1. science
2. biology
3. molecular biology

COVER SHEET FOR PROPOSAL TO THE NATIONAL SCIENCE FOUNDATION
PROGRAM ANNOUNCEMENT/SOLICITATION NO./CLOSING DATE/If not in response to a program announcement/solicitation enter NSF 00-2
FOR NSF USE ONLY
NSF PROPOSAL NUMBER
FOR CONSIDERATION BY NSF ORGANIZATIONAL UNIT(S) (Indicate the most specific unit known, i.e., program, division, etc.)
DATE RECEIVED
NUMBER OF COPIES
DIVISION
ASSIGNED
EMPLOYER IDENTIFICATION NUMBER (EIN) OR
TAXPAYER IDENTIFICATION NUMBER (TIN)
FUND CODE
DUNS # (Data Universal Numbering System)
SHOW PREVIOUS AWARD NO. IF THIS IS
FILE LOCATION
IS THIS PROPOSAL BEING SUBMITTED TO ANOTHER FEDERAL
A RENEWAL
AGENCY?
YES
NO
IF YES, LIST ACRONYM(S)
AN ACCOMPLISHMENT-BASED RENEWAL
NAME OF ORGANIZATION TO WHICH AWARD SHOULD BE MADE
ADDRESS OF AWARDEE ORGANIZATION, INCLUDING 9 DIGIT ZIP CODE
University of Florida, Dept. of Zoology
611A Bartram Hall, Dept. of Zoology, University of Florida
P.O. Box 118525
Gainesville, FL 32611-8525
AWARDEE ORGANIZATION CODE (IF KNOWN)
NAME OF PERFORMING ORGANIZATION, IF DIFFERENT FROM
ABOVE
ADDRESS OF PERFORMING ORGANIZATION, IF DIFFERENT, INCLUDING 9 DIGIT ZIP CODE
PERFORMING ORGANIZATION CODE (IF KNOWN)
IS AWARDEE ORGANIZATION (Check All That Apply)
(See GPG II.D.1 For Definitions)
FOR-PROFIT ORGANIZATION
SMALL BUSINESS
MINORITY BUSINESS
WOMAN-OWNED BUSINESS
TITLE OF PROPOSED PROJECT Small things matter: Corals, microbes & environmental stress
REQUESTED AMOUNT
PROPOSED DURATION (1-60 MONTHS)
REQUESTED STARTING DATE
36
5/2008
$
SHOW RELATED PREPROPOSAL NO.,
IF APPLICABLE
months
CHECK APPROPRIATE BOX(ES) IF THIS PROPOSAL INCLUDES ANY OF THE ITEMS LISTED BELOW
BEGINNING INVESTIGATOR (GPG I.A.3)
VERTEBRATE ANIMALS (GPG II.D.12) IACUC App. Date
DISCLOSURE OF LOBBYING ACTIVITIES (GPG II.D.1)
PROPRIETARY & PRIVILEGED INFORMATION (GPG I.B, II.D.7)
HUMAN SUBJECTS (GPG II.D.12)
Exemption Subsection
or IRB App. Date
NATIONAL ENVIRONMENTAL POLICY ACT (GPG II.D.10)
INTERNATIONAL COOPERATIVE ACTIVITIES: COUNTRY/COUNTRIES
HISTORIC PLACES (GPG II.D.10)
SMALL GRANT FOR EXPLOR. RESEARCH (SGER) (GPG II.D.12)
FACILITATION FOR SCIENTISTS/ENGINEERS WITH DISABILITIES (GPG V.G.)
RESEARCH OPPORTUNITY AWARD (GPG V.H)
PI/PD DEPARTMENT
PI/PD POSTAL ADDRESS
Zoology
PI/PD FAX NUMBER
611A Bartram Hall, Dept. of Zoology, University of Florida
P.O. Box 118525
Gainesville, FL 32611-8525
NAMES (TYPED)
High Degree
Yr of Degree
Telephone Number
Electronic Mail Address
MA
1990
352-443-9684
[email protected]
PI/PD NAME
Carol L. Chaffee
CO-PI/PD
CO-PI/PD
CO-PI/PD
CO-PI/PD
NSF Form 1207 (10/99)
Page 1 of 2
1
CERTIFICATION PAGE
Certification for Principal Investigators and Co-Principal Investigators
I certify to the best of my knowledge that:
(1) the statements herein (excluding scientific hypotheses and scientific opinions) are true and complete, and
(2) the text and graphics herein as well as any accompanying publications or other documents, unless otherwise indicated, are the original work of the
signatories or individuals working under their supervision. I agree to accept responsibility for the scientific conduct of the project and to provide the
required project reports if an award is made as a result of this proposal.
I understand that the willful provision of false information or concealing a material fact in this proposal or any other communication submitted to NSF is a
criminal offense (U.S.Code, Title 18, Section 1001).
Name (Typed)
PI/PD
Signature
Social Security No.*
Carol L. Chaffee
Date
3/7/2008
Co-PI/PD
Co-PI/PD
Co-PI/PD
Co-PI/PD
Certification for Authorized Organizational Representative or Individual Applicant
By signing and submitting this proposal, the individual applicant or the authorized official of the applicant institution is: (1) certifying that statements made herein
are true and complete to the best of his/her knowledge; and (2) agreeing to accept the obligation to comply with NSF award terms and conditions if an award is
made as a result of this application. Further, the applicant is hereby providing certifications regarding Federal debt status, debarment and suspension, drug-free
workplace, and lobbying activities (see below), as set forth in the Grant Proposal Guide (GPG), NSF 00-2. Willful provision of false information in this application
and its supporting documents or in reports required under an ensuing award is a criminal offense (U.S. Code, Title 18, Section 1001).
