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Features

Author’s Name

Features Green Penn

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Microorganisms: The Answer to the Energy Crisis?

Brian Laidlaw

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Renewable Energy: Achieving Technological Innovation through the Private Sector

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Sanders Chang and Isabel Fan

About PennScience PennScience is a peer reviewed journal of undergraduate research published by the Science and Technology Wing at the University of Pennsylvania.

PennScience is an undergraduate journal that is advised by a board of faculty members.

PennScience presents relevant science features, interviews, and research articles from many disciplines including biologial sciences, chemistry, physics, mathematics, geological sciences, and computer sciences.

PennScience is a SAC funded organization.

For additional information about the journal including submission guidelines, visit http://www.pennscience.org.

Peter H. Kwag

Interviews Wen K. Shieh, Ph.D.

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Cherie Kagan, Ph.D.

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Larry Sneddon, Ph.D.

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Chemical and Biomolecular Engineering

Department of Electrical and Systems Engineering, Department of Materials Science and Engineering

Department of Chemistry

Research Articles Is Individual Variation in Escape Behavior Driven by Differences in Escape Ability in Oophaga pumilio?

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Circulating Endothelial Progenitor Cells and Atherosclerosis in SLE

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Beach Erosion as a Factor in Nest Site Selection by the Leatherback Sea Turtle at Playa Gandoca, Costa Rica

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Julie Charbonnier and Trena Webb

Alexis Sharpe

Matthew J. Spanier

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Editors’ Note Dear PennScience Readers,

As citizens and scientists, environmental issues are increasingly present in our lives. For this reason we chose the theme ‘Green Science’ (including green headers—with the journal now in color) to celebrate environmental science and the development of green technology. This semester PennScience added a section, ‘Features,’ with articles by the new PennScience writing department.

We also present interviews with faculty doing exciting research in renewable energy and energy technology.

As always, we have an exciting collection of student research as well. Julie Charbonnier and Trena Webb discuss the escape behavior of a Costa Rican frog. Alexis Sharpe presents research on the implications of reduced circulating endothelial progenitor cell count in Systemic Lupus Erythematosus patients. Mathew J. Spanier writes about his research on the effect of beach erosion in leatherback turtle nest site selection.

Lastly, we have enjoyed our stay as co-Editors-in-Chief, but we are pleased to introduce AJ Argall and Vishesh Agrawal as the new leadership of PennScience. We hope you enjoy this issue of PennScience, and if you completed research last fall or in previous semesters, we hope you will consider submitting your work for publication in our journal.

Sincerely, Mathew Canver and Evan Daugharthy Co-Editors-in-Chief

Journal Staff Executive Board Editors-In-Chief Matthew C. Canver Evan Daugharthy Editing Managers AJ Argall Zen Liu

Layout Managers Vishesh Agrawal Hijoo Karen Kim

Publicity Manager Srini Sathyanarayanan

Business Managers Ningkun Nancy Li Michael Weintraub Peter H. Kwag

Writing Managers AJ Argall Peter Kwag

Staff Assistant Editing Managers Brian Laidlaw Nikhil Shankar Zhu Wang

Assistant Layout Managers Steven Chen Isabel Fan

Writing Sanders Chang Julia Enberg Isabelle Fan Brian Laidlaw

Website Raghav Puranmalka

Cover Design Maggie Edkins

Faculty Advisors Dr. M. Krimo Bokreta Dr. Jorge Santiago-Aviles

Editing Vishesh Agarwal Sanders Chang Maggie Hu Ming Hu Sarah Johnson Hijoo Kim Peter Kwag Jenny Lin Qinnan Lin Weiren Liu Susan Sheng

Copyright © 2010 PennScience Journal of Undergraduate Research. The authors of the individual research articles published in this journal retain all rights to their work. No part of PennScience Journal of Undergraduate Research may be reprinted, reproduced, or transmitted in any form or by any means without permission in writing from PennScience or the individual authors, whichever is appropriate.

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Features

Chang, S & Fan, I

Green Penn Sanders Chang & Isabel Fan University of Pennsylvania According to the World Commission on Environment and Development, sustainability is defined as “meeting the needs of the present without compromising the ability of future generations to meet their own needs” (1). Though a fairly simple definition, sustainability is no issue to be ignored. With the rise in concern over impending energy shortages and escalating pollution, many institutions across the United States have taken up measures to address sustainability within their communities. Penn is one of these institutions that included sustainability as part of its agenda.

In 2007, Penn joined the American College and University Presidents’ Climate Committee, a coalition of schools devoted to reducing their carbon emissions. From then on, university leaders, faculty, and students have been working arduously to develop ways to promote green energy on campus. In fact, the U.S. Environmental Protection Agency has named Penn as one of the top 25 leaders in energy sustainability (2). To better engage the public, Penn recently released the Climate Action Plan on September 16, 2009, detailing campus initiatives in research, academics, and student affairs (3). From providing reusable bags and water bottles in dining halls to groundbreaking research, Penn is committed to improving the lives of its students and faculty members through the promotion of sustainable energy on campus and beyond the university’s boundaries.

Penn’s various schools have also provided academic opportunities to mold students to be future environmental leaders. The Wharton School of Business has introduced the Environmental Policy and Management concentration and minor as a part of its Initiative for Global Environmental Leadership (4). This concentration focuses on the link between business and the natural environment through a curriculum consisting of courses in Wharton, the College, and the Engineering schools. Within the College’s Department of Earth and Environmental Science is the Environmental Studies major. This academic program is aimed toward shaping students to become well-versed in politics, science, and humanities so that they can tackle environmental issues from a diverse array of standpoints. There are seven concentrations within the Environmental Studies Major that allow students to mold their own environmental ambitions into their curriculum, whether they want to go into environmental research, business, or law (15). The Department of Earth and Environmental Science has also hosted a seminar series every Wednesday of last semester, bringing in Penn professors as well as prominent figures in environmental studies from around the world to lead discussions on current environmental issues with the Penn community.

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In addition to adding to the curriculum, Penn is altering the campus dining menu in accordance with the Climate Action Plan. As part of its newly initiated Farm-to-Institution program with White Dog Community Enterprises, Penn Dining has been purchasing more produce grown on local farms as well as offering more organic food options to students on campus (5). Students are therefore encouraged to make wiser dining choices that not only support a healthier lifestyle but also, in the end, benefit the surrounding environment that they live in. Along with these changes, Penn has begun working toward improving waste management and recycling conditions in dining halls and retail dining places, eliminating trays and takeout plastic bags in exchange for distributing reusable bags and water bottles (6).

“the EPA has named Penn one of the top 25 leaders in energy sustainability” Aside from direct changes made at Penn, there has been much ongoing faculty research in sustainable energy with the Penn Energy Research Group, a coalition of researchers collaborating with other universities, such as Drexel University and the University of Albany, and prestigious national laboratories like the Los Alamos Laboratories to find alternative energy sources. There are four major research sectors of the Penn Energy Research Group. One sector focuses on increasing fuel cell and ceramic sensor performance, while another aims to find more efficient ways to convert solar energy into electrical and fuel means. The Hydrogen Storage sector is currently exploring ways to incorporate hydrogen storage into fuel cells and improve their efficiency. The last sector’s research objective is to optimize the strength and purity of advanced structural materials, particularly steel, and to implement these materials into energy endeavors (13).

The University of Pennsylvania also opens research opportunities to the rest of the community to promote a greener Penn. As part of the Climate Action Plan, Penn has created the Green Fund to award research grants provided by the Division of Facilities and Real Estate Services and the Office of the Provost. The Green Fund welcomes anyone with innovative ideas to reduce Penn’s carbon footprint and make Penn a more environmentally-friendly campus (14).

Research at Penn has also gone hand in hand with the task of redesigning buildings to minimize pollution and energy consumption on campus. Starting last fall, buildings such as Kings Court College House and the Radian apartment complex have begun installing green roofs comprised of various greenery and grasses to help cool and insulate buildings as well as provide fertile habitats for birds and insects. Green roofs also serve an important role in slowing down the rate of rain water entering Penn’s

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Chang, S & Fan, I sewage system via the roofs, minimizing the chances of flooding and pollution of rivers by untreated waste (7).

Along with academics and research, student involvement is integral in the promotion of sustainability at Penn. Various student groups on campus have sought out ways to encourage students to get more in touch with the environment.

Penn’s Undergraduate Assembly is one group making progress in addressing Penn’s sustainability initiative by forming the Housing, Sustainability and Facilities Committee. This new committee is primarily focused on carrying out and raising awareness on initiatives proposed in the Climate Action Plan. This ultimately gives students a chance to not only work directly with the university on environmental issues, but also to gain valuable leadership experience in this field (8). Along with the Undergraduate Assembly, Penn’s chapter of Engineers Without Borders has provided students with many opportunities to directly address environmental is-

“Research has gone hand in hand with the task of redesigning buildings to reduce pollution and energy consumption.”

sues affecting the city of Philadelphia as well as the world.

Opportunities range from reaching out and educating local high schools in sustainable development (9) to implementing hands-on projects in developing communities around the world (10). When asked about ways to further increase student awareness of environmental issues, PennEWB president Alex Yen comments, “We are currently working with the Sustainability Team here at Penn to get members of Engineers Without Borders involved in more initiatives -- research, speaking events, building projects on campus to name a few. That said, I think we can still make an effort to increase Penn’s awareness about the merits of a sustainable environment [through] university-supported programs and coursework, such as PennEWB’s sustainable development implementations abroad.” Along with PennEWB, the Penn Environmental Group is aimed at directly impacting Penn’s campus and community through on-going projects that any Penn student can join and participate in. One beneficial project is the Light Bulb Exchange, which, since its inception in 2007, has significantly helped reduce university energy costs by replacing over 1000 incandescent light bulbs with compact fluorescent light bulbs (11). The Penn Environmental Group has also initiated the Green Acorn Business Certification Program, which aims at encouraging local businesses to adopt greener business operations for the purpose of reducing costs, improving efficiency, and making Philadelphia one step closer toward reaching its goals in sustainability (12).

Penn has proven to make its mark on the national and international spheres; it continues to make great strides

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in promoting environmental sustainability through academics, research, student affairs, and more direct means.

With all the progress and achievements Penn has made in various areas, students, regardless of what school they are in, can further contribute to this Green Campus Initiative, whether by simply recycling or striving to use their Penn education to make a global environmental impact.

References 1. “Basic Information.” Sustainability - US EPA. 24 Aug 2009. U.S. Environmental Protection Agency, Web. 7 Nov 2009. <http://www. epa.gov/Sustainability/index.htm>.

2. “Penn is only university to earn ranking in EPA’s list of nation’s top 25 green-energy leaders.” Penn Current. 16 Jun 2009. Penn Current, Web. 1 Nov 2009. <http://www.upenn.edu/pennnews/current/latestnews/061609-2.html>.

3. “Climate Action Plan.” Penn Green Campus Partnership. 2009.

University of Pennsylvania, Web. 1 Nov 2009. <http://www.upenn. edu/sustainability/cap.html>.

4. “Learning Sustainability - Learning Sustainability at Penn.” Penn Green Campus Partnership. 2009. University of Pennsylvania, Web. 1 Nov 2009. <www.upenn.edu/sustainability/academics.html>.

5. “Local Foods - Local Foods Initiatives.” Penn Green Campus Partnership. 2009. University of Pennsylvania, Web. 1 Nov 2009. <http:// www.upenn.edu/sustainability/food.html>.

6. “Minimizing Waste - Waste Minimization and Recycling at Penn.”

Penn Green Campus Partnership. 2009. University of Pennsylvania, Web. 1 Nov 2009. <www.upenn.edu/sustainability/waste.html>.

7. “Designing Green - The Physical Environment at Penn.” Penn Green Campus Partnership. 2009. University of Pennsylvania, Web. 1 Nov 2009. <www.upenn.edu/sustainability/environment.html>.

8. Sanchez, Dan. “Housing, Sustainability, and Facilities.” Undergraduate Assembly - UPenn. 30 Sep 2009. Penn Undergraduate Assembly, Web. 7 Nov 2009. <pennua.org/category/hsf/>.

9. “Local Outreach Projects.” University of Pennsylvania Chapter Penn Engineers Without Borders. 2009. PennEWB, Web. 7 Nov 2009. <http://www.pennewb.org/local.php>.

10. “International Projects.” University of Pennsylvania Chapter Penn Engineers Without Borders. 2009. PennEWB, Web. 7 Nov 2009. <http://www.pennewb.org/international.php>.

11. “Projects - Lightbulb Exchange” Penn Environmental Group University of Pennsylvania. 2009. Penn Environmental Group, Web. 7 Nov 2009. <www.dolphin.upenn.edu/pennenv/projects.html>.

12. “Penn Environmental Group (PEG) - Green Acorn Business Certification” Penn Environmental Group - University of Pennsylvania.

2009. Penn Environmental Group, Web. 7 Nov 2009. <http://www. dolphin.upenn.edu/pennenv/Green%20Acorn/Green%20Acorn%20 Program%20Overview.pdf>.

13. “Research.” Energy Research Group at Penn. 2009. Energy Research Group at Penn, Web. 1 Nov 2009. <http://www.energy. upenn.edu/research.html>.

14. “Penn Green Fund.” Penn Green Campus Partnership. 2009. University of Pennsylvania, Web. 1 Nov 2009. <http://www.upenn.edu/ sustainability/greenfund.html>.

15. “Environmental Policy and Management.” 2009. Wharton School of Business, Web. 1 Nov 2009. <http://spike.wharton.upenn.edu/ ugrprogram/advising/concentrations/environmental.cfm>.

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Microorganisms: The Answer to the Energy Crisis?

Brian Laidlaw University of Pennsylvania The problem of the energy crisis is one that grows larger and more apparent with each passing year. As new studies detailing the extent of the crisis are released and the international demand for fuels continues to increase it is clear that this problem is here to stay. In fact, as growing industrial powers, such as China and India require more energy to run their economies, the energy problem is likely to get worse. When you consider that fossil fuels are a limited resource requiring millions of years to create, it is inevitable that there will come a time when the supply of fossil fuels is no longer able to meet the demand. Already, we are being forced to look to ever more exotic locales to find the remaining deposits of fossil fuels, which come with an added cost in time and money to extract-a cost that is passed on to the consumer. In addition, the countries with control over these sources wield a disproportionate amount of power compared to other countries. This puts our national security in question. Making matters worse, the use of these fossil fuels releases vast amounts of greenhouse gases into the atmosphere, which in turn can damage the environment and speed up the process of global warming (3).

