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Do varying levels of sperm competition affect sperm investment, sperm attributes, and sperm allocation
in the American Horseshoe Crab, Limulus polyphemus?
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Page 2 of 2
Project Summary
The proposed study will examine how varying levels of sperm competition affect
sperm production, sperm investment, and sperm attributes in the American horseshoe
crab, Limulus polyphemus. These experiments will test the validity of models of sperm
competition and will provide information essential for enacting conservation measures
meant to protect both horseshoe crab and migratory shorebird populations.
According to game theory models of sperm competition, males that experience
high levels of sperm competition should increase sperm production and investment as
well as show adaptations to sperm morphology that increase sperm speed. Additionally
the models predict that, at any given mating event, males should conserve sperm when
facing fertilization competition from multiple males. Conducting electro-ejaculations,
measuring sperm attributes, and using quantitative PCR for samples taken during mating
events of various sizes, this project will test predictions made by game theory models of
sperm competition.
Harvesting of horseshoe crabs for eel and whelk bait has resulted in large declines
in horseshoe crab populations along the East Coast of the United States. L. polyphemus
eggs are an essential part of the diet of shorebirds migrating to summer nesting grounds
in the arctic. Recent studies have shown that the decreased number of horseshoe crabs
has taken a toll on these migratory birds; the lack of the nutritious eggs has caused the
populations of these shorebirds to fall precipitously as the birds can not gain the energy
necessary to continue their migration.
Recent policies enacted in order to stabilize populations of L. polyphemus have
restricted harvesting to male horseshoe crabs. It is unclear, however, if reducing the
amount of polyandry and thus sperm competition will have a negative effect on
horseshoe crab survivorship. If, for example, the highest quality males have the fastest
sperm or can produce more sperm than lesser quality males, culling males and reducing
sperm competition may lead to decreased offspring fitness. Understanding the factors
affecting reproductive success is a necessary step before enacting conservational policies
meant to stabilize horseshoe crab populations.
TABLE OF CONTENTS
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NSF Form 1359 (10/99)
46
Sperm Wars
Do varying levels of sperm competition affect sperm investment, sperm attributes,
and sperm allocation in the American Horseshoe Crab, Limulus polyphemus?
Variation in fertilization success between competing males is determined by
sperm production and sperm attributes. For externally fertilizing organisms, game theory
models of sperm competition predict that males who experience high levels of sperm
competition should invest more resources into sperm development and expenditure than
males living under low levels of competition (Ball & Parker, 1996). Recent studies have
supported the theory by showing that males of species where fertilization competition is
common generally have larger testes relative to body mass (GSI) than related species
without sperm competition (Byrne et al, 2002, Prado et al, 2003). Furthermore, within
species sperm morphological differences are often found when males have multiple
reproductive tactics: the males that experience the most sperm competition have more
densely concentrated and faster sperm than those with less competition (Neff et al 2003,
Stoltz & Neff, 2006). These sperm characteristics lead to increased success when males
are competing to fertilize the same egg (Fu et al. 2001). Additionally, game theory
models predict that organisms should vary the amount of sperm released during a
spawning event. When two males compete for one clutch of eggs, each male should
release more sperm. However, in mating groups with more than two males, it benefits
each male to reduce his sperm investment. In the proposed study, I will study how sperm
morphology, sperm production, and sperm allocation affect the fertilization success of
competing males in the American horseshoe crab, Limulus polyphemus. I will also
examine if varying levels of male competition in genetically isolated populations have
resulted in adaptations to sperm investment and attributes.
Study System
On the Gulf Coast of Florida, L. polyphemus spawn on the shore from late
February to early May and then from August through November. Females approach the
beach at high tide with an attached male and lay multiple clutches of eggs that are left in
the sand. As the female deposits her eggs in the sand, non-attached males roaming the
beach sometimes surround the female and spawn onto the eggs. The female returns to the
ocean once spawning is complete. Close to 100% of the eggs are fertilized (Brockmann,
1990).
When females arrive at
the beach, they are always
followed by a male holding
spines on the back of her
opisthosoma. This attached male
normally stays with the female
throughout the spawning event
and leaves the beach when she is
finished, nearly always still
attached. During and throughout
a spawning event, unattached males roam the beach and join various mating pairs. These
satellite males, who are usually older and in poorer condition than the attached male
(Brockmann & Penn, 1992), compete for optimal positions around the female. The males
that obtain preferred positions show high levels of fertilization success (Brockmann et al,
2000). The number of males during a spawning event is highly variable, with mating
pairs consisting of just the female and the attached male to large mating groups with 10+
males (personal observation).