In addition, if the applicant institution employs more than fifty persons, the authorized official of the applicant institution is certifying that the institution has
implemented a written and enforced conflict of interest policy that is consistent with the provisions of Grant Policy Manual Section 510; that to the best of his/her
knowledge, all financial disclosures required by that conflict of interest policy have been made; and that all identified conflicts of interest will have been
satisfactorily managed, reduced or eliminated prior to the institution’s expenditure of any funds under the award, in accordance with the institution’s conflict of
interest policy. Conflicts that cannot be satisfactorily managed, reduced or eliminated must be disclosed to NSF.
Debt and Debarment Certifications
(If answer “yes” to either, please provide explanation.)
Is the organization delinquent on any Federal debt?
Is the organization or its principals presently debarred, suspended, proposed for debarment, declared ineligible,
or voluntarily excluded from covered transactions by any Federal Department or agency?
Yes
No
Yes
No
Certification Regarding Lobbying
This certification is required for an award of a Federal contract, grant or cooperative agreement exceeding $100,000 and for an award of a Federal loan or
a commitment providing for the United States to insure or guarantee a loan exceeding $150,000.
Certification for Contracts, Grants, Loans and Cooperative Agreements
The undersigned certifies, to the best of his or her knowledge and belief, that:
(1) No Federal appropriated funds have been paid or will be paid, by or on behalf of the undersigned, to any person for influencing or attempting to
influence an officer or employee of any agency, a Member of Congress, an officer or employee of Congress, or an employee of a Member of Congress in
connection with the awarding of any federal contract, the making of any Federal grant, the making of any Federal loan, the entering into of any cooperative
agreement, and the extension, continuation, renewal, amendment, or modification of any Federal contract, grant, loan, or cooperative agreement.
(2) If any funds other than Federal appropriated funds have been paid or will be paid to any person for influencing or attempting to influence an officer or
employee of any agency, a Member of Congress, and officer or employee of Congress, or an employee of a Member of Congress in connection with this
Federal contract, grant, loan, or cooperative agreement, the undersigned shall complete and submit Standard Form LLL, “Disclosure of Lobbying
Activities,” in accordance with its instructions.
(3) The undersigned shall require that the language of this certification be included in the award documents for all subawards at all tiers including
subcontracts, subgrants, and contracts under grants, loans, and cooperative agreements and that all subrecipients shall certify and disclose accordingly.
This certification is a material representation of fact upon which reliance was placed when this transaction was made or entered into. Submission of this
certification is a prerequisite for making or entering into this transaction imposed by Section 1352, Title 31, U.S. Code. Any person who fails to file the
required certification shall be subject to a civil penalty of not less than $10,000 and not more than $100,000 for each such failure.
AUTHORIZED ORGANIZATIONAL REPRESENTATIVE
NAME/TITLE (TYPED)
TELEPHONE NUMBER
SIGNATURE
ELECTRONIC MAIL ADDRESS
DATE
FAX NUMBER
*SUBMISSION OF SOCIAL SECURITY NUMBERS IS VOLUNTARY AND WILL NOT AFFECT THE ORGANIZATION’S ELIGIBILITY FOR AN AWARD. HOWEVER, THEY ARE AN
INTEGRAL PART OF THE NSF INFORMATION SYSTEM AND ASSIST IN PROCESSING THE PROPOSAL. SSN SOLICITED UNDER NSF ACT OF 1950, AS AMENDED.
Page 2 of 2
2
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Project Summary
Stress has been an omnipresent evolutionary force, but human development has altered
both the nature and level of stress that many organisms face in their environments.
Changes in temperatures, nutrient spikes due to agricultural runoff and acidification of
the oceans due to changes in the concentration of carbon dioxide in the atmosphere are
a few examples of how development has altered the levels of stress in many ecosystems.
The coral reef system is one that is both particularly valuable and particularly fragile,
although susceptibility to stress-related damage appears to be variable. This project will
explore how interactions between corals and the microbial organisms that exist in close
proximity to them influence this variability.
Two broad questions drive this project:
1. What characteristics of microbial assemblages do corals that are more resistant to a
particular stressor share?
2. Are there particular microbial markers that indicate resistance or susceptibility?
To explore these questions, I will use a combination of field experiments and molecular
techniques to explore different stress responses of corals found in Moorea, French
Polynesia. The study will examine variable responses to four stressors that span a range
of human-influenced factors:
1. Turf and macro-algae appear to vary considerably based on a number of factors,
including nutrient availability.
2. Vermetid gastropods have been observed to adversely affect the growth of massive
corals, which are key reef-builders.
3. Temperature has a strong influence on corals, with many able to function in only a
narrow range.
4. Acidity affects how well corals are able to deposit the calcium carbonate that
forms their skeletons.
Samples will be collected in Moorea during different seasons and at different levels of
stressor exposure, then molecular genotyping techniques will be used to characterize
both the symbiotic algae and bacterial communities.
