Grazing Dynamics of the Pinfish (Lagodon Rhomboides) on Thalassia Testudinum and Halimeda Incrassata Across a Temperature Gradie

Home , Gambierdiscus carpenteri, Lagodon

Grazing dynamics of the pinfish (Lagodon rhomboides) on Thalassia testudinum and Halimeda incrassata across a temperature gradient in the Florida Keys and implications for Ciguatera Fish Poisoning

A Thesis Presented to

The Faculty of the College of Arts and Sciences Florida Gulf Coast University

In Partial Fulfilment of the Requirement for the Degree of Master of Science

By Kathryn Alissa Ribble 2019

Florida Gulf Coast University Thesis

APPROVAL SHEET

This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science

Kathryn Alissa Ribble

Approved: December 2, 2019

______

Michael Parsons, Ph.D., Advisor

______James Douglass, Ph.D., Committee Member

______Brian Bovard, Ph.D., Committee Member

The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above-mentioned discipline.

ACKNOWLEDGMENTS

Writing these acknowledgments allows me the time to reflect on the knowledge and experience I have gained from pursuing my Master of Science degree. When I started this program, I was unaware of how immense some of the challenges and opportunities would be that came from attending graduate school. I have learned just how much effort goes into completing a masters degree and how willing people are to help you pursue your endeavors. Through hard work and with the support of many others I was able to successfully earn my Masters of Science degree!

I would first like to thank my advisor Dr. Mike Parsons who initially gave me a job in his lab years ago and ultimately inspired me to get my masters degree. He has guided me through the challenges of creating a research project and enhanced my scientific writing skills in addition to providing myself and lab mates with awesome places to conduct field research and giving us the opportunities to experience and participate in scientific conferences around the country. Additionally, my committee members Dr. James Douglass and Dr. Brian Bovard were an integral part in helping me write and interpret the data I collected for this project.

Funding and support were provided by the NOAA NCCOS ECOHAB program

(award N1A17NOS4780181), the Florida Gulf Coast University Department of Marine and Earth Sciences, the Jean and Bill Wilshere Endowment for Innovative Research in

Environmental Science scholarship, the Wanda G. Tolley Scholarship Endowed Fund, the

Dan Cassani Memorial Fund for Marine Fish Research and Conservation, the Community

Foundation of Sarasota County, and the Angler Action Foundation for providing me the

iii funding to go back to school for my masters degree. These organizations made it financially possible for me to achieve this goal.

There are many individuals who were a large part in helping me throughout this experience. Of them, Nicholas Culligan has been my rock. Whether it was helping me set up and break down the same 50-gallon seagrass tank for the 7th time, collect samples for my many attempts at setting up my experiment, making me laugh when I had a bad day, or inspiring me when I felt uninspired, he helped make the difficult parts of this experience a little less difficult. I would also like to acknowledge the help and support that my family gave me throughout this process; thank you Andrea, Steven, and Kimbra Ribble. Lastly, I would like to thank Anne Smiley, Andrea James, Adam Catasus, Jeff Zingre, and Allie

Bury for being such good friends and helping me with this project in various ways.

Attending graduate school has been a rewarding challenge that I would not have wanted to do without the help and support of all the people aforementioned. As the quote by Helen Keller goes, “alone we can do so little; together we can do so much”, I too believe that this project and experience would not have been as successful without the support from all of them. Thank you all so much!

ABSTRACT

Seagrass beds are an important component of coastal environments and serve as important nursery grounds for juvenile and commercially important fish species. Seagrass beds are often monospecific, composed of just one species of seagrass. However, macroalgal species such as Halimeda, Laurencia, and Dictyota (among others) can also be found in seagrass beds in quantities that vary depending on many factors including grazing pressures. This study focuses on the grazing dynamics of Lagodon rhomboides (commonly referred to as pinfish) between two different macrophytes (Thalassia testudinum and

Halimeda incrassata) over a temperature gradient in a laboratory setting representative of a seagrass bed from the middle Florida Keys (Heine Grass Bed; HGB).

HGB, where samples were collected for this study, is a long-term study site for

Ciguatera Fish Poisoning (CFP) research as well. CFP is a form of food poisoning in humans who consume tropical reef fish that have accumulated high levels of ciguatoxins.

Ciguatoxins are naturally produced lipid-soluble toxins produced by the benthic dinoflagellate, Gambierdiscus, which enter reef food webs through the consumption of these epiphytic cells by herbivorous fishes. Thalassia testudinum and Halimeda incrassata are known to harbor high quantities of toxic Gambierdiscus species within their associated epiphytic community in areas such as HGB. The overall objective of this study is to examine the grazing dynamics of pinfish on Thalassia testudinum and Halimeda incrassata under varying temperatures within a seagrass bed environment (HGB) in the middle

Florida Keys. This study will also aid in determining how ciguatoxins are being introduced into the marine food web through grazing processes occurring within seagrass beds in the middle Florida Keys and assist modeling efforts for CFP outbreaks. Additionally, it will

v identify if specific temperatures influence the grazing behaviors of pinfish which will lend insight into how grazing pressures change seasonally and how they may change as the climate warms.

