was mediated by actin polymerization facilitated by the activity of the Arp2/3 complex. This complex is known to play a role in polymerization of the branched actin filament arrays present at the leading edge of broad, fan-shaped lamellipodia. Digitally-enhanced video phase contrast microscopy as well as immunofluorescent labeling employing conventional and confocal microscopy revealed that these processes are physical ridges in the cell surface that are actin-filled and distally-enriched in Arp3 (Figure 1). Two pharmacological treatments were utilized to further characterize the roles for actin and the Arp2/3 complex. Process motility was inhibited in coelomocytes treated with the actin polymerization disrupting drug Cytochalasin D. Treatment with Cytochalasin D halted the ridge mobility and also induced a curling of the distal tips driven in part by the centripetal movement associated with residual myosin tension on the actin cytoskeleton. In addition process movement was also interfered with by treatment with the drug 2,3- butanedione monoxime (BDM), which we have previously demonstrated to delocalize the Arp2/3 complex from the coelomocyte lamellipodial edge and to therefore alter the nature of actin organization 1. Interestingly, upon washout of the BDM drug a subset of cells exhibit spontaneous regeneration of the ridge processes. Note that BDM does not appear to inhibit myosin II contractility in the coelomocyte system 1. Microtubules are known to regulate aspects of the actin cytoskeleton in other cell types, however they were not physically associated with the actin and Arp2/3 complex-rich ridges. Taken together these results suggest that Arp2/3 complex-facilitated actin polymerization can play a role in the generation of the rocketing activity of ridges of actin-filled peripheral cytoplasm involved in coelomocyte spreading.
Figure 1: Double immunolabeling of actin (A) and Arp3 (B) in coelomocytes shows the clear enrichment of each in the cytoplasmic ridges (arrows in A) present in spreading cells. This cell is 40 microns in diameter.
Supported by a MDIBL Forster Fund Scientific Team Visiting Fellowship and a student/faculty research grant from Dickinson College.
1. Henson, JH, Cheung, D, Fried, CA, Shuster, CB, McClellan, MK, Voss, MK, Sheridan, JT and Oldenbourg, R. Structure and dynamics of an Arp2/3 complex-independent component of the lamellipodial actin network. Cell Motil. Cytoskel. 66: 679-692, 2009. Defining the ectopic expression pattern of nodal induced by nickel, zinc, and Dynasore in embryos of the sea urchin Strongylocentrotus purpuratus
Diane C. Saunders, Robin P. Ertl, and James A. Coffman Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
In sea urchin embryos, localized expression of the nodal gene specifies the oral-aboral axis of the embryo. Embryos exposed to nickel or zinc ions become radialized owing to ectopic expansion of nodal expression at blastula stage, when there are no morphological ‘landmarks’. Using whole mount in situ hybridization with multiple gene-specific probes that allow definition of the embryonic axes, this study defined the spatial pattern of this ectopic expression, and tested the hypothesis that localization of nodal expression requires dynamin-mediated endocytosis.
Early development establishes the axes that define the body plan. In the sea urchin, a nonchordate deuterostome, the secondary or oral-aboral (O-A) axis becomes evident at the gastrula stage and, together with the animal-vegetal (A-V) axis, defines the plane of bilateral symmetry in the later larval stages. The gene nodal controls specification of the O-A axis. The Nodal protein is a member of the TGF-β superfamily of extracellular signaling ligands that interacts with receptor serine-threonine kinases to activate Smad2/3, a transcription factor essential for the differentiation of cells in the oral territory. Via this signal transduction pathway, Nodal positively regulates its own production and also the gene encoding its competitive inhibitor, Lefty. The Lefty protein diffuses further than Nodal, establishing a boundary on the nodal expression domain, which marks the prospective oral ectoderm. Oral ectoderm gives rise to facial nerves and larval mouth, whereas aboral ectoderm differentiates into a simple epithelial tissue. Overexpression of nodal causes the entire ectoderm to adopt an oral fate, whereas blocking nodal expression has the opposite effect1.
Nickel ions have been shown to mimic phenotypic effects of nodal overexpression1, an effect that is also obtained with low micromolar zinc2. Exposure to these ions disrupts the formation of the O-A axis, inducing radial symmetry in the embryo by converting the entire ectoderm to oral territory. Whole mount in situ hybridization (WMISH) data obtained in the Coffman lab thus far suggest that both nickel and zinc cause expansion of nodal and lefty expression. However, in the early blastula stage embryo it is difficult to define the specific spatial pattern of this expansion, since there are no morphological landmarks.
The purpose of this study was to clarify the spatial effects of zinc and nickel ions on nodal expression, as well as to investigate the role of dynamin-mediated endocytosis in nodal regulation. The latter studies made use of Dynasore, a specific inhibitor of dyanamin3. Using fluorescent WMISH, expression profiles of nodal, as well as foxQ2, a gene specifying the animal pole4, were analyzed by confocal fluorescence microscopic imaging. Double-labeling the embryos enabled definitive orientation of the embryos in relation to the A-V and O-A axes.
Sequence-specific oligonucleotide primers were used to amplify cDNA fragments from 10-hour embryos. Purified PCR product (PureLink PCR Micro Kit, Invitrogen, Carlsbad, CA) was inserted into a vector at a 3:1 molar ratio using pGEM®-T Easy (Promega, Madison, WI). The vector was then transformed into DH5α E. coli cells using high efficiency electrotransformation. Following bacterial cells harvest (Plasmid Midi kit, Qiagen, Germantown, MD), purified plasmids were sequenced using SP6 primers. The GenBank accession numbers for the nodal and foxQ2 sequences are EF036514.1 and DQ286735, respectively. RNA probes were synthesized following a protocol adapted from mMessage mMachine kit (Ambion, Austin TX). Digoxigenin (nodal) and fluorescein (foxQ2) labeled probes (Roche, Indianapolis, IN) were synthesized with SP6 RNA polymerase after linearization of the plasmids by SpeI (foxQ2) and NotI (nodal) and purification (PureLink, Invitrogen).
Embryos cultured in natural filtered seawater were treated with either 2 µM ZnCl2 or 15 µM NiCl2 beginning 14 hours post-fertilization (hpf). Embryos were treated with Dynasore (Tocris, St. Louis, MO) beginning 6 hours (6 µM treatment) or 12 hours (12 µM treatment) post-fertilization. At 24 hours embryos were fixed in paraformaldehyde and permeablized by treatment with proteinase K and plunging into ice cold methanol. Methanol was replaced with PBS with 0.1% Tween-20 (PBST) buffer by 25% and then 4x50% volume, followed by 3 washes in PBST. After one wash with hybridization (hybe) buffer (50% formamide, 5x denhardts, 5X SSC, 1 mg/ml yeast RNA, and 50 µg/ml heparin), embryos were incubated with in hybe buffer at 65o C for one hour. Embryos were then incubated at 65o C for 7 hrs in hybe mixture containing 200 ng of each probe, ramped at 0.5o C/min to 60o C and held for 8 hours. The following buffer washes occurred for 15 minutes at 60o C: hybe; 50% hybe/50% 2X SSCT; 2X SSCT; 0.2X SSCT; 0.1XC. This was followed by 3x15 min washes in MABT (0.1M maelic acid pH 7.2, 0.15M NaCl, 0.1% Tween-20) and 30 minute incubation in blocking solution. The appropriate peroxidase conjugated antibodies (Roche) were incubated for 1 hr in blocking solution at room temperature, followed by staining at 37o C with fluorescent substrates (TSA Plus Cyanine 3/Fluorescein System, PerkinElmer, Waltham, MA). The Cyanine 3 substrate was added to the primary antibody for 20 minutes and terminated by 4x 5min PBST washes. Embryos were then treated with
0.15% H2O2 for 6 minutes, followed by washes for 5 min in PBST and 3x 5 min in MABT. After application of secondary antibody, the Fluorescein substrate was incubated for 30 minutes and terminated identically to cy3. Embryos were imaged using a Zeiss LSM Meta 510 (Carl Zeiss, Inc., Thornwood, NY) confocal microscope.
The control embryos exhibited a characteristic expression profile of nodal restricted to the prospective oral ectoderm (Fig. 1A, red). Zinc- and nickel-treated embryos showed expanded expression, with nodal appearing in both the oral and aboral regions (Fig. 1B, C). The nickel-treated embryos additionally displayed some coexpression with foxQ2 in the animal pole region (Fig. 1B).
Figure 1. Expression of nodal (red) in (A) control, (B) 15uM Ni- treated (B), and (C) 2 µM Zn-treated Stronglyocentrotus purpuratus embryos, with foxQ2 (green) marking the animal pole. Embryos fixed at 24 hpf were probed by WMISH using digoxigenin (nodal) and fluorescein (foxQ2) labeled probes with peroxidase conjugated antibodies and fluorescent substrates.
In Dynasore-treated embryos, nodal expression was similarly radialized (Fig. 2A-C), which is even more clearly shown in polar views (Fig. 2D-F), with the animal pole marked by foxQ2 expression.
Control 6 µM Dynasore 12 µM Dynasore
Figure 2. Lateral (A-C) and end-on (D-F) views of nodal expression (red) in control and Dynasore-treated Stronglyocentrotus purpuratus embryos, with foxQ2 (green) marking the animal pole. Embryos fixed 24hpf were probed by WMISH using digoxigenin (nodal) and fluorescein (foxQ2) labeled probes with peroxidase conjugated antibodies and fluorescent substrates.
The expanded expression of nodal in nickel and zinc-treated embryos is consistent with previous observations1, 2. The addition of a probe for foxQ2 provides a definitive orientation of the animal-vegetal axis, and because images are the projections of several sections (each 3.5um thick), we can be confident that nodal expression is occurring in both the oral and aboral territories of treated embryos (Fig. 1).
The slight expansion of foxQ2 from the animal pole in some treatments (Fig. 1B, 2B) is likely to reflect changes in developmental timing. Because the transcription factor is first expressed in all mesomeres during cleavage and becomes restricted to the animal pole during the blastula stage4, slightly younger embryos may exhibit less localized expression. As zinc, nickel, and Dynasore cause a slight delay in development in addition to inducing radialization, it is not surprising to see that in these embryos foxQ2 expression is more diffuse.
Interestingly, nickel-treated embryos show some nodal expansion in the animal pole region (Fig. 1B) as compared to the zinc-treated embryos, where nodal appears relatively absent from this territory (Fig. 1C). This phenomenon is consistent with previous data suggesting that foxQ2 expression is depressed in nickel-treated embryos2. Further investigation of both nodal and foxQ2 expression, perhaps by using another marker of the animal pole, six3, will be necessary to establish whether nodal expansion in the animal pole territory is facilitated by lower foxQ2 expression.
The expression of nodal in the end-on view of embryos, in which foxQ2 expression clearly marks the animal pole in the topmost image plane, offers convincing evidence for radialization as compared to the control in the Dynasore-treated embryos (Fig. 2D-F). Nodal expansion observed in the Dynasore-treated embryos (Fig. 2) supports the hypothesis that Nodal expression is regulated by a reaction-diffusion mechanism, which requires a sink (endocytosis) to limit accumulation of Nodal protein. The expansion of nodal in embryos with blocked endocytotic capabilities is consistent with a reaction-diffusion mechanism of pattern formation similar to that which has been described for other morphogens5.
This work was supported by the NCRR Maine IDeA Network of Biomedical Research Excellence (NIH P20-RR016463) and by a research grant from the NIH (R01-ES016722) to J.A.C.
1. Duboc V, Rottinger E, Besnardeau L, and Lepage T. Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. Dev Cell 6: 397-410, 2004. 2. Ertl RP, Robertson AJ, Saunders D, and Coffman JA. Nodal-mediated epigenesis requires dynamin-mediated endocytosis. Dev Dyn 240: 704-711, 2011. 3. Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, and Kirchhausen T. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10: 839-850, 2006. 4. Tu Q, Brown CT, Davidson EH, and Oliveri P. Sea urchin Forkhead gene family: phylogeny and embryonic expression. Dev Biol 300: 49-62, 2006. 5. Yu SR, Burkhardt M, Nowak M, Ries J, Petrasek Z, Scholpp S, Schwille P, and Brand M. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461: 533-536, 2009.
Developmental plasticity of sea urchin (Strongylocentrotus purpuratus) larvae in response to varying diet
Benjamin Davis and James A. Coffman Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
Larvae of sea urchins and other echinoderms are well known for their developmental plasticity as evidenced by their ability to regenerate organs, reproduce asexually through clonal budding, and suppress development of the juvenile rudiment when faced with low nutrient availability. This phenotype can be reproduced in the laboratory through dietary restriction, and such larvae can be maintained in a state of arrested development for many months. Here we show that six-month old developmentally arrested sea urchin larvae retain the capacity to resume development and complete metamorphosis when feeding rate is increased, and that this response is inhibited by rapamycin, indicating that juvenile development is controlled by evolutionarily conserved nutrient-sensing pathways involving the target of rapamycin (TOR) kinase.
Echinoid (sea urchin and sand dollar) life history involves a benthic adult stage and a pelagic larval stage. Maintaining echinoid larval cultures below certain feeding thresholds prevents development of the juvenile (‘echinus’) rudiment and subsequent metamorphosis from pluteus larvae to juvenile adult.1 Larvae cultured under conditions of diet restriction (DR) develop an alternate phenotype that has been observed in natural planktonic populations experiencing low nutrient availability.12 This phenotype is characterized by reduced allocation of tissue to the echinus rudiment and increased allocation to larval feeding apparatuses, suggesting a mechanism to favor larval survival over progression through metamorphosis.
Larvae are obligate feeders and must overcome fluctuations in nutrient availability in addition to variability in temperature and other environmental factors in order to reach maturity.12 The phenotypic plasticity demonstrated in response to food levels is one adaptive mechanism by which larvae could respond to environmental cues and thereby enhance species survival. To evaluate the viability of such a mechanism it must be established whether individuals suspended long-term in this state of indefinite immaturity can re-enter the ‘normal’ life cycle and undergo metamorphosis. While following planktonic larvae in situ over great distances and varied environments presents significant logistical challenges, the basic question can be addressed in the laboratory using long-term cultures maintained under DR. A capacity for larvae that have remained in a developmentally arrested state for significant periods of time to regain metamorphic competency upon return to favorable conditions could represent a dispersive ‘life raft’ for the species.
Larvae of the sea urchin Strongylocentrotus purpuratus were cultured long-term (> 6 months) on a restricted diet using standard methods12: Embryos at early pluteus stage where diluted to a density of approximately 1 larva/ml in a culture medium of sterile filtered sea water (SFSW) that was changed bi-weekly to reduce accumulation of waste products. Cultures were maintained on bi-weekly feedings of a 1:1 mixture of the algae Dunaliella tertiolecta and Rhodamonas salina. DR conditions were established by feeding at an algal concentration estimated at less than 2,000 cells/ml. No juvenile or transitional individuals were identified when sampling from the primary culture prior to transitioning to a more robust diet, and all larvae displayed the long arms and diminutive or absent rudiments characteristic of the DR phenotype. At 198 days post fertilization, two dishes of 50 larvae each were transitioned to an ad libitum (AL) feeding regimen of approximately 40,000 cells/ml. In one of the dishes 100 µM rapamycin was added to the SFSW. The cultures were monitored by hand transferring individuals to fresh media. Competency was gauged by scoring individuals as retaining the pluteus form (P), demonstrating a transitional form (T) as evidenced by emerging juvenile structures such as tube feet and spines that were readily apparent at low magnification, or finally as metamorphosed (M) if the larval body was no longer visible and had been replaced by a radially symmetric juvenile form.
Larvae cultured long-term under DR attained metamorphic competency upon transfer to AL feeding. In the untreated AL culture, the first transitional individual was observed on the sixth day post transfer and by day 20, 28% of the culture had completely metamorphosed. At day 24, the conclusion of the study, 36% had metamorphosed, 46% were in transition and only 18% retained a pluteus form. In contrast, rapamycin significantly inhibited development to competency following transfer to AL conditions. No transitional individuals were identified until day 13 and at the conclusion of the observation period no individuals had metamorphosed. These results are summarized in Table 1.
Rap (+) Rap (-) Day P T M P T M Table 1. Effects of rapamycin and AL 0 100 - - 100 - - diets on metamorphic competency. 3 100 - - 100 - - Percent individuals demontstrating 6 100 - - 98 2 - pluteus (P), transitional (T), or 9 100 - - 98 2 - juvenile (M) morphology in AL cultures, with and without exposure to 13 94 6 - 80 20 - rapamycin, over time. 20 87 13 - 47 25 28 22 75 25 - 42 25 33 24 66 34 - 18 46 36
These results suggest that the DR phenotype is indeed a viable alternate developmental trajectory. The extent to which such viability can be maintained in terms of duration and nutrient deprivation will be further explored in future studies.
In addition to shedding light on novel aspects of echinoid life history, exploration of this phenomenon is warranted as it speaks to fundamental biological questions such as the nature of temporal aging and the limits on longevity. If cultures maintained in controlled conditions can be sustained indefinitely, it would suggest that echinoid lifespan is determined not merely by environmental factors but by an interaction between the environment and genetically based programs that have been shaped by selection to regulate the series of events that define a finite life cycle. The marked affect of rapamycin on the ability of DR plutei to metamorphose has corollaries in two classic models of aging and stress biology, the mouse and C. elegans. The life span of mice exposed to rapamycin at advanced age has been shown to be significantly increased.9 Perhaps more relevant is the alternate developmental trajectory known as the dauer in C. elegans, which can be induced both through diet restriction and suppression of the target of rapamycin (TOR) signaling pathways.6 In addition to increased longevity, dauer specimens have greater resistance to environmental stressors due to the regulation of such responses by TOR.
An interesting aspect of this particular model of aging and longevity are the ties it presents to the study of how temporal age affects developmental plasticity, a hallmark of immature systems that is generally lost through the aging process. It is well known that echinoid larvae possess a high degree of developmental plasticity as evidenced by their capacities for phenotypic plasticity, organ regeneration, and asexual reproduction through clonal budding. 2-5,7,12 Investigating the gain or loss of developmental plasticity in long term cultures could prove informative to our understanding of the effects of age on developmental plasticity in other deuterostomes, such as humans. A necessary first step in establishing this model is to evaluate the extent to which hallmarks of developmental plasticity such as regeneration and cloning are affected by long term culture under DR.