Three initiatives are included in this project to address broader impacts:
1. Undergraduates will be integral members of the project team, and will be involved
with both field collection and molecular analysis.
2. Screening tools designed to identify the presence or absence of key microbial
markers will also be developed, with the goal of disseminating these for use in
managing coral reef ecosystems.
3. Production of a “virtual experiment” web site aimed at middle to high school
students that will expose them to both the process and results of this study.
3
TABLE OF CONTENTS
For font-size and page-formatting specifications, see GPG Section II.C.
Total No. of
Pages in Section
Section
Page No.*
(Optional)*
Cover Sheet (NSF Form 1207) (Submit Page 2 with original proposal only)
A
Project Summary (not to exceed 1 page)
1
3
B
Table of Contents (NSF Form 1359)
1
4
C
Project Description (including Results from Prior NSF Support)
(not to exceed 15 pages) (Exceed only if allowed by a specific
program announcement/solicitation or if approved in advance by the
appropriate NSF Assistant Director or designee)
7
5
D
References Cited
2
12
E
Biographical Sketches (Not to exceed 2 pages each)
1
14
F
Budget
(NSF Form 1030, plus up to 3 pages of budget justification)
2
15
G
Current and Pending Support (NSF Form 1239)
1
16
H
Facilities, Equipment and Other Resources (NSF Form 1363)
1
17
I
Special Information/Supplementary Documentation
0
J
Appendix (List below)
Include only if allowed by a specific program announcement/
solicitation or if approved in advance by the appropriate NSF
Assistant Director or designee)
0
Appendix Items:
*Proposers may select any numbering mechanism for the proposal. The entire proposal, however, must be paginated. Complete
both columns only if the proposal is numbered consecutively.
NSF Form 1359 (10/99)
46
4
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Small things matter: Corals, microbes & environmental stress
Introduction
Stress is a daily part of life for many, if not most, organisms. Stress may be chronic, such
as the daily fluctuations in temperature and salinity faced by intertidal organisms
(Gilman et al. 2006) or it may be acute, such as the disturbance caused by a hurricane.
As such, stress has been an evolutionary force throughout history. In recent years,
however, human development has increased both the nature and the level of stress that
organisms face in their environments. The latest report (2007) issued by the
Intergovernmental Panel on Climate Change (IPCC) details a wide variety of
environmental stressors that are attributable to anthropogenic impacts, including
increased temperatures, eutrophication and acidification of the oceans due to changes in
the concentration of carbon dioxide in the atmosphere. Recent reports on the effects of
these factors (Coelho and Manfrino 2007; Millar et al. 2007; Gillespie et al. 2008; Jin 2008)
show that a diverse array of ecosystems is threatened.
Among the ecosystems affected by these anthropogenically-driven stressors, coral reefs
are one of the most biologically diverse (Sebens 1994). Coral reefs are also economically
important (van Oppen and Gates 2006; Rosenberg et al. 2007), with annual global
economic contributions exceeding US$30 billion (Rosenberg et al. 2007). These systems,
however, are highly susceptible to damage from a wide range of stressors, both natural
and anthropogenic. For example, crown-of-thorns starfish (Acanthaster planci) outbreaks
linked to nutrient spikes (Brodie et al. 2005), coral bleaching caused by elevated
temperatures (Penin et al. 2007) and disease outbreaks linked to a variety of
environmental changes (Ainsworth et al. 2007; Hall-Spencer et al. 2007; Luna et al. 2007;
Toledo-Hernandez et al. 2007; Yarden et al. 2007) also appear to have been increasing in
recent years.
Some studies have shown, however, that certain corals, even different representatives of
the same species, are more resilient in the face of various stressors (van Oppen and
Gates 2006). Because corals have unique relationships with various microbial species,
especially their endosymbiotic dinoflagellate algae, Symbiodinium sp., exploration of
how variation in coral-associated microbial species may help explain some of the
variability in this resilience. For example, there is evidence, as reviewed by Baker (2003),
indicating that the same coral species may harbor different Symbiodinium clades under
different environmental conditions. Given that certain clades have been shown to
provide thermal resilience to their hosts (Berkelmans and van Oppen 2006), a detailed
study of algal symbiont variation in relation to resistance to environmental stressors
will indicate if certain symbiont clades generally improve the resilience of their hosts.
Alternatively, some clades may improve responses to one type of stressor, whereas
other clades improve responses to a different type of stressor.
Bacteria that are closely associated with corals have also been shown to mediate coral
interactions with the surrounding environment and with other organisms. Bacterial
have been shown to play both beneficial and detrimental roles in their associations with
5
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
corals, but a detailed understanding of the specific mechanisms involved is generally
lacking. Are microbes altering nutrient cycling? Changing the micro-scale
environmental conditions, such as pH or oxygen concentration? Preventing or
promoting growth of other organisms, which, in turn, may be either beneficial or
harmful? In order to uncover the answers to questions such as these, however, the types
of microbes present under a variety conditions (healthy and stressed) must first be
identified. The composition of microbial biofilms covering substrates has been shown as
factor in the settlement and metamorphosis of coral larvae (Negri et al. 2001; Webster et
al. 2004). Bacterial communities may also mediate interactions with possible coral
competitors, as it has been demonstrated that surface microbes on some corals may
prevent settlement of larvae of other invertebrate species (Dobretsov and Qian 2004). As
with the zooxanthellae, the range of interactions that may be mediated by these
bacterial communities make them an appropriate target for the study of variation in the
resilience of corals to environmental stressors.