This study found that pinfish did not consume one macrophyte agar (Thalassia testudinum or Halimeda incrassata) at a significantly higher amount than the other at ambient temperature. In addition, the results also suggest that pinfish are not significantly increasing their grazing intensity on Thalassia testudinum agar across a temperature gradient (22°C – 30°C). However, the grazing intensity of pinfish on Halimeda incrassata agar did significantly increase with increasing temperature but the strength of this linear correlation is fairly weak (R2 = 0.2154). Finally, pinfish ate a marginally significantly larger amount of the Halimeda incrassata agar cubes than the Thalassia testudinum agar cubes over the temperature gradient tested (22°C – 30°C; p = 0.052).

TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iii ABSTRACT ...... v TABLE OF CONTENTS ...... vii LIST OF FIGURES ...... ix LIST OF TABLES ...... xii 1. INTRODUCTION ...... 1 1.1. Importance of Seagrass Ecosystems ...... 1 1.2. Role of Grazers in Seagrass Ecosystems ...... 3 1.3. Target Species ...... 6 1.3.1. Thalassia testudinum ...... 6 1.3.2. Halimeda incrassata ...... 8 1.3.3. Lagodon rhomboides (Pinfish) ...... 10 1.4. Ciguatera Fish Poisoning ...... 12 1.5. Study Overview ...... 20 2. RESEARCH PURPOSE ...... 21 3. METHODS ...... 22 3.1. Study Site ...... 22 3.2. Field Collection Procedures ...... 23 3.3. Experimental Set Up ...... 27 3.3.1. Control Trial ...... 31 3.3.2. Experimental Trials ...... 32 3.4. Data Analysis ...... 34 4. RESULTS ...... 36 4.1. Overview ...... 36 4.1.1. Control Trial ...... 37 4.1.2. Experimental Trial 1 ...... 37 4.1.3. Experimental Trial 2 ...... 39 4.1.4. Experimental Trial 3 ...... 40 4.2. Results of Statistical Analysis ...... 42

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4.2.1. Control Trial ...... 42 4.2.2. Experimental Trials ...... 42 4.2.3. Hypothesis 1 ...... 43 4.2.4. Hypothesis 2 ...... 45 4.2.5. Hypothesis 3 ...... 46 4.2.6. Hypothesis 4 ...... 54 5. DISCUSSION ...... 57 5.1. Significant Conclusions ...... 57 5.1.1. Hypothesis 1 ...... 57 5.1.2. Hypothesis 2 ...... 61 5.1.3. Hypothesis 3 ...... 62 5.1.4. Hypothesis 4 ...... 65 5.2. Implications for Ciguatera Fish Poisoning ...... 68 5.3. Implications for Pinfish Feeding Behaviors ...... 71 5.3.1. Laboratory Environment Pinfish Were In ...... 71 5.3.2. Reconstituted Macrophytes Used for Feeding Trials ...... 72 5.4. Future Research ...... 73 5.5. Studies on Other Herbivorous Species and Macrophytes ...... 74 5.6. Improved Experimental Design ...... 75 REFERENCES ...... 76

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LIST OF FIGURES Figure 1.1: The geographic distribution of Thalassia testudinum found in the Caribbean. (Figure adapted from Short et al., 2010) ...... 6 Figure 1.2: Depicts the different features of the seagrass Thalassia testudinum. (Figure adapted from https://drawnbydawn.com/collections/scientific-illustration-anatomy/ products/turtle-grass-anatomy) ...... 7 Figure 1.3: Depicts the basic characteristics (holdfasts, segments, and nodes) of Halimeda plants. The species of Halimeda and the location it resides determines the type of holdfast the plant will have (i.e. the “sprawler”, “rock-grower”, or “sand-grower”). (Figure adapted from Hillis-Colinvaux, 1980) ...... 9 Figure 1.4: The geographical distribution of ciguatera (light gray band: range from 35°N to 35°S latitudes). The medium gray markings indicate coral reef regions within the range where ciguatera is found. The darkest gray markings indicate disease-endemic areas of ciguatera. (Figure adapted from Perez-Arellano et al., 2005) ...... 15 Figure 1.5: Depicts how ciguatoxins are introduced to marine food webs via grazing by herbivorous fish and subsequently how the toxins move up the food web to larger carnivorous fish that people consume thus causing CFP outbreaks. (Figure adapted from https://www.nbc-2.com/story/39239441/poisonous-toxins-could-be-in-local-seafood) ..17 Figure 1.6: HGB Coordinates: N. 24°51’38.4”, W. 80°44’17.4” a) Identifies the location of the study site, HGB (marked with red circle), in relation to Florida and the Florida Keys. The largest image (colorful image) depicts the site at a closer angle, on the Florida Bay side of Lower Matecumbe Key in the middle Florida Keys. (Figure adapted from Google Earth) ...... 21 Figure 3.1: Timeline of the preformed study. This figure includes dates that pinfish and macrophytes were collected in the Florida Keys, when both macrophytes (Thalassia testudinum and Halimeda incrassata) were made into agar for the control trial and each experimental trial, when the 24-hour soaking of the agar occurred and the 24 hour food deprivation periods occurred for pinfish, the start and end dates for the control trial and experimental trials, and what days (represented by “D” followed by the day number) the wet weights (ww) were recorded during the control trial and each experimental trial. ....26 Figure 3.2: Pinfish Holding tank set up a) Shows where water is removed from the tank. b) Carbon filter that dirty water passes through c) UV sterilizer d) Bio Ball filtration tank e) & f) Show the entire tank set up from the front and side angles. Looking at (f) water is removed from the left side of the tank filtered and sterilized then flows back into the right side of the tank. Figures (e) and (f) also show the mesh barrier that was put into the holding tank creating a separation device for pinfish once they had undergone a feeding trial (discussed further in section 3.3.2)...... 28