The proposed study will be conducted at the University of Florida Marine Lab at
Seahorse Key where large numbers of horseshoe crabs mate during the spring. This
population has been studied over a number of years and its basic patterns of activity are
known. Horseshoe crabs used in this study will be kept in flow-through seawater tanks at
the Marine Lab and they will be returned to the ocean once no longer needed for the
study.
Horseshoe crabs are an ideal system for studying sperm competition. Unlike
organisms with internal fertilization, cryptic female choice, such as differential sperm
storage or utilization, is unlikely to be a factor. Furthermore, males do not have the ability
to remove or displace the sperm of a competing male from the reproductive tract. Male
fertilization success is likely attributable to only three factors: 1) male positioning around
the female, 2) sperm output, and 3) sperm attributes. The importance of male position has
been previously shown to influence reproductive success (Penn & Brockmann, 1994,
Brockmann, 2000), but the role of sperm output and sperm attributes in fertilization
success has not been examined.
Proposed Research
Hypothesis 1
Sperm investment is higher in satellite males than in attached males.
Within a species, it is not uncommon for certain males to face higher levels of
sperm competition than other males. Such varying levels of sperm competition are often
found in systems with alternative reproductive tactics. For example, in systems with
parental (males that guard a territory and spawning females) and sneaker males (males
that do not guard a territory and attempt to fertilize eggs of females in a parental male’s
territory), sneaker males always compete for fertilizations with at least one other male
whereas parental males do not. Research into these systems has shown that males facing
intense sperm competition display increased sperm production when compared to males
that do not always face sperm competition (Macronata & Shapiro, 1995, Fu et al, 2001,
Neff et al. 2003).
Satellite male horseshoe crabs, by definition, never mate without at least one other
male present. They are therefore subject to sperm competition with every mating. The
game theory model of sperm competition suggests satellite males, as a response to more
intense sperm competition, should produce sperm more quickly than attached male
horseshoe crabs.
Methods
To evaluate hypothesis 1, I will collect 30 attached males and 30 satellite males.
Through electro-ejaculation (see Brockmann et al, 2000), I will clear the spermiducts of
any ejaculatory fluid. After a refractory period of a few hours (the exact amount of time
will be determined by a pilot study) during which the horseshoe crabs will remain in the
flow-through seawater tanks, I will again electro-ejaculate the males until the spermiducts
no longer produce fluid. This procedure will ensure that any seminal fluid released will
be recently produced. By comparing the amount of ejaculate from the attached and
satellite males, I will determine whether the satellite and attached males differ in the
amount they invest in ejaculates. The amount of ejaculate will be measured by capturing
the fluid in a clean pipette and measuring the volume of fluid produced.
Hypothesis 2
The sperm of satellite males are not more densely concentrated and faster than the sperm
of attached males.
In addition to showing increased sperm production, males with alternative
reproductive tactics often show differences in sperm attributes. A number of studies in
externally fertilizing fish have observed that the sperm of sneaker males are more densely
concentrated and faster than the sperm of parental/guarder males (Stoltz & Neff 2006,
Burness et al. 2004). Both characteristics give sneaker males a fertilization advantage
over competing males. In a fair raffle model of sperm competition (Parker, 1990), any
male with more spermatozoa has an advantage. Densely concentrated sperm allows a
male to release more sperm per unit of ejaculate than a male with less concentrated
sperm.
Spermatozoa speed can also affect the ability of an individual sperm to compete.
Through a chemical cue (Shoger & Bishop, 1967), L. polyphemus sperm capacitate in the
presence of an unfertilized egg and race towards the egg. Since only one sperm can
fertilize any egg, the fastest sperm have a clear advantage. If two males compete for
fertilizations and all attributes are equal except for sperm speed, the male with the faster
sperm should show a higher level of fertilization success given equal compatibility with
the egg. Sperm speed is primarily affected by sperm morphology; sperm with more
narrow heads and longer principle flagella swim at faster speeds than competing sperm
(Malo et al., 2006).