Study System
Field collection sites are located in
Moorea, French Polynesia (Figure 1). I
will be working out of UC Berkeley’s
Richard B. Gump South Pacific
Research Station, where my lab group
at the University of Florida, the
St.Mary-Osenberg-Bolker lab, has
several ongoing research projects.
Adrian Stier and Jada-Simone White,
who are fellow graduate students in
this lab group, have performed
preliminary work showing some of the
effects of environmental stress on
corals in this area. Both Adrian and
Jada are studying different aspects of
the reef system that pair with my
study. As such, we are collaborating to
develop a more complete picture of the
interactions occurring in this system
than any one of our projects could
provide.
Figure 1. Map of Moorea, French
Polynesia, showing location of study site
in lagoon near Gump Station.
6
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Research Questions
1. Is there variation in the response of corals to environmental stressors that is related to the
identity of associated bacteria or zooxanthellae? The fundamental biological process on
which this research aims to shed light is the response of corals to environmental
stress. In particular, I will examine how two types of coral-associated microbes
(symbiotic algae and coral-mucus bacterial communities) differ between unstressed
conditions and several types of environmental stress. There are two possible
scenarios that may explain observed differences in the microbial communities: 1) the
community structure actually changed, but the corals did not, or 2) corals associated
with certain microbial strains survived either better or worse than other corals,
which thus changed the observed community composition. By testing how the
microbial communities change when stressors are introduced or removed, I
hypothesize that it will be possible to determine which of these two scenarios
applies.
2. Are there particular
microbial markers that
indicate resistance or
susceptibility? A
secondary goal of this
project is to use the
genetic data collected
to identify particular
microbial species,
either algae, bacteria,
or both, that are
correlated with the
coral response. If such
markers can be
identified, then
diagnostic tools may
be developed that
would allow
Figure 2. Bleaching front associated with turf algae on
stakeholders such as
Porites in the lagoon of Moorea, French Polynesia.
managers of marine
Photo provided by Jada-Simone White.
protected areas to
identify areas where
adverse responses appear likely. This information could then be used to help
develop plans to mitigate the response.
Three particular environmental stressors will be evaluated:
1. Turf and macro-algae. As shown in Figure 2, massive Porites sp. exhibit a bleaching
front where they come in contact with turf algae. Based on initial evidence that both
flow rate and the presence of sediment influence this response (J.-S. White, pers.
7
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
com.), I hypothesize that the mucus of corals adjacent to turf algae will harbor
pathogenic bacterial strains.
2. Vermetid gastropods. Some massive Porites sp. show significantly reduced growth in
the presence of Dendropoma maxima (A. Stier, pers. com.). Because this pattern does
not hold for all Porites colonies that have been observed, I will compare resistant
with susceptible colonies to determine if any consistent patterns in the microbial
assemblages occur.
3. Temperature. Corals are particularly susceptible to temperature stress (HoeghGuldberg et al. 2007), but sensitivity to temperature fluctions has been shown to
vary at both the individual and the population level (Smith-Keune and van Oppen
2006). Certain Symbiodinium clades have also been shown to reduce the susceptibility
of their coral hosts to bleaching (van Oppen et al. 2005). Thus, I hypothesize that
Symbiodinium clades will exhibit a predictable pattern in resistant versus susceptible
corals. In addition, I will determine how the bacterial community composition varies
with resistance to these stressors.
Research Activities
The following series of studies will be performed for each of the above stressors. Both
the field and laboratory experiments will be conducted for a period of four weeks, so
that bacterial communities have adequate time to form and mature into a relatively
steady-state.
1. Field Survey. I will sample sites that naturally vary in their response to the stressor
being evaluated, then analyze both the algal symbionts and mucus-associated
bacterial communities as described below. The goal of these surveys will be to
determine the broad patterns that are observable on the colony level. Sites will be
selected to reduce the variability of other environmental factors as much as possible,
e.g., samples will be taken from both stressed and unstressed locations on the same
coral head as a control for genetic variability of the corals.
2. Field Experiment. Coral nubbins (juveniles) will be used to perform manipulative
experiments in the field. These experiments are designed to evaluate microbial
changes that occur at the time a stressor is introduced or removed. Nubbins will be
prepared, then allowed to acclimate in situ for a period of two weeks. After
acclimation, nubbins will be moved to the experimental sites, and the algal
symbionts and bacterial communities will be analyzed as described below.
3. Laboratory Experiment. To provide a means for detailed manipulations that are either
not practical or not ethical to perform in the field, I will also manipulate coral
nubbins in laboratory tanks with flow-through seawater. In each case, nubbins will
be introduced into the tanks, allowed to acclimate for two weeks, then the stressor
will be applied, and the algal symbionts and bacterial communities will be analyzed
as described below.
8
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Two basic sets of activities will be performed for each study described above: analysis
of the algal symbionts and characterization of the microbial community found in the
coral mucus.
1. Algal Symbiont Analysis. To assess how genetic variation of the symbiotic
dinoflagellates (Symbiodinium sp.) affects the interaction of corals with each of the
environmental stressors, the phylotype of these zooxanthellae will be determined.