Considering all of these issues, it is no surprise that many are looking towards alternative energy sources to help alleviate the problem. The use of biofuels, specifically the production of bioethanol, is one of the more promising avenues of alternative energy research (2). The production of bioethanol from cellulosic biomass-plant biomass coming from wood residues, paper waste, agricultural residues, or energy crops-offers a number of advantages over other alternative energy strategies such as wind and solar power (1). For instance, the cellulosic biomass needed for bioethanol production is one of the primary renewable resources on the planet. It is widely available throughout the world, with a significant portion found in the United States, reducing America’s reliance on foreign energy sources. Bioethanol production also emits almost no green house gases and has a decisively positive energy balance, making it an environmentally-friendly energy source. In addition, ethanol is a liquid fuel, ideal for the storage and transport of energy. Furthermore, it is a clean burning fuel with a favorable energy input to output ratio (2). Thus, bioethanol is a very promising candidate for the fuel of the future.

Despite all of these advantages there are still a number

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of economic and technological barriers to be overcome before bioethanol can feasibly be produced on an industrial scale. These issues include finding enough land to produce substantial amounts of cellulosic biomass with a high land fuel yield, managing this land efficiently to minimize any environmental abuse, and adapting our technologies for use with bioethanol. However, the primary problem associated with bioethanol production is finding a way to increase the cost effectiveness of this process so that it can be used on a large scale (5). With this is in mind, a large amount of research is being conducted to find a way to optimize bioethanol production efficiency by improving the conversion of biomass to sugars, and sugars to fuel. Much effort is also being put into streamlining the four main steps of the production process: 1) Pretreatment, 2) Enzymatic hydrolysis, 3) Fermentation, and 4) Ethanol purification (4).

“we are being forced to look to ever more exotic locales to find remaining deposits of fossil fuels” While improving the conventional bioethanol production process in the ways already mentioned may one day create a cost-effective process, progress is slow with improvements occurring only incrementally. However, some cutting-edge scientists are using novel techniques to reduce the number of steps in this production process to one. With only a single step required these new techniques have the potential to be extremely economical and workable on an industrial scale. This one-step process, widely known as consolidated bioprocessing (CBP), employs engineered microorganisms to convert biomass to biofuels without the need to use any additional costly enzymes (1). In a recent Forbes article, biofuels expert Helena Chum of the National Renewable Energy Laboratory in Golden, Colorado, commented on CBP, saying “This is the golden dream. All of the processes in one super-organism. That would be the lowest cost possible.” A prominent DOE/USDA research agenda also states that “CBP is widely considered to be the ultimate low-cost configuration for cellulose hydrolysis and fermentation” (8).

The conditions needed for industrial conversion of biomass to biofuels differs greatly from those found in native biomes. This makes it very unlikely that a microorganism capable of CBP will be found in any native population, and thus metabolic engineering is required to create such an organism (7). Currently there are two approaches to carrying out the functions needed for CBP (1). The first approach involves the improvement of native, isolated microorganisms that already have some of the function needed for CBP. By optimizing the function of these native microorganisms, many common problems associated with creating new functions are eliminated. For instance, a pathway that

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Laidlaw, B might have one function in a certain organism can have a completely different function in another host, since this host may have different environmental needs and metabolic functions than the original organism. Also, the importation of genes can at times reduce the capabilities of recombinant organisms in comparison with native ones (7).

Thus, exploiting the natural capacity of microorganisms is a very attractive option in creating a CBP microorganism.

However, since no organism has all the functions needed to achieve CBP, it is necessary to employ the second approach for metabolically engineering microorganisms: importing biosynthetic function. This pathway allows for the creation of microorganisms that can be employed on an industrial scale, since the most efficient pathways for breaking down biomass into ethanol can be introduced into the host microorganism of the scientist’s choice. If robust genes and pathways, which have similar functions in different hosts, can be found, the introduction of desired functions into host microorganisms can be a very powerful tool in the creation of a CBP organism (9).

Utilizing these two alternative approaches in a way that maximizes the efficiency of the engineered microorganism is not the only problem currently facing scientists. One of the biggest issues associated with CBP is whether a consortia or single organism should be used for bioprocessing. The

“With only a single step required new techniques have the potential to be extremely economical” single-organism solution is being heavily researched due to the bioprocessing simplicity of only using a single organism avoiding the stability problems associated with the use of a mixed culture of microorganisms. However, current evidence indicates a single organism is incapable of outcompeting a consortium of microorganisms and that a mixed culture is capable of higher ethanol yields and faster consumption of sugars. The consortium approach to CBP also allows a wider range of operations to be achieved more efficiently then a lone microorganism would allow.

So a culture of several microorganisms together that can breakdown cellulosic biomass and convert all sugars in a single step may represent the best hope for efficient CBP (10).

Despite the great potential of consolidated bioprocessing there are still a number of hurdles that must be overcome before it becomes technologically feasible. The primary challenge is the creation of microorganisms that are sufficiently efficient at CBP to be cost effective on an industrial scale. Another area of concern is that the number of functions that can be consolidated in a single organism may be limited. A delicate balance needs to be achieved between creating a microorganism that can efficiently

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New types of recombinant bacteria (such as these cellulase producing streptomycetes) are needed to address the bottleneck in conversion of cellulosic biomass into sugars for fermentation (3)

perform CBP while avoiding the adverse effects that can come with engineering an organism (10). Also the temperatures required for the optimum breakdown of biomass and ethanol production for CBP are very different, which can affect the overall rate of the reaction and have an adverse effect on the yield (6). These issues, among others, were thought to leave the dream of CBP well in the future.

However, recent breakthroughs may make this future much closer than once thought possible. Mascoma Corporation, a biotechnology company founded by Dartmouth faculty Charles Wyman and Lee Lynd, recently presented evidence of several key discoveries that demonstrate the capabilities of CBP. Using thermophiles, bacteria that grow at high temperatures, they were able to increase the concentration of ethanol produced per unit volume by 60% from the amount possible a year ago. Mascoma also created thermophiles that could break down cellulosic biomass with a minimum of side products, even in the presence of commercial levels of ethanol. In addition, Mascoma reported the creation of recombinant cellulolytic yeast with a 3000% increase in production of cellulose, which is used for breaking down biomass. This yeast also was able to convert pretreated hardwood to ethanol with a 2.5 fold reduction in added cellulose and waste paper sludge to ethanol with no added cellulose. The ability to break down forms of plant biomass such as hardwood and paper sludge using little to no additional enzymes is vital in making CBP an economically feasible process, as enzymes can often be one of the more expensive elements of the biomass to biofuel process (8).

Of these innovations Bruce Dale, faculty at the University of Michigan State and editor of the journal Biofuels, Bioproducts, and Biorefineries, said “This is a true breakthrough that takes us much, much closer to billions of gallons of low cost cellulosic biofuels. Many had thought

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Laidlaw, B 5. Shaw, A.J., K. Podkaminer, S.G. Desai, J.S. Bardsley, S.R. Rogers, P.G. Thorne, D.A. Hogsett, L.R. Lynd. 2008. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. PNAS. 105:13769-13774 6. Wilson D.B. 2009. Cellulases and Biofuels. Current Opinion In Biotechnology, 20:295-299 7. Xu, Q.; Singh, A.; Himmel, M. E. 2009. Perspectives and New Directions for the Production of Bioethanol Using Consolidated Bioprocessing of Lignocellulose. Current Opinion in Biotechnology.

20:364-371 8. Mascoma Corporation. 7 May 2009. Mascoma Announces Major Cellulosic Biofuel Technology Breakthrough. < www.masoma.com>

Mascomaâ&#x20AC;&#x2122;s pilot cellulosic ethanol plant in Rome, NY

that CBP was years or even decades away, but the future just arrived.â&#x20AC;? So the day we can switch from fossil fuels to a clean burning, renewable energy source may be closer than we all think. Consolidated bioprocessing and the microorganisms that it employs just may prove to be the answer to the specter of the impending Energy Crisis (8).

9. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev. 66:506-577.

10. Alper A, Stephanopoulos. 2009. Engineering for Biofuels: exploiting innate microbial capacity or importing biosynthetic potential. Nature Reviews Microbiology. 7(11):715-723.

References 1. Lynd, L.R., W.H. van Zyl, J.E. McBride, M. Laser. 2005. Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16:577-583.

2. Lynd, L.R. 1996. Overview and evaluation of fuel ethanol from cellulosic biomass: Technology, economics, the environment, and policy. Ann. Rev. Energy Environ. 21:403-465.

3. Lynd, L.R., M.S. Laser, D. Bransby, B.E. Dale, B. Davison, R. Hamilton, M. Himmel, M. Keller, J. D. McMillan, J. Sheehan, C.E. Wyman.

2008. How biotech can transform biofuels. Nature Biotechnology 26:169-172 4. Antonie Margeot, Barbel Hahn-Hagerdal, Maria Edlund, Rapheal slade, Frederic Monot, 2009, New improvements for lignocellulosic ethanol, Current Opinion In Biotechnology, 20:372-380

Informing and supporting students interested in conducting research and applying for competitive fellowships.

http://www.upenn.edu/curf/

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Renewable Energy: Achieving Technological Innovation through the Private Sector Peter H. Kwag University of Pennsylvania In order to stimulate investment and promote the utilization of renewable energy technology within the private sector, many countries around the world have implemented a wide range of financial incentives. Federal and state initiatives within the United States usually take the form of tax subsidies, but also include loan guarantees and rebates to make renewable energy more appealing in comparison to traditional sources of energy. The broad term, “renewable energy,” comprises a diverse array of technological advancements which includes solar energy, wind energy, geothermal energy, hydroelectricity and various types of biofuel and biomass. In the United States, the utilization of renewable energy for the generation of electricity has risen significantly in recent years. Much of this remarkable growth has been attributable to increased interest within the private sector, among both personal and corporate entities.

The Energy Policy Act of 2005 (6) was passed by Congress on July 29, 2005. It was subsequently signed into law

by President George W. Bush on August 8, 2005. It included provisions that authorized loan guarantees for qualified investments in renewable energy facilities, in addition to other systems that improve energy efficiency (12). In addition, it mandated that the Department of Energy and the Environmental Protection Agency initiate the Energy Star Program in order to promote energy conservation within the private sector (7). This federal legislation also established the Residential Energy Efficient Property Credit (1), a personal tax subsidy. Under this statute, taxpayers are able to claim a 30% credit for renewable energy equipment placed in service within residential buildings (10).

The Energy Improvement and Extension Act of 2008 (14) was enacted on October 3, 2008, and constituted Division B of Public Law 110-343. It was passed in conjunction with the Emergency Economic Stabilization Act of 2008 (Division A) and the Tax Extenders and Alternative Minimum Tax Relief Act of 2008 (Division C). Under this federal legislation, the Residential Energy Efficient Property Credit received an eight year extension (16). The Energy Improvement and Extension Act of 2008 also enabled taxpayers to claim the credit against the alternative minimum tax (15).

The estimated cost of the extension of the Residential En-

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ergy Efficient Property Credit is $1.294 billion over 10 years (18). Another important provision of this act was the authorization of up to $800 million of Clean Renewable Energy Bonds (17). Proceeds from the issuance of these bonds are utilized to finance qualified renewable energy projects.

The American Recovery and Reinvestment Act of 2009 (19) further enhanced previous initiatives aimed at increasing private investment in renewable energy technology. It effectively removed the Residential Energy Efficient Property Credit limit for all eligible renewable energy systems installed on residential property after 2008, with the exception of fuel cell technology (21). The maximum tax credit an individual may claim on fuel cell equipment is contingent on electrical generation capacity, and is limited to $500 per 0.5 kW. With the credit limit removed for most categories of renewable energy technology, private consumers would be more likely to make larger equipment purchases in order to receive the additional tax benefit.

There have also been a number of federal initiatives aimed directly at encouraging corporate entities to utilize renewable energy technology. Under the Modified Accelerated Cost Recovery System (4), many types of renewable energy equipment are classified under property lives of five or seven years. This allows businesses to record depreciation expenses at a faster rate, and allows them to recover a portion of the equipment cost more quickly through tax deductions. Qualifying types of renewable energy equip-

Source: Energy Information Administration “Updated Annual Energy Outlook 2009 Reference Case Service Report”

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ment include solar electric, solar thermal, fuel cell, geothermal and wind energy systems. With the passage of the Economic Stimulus Act of 2008 (13), Congress authorized businesses to take an additional 50% in depreciation expenses (4) for eligible equipment. The allowance for additional depreciation expenses was further extended through the American Recovery and Reinvestment Act of 2009 (22).

Corporations may also claim investment tax credit for eligible expenditures associated with renewable energy technology (3). Businesses are able to claim a 30% tax credit for investments made in solar, wind and fuel cell technology. They are also able to claim a 10% tax credit for expenditures on geothermal, microturbine and combined heat and power systems. The Energy Improvement and Extension Act of 2008 extended the investment tax credit for an additional eight years, and also gave corporations the ability to take the credit against the alternative minimum tax (15). It was then further extended by the American Recovery and Reinvestment Act of 2009, which also removed the limitation on the use of tax credits for projects receiving assistance from subsidized energy financing (20).

Another key incentive that spurs private investment in renewable energy technology is the ability to transfer excess electricity back into the power grid. Corporations may claim production tax credit for generating surplus electricity from renewable sources of energy (2). This tax subsidy was originally established under the Energy Policy Act of 1992 (5), and has been subsequently modified and extended. Currently, businesses may receive approximately 2.1¢ per kWh of electricity generated through wind, geothermal and closedloop biomass technology. For all other eligible renewable energy systems, businesses may receive approximately 1.1¢.