From an evolutionary standpoint, these abilities present mechanisms by which larvae might be able to meet some of the selective pressures to which they are subjected. Regeneration presents obvious advantages to individuals that incur injuries due to predation. Further, the incidence of cloning has been shown to increase in the presence of predatory cues such as fish mucus.7,12 The cloning response has been demonstrated to produce larvae of decreased size, and it has been suggested that this may thwart predation by visual feeders such as fish.3,5,7,12 Additionally, echinoid larvae are capable of somatic nutrient exchange with surrounding sea water,9 a process that would be augmented by size reduction and the accompanying increase in surface to volume ratio. Other studies have indicated that the incidence of cloning of asteroid larvae is maximized under ideal conditions of nutrient availability and temperature.3 If this level of plasticity persists in long term DR cultures it would represent a mechanism by which larval populations could be rapidly increased upon meeting with environments conducive to growth and survival of mature colonies. This work was supported by the NSF REU program at MDIBL (DBI 0453391).
1. Strathmann RR, Fenaux L, Starthmann MF. Heterochronic developmental plasticity in larval sea urchins and its implications for evolution of nonfeeding larvae. Evolution 46(4): 972-986, 1992. 2. Vickery MS, McClintock JB. Regeneration in metazoan larvae. Nature 394: 140. 1998. 3. Vicker MS, McClintock JB. Effects of food concentration and availability on the incidence of cloning in the planktotrophic larvae of the sea star Pisaster ochraceus. Bio Bull 199: 298-304, 2000. 4. Vickery MS, Vickery MCL, McClintock JB. Morphogenesis and organogenesis in the regenerating planktotrophic larvae of Asteroids and Echinoids. Biol Bull 203: 121-133, 2002. 5. Eaves AA, Palmer AR. Widespread cloning in echinoderm larvae. Nature 425: 146, 2003. 6. Vellai T, Takacs-Vellai K, Zhang Y, Kavacs AL, Orosz L, Muller F. Influence of TOR kinase on lifespan in C.elegans. Nature 426:620, 2003. 7. Vaughn D, Strathmann RR. Predators induce cloning in Echinoderm larvae. Science 319: 1503, 2008. 8. Chera S, Ghila L, Dobertz K, Wenger Y, Bauer C, Buzgariu W, Martinou JC, Galliot B. Apoptotic cells provide an unexpected source of Wnt3 signalinh to drive Hydra head regeneration. Developmental Cell 17: 279-289, 2009. 9. Harrison DE, Strong ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA. Rapamycin fed late in life extends lifespan in genetically heterogenous mice. Nature 460: 392-396, 2009. 10. Meyer E, Manahan DT. Nutrient uptake by marine invertebrates: Cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus). Bio Bull 217: 6-24, 2009. 11. Pelletieri J, Fitzgerald P, Wantanabe S, Mancuso J, Green DR, Sanchez Alvarado A. Cell death and tissue remodeling in planarian regeneration. Dev Biol doi:10.1016/j.ydbio.2009.09.015, 2009 12. Vaughn D. Predator-induced larval cloning in the sand dollar Dendraster excentricus: Might mothers matter? Biol Bull 217: 103-114, 2009.
Development and partial characterization of two cell lines derived from pituitaries of adult Atlantic salmon, Salmo salar
Nguyen T.K. Vo, Mike S. Mikhaeil, and Lucy E.J. Lee Department of Biology, Wilfrid Laurier University, ON, CANADA N2L 3C5
Atlantic salmon, Salmo salar, are highly valued fish for both fisheries and aquaculture in Canada and worldwide. Growth and reproduction in this species have been widely studied and factors regulating their life cycle including somatic growth, sexual maturation and reproduction, spawning, smoltification have been extensively investigated. However, the molecular mechanisms controlling expression of the hormones involved in these processes are largely unknown. Pituitary cell cultures could be valuable for elucidating these mechanisms, and cell lines derived from this teleost‘s pituitary could make significant impacts in understanding growth and hormonal regulation as well as endocrine disruption by environmental contaminants. Here we report on the development of two continuous cell lines derived from adult Atlantic salmon pituitary and their preliminary characteristics.
Atlantic salmon, Salmo salar, is the predominant fish species farmed in Canada as well as in Maine. Research on these species is of high priority for enhancing their output and minimizing disease, and cell lines are being sought to assist in this quest. In the past year, we reported on the establishment of an Atlantic salmon intestinal myofibroblast cell line, ASimf20, that was established in an initial attempt to study inflammatory and cytotoxic responses caused by plant-derived fish feeds in the gastrointestinal tract8. In this article, we report on the development and initial characterization of two Atlantic salmon pituitary cell lines.
Primary cell cultures were initiated from pituitaries of several mature female Atlantic salmon that were obtained from National Cold Water Marine Aquaculture Center in Franklin, ME. The pituitary tissues were aseptically removed, minced into smaller pieces, and rinsed twice in sterile Hank’s Buffered Salt Solution with 1% penicillin/streptomycin. The minced tissues were placed in 12.5-cm2 tissue culture flasks and allowed to attach to the culture surface in Leibovitz’ L-15 media supplemented with 10% fetal bovine serum (FBS) at 15°C. Cell outgrowth directly from the pituitary explants was observed after 7-10 days in culture. However, confluent monolayers of cells were obtained after more than 4 months in culture only for two out of four attempted cultures, resulting in the establishment of two cell lines, designated as ASP309 and ASP409.
ASP309 ASP409 A B C D
E F G H
Figure 1. Phase-contrast morphologies and some culture properties of ASP309 and ASP409 cell lines. Mitotic figures (arrow) are seen in confluent monolayers of ASP309 at passage 12 (Panel A) and ASP409 at passage 5 (Panel C). ASP309 readily attach and spread out to the culture surface after treatment with TryplE and being re-plated onto a new 24-well microplate within an hour (Panel B) whereas ASP409 cells or cell clumps are still in suspension (Panel D). Pituitary cells were incubated in either serum-free L-15 (Panels E and G) media or L15/ex (Panels F and H). Unlike ASP309, ASP409 experience morphological stresses (presence of apoptotic bodies) in 24-h exposure to L15/ex. Scale bars = 100 µm.
Table 1. Comparison of two Atlantic salmon pituitary-derived ASP309 and ASP409 cell lines Pituitary cell line Properties ASP309 ASP409 Morphology Fibroblastic (predominant) Epithelial Cell sizea (µm) 20.5 ± 0.5 9.5 ± 0.5 Enzymatic dissociation Cells readily dissociate (in 1 min) into Cells take longer to dissociate and form individual cells small clumps/sheets Culture properties after Cells readily attach and spread out within Cells take longer (more than 4 hours) to dissociation an hour on cell culture surfaces spread out Doubling timeb (estimated) 9 days 3 days Current passage 10 14
Toptimal growth (°C) 18-20 18-20 Cells show clear signs of stresses even Acute tolerance in minimal salt No morphological sign of stresses only after 24 hours (viability decreases to solution L15/exc 70% compared to cells exposed to L15 alone) Phagocytotic capacityd Yes Yes β-galatosidase activity 16.89% 1.82% Spheroid body formatione Tightly packed Loosely packed a calculated from two independent counts by the TC10TM automated cell counter (BioRad) b cell proliferation was assayed by using fluorometric dye Alamar Blue® (Invitrogen) c L15/ex is a simplified solution of L-15 media that excludes all the amino acids, vitamins, and phenol red d determined by the ability of cells to uptake fluorescent carboxylate-modified latex beads of 2 µm in diameter after 24 hrs e Hanging drop method employed to form cell aggregates called spheroid bodies
These cell lines have been maintained in L-15 media supplemented with 10% FBS at 18-20°C, have been routinely subcultured for over 15 generations by TryplE (a type of recombinant trypsin trademarked by Invitrogen) in a span of a year, and successfully cryopreserved at passages 3-14. ASP309 are predominantly fibroblastic surrounding small islands of polygonal-like large epithelial cells, whereas the ASP409 cell line consists of mostly smaller-sized epithelial cells containing dark condense nuclei with several nucleoli (Fig. 1- A&C, Table 1). Interestingly, in contrast to ASP309, ASP409 cell proliferation is relatively fast. DAPI staining revealed that a small population of cells underwent triploid and tetraploid mitotic divisions (Fig. 2). Moreover, by the hanging drop method9, ASP409 formed loosely-packed spheroid bodies, an in vitro characteristic of tumor-derived cell cultures, whereas ASP309 formed well-defined spheroid bodies, a stem cell characteristic (Fig. 3). Whether ASP409 arose from a neoplastic or malignant pituitary, or was a result of oncogenic transformation was not known. Karyotyping is currently underway to determine the chromosome distribution of ASP409 cell line.
In addition, about 17% of ASP309 cells within a monolayer stained positive for β-galactosidase activity whereas only 2% of ASP409 was stained positive (Table 1). This result indicates that ASP309 has not yet been completely spontaneously immortalized (a common phenomenon in fish cell cultures10) or continues to develop terminally differentiated specialized cells such as gonadotrophs from progenitor cell populations. On the other hand, low senescence detection in ASP409 suggests this cell line has become an immortal cell line, which could account for the high mitotic activities and proliferation rate.
A B C D
Figure 2. Phase-contrast and fluorescence pictures of DAPI staining of confluent ASP409 cultures at passage 13. Triploid (Panels A and B) and tetraploid (Panels C and D) mitotic divisions were randomly distributed in cultures. Scale bars = 50 µm.
Figure 3. Formation of spheroid bodies by the hanging drop method. Pituitary cell suspensions were allowed to “hang” in tiny droplets on non-adherence culture surface for 3 days. ASP309 form nicely compact spheroid bodies whereas ASP409 form loosely packed cell aggregates and failed to form well-defined spheroid bodies.
The pituitary gland produces growth hormones, endocrine-related peptides, and neuropeptides that can have immunogenic roles. Thus the effects of conditioned media (CM) of ASP309 and ASP409 cultures on the immune cells such as professional macrophages were also investigated. Preliminary results suggested that seven-day-old CM from ASP309 cultures caused the round rainbow trout monocyte/macrophage cells RTS114 to enhance adherence onto culture surface and become more spindle-like only after 24 h (Fig. 4). In contrast, CM from ASP409 cultures did not induce any noticeable morphological changes. Neuropeptides such as pituitary adenylate cyclase-activating polypeptides (PACAP-38) have been shown to enhance adherence of peritoneal rat macrophages5. Since PACAP-38 is highly conserved in vertebrates including fish7, it is possible that ASP309 are capable of producing and releasing PACAP-38 into the culture media in vitro, thus accounting for previously mentioned observations. Moreover, growth hormones and prolactin have been reported to stimulate the activation of mammalian and teleost professional phagocytes2,6. Thus, both enhancement in adherence and the quick change in cell morphologies could also imply that RTS11 cells had differentiated into more mature macrophages. Molecular characterizations are currently underway to understand which factors in ASP309 CM induce these observations in RTS11.
Further molecular and biochemical characterizations are needed to clearly identify the cell type(s) present within these Atlantic salmon pituitary cell lines. Whether these cells produce pituitary hormones needs to be further investigated. Subsequently, studies of how environmental contaminants affects endocrine functions and induce cytotoxic insults would be promising. Pituitary is one of the tissues that have been reported to express cytochrome P450 1A1 in fish through EROD activity both in vivo and in vitro1,11,12. In particular, gonadotrophs in the anterior pituitary is believed to serve as “target cells” for CYP450-inducing aromatic hydrocarbon compounds (AHCs)1. Thus we are currently investigating whether these two pituitary cell lines are inducible by a variety of AHCs by measuring EROD activities.
A B C
Figure 4. Effect of conditioned media (CM) from ASP309 and ASP409 cultures on rainbow trout macrophage (RTS11) cells. RTS11 were exposed to 10% FBS as a control (panel A), ASP309 CM (panel B), or ASP409 CM (panel C). Morphological change was observed 24-h post-exposure. Scale bar = 50 µm.
ASP309 and ASP409 join a very modest list of pituitary cell lines originating from poikilothermic vertebrates3, and could become invaluable tools for mechanistic, endocrinological, toxicological and fish nutrition studies in Atlantic salmon.
This research was supported by a 2009 New Investigator Award from MDIBL and Discovery and Strategic grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to LEJL. We would like to acknowledge Dr. William R. Wolters from the USDA, ARS National Cold Water Marine Aquaculture Center, Franklin, ME, for providing the fish used in this study. Assistance from Brooke Beggs, Neil McCaffrey and Krista Schleicher for maintaining the cell lines is acknowledged. The authors are also grateful to Wilfrid Laurier University for a STEP travel award to NTKV that facilitated his travel and accommodation at MDIBL where this study was initiated.
1. Andersson, T, Forlin, L, Olsen, S, Fostler, A and Breton, B. Pituitary as a target organ for toxic effects of P4501A1 inducing chemicals. Mol. Cell. Endocrinol. 91: 99-105, 1993. 2. Auernhammer, CJ and Strasburger, CJ. Effects of growth hormone and insulin-like growth factor I on the immune system. Eur. J. Endocrinol. 133: 635-645, 1995. 3. Bols, NC, Yang, BY, Lee, LE and Chen, TT. Development of a rainbow trout pituitary cell line that expresses growth hormone, prolactin, and somatolactin. Mol. Mar. Biol. Biotechnol. 4: 154-163, 1995. 4. Ganassin, RC and Bols, NC. Development of a monocyte/macrophage-like cell line, RTS11, from rainbow trout spleen. Fish Shellfish Immunol. 8: 457-476, 1998. 5. Garrido, E, Delgado, M, Martinez, C, Gomariz, RP and De la Fuente, M. Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility. Neuropeptides 30: 583-95, 1996. 6. Harris, J and Bird, DJ. Modulation of fish immune systems by hormones. Vet. Immunol. Immunopathol. 77: 163-176, 2000. 7. Jakab, B, Reglodi, D, Jozsa, R, Hollosy, T, Tamas, A, Lubics, A, Lengvari, I, Oroszi, G, Szilvassy, Z, Szolcsanyl, J and Nemeth, J. Distribution of PACAP-38 in the central nervous system of various species determined by a novel radioimmunoassay. J. Biochem. Biophys. Methods 61: 189-198, 2004. 8. Kawano, A, Dixon, B, Bols, NC and Lee, LEJ. Establishment of a myofibroblast cell line from the gastrointestinal tract of Atlantic salmon. MDIBL Bulletin 49: 87-90, 2010. 9. Kurosawa, H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng. 103: 389-398, 2007 10. Lakra, WS, Swaminathan, TR and Joy, KP. Development, characterization, conservation and storage of fish cell lines: a review. Fish Physiol. Biochem. (In Press) 11. Sarasquete, C and Segner, H. Cytochrome P4501A (CYP1A) in teleostean fishes. A review of immunohistochemical studies. Sci. Total. Environ. 247: 313-332, 2000. 12. Tom, DJ, Lee, LEJ, Lew, J and Bols, NC. Induction of 7-ethoxyresorufin-O-deethylase activity by planar chlorinated hydrocarbons and polycyclic aromatic hydrocarbons in cell lines from the rainbow trout pituitary. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 128: 185-198, 2001. Surface Seawater Temperature at MDIBL Dock - 2009 and 2010
George Kidder1 and Wendy Norden2 1 Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672 2 University of Maine at Machias, Machias, ME 04654
During the summers of 2009 and 2010, hourly temperature was recorded by sensors suspended from the MDIBL floating dock. The data show diurnal and seasonal trends, and suggest the utility of a permanent seawater monitoring system.
In June of 2009 and again in 2010, a set of marine inver- 20 tebrate larval collectors were suspended from the floating 19 dock at MDIBL at a depth of ~60 cm. One collector in each 1 18 set contained a temperature logger , recording water tempera-
C 17 ture every hour. Each day was averaged to produce Figure 1, o 16 suppressing diurnal variation. Figure 2 plots the average read-
15 ing for each hour of the day, emphasizing diurnal variation.
14 The diurnal timing is quite constant, leading to high correla- 2 13 tion coefficients between the two year's data for June (r = 2 2 12 0.939), July (r = 0.876) and August (r = 0.959). June and Filled dots - 2009 August patterns are similar between years, but the July values 11 Open dots - 2010 Water Temperature, for 2010 are considerably elevated, closely paralleling the Au- 10 gust values for this year. The mean temperatures for July and 9 JUNE JULY AUGUST SEPT. August were very significantly different (P = 1.16 x 10-17) in 8 0 204060801001202009, but only marginally different (P = 0.027) in 2010. Days after June 1 Figure 1. Daily average temperature. Table I shows the average temperature for three months. The hourly averages were themselves averaged for each month, giving a mean and SE (N = 30 or 31) for that period. 18 It should be noted that only for the month of July was there a mean difference between years greater than 1°C, and that the 17 difference for August is -0.34°C; 2010 was cooler than 2009 during this month. 16 Table I. Average daily temperatures by month 15 Year Mean ± Standard Error June July August Deg. C 14 2009 12.43 ± 0.103 14.58 ± 0.072 16.24 ± 0.103
13 2010 12.25 ± 0.048 16.04 ± 0.044 15.90 ± 0.046 P of dif 5.65 x 10-3 3.45 x 10-22 6.81 x 10-6 12 Differences both between years and between months are all highly sig- nificant (P<0.01) except for the difference between July and August 2010
11 where 0.05>P>0.01. 0 6 12 18 24 It is probable that the water temperature reflects terrestrial Hour weather patterns that are different between years. Daily tem- Figure 2. Average temperature by hour, for June perature data for 2010 are available from Acadia National (circles), July (squares) and August (triangles). Filled Park's McFarland Hill station, but 2009 data was taken atop symbols = 2009, open symbols = 2010. Note that July and August of 2010 are nearly identical. Cadillac Mt., and is thus not comparable. Local temperature at MDIBL has never been systematically recorded, and these and other weather data would be useful as a foundation for various comparisons such as this.
Supported by Jesse B. Cox Foundation and Davis Conservation Foundation grants to WN.
1 Onset HOBO Underwater Temperature Pro v2 temperature Data Logger, purchased with a grant from the Jesse B Cox Foundation First step in establishing a new marine model organism: Induced spawning, rearing, and surface reactivity of Mytilus edulis embryos
David Brann1 and Gary Conrad2 1Mt. Ararat High School, Topsham, ME 04086 2Division of Biology, Kansas State University, Manhattan, KS 66506-4901
Biofouling, the attachment of organisms, such as the blue mussel, Mytilus edulis, to the hulls of ships and other surfaces, is responsible for increased drag, fuel use, and high costs in cleaning and preventing biofouling. A practical protocol for induced spawning and development of Mytilus edulis embryos is presented. Such embryos can become a useful model to study biofouling and test the effectiveness of new compounds using settlement assays, in hopes of future development of effective alternate antifoulants.
