Effects of Trichechus manatus latirostris on Blue Spring Food Web Using Stable Isotope Analysis

Stetson University Effect of Trichechus manatus latirostris on Blue Spring food web using stable isotope analysis Anja Erwin Mentor: Dr. Work Biology Department Erwin 2 Abstract Food webs depict the interactions between producers and consumers and are important tools in understanding the dynamic of an ecosystem. With an understanding of the food web, we can better study how exotic species and other disturbances may affect the ecosystem. Florida manatees, Trichechus manatus latirostris, are seasonal disturbances at Blue Spring in Volusia County, FL, where they seek thermal refuge during colder seasons. Due to a lack of vegetation in the spring, the manatees venture to the St. Johns River to forage for food before returning to the spring to digest and consequently release large amounts of organic matter into the spring. Stable isotope analysis (δ13C and δ15N) was used to determine isotopic signatures for algae, amphipods, Sailfin Mollies, and Mosquitofish during spring and fall seasons. Grab samples were gathered near the boil and mid-run of Blue Spring using a seine net and tested at the UC Davis Stable Isotope Facility. The data revealed a significant difference in δ15N signatures for all samples when the manatees were present, but no significant differences between the boil and mid-run. Alternately, there was no significant change in δ13C signatures when manatees were present and only δ13C signatures for algae were significant between the boil and mid-run. These data suggested that a disturbance of δ15N signatures in the spring might have been caused by Florida manatees releasing lighter nitrogen isotopes from plants consumed in the St. Johns River into the spring run. Introduction Food webs provide a visual of the flow of energy through a system and give us insight into the relationships between producers and consumers (Pimm, 1982). By following trends in food webs, we are able to get a better picture of what animals are eating and how this impacts the rest of the food web. We are able to recognize commensal and mutualistic relationships between members of a system and which species are limiting factors within the food web. Most importantly, Erwin 3 understanding the dynamic of an ecosystem allows us to study the effects that disturbances may have on the rest of the community (Nystrom and McIntosh, 2003). Stable isotope analysis is used to trace ratios of heavy and light carbon (δ13C) and nitrogen (δ15N) isotopes and help identify food web linkages in ecosystems (Cloern et al., 2002). The δ13C signatures can predict diet composition of aquatic animals through identifying the primary energy source, whereas the δ15N signatures determine the consumer’s trophic level (Alves-Stanley et al., 2010 and O’Reilly and Hecky, 2002). Together, these isotope signatures can be arranged in a bi-plot showing the increase in trophic levels. Figure 2 depicts a simplified bi-plot of a stereotypical freshwater ecosystem with trophic levels increasing as you go up the food chain. Comparing bi-plots before and after a disturbance gives us a better picture of how the disturbance may affect other species in the food web. For manatees in particular, isotope analysis using fecal samples is an ideal, non-invasive method to track their feeding ecology as compared to traditional methods of using muscle and skin samples or stomach content analysis (Salvarina et al., 2013 and Alves-Stanley et al., 2010). Additionally, most stable isotope studies have analyzed the feeding habits of deceased Florida manatees or those in captivity, so this study will add to the literature of the feeding ecology of free-living Florida manatees (Alves-Stanley et al., 2010). Freshwater springs are important systems to study for the impacts of disturbances because they release low-nutrient water directly from the aquifer, making it easier to analyze how outside factors influence the water quality. A majority of freshwater systems in Florida are affected by anthropogenic impacts, which have been shown to influence water flow and cause a decrease in biological diversity within the ecosystem (Harrington et al., 2010). Notably, an increase of urbanization and agriculture surrounding many of our freshwater systems introduce additional Erwin 4 nutrients from run-off and fertilizers (USGS, 2008). Recreational impacts can also be destructive to an ecosystem by destroying algal growth and disturbing the feeding of grazers (Stevenson et al., 2004). Florida manatees, Trichechus manatus latirostris, could be considered a unique type of freshwater disturbance because of their seasonal changes in distribution (Deutsch et al., 2003). T. manatus latirostris are sensitive to water temperatures below 20⁰C due to a low metabolic rate, so approximately 75% of the manatee population find refuge from cold water between November and February