Both of these figures have been adjusted for inflation since 1993, when the credit value was first established. The pro-

duction tax credit encourages companies not only to generate enough energy for their own operations, but incentivizes them to generate additional electricity for the power grid.

“Much of this remarkable growth has been attributable to increased interest within the private sector, among both personal and corporate entities.”

Private residences also possess the ability to sell energy to the power grid through net metering, and are eligible to receive compensation in retail credit. The Energy Policy Act of 2005 mandated that all public electric utilities offer net metering to their customers upon request (8). The Energy Policy Act of 2005 also extended the production tax credit until 2008 and increased the credit period from 5 to 10 years (9). As a result, private investment in wind energy facilities rose precipitously in the following years. Between 2005 and 2007, U.S. wind power capacity increased by 8,438 MW (23). As a result, cumulative wind power capacity additions within the U.S. more than doubled from under 8,000 MW to about 16,000 MW during this two year period. Such a dramatic increase conveys the importance of financial incentives in attracting investment from the private sector. According to the Energy Information Administration, it has been estimated that production tax credit may reduce the cost of wind energy by about one-third. In addition to extending the production tax credit, the Energy Policy Act of 2005 also broadened existing tax subsidies for research expenses incurred through contractual payments made to small businesses, federal laboratories and universities (11). This measure serves to lower the costs associated with research and development, which may ultimately translate into a higher rate of technological advancement.

The renewable energy industry is rapidly expanding, fueled by both private investment and publicly-funded scientific innovation. The financial incentives currently in place significantly lower the costs realized directly by the private sector, increasing the potential return on investments in renewable energy technology. According to the Energy Information Administration, an independent agency within the Department of Energy, domestic electricity generation from non-hydroelectric renewable sources is projected to rise from approximately 100 billion kWh in 2007 to over 400 billion kWh by 2030 (24). In 2007, renewable energy sources accounted for about 9% of domestic electricity production. By 2030, this figure is projected to increase to approximately 16%. The majority of this growth will be atSource: Energy Information Administration “Federal Financial tributable to the increased utilization of non-hydroelectric Interventions and Subsidies in Energy Markets 2007” (4/08) renewable energy sources including solar, wind, biomass

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Kwag, P H and geothermal energy. Increased interest within the private sector will continue to facilitate and drive this trend in the future. Federal subsidies for renewable energy amounted to approximately $4.9 billion in fiscal year 2007, which was substantially higher than any other energy category (23). Many advocates view renewable energy technology as a fundamental step toward achieving energy independence and security. Because renewable sources of energy also have a low carbon footprint, unlocking their potential may also be essential to ensuring environmental conservation and impeding the progression of climate change.

References 1. United States Code. Title 26, Subtitle A, Chapter 1, Subchapter A, Part IV, Subpart A, Section 25D- Residential Energy Efficient Property. January 8, 2008.

2. United States Code. Title 26, Subtitle A, Chapter 1, Subchapter A, Part IV, Subpart D, Section 45- Electricity Produced from Certain Renewable Resources. January 8, 2008.

3. United States Code. Title 26, Subtitle A, Chapter 1, Subchapter A, Part IV, Subpart E, Section 48- Energy Credit. January 8, 2008.

4. United States Code. Title 26, Subtitle A, Chapter 1, Subchapter B, Part VI, Section 168- Accelerated Cost Recovery System. January 8, 2008.

5. Energy Policy Act of 1992. Public Law 102-486. House Resolution 776. October 24, 1992.

6. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. August 8, 2005.

7. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title I, Subtitle C, Section 131- Energy Star Program. August 8, 2005.

8. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title XII, Subtitle E, Section 1251- Net Metering and Additional Standards. August 8, 2005.

9. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title XIII, Subtitle A, Section 1301- Extension and Modification of Renewable Electricity Production Credit. August 8, 2005.

10. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title XIII, Subtitle C, Section 1335- Credit for Residential Energy Efficient Property. August 8, 2005.

11. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title XIII, Subtitle E, Section 1351- Expansion of Research Credit.

August 8, 2005.

12. Energy Policy Act of 2005. Public Law 109-58. House Resolution 6. Title XVII, Section 1703- Eligible Projects. August 8, 2005.

13. Economic Stimulus Act of 2008. Public Law 110-185. House Resolution 5140. February 13, 2008.

14. Energy Improvement and Extension Act of 2008. Public Law 110-343. House Resolution 1424. Division B. October 3, 2008.

15. Energy Improvement and Extension Act of 2008. Public Law 110-343. House Resolution 1424. Division B, Title I, Subtitle A, Section 103- Energy Credit. October 3, 2008.

16. Energy Improvement and Extension Act of 2008. Public Law 110-343. House Resolution 1424. Division B, Title I, Subtitle A, Section 106- Credit for Residential Energy Efficient Property. October 3, 2008.

17. Energy Improvement and Extension Act of 2008. Public Law 110-343. House Resolution 1424. Division B, Title I, Subtitle A, Section 107- New Clean Renewable Energy Bonds. October 3, 2008.

18. United States Senate Committee on Finance Report- The Energy Improvement and Extension Act of 2008. Long-term Extension and Modification of the Residential Energy Efficient Property Credit.

September 17, 2008.

19. American Recovery and Reinvestment Act of 2009. Public Law 111-5. House Resolution 1. February 17, 2009.

20. American Recovery and Reinvestment Act of 2009. Public Law 111-5. House Resolution 1. Division B, Title I, Subtitle B, Part I, Section 1103- Repeal of Certain Limitations on Credit for Renewable Energy Property. February 17, 2009.

21. American Recovery and Reinvestment Act of 2009. Public Law 111-5. House Resolution 1. Division B, Title I, Subtitle B, Part III, Section 1122- Modification of Credit for Residential Energy Efficient Property. February 17, 2009.

22. American Recovery and Reinvestment Act of 2009. Public Law 111-5. House Resolution 1. Division B, Title I, Subtitle C, Part I, Section 1201- Special Allowance for Certain Property Acquired During 2009. February 17, 2009.

23. United States Department of Energy- Energy Information Administration. Office of Coal, Nuclear, Electric, and Alternate Fuels.

Federal Financial Interventions and Subsidies in Energy Markets 2007. April 2008.

24. United States Department of Energy- Energy Information Administration. National Energy Information Center. Annual Energy Outlook 2009 with Projections to 2030. March 2009.

< ! - - S T Wing - - > The Science and Technology Wing at the University of Pennsylvania A project-based community, pursuing a wide variety of research projects for formal academic credit or personal interest, hosting dinner discussions with University faculty and staff, and holding social events throughout the year.

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Vol 8 Issue 2, Spring 2010

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Interviews

Wen K. Shieh, Ph.D. Dr. Wen K. Shieh is a Professor of Chemical and Biomolecular Engineering at the University of Pennsylvania. His research interests focus on the production of liquid and gaseous biofuels from waste materials. He has developed the biofluidized bed technology to support fuel conversion at high rates. The biofluidized bed technology developed in his laboratory has recently been adopted in Finland to biologically oxidize ferrous ion to ferric ion to facilitate the bioleaching process at mine sites.

What are your thoughts on renewable energy?

How do you define bioenvironmental engineering?

Well, to answer that, let me begin by talking about my research interests. I am in the process of working with people from the veterinary medicine school. We’ve already submitted a proposal to produce bio-gas from the animal waste produced. Basically, instead of just dumping the waste on the ground, we want to try to collect the animal waste, put it in a facility, and produce this bio-gas. When I talk about “producing bio-gas,” I mean that we use a microbial process to convert the organic matter in the waste into methane gas and CO2. Usually in the bio-gas, 65-70% of the composition is methane gas and CO2. Once we produce the methane gas, we collect it and try to produce electricity via the gas/steam on site. By doing so, not only do we solve the energy problems for the farmers (to some extent), we are also helping to solve the waste problem. This is a one stone, two birds approach.

When you are doing this kind of energy conversion (renewable energy production), we recycle some of the carbons through the system. Based on the carbon intensity, if we can use the energy produced from the methane gas to replace the energy you produce from the fossil fuel power plants, we calculate that we can probably recycle as much as 4050% of the spent carbon. This amount would otherwise be released into the atmosphere. By recycling, we can create a loop and maintain energy in the system. This is one of the ways we’re looking at the alternative energy source problem.

This is only usable to solve the local problems, not the national problems. Bio-gas production is a very economical way to deal with animal waste problems and energy deficiencies in Pennsylvania. It’s hard to use it elsewhere because we don’t necessarily have enough waste. Pennsylvania is one of the largest agricultural states in the country.

If you go to other places you don’t have enough waste being produced so the rich organic matter cannot even be used.

To produce alternative energy you have to worry about how much you can produce, and also, the economy. For our research we are trying to solve the environmental problem while also considering the appeal to policymakers.

The discipline looks into the environmental problems and tries to design biological processes to solve these problems. For instance, my bio-gas production research is one such example. To produce the bio-gas we need to select specific microbial species as the active agents that perform the conversion reactions. We can also use microbial species to treat wastewater, thereby removing pollutants in the wastewater. We try to select and enrich the microbial species and incorporate them into the engineering design. In this manner we can make them do whatever we want them to do.

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“The discipline looks at environmental problems and tries to design biological processes to solve these problems.”

What do you think is your most exciting project?

Well, beyond the environmental aspect, I am excited about the money that can be saved with my approaches.

Waste treatment, traditionally speaking, is not a profitmaking enterprise. In this country if you want to treat waste water or dispose of garbage, the whole operation is supported by taxes. Revenues need to come from taxes. But now, we’re trying to design alternative approaches which still solve the environmental problem, but also allow us to produce something that deflates the cost. We’re just talking about a 20, 30, or 40% lowering of the costs, not 100%!

And then, if and when we succeed, we do two things at the same time. By looking at the economics this way, the option becomes more attractive to policymakers. We can answer “No!” when policymakers ask, “Do we have to increase taxes?” So this is a more convincing argument for them.

Once again, the ultimate goal is to do good things.

It is also cost-effective. Bio-gas production is just one of many examples; you can still do other things, such as reusing the waste water. You can use the clean wa-

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Interviews ter for certain applications. You want to try to reduce the cost as much as you can without sacrificing efficiency.

How does your research affect everyday people?

Producing bio-gas will reduce the burden of the farmer. Their operation already is not that profitable. They have to purchase the fertilizer seed and the electricity to sustain the operation. If you can recycle something back, that will make the operation better.

As to how expensive our procedure would be: we’re trying to figure that out now. We want to design the system such that it is highly efficient and robust, but not expensive. Of course nobody will use an expensive system, even if its efficiency is really high. We need a tradeoff.

We’re also looking into other possibilities. For instance, we’re talking to the state and they’re considering our proposal. Basically, we’re looking into the possibility of mixing the food waste produced in the city with the animal waste; we can then use it to produce usable things such as mulch. We submitted a proposal to do a feasibility study here in Philadelphia. We do want to try and use the food waste from the dining services in different dormitories. We want to see if we can use that with animal waste to produce a residual bio-gas, or compost, or mulch. Maybe we can then give it to citizens in the city, for gardening or landscaping. So those kinds of things, even though we start from the small scale, may have amazing applications if we can successfully promote them.

“It’s easy to get students involved!

We should encourage undergraduate students to participate in this kind of activity as early as possible, so they can practice something they learn from the textbook.”

Does your work involve undergrads? How can they get involved?

Well, if they’re interested in environmental topics like reusable energy or wastewater, I usually talk to the student and find some ideas they may have. If we can come to a mutual agreement, then we can always arrange a project through independent study or through summer research. It’s easy to get students involved! We should encourage undergraduate students to participate in this kind of activity as early as possible, so they can practice something they learn from the textbook.

For instance, I have a student working in my lab from last summer, trying to figure out how to remove some nitrogenous compounds from waste water (the drinking water) to make it cleaner. After we remove those compounds we can discharge it as purer water. We’re trying

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to design different reactive systems to accomplish this.

What is your long-term goal?

I’d like to see my work in the laboratory finished and I’d really like to see it applied in practice. Nothing will work if it isn’t applied! People can see it and experience it and then share the benefit, and only at that time can you convince people that this is the right way to go. Things you do in the laboratory are fine, but they always have to be applied in practice, so people can see and benefit from what we have done. I’m excited about the bio-gas because if it is successful, the farmers will benefit from that tremendously.

Do you have any words of wisdom for those interested in pursuing bioenvironmental engineering?

Well, the most important thing is that you have to enjoy it. Don’t force yourself. Convince yourself that this is the right thing to do. The discipline contributes to human society in a very reliable way and you will feel proud of yourself. Ask yourself, do you really want to participate and devote your professional life to bioenvironmental techniques?

What other fields need attention in bioenvironmental engineering?

Water is one of the most urgent issues. This might not be true in this country, but it certainly is in other ones. Two billion people in the world have no access to clean drinking water! As long as you can provide safe, clean drinking water, very often you can eliminate water-borne diseases by as much as 80%. You don’t even have to resort to medication; if people simply have access to clean drinking water, the probability of contracting water-borne diseases is reduced tremendously.

Even in this country we still have the episodes of E. coli and Salmonella poisonings and things like that, even with our modern infrastructure. We still have to do something to safeguard the quality of our drinking water. So, water is obviously one of the areas that still requires much attention.

The other topic of interest is energy, but the topic is so broad. The things we are doing right now only address a small portion. If we can address the regional issues, that’s adequate. We don’t have to come up with solutions for the entire world. That’s not the purpose; that is too broad. We can afford to focus on small, local effective solutions for now, and hopefully we will find broader solutions as time goes on.

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Vol 8 Issue 2, Spring 2010

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Interviews

Cherie Kagan, Ph.D. Cherie Kagan is an Associate Professor in the Department of Electrical and Systems Engineering and the Department of Materials Science and Engineering. She is co-Director The Penn Center for Energy Innovation and Director of the Penn’s Wolf Nanofabrication facility. She earned both a B.S.E. in Material Science and Engineering and a B.A. in Mathematics from the University of Pennsylvania in 1991, and received her Ph.D. in Electronic Materials from Massachusetts Institute of Technology in 1996. Later, she went to Bell Laboratories as a Postdoctoral Fellow. Kagan joined the faculty of the University of Pennsylvania in January of 2007.