Unlike most other systems with alternative reproductive tactics where sperm
attributes have been described, the reproductive strategy of male horseshoe crabs is not a
fixed behavior. In previous studies comparing the sperm characteristics of male
phenotypes in systems with alternative reproductive strategies, the body and testes of
males with faster and more densely concentrated sperm are morphologically different
than the other male types. Similar morphological differences have not been described
between attached and satellite male horseshoe crabs. While attached males are normally
younger and in better condition than satellite males, individual males have been seen
alternating between strategies within a breeding season (Brockmann & Penn, 1992,
Brockmann, 2002). For this reason, I predict that there will be no difference between
attached and satellite males in the characteristics of their sperm.
Methods
To measure sperm attributes, I will electro-ejaculate 30 attached and satellite
males. 2µl of sperm will be fixed in a 2% gluteraldehyde mixture for preservation and
brought back to the lab. Sperm density can be measured by counting the number of
spermatozoa in a given area using a compound microscope with a hemacytometer. An
average of three independent counts for each sample will taken with the counting
researchers blind as to whether the sample came from an attached or satellite male.
Sperm density will be calculated by multiplying the mean sperm per hemacytometer grid
by the dilution factor and volume (Neff et al, 2003).
Using the same sperm taken to measure sperm concentration density, I will
examine sperm speed. First, I will measure the ratio of sperm head length to flagellum
length. By taking a digital picture of the sperm at 400x magnification the sperm head and
flagellum length can be measured with a computer program. If the ratio is not
significantly different between the attached and satellite males, it is unlikely that one
group of sperm is faster than the other.
Hypothesis 3
The sperm of satellite males will not show increased fertilization success when compared
to the sperm of attached males.
Any variation in sperm attributes between satellite and attached males should
result in differential fertilization success. Faster or more densely concentrated sperm
would give an advantage to one male over the other if directly competing for
fertilizations. Measuring the sperm characteristics previously described will allow me to
predict whether satellite males do have an advantage over attached males; however, it is
possible that there is an attribute of sperm morphology or physiology that may affect
fertilization success. For this reason, it is necessary to test directly whether satellite males
show higher fertilization success than attached males given the same volume of sperm.
Methods
Through electro-ejaculation, I will collect the sperm of 30 attached and 30
satellite males. Each attached male will be paired with a randomly selected satellite male
and 1ml of seminal fluid from each male will be mixed together in centrifuge tube with
8ml of seawater. Once the sperm and water are thoroughly mixed, I will drop a recently
collected unfertilized egg from a female into the centrifuge tube. For each male pair, this
procedure will be repeated 10 times. The egg will be kept until hatching at which point a
paternity test will be conducted using tissue collected from each male. Any significant
difference in paternity between attached and satellite males will be indicative of
differential fertilization success.
Hypothesis 4
Attached males allocate sperm based on the amount of male competition.
Brockmann et al. (2000) found that attached males had lower fertilization success
than satellite males if two or more satellite males were present during a mating (see
Figure 2). It is unknown whether this decreased paternity was due to some advantage the
satellite males had over the attached male in terms of position or water flow or whether
the attached male acted differently in the presence of competition. One possible
explanation for the observed change in fertilization success is that the attached male
released less sperm when facing high competition.
Figure 2: The effect of the number
of satellites on the mean percentage
of paternity per male for attached
and satellite males. Sample sizes are
number of nests. (Taken from
Brockmann et al 2000)
A model describing
sperm allocation under
conditions of sperm
competition predicts exactly
such a scenario (Parker, G.A.
et al, 1996. Figure 3). When
only one competitor is present
at a spawning event, the
model predicts that a male should increase the amount of sperm released. However, if
more than one competitor is attempting to fertilize the eggs, the male should reduce the
amount of sperm released. Female horseshoe crabs do not always lay all of their eggs
during a single spawning event; they have been observed returning to the beach over
multiple nights to lay eggs, often with the same attached male (Brockmann & Penn,
1992). Thus it might be reproductively beneficial for an attached male to conserve his
sperm if there is a reasonable chance that he will have a less competitive mating
opportunity in the near future. Since attached males have the future possibility of mating
in a less competitive spawning, I
expect to find that attached males will
reduce their ejaculatory expenditure
during highly competitive matings.
Figure 3: The expenditure on the ejaculate
(relative to the total reproductive effort per
spawning) plotted against the number, Ni, of
males competing at spawning of type i. The
curves represent three different hypothetical
conditions based on the mean number of males
present at a spawning event, N = 1, 2, and 5.