in warmer freshwater springs that remain 22⁰C year round (Alves-Stanley et al., 2010, Gibbs et al., 2010, Laist and Reynolds, 2005). Consequently, Florida manatees temporarily relocate to warm water discharges, including freshwater springs and power plant outflows, or thermal basins to conserve the increased energetic costs of thermoregulation (Alves-Stanley et al., 2010, Irvine, 1983, Laist and Reynolds, 2005). Blue Spring is a designated thermal refuge for Florida manatees, making it an interesting location to study the effect of manatee disturbances to the Blue Spring food web (USGS, 2008). Blue Spring is classified as a first-magnitude spring, meaning it releases at least 65 million gallons of water per day into the run, and supports 30+ fish species, including seasonal tarpon and several exotic species. (USGS, 2008 and Work et al., 2010). Since Blue Spring is also the largest spring connected to the St. Johns River, its location provides a centrally-located thermal refuge for manatees throughout the state of Florida (Figure 1). Blue Spring is one of 14 major locations for manatee migration in Florida, and the population of manatees at Blue Spring has increased fivefold since the mid-1980’s (Laist and Reynolds, 2005). Blue Spring Park Ranger and manatee specialist Wayne Hartley recorded 471 manatees in 2016 (Wayne Hartley, pers. comm.). Erwin 5 With an ever-increasing presence of Florida manatees, the Blue Spring ecosystem experiences noticeable pressures during cold seasons due to the food demands of the manatees, physical disturbance, and sudden influx of large animals in a confined space. Estimates of the daily amount of aquatic vegetation consumed by Trichechus manatus latirostris revealed that the manatees consume between 4-9% of their body weight, which is mostly fiber (Bengtson, 1983). Blue Spring, however, does not supply the necessary amount of vegetation within the spring run to satisfy the food demands of the manatees, forcing them to venture into the St. Johns River to forage for food (Gibbs et al., 2010). Florida manatees are generalist herbivores and consume roughly 60 different species of aquatic plants; however, limited food supply at Blue Spring forces the manatees to rely heavily on various species of algae including Vaucheria and any other vegetation found in the St. Johns River (Alves-Stanley et al., 2010 and Bengtson, 1983). This paper sought to address whether stable isotope signatures of select members of the Blue Spring food web were affected when manatees were present. Considering the large number of manatees in the spring and the large amount of daily food intake per manatee, we can infer that there would be a significant increase of manatee feces deposited into the spring during winter months. As a result, we expect that the spring undergoes nutrient enrichment from the manatee feces, which could impact the food web. This study quantitatively analyzed the effect that manatee presence has on the Blue Spring food web. We hypothesized that the δ13C and δ15N isotope signatures of algae, amphipods, Sailfin Mollies, and Mosquitofish would be changed when manatees were present at Blue Spring. Further, we predicted that there would be a change of δ13C and δ15N isotope signatures at the mid-run, but expected δ13C and δ15N isotope signatures to have no change near the boil since the manatee activity may be lower near the boil and the water flow may carry any nutrients toward the mid-run. Erwin 6 Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue Figure 1. A map depicting the thermal refuges of Florida. Blue SprinSprin g (upper rightg (upper right g (upper rightg (upper right g (upper rightg (upper rightg (upper right ) is a key refuge ) is a key refuge) is a key refuge ) is a key refuge) is a key refuge) is a key refuge ) is a key refuge ) is a key refuge (Laist and Reynolds, 2005)(Laist and Reynolds, 2005)(Laist and Reynolds, 2005) (Laist and Reynolds, 2005) (Laist and Reynolds, 2005) (Laist and Reynolds, 2005)(Laist and Reynolds, 2005)(Laist and Reynolds, 2005)(Laist and Reynolds, 2005) (Laist and Reynolds, 2005) (Laist and Reynolds, 2005) (Laist and Reynolds, 2005) (Laist and Reynolds, 2005). P. = Power P. = Power P. = Power P. = Power P. = Power P. = Power P. = Power plant, T.B. = plant, T.B. = plant, T.B. = plant, T.B. = plant, T.B. = plant, T.B. = thermal basin thermal basin thermal basinthermal basinthermal basinthermal basin δ13 C δ 15 N Figure 2. AFigure 2. A Figure 2. AFigure 2. A Figure 2. A stereotypic stereotypic stereotypic stereotypicstereotypic bi -plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring plot of carbon and nitrogen isotope signatures Blue Spring showing showing showing an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae an increase in trophic levels (Algae  Amphipods Amphipods  Mollies Mollies andand Mosquitofish MosquitofishMosquitofish  BassBassBass ). Erwin 7 Methods Study area: Blue Spring