watch videos on butterflies or observe butterflies…
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