The psbA gene of the chloroplast will be used, based on previous work (Barbrook et
al. 2006) that has shown this gene as a reliable indicator of Symbiodinium phylotypes.
A particular benefit of using a chloroplast marker is that corals themselves do not
have chloroplasts, so there is no need to perform special steps to isolate the
dinoflagellate DNA from the coral DNA. If any coral DNA is included in the PCR
reaction mixture, it will not be amplified, so it will not affect the downstream
sequencing and analysis.
At the conclusion of each study described above, small coral samples will be
collected from coral colonies that show resistance to a stressor, those that show
susceptibility to that stressor, and control colonies that are not exposed to the
stressor. Symbiodinium will be separated from coral samples using centrifugation
and washes with filtered seawater. The algal DNA will extracted, and an
approximately 400 base pair fragment of the psbA chloroplast gene will be amplified
using the IA2F/IA2R primer pair and thermocycler conditions described by
Barbrook and colleagues (2006). After purification, these amplicons will be
sequenced using the automated sequencers at the University of Florida’s
Interdisciplinary Center for Biotechnology Research (ICBR). All sequences produced
will be submitted to NCBI’s GenBank (Benson et al. 2007),
NCBI BLAST (Altschul et al. 1997) queries for all sequences will be performed to
determine the phylotypes present in the sampled corals. Sequences (including the
dinoflagellate alga Gymnodinium simplex (Acc. no. AB096158) as an outgroup) will be
aligned using ClustalX (Thompson et al. 1997), then manually adjusted as necessary
in MacClade (Maddison and Maddison 2003). The most appropriate model of
nucleotide evolution will be determined using ModelTest (Posada and Crandall
1998). Maximum-likelihood trees, including a 1000-replicate bootstrap, will be built
using GARLI (Zwickl 2006). The pattern of phylotypes associated with either
resistance or susceptibility will then be analyzed with MacClade by treating this as a
character with two possible states.
2. Microbial Community Characterization. The microbial communities inhabiting the
mucus covering the corals included in this project will be characterized to determine
what, if any, alterations occur under the conditions of environmental stress being
studied. Because of the significant biases that may be introduced by using culturedependent methods to identify the bacteria and archaea present in marine samples
(Amann et al. 1995), identification will be performed using a fragment of 16S rRNA
gene. For manipulative field experiments, where the stressor will be introduced to
the study corals, mucus samples will be collected using disposable plastic pipets
prior to application of the stressor, then every three days for the first two weeks of
9
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
the study, so that the alterations of the microbial community in relation to the onset
of a disturbance may be detected. Following this initial period, samples will be
collected weekly for the remainder of the four-week study. DNA will be extracted,
and an internal fragment of the 16S rRNA gene will be amplified using the universal
bacterial primers 517F and 1492R, then cloned and prepared for sequencing as
described by Uthicke and McGuire (2007). The purified PCR products after cloning
will be sequenced using the automated sequencers at the University of Florida’s
ICBR. All sequences produced will be submitted to NCBI’s GenBank.
NCBI BLAST queries for all sequences will be performed to determine the nearest
matches. Sequences (including the α-proteobacterium Defluvicoccus vanus (Acc. no
AF179678) as an outgroup) will be aligned using ClustalX, then manually adjusted
as necessary using MacClade. The most appropriate model of nucleotide evolution
will be determined using Model Test. Maximum-likelihood trees, including a 1000replicate bootstrap, will be built using GARLI. Comparisons between the
communities found will be compared (resistant versus susceptible) using PRIMER 6
for Windows (PRIMER-E Ltd., Plymouth, UK), as described by Uthicke and
McGuire (2007). Longitudinal patterns based on the time series data will also be
evaluated.
Broader Impacts
Beyond dissemination of my results to the scientific community, I will include two
particular activities aimed at involve a broader audience in understanding both this
study’s results and the process used to obtain them.
1. Undergraduate research mentoring. Because the volume of lab work will be extensive,
undergraduate research assistants will be involved in all laboratory phases
performed at the University of Florida in Gainesville. I anticipate that additional
side projects for these undergraduates will be developed as they learn the skills
required to perform molecular analyses on projects of their own design, so time for
mentoring will be built into the project plans. An undergraduate field assistant will
also participate in at least one field experiment in Moorea, so that s/he will have an
opportunity to participate in the complete set of activities that are included in this
project.
2. Production of a “virtual experiment” web site. By the time students reach the university
level, many have already decided whether or not to pursue study in the sciences.
Thus, exposing middle and high school students to cutting-edge scientific
techniques is one method of increasing the number of students who enter college
with a positive view of scientific study. I will use this project to develop a web site
designed to walk students in grades 6-12 through the steps involved in exploring an
ecological question. Santa Fe Community College in Gainesville has an associate of
applied arts program in web design. One requirement of this program is
development of a professional portfolio, so I will partner with a student in this
program to implement this web project. Because of its popular appeal, the coral reef
ecosystem provides a platform on which some of the more advanced scientific
10
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
techniques may be presented in a way that is interesting and fun for students in this
age range. This web tool will be presented at forums aimed at middle and high
school teachers, so that they may incorporate it into classroom activities.