Figure 3.3: Schematic of the control and experimental tank setup. Blue boxes represent the individual 19-liter tanks and are labeled with tank numbers and corresponding temperatures. The black boxes surrounding sets of four tanks represent the fluorescent light that the tanks were placed under. Tanks were separated into blocks of four because of the size of the lights and by splitting the tanks into two groups of four it ensured all tanks would receive equal amounts of light...... 30 Figure 3.4: Schematic of the control trial. The experimental tanks are represented by the blue rectangles and labeled with the tank number and temperature. The larger white rectangles that encompass the tanks represent the lights that the tanks were placed under during the experiment. Dark green squares represent the Thalassia testudinum agar cubes and light green squares represent the Halimeda incrassata agar cubes...... 32 Figure 3.5: Schematic of the experimental trials. The experimental tanks are represented by the blue rectangles and labeled with the tank number and temperature. The larger white rectangles that encompass the tanks represent the lights that the tanks were placed under during the experiment. The dark green squares represent the Thalassia testudinum agar cubes and the light green squares represent the Halimeda incrassata agar cubes. Pinfish are represented by the gray fish shape...... 33 Figure 4.1: Bite marks on agar cubes made by pinfish. a) Shows the Halimeda incrassata agar cube from tank 2 (28°C) on Day 3 before the wet weight was recorded. b) Shows the same Halimeda incrassata agar cube from Tank 2 (28°C) after the wet weight was recorded on Day 3. c) Shows both the Thalassia testudinum and Halimeda incrassata agar cubes from tank 2 (28°C) on Day 6...... 38 Figure 4.2: Bite marks on agar cubes made by pinfish. a.) Shows a comparison of the bite marks on the Thalassia testudinum agar cube (left) compared to the Halimeda incrassata agar cube (right) from tank 6 (24°C) on Day 2. b.) Shows a bite mark in the Thalassia testudinum agar cube from tank 5 (25°C) on Day 3. c.) Shows multiple bite marks in the Thalassia testudinum agar cube from tank 7 (23°C) on Day 3. d.) Shows multiple bite marks on both the Thalassia testudinum (back) and Halimeda incrassata (front) agar cubes from tank 2 (28°C) on Day 7...... 40 Figure 4.3: Bite marks in agar cubes made by pinfish. a) Shows bite marks in Halimeda incrassata agar cube (right) made by the pinfish in tank 3 (27°C) on Day 5. b) Shows the bite marks in both the Thalassia testudinum agar cube (front) and Halimeda incrassata agar cube (back) made by the pinfish in tank 8 (22°C) on Day 5...... 41 Figure 4.4: The average percent change in wet weight for the control agar cubes at each temperature tested in this study for both macrophytes (Thalassia testudinum in dark green and Halimeda incrassata in light green). Linear equations, r squared values, and p values are listed for both species under the corresponding macrophyte they are for...... 43 Figure 4.5: The total amount of Thalassia testudinum agar and Halimeda incrassata agar consumed by the individual pinfish in each experimental trial at ambient temperature (26.8°C, tank 4). This figure represents the data used for both Hypothesis 1 and Hypothesis 2...... 44

Figure 4.6: Both graphs in this figure are looking at the ranked amount of Thalassia testudinum (a) or Halimeda incrassata (b) consumed by pinfish from all experimental trials at each temperature tested in this study. a) The ranked amount of Thalassia testudinum agar consumed by each pinfish in each experimental trial for all temperatures tested in this study. b) The ranked amount of Halimeda incrassata agar consumed by each pinfish in each experimental trial for all temperatures tested in this study. The linear equation, r squared value and p value from the linear regression analysis for each macrophyte are listed in the top right corner of each graph...... 47 Figure 4.7: The average amount of each macrophyte agar cube consumed by pinfish (in grams wet weight) from all three experimental trials preformed across the temperature gradient (22°C – 30°C) used in this study. Standard error bars represent the error associated with the average consumption amounts for each macrophyte at each temperature. The dark green bars show the average amount of agar consumption by pinfish for Thalassia testudinum and the light green bars show the average amount of agar consumption by pinfish for Halimeda incrassata...... 50 Figure 4.8: Boxplots from the Non-Parametric Kruskal-Wallis analysis for Thalassia testudinum (top) and Halimeda incrassata (bottom)...... 53 Figure 4.9: The average ranked amount of each macrophyte agar consumed by pinfish from all three experimental trials preformed across the temperature gradient (22°C – 30°C) used in this study. Standard error bars represent the error associated with the ranked average amount of agar consumed by pinfish for each macrophyte at each temperature. The dark green bars show the ranked average consumption by pinfish for Thalassia testudinum agar and the light green bars show the ranked average consumption by pinfish for Halimeda incrassata agar...... 54