is located within Blue Spring State Park roughly 10 miles southwest of Deland in Volusia County, FL. Samples were obtained between the fall of 2013 and fall of 2015 from two predetermined locations: the boil of Blue Spring and a mid-run site located halfway down the run (Figure 3). The spring boil is composed of limestone whereas the rest of the run is sandy with some submerged underwater vegetation. Blue Spring is a prime example of nutrient contamination since over 45% of its springshed is located on residential land (Harrington et al., 2010). Further, Blue Spring supports 30+ fish species, including seasonal tarpon and several exotic species (Work et al., 2010). These exotic and migrating species may cause major disturbances when introduced to the Blue Spring ecosystem. Boil BoilBoil Mid Mid-runrun Figure Figure Figure Figure Figure 3. An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue . An image of the two sampling locations at Blue Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Spring in Volusia County, FL. Erwin 8 Sample Collection: We collected samples from two locations of the spring, the boil and mid-run, and immediately placed them in Ziploc bags. We removed a generous handful of leaves and algae samples from the submerged edge of the spring. We used a pipette dropper to separate amphipods from the algae samples and placed roughly 200 amphipods in a separate vial. We used a seine net to catch 10 Sailfin Mollies (Poecilia latipinna) and 10 Mosquitofish (Gambusia affinis holbrooki) at each location, which were immediately kept on ice in a portable cooler. We manually collected manatee feces, which were easy to spot because of their orange color. We labeled all of the samples, and they were frozen within two hours of collection. We thawed out the samples and distributed them evenly into glass vials using sterile forceps. Once the samples were thawed, we minimized the time they were spent uncovered to prevent contamination. We measured the length of the fish in millimeters before putting them into separate glass vials. We labeled the tops and sides of the glass vials with a number representing the location of the sample followed by the sample number (1= boil, 4=mid-run). We placed the vials in the laboratory drying oven at ~55°C for a minimum of three days to dehydrate the samples. We used an electric coffee grinder to pulverize the manatee feces and a glass rod or mortar and pestle to pulverize the remaining samples. We used a metal micro spatula to distribute the samples into standard 5x8 mm tin capsules weighing 30mg. We weighed the samples on an analytical balance at Stetson University. The animal samples (amphipods, Sailfin molly, and Mosquitofish) weighed between 1.15 and 1.40 mg whereas plant samples (algae and manatee feces) weighed between 3.50 and 4.50 mg. I used sterile forceps to fold the tin capsules into round, compact packets so the sample was tightly secured. I Erwin 9 prepared two tin capsules per sample and loaded them into individual wells of a 96-well plastic container. I recorded the placement of the samples on a separate spreadsheet. Mass Spectrometry: We determined the isotopic composition of the samples using mass spectrometry. We sent the tin capsules containing the samples to the UC Davis Stable Isotope Facility and the raw data was sent back via email. Data Analysis: We used Excel to order the data into two groups to reflect seasons with recent manatee activity (spring) and seasons with no recent manatee activity (fall). These groups were also separated into boil and mid-run samples. A two-way ANOVA test was run with the δ13C and δ15N signatures for each sample as the dependent variable and the testing site and manatee presence as independent variables. Stable isotope bi-plots were created with δ13C on the x-axis and δ15N on the y-axis comparing manatee presence and no manatee presence for each location. These bi-plots provided a visual of the food web of the Blue Spring ecosystem and distinguished the trophic levels of our data set. Results There was significant overlap of food consumed by animals of the Blue Spring food web. We expected the food chain to have a linear trend, similar to the stereotypical bi-plot in Figure 2; however, the only bi-plot that exhibited this trend was the mid-run when manatees were not present (Figure 6). The rest of the bi-plots did not have such defined trophic levels, which might be accounted for by the variability of the species’ diets (Figure 4, 5 and 7). Erwin 10 The δ15N signatures were significantly lowered when manatees were present compared to when the manatees were not present for all samples (Algae p=0.12; amphipods p=0.001; mollies p<0.001; mosquitofish p<0.001) (Figure 8 & 9). There was no significant difference between δ15N signatures at the boil and mid-run for all samples (Algae p=0.402; amphipods p=0.222; mollies p=0.470; mosquitofish p=0.381). On the other hand, there was no significant change in δ13C signatures when manatees were present with the exception of algae. The δ13C signatures for algae were significantly lower at the boil compared to the mid-run (p=0.009). Further, there was no significant change in δ13C signatures between sites with the exception of Mosquitofish (Figure 10 & 11). The δ13C signatures for Mosquitofish were significantly increased when manatees were present (p=0.005). Figure 4. The stable isotope food web of Blue Spring at the boil when manatees were not present during the fall season. 