3. Involvement of stakeholders in tool development. The high number of DNA sequences
that will be generated over the course of this project, as well as the assessment of
whether those sequences were taken from “resistant” or “susceptible” corals, means
that it will be possible to develop a screening tool. For this tool to provide the
maximum benefit to those responsible for managing coral reefs, stakeholders, such
as managers of marine protected areas, will be actively included in the development
process. It is anticipated that development of this tool will be in concert with a nongovernmental organization that is involved in protection and management of coral
reefs. Identification of possible partner organizations is a current priority.
11
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Literature Cited
Ainsworth TD, Kramasky-Winter E, Loya Y, Hoegh-Guldberg O, Fine M (2007) Coral disease diagnostics: What's
between a plague and a band? Applied and Environmental Microbiology 73:981-992
Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research 25:3389-3402
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in-situ detection of individual microbial
cells without cultivation. Microbiological Reviews 59:143-169
Baker AC (2003) Flexibility and specificity in coral-algal symbiosis: Diversity, ecology, and biogeography of
Symbiodinium. Annual Review of Ecology Evolution and Systematics 34:661-689
Barbrook AC, Visram S, Douglas AE, Howe CJ (2006) Molecular diversity of dinoflagellate symbionts of Cnidaria:
The psbA minicircle of Symbiodinium. Protist 157:159-171
Benson D, Karsch-Mizrachi I, Lipman D, Ostell J, Wheeler D (2007) GenBank. Nucleic Acids Res 35:D21-25
Berkelmans R, van Oppen M (2006) The role of zooxanthellae in the thermal tolerance of corals: a 'nugget of hope'
for coral reefs in an era of climate change. Proceedings of the Royal Society B-Biological Sciences
273:2305-2312
Brodie J, Fabricius K, De'ath G, Okaji K (2005) Are increased nutrient inputs responsible for more outbreaks of
crown-of-thorns starfish? An appraisal of the evidence. Marine Pollution Bulletin 51:266-278
Change IPoC (2007) Impacts, Adaptation and Vulnerability: Contribution of Working Group II to the Fourth
Assessment Report of the IPCC. Cambridge University Press, New York, NY
Coelho VR, Manfrino C (2007) Coral community decline at a remote Caribhean island: Marine no-take reserves are
not enough. Aquatic Conservation-Marine and Freshwater Ecosystems 17:666-685
Dobretsov S, Qian PY (2004) The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp in
the inhibition of larval settlement. J Exp Mar Biol Ecol 299:35-50
Gillespie RG, Claridge EM, Roderick GK (2008) Biodiversity dynamics in isolated island communities: interaction
between natural and human-mediated processes. Mol Ecol 17:45-57
Gilman SE, Harley CDG, Strickland DC, Vanderstraeten O, O'Donnell MJ, Helmuth B (2006) Evaluation of
effective shore level as a method of characterizing intertidal wave exposure regimes. Limnology and
Oceanography-Methods 4:448-457
Hall-Spencer JM, Pike J, Munn CB (2007) Diseases affect cold-water corals too: Eunicella verrucosa (Cnidaria :
Gorgonacea) necrosis in SW England. Dis Aquat Org 76:87-97
Hoegh-Guldberg O, Mumby PJ, Hooten AJ, Steneck RS, Greenfield P, Gomez E, Harvell CD, Sale PF, Edwards AJ,
Caldeira K, Knowlton N, Eakin CM, Iglesias-Prieto R, Muthiga N, Bradbury RH, Dubi A, Hatziolos ME
(2007) Coral reefs under rapid climate change and ocean acidification. Science 318:1737-1742
Jin CH (2008) Biodiversity dynamics of freshwater wetland ecosystems affected by secondary salinisation and
seasonal hydrology variation: a model-based study. Hydrobiologia 598:257-270
Luna GM, Biavasco F, Danovaro R (2007) Bacteria associated with the rapid tissue necrosis of stony corals.
Environmental Microbiology 9:1851-1857
Maddison D, Maddison W (2003) MacClade 4: Analysis of phylogeny and character evolution. Sinauer Associates,
Sunderland, MA
Millar CI, Stephenson NL, Stephens SL (2007) Climate change and forests of the future: Managing in the face of
uncertainty. Ecological Applications 17:2145-2151
Negri AP, Webster NS, Hill RT, Heyward AJ (2001) Metamorphosis of broadcast spawning corals in response to
bacteria isolated from crustose algae. Marine Ecology-Progress Series 223:121-131
Penin L, Adjeroud M, Schrimm M, Lenihan HS (2007) High spatial variability in coral bleaching around Moorea
(French Polynesia): patterns across locations and water depths. Comptes Rendus Biologies 330:171-181
Posada D, Crandall K (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817-818
Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I (2007) The role of microorganisms in coral health,
disease and evolution. Nature Reviews Microbiology 5:355-362
Sebens KP (1994) Biodiversity of coral reefs: What are we losing and why? Am Zool 34:115-133
Smith-Keune C, van Oppen M (2006) Genetic structure of a reef-building coral from thermally distinct
environments on the Great Barrier Reef. Coral Reefs 25:493-502
Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The CLUSTAL_X windows interface:
flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research
25:4876-4882
12
Carol Chaffee Doctoral Dissertation Improvement Grant