LIST OF TABLES Table 1: A table of all statistical tests being run by hypothesis. The table includes the hypothesis number and hypothesis, the variables that were compared, the statistical test that was used for each hypothesis, and the purpose of each statistical test being run...... 35 Table 2: Results of both One Sample T-Tests comparing the actual amount consumed of each macrophyte agar by pinfish to zero consumption, individually, at ambient temperature (26.8°C)...... 45 Table 3: Results of the Independent Samples T-Test comparing the actual amount consumed of each macrophyte by pinfish to identify if there was a preference between the two macrophytes by pinfish at ambient temperature...... 46 Table 4: Results of the Linear Regression analyses (with all data points) identifying if a linear relationship was or was not present between the amount of Thalassia testudinum agar or Halimeda incrassata agar consumed by pinfish with temperature...... 49 Table 5: Results of the Non-Parametric Kruskal-Wallis analyses identifying if there was a significant difference in the amount of consumption of either macrophyte by pinfish at a specific temperature. Top table (1) is for Thalassia testudinum and bottom table (2) is for Halimeda incrassata...... 52 Table 6: Results of the Linear Mixed Model analysis identifying if pinfish consume one macrophyte over the other consistently across all temperatures...... 55 Table 7: An overview of the results from the statistical analyses that were run for this study. This table includes the hypotheses tested, the null hypothesis of each hypothesis, whether or not there was a significant preference identified, and the possible reasons for the significance or insignificance of each analysis...... 56

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1. INTRODUCTION

1.1. Importance of Seagrass Ecosystems

Seagrasses are submerged marine angiosperms that thrive in shallow subtidal zones and cover large areas referred to as seagrass meadows or beds (Bury, 2017; Handley et al.,

2007; Larkum et al., 2006; Nakaoka, 2005). Seagrass beds can be found throughout shallow marine and estuarine environments globally with the exception of Antarctica and have been identified as one of the most productive coastal ecosystems in the world (Duffy, 2006;

Green et al., 2003; Hemminga & Duarte, 2000; Nakaoka, 2005; Valentine & Heck, 1999).

The primary productivity of seagrass beds has been compared to that of tropical rainforests indicating they are an important component of coastal environments (Nakaoka, 2005). In addition, seagrass beds create biogenic habitat structure in an otherwise predominantly featureless sand/mud environment (Duffy, 2006). Seagrass environments support high diversity and secondary productivity of associated organisms by providing food and shelter for both permanent and part time residents (Gambi et al., 1990; Worcester, 1995). In

Florida alone, seagrass beds make up 919,962 hectares of nearshore environments and serve as important nursing grounds for juvenile and commercially important fish species

(Dawes & Mathieson, 2008; Heck & Valentine, 2006; Zieman et al., 1989; Zieman &

Zieman, 1989).

The sedimentary makeup of seagrass meadows is primarily composed of fine- grained sediments and tends to be high in organic matter (Gacia et al., 1999; Gambi et al.,

1990; Hemminga & Duarte, 2000; Kenworthy et al., 1982; Worcester, 1995). This is assumed to be in part, a result of both the leaf canopy of the seagrass meadow trapping particles from the water column and the detrital material produced by seagrass and algal

1 species and other organisms living within the ecosystem (Hemminga & Duarte, 2000). It should also be noted that a portion of the leaf litter from seagrass beds can be removed from the system through tidal or wave influences; the heightened deposition of organic matter within seagrass beds is not exclusive to leaf litter from seagrasses (Hemminga &

Duarte, 2000; Slim et al., 1996). In addition, it has also been documented that seagrass beds can reduce the kinetic energy of water moving over the bed; this occurrence results in rather still benthic environments within seagrass beds allowing particulate matter to settle out of the water column and into the seagrass bed (Garcia et al., 1999; Gambi et al., 1990;

Hemminga & Duarte, 2000; Kenworthy et al., 1982; Worcester, 1995). Once sediment has settled onto the floor of the seagrass ecosystem, the complex root system prevents those sediments from being easily eroded or resuspended (Garcia et al., 1999; Hemminga &

Duarte, 2000; Kenworthy et al., 1982).

There are approximately 58 species of seagrass in the world, and seven species native to Florida (Dawes & Mathieson, 2008). Seagrass beds are often monospecific, composed of just one species of seagrass (Duffy, 2006; Hemminga & Duarte, 2000).