Biofouling is the attachment of organisms to the hulls of ships and other surfaces such as pipes and water intake systems2. One of the most detrimental effects of biofouling is the increased drag from increased roughness, and increased fuel consumption to maintain specific speeds3. Due to its strong byssal threads, Mytilus edulis, is a major biofouler. Barnacles, mussels, tubeworms, and oysters are the major macrofoulers. The ships of the Navy and those of commercial supertankers have the biggest problems with biofouling. Since the ban of Tributyltin (TBT) in 2001, there exists a need for effective alternate antifoulants4.
To study this process, specifically the attachment of Mytilus edulis (blue mussel) larvae to various surfaces, a practical protocol for induced spawning and development of Mytilus edulis embryos is needed. Protocols cited to induce spawning in Mytilus edulis were found to be unreliable at inducing spawning in the mussels we collected. Our research found that bathing the mantle tissue in isotonic KCl consistently induced spawning in both males and females, and that the sex ratio of Mytilus edulis was fairly close to fifty percent. Fertilization was confirmed by the emergence of a polar lobe, indicating that the mussels collected were indeed gravid, and can be induced to shed sperm or eggs. Beginning developmental stages were observed, though the division was not always synchronous.
One can use these embryos in new techniques to study biofouling and test new antifoulants. One can test the effectiveness of antifoulants by using settlement assays, bringing Mytilus edulis embryos to surfaces in the same way barnacles are currently used1 instead of hanging surfaces in the water and seeing which organisms attach. Future studies could include determining the surface reactivity of coatings and embryos, determining when during its development Mytilus edulis attaches, studying the attachment mechanisms of late stage larvae and adult mussels via byssal threads, and testing the effectiveness of new compounds using settlement assays with these embryos. Lastly, when the sequencing of Mytilus edulis genomic DNA is completed, DNA analysis, as in forensics, could also be used to sequence and identify which organisms attach, and hopefully help improve antifoulants and reduce biofouling.
1. Chambers, L. D.; Stokes, K. R.; Walsh F. C. and Wood, R. J. K. Modern approaches to marine antifouling coatings. Surf. Coat. Technol. 201: 3642-3652, 2006 2. Hellio, C. The potential of marine biotechnology for the development of new antifouling solutions J. Sci. Hal. Aquat., 2:35-41, 2010 3. Schultz MP. Effects of coating roughness and biofouling on ship resistance and powering. Biofouling 23:331–341, 2007 4. Yebra DM, Kiil S, Dam-Johansen K. Antifouling technology– past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog Organic Coatings 50:75 – 104, 2004
Variable spawning periodicity in Fundulus heteroclitus within in a New England salt marsh
Dale Quinby1,and Chris W. Petersen College of the Atlantic, Bar Harbor, ME 04609 Mount Desert Biological Laboratory, Salisbury Cove, ME 04672
Fundulus heteroclitus has been documented to exhibit differential reproductive periodicity between geographic regions. This preliminary study found that in the absence of tidal cues, fish in the upper Northeast Creek estuary stopped spawning during the neap tide series, limiting the number of days in the spawning season that they reproduced compared with downstream sites. Field observations suggest that this variation in reproductive periodicity is due to limited access to intertidal spawning habitat in the upper estuary.
Fundulus heteroclitus is a common estuarine fish whose range spans the east coast of the United States. Over its range its spawning periodicity appears to be variable, with semi-lunar spawning during the extreme spring tides south of Cape Cod1,3,4, and populations that lack semi-lunar periodicity during a shortened spawning season north of Cape Cod2,5,6. To examine this pattern further, we studied reproductive behavior of F. heteroclitus at an upstream portion of Northeast Creek, Mount Desert Island, where tidal variation was less than 30cm during spring tides and tidal height variation was minimal or absent during neap tides. For this study, we defined a spring tide series to include any day with a high tide predicted to be 10.7 feet above Mean Low Low Water (MLLW) or higher, which were typically the days surrounding the new and full moons. We also estimated tidal height at the site with a temporary stake marked in cm. Measured levels varied by a total of 29.8cm over the course of the study. These results were compared to a site approximately 1km downstream in the same estuary where tidal fluctuation average approximately 1.2m and F. heteroclitus spawned daily during the reproductive season2.
Fundulus heteroclitus is an estuarine spawner, and egg deposition is largely restricted to the intertidal zone, with individuals spawning near the water line immediately after high tide2. Eggs are deposited in the sediment and externally fertilized by males. The eggs develop with emersion in air for at least part of their time before hatching. In the upper estuary there is little or not tidal height variation during neap tides, so it is unclear if fish living in the upper estuary spawn subtidally during the periods of no tidal height variation or if they restrict their spawning to spring tides when intertidal habitat is accessible.
For data collection at the upstream site, observers were stationed on the shore, monitoring an area of approximately 5m of shoreline from the water line to their limits of visibility (approximately 2m offshore). All spawning observed within the area was recorded, as well as relative tidal height. At night, small flashlights were used to monitor fish, and they quickly became acclimated to the lights. Over 110 man-hours were devoted to these field observations between June 18, 2010 and July 21, 2010 during all phases of the tidal cycle and at all times of day and night.
At the upstream site during spring tides F. heteroclitus spawned on high tides similarly to the downstream site. Although the majority of spawning occurred during the 100min after the highest tide, on days of the most extreme spring tides, low levels of spawning activity continued throughout the tidal cycle. These included both day and night high tides. At this upstream site, water levels only gradually decreased after high tide, so intertidal habitat was accessible to fish during most of the day during extreme spring tides. Unlike the more tidal site downstream, there was very limited spawning activity during neap tides at the upstream site. Only one spawn was observed over 6 neap-series high tides, while spawning was observed in 7 of 12 spring-series high tides, with an average of 40.6 spawns (range 13-90 spawns) observed per high tide during those days when spawning occurred. Spawning intensity over a tidal cycle was positively correlated with the measured height of the high tide at the site (Spearman rank correlation, P < 0.001).
The implication of the lack of spawning at the less tidal site during neap tides is that either F. heteroclitus in that area move downstream to spawn or do not spawn during those times. If a sub-population upstream does not spawn during part of the lunar cycle, this will have implications for the reproductive productivity for this species in this estuary and the genetic composition of the estuarine population. Alternatively, individuals may be moving freely between upstream and downstream locations and changing their spawning patterns to match the tidal cues from the environment that they find themselves in. We have collected fin clips for genetic analysis of individuals from both parts of the estuary and plan to follow up with a mark-recapture study to determine the level of individual movement within the estuary during the reproductive period.
D. Quniby was supported by an INBRE summer fellowship from the Maine IDeA Network of Biomedical Research Excellence (2-P20-RR016463) and the Maine Space Grant Consortium (NASA) program. CP was supported by a New Investigators award from MDIBL.
1. Able, KW and Hata, D. Reproductive behavior in the Fundulus heteroclitus–F. grandis complex. Copeia, 1984:820– 825. 1984. 2. Petersen, CW, Salinas, S, Preston, RL and Kidder III, GW. Spawning periodicity and reproductive behavior of Fundulus heteroclitus in a New England salt marsh. Copeia, 2010:203-210. 2010. 3. Taylor, MH. A suite of adaptations for intertidal spawning. American Zoologist, 39:313-320. 1999. 4. Taylor, MH, Leach, GH, DiMichele L, Levitan, WM and Jacob, WF. Lunar spawning cycle in the mummichog, Fundulus heteroclitus (Pisces: Cyprinodontidae). Copeia, 1979:291-297. 1979. 5. Tewksbury, HT, and Conover, DO. Adaptive significance of intertidal egg deposition in the Atlantic silverside Menidia menidia. Copeia, 1987:76-83. 1987. 6. Wallace, RA, and Selman, K. The reproductive activity of Fundulus heteroclitus females from Woods Hole, Massachusetts, as compared with more southern locations. Copeia, 1981: 212-214. 1981.
Patterns of Trematode Parasite Prevalence in Littorina spp.
Robin Van Dyke1, 2, Helen Hess1, and Chris W. Petersen1, 2 1College of the Atlantic, Bar Harbor, ME 04609 2Mount Desert Biological Laboratory, Salisbury Cove, ME 04672
Parasitic flatworms commonly infect three species of Littorina, intertidal snails found in the Mount Desert Island area. Infection is non-fatal, but typically castrates the snail, as worms grow and reproduce relying on energy from host tissues. Both the prevalence of flatworm parasitism and the species of parasites infecting the snails varied among Littorina species and among sites on a very fine geographical scale. Even within a site, parasite prevalence varied, with higher infection rates in the lower reaches of the intertidal.
Parasitism is an important factor in ecosystem structure8,13, yet patterns of parasite prevalence remain largely unexplored in most systems. In this study, we examine trematode (flatworm) infection in three common species of intertidal snail, Littorina littorea, L. obtusata, and L. saxatilis on Mount Desert Island (MDI) and vicinity. Snails are infected by ingesting trematode eggs while grazing on algae in the intertidal zone. The snail sheds cercaria larvae, infective stages that penetrate fish. The fish are eaten by seabirds, and the trematodes mature in the bird’s gut and release eggs with the host’s feces. Trematode infection is not lethal, but typically castrates the snail hosts6.
Littorina spp. were collected from 9 sites in the MDI area. Sites were located at College of the Atlantic, Mount Desert Island Biological Laboratory, Bar Harbor Town Hill Landing, Bartlett's Landing, two small islands off Bartlett's Landing, Mount Desert Rock, and two locations on Great Duck Island. Within each site, collections were categorized as from the high or low intertidal. High tide collections were made at the upper reaches of each species’ vertical range, well above the fucoid zone in the intertidal, and low tide collections were made within and below the fucoid zone. Not all species of Littorina were present at all sites, and in some cases species were not present in broad enough intertidal ranges for both high and low collections. The snails' shells were cracked open and the soft body of each snail was censused for trematode infection under a dissecting microscope. Trematodes were identified with keys from James4, Stunkard11, and Blakeslee and Byers1, and confirmed from digital photographs by April Blakeslee (Smithsonian Institute). Comparisons of trematode prevalence (the percent of hosts infected) among snail species, collection sites, and tidal heights were made using G-tests.
A total of 2,998 snails, composed of 2,074 L. littorea, 544 L. obtusata, and 380 L. saxatilis, were collected from 9 sites in the Mount Desert Island area. Average trematode prevalences among the three snail species ranged from 6 to 11%. The Littorina species varied significantly in both overall trematode prevalence, and in the prevalence of individual trematode species (Fig. 1). Although some trematode species may be able to use multiple Littorina species as suitable hosts, each Littorina species had a distinct most-common trematode species. The variation in overall trematode prevalence and most-common trematode species among the Littorina species suggests that these trematodes exhibit some host specificity, although there is overlap among host species. The extent of overlap among hosts depended on the species of trematode, with pygmaeus microphallids found only in L. saxatilis, and Microphallus similis prevalence not significantly differing between L. obtusata and L. saxatilis.
Other studies of trematode infection in Littorina often refer to infected snails tending to have a yellow or tan foot, while the foot in uninfected snails is white12. In our study, the color of the foot exhibited continuous variation from white to very light yellow, and coloration was not correlated with trematode infection. In all three species the overall prevalence of trematodes varied among sites (Fig. 2), and this pattern was statistically significant in L. littorea and L. saxatilis. While the spatial patterns of parasite prevalence were similar in L. littorea and L. obtusata, with the site with highest prevalence being Town Hill Landing for both species, L. saxatilis had the highest prevalence (>25%) on the offshore site at Mt Desert Rock, while L. obtusata had no infection at this site.
When trematode prevalences from the high and low intertidal were compared at a site for the two Littorina species with broad vertical ranges, snails from the low intertidal were consistently more likely to be infected. In 4 of 8 comparisons between L. littorea from the high and low intertidal, snails in the low intertidal had a significantly (p<0.01) higher trematode prevalence both overall and for the most-common trematode species (Fig. 3). In the other 4 comparisons, prevalence was very low, yet there was still a trend of higher trematode prevalence in the low intertidal. In L. obtusata 1 of 3 comparisons showed significantly higher prevalence of the most common trematode in the low intertidal, while the other comparisons exhibited no trend in the prevalence of the most-common trematode species between the high and low intertidal.
A mark-recapture experiment was conducted to examine differential patterns of movement between infected and uninfected snails that might explain the correlation between prevalence and intertidal height. During a low tide, four hundred L. littorea from the high and 200 from the low intertidal were marked with a small dot of nail polish and moved to the mid intertidal. At the next day’s low tide, marked snails were located, and the distance and direction traveled were noted. Recaptured snails were censused for parasites. A total of 509 (85%) snails were recaptured, and recaptured snails traveled an average of 1.05 meters. This experiment detected no significant differential movement of infected snails towards the low intertidal.
The snail species did not show predictable trematode prevalence based on life-history strategy. Littorina littorea, which has dispersive planktonic larvae10, had intermediate trematode prevalence (8% infected) compared to L. obtusata (6% infected) and L. saxatilis (11% infected), both of which have young that stay in the locality of their parents8, 10. Given the intermediate parasitism in L. littorea, this does not seem likely to reflect a phylogenetic bias, as L. obtusata and L. saxatilis are more closely related to each other than either is to L. littorea3, nor the shorter history of L. littorea in North America than its congeners1,2.
While trematode prevalence varied among sites, it did not follow a pattern reported for L. littorea. Byers et al.3 found a correlation between higher trematode prevalence and greater abundance of seabirds, the final hosts of these trematodes. Based on observations of relatively low seabird abundance at the mainland sites and high abundance (breeding colonies) at the two offshore islands (Great Duck Island and Mount Desert Rock), seabird abundance does not appear to explain differences in trematode prevalence among these sites.
There was consistently higher prevalence of trematodes in the low intertidal within sites (Fig. 3). This pattern occurred in both Littorina species that have a large tidal range. Snails may become differentially more infected in the low intertidal, or snails may move lower in the intertidal after infection. Mark-recapture results suggest that snails infected by trematodes are not more likely to move more towards the low intertidal than uninfected snails. This suggests that the higher trematode prevalence in the low intertidal is due to greater infection, rather than differential movement of infected snails to the low intertidal.
The authors thank Jeb Byers and April Blakeslee for their advice and assistance in identifying trematodes, and Marissa Altmann and Dale Quinby for their assistance in data collection. R. Van Dyke was supported by the Maine IDeA Network for Biomedical Research Excellence (2-P20-RR016463) and the Maine Space Grant Consortium (NASA) programs. C. Petersen was supported by a New Investigators award from MDIBL.
1. Blakeslee, AH and Byers, JE. Using parasites to inform ecological history: comparisons among three congeneric marine snails. Ecology 89:1068-1078, 2008. 2. Blakeslee, AMH, Byers, JE and Lesser, MP. Solving cryptogenic histories using host and parasite molecular genetics: the resolution of Littorina littorea's North American origin. Mol Ecol 17:3684-3696, 2008. 3. Byers, JE, Blakeslee, AMH, Linder, E, Cooper, AB and McGuire, TJ. Controls of spatial variation in the prevalence of trematode parasites infecting a marine snail. Ecology 89:439-451, 2008. 4. James, B. 1968. The distribution and keys of species in the family Littorinidae and of their digenean parasites, in the region of Dale, Pembrokeshire. Field Studies 2:615-650, 1968. 5. Johannesson, K. The paradox of Rockall: why is a brooding gastropod (Littorina saxatilis) more widespread than one having a planktonic larval dispersal stage (L. littorea)? Marine Biol 99:507-513, 1988. 6. Kuris, AM and Lafferty, KD. Community structure: larval trematodes in snail hosts. Ann Rev Ecol Sys 25:189- 217,1994. 7. Lively, CM. Parthenogenesis in a freshwater snail: reproductive assurance versus parasitic release. Evolution 46:907- 913, 1992. 8. Poulin, R and Mouritsen, KN. Climate change, parasitism and the structure of intertidal ecosystems. J Helminthology 80:183-191, 2006. 9. Reid, DG. The comparative morphology, phylogeny and evolution of the gastropod family Littorinidae. Phil Trans R Soc London B 324:1-110, 1989. 10. Reid, DG. A cladistic phylogeny of the genus Littorina (Gastropoda): implications for evolution of reproductive strategies and for classification. Hydrobiologia 193:1-19, 1990. 11. Stunkard, H. The marine cercariae of the Woods Hole, Massachusetts region, a review and a revision. Biol Bull 164:143-162, 1983. 12. Willey, CH and Gross, PR. Pigmentation in the foot of Littorina littorea as a means of recognition of infection with trematode. The Journal of Parasitology 43:324-327, 1957. 13. Wood, CL, Byers, JE, Cottingham, KL, Altman, I, Donahue, MJ and Blakeslee, AMH. Parasites alter community structure. Proc Nat Acad Sci 104:9335-9339, 2007.
Blue mussel (Mytilus edulis) settlement on restored eelgrass (Zostera marina) is not related to proximity of eelgrass beds to a bottom mussel aquaculture lease site in Frenchman Bay.
Jane Disney1, George W. Kidder1, Kavita Balkaran1,2, Chase Brestle1,3 and Grant Brestle1,3 1Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672 2University of the Virgin Islands, St. Thomas, USVI 00802 3Jupiter High School, Jupiter, FL 33458
Eelgrass beds in Frenchman Bay, Hancock County, ME are rich in species diversity, but the predominant organisms on eelgrass blades during the summer months are blue mussel larvae. We found larval mussels in high abundance, with no difference in their prevalence, when comparing eelgrass blades from a restored area near a bottom mussel aquaculture lease site at Hadley Point in Frenchman Bay and self-established eelgrass beds near-by and elsewhere. Serving as substrate is one important function of eelgrass, among a myriad of services it provides in subtidal areas of estuaries and bays. Another important function of eelgrass is water clarification through sediment stabilization and nutrient uptake. We found increasing water clarity in the years after eelgrass restoration at Hadley Point. These results indicate that at least some habitat functions are restored as an outcome of restoring eelgrass.