2008
Toledo-Hernandez C, Bones-Gonzalez A, Ortiz-Vazquez OE, Sabat AM, Bayman P (2007) Fungi in the sea fan
Gorgonia ventalina: diversity and sampling strategies. Coral Reefs 26:725-730
Uthicke S, McGuire K (2007) Bacterial communities in Great Barrier Reef calcareous sediments: Contrasting 16S
rDNA libraries from nearshore and outer shelf reefs. Estuar Coast Shelf Sci 72:188-200
van Oppen M, Gates R (2006) Conservation genetics and the resilience of reef-building corals. Mol Ecol 15:38633883
van Oppen M, Mahiny A, Done T (2005) Geographic distribution of zooxanthella types in three coral species on the
Great Barrier Reef sampled after the 2002 bleaching event. Coral Reefs 24:482-487
Webster NS, Smith LD, Heyward AJ, Watts JEM, Webb RI, Blackall LL, Negri AP (2004) Metamorphosis of a
scleractinian coral in response to microbial biofilms. Applied and Environmental Microbiology 70:12131221
Yarden O, Ainsworth TD, Roff G, Leggat W, Fine M, Hoegh-Guldberg O (2007) Increased prevalence of ubiquitous
ascomycetes in an acropoid coral (Acropora formosa) exhibiting symptoms of brown band syndrome and
skeletal eroding band disease. Applied and Environmental Microbiology 73:2755-2757
Zwickl D (2006) Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets
under the maximum likelihood criterion. Ph.D. dissertation. The University of Texas at Austin
13
Biographical Sketch
Carol L. Chaffee
Professional Preparation
University of California, Berkeley
San Jose State University
University of Southern California
University of Florida
Social Science
Molecular Biology
Communications Management
Zoology
BA 1989
BS 2007
MA 1990
PhD in progress
Appointments
2007 – present
Spring 2008
Fall 2007
2006 – 2007
2003 – 2007
Summer, 2006
PhD student, University of Florida
Lab Instructor, Introduction to Biology, University of Florida
Graduate Assistant, R/V Bellows, University of Florida
Research Assistant, Ouverney Lab, San Jose State University
Volunteer Naturalist, Fitzgerald Marine Life Reserve, Moss Beach, CA
Student (MARS 3102), Marine Microscopy, University of Queensland
Synergistic Activities
(i) Ouverney Lab. Because Dr. Ouverney was in the process of establishing his lab when
I began working with him, I had the opportunity to assist in developing the day-today lab operations protocols in consultation with Dr. Ouverney and the two other
initial undergraduate researchers. Our lab participated in RUMBA (Research by
Undergraduates using Molecular Biology Applications), San Jose State’s NSF-REU
program. My role in this effort was to train the participants in how quantitative PCR
could be used in their research projects, and to assist those who chose to use this tool
to accomplish their goals within the short time available in the summer program.
(ii) Fitzgerald Marine Life Reserve. As a volunteer naturalist at this intertidal marine
reserve, I worked with elementary school teachers to tailor our guided tours of the
reserve to fit with their classroom activities. Tours were given to groups of students
ranging from first grade through high school. In addition to interpreting the various
sub-habitats of the intertidal, we also worked to give a broader understanding of
this coastal area, by discussing how native Ohlone people used the reserve area in a
sustainable manner for thousands of years, but how modern development has
severely impacted this area within 100 years.
Collaborators & Other Affiliations
Collaborators
Adrien Stier, University of Florida
Jada-Simone White, University of Florida
Doctoral Advisor
Craig W. Osenberg, University of Florida
14
FOR NSF USE ONLY
5
4
SUMMARY PROPOSAL BUDGET
ORGANIZATION
PROPOSAL NO.
DURATION (MONTHS)
University of Florida
Proposed
PRINCIPAL INVESTIGATOR/PROJECT DIRECTOR
Granted
AWARD NO.
Carol L. Chaffee
A. SENIOR PERSONNEL: PI/PD, Co-PIs, Faculty and Other Senior Associates
NSF-Funded
List each separately with name and title. (A.7. Show number in brackets)
Person-months
CAL ACAD SUMR
1. Carol L. Chaffee
2.
3.
4.
5.
6. (
) OTHERS (LIST INDIVIDUALLY ON BUDGET EXPLANATION PAGE)
7. (1) TOTAL SENIOR PERSONNEL (1-6)
B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)
1. (
) POSTDOCTORAL ASSOCIATES
2. (
) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.)
3. (
) GRADUATE STUDENTS
4. (2) UNDERGRADUATE STUDENTS
5. (
) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY)
6. (
) OTHER
TOTAL SALARIES AND WAGES (A + B)
C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS)
TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C)
D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)
Funds
Granted by NSF
Proposer
$0
(If Different)
$
0
10,400
10,400
10,400
TOTAL EQUIPMENT
E. TRAVEL
1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS)
2. FOREIGN
F. PARTICIPANT SUPPORT
1. STIPENDS
$
2. TRAVEL
3. SUBSISTENCE
4. OTHER
TOTAL NUMBER OF PARTICIPANTS (
)
COSTS
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES
2. PUBLICATION/DOCUMENTATION/DISSEMINATION
3. CONSULTANT SERVICES
4. COMPUTER SERVICES
5. SUBAWARDS
Funds
Requested By
14,560
TOTAL PARTICIPANT
12,000
4,800
30,000
6. OTHER
TOTAL OTHER DIRECT COSTS
H. TOTAL DIRECT COSTS (A THROUGH G)
I. INDIRECT COSTS (F&A) (SPECIFY RATE AND BASE)
71,760
TOTAL INDIRECT COSTS (F&A)