However, macroalgal species such as Halimeda, Laurencia, and Dictyota (among others) can also be found in seagrass beds in quantities that vary depending on substrate type, water column nutrient concentrations, grazing, and other factors (Dawes & Mathieson, 2008;

Lirman et al., 2019). In the Florida Keys, seagrasses frequently co-occur with calcareous green macroalgae like Halimeda, Penicillus, Rhipocephalus, and Udotea, which attach to the coarse carbonate sediments with root-like rhizoids (Dawes & Mathieson, 2008; Lirman, et al., 2019). In this experiment, the seagrass bed studied is predominantly composed of

Thalassia testudinum. The next most abundant macrophyte species found in the seagrass

2 bed is Halimeda incrassata. Due to the fact that these two species (Thalassia testudinum and Halimeda incrassata) make up the majority of marine plants within the seagrass bed they were chosen as the best representative species of this ecosystem for this study.

1.2. Role of Grazers in Seagrass Ecosystems

Marine systems, especially those in tropical regions, experience the highest grazing pressures in the world (Cruz-Rivera & Villareal, 2005; Hay, 1991). Therefore, marine macrophytes, such as seagrasses and algae, have adapted over time to cope with these intense grazing pressures (Thayer et al., 1984; Valentine & Heck, 1999, Cruz-Rivera &

Villareal, 2005). In seagrass beds, herbivores will consume live blades of seagrass, macroalgae, and/or epiphytes (Hemminga & Duarte, 2000). Herbivory can be a controlling factor in the abundance of different algal, epiphytic, or seagrass species found in a particular marine environment (Valentine & Duff, 2005; Valentine & Heck, 1999). For example, increased grazing pressure from herbivorous marine organisms can cause shifts in the community structure of a marine environment (Valentine & Heck, 1999). Under high grazing pressure, fast growing algae that normally have an advantage can be out-competed by slower growing algae or seagrasses that have chemical and/or structural defenses to deter herbivory (Cruz-Rivera & Villareal, 2005; Hay & Steinberg, 1992; Valentine &

Heck, 1999).

Key grazers in seagrass beds include crustaceans, echinoderms, fish, sea turtles, and some marine mammals and birds (Hemminga & Duarte, 2000; Nakaoka, 2005). This study focuses on the interactions occurring by a major prey species, Lagodon rhomboides

(hereafter referred to as pinfish) at the primary consumer level within a Florida seagrass

3 bed. Most organisms living in seagrass meadows do not graze exclusively on living seagrass leaves and those that do graze extensively on seagrass leaves are also dependent on other food sources as well (Hemminga & Duarte, 2000; Klumpp et al., 1992). Due to the complexity and robustness of seagrass blades, many grazers lack the ability to break down and digest the structural carbohydrates such as cellulose and phenolic compounds found in seagrass and thus target epiphytes as their main source of food in these communities (Klumpp et al., 1992; Nakaoka, 2005; Valentine & Heck, 1999; Zapata &

McMillan, 1979). Those species that can breakdown seagrass cell walls must be specialized to be able to consume seagrasses through either chemical (e.g. low stomach pH; Moriarty,

1973; Lobel, 1980, 1981) or mechanical (e.g. pharyngeal mill or a muscular triturating stomach; Thayer et al., 1984; Valentine & Heck, 1999) processes. Some species of fish from the Families Scaridae (e.g. parrotfish), Sparidae (e.g. pinfish), Monacanthidae (e.g. filefish), and Siganidae (e.g. rabbitfish) have been found to be important consumers of living seagrass leaves (Hemminga & Duarte, 2000). In particular, pinfish have been reported to consume large amounts of seagrasses such as Thalassia testudinum,

Syringodium filiforme, and Zostera marina L. once they are greater than 120mm in length

(Darcy, 1985; Livingston, 1980; Montgomery & Targett, 1992). Epiphytes are also an important part of the diets of pinfish with studies finding omnivorous pinfish (36 – 80 mm in length) having around thirty percent of their diet made up of epiphytes (Darcy, 1985;

Stoner, 1980; Stoner & Livingston, 1984).

Benthic and epiphytic algal species are important autotrophs that also contribute to the primary production of seagrass beds (Hemminga & Duarte, 2000; Klumpp et al., 1992).

These algal species also appear to be important food sources for grazers living in seagrass

4 meadows. Previous studies have shown they are likely consumed at a much higher percentage than seagrasses alone in these systems (Hemminga & Duarte, 2000). However, similar to some seagrasses, certain algal species have defense mechanisms against herbivory such as increased concentrations of calcium carbonate (i.e. Halimeda species) within the algae tissues (Cruz-Rivera & Villareal, 2005; Hay et al., 1994). Such defense mechanisms increase the plants toughness and non-digestive material and decrease its nutritional value (Cruz-Rivera & Villareal, 2005; Hay et al., 1994). Additionally, calcium carbonate can also act as a chemical defense by interfering with the digestion of such plants by some herbivorous fishes that don’t have gastric acidity low enough to lyse algal cell walls; calcium carbonate can cause a buffering effect on such fishes that rely on acid- mediated digestion (Cruz-Rivera & Villareal, 2005; Hay et al., 1994; Horn,1989; Lobel,

1981).