Eelgrass plants (Zostera marina) form important biogenic habitat for many marine organisms in the Gulf of Maine, serving as nursery, refuge, and feeding grounds for organisms such as fish, crustaceans, gastropods, bivalves and other invertebrates1. Eelgrass beds help protect the shoreline and clarify the water by stabilizing sediments and taking up nutrients from land-based activities1. Eelgrass in Frenchman Bay has declined significantly since 1996 (Figure 1), possibly due to dragging for shellfish and other commercially important sessile species. We commenced eelgrass restoration efforts at Hadley Point in 2007 and have documented a steady increase in eelgrass coverage at Hadley Point each year since then2. In 2008, a single day study of eelgrass colonizers revealed that the dominant species on eelgrass plants was the blue mussel (Mytilus edulis) in both our recently restored eelgrass area and in a near-by, self-established eelgrass bed at Hadley Point. Mussel larvae accounted for the majority of organisms on young and old blades (98% for the restored eelgrass area and 95% for the self-established eelgrass area). We did find more mussel larvae on the older (longer, outer) blades, and decided to focus on the outer blades of the plant in subsequent studies. The mussels were present as pediveligers, the larval stage that precedes metamorphosis into the first post- larval or spat stage called the plantigrade3. Blue mussel pediveligers use the eelgrass blades to suspend themselves in the water column where they feed on phytoplankton and suspended organic matter.
We hypothesized that proximity of the restored and self established eelgrass areas to a bottom mussel Figure 1: Sample sites were located at Hadley Point near a 47 acre bottom aquaculture lease site might account mussel aquaculture lease site, and near Bar Island in Bar Harbor, Maine. for the abundance of mussel larvae Note the decline of eelgrass between 1996 and 2008 in the upper bay. in the 2008 study.
During the summers of 2009 and 2010, we tested the hypothesis that proximity to a mussel aquaculture lease site influenced the abundance of mussel larvae on eelgrass by setting up a comparison site inside a self-established eelgrass bed near Bar Island, approximately 7.5 nautical miles from the restoration site at Hadley Point (Figure 1). During low tide we collected six older (longer, outer) eelgrass blades from both Hadley Point and Bar Island. The blades were placed in separate sample bags or collection jars. Salinity and temperature were measured at each site on each sample day. Samples were taken back to the laboratory; blade length was measured and the numbers of blue mussel larvae and other organisms were determined.
We calculated the number Figure 2: Average mussel larvae on eelgrass blades (leaves) throughout the summers of 2009 and 2010. There is no significant difference between Hadley of organisms per centimeter Point and Bar Island sites until the end of summer 2010. * denotes significant of blade in order to make difference between Hadley Point and Bar Island average mussel larvae (t-test, p<.05, comparisons between sites n=6 for each sample week). (Figure 2). Mussel larvae were the predominant organism inhabiting the surface of eelgrass blades both from plants in the restored eelgrass area at Hadley Point and the self-established area near Bar Island (Table 1, Figure 3). There was no significant difference in the overall number of mussel larvae per cm. of blade at the two sites. However, there was a difference in how long mussel larvae persisted on plants in the two locations. 2009 2010 There were significantly more mussel larvae on eelgrass blades at the Bar Island site than on eelgrass blades in the Hadley Hadley Point 92.9 ±1.8% 92.6 ± 3.9% Point eelgrass restoration area as the summer progressed (p<0.05). The length of time that blue mussels remain in the Bar Island 94.0 ± 0.7% 97.6 ± 0.67% pediveliger stage ranges from 2 to 46 days in an inverse relation to temperature6. Table 1: Average relative abundance of mussel larvae (total Salinity can also affect the length of time mussels/ total organisms) on eelgrass blades. spent as a pediveliger, the delay being longer at lower salinities6. However, we
cannot attribute the difference in the number of mussel pediveligers on eelgrass blades at the end of the summer in 2010 to variances in temperature or salinity, as there were no significant differences between these variables at the two sites.
The numbers of mussel larvae were significantly higher than the combined numbers of other organisms (periwinkles, whelks, chink shells, mysid shrimp, nudibranchs, amphipods, polychaetes, and nemotodes) per cm. of blade in both areas (p< 0.05). When mussel larvae decreased over the course of the summer, they were not replaced by other organisms. In other words, mussel larvae did not compete 500 µm with other organisms for space; they were most likely more plentiful in the water column.
In Frenchman Bay, mussel larvae and other suspension feeders Figure 3: Mussel larvae on an may be serving the function of clearing the overlying water column in eelgrass eelgrass beds, controlling the amount of suspended organic matter in blade. the eelgrass areas. This has been documented for other types of seagrass beds in other locations4,5. We measured turbidity in triplicate at Hadley Point on two occasions at two sites over three summers, beginning with the first year of eelgrass restoration. In 2007, turbidity was measured at 1.11 ± 0.17 Nephelometric Turbidity Units (NTU); in 2008, turbidity was measured at 1.11 ± 0.05 NTU. In 2009, we measured 0.80 ± 0.07 NTU, while in 2010 the value was 0.79 ± 0.02 NTU; these latter measurements (n = 12 in 2009 and n = 3 in 2010) were not different from each other, but were significantly (p<0.01) lower than in previous years. The decrease in turbidity may be related to the increased presence of suspension feeders supported by re-established eelgrass combined with sediment stabilization by spreading eelgrass rhizomes. Eelgrass coverage increased from <1% in 2007 when we commenced eelgrass restoration, to 8% by 20092 and jumped to over 30% in 2010.
Comparison of organisms on eelgrass blades at our two study sites suggests that large numbers of mussel larvae might be a naturally occurring phenomenon throughout Frenchman Bay in the summer. There was no relationship between proximity to a mussel aquaculture site and the abundance of mussels on eelgrass plants. Future studies include assessing the distribution of organisms in the water column as compared to organisms settling on eelgrass blades, documenting juvenile mussel settlement in and near restored eelgrass beds, and continued monitoring of water quality both inside and outside our restoration area.
Project funding came from Gulf of Maine Council on the Marine Environment, US Fish and Wildlife Foundation and private sources. We thank undergraduate interns Molly Miller, Casie Reed, Kevin Lanza, and Hannah Clemente (supported by NSF REU DBI-0453391) and high school interns Ellen Daily and Dacie Manion (supported by NIEHS STEER R25-E5016254).
1. Moore, KA, Short, FT. Zostera: Biology, Ecology, and Management in Seasgrasses: Biology, Ecology, and Conservation Larkum, AWD, Orth, RJ, and Duarte, CM, eds. Springer, 2006, p. 370-371. 2. Disney, JE, Kidder, GW. Community-based eelgrass (Zostera marina) restoration in Frenchman Bay Bull. Mt. Desert. Isl. Biol. Lab. 108-109, 2010. 3. Wildish, D, Kristmanson, D. Benthic Suspension Feeders and Flow Cambridge University Press, 1997, p. 75. 4. Lemmens, JWTJ , Clapin G, Lavery P, Cary, J. Filtering capacity of seagrass meadows and other habitats of Cockburn Sound, Western Australia, Mar. Ecol. Prog. Ser. 143:187-200, 1996. 5. Bologna, PAX, Fetzer, ML, McDonnell, S, Moody, EM. Assessing the potential benthic-pelagic coupling in episodic blue mussel (Mytilus edulis) settlement events within eelgrass (Zostera marina) communities. J. Exp. Mar. Biol. Ecol. 316:117-131, 2005. 6. Bayne BL. Growth and the delay of metamorphosis of the larvae of Mytilus edulis (L.) Ophelia 2(1): 1-47, 1965. Population genetics of estuarine fish of Acadia National Park: connectivity among estuaries in Downeast Maine
Elena Correa1, Chris Petersen2 and Charles Wray3 1The Colorado College, Colorado Springs, CO, 80903 2College of the Atlantic, Bar Harbor, ME, 04679 3Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
This preliminary study investigates genetic connections between estuaries across Acadia National Park. Based upon genetic markers, killifish and Atlantic silversides exhibit different levels of population mixing in the four estuaries investigated.
Connectivity is an emergent property of natural populations1. Understanding this connectivity is critical to managing any ecosystem, as the most suitable management plan is different for population landscapes with different structures and different amounts of connectivity. While isolated populations should be managed separately, populations with a high level of connectivity merit a comprehensive management scheme2,3,4. The goal of this preliminary study is to determine whether the estuaries of Acadia National Park (ANP) are genetically distinct or connected.
Two of the most common estuarine fishes in Maine were chosen to investigate genetic connectivity. Fundulus heteroclitus (killifish) exhibits strong patterns of increasing isolation by distance and has limited dispersal ability; it is known as one of the most stationary species of marine fishes5. Menidia menidia (Atlantic silverside) migrates out of estuaries to congregate offshore during the winter5,6. The life history differences between Fundulus and Menidia represent the range of life histories found in estuarine communities.
Four sites within different estuaries in ANP were chosen for assessment in this study: Bass Harbor Marsh, Babson Creek, Mosquito Cove, and Northeast Creek (Figure 1). Thirty fin clips each from F. heteroclitus and M. menidia were taken from each field site. DNA was extracted from all samples using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), then amplified at eight different species specific microsatellite loci7,8 using the polymerase chain reaction (PCR). Forward amplification primers were fluorescently labeled for subsequent detection by the ABI 3130XL sequencer at MDIBL.
Statistical analyses of the microsatellite data yielded the following results. M. menidia had higher values than F. heteroclitus for every measure of genetic diversity in every population (Table 1).
Population n HZ HO A Bass Harbor Marsh 30 0.3460 0.3042 3.38 (Tremont) 30 0.8501 0.6453 12.75 Babson Creek 30 0.3062 0.3292 3.00 (Mount Desert) 29 0.8588 0.7821 11.13
Mosquito Cove 30 0.2286 0.1895 2.38
(Winter Harbor) 28 0.8485 0.6034 13.88
Northeast Creek 25 0.4246 0.3914 3.38
(Bar Harbor) 30 0.8059 0.6096 11.50
Average 28.75 0.3263 0.2784 3.035
29.25 0.8408 0.6601 12.31
Table 1. Sample sizes and three measures of genetic diversity for F. heteroclitus and M. menidia. HZ (expected heterozygosity) and HO (observed heterozygosity) are measured from 0 to 1; A = allelic richness. The results for F. heteroclitus are reported in bold and those of M. menidia are italicized.
In addition to measures of genetic diversity, FSTAT9 was used to calculate Wright’s fixation index between populations or Fst. By measuring reduction in genetic diversity due to drift, Fst measures genetic substructure; Fst values up to 0.05 indicate no genetic differentiation between groups, while Fst values >0.2 indicate genetic differentiation is ongoing. Fst values between all sampled populations are presented in figure 1. In five of six pairwise comparisons Fst values for Fundulus are greater than those for Menidia. Indicating greater levels of genetic connectivity for Menidia. With the exception of Fundulus at Mosquito Cove, Fst values suggest low levels of population substructure, indicating that ANP estuaries are connected by gene flow.
Figure 1. Pairwise differentiation between four sites. Differentiation between populations at Bass Harbor Marsh, Babson
Creek, Mosquito Cove, and Northeast Creek for both F. heteroclitus and M. menidia. FST values for F. heteroclitus are in bold, and those for M. menidia are italicized (map: Harvard Digital Map Collection) .
This work was supported by an L.L. Bean Acadia national Park research Grant to Chris Petersen and Charles Wray and a 2010 NSF REU (0453391) award to E. Correa.
1. Doherty PJ and Fowler T. An empirical test of recruitment limitation in a coral reef fish. Science, 263: 935-939, 1994. 2. Lipcius RN, Crowder LB, and Morgan LE. Metapopulation structure and marine reserves. In Marine Conservation Biology: The Science of Maintaining the Sea’s Biodiversity. P 328-345. Norse EA and Crowder LB, Eds. Island Press, Washington, DC, 2005. 3. Palumbi SR. Population genetics, demographic connectivity, and the design of marine reserves. Ecological Applications, S13: S146-S158, 2003. 4. Rosenberg DK, Noon BR, and Meslow EC. Biological corridors: Form, function, and efficacy. Bioscience, 47: 677- 687, 1997. 5. Bigelow HB and Schroeder WC. Fishes of the Gulf of Maine. U.S. Fish and Wildlife Service Fishery Bulletin, 53: 162-164, 1953 6. Collette BB and Klein-MacPhee G. eds. Bigelow and Schroeder’s Fishes of the Gulf of Maine. Washington and London: Smithsonian Institution Press, 2002. 7. Adams, SM, Olesiak, MF, and Duvernell, DD. Microsatellite primers for the Atlantic Coastal killifish, Fundulus heteroclitus, with applicability to related Fundulus species. Mol. Ecol. Notes, 2005, 5, 275-277. 8. Sbrocco, EJ. Unpublished Menidia menidia microsatellite primers, used by permission, Boston University Dept or Biology, 2010. 9. Goudet J. Fstat version 1.2: a computer program to calculate Fstatistics. Jour Hered. 86(6): 485-486, 1995.
Preliminary population genetic analysis of eelgrass and Fundulus at the Callahan Mine Superfund Site
Chris Petersen1 and Charles Wray2 1College of the Atlantic, Bar Harbor, ME, 04679 2Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
The Callahan Mine Superfund site occupies a disrupted estuary, flooded after mining operations ceased. Preliminary genetic analyses suggest that eelgrass and killifish are genetically connected to populations outside of the mine site.
The Callahan Mine in Brooksville, Maine is an EPA Superfund (MED980524128) site that includes a large estuary, Goose Cove. From 1968 to 1972 a 320’-deep, copper and zinc open pit mine was dug, and today the mine pit is submerged beneath Goose Cove. During mining operations the estuary was dammed and drained; in late 1972 two dams were removed, and the tidal estuary naturally refilled. Previous studies by the Maine Department of Environmental Protection and EPA sub-contractors have demonstrated that sediments and water discharging from mine wastes contain toxic levels of copper, lead, and zinc5. Studies have also demonstrated that metals are accumulating in biota at the site, including fish, crabs, clams and sea grass. Today, the mine site estuary supports floral and faunal diversity that reflects the Acadian biogeographic location of the site.
In the summers or 2009 and 2010, samples of eelgrass (Zostera marina) and killifish (Fundulus heteroclitus) were collected from the Goose Cove estuary. 30 eelgrass samples were taken along a transect within the Dyer Cove section of the mine. 40 killifish were collected using minnow traps placed within the estuary at the base of the tailings pile.
Following standard CTAB (eelgrass) or Qiagen (killifish tissue) DNA extraction, polymorphic microsatellite loci1,4 (6 loci for eelgrass and 8 loci for killifish) which exhibit high levels of polymorphism between genetic individuals3, were individually PCR amplified using fluorescently labeled forward primers and unlabeled reverse primers. Amplified samples were multiplexed on the ABI 3130XL sequencers to determine size in base pairs. MSToolkit software within Excel 2004 was used to quantify genetic diversity within sampled populations and to compare Goose Cove samples to nearby populations of eelgrass and killifish. FSTAT2 was used to measure allelic diversity and generate Fst population differentiation measures. Genetic diversity measures and comparative data from Mount Desert Island area samples are presented in table 1.
Population n HZ HO A Goose Cove Z. marina 29 0.6391 0.5716 3.66 Goose Cove F. heretoclitus 25 0.3025 0.2751 2.63 Jordan River Z. marina 72 0.5581 0.6541 4.546 Northeast Creek F. heteroclitus 25 0.4246 0.3914 3.38
Table 1. Sample sizes and three measures of genetic diversity for Z. marina and F. heteroclitus. HZ (expected heterozygosity) and HO (observed heterozygosity) are measured from 0 to 1; A = allelic richness. The results for Z. marina are reported in bold and those of F. heteroclitus are italicized.
Fundulus Population Differentiation Zostera Population Differentiation
Fst pairwise Camp Stream NE Creek Hadley Point Jordan River differentiation Population Population Population Population
Callahan Mine 0.088 0.105 0.1815 0.2037 Population
Table 2. Fst measures between nearby populations.
Wright’s fixation index, or Fst, measures reduction in genetic diversity due to drift. In general, Fst values up to 0.05 indicate no genetic differentiation between groups, while Fst values >0.2 indicate genetic differentiation is ongoing. Measurements of Fst were made between killifish at the mine site and killifish sampled ~20 km distant within the Bagaduce River estuary at Camp Stream, and between eelgrass from the mine site and eelgrass
sampled in the Jordan River, ~50 km distant. For killifish, low Fst values suggest that gene flow is occurring between Goose Cove and adjacent populations. Higher Fst values for eelgrass indicate genetic differentiation is underway; however, Z. marina dispersal capacity and the lack of a comparative sample from the Bagaduce
estuary likely both contribute to higher relative Fst values. This preliminary analysis indicates that gene flow in and out of the Superfund site is active, and EPA efforts may affect dynamic populations of killifish and eelgrass.
This work was supported by a Dartmouth Superfund Basic Research Program (NIEHS, P42 ES007373) Pilot Project Grant to Celia Chen and Charles Wray.
1. Adams, SM, Olesiak, MF and Duvernell, DD. Microsatellite primers for the Atlantic Coastal killifish, Fundulus heteroclitus, with applicability to related Fundulus species. Mol. Ecol. Notes, 5,275-277, 2005. 2. Goudet J. Fstat version 1.2: a computer program to calculate F statistics. Jour Hered. 86(6): 485-486, 1995. 3. Olsen, JL, Stam, WT, Coyer, JA, Reusch, TBH, Billingham, M, Boström, C, Calvert, E, Christie, H, Granger, S, La Lumière, R, Milchakova, N, Oudot-le Secq, M, Procaccina, G, Sanjabi, B, Serrão, E, Veldsink, J, Widdicombe, S and Wyllie-Echeverria, S. North Atlantic phylogeography and large-scale population differentiation of the seagrass Zostera marina L., Molecular Ecology, 13, 1923-1941, 2004. 4. Reusch, TBH, Stam, WT and Olsen, JL. A microsatellite-based estimation of clonal diversity and population subdivision in Zostera marina, a marine flowering plant. Molecular Ecology, 9, 127-140, 2000. 5. U.S. Environmental Protection Agency, Conceptual Model and RI/FS SOW, Callahan Mining Superfund Site, EPA Contract #68-W6-0042, 2003. Second-year study of water quality parameters along a north to south transect in Frenchman Bay, ME
Brett Rabeneck and James Claiborne Department of Biology, Georgia Southern University, Statesboro, GA 30460 Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
In summer 2009 we began baseline measurements of pH and CO2 in Frenchman Bay. In summer 2010, we replicated our methods and location of water sample collection, and this article discusses the results. Our measurements again showed that the bay was more acidic than “typical” ocean pH.
In the 2010 MDIBL bulletin3 we reported pH measurements in Frenchman Bay that were already at ocean acidity values projected to occur by the end of the century1. pH values measured from water samples collected at the bottom of the bay during summer 2009 were significantly lower than the surface pH3. There were no differences between salinity and temperature taken from surface and bottom depths.