What is the focus of your current research?

My current research is at the interface between chemistry and material science and electrical engineering. We work to understand very fundamental questions about the physics and chemistry of materials and their integration into devices. We are interested in questions of how electrons and energy flow in materials and how we can take advantage of those properties in making different types of devices, including electronics, photovoltaics, and photonics. We think about new materials as opposed to the traditional ones; how we might build them using cheap methods, and how we can fabricate them on materials like plastic, but also optimize their properties for different applications.

But we think about very intriguing fundamental questions and then take them all the way to potential applications.

For example, we are interested in how photons and electrons move in nanorods and nanowires, and how that can be taken advantage of in devices. If we are working on transistors, we ask how well charges move through very small devices and can we make them move faster? Can we use new materials, can we deposit them on things like plastics? And can we use them to make things like electronics that are fast yet flexible? For example, we want to make good solar cells from these flexible electronics. They will be low enough in cost to make solar photovoltaic panels one day competitive with more traditional sources.

What interested you in this kind of research?

I was a materials science major so I guess I was always very interested in the intersection between physics and chemistry. I have always incorporated combinations of science and engineering into my research. So I think it’s very interesting to go all the way from thinking about how to take advantage of physical phenomena to applying them to devices.

What is the goal of your research?

Because we go from fundamental questions to their applications, there are some overarching questions that we can shed light on to open new directions. We wish to bring something new. We also hope to understand the behavior of charge

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and energy and our ability to manipulate them in materials.

We also hope to harness systems to make novel devices, for either electronics or photonics, that will offer us something that is low in cost but high performing to be competitive and transform the way we use energy. So it would nice to be able to make a significant contribution to help alternative energy play a more significant role in the way we look at energy.

“we think about ways to build devices that lessen the demand for energy” What are some of the impacts of your research?

We hope that we can make a number of impacts that will change how we supply energy, and develop new materials that will impact how we make, process, and define architectures for devices. This will provide us with low cost competitive alternative energy, such as photovoltaics. We also hope to provide new directions towards energy efficiency, where we think about ways to build devices that lessen the demand for energy. We also think about smart designs of devices where we try to think about ways to operate quickly but at much lower power.

What is the future of these devices?

They each have their niche applications; some molecular devices are potentially making their way into the market.

There are potential challenges but there are certainly exciting opportunities for each kind of material. For example, some molecular systems have challenges in terms of stability depending on the application in which they are used.

What is the stability of these materials when subjected to light? The conditions in which the systems are used always matter. In reality some devices probably will make it into the market and some won’t. But I think there are a number of opportunities to make exciting devices. For society, they offer new opportunities for low cost electronics. They also offer new opportunities for health care, energy, and security, as well as an appreciation for new technology.

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Larry Sneddon, Ph.D. Larry Sneddon is a Blanchard Professor of Chemistry and a leading inorganic and materials chemist. He received his PhD from Indiana University in 1973 and completed post doctoral fellowships at the University of Virginia and the Massachusetts Institute of Technology prior to his arrival at Penn in 1974. Dr. Sneddon has coauthorized nearly 200 publications and 10 patents and has served as a longtime member of the Laboratory for Research on the Structure of Matter.

Why are people interested in hydrogen fuel?

The reason why people are interested in hydrogen is because beginning in the 1950â&#x20AC;&#x2122;s the production of domestic energy was not keeping up with consumption. Today there is a big difference between production and consumption.

Projections to the future show that the use of petroleum will continue to increase with the biggest use of petroleum being transportation. We now import 60% of our oil and this number is projected to go up to about 68% by 2025. This is a cost issue for us but also a national security issue because we need to have a stable supply of energy that does not depend on imports from the rest of the world. That is why in 2003 President Bush announced the Presidential Hydrogen Fuel Initiative. The key goals of the initiative were to develop new technology for the production, storage and distribution of hydrogen and to make hydrogen fuel cells competitive by 2020.

Cars today burn gasoline and combustion engines are not very efficient and make greenhouse gases such as CO2 and carbon monoxide, which is also toxic. A hydrogen fuel cell reacts hydrogen with oxygen, oxidizing hydrogen to make water as a product and this process liberates electrons that can be used to drive an electric motor. The advantages of fuel cell are that it is a pollution free process and has higher efficiency than a combustion engine. It is also portable allowing it to replace the petroleum engines in your car.

That is why people are interested in the hydrogen economy.

â&#x20AC;&#x153;We now import 60% of our oil and this number is projected to go up to about 68% by 2025.â&#x20AC;?

What are some barriers to using hydrogen fuel?

Today we get 85-95% of our hydrogen from a process called steam reforming of natural gas where methane and steam are reacted to give hydrogen gas and CO2. However, what is the point of making hydrogen from methane if we are still making CO2, which is a greenhouse gas? So production is the biggest barrier to the hydrogen economy. There is also

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a problem with delivery. Once we make the hydrogen how are we going to deliver it to the filling station? Right now we use pipelines for oil but hydrogen is a gas so this is a big infrastructure problem. Suppose we can make it, we can deliver it, we get it to the gas station; the question is how are we going to put it in our car and store enough for a 300 mile drive?

This is a problem because this hydrogen is a very volatile gas.

There are a number of approaches to hydrogen storage that have been looked at. One is to use high pressured cylinders, which is what the hydrogen cars that people drive around today use. The problem is that the cylinders weigh too much, so dragging that weight around is not very energy efficient. So that is not going to be a solution using the tanks we presently have. A lot of research is going on to find ways to make lighter weight and higher pressure tanks. Storing hydrogen as a liquid is the worst solution because hydrogen is a very low temperature liquid, so you waste all that energy liquefying it and you will have to have a cryogenic tank in your car which is not going to be very feasible. The third alternative is to use a materials based hydrogen storage system that stores hydrogen either bound covalently or absorbed onto a material to store larger volumes of it.

What material-based storage system are currently being explored?

So back in 2003, the Department Of Energy (DOE) issued a Grand Challenge where they asked groups to come together to form centers to explore hydrogen storage. I was in one of the groups that won one of the competitions. So there are three centers which have been established through this initiative. One center is on reversible metal hydrides such as aluminum hydrides. Another one is based on adsorption on carbon materials with a lot of the work now focused on metal organic framework materials. In the center that I am in, we are looking at chemical hydrides which are compounds that have hydrogen covalently bound to them and can be heated up to release hydrogen on demand.

The DOE requirements for the initiative include a weight and volume of hydrogen that must be stored. So to be able to drive your car 300 miles you need to be able to store about

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Interviews 9 weight percent hydrogen. This weight includes the weight of the compound plus any heaters, gas tanks, and anything else you might need so it is a very stringent requirement.

There is also a volume requirement because you cannot have your gas tank much bigger then it is today. The DOE has looked at all sorts of different compounds to use for hydrogen storage. Some materials can store lots of hydrogen but you have to go to 400 degrees to release it which is not feasible since it costs too much energy. Other materials store hydrogen but they release it at too low a temperature which is not good either. What you really want is to have hydrogen release around 85 degrees, which is the temperature of the fuel cell. So a hydrogen release temperature between 80-120 degrees can use the waste heat of the fuel cell to release the hydrogen which is very energy efficient.

The objectives of our center are to develop new methods for low temperature on demand hydrogen release from chemical hydrides and to develop ways of regenerating them.

There are very few compounds that can actually store enough hydrogen to meet the DOE targets and it is these compounds we are looking at. Some of the compounds we are looking at in the center are amineboranes such as ammonia borane. If I were to liberate all of the hydrogen from ammonia borane I could get three equivalents, equal to about 20 weight percent of hydrogen, which exceeds the total DOE requirement of 9. The reason we need to start with compounds that have much higher than DOE weight requirements of hydrogen is that once the heaters and other equipment are added we can still meet the DOE requirement.

“There is enough of the element boron in the world to fuel one billion cars one time” Why is it important to regenerate the compounds you use?

The key goal of the project is really to find a way to liberate the hydrogen when we want to and then to be able to convert the spent fuel product back to the chemical hydride. There is enough of the element boron in the world to fuel one billion cars one time, which means that once we use a boron hydride compound to liberate hydrogen we need a way to get back. If we can’t go backwards from the spent fuel then this process is really of no value since the supply of boron would quickly run out.

Where does the project currently stand?

Currently we have created systems with about 11 weight percent hydrogen release. The problem is while these systems are fine in meeting the weight and volume requirements, they are not yet practical at this point because there are many other engineering hurdles which must first be

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overcome. This center, which is targeted to end in March, has discovered a tremendous amount of knowledge about how to release hydrogen from these covalent hydrides.

We have also discovered a tremendous amount about regeneration. Scientists at Los Alamos have recently published a very important paper about regeneration which showed a complete cycle. Rohm and Haas have shown how this could potentially be done on a plant scale, but it is not yet feasible to begin production on a plant scale.

That is the way research is. You get a first generation of something and then you discover what problem you need to solve next. When we started 5 years ago there was almost no knowledge base so we have discovered a tremendous amount of information but whether it is going to become practical is hard to say. A lot of that depends upon whether the engineering problems can be solved. There is a long way from discoveries in the lab to the real thing and a lot of it is tied up with economics. Some technologies are feasible when gas is a certain price and others are feasible when gas is at a much higher price. So while we are not yet at the point where the technology is practical, the innovations that are going to come in the next 10 years are going to be fantastic. People are going to look back and say “why were you doing this?”, but you have to do the basic research to get to this point.

How did you become involved in this research?

I have a lot of different interests in chemistry and some of the areas that my research group are looking into are new synthetic methods and how to make compounds better. I also have an interest that is supported by the air force on ultrahigh temperature materials and things that can be useful for hypersonic vehicles. One of the areas that I worked in over many years is boron chemistry. These compounds are lightweight and can store large amounts of hydrogen. So when people started looking into hydrogen storage they were talking about a lot of the compounds I had been looking at for many years and I had the expertise to know how to manipulate them.

What advice would you give to undergraduates?

I think the best thing to do is to do some research. So beginning in freshman year whether you are interested in chemistry, physics, or biology you should go knock on someone’s door and ask if there are any opportunities to do research in their group. Even if there are not any opportunities that year there may be the following year.

There are usually always opportunities, but people just need to ask. There are also a lot of opportunities for doing research in the summer. I have helped students get internships at DuPont and other places, but you have to be persistent because people do not just come to seek you out.

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Articles

Charbonnier, J & Webb, T

Is Individual Variation in Escape Behavior Driven by Differences in Escape Ability in Oophaga pumilio?

Julie Charbonnier and Trena Webb La Selva Biological Research Station Numerous studies have suggested that animals may assess risk and adjust their behavior accordingly. We sought to examine whether difference in ability to escape predators could account for individual variation in latency to escape a predator in Oophaga pumilio, an aposematically colored frog which has remained highly responsive to predators. We hypothesized that individuals capable of jumping further and having higher endurance capacities were more likely to allow an artificial predator to approach closer or display longer periods of time between hops. While O. pumilio responds differently to predators, this response is independent of our measures of their ability to escape, SVL or sex in O. pumilio. Our results suggest we have identified a continuum of behavior types within the population which is independent of escape ability. Frogs that are “courageous” will allow the predator to get close and hop short distances slowly. Frogs that are “timid” will not allow the predator to get close and hop long distances in quick succession. Our results suggest that variation in escape behavior in O. pumilio is not determined by predator escape ability, but rather by behavioral types within the population. (The lack of correlation between ability (endurance and size) and escape behavior may reflect lack of predation pressure.) However, future studies should explore the escape behavior of Oophaga pumilio in the field in order to test whether males and females differ in their escape behavior, and whether our results are driven by individual stressresponses to laboratory conditions.

Introduction Predator avoidance is an important component of survivorship and thus fitness (Arnold 1983). Species have evolved a wide array of strategies that permit prey to avoid or escape predators effectively. For example, potential prey may be toxic, cryptic, assume defensive positions or flee (Edmunds 1974). Many studies have quantified physiological (ie. differences in muscle types) and behavioral differences in various species that employ different anti-predator strategies (Putman & Bennett 1983; Sherratt et al . 2005).

For instance, cryptic species are generally thought to remain immobile but to flee quickly once detected by a predator (Sherratt et al. 2003). On the other hand aposematic species, which advertise their chemical defenses by bold colors pattern, (Ruxteon et al. 2004), are generally thought to remain immobile and slowly move away from a predator. This behavior allows them to

effectively display their toxic coloration (Hatle et al. 2002; Sherratt et al. 2003).

The escape strategy of a species can also be related to a species morphology and locomotor performance (ie. sprint speed and endurance capacities) (Losos et. al 2002). For example, different species of skinks (Niveoscincus: Lygosominae) using different escape tactics show differences in sprint, jump and climbing abilities (Melville & Swain 2003). However, few studies have examined how individual variation in locomotor performance within a species may affect predator escape strategies.

Within a species, natural selection should favor fleeing when the benefits outweigh the costs of fleeing. Although the benefits of fleeing a predator may appear more evident, there may also be significant costs to fleeing even if successful in thwarting the predation attempt (Sherratt et al. 2003). In the cryptic Craugastor frogs, survivorship may be higher for individual Crau-

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gastor frogs which do not attempt to escape (Cooper et. al 2008). The increased probability of being detected when moving and their limited escape ability may raise the probability of capture for frogs that attempt to escape (Cooper et. al 2008). In aposematic species, rapid escape attempts may increase the probability of attack if the predators fails to perceive the aposematic coloration (Cooper et al. 2009a).

Different escape tactics can entail significant energetic and fitness costs.

(Dudley 1991; Sherratt et al. 2003). For example, Rodriguez-Prieto et al. (2008) suggests that when energetic resources are lower, the blackbird Turdus merula is not able to use costly escape strategies like flying and will instead run away from a predator. Animals may experience a foraging cost if fleeing causes an animal to leave a profitable patch (Brown & Kotler 2004). Likewise, males which defend territories may experience additional costs, since

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they risk losing their territory while escaping predators. Predator avoidance is thus a dynamic process and decisions made by prey regarding when to flee should be determined by both the costs and benefits of fleeing at any given moment. This dynamic cost and benefit tradeoff is in turn influenced by an individualâ&#x20AC;&#x2122;s ability to escape rapidly versus utilizing more stationary anti-predator strategies.