Many studies have identified that grazing rates by herbivores in marine systems vary with water temperature (Bruno et at., 2015; Carpenter, 1986; Ferriera et al., 1998;

Polunin & Klumpp, 1992; Smith, 2008). It is theorized this could be a result of concomitant changes in the productivity of food sources such as algae or seagrass (Carpenter, 1986;

Klumpp & McKinnon, 1989, 1992). Another thought is that metabolic demand could explain changes in grazing pressures with changing temperature (Bruno et al., 2015). A study by Carpenter (1986) done off of St. Croix, U.S. Virgin Islands, found that there was an increase in grazing rates by herbivorous fish in summer and fall (June – November) with the highest grazing rates found in September; that trend was strongly correlated with fish abundance. Additionally, Ferriera et al. (1998) found the grazing rate of damselfish positively correlated with changes in ambient water temperature (over minutes – hours) in

5 subtropical upwelling environments off of Cabo Frio Island, Brazil. As global sea surface temperatures rise in association with climate change, it creates warmer marine environments for fish which could also influence the rate they are consuming food and/or the availability of their food sources.

1.3. Target Species

1.3.1. Thalassia testudinum

Thalassia testudinum is the most abundant of the seven seagrass species found in

Florida (Dawes & Mathieson, 2008; Phillips, 1960). The geographical range for Thalassia testudinum includes the entire coast of the Gulf of Mexico, the coasts of the Atlantic Ocean starting around the eastern coast of Central Florida, and the coasts of the Caribbean Sea through Venezuela and Bermuda (Figure 1.1; Short et al., 2010). The lower depth limit of

Thalassia testudinum is set by its relatively high light requirements (it requires about 25% of surface irradiance), but it can grow to 10 meters deep or so in clear waters (Short et al.,

2010).

Figure 1.1: The geographic distribution of Thalassia testudinum found in the Caribbean. (Figure adapted from Short et al., 2010)

Figure 1.2: Depicts the different features of the seagrass Thalassia testudinum. (Figure adapted from https://drawnbydawn.com/collections/scientific- illustration-anatomy/ products/turtle-grass-anatomy)

Thalassia testudinum produces rhizomes that expand horizontally through the sediment and are located between 5 – 25 cm under the sediment (Figure 1.2; Davis &

Fourqurean, 2001). From these rhizomes, the plant produces vertical shoots that hold leaves that can reach lengths of 10 – 25 cm (Figure 1.2; Davis & Fourqurean, 2001). Thalassia testudinum is a slower growing seagrass species (Gallegos et al., 1993) with greater longevity (Davis & Fourqurean, 2001) compared with other seagrass species. Gallegos et al. (1993) calculated that the shoot life span of Thalassia testudinum could range between

6 and 9 years. However, the turnover rate of individual blades produced by the shoot is much shorter, on the order of weeks. New blades must constantly be produced, and old ones shed as they become damaged and/or too epiphytized to efficiently collect light

(Gallegos et al., 1993). Thalassia testudinum was chosen as one of the two macrophyte species for this experiment because of its palatability to pinfish and because it is the most abundant seagrass in Florida (and common in seagrass beds in the Florida Keys; Darcy,

1985; Hemminga & Duarte, 2000; Lirma et al., 2019; Livingston, 1980; Montgomery &

Targett, 1992). Additionally, Thalassia testudinum found in the middle Florida Keys (the area that this study is representative of) is known to harbor species of the toxic dinoflagellate Gambierdiscus within its epiphytic community (Cruz-Rivera & Villareal,

2005; Parsons et al., 2017). The effects of Gambierdiscus species and its implication to this study will be discussed in further detail in section 1.4. Ciguatera Fish Poisoning.

1.3.2. Halimeda incrassata

There are 33 species of Halimeda found in tropical Atlantic and tropical Indo-

Pacific oceanic regions (Kooistra et al., 2002). All Halimeda species consist of a holdfast and a thallus; the thallus consists of calcified branching segments and nodes giving the plant a similar resemblance to an underwater cactus (Figure 1.3; Drew & Abel, 1988;

Kooistra et al., 2002; Verbruggen et al., 2009). Halimeda species are a source of both organic material and calcareous sediment in the marine environments where they are found

(Castro-Sanquino et al., 2017; Drew & Abel, 1988; Verbruggen et al., 2009). The toughness and ability for Halimeda species to incorporate calcium carbonate into its tissues at a particularly high rate (sometimes >90% CaCO3 per dry mass of tissue) is thought to be

8 a result of both structural and chemical defenses adapted by the plant to deter grazers

(Castro-Sanquino et al., 2017; Cruz-Rivera & Villareal, 2005; Hay, 1997; Hay & Fenical,

1988; Hay et al., 1994; Pennings & Paul, 1992). Nevertheless, organisms such as larger fish and/or small invertebrates have been observed to “intensely graze” on Halimeda

(Castro-Sanquino et al., 2017). The understory of Thalassia testudinum beds is often comprised of macroalgae species such as Halimeda among others (Lirman et al., 2019). In certain locations, Thalassia beds that contained Halimeda were found to be denser with higher biomass and increased productivity relative to beds without Halimeda (Duffy,

2006).