We again collected surface and bottom water samples on a transect at the same three coordinates as 2009. The 2010 measurements indicated pH values were still lower than the current average ocean pH (8.1), but significantly higher than summer 2009 pH values (Table 1; surface 2009 & 2010 p<0.001; bottom 2009 & 2010 p<0.02). Wootton et al.4 suggested that seawater pH can fluctuate by a full unit or more, year-to-year, and by as much as 0.24 units diurnally. 2010 bottom pH was significantly lower than the pH within one meter of the surface, agreeing with the pattern seen in 2009 (Table 1; p<0.001). A biyearly temperature comparison demonstrates that surface temperature was significantly higher than bottom temperatures in 2010, and bottom temperatures in 2010 were higher than bottom temperatures in the previous year (Table 1; p=0.005).
Clearly, a two year “snap-shot” of water pH variability is not enough to show long term changes, but these base line measurements will provide a basis for comparison in future studies. This research was funded by NSF IOB-061687 to JBC and GSU Allen E. Paulson Academic Excellence Award to BR.
Table 1: Mean water quality parameters measured in Frenchman Bay, ME, taken within 1 m of the surface and at the bottom of the bay at three different locations. pH surface and bottom measured in 2009 U=222, df = 1, p<0.001 (one- tailed test). pH difference between 2010 surface and bottom values when corrected for ties t=10.602, df = 1, p<0.001 (two-tailed). pH comparison between surface 2009 and 2010 when corrected for ties t=3.46, df = 1, p<0.001 (one- tailed). pH bottom 2009 and 2010 when corrected for ties t=6.046, df=1, p=0.0155 (two-tailed). Salinity F=7.2885, df=3, p<0.001, Tukey-Kramer test surface 2010 significantly different surface 2009. Temperature F=5.3410, df=3, p=0.0047. Mann-Whitney-U test for pH, 1-way ANOVA for salinity and temperature, mean ± SE.
Year Depth pH Salinity (ppt) Temperature (° C) Mean N Mean N Mean N 2009 Surface 7.87 ±0.01 30 31.0 ±0.1 7 14.9 ±0.5 7 Bottom 7.76 ±0.02 19 31.4 ±0.2 8 12.6 ±0.7 8 2010 Surface 7.93 ±0.01 26 31.8 ±0.0 9 15.8 ±0.8 9 Bottom 7.82 ±0.01 27 31.7 ±0.1 9 15.4 ±0.4 9
Note: 2009 surface and bottom pH data were calculated in a slightly different form from the previous year3 (bottom = 7.75 ±0.41, surface = 7.75 ±0.08, mean ±SD). We have used the average pH of each replicate in the present data analysis. pH values in Table 1 and discussed in the text are directly measured pH with a probe. Extrapolating pH from dissolved inorganic carbon (DIC) and total alkalinity (TA) with CO2Sys Excel Macro2 results in an average surface pH 8.09 ±0.01 and a bottom pH of 8.01 ±0.01 for 2010. This method was not employed for 2009 data because sufficient DIC and TA values were not obtained for that year.
1. Haugan PM and Drange H. Effects of CO2 on the ocean environment. Energy Conversion and Management 37: 1019-1022, 1996. 2. Lewis E and Wallace WR. Program developed for CO2 System Calculations. (ORNL/CDIAC-105 ed.). Oak Ridge, TE: Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, 1998. 3. Rabeneck B, Diamanduros A, and Claiborne J. Characterization of pH and total alkalinity in waters along a north to south transect in Frenchman Bay, ME The Bulletin, MDI Biological Laboratory 49: 106-107, 2010. 4. Wootton JT, Pfister CA, and Forester JD. Dynamic patterns and ecological impacts of declining ocean pH in a high- resolution multi-year dataset. Proceedings of the National Academy of Sciences 105: 18848-18853, 2008. Toward A New Online Computational Mining Tool for Peptide Precursor Prediction
Clare Bates Congdon1, Daniel H. Nolan2, Ingrid Olson1, Christine Shea1, and Andrew E. Christie2 1Department of Computer Science, University of Southern Maine, Portland ME 04104 2Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
Numerous large-scale studies have identified many hormones from among arthropods and nematodes. In contrast, little is known about related phyla, such as Onychophora (velvet worm), Priapulida (priapulid worms), and Tardigrada (water bears). In this work, we are developing a web-based computational tool that allows the user to search online databases for evidence of peptides that are similar to known peptides. This computational search will significantly hasten our ability to identify peptides in the related species.
Much is known about the physiological control systems present in both the arthropods and the nematodes, including the identity of many peptide paracrines/hormones that are used by them to modulate their behavior2. In contrast little is known about the peptidergic signaling systems used by members of the other ecdysozoan phyla, despite the close phylogenetic relationships among these species. Previous work4 has reported on a methodology for extensive database searches to use tblastn [National Center for Biotechnology Resources (NCBI) http:// www.ncbi.nlm.nih.gov/BLAST/] to mine for ESTs encoding putative peptide precursors, based on known peptides for related species. The work described here seeks to automate many of the steps of this workflow, in order to both hasten identification of putative peptide precursors, and also to reduce human errors in the process.
The workflow procedure is described in previous work2,3. Briefly, known peptide precursors are used for a tblastn search to identify candidate sequences in related species. Each EST was translated and checked for homology to the target query, and its deduced protein assessed for typical neuropeptide precursor features, including start and stop codons. Signal peptide prediction was done using the online program SignalP 3.01. This workflow requires human researchers to visually assess the quality of match, to identify start and stop codons, and to identify putative cleavage sites. In the first phase of development, our computational tool now does these error-prone steps, making recommendations to the human users on the putative peptide precursors most likely to be of interest. The next steps of program development will further automate the workflow, so that our program can invoke others to do parts of the work, and to reduce effort and cut and paste errors, and further streamline the process.
This project was supported by NIH Grant Number P20 RR-016463 from the INBRE Program of the National Center for Research Resources, institutional funds provided by MDIBL to AEC, as well as an NSF CAREER award (NSF 08-557) to CBC. Additional student support has been received from Maine Economic Improvements Funds, as well as the Eastern Alliance in Science, Technology, Engineering and Mathematics for Students With Disabilities-2 (EAST-2), funded under the NSF Cooperative Agreement No. HRD-0833567.
1. Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783-795, 2004. 2. Christie, A.E., Stemmler, E.A., and Dickinson, P.S. Crustacean neuropeptides. Cell. Mol. Life. Sci. 67, 4135-4169, 2010. 3. Christie, A.E., Nolan, D.N., Garcia, Z.A., McCoole, M.D., Harmon, S.D., Congdon-Jones, B., Ohno, P., Hartline, N., Congdon, C.B., Baer, K.N., Lenz, P.H. Bioinformatic prediction of arthorpod/nematode-like peptides in non- arthropod, non-nematode members of the Ecdysozoa. Gen. Comp. Endocrinol. 170, 480-86, 2011. 4. Gard, A.L., Lenz, P.H., Shaw, J.R., and Christie, A.E. Identification of putative peptide paracrines/hormones in the water flea Daphnia pulex (Crustacea; Branchiopoda; Cladocera) using transcriptomics and immunohistochemistry. Gen. Comp. Endocrinol. 160, 271-87, 2009.
A screen for bacteria that induce a gut-associated immune response in the larval sea urchin
Eric C.H. Ho, Katherine M. Buckley, Cynthia M. Solek and Jonathan P. Rast Department of Medical Biophysics and Department of Immunology, University of Toronto Sunnybrook Research Institute, Toronto ON M4N 3M5 Canada
The close relationship to vertebrates and the simple feeding larval stage of the sea urchin make this group an ideal model for systems-level investigations of gut-associated immunity. Since little is known about the natural pathogens of these larvae we have set up a screen to isolate culturable seawater bacteria that can be used in immune investigations. A pilot survey of the stocks that we collected at MDIBL was successful in isolating several new immunogenic bacteria that will enable more varied and biologically relevant strategies to investigating animal immunity in this model.
The larva of the purple sea urchin, Strongylocentrotus purpuratus, provides a simple gene regulatory network model for immunity1. Characterization of the sea urchin genome sequence reveals a complex immune system with important regulatory affinities to vertebrates 1,2. Since little is known about the natural pathogens of this developmental stage, we have characterized the larval immune response to an intestinal isolate from adult S. droebachiensis. This bacterium, Vibrio diazotrophicus, is not a true larval pathogen but exposure induces a controllable immune response in which stereotypic immune gene activation is coupled with pigment cell migration to the inner gut epithelial wall (Fig 1). We exploited this observation to identify new larval pathogens from immune activated larvae in cultures fed from natural sea water with its associated microbiota. Larvae that exhibited this response were isolated, and associated bacteria were analyzed.
For these investigations we cultured larvae in daily changes of freshly collected, 25 μm-filtered seawater. This provided a source of food and a complex microbiota. Embryos and larvae were raised from fertilization to eight days under these conditions (three days past the onset of feeding). Larvae that displayed pigment cell accumulation in the region of the gut were individually isolated and thoroughly washed in filter-sterilized sea water to remove non- adherent bacteria. They were then triturated and spread onto marine Figure 1. Immune response in the sea agar plates. After culture at 15ºC, bacterial colonies were pooled urchin larva. Pigment cells migrate from their usual position at the ectoderm into 10% Table 1. 16S ribosomal sequence analysis of (arrowhead) to the gut epithelium (arrow) bacterial isolates associated with a gut response. glycerol stocks after exposure to some bacteria. Scale bar Nearest identified* Larval response and stored at - is 100 μm. Vibrio cyclitrophicus ++ 80ºC for future Pseudoalteromonas + analyses. Responsive larvae were also frozen in glycerol Marinomonas blandensis ++† supplemented homogenates. Algicola bacteriolytica (1) + Algicola bacteriolytica (2) + After replating glycerol stocks, colonies were Psychrosphaera saromensis n.t. individually streaked and DNA was extracted and used as Vibrio sp. n.t. template for 16S ribosomal RNA sequence analysis Vibrio splendidus + employing universal eubacterial primers4. In our pilot Marinomonas sp. - studies we have analyzed 16S sequences to identify nine * Closest identifiable taxonomic category; † High separate isolates. The taxa identified in this sequencing larval mortality unrelated to the gut.; n.t.: Not project are listed in Table 1. tested.
Once characterized, monocultures of these bacterial strains were incubated with feeding larva at varying concentrations, and the level of larval immune response was characterized by counting the number of pigment cells that migrated to the gut epithelium. Six of the isolates induce an immune response (Fig. 2). One of these, a Vibrio cyclitrophicus-like strain appears to be more effective at inducing immunocyte migration than our standard V. diazotrophicus model.
Studies at MDIBL over three summers have significantly advanced our understanding of sea urchin larval- bacterial associations. The approach that we outline here provides an extensive and stable bank of larval associated bacteria from which we can isolate further strains for analysis and experimentation in our immunity model. Notably we find that in a complex microbial environment such as the one that we exposed larvae to in these investigations, pigment cells are found at the gut in a large proportion of the larvae examined. Similar observations were made for the larvae of the sand dollar, Echinarachnius parma. This is rare in laboratory cultures. Thus, the relatively sterile culture conditions of larvae raised in standard laboratory circumstances do not reflect the typical state of immune challenge in the wild and studies such as these are necessary to investigate immune potential in the larva. We expect to see the activation of additional immune response programs as we analyze additional bacteria in these microbial challenge models.
Figure 2. Immune response as measured by the extent of pigment cell migration to the gut epithelium after exposure to different bacterial isolates at different levels.
Supported by a MDIBL New Investigator Award, the Sunnybrook Research Institute and a grant from the National Sciences and Engineering Research Council of Canada (NSERC) to JPR.
1. Hibino, T., Loza-Coll, M., Messier, C., Majeske, A. J., Cohen, A. H., Terwilliger, D. P., Buckley, K. M., Brockton, V., Nair, S. V., Berney, K., Fugmann, S. D., Anderson, M. K., Pancer, Z., Cameron, R. A., Smith, L. C., and Rast, J. P. The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. 300: 349-365, 2006. 2. Rast, J. P., Smith, L. C., Loza-Coll, M., Hibino, T., and Litman, G. W. Genomic insights into the immune system of the sea urchin. Science 314: 952-956, 2006. 3. Messier-Solek C, Buckley KM, Rast JP. 2010. Highly diversified innate receptor systems and new forms of animal immunity. Semin Immunol. 22:39-47. 4. Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703, 1991.
MDIBL REGISTER
PAST PRESIDENTS/CHAIRMEN PAST DIRECTORS Dr. John S. Kingsley 1910-1922 Dr. Ulrich Dahlgren 1920-1926 Dr. Harold D. Senior 1922-1926 Dr. Herbert V. Neal 1926-1931 Dr. William Proctor 1926-1927 Dr. William H. Cole 1931-1940 Dr. Hermon C. Bumpus 1927-1932 Dr. Roy P. Forster 1940-1947 Dr. Warren H. Lewis 1932-1937 Dr. J. Wendell Burger 1947-1950 Dr. Ulrich Dahlgren 1937-1946 Dr. Warner F. Sheldon 1950-1956 Dr. Dwight Minnich 1946-1950 Dr. Raymond Rappaport 1956-1959 Dr. William C. Cole 1950-1951 Dr. Alvin F. Rieck 1959-1964 Dr. Homer W. Smith 1951-1960 Dr. William L. Doyle 1964-1967 Dr. Eli K. Marshall 1960-1964 Dr. Charles E. Wilde 1967-1970 Dr. Roy P. Forster 1964-1970 Dr. H. Victor Murdaugh 1970-1975 Dr. William L. Doyle 1970-1975 Dr. Richard M. Hays 1975-1983 Dr. Jack D. Myers 1975-1978 Dr. Leon Goldstein 1979-1983 Dr. Charles E. Wilde 1978-1979 Dr. David H. Evans 1983-1992 Dr. Raymond Rappaport 1979-1981 Dr. David C. Dawson 1992-1998 Dr. Bodil Schmidt-Nielson 1981-1985 Dr. John N. Forrest, Jr. 1998-2009 Dr. Franklin H. Epstein 1985-1995 Dr. James L. Boyer 1995-2003
2009-2010 OFFICERS
Chair, Board of Trustees Mr. Terence C. Boylan Vice Chair Dr. Edward J. Benz, Jr. Director Dr. Kevin Strange Secretary Dr. John H. Henson Treasurer Mr. Maximiliaan J. Brenninkmeyer Clerk Nathaniel I. Fenton, Esq.