Animals have been shown to assess risk and respond according to their ability during interactions between competing individuals (Enquist & Jacobsson 1986; Jenssen et al. 2005). Numerous studies have demonstrated that prey are able to assess risk when faced with a predator and respond to change in risk accordingly (Ydenberg & Dill 1986). For example, the fiddler crab Uca vomeris responded more often and earlier to an artificial predator, the further away they were from their refuge (Hemmi 2005). The common chameleon, Chamaeleo chamaeleon were able to assess the risk of being detected by potential predators in relation to their conspicuousness in different trees and adjusted their escape behaviors accordingly (Cuadrodo et al. 2001). The behavior of the predator may also influence risk and escape behavior. For example, the lizard Anolis lineatopus was shown to adjust flight initiation distance based on changes in risk associated with predator speed (Cooper 2005).

The salamander Eurycea bislineata remains immobile when approached by the head and body of a snake predator, but flees when contacted by a tongue flick (Ducey & Brodie 1983).

A potential cause of intrapopulational variation in escape behavior may be that individuals assess risk not only based on their surroundings and the behavior of the predator, but on their ability to escape at a particular time.

Individuals may take into account their own sprint, jumping or endurance capacities when assessing the risk of an approaching predator, leading to different escape behaviors. For in-

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stance, many reptiles are less likely to flee a potential predator when their locomotor abilities are reduced in cooler temperatures (Mori &Burghardt 2004).

An ideal organism for studying the variation in escape behavior within species is Oophaga pumilio (Dendrobatidae) because it is both aposematically colored (Summer & Clough 2001), toxic (Darst et al. 2006) yet has remained highly responsive to attack by predators (Cooper et al. 2009a; Cooper et al. 2009b). Recent studies suggest that the bright coloration of Oophaga pumilio is an effective visual signal that deters predators (Saporito et al. 2007; Nooman et al. 2009). Additionally, Nooman et al. (2009) found that clay models with novel coloration forms were attacked more frequently than local aposematic Dendrobates which provides further evidence for the effectiveness of the local aposematic signal.

The reluctance of predators to attack brightly-colored prey may explain why aposematic species move more slowly in relation to palatable prey and are less likely to flee (Hatle et al. 2002; Sherratt et. al 2004; Cooper et al. 2009a). However, O. pumilio has been shown to be highly responsive to attack by predators (Cooper et al. 2009b). A field experiment noted that O. pumilio consistently escaped to the closest side of the forest when approached by a human predator (Cooper et al. 2009b). Thus, while there is a visual component to their predator avoidance strategy, O. pumilio have retained their ability to assess certain aspects of risks and respond accordingly. Predator escape behavior is particularly dynamic in O. pumilio since they are both aposematic (which would favor a more static response to predators) yet have retained their capacity to rapidly and safely escape from potential predators.

Various factors may influence the escape behavior of O. pumilio. Remaining immobile and moving slowly may allow O. pumilio to advertise its unprofitability to predators (Sherratt et al.

2004). Cooper et al. (2009a) report that

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in O. pumilio displayed reduced escape ability in an inter-species comparison and hypothesized this was due to an increase reliance on aposematism. Thus, O. pumilio should delay responding to a predator until the last minute in order to allow the predator to maximize the effectiveness of their aposematic coloration. Male O. pumilio may also avoid or delay escape, since they actively defend territories in order to secure mates (Gardner & Graves 2005).

Thus, adult males may be less likely to escape since they may experience the additional cost of losing their territory and potentially decreasing their reproductive output by attempting to escape Forsman & Hagman 2006).

In our study, we examined whether difference in ability to escape predators could account for individual behavior variation in latency to escape a predator in O. pumilio. We used maximum hop length and endurance capacities as proxies for whole-organism escape capacities. We hypothesized that individuals which were able to jump farther and had greater endurance are more likely to allow the predator to approach closer and wait longer between hops. Because males in this species are territorial and may delay responding to predator for this reason, we also assessed the effect of sex on escape responses. Finally, since size may reflect physiological differences in escape ability (Martin at al. 2005), or may indicate age and experience, we examined individual escape responses as a function of snout-vent length.

Methods This study was conducted at La Selva Biological Station, Heredia Province, Costa Rica from 26-30 April, 2009. La Selva Biological Station is classified as lowland tropical wet forest dominated by Pentaclethra macroloba and receives approximately 4000 mm of rain annually (sensu Holdrige 1947).

O. pumilio were collected from primary and secondary forests on the Sendero Tres Rios (750 meters) and Cami-

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Charbonnier, J & Webb, T no Experimantal Sur (150 meters).

Oophaga pumilio is a small, diurnal frog abundant at la Selva. It displays aposematic coloring that varies through its range (Summer & Clough 2001). Adult O. pumilio have a complex mating system and reproduce seasonally (Donnelly 1989). They do not utilize breeding pools and are thus restricted to leaf-litter forest habitat (Donnelly 1989). Male are territorial and defend territories which include foraging sites, calling sites and tadpole rearing sites (Donnelly 1989). In the Neotropics, birds, snakes and spiders are common predators of small frogs (Poulin et al. 2001). In an experimental study using plastic models of O. pumilio, birds attacked a small number of red models which suggests that some birds prey on O. pumilio or that naïve birds may attack (Saporito et al. 2007). The tarantula Sericopelma rubronitens is a predator of Dendrobates auratus, suggesting that tarantulas may also prey on O. pumilio (Saporito et al. 2007). The snake Liophis epinephelus and Coniophanes fissidens have been observed attacking O. pumilio at La Selva (see Saporito et al. 2007).

To determine whether escape behavior is related to escape ability, 69 frogs were captured and tested under laboratory conditions. Prior to testing, frogs were kept at room temperature (28º C). Each frog was housed in separate plastic bags with a humid cotton ball and leaf litter. Snout-vent length (SVL) was measured to the nearest 0.01 mm using dial calipers and the sex of each individual was scored.

Males were distinguished from females by the presence of a pigmented gular sac (Gardner & Graves 2005).

After acclimating to laboratory conditions for 24 hours, individual predator escape responses were tested in a cardboard arena with four lanes. Each lane (1.0 x 0.3 m) had two gates at opposite ends to allow our artificial predator to attack the frog head on. Each frog was placed in the exact center of the arena under a plastic cup. Each cup

had a small window which allowed us to view the position of the frog without disturbing the frog. Frogs were allowed to acclimate for 30 min. Following acclimation, the cup was lifted and the artificial predator was moved towards the frog at 2 m/s. The artificial predator was a meter stick with an attached cone cardboard head. This cardboard head was painted green and with black and yellow eyes to effectively imitate the head of a snake predator. A similar design (with a yellow, rather than green head) was used in Cooper et al. (2009a). O. pumilio’s escape response to the artificial predator was positively correlated with the strength of the attack by the stick, which suggests a stick can effectively simulate a snake predator (Cooper et al. 2009a).

The frog was approached from whatever end of the arena it was facing in order to keep the angle of attack constant. Predators approached frogs until the first jump by the frog away from the predator. At that point, how close the frog allowed the predator to come was measured to the 0.5 cm using a standard meter stick. The time delay between the first and second jumps was also recorded to the nearest 0.01 seconds.

Following the trials to assess latency in response to approaching predators, two measures of performance were assessed: maximum hop length and endurance. All performance trials were conducted at room temperature (28º C). To reduce the likelihood of exhaustion influencing hop length, hop length was assessed first. Each frog was dipped in water and both hindlegs of the frogs were dipped in florescent powder. Each frog was stimulated to move by lightly tapping its foot or backside for two consecutive trials onto a 6.48 cm x 8.37 cm paper. To measure hop distance, we measured from the midpoint of the imprint of the body of the frog to the next midpoint of the imprint of the body of the frog. The maximum hop length was recorded as the longest hop in the two trials.

Endurance was assessed in a sub-

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set of frogs (n=17). Trials were conducted on a wet cloth to reduce the risk of desiccation. We stimulated the frogs to hop forward with a wooden stick. Frogs were considered exhausted when they no longer responded to thirty consecutive stimuli. To ensure the frogs were exhausted, we flipped the frogs over on their back at the end of the thirty stimuli. Frogs which responded by righting themselves were not considered exhausted, and were stimulated to continue hopping.

To determine whether frogs with greater escape ability delay responding to approaching predation, three stepwise multiple regressions in the forward direction with a probability to enter of 0.250 and probability to leave of 0.100, were performed with sex, SVL, maximum hop length as explanatory variables and distance from the predator and time between hops as response variables. If one of the variables entered the model, a simple linear regression was performed. If both variables entered the model, a multiple regression was performed. To assess the effect of the variables, an effects test was conducted. To determine whether endurance was a predictor of distance from predator, time to response, and time between hops, three simple linear regressions were performed. All statistical analyses were performed using JMP (Version 5.1; SAS Institute 1997).

Results In total, 38 male and 31 female frogs were collected. Mean snoutvent length was 1.5993 ± 0.1394 cm.

Mean maximum hop length was 19.83 ± 3.415435 cm. Mean endurance was 822.29 ± 241.77 seconds.

Maximum hop length entered the stepwise multiple regression model (p=0.0865) as a predictor of distance from the predator at which frogs first jump. Frogs that initiated behavior when the predator was far away had higher maximum hop length than frogs that allowed the artificial predator to approach more (F=3.0272,

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Charbonnier, J & Webb, T four male frogs and 13 female frogs.

Endurance was not a determinant of time to response (F = 0.01, d.f. = 1, p = 0.93; R2 = 0.0005), distance from predator (F = 1.33, d.f. = 1, p = 0.27; R2 = 0.08) or time between hops (F = 2.38, d.f. = 1, p = 0.14; R2 = 0.14).

20 18 16

Distance from predator (cm)

14 12

Discussion

10 8 6 4 2 0 0

5

10

15 Max Hop Length (cm)

20

25

30

Figure 1. Distance from predator as a function of the maximum hop length in Oophaga pumilio when responding to an artificial predator approach at la Selva Biological Station. (N= 69) 90 80

Time Between Jumps (seconds)

70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

Max Hop Length (cm)

Figure 2. Time between jumps as a function of the maximum hop length in Oophaga pumilio when responding to an artificial predator approach at La Selva Biological Station. (N=69)

d.f.=1, p=0.0865; R2=0.043; Figure 1).

Sex, SVL and maximum hop length all entered the stepwise multiple regression mode (p=0.20, p=0.15 and p=0.06 respectively) when time between hops was the response variable.

However, only maximum hop length

20

was a significant predictor of time between hops (F= 2.71, d.f. =3, p=0.053; R2=0.12; p=0.05; Figure 2). Frogs that made hops in quick succession when escaping the artificial predator had longer maximum hop distances.

We conducted endurance trials on

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Animals should adjust their escape response according to the risk of predation in order to minimize the costs associated with fleeing. Numerous studies have suggested that animals may assess risk and adjust their behavior accordingly (Ydenberg & Dill 1986). Such studies have focused on how the environment and the behavior of the predator influence the assessment of risk (Hammi 2005; Cooper et al. 2009a). However, few studies have examined how prey abilities, such as maximum hop length and endurance, may influence risk assessment, and thus escape behavior. In this study, we presented a model predator approaching at a constant speed to O. pumilio in order to ask whether individuals with greater predator escape ability would reduce the costs associated with escape by delaying flight and reducing response. Specifically, we predicted that frogs which performed longer jumps and had greater endurance would allow the predator to get closer, and would move away only slowly.

Our results suggest we have identified a continuum of behavior types within the population which is independent of escape ability. Frogs that are “courageous” will allow the predator to get close and hop short distances slowly. Frogs that are “timid” will not allow the predator to get close and hop long distances in quick succession. (For example, the frogs which had the longest jumps initiated escape when the predator was farther away, and waited the least amount of time between jumps thus moving quickly rather than slowly.) Thus, while O. pumilio responds differently to predators, this response is independent of

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Charbonnier, J & Webb, T our measures of their ability to escape. Neither endurance, SVL or sex were significant predictors of escape behavior in O. pumilio. Our results suggest that variation in escape behavior in O. pumilio is not determined by predator escape ability, but rather by behavioral types within a population.

Implicit in this interpretation is a recategorizing of the variable maximum hop length. Rather than indicating individual hop ability in an absolute sense, we suggest here that the maximum hop reflects a frog’s willingness, or motivation to hop at that time. Such behavior differences may be explained by differences in stress responses associated with how much danger the animals feel they are in. In the lizard Urosaurus ornatus, total corticosterone levels after a predator encounter were strongly correlated with both their flight initiation distance and hiding duration during the attack (Thaker et al. 2009) Likewise, the continuum of behaviors of O. pumilio we observed may reflect differences in baseline corticosteroid levels.

Therefore, individual stress-response to laboratory conditions may also explain the escape behaviors we observed.

Generally, larger anurans display better whole animal level performance in both maximum hop length and endurance capacities (Beck & Congdon 1997). Bigger frogs are thought to possess larger hindlimb muscles, which would allow them to hop longer distances (Choi 1996). Additionally, larger animals tend to have a higher metabolic capacity, thus, better endurance (Galliard 2008). Both size and endurance have been implicated in predator-prey interactions (Arendt 2009). For example, large body size in tadpoles of the New Mexico spadefoot toad (Spea multiplicata) decreases predation risk (Arendt 2009). In Anolis cristatellus lizards, endurance capacities have been shown to accurately signal an individual’s ability to escape (Leal 1999). In O. pumilio however, SVL was not a determinant of escape

behavior. This is consistent with our hypothesis that “behavioral types” are the main determinants of escape behaviors we observed. Additionally, the lack of correlation between escape ability and escape behavior may reflect lack of predation pressure (see Blazquez et al. 1997). Aposematism and toxicity in O. pumilio may be effective at deterring most predators and thus selection for speed may be low. This is an agreement with Cooper et al. (2009a) which found that O. pumilio exhibited reduced escape speeds when compared to the palatable Craugastor frogs.