(Taken from Parker, G.A. et al. 1996)
Methods
To test this hypothesis, I will measure the proportion of attached male sperm to
satellite sperm during spawning events. Sperm will be collected by inserting a large
pipette underneath the mating groups through the female’s incurrent canal, a space
separating the prosoma from the opisthosoma through which water enters and flows over
the book gills. Sand will be allowed to settle from each sample and the supernatant will
be removed. A tissue sample will be collected from the female and from each male. The
sperm will be preserved in a solution of 2% glutaraldehyde. Using quantitative PCR and
established microsatellite loci, I will quantify the proportion of sperm from each male in
the collected sample. Samples will be taken from 20 mating events with one satellite
male, 20 mating events with two satellite males, and 20 mating events with three satellite
males.
Hypothesis 5
Genetically isolated populations of L. polyphemus that vary in intensity of sperm
competition will show differences in sperm morphology and/or sperm investment.
L. polyphemus range from the coast of Maine to the Yucatan Peninsula with
populations that are genetically isolated (King, T.L. et al, 2005). Due to differences in
climate and breeding season length, the operational sex ratio (OSR) varies amongst the
horseshoe crab populations (Brockmann & Smith, in revision) with colder climates
leading to a more male biased OSR and thus a higher number of males per spawning
event. Because sperm characteristics are largely heritable and because any differences in
sperm will affect a male’s fertilization success, the horseshoe crab population with a
highly male biased OSR should have faster and more densely concentrated sperm than
populations with a low OSR. Additionally, the males in populations that have more
mating competition should invest more into sperm production than the males in
populations with little competition.
Methods
The methods to test this hypothesis are similar to the methods from hypothesis 2.
I will collect ejaculatory fluid from 30 males from the Yucatan population (low OSR) and
from 30 males from the Delaware Bay population (high OSR). I will count the number of
sperm per 2µl of ejaculate using a hemacytometer and multiply that number by the
dilution factor and volume. As a proxy for sperm speed, I will measure the sperm head to
flagellum length ratio for each male.
The methods to measure sperm investment are similar to the methods described
for hypothesis 1.Thirty males from each population will be electro-ejaculated until their
seminal tubules are empty. After a refractory period, the males will be electro-ejaculated
again and the amount of seminal fluid released will be measured. Since horseshoe crabs
in the Yucatan are significantly smaller than horseshoe crabs in Delaware Bay, the ratio
of seminal fluid to body size will be calculated before comparing the two populations.
Conclusion
Taken individually, each experiment answers a particular question; however,
when pooled together, the experiments laid out in this proposal will provide an
overarching picture of how organisms adapt to varying levels of sperm competition. Such
a finding will not only enhance our understanding of horseshoe crab reproduction, but it
will potentially elucidate broadly applicable principles for externally fertilizing species.
Additionally, the proposed experiments will examine widely accepted, though rarely
tested, predictions made by game theory models of sperm competition.
Broader Impacts
Horseshoe crab eggs play a vital role in the diet of shorebirds migrating along the
Atlantic coast to Northern grounds. Since the early 1990s, fishermen from Maine to
Florida have harvested horseshoe crabs by the hundreds of thousand to use as bait for eel
and whelk (Clarke, WM 2008). These harvests have depleted horseshoe crab numbers
which in turn have seriously
affected bird survivorship
during migration (Baker AJ
et al. 2004). Over the last few
years, a number of measures
have been put into place to
limit the number of horseshoe
crabs that can be culled
annually. Additionally, many
measures have restricted the
harvesting of female
horseshoe crabs; for example,
in 2006 the Atlantic States
Marine Fisheries
Commission restricted
Figure 4: Horseshoe crab egg density in Delaware Bay. Taken
catches of horseshoe crabs in
from http://www.nj.gov/dep/dsr/trends2005/pdfs/wildlifehorseshoe.pdf
Delaware to 100,000 males
(Clarke, WM 2008).