EXECUTIVE COMMITTEE DIRECTOR’S ADVISORY COMMITTEE Mr. Terence Boylan, Chair Dr. Kevin Strange, Chair Dr. James L. Boyer Dr. Barbara Beltz Dr. Edward J. Benz, Jr. Dr. Edward J. Benz, Jr. Dr. Kevin Strange, Ex Officio Ms. Jerilyn Bowers Dr. Bruce Stanton, Ph.D. Dr. James L. Boyer Dr. John H. Henson Mr. Terence C. Boylan Dr. James Coffman Dr. David Dawson Dr. Susan Fellner Dr. Raymond A. Frizzell Dr. Patricia H. Hand Dr. Petra Lenz Dr. Carolyn Mattingly Mr. Michael McKernan Dr. Charles Wray
Administrative Director Patricia H. Hand, Ph.D. TRUSTEES
Class of 2010
Peter J. Allen, M.D. Frank L. Hohmann III Memorial Sloan-Kettering Cancer Center New York, NY
James L. Boyer, M.D. Alan B. Miller Yale University School of Medicine New York, NY
David H. Evans, Ph.D. Kevin Strange, Ph.D. University of Florida Mount Desert Island Biological Laboratory
John A. Hays New York, NY
Class of 2011
Edward J. Benz, Jr., M.D. Richard M. Hays, M.D. Dana Farber Cancer Institute Greenwich, CT
Phoebe C. Boyer Emily Leeser New York, NY New York, NY
Ruth Cserr Bruce Stanton, Ph.D. Orford, NH Dartmouth School of Medicine
Class of 2012
Thomas Cech, Ph.D. Barbara Kent, Ph.D. University of Colorado - Boulder Hancock Point, ME
James B. Claiborne, Ph.D. Steen L. Meryweather Georgia Southern University Salisbury Cove, ME
J. William Freytag John Blair Overton Longmont, CO Honolulu, HI
Class of 2013
Terence Boylan Max Brenninkmeyer Rhinebeck, NY Surry, ME
Spencer Ervin Thomas Cech, Ph.D. Bass Harbor, ME Boulder, CO
I. Wistar Morris III Margaret Myers, M.D. West Conshoken, PA Radnor, PA
SCIENTIFIC PERSONNEL
Principal Investigators Associates
Sharon Ashworth, Ph.D. Justine Cyr Assistant Professor Sharon Perrone Department of Biochemistry, Microbiology, and Molecular Biology Christopher Preziosi The University of Maine
David W. Barnes, Ph.D. Senior Staff Scientist Mount Desert Island Biological Laboratory
Edward J. Benz, Jr., M.D. President Professor of Medicine Dana Farber Cancer Institute
Nancy Berliner, M.D. Navid Nia Professor Department of Medicine Harvard Medical School Senior Attending Physician Medicine/Hematology Brigham and Women’s Hospital
James L. Boyer, M.D. Shi-Ying Cai, Ph.D. Ensign Professor of Medicine Maya DeGroote Director, Liver Center Daniel Lionarons Yale University School of Medicine Victoria Smith
Keith Choe, Ph.D. Assistant Professor Department of Biology University of Florida
Jon Chorover, Ph.D. Professor of Environmental Chemistry Department of Soil, Water, and Environmental Science University of Arizona
Andrew E. Christie, Ph.D. Zachery Garcia Staff Scientist and Director of Imaging Core Sarah Harmon Mount Desert Island Biological Laboratory Daniel Nolan
James B. Claiborne, Ph.D. Sarah Cullen Professor of Biology Andrew W. Diamanduros Georgia Southern University Susan Edwards Ph.D. Hana Kratochvilova Brett Rabeneck
James Coffman, Ph.D. Benjamin Davis Associate Professor Chris McCarty Mount Desert Island Biological Laboratory Diane Saunders
Clare Bates Congdon, Ph.D. Christine Shea Assistant Research Professor University of Southern Maine
Gary W. Conrad, Ph.D. David Brann University Distinguished Professor Gage Brummer Division of Biology Kansas State University
Elizabeth Crockett, Ph.D. Kathleen Kelley Associate Professor David Tapley Department of Biological Sciences Mark Wheeler Ohio University
Suzie Currie, Ph.D. Ashra Kolhatkar Assistant Professor Nathan Walker Department of Biology Mount Allison University
Christopher Cutler, Ph.D. Christopher Katsekis Assistant Professor Marcela Kuijpers Department of Biology Jonathan Walsh Georgia Southern University
Randall D. Dahn, Ph.D. Heather Carlisle Investigator Kathryn Dirks Mount Desert Island Biological Laboratory
David C. Dawson, Ph.D. Professor and Chair Department of Physiology and Pharmacology Oregon Health & Science University
Hugo de Jonge, Ph.D. Thomas Flynn Professor Erasmus Medical Center
Jane Disney, Ph.D. Hannah Clemente Staff Scientist Dacie Manion Director Jake Van Gorder Community Environmental Health Laboratory Mount Desert Island Biological Laboratory
David H. Evans, Ph.D. Professor Department of Zoology University of Florida
Susan K. Fellner, M.D. Research Professor Department of Cellular and Molecular Physiology University of North Carolina at Chapel Hill
John N. Forrest, Jr., M.D. Kentrell Burks Professor of Medicine Samuel Duffy Director of Student Research Megan Kelley Department of Internal Medicine August Melita Yale University School of Medicine Montana Morris
Gert Fricker, Ph.D. Juliane Klaes Professor Valeska Reichel, Ph.D. Institute of Pharmacology and Molecular Biology University of Heidelberg
Fernando Galvez, Ph.D. Charlotte Bodinier Assistant Professor of Biology Christine Savolainen Louisiana State University Martin Tresguerres
Wolfram Goessling, M.D., Ph.D. Trista North, Ph.D. Assistant Professor of Medicine Harvard Medical School Associate Physician Dana-Farber Cancer Institute and Brigham Young Women’s Hospital
Hermann Haller, M.D. Lynn Beverly-Staggs Professor of Medicine Danny Claeys Dean of Medical Education Hannover Medical School
Daniel K. Hartline, Ph.D. Mareike Herzog Research Professor and Director Bekesy Laboratory for Neurobiology Pacific Biosciences Research Center University of Hawaii, Manoa
R. Patrick Hassett, Ph.D. Assistant Professor Dept. of Biological Sciences Ohio University
John Henson, Ph.D. Anastasia Gianakas Charles A. Dana Professor of Biology Dickenson College
Billy Hudson, Ph.D. Aaron Fidler Elliott V. Newman Professor of Medicine Richard Harris Professor of Biochemistry and Pathology Keith Wade Director Lili Zarzycki Center for Matrix Biology Stephanie Zeiger, Ph.D. Vanderbilt University
David Julian, Ph.D. Tim Crombie Associate Professor Maria Duarte Department of Biology Lauren Gravois University of Florida Breanna Sipley Maria Christina Vasquez
Christine Keating, Ph.D. Associate Professor of Chemistry Pennsylvania State University
George W. Kidder, III, Ph.D. Kevin Lanza Senior Staff Scientist Mount Desert Island Biological Laboratory
Rolf K.H. Kinne, M.D., Ph.D. Director Emeritus, Max-Planck Institute of Mol. Physiology Director, Con Ruhr Academic Exchange Office
Seth Kullman, Ph.D. Erin Kollitz Assistant Professor Erin Yost North Carolina State University
Christopher Lage, Ph.D. Assistant Professor of Biology The University of Maine - Augusta
Petra H. Lenz, Ph.D. Monica Orcine Associate Research Professor Ebru Unal Bekesy Laboratory of Neurobiology Pacific Biomedical Research Center University of Hawaii at Manoa
Weiming Li, Ph.D Chu-Yin Yeh Professor Department of Fisheries and Wildlife Michigan State University
Rosalinde Masereeuw, Ph.D. Associate Professor Department of Pharmacology and Toxicology Radbound University Nijmegen Medical Center
Carolyn Mattingly, Ph.D. Allan Peter Davis, PhD Associate Professor Heather Keating, PhD Director of Bioinformatics Benjamin King, MS Comparative Toxicogenomics Database Jean Lay, PhD Mount Desert Island Biological Laboratory Kelley Lennon-Hopkins, PhD Robin Johnson, PhD Roy McMorran Susan Mockus, PhD Cindy Murphy, PhD Cynthia Richards, PhD Michael C. Rosenstein, JD Daniela Sciaky, PhD Thomas Wiegers
Gregory Mayer, Ph.D. Kaylyn Germ Assistant Professor of molecular Toxicology Katarina Rydlizky The Institute of Environmental and Human Health Song Tang Texas Tech University
Francis McGowan, M.D. Dimitrios Poutias Professor of Anesthesia Harvard Medical School Chief Division of Cardiac Anesthesia Boston Children’s Hospital
David S. Miller, Ph.D. Anne Mahringer, Ph.D. Research Physiologist Brigitte Prevoo Laboratory of Pharmacology and Chemistry NIH/NIEHS
Robert Morris, Ph.D. Ian Greenstein Professor and Chair Tstsushi Shintaku Biology Department Wheaton College
Antonio Planchart, Ph.D. Samuel Entwisle Assistant Professor Michael J. Jose Mount Desert Island Biological Laboratory Dale Quinby Christopher Petersen, Ph.D Timothy Webber Professor Robin Van Dyke College of the Atlantic
Ken Poss, Ph.D. Greg Nachtrab Associate Professor of Cell Biology Duke University Medical Center Early Career Scientist Howard Hughes Medical Institute
Robert L. Preston, Ph.D. Sirilak Chuaypanang Professor of Physiology Arhea Marshall Department of Biological Sciences Michaela Petit Illinois State University Jennifer Willis
Jonathan Rast, Ph.D. Kate Buckley Associate Professor Eric Ho Department of Immunology Cynthia Messier Solek University of Toronto
J. Larry Renfro, Ph.D. Pedro M. Guerreiro, Ph.D. Professor Department of Physiology/Neurobiology University of Connecticut
Jack Riordan, Ph.D. Timothy Jensen Distinguished Professor Department of Biochemistry and Biophysics University of North Carolina School of Medicine
J. Denry Sato, D. Phil. Christine Chapline Investigator Mount Desert Island Biological Laboratory
Mario Schiffer, M.D. Jessica Kaufeld, Ph.D. Assistant Professor Philipp Niggemann Nephrology Hannover Medical School
Joseph Shaw, Ph.D. Assistant Professor School of Public and Environmental Affairs Indiana University
Patricio Silva, M.D. Katherine C. Spokes Professor of Medicine Section Nephrology and Kidney Transplant Temple University Health Science Center
Bruce A. Stanton, Ph.D. Marisa Dzioba Professor of Physiology Dawoon Jung, Ph.D. Dartmouth Medical School Emily Notch, Ph.D.
Kevin Strange, Ph.D. Kristopher Burkewitz Director, Anesthesiology Research Division Rebecca Falin, Ph.D. Vanderbilt University Medical Center Elaine Lee, Ph.D. Hiroaki Miyazaki, Ph.D. Rebecca Morrison Angela Parton
Nicole Theodosiou, Ph.D. Assistant Professor Department of Biology Union College
Alice Villalobos, Ph.D. Assistant Professor Department of Nutrition and Food Science Texas A&M University
Mary Kate Worden, Ph.D. Assistant Professor Department of Neuroscience University of Virginia Health Sciences Center
Charles Wray, Ph.D. Elena Correa Staff Scientist Erin Schnettler Director of Scientific Resources Mount Desert Island Biological Laboratory
Viravuth P. Yin, Ph.D. Assistant Professor Mount Desert Island Biological Laboratory
2010 FELLOWSHIP RECIPIENTS
HIGH SCHOOL FELLOWSHIP RECIPIENTS
High School Research Fellowship: Mentors: Lili Zarzycki, St. Paul’s Girls’ School Billy Hudson, Ph.D. Arhea Marshall, Math, Science and Engineering High School Robert Preston, Ph.D.
Aspirnaut Fellowship: Richard Harris, KIPP Delta Collegiate High School Billy Hudson, Ph.D. Keith Wade, KIPP Delta Collegiate High School Billy Hudson, Ph.D.
NIEHS Short Term Educational Experiences for Research (STEER): David Brann, Mt. Ararat High School Gary Conrad, Ph.D. Maya DeGroote, Boothbay Region High School James L. Boyer, M.D. Thomas Flynn, John Bapst High School John N. Forrest, Jr., M.D. Dacie Manion, Old Town High School Jane Disney, Ph.D. Katarina Rydlizky, Ellsworth High School Greg Mayer, Ph.D. Timothy Weber, Portland High School Antonio Planchart, Ph.D.
UNDERGRADUATE RESEARCH FELLOWSHIP RECIPIENTS
NSF Research Experience for Undergraduates (REU)
Hannah Clemente, Smith College Jane Disney, Ph.D. Elena Correa, Colorado College Charles Wray, Ph.D. Sarah Cullen, Georgia Southern University J.B. Claiborne, Ph.D. Benjamin Davis, Boise State University James Coffman, Ph.D. Zachary Garcia, The University of Maine Andrew Christie, Ph.D. Megan Kelley, University of Vermont John N. Forrest, Jr., M.D. Kevin Lanza, Emory University George Kidder, Ph.D. Monica Orcine, University of Hawaii – Manoa Petra Lenz, Ph.D. and Dan Hartline, Ph.D. Sharon Perrone, Dickinson College Sharon Ashworth, Ph.D. Michaela Petit, Arcadia University Robert Preston, Ph.D. Breanna Sipley, University of Florida David Julian, Ph.D.
NIH/NCRR Maine IDeA Network of Biomedical Research Excellence (INBRE-ME)
Andrew Albert, UMaine – Fort Kent Carol Kim, Ph.D. The University of Maine Emily Bradford, Colby College Joshua Kavaler, Ph.D. Colby College Michaela Calnan, Bowdoin College Patsy Dickinson Bowdoin College Justine Cyr, UMaine – Presque Isle Sharon Ashworth The University of Maine Ryan Dawes, The University of Maine Hadley Horch Bowdoin College Samuel Entwisle, The University of Maine Antonio Planchart, Ph.D. MDI Biological Laboratory Sarah Harmon, Colby College Andrew Christie, Ph.D. MDI Biological Laboratory Kathleen Kelley, UMaine – Farmington Elizabeth Crockett, Ph.D. Ohio University John Leso, UMaine – Farmington Ryan Bavis, Ph.D. Bates College Amy Luce, The University of Maine Richmond Thompson, Ph.D. Bowdoin College Jame Nickerson, Bates College Pamela Baker, Ph.D. Bates College Dale Quinby, College of the Atlantic Chris Petersen, Ph.D. College of the Atlantic Rohit Sangal, Bowdoin College Hadley Horch, Ph.D. Bowdoin College Diane Saunders, Bates College James Coffman, Ph.D. MDI Biological Laboratory Robin VanDyke, College of the Atlantic Chris Petersen, Ph.D. College of the Atlantic Aaron Whitman, UMaine – Machias Melissa Glenn, Ph.D. Colby College Eric Williams, Southern Maine Community College Andrea Tilden Colby College Jennifer Willis, Southern Maine Community College Robert L. Preston, Ph.D.
OTHER FELLOWSHIP RECIPIENTS
Erin Schnettler, Colby College Charles Wray, Ph.D. NEW INVESTIGATOR AWARDS
Salisbury Cove Research Fund:
Suzie Currie, Ph.D., Mt. Alison University Hugo de Jonge, Ph.D., Erasmus University Billy Hudson, Ph.D., Vanderbilt University Seth Kullman, Ph.D., North Carolina State University Weiming Li, Ph.D., Michigan State University Francis McGowan, M.D., Harvard Medical School Ken Poss, Ph.D., Duke University Jonathan Rast, Ph.D., University of Toronto
MDIBL Named Fellowships:
John Henson, Ph.D., Dickinson College, Forster Fellowship Christine Keating, Ph.D., Pennsylvania State University, Bodil Schmidt Nielsen Fellowship & Bioengineer in Residence Award Gregory Mayer, Ph.D., Texas Tech University, Maren Fellowship; Blum Halsey Fellowship Robert Morris, Ph.D., Wheaton College, Forster Fellowship Alice Villalobos, Ph.D., Texas A&M university, Forrest Fellowship
NIH/NCRR Maine IDeA Network of Biomedical Research Excellence (INBRE-ME):
Sharon Ashworth, Ph.D., University of Maine Fernando Galvez, Ph.D., University of Louisiana Christopher Petersen, Ph.D. College of the Atlantic
NIH/NCRR Maine IDeA Network of Biomedical Research Excellence Junior Faculty:
Jack Bateman, Ph.D., Bowdoin College Randall Dahn, Ph.D., MDI Biological Laboratory Melissa Glenn, Ph.D., Colby College Ellen Hostert, Ph.D., University of Maine – Machias William Jackman, Ph.D., Bowdoin College Kevin Rice, Ph.D., Colby College Paula Schlax, Ph.D., Bates College Robert Wheeler, Ph.D., The University of Maine Voot Yin, Ph.D., MDI Biological Laboratory
2010 SEMINARS
Monday Morning Science Seminars
June 14 “Actin dynamics: A cellular balancing act”, Sharon Ashworth, Ph.D., Assistant Research Professor, Biochemistry, Microbiology, and Molecular Biology, School of Biology and Ecology, the University of Maine.
June 21 “Materials science/bioengineering approaches to biological questions”, Christine Dolan Keating, Ph.D., Associate Professor of Chemistry, Penn State University
June 28 “Physiological stress at the population distribution edge”, David Julian, Ph.D., Associate Professor, Department of Biology, University of Florida
July 12 “The evolution of myelin”, Daniel Hartline, Ph.D., Research Professor, The University of Hawaii at Manoa
July 19 “The physiological plasticity and functional genomics of osmotic tolerance in Fundulus species”, Fernando Galvez, Ph.D., Assistant Professor of Biology, Louisiana State University
July 26 “Multiple mechanisms of dioxin toxicity: Classical vs. Non-Classical actions of a prototypic AhR agonist”, Seth Kullman, Ph.D., Assistant Professor, North Carolina State University
August 2 “Search for the sulfilimine bond (S=N) in the animal kingdom”, Billy Hudson, Ph.D., Elliott V. Newman Professor of Medicine, Professor of Biochemistry and Pathology; Director, Center for Matrix Biology, Vanderbilt University
August 9 “The sea lamprey- a model of biliary atresia”, James Boyer, M.D., Professor, Department of Internal Medicine, Yale University School of Medicine
August 16 “A simple animal model for gut associated immunobiology”, Jonathan Rast, Ph.D., Associate Professor, Department of Immunology, University of Toronto, Canada
August 23 “Xiphophorus: a fish model of melanoma”, David W. Barnes, Ph.D., Senior Investigator, MDIBL
Friday Noon Brown Bag Seminars
June 25 Introductory Five Minute Talks by MDIBL PIs and Staff Introductions
July 2 Introductory Five Minute Talks by Faculty
July 9 Chalk Talk by Mario Schiffer, M.D. and Additional Talks by Students
July 16 Chalk Talk by Joseph Shaw, Ph.D. and Additional Talks by Students
July 23 Chalk Talk by Andy Christie, Ph.D. and Additional Talks by Students
July 30 Chalk Talk by Stephanie Zeiger, Ph.D. and Additional Talks by Students
August 6 Chalk Talk by Jonathan Rast and Alice Villalobos with Additional Talks by Students
August 13 Chalk Talk by Benjamin King and Additional Talks by Students
August 20 Chalk Talk by David Miller, Ph.D. and Additional Talks by Students
August 27 Chalk Talk by Nicole Theodosiou, Ph.D. and Additional Talks by Students
Lectureships
July 7 William B. Kinter Memorial Lecture – “Integrative genetic approaches to understanding complex lung diseases: Susceptibility to environmental pollutants", Steven Kleeberger, Ph.D., Acting Deputy Director of the National Institute of Environmental Health Sciences
July 26 Thomas H. Maren Memorial Lecture – “Complex roles for microglia in glaucoma", Monica Vetter, Ph.D., Professor and Interim Chair of Neurobiology and Anatomy, The University of Utah
July 30 Helen F. Cserr Memorial Lecture – “Genes, behavior, and the sense of smell: Generating flexible behaviors with a fixed nervous system”, Cornelia Bargmann, Ph.D., Howard Hughes Medical Institute Investigator, Torsten N. Wiesel Professor, Laboratory of Neural Circuits and Behavior, The Rockefeller University
August 9 Lewis Science Lecture – “Stem cells, epigenetics and other stories: An introduction to the New Biology”, Jonathan A. Epstein, M.D., William Wikoff Smith Professor of Cardiovascular Research, Scientific Director, Cardiovascular Institute, University of Pennsylvania
Thursday Afternoon Seminars
April 1 “Dynamic modulation of cell adhesion during zebrafish muscle morphogenesis”, Clarissa Henry, Ph.D., Assistant Professor in Biological Sciences, The University of Maine, Orono.
May 6 “Signaling pathways regulating identity and patterning of the lower jaw”, David Clouthier, Ph.D., Associate Professor, Department of Craniofacial Biology, School of Dental Medicine, University of Colorado, Denver.
July 1 “The brave new world of marine genomics: From evolution to memory mechanisms”, Leonid Moroz, Ph.D., Professor of Neuroscience, Zoology and Chemistry, The Whitney Laboratory for Marine Bioscience July 29 “Novel chemical probes for integrative potassium channel physiology”, Jerod Denton, Ph.D., Associate Professor of Anesthesiology and Pharmacology, Vanderbilt University Medical Center
August 5 “The dynamics of sea urchin gastrulation: a systems approach”, David McClay, Ph.D., Professor, Duke University
Special Seminars and Presentations
January 19 “Programmed cell death and regenerative tissue remodeling in planarians”, Jason Pelletieri, Ph.D., Howard Hughes Medical Institute Research Fellow, University of Utah School of Medicine
February 2 “To regenerate or not to regenerate? A tale in the zebrafish lateral line”, Martine L. Behra, Ph.D., Research Fellow, National Human Genome Research Institute (NHGRI)
March 12 “Transcriptional analysis of trait deterioration and stress survival in nematodes”, Bishwo Adhikari, Ph.D. candidate, Brigham Young University
March 16 “Distinct populations of pancreatic beta-cells in zebrafish: Implications for regenerative medicine”, Dan Hesselson, Ph.D., Department of Biochemistry, Diabetes Center, University of California, San Francisco
March 17 “The molecular regulation of neural patterning”, Peter Fuerst, Ph.D., Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University
March 24 “Sponges, choanoflagellates and the foundations of animal origins”, Scott Nichols, Ph.D., Postdoctoral Fellow, University of California, Berkeley, Department of Molecular and Cell Biology
March 31 “MicroRNAs control organ regeneration in adult zebrafish”, Viravuth Pho Yin, Ph.D., Postdoctoral Fellow, Duke University Medical Center, Department of Cell Biology, Durham, NC
April 13 “Molecular mechanisms of dendrite and axon development”, Yi Zheng, Ph.D., Howard Hughes Medical Institute, University of California, San Francisco, CA
June 21 “Effect of exercise, heat and dehydration stress on lymphocyte cytoprotective responses in trained males”, Elaine Lee, Ph.D., Postdoctoral Fellow, Yale University
June 21 “CO2/HCO3-/pH sensing via soluble adenylyl cyclase (sAC)”, Martin Tresguerres, Ph.D., Weill Cornell Medical College
June 23 “How to give a talk” Student Workshop, Susan Fellner, Ph.D., Professor, University of North Carolina at Chapel Hill June 28 “Identification of protein that form sulfenic acid in response to oxidation”, Christina Takanishi, Ph.D., University of California, Davis
July 6 “Hyperactive ras in developmental diseases and cancer: Insights from zebrafish”, Marina Mione, Ph.D., Assistant Professor, Firc Institute of Molecular Oncology, Milan, Italy
July 16 “How to get started on a new grant”, David Dawson, Ph.D., Professor and Chair, Dept. of Physiology and Pharmacology, Oregon Health & Science University
July 20 “Heart and fin regeneration in zebrafish", Kenneth Poss, Ph.D., HHMI, Associate Professor of Cell Biology, Duke University Medical Center
July 28 “Between mice and men – searching for proteinuria genes in zebrafish”, Herman Maller, M.D., Hannover Medical School, Germany, Ron Korstanje, Ph.D., The Jackson Laboratory, Mario Schiffer, M.D., Hannover Medical School, Germany
August 10 “Imaging mass spectrometry: A new view of biology”, Richard Caprioli, Ph.D., Professor, Vanderbilt-Ingram Cancer Center
August 13 “Cancer care and research in the era of genomics: Pursuit of personalized medicine for cancer patients", Edward J. Benz, Jr., M.D., President, Dana Farber Cancer Institute
August 16 “The Xiphophorus melanoma model: Unappreciated complexities”, Rodney Nairn, Ph.D., Professor, Carcinogenesis, The University of Texas M. D. Anderson Cancer Center
October 19 “MicroRNAs and metazoan macroevolution”, Kevin J. Peterson, Ph.D., Associate Professor of Biological Sciences, Dartmouth College
2010 CONFERENCES, SYMPOSIA, AND WORKSHOPS
January 26 MDI Regional School System Eelgrass Summit
April 23-24 37th Maine Biological and Medical Sciences Symposium
SESSION I: MOLECULAR GENETICS
Keynote Address: Kevin Strange, Ph.D., MDI Biological Laboratory The effects of osmotic stress on cells and their proteins Maine New Investigator: Rick Maser, Ph.D., The Jackson Laboratory The role of telomere dysfunction in aging and cancer Maine New Investigator: Chengkai Dai, Ph.D., The Jackson Laboratory Stress phenotype of cancer: Implication of the stress response in tumorigenesis
Joshua Boucher, GSBS and Maine Medical Center Research Institute Notch and miR-145: A novel mechanism controlling VSMC fate Maine New Investigator: Danielle Dube, Ph.D., Bowdoin College Metabolic profiling of Helicobacter pylori glycosylation Natalie Fredette, Maine Medical Center Research Institute The role of Delta-like1 (Dll1) signaling in endothelial senescence Maine New Investigator: Julie Gosse, Ph.D., The University of Maine Inorganic arsenite inhibits IgE receptor-mediated degranulation of mast cells Xiong Li, Ph.D., Maine Institute for Human Genetics and Health The molecular signaling involved in low doses of arsenic-promoted cell proliferation Maine New Investigator: Peter Woodruff, Ph.D., Bowdoin College Trehalose production expedites bioremediation of hexavalent chromium Justin Guay, GSBS and Maine Medical Center Research Institute Death associated protein Kinase 2 promotes cell death in renal cortical interstitial cells following acute nephrotoxic injury Lara Carlson, DPE, FACSM, CSCS, University of New England Resistance exercise-induced changes in gene expression of human blood mononuclear cells Caroline McPhee, DVM, The Jackson Laboratory CD8 T suppressor cells protect from Lupus-like autoimmunity
SESSION 2: GENERAL BIOLOGY AND ECOLOGY
Maine New Investigator: Daniel Thornhill, Ph.D., Bowdoin College The evolution of bacterial symbiosis in siboglinid annelids Karen Pelletreau, Ph.D., The University of Maine The feeding behavior of the highly selective gastropod Elysia chlorotica and the surface composition of its algal prey Vaucheria litorea: Do lectin/glycan interactions play a role in recognition? Maine New Investigator: Ursula Roese, Ph.D., University of New England Biosynthesis of constitutive and herbivore-inducible terpenes and their role in multitrophic interactions Aaron Whitman, University of Maine - Machias Patterns of Ambylomma dissimile parasitism on insular dwarfed Boa constrictor imperator in the Cayos Cochinos, Honduras George Kidder, Ph.D., MDI Biological Laboratory Fine-scale current mapping in Eastern Bay (upper Frenchman Bay) as a guide to eelgrass restoration Emily Argo, College of the Atlantic Sex ratios and reproduction in spiny dogfish Squalus acanthias found in the Gulf of Maine
SESSION 3: DEVELOPMENTAL BIOLOGY
Michelle Goody, The University of Maine Nrk2b regulates cell adhesion and is required for muscle morphogenesis in vivo Chris McCarty, MDI Biological Laboratory Characterization of selected cis-regulatory elements in the cyclin D gene of the purple sea urchin Strongylocentrotus purpuratus Shelby Davies, Bowdoin College Targeted functional analysis of FGF/BMP antagonistic signaling during tooth development in Danio rerio SESSION 4: NEUROSCIENCE
Maine New Investigator: Tariq Ahmad, Ph.D., Colby College Serpin5 regulates Toll pathway mediated melanization upon mutant CHMP2B expression in the Drosophila model of Frontotemporal Dementia Lura Caddle, The Jackson Laboratory Radiation therapy may reduce plaque formation to prevent the neurodegeration associated with Alzheimers Disease Jennifer Corriveau, Colby College Preventing severe psychosis: Anti-Schizophrenia properties of choline supplementation Daniel Swett, University of Southern Maine Arsenic exposure alters the regulation of the neuronal cytoskeleton during neuritogenesis Matthew Bowers, Bowdoin College Effects and distribution of Homarus americanus calcitonin-like diuretic hormone (Homam CLDH), an intrinsic neuromodulator in the cardiac system of the American lobster, Homarus americanus Maine New Investigator: Stephanie Taylor, Ph.D., Colby College Clarifying the role of damped oscillators in the mammalian circadian clock Alan Rosenwasser, Ph.D., The University of Maine Effects of ethanol withdrawal on the level and circadian periodicity of locomotor activity in inbred mice Donna Bass, University of New England The effects of a diet rich in Omega-3 essential fatty acids on behavior and medial prefrontal cortex neurotransmitters in the adolescent rat
July 27 2010 INBRE and MDIBL Student Symposium
DEVELOPMENT AND REGENERATION Michaela Calnan*, Bowdoin College Regenerative response of cricket auditory neurons to injury: What role is played by activity in sensory neurons? Mentor: Patsy Dickinson, Ph.D., Bowdoin College
Benjamin Davis, Boise State University Characterization studies of diet restricted Echinoid larvae Mentor: James Coffman, Ph.D., Mount Desert Island Biological Laboratory
Samuel Entwisle*, The University of Maine Role of Groucho corepressors in FoxQ1b-mediated transcriptional regulation during zebrafish jaw development Mentors: Carolyn Mattingly, Ph.D. and Antonio Planchart, Ph.D., Mount Desert Island Biological Laboratory
Timothy Weber, Portland High School The effects of misregulated connective tissue growth factor in craniofacial development Mentors: Carolyn Mattingly, Ph.D. and Antonio Planchart, Ph.D., Mount Desert Island Biological Laboratory
David Brann, Mt. Ararat High School First step toward using a marine model organism: Induced spawning, rearing and surface reactivity of Mytilus edulis embryos Mentor: Gary Conrad, Ph.D., Kansas State University
PHYSIOLOGY AND STRESS BIOLOGY Megan Kelley, University of Vermont Thomas Flynn, John Bapst Memorial High School Study of the presence of specific phosphodiesterase types and effects of their inhibition on chloride secretion in Squalus acanthias rectal gland Mentor (Kelley): John N. Forrest, Jr., M.D., Yale University School of Medicine Mentor (Flynn): Hugo de Jonge, Ph.D., Erasmus University Medical Center
Daniel Lionarons, Yale University School of Medicine Characterization of bile salt uptake in the skate intestine Mentor: James Boyer, M.D., Yale University School of Medicine
Victoria Smith, Willamette University Maya DeGroote, Boothbay Regional High School Mechanisms of xenobiotic and bile salt transportation and excretion in the sea lamprey Petromyzon marinus: A unique cholestatic model for biliary atresia Mentor: James Boyer, M.D., Yale University School of Medicine
Kathleen Kelley*, University of Maine at Farmington Production of ROS (reactive oxygen species) in mitochondria from cold- and warm- acclimated Fundulus heteroclitus Mentor: Elizabeth Crockett, Ph.D., Ohio University
Nathan Walker, Mount Allison University Physiological effects of chemical and molecular chaperones following hypo-osmotic stress in red blood cells of the spiny dogfish shark, Squalus acanthias Mentor: Suzanne Currie, Ph.D., Mount Allison University
Katarina Rydlizky, Ellsworth High School Estrogen's effect on DNA repair in killifish Mentor: Gregory Mayer, Ph.D., Texas Tech University
Michaela Petit, Arcadia University Possible role of HSP90 in desiccation resistance in Fundulus heteroclitus embryos Mentor: Robert Preston, Ph.D., Illinois State University
Ashra Kolhatkar, Mount Allison University Can sharks take the heat? The role of trimethylamine oxide and heat shock proteins following heat stress in the spiny dogfish shark, Squalus acanthias Mentor: Suzanne Currie, Ph.D., Mount Allison University
ECOLOGY Dacie Manion, Old Town High School Effects of temperature variation on eelgrass (Zostera marina) flowering Mentor: Jane Disney, Ph.D., Mount Desert Island Biological Laboratory
Kevin Lanza, Emory University Fish and invertebrate richness and abundance inside versus outside of Zostera marina. Mentor: George Kidder, Ph.D., Mount Desert Island Biological Laboratory
Hannah Clemente, Smith College An investigation of the interactions between eelgrass (Zostera marina) and red tide phytoplankton Mentor: Jane Disney, Ph.D., Mount Desert Island Biological Laboratory
Elena Correa, Colorado College Population genetics of estuarine fish of Acadia National Park: Connectedness among estuaries in downeast Maine Mentor: Charles Wray, Ph.D., Mount Desert Island Biological Laboratory
SESSION 4: GENETICS AND NEUROSCIENCE Andrew Albert*, University of Maine at Fort Kent Functional analysis of zebrafish MDA5 during viral infection Mentor: Carol Kim, Ph.D., The University of Maine
Ryan Dawes*, The University of Maine Immunohistochemical observation of synaptic localization after deafferentation in the cricket Gryllus bimaculatus Mentor: Hadley Horch, Ph.D., Bowdoin College
Monica Orcine, University of Hawaii at Manoa Neurophylogeny of histaminergic neurons in copepods Mentor: Daniel Hartline, Ph.D., University of Hawaii at Manoa
Rohit Sangal*, Bowdoin College Investigation of the differential expression of Sema-2a due to deafferentation in the cricket, Gryllus bimaculatus Mentor: Hadley Horch, Ph.D., Bowdoin College
Zachary Garcia, The University of Maine Mapping of calcitonin-like diuretic hormone immunoreactivity in cardiac and stomatogastric nervous systems of Homarus americanus and Cancer borealis Mentor: Andrew Christie, Ph.D., Mount Desert Island Biological Laboratory
Sarah Harmon*, Colby College Towards the identification of the Daphnia pulex circadian system Mentor: Andrew Christie, Ph.D., Mount Desert Island Biological Laboratory
Emily Bradford*, Colby College Regulation of d-Pax2 expression in D. melanogaster by RNAi Mentor: Joshua Kavaler, Ph.D., Colby College
Sarah Cullen, Georgia Southern University Expression of rhesus proteins in Fundulus heteroclitus Mentor: J.B. Claiborne, Ph.D., Georgia Southern University
Justine Cyr*, The University of Maine at Presque Isle Localization of cofilin 1 in the zebrafish using in situ hybridization Mentor: Sharon Ashworth, Ph.D., The University of Maine
Anastasia Gianakas, Dickinson College Arp2/3 complex-facilitated rocketing of actin-filled processes during sea urchin coelomocyte spreading Mentor: John Henson, Ph.D., Dickinson College
Amy Luce*, The University of Maine Neuroendocrine influences on social behavior Mentor: Rick Thompson, Ph.D., Bowdoin College
Arhea Marshall, High School for Mathematics, Science & Engineering Predation of killifish embryos by adult killifish Mentor: Robert Preston, Ph.D., Illinois State University
Jamie Nickerson*, Bates College Genetics of susceptibility to bone loss related to periodontal disease Mentor: Pam Baker, Ph.D., Bates College
Sharon Perrone, Dickinson College Role of Cofilin 1-like in Danio rerio development and kidney function Mentor: Sharon Ashworth, Ph.D., The University of Maine
Dale Quinby*, College of the Atlantic Apparent phenotypic plasticity in reproductive timing of Fundulus heteroclitus Mentor: Christopher Petersen, Ph.D., College of the Atlantic
Diane Saunders*, Bates College Effects of zinc, nickel, and hypoxic conditions on genetic regulation of oral-aboral axis formation in developing Strongylocentrotus purpuratus embryos Mentor: James Coffman, Ph.D., Mount Desert Island Biological Laboratory
Robyn Van Dyke*, College of the Atlantic Patterns of trematode prevalence and local adaptation in Littorina spp. Mentor: Christopher Petersen, Ph.D., College of the Atlantic
Aaron Whitman*, The University of Maine at Machias Neural degeneration in the frontal cortex of aged rats as a function of in utero choline levels Mentor: Melissa Glenn, Ph.D., Colby College
Eric Williams*, Southern Maine Community College Melatonin: neuritogenesis and neuroprotective effects in Uca pugilator x-organ cells Mentor: Andrea Tilden, Ph.D., Colby College
Jennifer Willis*, Southern Maine Community College Glucose content in aerially incubated mid-stage killifish embryos Mentor: Robert Preston, Ph.D., Illinois State University
*INBRE student
August 6-7 9th Annual Mount Desert Island Stem Cell Symposium
EPIGENETIC REGULATION OF TRANSCRIPTION AND EXPRESSION Andrew Xiao, Ph.D., Yale University New functions of histone variant H2A.X in maintaining genome integrity
Richard Gregory, Ph.D., Children's Hospital, Boston Regulation of microRNA biogenesis in embryonic stem cells and cancer
John Rinn, Ph.D., Beth Israel Deaconess Medical Center The role of large non-coding RNA in establishing epigenetic states of adult and embryonic cells and their misregulation in cancer
Carla Kim, Ph.D., Children’s Hospital and Harvard Medical School Stem cells in normal lung and lung cancer
Stuart Orkin, M.D., HHMI / Children's Hospital, Boston Embryonic stem cell signatures and cancer
Alex Schier, Ph.D., Harvard University The maternal-zygotic transition
Leonard Zon, M.D., HHMI / The Children's Hospital Transcriptional elongation and hematopoiesis
Alex Meissner, Ph.D., The Broad Institute, Harvard University Epigenetic reprogramming and cellular states