Figure 1.3: Depicts the basic characteristics (holdfasts, segments, and nodes) of Halimeda plants. The species of Halimeda and the location it resides determines the type of holdfast the plant will have (i.e. the “sprawler”, “rock-grower”, or “sand-grower”). (Figure adapted from Hillis-Colinvaux, 1980)

Halimeda incrassata is the specific Halimeda species used in this study, as it is the most abundant primary producer after Thalassia testudinum at the long-term study site located in the middle Florida Keys. In addition, as with Thalassia testudinum, Halimeda incrassata found in the middle Florida Keys is also known to harbor species of the toxic dinoflagellate Gambierdiscus within its epiphytic community (Cruz-Rivera &Villareal,

2005; Parsons et al., 2017). Halimeda incrassata is most commonly found in shallow sandy and mixed sandy-muddy areas such as seagrass beds or mangrove habitats. The distribution of the species includes South Florida, the Caribbean, South America, and the Indo-Pacific

(Guiry & Guiry, 2019; Wysor & Kooistra, 2003). Occasionally this species can also be found near coral reefs if the sediment conditions are right (Guiry & Guiry, 2019).

1.3.3. Lagodon rhomboides (Pinfish)

Pinfish are an important estuarine species and one of the most abundant inshore fish found in the Gulf of Mexico and southeastern United States up to Virginia (Darcy,

1985; Chacin, 2014). Pinfish can be found in temperate zones and subtropical seagrass meadows, submerged mangrove systems, oyster beds, and salt marshes along the southeastern Atlantic and Gulf coasts of North America (Darcy, 1985; Chacin, 2014;

Nelson, 2002; Montgomery & Targett, 1992; Potthoff & Allen, 2003; Shervette et al.,

2007). In the northeastern Gulf of Mexico pinfish have been found to be the most abundant vertebrates in seagrass beds and a common prey item for commercially important fish species in the Florida Keys. (Chacin, 2014).

Pinfish are omnivores that undergo transitions in feeding habits throughout their life cycle (Chacin, 2014; Darcy, 1985; Luczkovich, 1988; Montgomery & Targett, 1992;

Stoner, 1980; Stoner & Livingston, 1984). Studies have found that small juvenile pinfish

(between 20 – 35 mm) tend to be predominantly carnivorous while larger juvenile pinfish

(between 36 – 80 mm) are more omnivorous and have been observed consuming items such as small crustaceans, shrimp, fish, and epiphytes depending on their environment

(Darcy, 1985; Nelson, 2002; Luczkovich, 1988; Stoner, 1980; Stoner & Livingston, 1984).

Studies have also noted that epiphyte consumption by larger juvenile pinfish (36 – 80 mm) made up about thirty percent of their diets (Darcy, 1985; Stoner, 1980; Stoner &

Livingston, 1984). As pinfish continue to grow (between 80 – 120 mm) the abundance of plant material in their diets has been observed to increase (Darcy, 1985; Stoner, 1980;

Stoner & Livingston, 1984). Once larger than 120 mm, the diet of pinfish becomes predominantly herbivorous (studies noted that invertebrates were still consumed some of the time though; Darcy, 1985; Luczkovich, 1988; Stoner, 1980; Stoner & Livingston,

1984).

Pinfish have been identified as generalist feeders, but some individual have been observed to be selective at times (Darcy, 1985; Luczkovich, 1988; Stoner, 1980). Both juveniles and adult pinfish feed diurnally and are found to primarily feed along the bottom of the water column with the exception of larval and small juveniles that have been found feeding higher in the water column (Darcy, 1985). Pinfish use both vision and scent to locate food (Darcy, 1985; Luczkovich, 1988; Stoner & Livingston, 1984). Their diet can consist of organisms such as amphipods, isopods, polychaetes, barnacles, small crustaceans, smaller fishes, epiphytes and they are major consumers of vegetation in seagrass meadows (Nelson, 2002; Montgomery & Targett, 1992; Shervette et al., 2007;

Stoner & Livingston, 1984). In previous studies, the diet of pinfish has been observed to

11 consist of 18 – 90% seagrasses such as Thalassia testudinum, Syringodium filiforme, and

Zostera marina L. (Adams, 1976; Stoner & Livingston, 1984) and can consist of up to 50% macroalgae (primarily unspecified green algae species; Darnell, 1958; Hansen, 1969;

Montgomery & Targett, 1992; Stoner, 1980). As a result, this species can give good insight into the links between primary and secondary production within seagrass ecosystems.