In order to manage any population effectively, however, it is essential to
understand the life-history and reproductive behavior of the organism. On the surface,
harvesting only male horseshoe crabs seems like an ideal solution. However, we do not
yet know the importance of female polyandry and male-male competition to the
continued success of the species. Preferentially eliminating males will lower the male
biased OSR and reduce competition. If male-male competition, whether through sperm
competition or direct aggression, provides a benefit to offspring viability or survivorship,
culling males may have an unintended negative effect on future horseshoe crab
populations. Without a firm grasp on how reproductive behavior works in Limulus
polyphemus, it is potentially dangerous to make conservation decisions based on possible
incorrect assumptions. Sexual selection has undoubtedly played a large role in the
evolution and maintenance of horseshoe crab behavior. My research into sperm
competition in the American horseshoe crab will provide us with essential knowledge for
making informed policy decisions.
The proposed research will also play a role in educating and mentoring future
scientists. Our lab currently has 15 undergraduate student volunteers who are involved in
every aspect of the research. In the field, these students assist with all aspects of the
experiments; in the lab they will have the opportunity to prepare and analyze the samples.
My hope is that such hands on experience will inspire our undergraduate assistants to
begin their own independent research projects.
Literature Cited
Baker, A. L. et al. (2004) Rapid population decline in red knots: fitness consequences of
decreased refueling rates and late arrival in Delaware Bay. Proc. R. Soc. Lond. B
271, 875 - 882
Ball, M.A. and Parker, G.A. (1996) Sperm Competition Games: External Fertilization
and “Adaptive” Infertility. J.Theor. Biol. 180, 141 - 150
Brockmann, H.J. & Smith, M.D. (In revision) Reproductive competition and sexual
selection in horseshoe crabs. In: Biology and Conservation of Horseshoe Crabs,
eds. J. Tanacredi, M.L. Botton, & D. Smith. Springer Publishers.
Brockmann, H.J. (1990) Mating behavior of horseshoe crabs, Limulus polyphemus.
Behaviour, 114, 206 - 220
Brockmann, H.J. and Penn, D. (1992) Male mating tactics in the horseshoe-crab,
Limulus-polyphemus. Anim. Behav. 44, 653 - 665
Brockmann, H.J. et al. (2000) Paternity in horseshoe-crabs when spawning in multiplemale groups. Anim. Behav. 60, 837 - 849
Brockmann, H.J. (2002) An experimental approach to altering mating tactics in male
horseshoe crabs (Limulus polyphemus). Behavioral Ecology, 13, 232 - 238
Burness, G. et al. (2004) Sperm swimming speed and energetics vary with sperm
competition risk in bluegill (Lepomis macrochirus). Behav. Ecol. Sociobiol. 56,
65 - 70
Byrne, P.G. et al. (2002) Sperm competition selects for increased testes mass in
Australian frogs. Jour. Evol. Biol. 15, 347 - 355
Clarke, W.M. (2008) Limulus Lately. Chesapeake Bay Magazine.
Fu, P. et al. (2001) Tactic-specific success in sperm competition. Proc. R. Soc. Lond. B
268, 1105 - 1112
King, T.L. et al. (2005) Regional differentiation and sex-biased dispersal among
populations of the horseshoe crab Limulus polyphemus. Transactions of the
American Fisheries Society. 134 (2), 441 – 465
Malo, et al. (2006) Sperm design and sperm function. Biology Letters, 2, 246 - 249
Marconato, A. and Shapiro, D.Y. (1995) Sperm allocation, sperm production and
fertilization rates in the bucktooth parrotfish. Anim. Behav. 52, 971 - 980
Neff, B.D. et al. (2003) Sperm investment and alternative mating tactics in bluegill
sunfish (Lepomis macrochirus). Behav. Ecol. 14, 634 - 641
Parker, G.A. (1990) Sperm competition games: raffles and roles. Proc. R. Soc. Lond. B
242, 120 – 126
Parker, G.A. et al. (1996) Sperm competition games: individual assessment of sperm
competition intensity by group spawners. Proc. R. Soc. Lond. B 263, 1291 -1297
Prado, CPA and Haddad, CFB. (2003) Testes size in leptodactylid frogs and occurrence
of multimale spawning in the genus Leptodactylus in brazil. Jour. Herp. 37, 354 362
Shoger, R.L. & Bishop, G.G. Sperm activation and fertilization in Limulus polyphemus.
Biological Bulletin, 133, 485
Widelife Populations – Horseshoe crabs
http://www.nj.gov/dep/dsr/trends2005/pdfs/wildlife-horseshoe.pdf
FOR NSF USE ONLY
54
SUMMARY PROPOSAL BUDGET
ORGANIZATION
University of Florida
PROPOSAL NO.