Contrary to our predictions, sex was not a determinant of escape behavior. Since fleeing entails additional costs for males who are defending territory, we had hypothesized that males would be less likely to flee a predator. It is likely we did not find differences between male and female escape behavior because we measured escape latency in the lab. We predict that a similar study conducted in the field within male territories would find differences in behavior between males and females. Chang et al. 2009 (unpublished data) has observed differences in males and female O. pumilio escape behavior. Males in the field may allow the predator to approach at a closer distance and wait longer between jumps than females or simply rely more on stationary antipredator behaviors. Future studies should explore the escape behavior of O. pumilio in the field in order to test whether males and females differ in their escape behavior, and whether our results are driven by individual stressresponses to laboratory conditions.

Acknowledgments This project would not have been possible without the facilitation of the Organization for Tropical Studies and La Selva Biological Station.

We thank Dr. Erika Deinert for her technical support. We especially thank Dr. Gary Gerald for providing helpful comments on this paper.

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Circulating Endothelial Progenitor Cells and Atherosclerosis in SLE Alexis Sharpe University of Pennsylvania, Department of Rheumatology Circulating endothelial progenitor cells are more infrequent in patients with Systemic Lupus Erythematosus (SLE) and have been strongly associated with the presence of atherosclerosis. This study sought to resolve if reduced numbers of EPCs in SLE patients is related to advanced coronary artery calcification or to lupus itself. It was found that patients with SLE had significant decreases in EPC numbers when compared to healthy controls (p<0.0001), and those without evidence of coronary artery calcification also had significantly lower numbers of EPCs (p=0.02). Increased carotid intima-media thickness did not associate with coronary artery calcification or decreased numbers of EPCs in SLE patients. This study was able to show that reduced numbers of EPCs in SLE patients may be seen even without coronary artery calcification.

Introduction Systemic Lupus Erythematosus (SLE) is a systemic autoimmune inflammatory disease that is associated with the pathologic involvement of many organ systems. Patients with SLE are known to be at higher risk of atherosclerotic cardiovascular disease (ASCVD). Electron Beam Computed Tomography (EBCT) has been established as a method to determine the degree of calcified coronary atherosclerotic disease. This method can also be used to identify increased Coronary Artery Calcification (CAC) in women with SLE compared to age-matched controls (1). Circulating endothelial progenitor cells have also been found to play a role in endothelial repair (2, 3). Reduced numbers of circulating endothelial progenitor cells (EPCs) have been correlate strongly with atherosclerotic cardiovascular disease (4, 5). Studies of SLE patients have shown that EPCs are less abundant in these patients when compared to healthy controls (6, 7). Since multi-vessel coronary disease is known to be associated with decreases in EPCs, we proposed to examine whether this reduced number is present only for patients with ASCVD or if it is an indicator, preceding ASCVD development. This study

Methods

tients were compared with a group of healthy controls of similar ages, without histories of hypertension, diabetes, smoking, or cardiovascular disease. These control patients did not have EBCT or CIMT performed.

Subjects Patients were recruited from a previous cross-sectional study performed at the University of Pennsylvania for which EBCT scans were performed within the past 7 years.Patient inclusion required informed consent from females between the ages of 25 and 65 who meet at least four American College of Rheumatology criteria for the SLE diagnosis, along with age, race, and weight-matched controls. They must have previous coronary calcium scores over the 75th percentile or below the 25th percentile for their ages.

Scores were taken from those who had given consent to be contacted based on results from previous cross-sectional study. Patients were excluded if they currently use over ten milligrams of prednisone since steroids are known to alter numbers of EPCs and known cardiovascular risk factors (8, 9). Patient exclusion also included those who have a history of advanced renal disease or cardiovascular events. Pa-

Flow Cytometric Analysis and Fluorescence-activated Cell Sorting (FACS) Analysis Fluorescence-Activated Cell Sorting was performed on the samples, and EPC populations were identified and quantified as previously outlined (11).

A protocol that successfully identifies

therefore sought to resolve if reduced numbers of endothelial progenitor cells in Systemic Lupus Erythematosus patients is related to advanced coronary artery calcification or to lupus itself.

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Patient Visit and Collection and Processing of Blood Samples Patients underwent detailed histories concerning their medical pasts.

A thirty milliliter syringe was coated with approximately 0.5 mL of heparin.

One tube was filled for blood count, and another was filled for stored serum.

Subsequently after a thirty mL syringe was filled through a 21-gauge butterfly neele. All blood samples were drawn in the morning. Twenty milliliters of blood was drawn from fasting participants.

Blood samples were used for flow cytometric analyses. White blood cells were isolated from approximately ten milliliters of blood using ammonium chloride lysis as previously described (10).

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mature circulating EPCs was used. Five million leukocytes were stained with a 5-color antibody panel: FITC-antiCD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3, -CD19, -CD22, PE-Cy7-anti-CD34, and APCati-VEGF-R2. Fluorescence minus-one (FMO) samples were used as negative controls. Cell populations were grouped using cell surface markers for progenitor cells, EPCs, and mature endothelial cells. Data analysis was performed that allowed complex cell marker analysis

Electron Beam Computed Tomography (EBCT) EBCT has been widely accepted to determine cardiovascular risk in CAC (12, 13). Patients were classified and clustered according to percentile rankings in previous CAC scores.

EBCT scans were repeated to confirm that their scores had not significantly changed from previous study results. Results were reported as calculated numeric values, along with percentiles matched for age and gender.

Carotid Plaque and Intima Media Thickness (CIMT) Carotid plaque and intima-media Group

Healthy Controls

N Age (Mean +/- SD) Women (n) Active Smoking (n) History of HTN (n) Diabetes (n) ALC/uL

13 55.1 + 12.8 5 0 0 0 1526 + 583 N/A N/A N/A N/A

GFR (mL/min/1.73m2) Disease Duration (years) SLEDAI SLICC

thickness was measured on patients, as CIMT irregularities have been noticed in SLE patients (14). Carotid plaque is a predictor of ASCVD, and increases in the measured thickness of the intima and media of the carotid artery are directly related with an increase risk of cardiovascular disease (15, 16). This method was used to assess patient risk for ASCVD and correlate these findings with EBCT scores and EPC numbers.

Sample Size and Statistical Analysis Eighteen patients were tested, and their results were analyzed with biostatistics and Stata 10.1 software.

The analyses included comparison mean levels in EPC numbers, CIMP, and CAC among the three CACpercentile-based groups and association of cardiovascular risk variables.

Results No patients needed to be excluded based on renal disease or cardiovascular events since the initial study. No patients were excluded based on dosages of prednisone greater than 10 milligrams. Two patients initially without CAC were found to have developed SLE patients without CAC 7 51.1 + 8.7 (p=0.2) 7 0 3 0 1168 + 507 (p=0.09) 92.6 + 17.6 11.4 + 4.4 5.6 + 4.5 1.9 + 0.3

SLE patients with CAC >75% for age (p*) 10 53.2 + 5.7 (p=0.7) 10 5 6 0 1643 + 740 (p=0.08) 85 + 25.5 (p=0.26) 18.3 + 10.9 (p=0.07) 8 + 8.9 (p=0.26) 3.3 + 0.6 (p=0.05)

Table 1. Group characteristics in healthy controls, SLE patients without CAC and SLE patients with >75th percentile CAC for age. *value for test of significance between SLE groups CAC, Coronary Artery Calcification; HTN, Hypertension; ALC, absolute lymphocyte count; GFR, Glomerular Filtration Rate; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; SLICC, Systemic Lupus International Collaborating Clinics damage score

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significant CAC on repeat carotid ultrasound imaging, which moved them into over the 75th percentile for age.

Characteristics of the three groups are described in Table 1. SLE patients were more likely to have hypertension and had non-significant decreases in total number of lymphocytes. Those with CAC were more likely to smoke and have hypertension than those without CAC, and they had longer disease duration than SLE patients without CAC. CAC patients therefore had an increased Systemic Lupus International Collaborating Clinics damage score (SLICC) (p=0.05).

Hypertension was more common in patients with SLE than in the healthy controls, but within SLE patients without CAC, there was no found association between a reported history of hypertension and low EPC number. Steroid and other medication use did not correlate with EPC number or presence of CAC. It was found that patients with SLE had significant decreases in EPC numbers when compared to healthy controls (Mean: 9.7 v. 0.9 (p<0.0001)) and (Median: 10.2 vs. 0 (p<0.0001)).

SLE patients lacked CD133+/CD34+/ VEGFR+ cells (p<0.0001). Compared to healthy controls, SLE patients without evidence of CAC on the EBCT also had significantly lower numbers of EPCs (Median: 10.2 v. 0 (p=0.02)).

SLE patients were not found to have significantly reduced numbers of progenitor cells (CD133+, CD34+), however (Mean: 1007 v. 824 (p=0.2)).

In the analysis of healthy controls, men had non-significant increases in EPC numbers in comparison to women (Median: 119 v. 5.8 (p=0.09)).

Men, however, were significantly younger (p=0.01), and age correlated with EPC number (p=0.02) among healthy controls. When men were excluded from the analysis, however, differences in EPC number between the healthy controls and SLE patients without CAC were still significant for CD+133/CD34+ EPCs (p=0.02)

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Sharpe, A Pro-angiogenic Cells

Healthy Controls

CD133+, VEGFR+

11.5 (6.7, 18.9)

CD34+, VEGFR+

21.2 (11.5, 28.8)

CD34+, CD133+, VEGFR+

10.2 (5.8, 12.3)

SLE patients without CAC 0 (0, 0) (p=0.005) 0 (0, 20.9) (p=0.05)

SLE patients with advanced CAC 0 (0, 0) (*p=0.6) 0 (0, 4.9) (*p=0.9)

0 (0, 6.7)

0 (0, 0)

(p=0.02)

(*p=0.1)

638 (364, 1024) (p=0.10)

800 (365, 1062) (*p=0.63)

2134 (1260, 3912)

2031 (980, 3320)

(p=0.19)

(*p=0.49)

533 (161, 1164)

766 (302, 1743)

(p=0.19)

(*p=0.59)

Total Progenitor Cells CD133+

1028 (693, 1609)

CD34+

3315 (2648, 4743)

CD133+, CD34+

832 (639, 1344)

Table 2. Median value for PAC numbers between groups (Median (IQR)) *test of significance between SLE groups

but non-significant for CD133+/ CD34+/VEGFR+EPCs (p=0.12) and CD34+/VEGFR+ EPCs (p=0.36).

No significant difference was found in EPC numbers between SLE patients without coronary artery calcification and those with advance coronary artery calcification (Median: 0 v. 0 (p=0.1)). An increased carotid intima-media thickness did not associate with coronary artery calcification or decreased numbers of EPCs in SLE patients (p=0.45). There also was no difference in carotid intima-media thickness between SLE patients with CAC and SLE patients without CAC (Mean: 0.740 v. 0.722mm (p=0.40)).

Discussion This pilot study is the first to show that SLE patients both with and without evidence of atherosclerosis on EBCT scan have decreased numbers of EPCs in comparison to age-matched healthy controls. These findings may imply that EPC depletion may be a precursor for the development of atherosclerosis.

This suggests that SLE numbers may serve as a useful marker for the early risk of cardiovascular disease independent of other risk factors evaluated in our study. The non-significant decrease in the number of progenitor cells could in part contribute to the reduced number of EPCs identified, however.

Carotid intima-media thickness did not correlate with CAC or EPC number in this study. This may imply that CIMT is not as sensitive to the presence of atherosclerosis in patients as previously thought or that this measurement of CAC did not fully represent the overall risk of vascular disease.

Though this study is not the first to prove that SLE patients have reductions in EPC numbers in comparison to healthy controls, it included an evaluation of patients for atherosclerosis. This has been highly related with low EPC numbers in other populations. Our study therefore implies that decreased numbers of EPCs in SLE patients may precede the development of advanced vascular disease.

The finding in turn may prove to be relevant in terms of future treatments.

This study included many limitations. The healthy control population included men, yet the lupus study was composed solely of women. While previous data does not imply a difference in EPC by gender, men in this study did show a non-significant increase in EPC numbers in comparison to women (17, 18). This difference was accounted for by a significant age difference, however. Nevertheless, if a gender difference does exist, the applicability of our findings may be limited.

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The risk factor profiles between groups also limited the study because not all profiles were available for SLE patients. Thus, there may be other confounding factors present.

Conclusions This study ultimately suggests that the reduction of EPCs in SLE patients is independent of atherosclerosis as evidenced by an EBCT scan (Median: 0 v. 0 (See Table 1)). It therefore supports the idea that decreased numbers of EPC in patients may be an early risk factor for the development of atherosclerosis. These results may have implications for populations at increased cardiovascular risks. It can also help later identify mechanisms to account for this reduction in number.

Further study is needed to determine if reduction in EPC numbers within SLE populations increases cardiovascular risk in a similar manner to that in other populations. In order to formulate more definite conclusions, a larger study must be conducted that includes male and female populations to further assess the applicability of our findings. Further study may also suggest whether active therapies may improve this destructive process.

Acknowledgements Figures and tables courtesy of Joshua Baker, MD

References 1. Von-Feldt JM, Scalzi LV, Cucchiara AJ, et al.: Homocysteine levels and disease duration independently correlate with coronary artery calcification in patients with systemic lupus erythematosus. Arthritis and Rheumatism 2006; 54 (7): 2220-2227.

2. Urbich C, Dimmeler S: Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 2004; 95(4): 343-353.

3. Asahara T, Murohara T, Sullivan A, Silver M, Zee Rvd, Li T: Isolation of putative progenitor endothelial cells for angiogenesis.

Science 1997; 275(5302): 964-967.

4. Urbich C, Dimmeler S: Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 2004; 95(4): 343-353.

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Sharpe, A 17. Werner N, Kosiol S, Schiegl T, et al.

Circulating endothelial progenitor cells and cardiovascular outcomes. New England Journal of Medicine 2005;353:999-1007.

18. Fadini GP, de Kreutzenberg S, Albiero M, et al. Gender differences in endothelial progenitor cells and cardiovascular risk profile: The role of female estrogens. Arterioscler Thromb Vasc Biol 2008;28:997-1004.