1.4. Ciguatera Fish Poisoning

Ciguatera Fish Poisoning (CFP) is a form of food poisoning in humans who consume tropical reef fish that have accumulated high levels of ciguatoxins (Banner, 1976;

Clausing et al., 2018; Dickey & Plakas, 2010; Erdner et al., 2008; Friedman et al., 2017;

Loeffler et al., 2015; Parsons et al., 2011; Yasumoto et al., 1977b). Ciguatoxins are lipid- soluble neurotoxins produced by some unicellular members of the benthic dinoflagellate genus, Gambierdiscus, that enter reef food webs through the consumption of these cells via herbivory (Clausing et al., 2018; Dickey & Plakas, 2010; Litaker et al., 2010; Loeffler et al., 2015; Parsons et al., 2011; Sparrow et al., 2017; Tester et al., 2010; Yasumoto et al.,

1977a,b; Adachi & Fukuyo, 1979). Ciguatoxins can bioaccumulate within fish tissues and subsequently biomagnify through trophic transfer up the food web (Clausing et al., 2018;

Erdner et al., 2008; Friedman et al., 2017). Over 400 species of tropical reef fish have been associated with CFP reports globally, including top predatory species such as barracuda

(Sphyraenidae), grouper (Serranidae), snapper (Lutjanidae), jacks (Carangidae), and mackerel (Scombrini; Catasus, 2019; Food and Drug Administration, 2013; Hossen et al.,

2015; Perez-Arellano et al., 2005; Randall, 1958; Tester et al., 2010).

CFP is rarely fatal and manifests itself through gastrointestinal, neurological, and cardiovascular symptoms in humans (Abraham et al., 2012; Banner, 1976; Catasus, 2019;

Erdner et al., 2008; Friedman et al., 2017; Hossen et al., 2015; Legrand, 1998; Lewis, 2001;

Litaker et al., 2010; Randall, 1958). Estimates for the number of people affected by CFP range from 10,000 to 500,000 per year (Chinain et al., 2010; Epelboin et al., 2014; Fleming et al., 1998; Friedman et al., 2017; Skinner et al., 2011). However, CFP is difficult to properly diagnose due to its similarity to other food-borne illnesses, so, in actuality, the amount of people affected annually is likely at the high end of this reported range (Epelboin et al., 2014; Fleming et al., 1998; Friedman et al., 2017; Lewis, 2001; Skinner et al., 2011).

Ciguatoxins cannot be removed from fish tissue prior to consumption by any known technique (including cooking, cleaning, or chemical treatments); they are colorless, tasteless, odorless, and stable through heating or freezing and acid treatments (Catasus,

2019; Abraham et al., 2012; Friedman et al., 2017; Randall, 1958). People that have contracted CFP can become hypersensitive to the toxin, causing symptoms to reemerge more easily than individuals not previously affected by CFP (Banner, 1976; Erdner et al.,

2008).

CFP is not a recently discovered food poisoning, with reports of CFP dating back to the early 1500’s (Legrand, 1998). CFP was historically known to predominantly affect island communities that relied heavily on fishing as their main food supply; however, in today’s society, the increased rates of international seafood trade and travel have expanded the areas that are affected by CFP (Develoux et al., 2008; Epelboin et al., 2014; Friedman et al., 2017; Skinner et al., 2011). Florida is an example of a popular global tourist destination with 126,981,000 out of state visitors in 2018 alone (Visit Florida, 2019). Much

13 of the tourism associated with the state revolves around the coasts and local seafood selection. Many of the top selling species of fish sold in markets and at restaurants (e.g., grouper, snapper, and mackerel) commonly cause CFP (Friedman et al., 2017; Hossen et al., 2015; Hsieh et al., 2009; Tester et al., 2010).

CFP outbreaks are predominantly recorded between the latitudes 35°N and 35°S

(Figure 1.4; Aligizaki & Nikolaidis, 2008; Epelboin et al., 2014; Food and Drug

Administration, 2016; Legrand, 1998; Lewis, 2001; Perez-Arellano et al., 2005). However,

Gambierdiscus species are being documented in new areas around the world, leading scientists to believe that their distribution is increasing globally (Aligizaki & Nikolaidis,

2008; Friedman et al., 2017; Nishimura et al., 2013; Perez-Arellano et al., 2005; Tawong et al., 2015; Villareal et al., 2007). These observations have not been scientifically linked to climate change although they have raised awareness on the importance of researching the role warming sea surface temperatures and other associated factors may play in CFP.

Changes in global temperatures could potentially create more viable environments for ciguatoxins to accumulate (Fleming et al., 1998; Friedman et al., 2017; Schlaich et al.,

2012; Villareal et al., 2007). Tester et al. (2010) found that all CFP occurrences observed in the Caribbean between 2002 and 2007 were in areas where average annual sea surface temperatures were 25°C or higher. They also noted that average sea surface temperatures in the majority of the Caribbean were at or above 25°C even in the coldest months of the year (Tester et al., 2010). At the site in the Florida Keys studied in the present experiment

(HGB), the average annual temperature from 2011 – 2016 was 26.8°C (Parsons, 2017).

Lastly, