watch videos on butterflies or observe butterflies and come up with a physiologically (thermoregulation, water balance, locomotion, energetics, etc) based question that could be experimentally tested using the butterflies. then write-up a short (limit of 350 words, not including references) mock grant proposal addressing their question, its significance, what is known, and how they plan to test their hypothesis. This proposal should include in-text citations of peer-reviewed articles that address the background information on the topic. Exemplary grant proposals (not on butterflies or necessarily physiology) are attached to illustrate how these types of proposals should be 
P.S The proposed experiment can have a wide range of focus (observational, behavioral, ecological, etc.) as long as it has, and addresses, physiological underpinnings. 

Example Proposal 1
Differences in Sperm Characteristics and Fertilization Success Across Populations
of the American Horseshoe Crab, Limulus polyphemus
The game model theory of sperm competition predicts that males who experience
high levels of sperm competition should invest more resources in sperm development and
expenditure1. Recent studies have supported this idea, showing both that species in which
sperm competition is common generally have larger testes relative to body mass than
species with little sperm competition,2 and that, within a population, males who
experience more sperm competition than competing males (which often occurs in species
with alternative reproductive tactics) show sperm characteristic adaptations that improve
fertilization success, specifically sperm length and concentration density. 3,4 No study,
however, has compared sperm characteristics and fertilization success between isolated
populations of the same species where levels of sperm competition differ. I propose to
conduct such a study with the American Horseshoe Crab, Limulus polyphemus.
Horseshoe crabs are the ideal subject for this experiment because males and
females spawn in the external medium. Since any male can join a spawning pair, external
fertilization limits the ability of the female to choose preferentially one male’s sperm
over another’s; therefore, sperm allocation and sperm characteristics are important factors
in determining male fertilization success. Limulus polyphemus range from the coast of
Maine to the Yucatan Peninsula with groups of populations that are genetically distinct.5
Due to differences in climate and thus breeding season length, the operational sex ratio
(OSR) amongst the horseshoe crab populations varies6 with colder climates leading to a
higher male biased OSR. Because sperm characteristics are largely heritable and because
any differences in sperm will affect a male’s fertilization success rate, I hypothesize that
horseshoe crabs from northern populations will have faster and more densely
concentrated sperm than males from Florida, which in turn will have faster and more
dense sperm than males from the Yucatan.
I will conduct the experiment in two phases. Through electro-stimulus7, 2ml of
sperm will be collected from 40 male horseshoe crabs in both the Yucatan Peninsula and
Delaware Bay (which has the highest male biased OSR6) along with a tissue sample. The
collected seminal fluid will be mixed with a cryoprotectant solution of diluted chicken
egg yolk, 0.4 M sucrose, 10mM sodium bicarbonate, and 2mM pentoxyfylline, frozen,
and sent to Zoology Department at the University of Florida. Upon thawing in a heated
bath, the number the spermatozoa per ml seminal fluid and the average length of the
spermatozoa will be measured using a hemacytometer.
Additionally, the sperm samples will be randomly paired so that each sample
from one site is matched with a sample from the other site. I will mix 0.5ml of each
sample with its pair and dilute the solution with 9ml of seawater.7 This solution will then
be squirted over an unfertilized egg collected at Seahorse Key, Fl. After the egg develops,
a paternity analysis using microsatellites will be conducted.
The findings of this study will be an integral part of a larger study examining
sperm characteristics and sperm allocation in horseshoe crabs.
Literature Cited

Ball, M.A. & Parker, G.A. (1996) Sperm Competition Games: External Fertilization and
“Adaptive” Infertility. Journal of Theoretical Biology. 180 (2), 141 - 150
2
Byrne, P.G. et al. (2002) Sperm competition selects for increased testes mass in Australian frogs.
Jour. Evol. Biol. 15, 347 - 355
3
Neff, B.D. et al (2003) Sperm investment and alternative mating tactics in bluegill sunfish
(Lepomis macrochirus). Behav. Ecol. 14, 634 - 641 
4
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 
5
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
6
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.
7
Brockmann, H.J. et al. (2000) Paternity in horseshoe crabs when spawning in multi-male groups.
Animal Behaviour. 60, 837-849.