8 9 10 11 12 13 14 15 16 -35 -33 -31 -29 -27 -25 Δ 15N Δ13 C NO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOILNO MANATEES: BOIL Algae Algae Amphipods Amphipods AmphipodsAmphipods Amphipods Molly Molly Molly MosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitofishMosquitfishMosquitofishErwin 11 Figure 5. The stable isotope food web of Blue Spring at the boil when manatees were present during the spring season. Figure 6. The stable isotope food web of Blue Spring at the mid-run when manatees were not present during the fall season. 3 4 5 6 7 8 9 10 11 12 13 -33 -31 -29 -27 -25 Δ 15N Δ13 C MANATEE: BOIL MANATEE: BOIL MANATEE: BOIL MANATEE: BOIL MANATEE: BOIL Algae Algae Amphipods Amphipods AmphipodsAmphipods Amphipods Molly Molly Molly Mosquitofish MosquitofishMosquitofish Mosquitofish MosquitofishMosquitofishMosquitofish Mosquitofish Manatee Manatee ManateeManateeManateepoop pooppoop 8 9 10 11 12 13 14 15 16 -36 -34 -32 -30 -28 -26 Δ 15N Δ13 C NO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MIDNO MANATEE: MID-RUN Algae Algae Amphipods Amphipods AmphipodsAmphipods Amphipods Molly Molly Molly Mosquitofish MosquitofishMosquitofish Mosquitofish MosquitofishMosquitofishMosquitofish Mosquitofish Manatee Manatee ManateeManateeManateepoop pooppoopErwin 12 Figure 7. The stable isotope food web of Blue Spring at the mid-run when manatees were present during the spring season. Figure 8. The difference of δ15N signatures for each sample when manatees were present and not present in the boil of Blue Spring. Values represent averages ±STDEV. 6 7 8 9 10 11 12 13 -32 -30 -28 -26 -24 Δ 15N Δ13 C MANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MIDMANATEES: MID-RUN Amphipods Amphipods AmphipodsAmphipods Amphipods Molly Molly Molly Mosquitofish MosquitofishMosquitofish Mosquitofish MosquitofishMosquitofishMosquitofish Mosquitofish Manatee Manatee ManateeManateeManateepoop pooppoop 0 5 10 15 20 Amphipods AmphipodsAmphipods Amphipods Amphipods MollyMollyMolly MosquitofishMosquitofishMosquitofish MosquitofishMosquitofish Mosquitofish Mosquitofish Manatee poopManatee poopManatee poopManatee poop Manatee poop Manatee poopManatee poopManatee poop Manatee poop δ 15N BOIL: BOIL: δ15 N Manatee NManatee NManatee NManatee N Manatee N Manatee NManatee N No Manatee NNo Manatee NNo Manatee NNo Manatee NNo Manatee N No Manatee N No Manatee N No Manatee NNo Manatee NErwin 13 Figure 9. The difference of δ15N signatures for each sample when manatees were present and not present in the mid-run of Blue Spring. Values represent averages ±STDEV. Figure 10. The difference of δ13C signatures for each sample when manatees were present and not present in the boil of Blue Spring. Values represent averages ±STDEV. -35-35-35 -30-30-30 -25-25-25 -20-20-20 -15-15-15 -10-10-10 -5 0 Amphipods AmphipodsAmphipods Amphipods Molly Molly Mosquitofish Mosquitofish MosquitofishMosquitofish MosquitofishMosquitofish Manatee poop Manatee poop Manatee poop Manatee poopManatee poopManatee poop δ 13C BOIL: BOIL: BOIL: δ13 C Manatee CManatee CManatee CManatee C Manatee C Manatee CManatee C No Manatee CNo Manatee CNo Manatee CNo Manatee CNo Manatee C No Manatee C No Manatee C No Manatee CNo Manatee C 0 2 4 6 8 10 12 14 16 Algae AlgaeAlgae Amphipods AmphipodsAmphipodsAmphipods AmphipodsAmphipodsAmphipods MollyMollyMolly Molly MosquitofishMosquitofishMosquitofish Mosquitofish Mosquitofish Manatee poopManatee poop Manatee poop Manatee poopManatee poop Manatee poopManatee poop δ 15N MIDRUN: δ15 N Manatee NManatee NManatee NManatee N Manatee N Manatee NManatee N No Manatee N No Manatee NNo Manatee NNo Manatee N No Manatee N No Manatee N No Manatee NErwin 14 Figure 11. The difference of δ13C signatures for each sample when manatees were present and not present in the mid-run of Blue Spring. Values represent averages ±STDEV. Discussion Our first hypothesis that δ13C and δ15N signatures would be lower when manatees were present was supported for the nitrogen measurements but not for the carbon measurements. These findings supported a previous study on the effect of manatee presence on the Blue Spring food web, which also suggested that δ15N signatures were decreased when manatees were present in the spring (Workman and Work, 2015 unpublished data). It was reasonable to expect a shift of δ15N signatures when manatees were present because the manatees were essentially