Figure 1. Total number of endothelial cells (EPCs) per mL in healthy controls, patients with SLE without CAC, and patients with SLE with >75th percentile CAC for age.

5. Asahara T, Murohara T, Sullivan A, Silver M, Zee Rvd, Li T: Isolation of putative progenitor endothelial cells for angiogenesis.

Science 1997; 275(5302): 964-967.

6. Westerweel PE, Liujten R, Hoefer E, Koomans HA, Derksen R, Verhaar MC: Haematopoietic and endothelial progenitor cells are deficient in quiescent in Systemic Lupus Erythematosus. Annals of Rheumatic Disease 2007; Doi: 10.1136/ ard.2006.065631: 1-16.

cytometric measurement of circulating endothelial cells: The effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations.

Cytometry Part B (Clinical Cytometry) 2006; 70B: 56-62.

12. Arad Y, Spadaro LA, Goodman K, Newstein D, Guerci AD: Prediction of coronary events with electron beam computed tomography. J Am Coll Cardiol 2000; 36(4): 1253-60.

7. Moonen JAJ, de-Leeuw K, van-Seijen XJGY, et al.: Reduced number and impaired function of circulating progenitor cells in patients with systemic lupus erythematosus. Arthritis Research and Therapy 2007; 9(4): R84.

13. Park R, Detrano R, Xiang M, et al.: Combined use of computed tomography coronary calcium scores and C-reactive protein levels in predicting cardiovascular events in nondiabetic individuals. Circulation 2002; 106(16): 2073-7.

8. Ablin JN, Boguslavski V, Aloush V, et al.: Effect of anti-TNFα treatment on circulating endothelial progenitor cells (EPCs) in rheumatoid arthritis. Life Sciences 2006; 79: 2364-2369.

14. Manzi S, al e: Prevalence and risk factors of carotid plaque in women with systemic lupus erythematosus. Arthritis Rheum 1999; 42(1): 51-60.

9. Grisar JC, Aletaha D, Steiner CW, et al.: Endothelial progenitor cells in active rheumatoid arthritis: Effects of TNF and of glucocorticoid therapy. Ann Rheum Dis 2007.

10. Cherian S, Moore J, Bantly A, al e: Peripheral blood MDS score: a new flow cytometric tool for the diagnosis of myelodysplastic syndromes. Cytometry B Clin Cytom 2005;64:9–17. 2005; 64: 9-17.

11. Shaffer RG, Greene S, Arshi A, et al.: Flow

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15. Amato M, Montorsi P, Ravani A, et al.: Carotid intima-media thickness by Bmode ultrasound as surrogate of coronary atherosclerosis: correlation with quantitative coronary angiography and coronary intravascular ultrasound findings. Eur Heart J 2007 28(17): 2094-2101.

16. O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Jr. SKW: Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. N Engl J Med 1999; 340(1): 14-22.

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Spanier, M J

Beach Erosion as a Factor in Nest Site Selection by the Leatherback Sea Turtle at Playa Gandoca, Costa Rica Matthew J. Spanier Associated Colleges of the Midwest, San José, Costa Rica Department of Biology, The Colorado College Leatherback sea turtles (Dermochelys coriacea) nest on dynamic, erosion-prone beaches. Erosive processes and resulting nest loss have long been presumed to be a hindrance to clutch survival. In order to better understand how leatherbacks cope with unstable nesting beaches, the role of beach erosion in leatherback nest site selection was investigated at Playa Gandoca, Costa Rica. The potential effect of nest relocation, a conservation strategy in place at Playa Gandoca to prevent nest loss to erosion, on the temperature of incubating clutches was also examined. Changes in beach structure as a result of erosion at natural nest sites were monitored during the time the nest was laid, as well as in subsequent weeks. To investigate slope as a cue for nest site selection, the slope of the beach was measured where turtles ascended from the sea to nest as well as the slopes at other random locations on the beach for comparison. Temperature differences between natural and relocated nest sites were examined with thermocouples placed in the sand at depths typical of leatherback nests. Nests were distributed nonrandomly in a clumped distribution along the length of the beach and laid at locations that were not undergoing erosion. The slope at nest sites was significantly different than at randomly chosen locations on the beach. The sand temperature at nest depths was significantly warmer at natural nest sites than at locations of relocated nests. The findings of this study suggest leatherbacks actively select nest sites that are not undergoing erosive processes, with slope potentially being used as a cue for site selection.

The relocation of nests appears to be inadvertently cooling the nest environment. Due to the fact that leatherback clutches undergo temperature-dependent sex determination, the relocation of nests may be producing an unnatural male biasing of hatchlings. The results of this study suggest that the necessity of relocation practices, largely in place to protect nests from erosion, should be reevaluated to ensure the proper conservation of this critically endangered species.

Introduction The leatherback sea turtle (Dermochelys coriacea), once among the most abundant of the world’s marine turtles (Pritchard 1982), has become listed as an IUCN critically endangered species (Sarti-Martínez 2000). Inhabiting both the Pacific and Atlantic Ocean basins, leatherback sea turtles are one of the most widely distributed reptiles in the world (Reina et al. 2002). Eastern Pacific populations have experienced serious decline, and extinction is now a looming reality (Sarti-Martínez et al.

2007, Spotila et al. 2000). Atlantic populations, on the other hand, are more stable and in some cases appear to be increasing (Dutton et al. 2005, Chacón & Eckert 2007). The offshore threats to

leatherback populations appear to come from incidental capture in ocean fisheries, ocean pollution primarily from plastics, and possible climate change driven alterations in ocean productivity (Saba et al. 2008, Sarti-Martínez 2000).

Onshore, leatherback nests are threatened by the illegal poaching of eggs (Sarti-Martínez 2000), which in recent decades has been widely curbed by conservation programs (Chacón & Eckert 2007, Spotila et al. 2000). Additionally, it has long been hypothesized that beach erosion poses a major risk to leatherback nest success (Chacón & Eckert 2007, Mrosovsky 1983). This threat stems from the dynamic, erosionprone nature of leatherback nesting beaches (Eckert 1987), where erosion

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has been estimated to be responsible for 36-50% of leatherback nest loss (Mrosovsky 1983). As a result, some management programs have implemented the practice of nest relocation, where leatherback eggs are physically relocated from natural nest sites to other areas of the beach deemed more stable.

Beach erosion will likely be exacerbated in the future as a result of climate change and rising sea levels. The International Panel on Climate Change forecasts a nearly 0.6 m rise in global sea levels during the next century (IPCC 2007). Worldwide, increased beach erosion and loss has already been attributed to climate change (Feagin et al. 2005, Perkins 2000, Zhang et al.

2004). Therefore, a better understand-

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nests closer to vegetation can disorient leatherback hatchlings (Mrosovsky 1983). Additionally, there appears to be a genetic component to nesting behavior (Kamel & Mrosovsky 2004), therefore relocating nests to safer areas may also affect natural selecN 3 km tion against females that nest in inappropriate locations.

Figure 1. The study site at Playa Gandoca, Costa Rica. This study was carried out in sectors A & B.Over 50% of natuFigure 1. The study site at Playa Gandoca, Costa Rica. rally deposited nests This study was carried out in sectors A & B. at Playa Gandoca are relocated to other ing of how erosive processes may inareas of the beach deemed more stafluence leatherback nest success could ble, largely in response to presumed be valuable to management programs.

The main objective of this study threats of erosion, as part of current was to test the hypothesis that beach conservation protocols (Chacón & erosion plays a significant role in the Eckert 2007, this study). To investiselection of nest sites by leatherback gate whether relocation practices were sea turtles. Research was carried out having an effect on sex ratios at Playa at Playa Gandoca, Costa Rica, a highly Gandoca, the hypothesis was tested dynamic beach where coastal erosion that nest relocation influences the is thought to be one of the largest ob- temperature of the nest environment. stacles nest hatching success (Chacón Materials and Methods & Eckert 2007). Specifically, it was predicted that beach slope would be Study Site Playa Gandoca (9°59.972’N, used by leatherbacks coming ashore as a cue for assessing nest site stability. 82°60.530’W) is an 8.85 km-long beach Another objective of this research located within the Gandoca-Manzawas to examine whether current nest nillo Wildlife Refuge on the southern relocation practices at Playa Gandoca Caribbean coast of Costa Rica (Fig 1). have the potential to affect sex ratios of A conservation program run by the hatchlings. All sea turtle species under- Wider Caribbean Sea Turtle Conservago temperature dependent sex determi- tion Network (WIDECAST) has been nation (Standora and Spotila 1985) and in place since 1990. Nightly patrols relocating nests has been shown to in- of the beach take place from 20:00fluence the temperature and subsequent 04:00 hours along the entire length of sex ratios in nests of the green sea turtle the beach, which is divided into three (Chelonia mydas) (Spotila et al. 1987) sectors (A = 1950 m, B = 2850, & C = and leatherbacks (Dutton et al. 1985). 2900 m). To aid in documenting the Relocating sea turtle nests may also distribution of turtle nests, sequential have other drawbacks in addition to markers run the length of the beach in potentially skewing sex ratios. Eggs 100 m increments along the forest edge.

Upon encountering a nesting turtle, run the risk of being damaged when they are transported across beaches. nightly patrols collect biometric data Changing light conditions by moving and record the location of the turtle

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on the beach relative to both tide line and sequential markers. Nests in areas of the beach deemed unsuitable by patrol leaders (i.e., below tide lines, high vulnerability to erosion, proximity to creeks or rivers that may flood, and high traffic areas of the beach) are relocated to areas of the beach thought to be more stable and suitable for nests or to two hatcheries constructed midway through each nesting season.

Erosion and slope data collection Research was carried out on a 4.8 km stretch of Playa Gandoca (sectors A & B) over the course of 54 days in the early part (24Feb–19Apr) of the 2008 nesting season (February–July). All nest locations where a nesting turtle had attempted to deposit eggs (some nests were relocated) as recorded by nightly patrols were visited within 14 hours of the nesting event. Since the physical presence of eggs at nest locations was not important for the research questions of this study, measurements were taken at all nest sites regardless of whether the eggs had been relocated.

The slope of the beach at nest sites was measured using a Sokkia® No.804710 Abney level with clinometer (Sokkia Corp; Olathe, KS) in the tracks left by the turtle as it ascended the beach from the ocean. Using two equal-length stakes, the clinometer was sighted from one stake located in the water below the low tide line to another located above at the high tide line (the high tide line was determined by a visible morphological change in beach slope) and the angle recorded. Measurements were never taken within 2 hours of a high tide to allow sufficient beach to be exposed for measurement. This method allowed for the accurate measurement of the angle of the beach up to the high tide line as was seen from the water, without being influenced by slight variation in beach slope between measuring stakes.

From the center of the body pit above each nest, the distance across the beach was measured from the vegetation line to the high tide line to the nearest one tenth of a meter. The loca-

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Spanier, M J 7.8

40

Berm Change (meters)

Berm Change (m) Berm Change

7.8

5.8

5.8 3.8

3.8

1.8 1.8

-0.2-0.2

30 20 10 0 -10 -20

-2.2-2.2 1

1

2 2 3 Period Period

0

3

2

4

6

8

10 12 14 16 18 20 22 24 Slope (degrees)

Figure 2. Left: The mean beach width of the entire study area during each of the three 18-day periods of the study. The third period showed the only significant change in mean width (F = 9.92; df = 2,123; p < 0.001). Right: Changes in beach width went from erosive to accretive processes with increasing slope. Negative numbers on the y-axis signify erosive processes Figure Left: The mean accretive beach width the entire study Rarea during (F each of thedfthree 18-day of the study.beach The 2 and2.positive numbers (y = of -0.2311 + 13.80/x; = 0.1436) = 20.78; = 1,124; p < periods 0.001). The average thirdslope period showed the only significant change in mean width (F = 9.92; df = 2,123; p < 0.001).

Right: Changes in beach at nest sites was 7.6°, which is correlated with an increase in beach width.

Number of nests within 50 m of marker

width went from erosive to accretive processes with increasing slope. Negative numbers on the y-axis signify erosive processes and positive numbers accretive (y = -0.2311 + 13.80/x; R2 = 0.1436) (F = 20.78; p < used 0.001). rdgdf+ =1°1,124; C)) were to The record sand 12 average beach slope at nest sites was 7.6°, which is correlated with an increase in beach width. temperature. Prior to use the accu11 racy of all thermocouples was tested 10 with a mercury thermometer, and only those units that varied < 1°C from the 9 mercury thermometer were accepted.

8 The homogenous color of the sand at 7 Playa Gandoca made unnecessary the consideration of sand color for tem6 perature measurement comparisons.

5 Four sample sites were established 4 to examine temperature difference in the nest environments of natural and 3 relocated nests. All four were located 2 within 50 m of both a natural and relocated nest along a 0.7 km length of 1 beach. Two sample sites were located at 0 1 6 11 16 21 26 31 36 41 areas of the beach where the vegetation Marker number (spaced every 100 m) line consisted of low-growing palms Figure 3.

Nests were distributed in a non-random, clumped distribution in the gure 3. Nests were distributed in a non-random, clumped distribution in the northern portion of Playa Gandoca. and the other two at areas characternorthern portion of Playa Gandoca.