DURATION (MONTHS)
Year 1
Proposed
PRINCIPAL INVESTIGATOR/PROJECT DIRECTOR
Granted
AWARD NO.
Daniel Sasson
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.
Daniel Sasson
12
2.
3.
4.
5.
6. (
) OTHERS (LIST INDIVIDUALLY ON BUDGET EXPLANATION PAGE)
7. (
) 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. (
) 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.)
9
3
Proposer
$21500
(If Different)
$
$1200
TOTAL PARTICIPANT COSTS
$250.81
6. OTHER Tuition
Quantitative PCR
TOTAL OTHER DIRECT COSTS
H. TOTAL DIRECT COSTS (A THROUGH G)
I. INDIRECT COSTS (F&A) (SPECIFY RATE AND BASE)
$10500
$7830
$41280.81
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)
M. COST SHARING: PROPOSED LEVEL $
PI/PD TYPED NAME AND SIGNATURE*
Funds
Granted by NSF
$21500
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 (
)
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES
2. PUBLICATION/DOCUMENTATION/DISSEMINATION
3. CONSULTANT SERVICES
4. COMPUTER SERVICES
5. SUBAWARDS
Funds
Requested By
$41280.81
$41280.81
$
AGREED LEVEL IF DIFFERENT: $
DATE
FOR NSF USE ONLY
INDIRECT COST RATE VERIFICATION
Daniel Sasson
ORG. REP. TYPED NAME & SIGNATURE*
DATE
Date Checked
Date of Rate Sheet
Initials-ORG
NSF Form 1030 (10/99) Supersedes All Previous Editions
*SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.C)
FOR NSF USE ONLY
54
SUMMARY PROPOSAL BUDGET
ORGANIZATION
University of Florida
PROPOSAL NO.
DURATION (MONTHS)
Cumulative
Proposed
PRINCIPAL INVESTIGATOR/PROJECT DIRECTOR
Granted
AWARD NO.
Daniel Sasson
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.
Daniel Sasson
12
2.
3.
4.
5.
6. (
) OTHERS (LIST INDIVIDUALLY ON BUDGET EXPLANATION PAGE)
7. (
) 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. (
) 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.)
9
3
Proposer
$21500
(If Different)
$
$1200
TOTAL PARTICIPANT COSTS
$250.81
6. OTHER Tuition
Quantitative PCR
TOTAL OTHER DIRECT COSTS
H. TOTAL DIRECT COSTS (A THROUGH G)
I. INDIRECT COSTS (F&A) (SPECIFY RATE AND BASE)
$10500
$7830
$41280.81
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)
M. COST SHARING: PROPOSED LEVEL $
PI/PD TYPED NAME AND SIGNATURE*
Funds
Granted by NSF
$21500
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 (
)
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES
2. PUBLICATION/DOCUMENTATION/DISSEMINATION
3. CONSULTANT SERVICES
4. COMPUTER SERVICES
5. SUBAWARDS
Funds
Requested By
$41280.81
$41280.81
$
AGREED LEVEL IF DIFFERENT: $
DATE
FOR NSF USE ONLY
INDIRECT COST RATE VERIFICATION
Daniel Sasson
ORG. REP. TYPED NAME & SIGNATURE*
DATE
Date Checked
Date of Rate Sheet
Initials-ORG
NSF Form 1030 (10/99) Supersedes All Previous Editions
*SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.C)
Budget Justification
Travel
Flights to Yucatan Peninsula, Mexico and to Delaware Bay, DE - $1200
Supplies
Hemacytometer for counting sperm density - $198.61
10ml gluteralderhyde for preserving sperm samples - $52.20
Other
Quantitative PCR done at University of Florida Genetics Institute - $43.5 per sample * 180
samples - $7830
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: Daniel Sasson No current support
Support:
Current
Pending
Submission Planned in Near Future
*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.
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)
55
USE ADDITIONAL SHEETS AS NECESSARY
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: University of Florida Genetics Institute
Will perform the quantitative PCR and paternity analyses.
Clinical:
Animal:
Computer:
Office:
Seahorse Key Marine Laboratory
Other: Field station
An island located in the Gulf of Mexico, 4 miles offshore from Cedar Key, FL. Facilities include a dormitory and a
marine laboratory. Field work will be conducted on the beach of the island. Permission to use the facilities has already
been granted.
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.
NSF Form 1363 (10/99)
56