Example proposal 3
Mapping Variation in Stable Isotope Composition of the American Alligator: Need
For Sample Site Validation
The use of stable isotope analysis in animal ecology has become increasingly
popular, following the discovery of the relationship between an organism’s isotopic
signature and that of their diet. Stable isotopes (mainly Carbon and Nitrogen) can be
used in ecological studies under two main observations: (1) an animal’s isotopic signature
resembles that of its diet 2, 3, 4 and (2) the isotopic signature of an animal does not
perfectly match that of its diet5. The first being used to determine the underlying source
of production and the later used to determine placement within food web of interest 1, 6.
Basically, the isotopic signature of an animal’s tissue is that of its diet plus or minus a
small amount (deemed the isotopic discrimination factor) 6. In general the isotopic
discrimination factor of an animal’s tissue is within a normal range of values, on average
3-4 ‰ per trophic transfer for 15N and 1 ‰ per trophic transfer for 14C 1, 6. These average
values are not always accurate for all species, especially for ectothermic organisms such
as sea turtles and alligators 7. In order to determine these values, detailed growth studies
should be performed on species on a tissue specific basis. In the case of the American
alligator no such studies exist which characterize isotopic values for any tissue type.
As a result this will be the first step in obtaining these important values for ecological
research.
The first step is to quantify any isotopic variation in the particular tissue of
interest through the replicated sampling of that tissue across the body plan of the
organism. This will validate sampling locations and provide a working map of variation
across the body plan of the target organism.
I am proposing to sample and analyze keratinized scute tissue from the skin of
two adult alligators, then quantify isotopic changes across the body plan. I have obtained
two hides from a private source and will not need to collect samples from any live
individuals. Sampling will consist of removing small sections of tissue using a biopsy
punch, taken in lateral and longitudinal series across body areas of interest (i.e. tail,
ventral-dorsal surfaces, and extremities). In addition, at each sampling location
representative sub-samples will be collected from an entire scute and measured for
changes in the thickness across the individual scute. Scute tissue is formed in layers over
time and may be able to provide a graded time series of dietary information; this method
has been used in sea turtle research and is of particular interest for use on alligators 7, 8.
Once samples have been analyzed, any variation in the isotopic signatures at
specific locations will be quantified and mapped out across the body plan. The
information gained here will inform all other future research utilizing stable isotope
analysis. This is the first step in being able to use stable isotope analysis in the ecological
research of alligators.

1.) Peterson, B.J., and Fry, B. (1987) Stable Isotopes in Ecosystem Studies. Ann. Rev.
Ecol. Syst. 18, 293-320
2.) Hobson KA, Clark RG (1992) Assessing avian diets using stable
isotopes I: turnover of 13C in tissues. Condor 94:181–188
3.) DeNiro MJ, Epstein S (1978) Influence of diet on the distribution of
carbon isotopes in animals. Geochim Cosmochim Acta 42:495–
506
4.) DeNiro MJ, Epstein S (1981) Influence of diet on the distribution of
nitrogen isotopes in animals. Geochim Cosmochim Acta
45:341–351
5.) Michener RH, Schell DM (1994) Stable isotope ratios as tracers in
marine aquatic food webs. In: Lathja K, Michener RH (eds)
Stable isotopes in ecology and environmental science. Blackwell,
New York, pp 138–157
6.) Post DM (2002) Using stable isotopes to estimate trophic position:
models, methods, and assumptions. Ecology 83:703–718
7.) Reich, K.J. et al. (2008) Effects of growth and tissue type on the kinetics of 13C
and 15N incorporation in a rapidly growing ectotherm. Oecologia 155(4), 651–663
8.) Phillips, D.L. and Eldridge, P.M. (2006) Estimating the timing of diet shifts using
stable isotopes. Oecologia 147, 195-203