Markers ran sequentially 100 starting arkers ran sequentially every 100 m, starting with marker 1 on the northern border ofevery sector A. (x2m, = 74.0; df = 20; pized < by high growing palms and other 001). with marker 1 on the northern border of sector A. (x2 = 74.0; df = 20; p < 0.001). trees to take into account differences in beach shading. At each site, three tion of each nest was triangulated us- racy: 10 m). To examine changes in thermocouples were placed in a single ing the two closest sequential beach the overall beach structure, the width hole at depths of 0.7, 0.6, and 0.5 m [the markers. In order to observe changes and slope of the beach was measured typical depth of leatherback egg chamin beach structure at nest sites dur- from the water to the high tide line bers (Chacón et al. 2007)]. Holes were ing the incubation period, the nest at all sequential markers four times dug at both the vegetation line where locations were revisited every 14 days. (every 18 days) during the study. current conservation protocols require Additionally, all nest and sequen- Sand Temperature relocated nests to be placed (to chartial beach marker locations were re® Omega Type K thermocouples acterize the sand conditions around corded using a Magellan® SporTrak and an Omega® HH501AJK Types J, a relocated nest) and the mid-beach Map handheld GPS (Magellan NaviK thermometer (Omega Engineering halfway between the high tide line gation Inc, San Dimas, CA; accuInc. Stamford, CT; accuracy: +/- (0.1% and the vegetation line (the most com-

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Spanier, M J Number of Beach Markers

Number of Nests

5 4 3 2 1

Positive berm change No berm change Negative berm change

10 8 6 4 2 0

0 -7

3 13 23 Berm Width Change (m)

1

33

Number of Beach Markers

5

Number of Nests

12

4 3 2 1

2 3 4 Number of Nests

5

5 Positive berm change No berm change Negative berm change

4 3 2 1 0

-5

-2

1 4 7 Berm Width Change (m)

10

1

2 3 4 Number of Nests

Figure 4. Top, left: A regression model in which beach growth is positively correlated with nesting frequency during the entire course of the study (positive x values are associated with accretion processes and negative erosion) (y = 1.698 + Figure 4. Top, left: A regression model in which beach growth is positively correlated with nesting frequency during the 0.0587*x; R2 = 0.1310) (F = 4.37; df = 1,29; p = 0.045). Top, right: Interaction plot with beach markers selected for nest entire course of the study (positive valuesthe are18-day associated accretion processes and negative erosion) (y =entire 1.698 + sites characterized by beach changexduring periodwith the nest was laid and the number of nests during the 2 0.0587*x; R =markers 0.1310)where (F = the 4.37; df =was 1,29; p = 0.045).

Top, right: Interaction plot with beach selected study. Beach beach undergoing accretion experienced the highest number of markers nests. Bottom, left:for A nest si regression model without data during from thethe third 18-day period when beach increasethe in beach characterized by beach change 18-day period the nestthe was laid was and undergoing the numberan ofoverall nests during entire study. width.

Increasing beach positively correlated with nesting frequency = 1/(0.8567 â&#x20AC;&#x201C; 0.0582*x); = 0.4708)left: (F = A Beach markers where thewidth beachis was undergoing accretion experienced the(yhighest number of nests.R2Bottom, 9.79; df = 1,28; p < 0.001).

Bottom, right: An interaction plot with the same variable characterized by the same factors regression model without data from the third 18-day period when the beach was undergoing an overall increaseas in beach above without the final 18-day period of the study. Leatherbacks still selected against eroding areas of the beach. 2

width. Increasing beach width is positively correlated with nesting frequency (y = 1/(0.8567 â&#x20AC;&#x201C; 0.0582*x); R = 0.4708) (F = 9.79; = 1,28;nest p <location 0.001). for Bottom, interaction with the same characterizedasbypositive the same factors as ed An using a one-wayplot ANOVA (Sokal & variable width characterized (accremondfnatural leather-right: above without the from final 18-day period of Rohlf the study.

Leatherbacks still selected eroding(erosion), areas of or thenobeach.

1981) with a Tukey multiple com- against tion), negative change backs).

Readings thermocouples, as well as the temperature 0.5 m above the sand and weather conditions, were recorded twice daily between 05:30-06:30 and 14:00-15:30 local time

Data Analysis All statistical analysis were performed using StatgraphicsÂŽ Plus for Windows 3.1 (StatPoint, Inc. Herdon, Virginia), with the exception of a simple chi-square goodness-of-fit test run with BIOM (Sokal & Rohlf 1981) to analyze nest distribution.

The variance in beach width between the 18-day periods was estimat-

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parison. To investigate the relationship between beach slope and change in beach width, the model was chosen with the most significant p-value among those offered in Statgraphics.

Changes in beach width during twoweek intervals at natural nest sites data were grouped by the total time monitored (two, four, or six weeks) without counting the same nest twice (i.e., if a nest was monitored for six weeks, it was not analyzed as a nest monitored for two or four weeks). Data from each interval period was then grouped according to the overall change in beach

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(less than a 0.1 m) over the time period it was monitored. A chi-squared goodness-of-fit test was then run on all three periods individually and pooled.

To further investigate erosion and accretion beach processes at turtle nesting sites, a regression analysis was performed on beach width change at the sequential beach marker nearest a nesting location during the 18-day period the nest was laid, and the number of nests near each marker. Additionally, markers that were within 50 m of a nesting site were categorized into three beach change categories: erosion,

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Spanier, M J stable, or accretion (deposition) and by nesting frequency (1 – 5 events) during a given 18-day period. A two-way ANOVA was performed on the number of beach markers within 50 m of a natural nest as the variable classified by these two factors (beach change category and nesting frequency). Both the ANOVA and regression tests were run on data from all three 18-day periods combined and on the first two 18-day periods only with the third thrown out (after data analysis indicated an overall positive change in beach width) to negate the confounding effect of an overall increase in beach width.

Differences in sand temperature at nest depths between mid-beach and vegetation line locations were compared using a two-way ANOVA. Sand temperature was the variable characterized by beach position (mid-beach or vegetation line) and depth (0.7, 0.6, or 0.5 m). Data from all sample sites were pooled. P-values for all analyses were considered significant if ≥ 0.05. All means are presented as χ ± ISD (standard deviation).

Results Beach changes at sequential markers Data collected from the 42 sequential beach markers in the study area showed no overall significant change in average beach width during the first two 18-day study periods. During the final 18-day period of the study the beach underwent an overall accretion process, significantly increasing the average beach width (F = 9.92; df = 2,123; p < 0.001, Fig. 2). Greater beach slopes were correlated with erosive processes in subsequent 18-day periods and lesser slopes with accretive processes (F = 20.78; df = 1,124; p < 0.001, Fig. 3).

Nest site selection & erosion A total of 60 nest sites were studied, at which 11 (18.3%) the eggs were left in their natural locations, 35 (58.3%) eggs were relocated to other parts of the beach, and 14 (23.3%) the eggs were moved to a hatchery. Nesting occurred in a non-random, clumped distribution along the length of the beach (x 2 = 74.0; df = 20; p < 0.001, Fig. 3). The mean slope of the beach from waterline to high tide line at nesting sites was 7.6 ± 1.8° (range = 4-11; n = 60). The mean beach width at nesting sites was 17.4 ±

Depth 0.7 m 0.6 m 0.5 m Pivotal temp.

Temperature (C)

31 30 29 28

1.0 m (range = 0-38.4; n = 60). A total of 55 nests (92%) were laid above the high tide line. Forty-two of the 55 nests above the high tide line (76%) were on the outer half of the beach closer to the high tide line than the vegetation line.

The mean slope of the beach at nest sites was significantly shallower than that of other randomly chosen locations (F = 4.22; df = 1,118; p = 0.042).

Beach width and slope changes in two-week intervals were measured for 28 natural nest sites (n two weeks = 14, n four weeks = 10, n six weeks = 4).

Four of these nests were located below the high tide line and three were lost to erosion during the study period. Nest sites monitored for two weeks were characterized by a marginally significant overall increase in beach width (x2 = 5.76; df = 2; p = 0.056). Nest sites monitored for four weeks showed a trend of increasing beach width (x2 = 5.44; df = 2; p = 0.066). Nest sites monitored for six weeks displayed no significant change in beach width (x2 = 1.90; df = 2; p = 0.388). The three time intervals pooled showed an overall increase in beach width at nesting sites (x2 = 12.56; df = 2; p = 0.002, Fig. 4).

Nest sites occurred within 50 m of half (21) of the beach markers in the study area. More nesting sites occurred near beach markers that were undergoing accretion processes, as opposed to erosion, during all three 18-day periods of the study (F = 4.37; df = 1,29; p = 0.045, Fig. 4). Additionally, more nesting sites from the first two 18-day periods (when the beach was not undergoing an overall beach building process) were located near beach markers experiencing accretion (F = 9.79; df = 1,11; p = 0.010, Fig. 4).

Temperature 27 A total of 689 temperature readMid-berm Veg. Line ings were taken over the course of 28 days. Temperatures were observed durPosition ing clear, partly cloudy, mostly cloudy, Figure 5. The sand temperature at the mid-beach was higher at all three nest overcast, and raining weather condidepths than at the vegetation line (F = 42.17; df = 2,665; p < 0.001). The dashed tions. Pooled data indicated that sand 5. The sand temperature the29.5° mid-beach was higher allsex three nest depths than at the vegetation line indicates at the C pivotal temp.atfor ratios in a developing clutch line (F = temperature at 0.7, 0.6, and 0.5 m depths df = 2,665;(Binckley p < 0.001).

The dashed line indicates the 29.5° C pivotal temp. for sex ratios in a developing et al. 1998).

(Binckley et al. 1998).

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was significantly higher at mid-beach locations than at the vegetation line (F = 42.17; df = 2, 665; p < 0.001, Fig. 5).

Discussion Leatherbacks at Playa Gandoca appeared to selectively avoid erosionprone areas of beach when selecting nest sites. Nests were laid in a clumped distribution that placed most nests at locations on the beach that were not eroding. The shallower slopes measured at nest sites support the prediction that beach slope is a potential means for leatherbacks to assess the stability of a nesting site. To the best of the author’s knowledge, this study was the first direct test of the relationship of beach slope and erosion to nest site selection. This study indicates that leatherbacks appear to have a mechanism to maximize nesting success on erosion prone beaches.

Beach slope has been shown to be a cue for nest site selection in loggerhead (Caretta caretta) and hawksbill (Eretmochelys imbricate) sea turtles (Wood & Bjorndal 2000). This study suggests that leatherbacks may also employ beach slope as a nest site selection cue, using it as a tool for selecting stable areas of the beach for their nests. In addition to the findings that nests were located on areas of the beach with shallow slope, the insertion of average nest site slope (7.6°) in the regression model which correlates slope and beach width change revealed the angle of ascent selected by leatherbacks corresponds to a 1.6 m increase in beach width per 18-day period. By having a range of acceptable beach slopes near the value found in this study, leatherbacks at Playa Gandoca may be selecting stable nest sites while avoiding those that are too low and risk being flooded as well as those that have beach slopes energetically unfavorable to ascend and are too steep to maintain beach stability.

While this study indicates the role erosion and beach slope may play in nest site selection, additional factors are

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likely to also influence nesting behavior. Sea floor topography has been reported as one component, with nesting turtles preferentially following trenches in the sea floor running perpendicular to the beach (Clune et al. 2008). The strong current running parallel to Playa Gandoca immediately offshore (Cortés et al. 1998) likely keeps this aspect of nest site selection in a dynamic state.

Offshore structures, such as reefs, are unnavigable to turtles as they approach the shore (Eckert 1987). The presence of a reef in the vicinity of Punta Mona at Playa Gandoca may explain why no turtles were observed nesting in the northern 0.8 km of the beach during this study, despite the fact that it appeared to meet ideal nest site criteria.

Some studies have suggested that leatherbacks engage in a scatter nesting strategy with nests randomly distributed along the length of a beach, which places at least a portion of nests at stable locations safe from erosion (Mrosovsky 1983, Tucker 1990). This is a seemingly maladaptive nesting strategy given the erosion-prone nature of leatherback beaches, as it would still lead to a relatively high rate of nest loss to erosion (Eckert 1987). Other evidence has also been gathered contrary to the scatter nesting hypothesis, such as at Grande Riviere Beach in Trinidad and Tobago where a general east to west shift in nesting on the beach was observed as an apparent response to increasing erosion on the eastern end (Lee Lum 2005).

The finding of this study are consistent with those of Lee Lum (2005), and contradict the scatter nesting hypothesis in support of a nesting strategy that takes into account the threat posed by erosion on nesting beaches.

This may be good news for the leatherbacks’ future, as nesting behavior that takes into account erosion may allow turtles to better adapt to the erosive effects of rising sea levels, rather than randomly losing more nests.

Conservation work at Playa Gandoca has had profound effects in helping

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maintain the vitality of the leatherback population in the rookery. Illegal poaching, which two decades ago affected nearly 100% of all eggs laid, now hovers around 1%-3% thanks to conservation efforts (Chacón & Eckert 2007). However, even if leatherbacks do not actively select stable areas of the beach, the results of this study indicate that they are still nesting at stable locations. If this is truly the case, nest relocation practices should be carefully reconsidered.

Slightly over three quarters of the nests recorded in this study were laid near the high tide line on the outer portion of the beach, nesting behavior typical of the species (Chacón et al.

2007). This indicates that differences found in sand temperature at nest depths between natural, mid-beach locations and sites of relocated nests along the vegetation may be an accurate representation of a negative effect of nest relocation practices. A one-half degree deviation from a 29.5° C pivotal incubation temperature can change the sex ratios of leatherback hatchlings to nearly 100% female (warmer temps) or male (cooler temps) (Binckley et al. 1998). In an attempt to conserve leatherbacks, nest relocation practices at Playa Gandoca may be inadvertently skewing the sex ratios of hatchlings towards an unnatural male bias.

Even nests relocated to hatcheries are not completely safe. During the 2008-2009 nesting season at Playa Gandoca, an entire hatchery full of nests was lost to erosion, and it was not the first time (D. Chacón & C. Figgener, pers. comm.). In light of the stability of leatherback nest sites discovered in this study, and given the potential risks of nest relocation previously discussed, the necessity of hatcheries and the widespread practice of nest relocation should be reevaluated.

Research into the natural leatherback sex ratios at Playa Gandoca, which includes the effects of global warming and the subsequent increase in nest temperature, is necessary for effective

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Spanier, M J management of the species. The removal of old growth forests and the beach shading they provide may have already altered the natural temperatures of turtle nests. This study should serve a springboard for future, more in-depth study of nest sites and erosion to ensure the proper conservation of the critically endangered leatherback sea turtle.

Acknowledgments Many thanks are due to Didiher Chacón, Judith Sénéchal, Christine Figgener, the supportive members of the Gandoca community, WIDECAST volunteers, and Scott Pentzer along with the entire staff of the Associated Colleges of the Midwest Costa Rica program for their help in the planning, execution, and writing of this research. I am also indebted to Brian Linkhart and Marc Snyder for their advisory work and help with the extensive revisions of this manuscript.

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