Don Wojchowski, Ph.D., Maine Medical Center Research Institute Novel regulators of progenitor cell survival discovered via global analyses of EPOR action
CELL PLURIPOTENCY AND REPROGRAMMING Jeanne Loring, Ph.D., The Scripps Research Institute Systems biology focus on pluripotence
Susan Mango, Ph.D., Harvard University Pluripotency and its loss during embryonic development
Konrad Hochedlinger, Ph.D., Massachusetts General Hospital Factors influencing nuclear reprogramming
Richard Young, Ph.D., Whitehead Institute Programming ES cell state
Kathrin Plath, Ph.D., UCLA Reprogramming to pluripotency - insights into the mechanism
Bradley Cairns, Ph.D., HHMI / University of Utah School of Medicine Germline chromatin: concepts shared in germ and ES cells
Elaine Fuchs, Ph.D., HHMI / The Rockefeller University Wnt signaling and stem cell biology
REGENERATION Fernando Camargo, Ph.D., Children’s Hospital Boston Hippo signaling pathway and the regulation of tissue/organ size and growth
Tom Rando, M.D., Ph.D., Stanford University Mechanisms of muscle stem cell ("satellite cell") activation in growth and regeneration of skeletal muscles
Amy Wagers, Ph.D., Joslin Diabetes Center Hematopoietic and skeletal muscle stem cells in mice
Ben Stanger, M.D., Ph.D., University of Pennsylvania Stem cells and progenitor cells in the vertebrate liver and pancreas
Daniel G. Anderson, Ph.D., Koch Institute for Integrative Cancer Research, MIT Combinatorial Development of Biomaterials for Stem Cells
Hal Broxmeyer, Ph.D., Indiana University School of Medicine Influence of SIRT1, a member of the Sirtuin family of deacetylases, on maintenance and differentiation of mouse embryonic stem cells
Nathan Lawson, Ph.D., UMass Medical School Control of neovascularization by microRNAs
Jayanta Roy-Chowdhury, M.D., Albert Einstein College of Medicine Transplantation of stem cell-derived hepatocytes for treating inherited liver diseases
August 11 CFTR Micro-Symposium: Dave Dawson: CFTR: Locating a bottle-neck in the pore; John Forrest: Species-dependent inhibition of CFTR by CFTRinhib-172; Hugo de Jonge: CFTR, WNKs and glutamate; Jack Riordan: Can a destabilizing, functionally enigmatic fragment of CFTR provide a therapeutic target?; Bruce Stanton: Ubiquitination and CFTR Trafficking
September 8 – 12 Getting Across: Cellular and Molecular Mechanisms of Vascular Permeability; Hermann Haller, M.D., Hannover Medical School
September 16 – 17 The Jackson Laboratory / MDIBL Joint Scientific Symposium
GRADUATE STUDENT AND POSTDOCTORAL FELLOW SYMPOSIUM
Cheryl Ackert-Bicknell, Ph.D., The Jackson Lab Loss of of Cappuccino (Cno), a member of the biogenesis of lysosome-related organelle complex 1, results in decreased adult bone mass
Kathy Snow, The Jackson Lab Nuclear positioning, higher-order folding, and gene expression of Mmu15 sequences are refractory to chromosomal translocation
Caroline McPhee, Ph.D., The Jackson Lab CD8+ T suppressor cells protect from lupus-like autoimmunity
Poster session
Jill Recla, The Jackson Lab In-silico haplotype association mapping in mice identifies candidate genes for human chronic pain susceptibility
Yichang Jia, Ph.D., The Jackson Lab RNA splicing abnormality and neurodegeneration
Ben Smith, The Jackson Lab 3D insights into why the blood-testis barrier is indestructible
Guest Speaker, Randolph Nesse, M.D., University of Michigan Evolutionary applications in medicine: The great opportunity
FACULTY SYMPOSIUM
Kevin Strange, Ph.D., MDIBL Science at MDIBL
Bob Braun, Ph.D., The Jackson Lab Science at The Jackson Laboratory
Ron Korstanje, Ph.D., The Jackson Lab and Mario Schiffer, M.D., MDIBL Identifying genes involved in proteinuria using mouse and zebra fish
Randy Dahn, Ph.D., MDIBL A comparative functional genomics approach to understanding vertebrate limb regeneration
Robert Burgess, Ph.D., The Jackson Lab Peripheral neuropathy: When housekeeping goes bad
Ray Frizzell, Ph.D., MDIBL The frustration of CFTR origami: Folding CFTR
Matt Hibbs, Ph.D., The Jackson Lab Using functional genomics to identify genotype-phenotype associations
Antonio Planchart, Ph.D., MDIBL A zebrafish model of a human craniofacial disorder
Patsy Nishina, Ph.D., The Jackson Lab Using chemical mutagenesis to elucidate pathways important in the retina
David Julian, Ph.D., MDIBL Understanding the limits of species' ranges: from model organisms to niche models
Elissa Chesler, Ph.D., The Jackson Lab A platform for integrative functional genomics: Understanding biological functions across species
2010 COURSES
January 4-8 Molecular Biology Research Techniques Southern Maine Community College INBRE course Elizabeth Ehrenfeld, Ph.D., Southern Maine Community College; Charles Wray, Ph.D., MDIBL
January 10-22 Crustacean Molecular Neurophysiology Colby College INBRE course Andrea Tilden, Ph.D., Colby College; Andrew Christie, Ph.D., MDIBL
February 14-19 Molecular Biology Research Techniques University of Maine- Machias, University of Maine- Farmington INBRE course Antonio Planchart, Ph.D., MDIBL; Rich Robinson, Ph.D., University of Maine – Farmington; Sherrie Sprangers, Ph.D., University of Maine - Machias
February 26-March 6 Molecular Mechanisms of Human Disease University of Maine Honors College INBRE course Bruce Stanton, Ph.D., Dartmouth Medical School; Denry Sato, Ph.D., MDIBL
January 17- 24, Part 1 Molecular Biology of Crustacean Neuropeptides March 12 – 18, Part 2 Bowdoin College INBRE course Hadley Horch, Ph.D., Bowdoin College; Andrew Christie, Ph.D., MDIBL
March 15-26 Molecular Evolutionary Genetics College of the Atlantic INBRE course Charles Wray, Ph.D., MDIBL; Christopher Petersen, Ph.D., College of the Atlantic
April 27- May 6 Prokaryotic Genetics Bates College INBRE course Paula Schlax, Ph.D., Bates College; Antonio Planchart, Ph.D., MDIBL
May 22-29 Structure and Function of Polarized Epithelial Cells University of Pittsburgh School of Medicine Ray Frizzell, Ph.D., University of Pittsburgh School of Medicine
June 6-13 Structure and Function of Polarized Epithelial Cells Yale University School of Medicine John N. Forrest, Jr., M.D., Yale University School of Medicine
June 13- 20 Twelfth Annual Intensive Course in Quantitative Fluorescent Microscopy Simon Watkins, Ph.D., University of Pittsburgh School of Medicine
July 31- August 7 Molecular Mechanisms of Human Disease Dartmouth Medical School and University of Vermont College of Medicine INBRE course Bruce Stanton, Ph.D., Dartmouth Medical School
August 9-12 Young Environmental Leaders Jane Disney, Ph.D., MDIBL
August 15-21 Health and Colony Management of Laboratory Fish Paul Bowser, Ph.D., Cornell University College of Veterinary Medicine
August 15- 20 Summer Academy in Genomics Charles Wray, Ph.D., MDIBL
August 21-28 Environmental Genomics John Colbourne, Ph.D., The Center for Genomics and Bioinformatics, Indiana University; Joe Shaw, Ph.D., Indiana University; Ben King, MDIBL
August 28- September 4 Comparative Physiology Beth Israel Deaconess Medical Center Mark Zeidel, M.D., Beth Israel Deaconess Medical Center; Bill Aird, M.D., Beth Israel Deaconess Medical Center
September 4-11 Origins of Renal Physiology Mark Zeidel, M.D., Beth Israel Deaconess Medical Center
October 8- 12 Molecular Biology and Bioinformatics University of Maine - Presque Isle, University of Maine- Fort Kent Charles Wray, Ph.D., MDIBL
October 11-16 Skate Genome Annotation Workshop Ben King, MDIBL; Carolyn Mattingly, Ph.D., MDIBL
PUBLICATIONS
Aleksandrov AA, Kota P, Aleksandrov LA, He L, Jensen T, Cui L, Gentzsch M, Dokholyan NV, Riordan JR. Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR. J Mol Biol. 2010 Aug 13;401(2):194-210. Epub 2010 Jun 16. PubMed PMID: 20561529.
Ashworth S, Teng B, Kaufeld J, Miller E, Tossidou I, Englert C, Bollig F, Staggs L, Roberts IS, Park JK, Haller H, Schiffer M. Cofilin-1 inactivation leads to proteinuria--studies in zebrafish, mice and humans. PLoS One. 2010 Sep 8;5(9):e12626. PubMed PMID: 20838616; PubMed Central PMCID: PMC2935884.
Christie AE, Nolan DH, Garcia ZA, McCoole MD, Harmon SM, Congdon-Jones B, Ohno P, Hartline N, Congdon CB, Baer KN, Lenz PH. Bioinformatic prediction of arthropod/nematode-like peptides in non-arthropod, non-nematode members of the Ecdysozoa. Gen Comp Endocrinol. 2011 Feb 1;170(3):480-6. Epub 2010 Nov 11. PubMed PMID: 21074533.
Coluccio AE, LaCasse TJ, Coffman JA. Oxygen, pH, and oral-aboral axis specification in the sea urchin embryo. Mol Reprod Dev. 2011 Feb;78(2):68. doi: 10.1002/mrd.21267. Epub 2011 Jan 20. PubMed PMID: 21254277.
Currie S, Edwards SL. The curious case of the chemical composition of hagfish tissues--50 years on. Comp Biochem Physiol A Mol Integr Physiol. 2010 Oct;157(2):111-5. Epub 2010 Jun 12. Review. PubMed PMID: 20547237.
Currie S, LeBlanc S, Watters MA, Gilmour KM. Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish. Proc Biol Sci. 2010 Mar 22;277(1683):905-13. Epub 2009 Nov 18. PubMed PMID: 19923129; PubMed Central PMCID: PMC2842717.
Edwards SL, Weakley JC, Diamanduros AW, Claiborne JB. Molecular identification of Na(+)-H(+) exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus. J Fish Biol. 2010 Feb;76(2):415-26. PubMed PMID: 20738718.
Ertl RP, Robertson AJ, Saunders D, Coffman JA. Nodal-mediated epigenesis requires dynamin- mediated endocytosis. Dev Dyn. 2011 Mar;240(3):704-11. doi: 10.1002/dvdy.22557. Epub 2011 Feb 8. PubMed PMID: 21337468; PubMed Central PMCID: PMC3079387.
Grim JM, Miles DR, Crockett EL. Temperature acclimation alters oxidative capacities and composition of membrane lipids without influencing activities of enzymatic antioxidants or susceptibility to lipid peroxidation in fish muscle. J Exp Biol. 2010 Feb 1;213(3):445-52. PubMed PMID: 20086129; PubMed Central PMCID: PMC2808215.
Guerreiro PM, Canario AV, Power DM, Renfro JL. Piscine PTHrP regulation of calcium and phosphate transport in winter flounder renal proximal tubule primary cultures. Am J Physiol Regul Integr Comp Physiol. 2010 Aug;299(2):R603-11. Epub 2010 May 19. PubMed PMID: 20484696.
He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR. Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR. FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. PubMed PMID: 20233947; PubMed Central PMCID: PMC2909275.
Lewis JM, Hori TS, Rise ML, Walsh PJ, Currie S. Transcriptome responses to heat stress in the nucleated red blood cells of the rainbow trout (Oncorhynchus mykiss). Physiol Genomics. 2010 Aug;42(3):361-73. Epub 2010 Jun 15. PubMed PMID: 20551145.
Messier-Solek C, Buckley KM, Rast JP. 2010. Highly diversified innate receptor systems and new forms of animal immunity.Semin Immunol. 22(1):39-47.
Monaghan SR, Rumney RL, Vo NT, Bols NC, Lee LE. In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish. In Vitro Cell Dev Biol Anim. 2011 Feb;47(2):104-13. Epub 2010 Nov 18. PubMed PMID: 21086187.
Prevoo B, Miller DS, Van de Water FM, Wever KE, Russel FG, Flik G, Masereeuw R. Rapid, nongenomic stimulation of multidrug resistance protein 2 (Mrp2) activity by glucocorticoids in renal proximal tubule. J Pharmacol Exp Ther. 2011 Apr 22. [Epub ahead of print] PubMed PMID: 21515814.
Smith LC, Ghosh J, Buckley KM, Clow LA, Dheilly NM, Haug T, Henson JH, Li C, Lun CM, Majeske AJ, Matranga V, Nair SV, Rast JP, Raftos DA, Roth M, Sacchi S, Schrankel CS, Stensvåg K. Echinoderm immunity. Adv Exp Med Biol. 2010;708:260-301. Review. PubMed PMID: 21528703.
Thomas DJ, Nava GM, Cai SY, Boyer JL, Hernández-Zavala A, Gaskins HR. Arsenic (+ 3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis. Toxicol Sci. 2010 Jan;113(1):70-6. Epub 2009 Oct 15. PubMed PMID: 19833739; PubMed Central PMCID: PMC2902911. AUTHORS
Abrell, Leif 59 Katsekis, Christopher J. 10 Aleksandrov, Andrei A. 14 Kelley, Kathleen M. 19 Amistadi, Mary Kay 59 Kelley, Megan H. 16 Ashworth, Sharon 42 Kidder, George W. 73, 80 Balkaran, Kavita 80 Kieffer, Allison 49 Bataille, Amy M. 13 Kinne, Rolf 24, 26, 28, Boyer, James L. 40, 57 30, 32 Brann, David 74 Kläs, Juliane 55 Brestle, Chase 80 Kolhatkar, Ashra 34 Brestle, Grant 80 Kratochvilova, Hana 11 Brummer, Gage 18 Kuijpers, Marcela V. 10 Brunk, Erin 42 Lee, Lucy E. J. 69 Buckley, Katherine M. 90 Li, Weiming 40 Cai, Shi-Ying 40, 57 Lionarons, Daniel A. 40, 57 Chorover, Jon 59 Littlechild, Stacy 18 Chorover, Nathan 59 Mahringer, Anne 55 Christie, Andrew E. 89 Marquis, Hannah 42 Chuaypanang, Sirilak 38 Marshall, Arhea V. 38, 48 Claiborne, James B. 11, 21, 87 Masereeuw, Rosalinde 53 Coffman, James A. 63, 66 Melita, August M. 16 Congdon, Clare Bates 89 Mikhaeil, Mike S. 69 Conrad, Gary W. 18, 74 Miller, David S. 53, 55 Correa, Elena 83 Morris, Montana 16 Crockett, Elizabeth L. 19, 36 Morris, Robert L. 62 Crombie, Timothy A. 45 Nolan, Daniel H. 89 Cui, Liying 14 Norden, Wendy 73 Currie, Suzanne 34 Olson, Ingrid 89 Cutler, Christopher 10 Orcine, Monica I. 44 Cyr, Justine 42 Perrone, Sharon 42 Danner, Russell 49 Perry, Deborah 51 Davis, Benjamin 66 Petersen, Chris W. 75, 77, 83, DeGroote, Maya 57 83 de Jonge, Hugo R. 16 Petit, Michaela K. 38 Diamanduros, Andrew 21 Preston, Robert L. 38,48 Disney, Jane 80 Prevoo, Brigitte 53 Edwards, Susan L. 11 Preziosi, Christopher 42 Ertl, Robin P. 63 Quinby, Dale 75 Flynn, Bob Tom 16 Rabeneck, Brett 21, 87 Forrest, John N., Jr. 1, 16 Rast, Jonathan P 90 Fricker, Gert 55 Reichel, Valeska 55 Gamperl, A. Kurt 34 Renfro, J. Larry 13, 51 Gianakas, Anastasia 62 Riordan, John R. 14 Guerreiro, Pedro M. 13, 51 Saunders, Diane C. 63 Hartline, Daniel K. 44 Schnettler, Erin 49 Henson, John H. 62 Shea, Christine 89 Hernandez-Ruiz, Selene 59 Silva, Anya 24 Hess, Helen 77 Silva, Patricio 24, 26, 28, Ho, Eric C. H. 90 30, 32 Jensen, Tim 14 Smith, Victoria 40 Julian, David 45 Solek, Cynthia Messier 90 Spokes, Katherine C. 24, 26, 28, 30, 32 Staggs, Lynn 42 Swanberg, Caleb 42 Tapley, David W. 36 Taylor, Robert J. 51 Van Dyke, Robin 77 Vasquez, M. Christina 45 Villalobos, Alice R. A. 51 Vo, Nguyen T. K. 69 Walker, Nathan S.B. 34 Walsh, Jonathon D. 10 Wheeler, Mark S. 36 Wickramasekara, Samanthi 59 Willis, Jennifer L. 38 Wray, Charles 49, 83, 85 Yeh, Chu-Yin 40 Young, Robin K. 51
SPECIES
Carassius carassius 11 Salmo salar 69 (koi) (Atlantic salmon)
Danio rerio 42 Squalus acanthias 18, 34, 13, (zebrafish) (spiny dogfish shark) 24, 26, 28, 30, 32, 51 Echinarachinius parma 90 (common sand dollar) Strongylocentrotus droebachiensis 62 (green sea urchin) Fundulus heteroclitus 75, 38, 48, (killifish) 83, 85 Strongylocentrotus purpuratus 63, 66, 90 (purple sea urchin) Glycera dibranchiata 45 (bloodworm) Zostera marina 80, 85 (eelgrass) Leucoraja erinacea 57 (little skate)
Littorina littorea 77 (common periwinkle)
Littorina obtusata 77 (smooth periwinkle)
Littorina saxatilis 77 (rough periwinkle)
Menidia menidia 83 (atlantic silverside)
Morone saxatilis 19, 36 (striped bass)
Myoxocephalus octodecemspinousus 21 (longhorn sculpin)
Mytilus edulis 74, 80 (blue mussel)
Oryctolagus cuniculus 18 (white albino rabbit)
Petromyzon marinus 40 (sea lamprey)
KEYWORDS actin 42 restoration 80 aerobic metabolism 24 salmon aquaculture 69 ASBT 57 semi-lunar periodicity 75 autophagy 45 spawning 74 bile salt transport 57 stress 34 biofouling 74 TMAO 34 cell line 69 trematode 77 cerebrospinal fluid 13 urea 32 cholestasis 40 urea transporter 32 choroid plexus 13, 51 water quality 87 cofilin 1-like isoform 42 zinc 51 cornea 18 disolved organic matter 59 ectotherm 36 eelgrass 80 endocrine distupting compounds 59 environmental mass spectrometry 59 Frenchman Bay 87 gastropod 77 gill 21 gill morphology 11 gut sacs 57 gycolysis 24 heat shock proteins 34 heavy metals 51 immunity 90 intertidal 45 intertidal spawning 75 ion regulation 11, 21 KCN 24 larva 90 LASIK 18 lysosome 45 marine bacteria 90 NHE 21 non-heme iron 36 Northeast Creek 75 organic anions 40 oxidative stress 36 oxygen concentration 11 parasitism 77 pH 87 phosphate 13 pituitary cell culture 69 rectal gland 32 renal excretion 40 RESEARCH SUPPORT
Dartmouth Superfund Basic Research Program 85
DFG (Deutsche Forschungsgemeinschaft) 55
Dickinson College 62
Dutch Society of Pharmacology 53
Dutch Digestive Foundation 57
Georga Southern University 10, 21
Gulf of Maine Council 80
Kansas St. University Terry C. Johnson Cancer Center 18
L.L. Bean 83
Maine Economic Improvement Fund 89
MDI Biological Laboratory Student Fellowship 45
Visiting Investigator Fellowship 11, 34, 42, 45, 51, 53, 62, 69, 75, 77, 90
Morris Animal Foundation 49
Mt. Sinai School of Medicine 38, 48
National Institutes of Health 14, 18, 40, 55, 57, 63 (NIH)
NIH/National Institute of 40, 53, 55 Environmental Health Sciences
NIH/National Center for Kansas INBRE 18 Research Resources
Maine INBRE 18, 19, 36, 38, 42, 63, 75, 77, 89
National Science Foundation 10, 11, 13, 19, 21, 38, 42, 44, 59, 66, 80, 83, 89
Natural Sciences and Engineering 34, 69, 90 Research Council of Canada (NSERC) Sunnybrook Tesearch Institute 90
United States Fish and Wildlife Foundation 80
University of Arizona 69
Water Research Foundation 59
Wilfreid Laurier University 69