depositing large amounts of nutrients from the St. Johns River into the spring run, which may have influenced the food web. The manatees left the -40 -30 -20 -10 0 Algae Amphipods AmphipodsAmphipods Amphipods Amphipods MollyMollyMolly MosquitofishMosquitofishMosquitofish MosquitofishMosquitofish Mosquitofish Mosquitofish ManateeManateeManateeManatee Manatee pooppooppoop δ 13C MIDRUN: δ13 C Manatee CManatee CManatee CManatee C Manatee C Manatee CManatee C No Manatee CNo Manatee CNo Manatee CNo Manatee CNo Manatee C No Manatee C No Manatee C No Manatee CNo Manatee CErwin 15 warmer spring water to forage for plant matter containing various ratios of heavy and light isotopic signatures. The manatees likely retained more of the heavy nitrogen isotopes and released the light nitrogen isotopes through their urine and feces (Peterson and Fry, 1987). We suspected that the algae then took up these lighter δ15N signatures from the fecal matter, which in turn generated a decrease in δ15N signatures for the remaining consumers in the food web. We were also able to deduce that the lower δ15N signatures were most likely not caused by fertilizer run-off percolating into the aquifer from surrounding farms and residential areas. Most inorganic fertilizers contain heavy δ15N isotopes, so we would have expected the signatures of the bi-plots to increase instead of decrease (Peterson and Fry, 1987). Additionally, the water discharge at Blue Spring was highest during manatee season, which was when we would have expected nutrient pollution to be the highest (Holland and Bridger, 2014). However, we were led to believe the change in δ15N signatures was due to nutrient enrichment from manatees because the signatures were lowered, reflecting the signatures from the plants they were consuming. We did not notice a shift in δ13C signatures when manatees were present, which led us to believe that manatee presence at Blue Spring had an effect on trophic level but did not influence consumers to change their food sources. The only exception we noticed was an increase in δ13C signatures for Mosquitofish when manatees were not present, which might have been due to feeding variability or the Mosquitofish might have selected a wider range of primary food sources when manatees crowded the spring run. Our second hypothesis stating that there would be a difference between isotope signatures at the spring boil and mid-run was not supported. There was no significant difference between δ13C and δ15N signatures at the boil and mid-run except for δ13C signatures for algae. The δ13C signatures for algae might have been lowered at the mid-run because the algae used different Erwin 16 primary carbon sources. The algae at the mid-run was most likely obtaining carbon from the CO2 that was being respired from aquatic animals in the spring, whereas the algae at the boil was most likely leaching carbon directly from the calcium carbonate from the limestone in the boil, which it does so more readily (Peterson and Fry, 1987). The biggest limitation to this study was the fact that we only had one sampling date when manatees were present (spring of 2014). Since this time period was one of the variables we were testing, it would have been ideal to have more sample dates when manatees were present to eliminate any seasonal variance (DeNiro and Epstein, 1978). Additionally, we did not have a complete set of samples for each sample date. In future studies, I would collect a larger sample size, particularly for the manatee feces samples, and collect a wider array of plants and larger fish to further expand the scope of this study. Nevertheless, this study was beneficial in understanding the influence of Florida manatees on the Blue Spring food web. Understanding this dynamic may help ensure the long-term stability of the Blue Spring ecosystem and help improve conservation efforts (Gibbs et al., 2010 and Alves-Stanley et al., 2010). Acknowledgements Dr. Kirsten Work, Dr. Melissa Gibbs, Genevieve Patrick, Stetson University Biology Department, Brown Scholar Program, and CUDARC Grant Erwin 17 Works Cited Alves-Stanley, C.D., Worthy, G.A.J. and Bonde, R.K. 2010. Feeding preferences of West Indian manatees in Florida, Belize, and Peurto Rico as indicated by stable isotope analysis. Marine Ecology Progress Series. 407:255-267. Bengtson, J.L. 1983. Estimating food consumption of free-ranging manatees in Florida. Journal of Wildlife Management. 47(4):1186-1192. Cloern, J.E., Canuel, E.A., and Harris, D. 2002. 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Accessed March 25, 2016 at http://www.floridasprings.org/downloads/florida_12_awhtxsw4.pdf Work, K., Gibbs, M., Peters, B., and French, L. 2010. Fish assemblage variability in a Florida spring. Southeastern Naturalist. 9(4):649-672. Workman, S. and Work, K. 2015. Variation in the Volusia Blue Spring food web as determined by stable isotope analysis. Unpublished manuscript.