J. TOTAL DIRECT AND INDIRECT COSTS (H + I)
K. RESIDUAL FUNDS (IF FOR FURTHER SUPPORT OF CURRENT PROJECT SEE GPG II.D.7.j.)
L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K)
0
$71,760
$
M. COST SHARING: PROPOSED LEVEL $
PI/PD TYPED NAME AND SIGNATURE*
AGREED LEVEL IF DIFFERENT: $
DATE
FOR NSF USE ONLY
Carol L. Chaffee
3/7/2008
NSF Form 1030 (10/99) Supersedes All Previous Editions
*SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.C)
ORG. REP. TYPED NAME & SIGNATURE*
DATE
INDIRECT COST RATE VERIFICATION
Date Checked
Date of Rate Sheet
Initials-ORG
15
Item #
B4
E2
G1
G2
G3
Description
Justification
Undergraduate funding Two undergraduate assistants at $6.50/hour, 10
hours/week for 40 weeks (16 week fall & spring
semesters & 8 week summer) for 2 years.
Round-trip travel to
Three field seasons for PI, and one field season for
Moorea, French
an undergraduate assistant.
Polynesia from
Gainesville, FL
Field station fees at
$40/day for three 8-week field seasons for PI, and
Gump Marine Station,
one 8-week field season for an undergraduate
Moorea.
assistant.
DNA collection &
Pipets, eppendorf tubes and FTA cards for
extraction
collecting and extracting DNA from both
symbionts (3 stressors x 3 experiments x 5
replicates = 45 samples) and coral mucus bacterial
communities (3 stressors x 3 experiments x 5
replicates x 5 samples = 225 samples).
PCR, cloning &
preparation for
sequencing
Presentations at
$800 in conference fees and travel to two
conferences
conferences per year for PI, and one conference for
each of the undergraduate assistants.
DNA sequencing
Sequencing of samples at the University of
Florida’s ICBR facility. Includes sequencing psbA
fragment for each symbiont sample (3 stressors x 3
experiments x 5 replicates = 45 samples), and
sequencing 48 clones for each bacterial community
sample (3 stressors x 3 experiments x 5 replicates x
5 samples = 225 samples).
16
Current and Pending Support
(See GPG Section II.D.8 for guidance on information to include on this form.)
The following information should be provided for each investigator and other senior personnel. Failure to provide this
information may delay consideration of this proposal.
Other agencies (including NSF) to which this proposal has been/will be submitted.
Investigator: Carol L. Chaffee
Support:
Current
Pending
Submission Planned in Near Future
*Transfer of Support
Project/Proposal Title:
Corals, algae and microbes: using molecular tools to illuminate an ecological interaction
Source of Support: Sigma Xi
Total Award Amount: $1,000
Total Award Period Covered: 2008
Location of Project: Moorea, French Polynesia & Gainesville, FL
Person-Months Per Year Committed to the Project.
Support:
Current
Pending
Cal: 5.5
Acad:4
Submission Planned in Near Future
Sumr: 1.5
*Transfer of Support
Project/Proposal Title:
No specific project, funds awarded to support general doctoral research.
Source of Support: University of Florida Frank Maturo Excellence Fund
Total Award Amount: $2,750
Total Award Period Covered: 2007-2008
Location of Project: Moorea, French Polynesia & Gainesville, FL
Person-Months Per Year Committed to the Project.
Support:
Current
Pending
Cal:
Acad:
Submission Planned in Near Future
Sumr:
*Transfer of Support
Project/Proposal Title:
Source of Support:
Total Award Amount: $
Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project.
Support:
Current
Pending
Cal:
Acad:
Submission Planned in Near Future
Sumr:
*Transfer of Support
Project/Proposal Title:
Source of Support:
Total Award Amount: $
Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project.
Support:
Current
Pending
Cal:
Acad:
Submission Planned in Near Future
Sumr:
*Transfer of Support
Project/Proposal Title:
Source of Support:
Total Award Amount: $
Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project.
Cal:
Acad:
Sumr:
*If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period.
NSF Form 1239 (10/99)
USE ADDITIONAL SHEETS AS NECESSARY
5
5
17
FACILITIES, EQUIPMENT & OTHER RESOURCES
FACILITIES: Identify the facilities to be used at each performance site listed and, as appropriate, indicate their capacities, pertinent
capabilities, relative proximity, and extent of availability to the project. Use “Other” to describe the facilities at any other
performance sites listed and at sites for field studies. Use additional pages if necessary.
Laboratory: Kimball Lab, Dept. of Zoology, University of Florida
Molecular genetics laboratory with full capabilities for performing DNA extraction, PCR, cloning and preparation for
bulk sequencing at ICBR.
Clinical:
Animal:
Computer:
Office:
Other:
Gump Marine Station,
Moorea, French Polynesida
Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida
MAJOR EQUIPMENT: List the most important items available for this project and, as appropriate, identify the location and
pertinent capabilities of each.
OTHER RESOURCES: Provide any information describing the other resources available for the project. Identify support services
such as consultant, secretarial, machine shop, and electronics shop, and the extent to which they will be available for the project.
Include an explanation of any consortium/contractual/subaward arrangements with other organizations.
5
6
NSF Form 1363 (10/99)
18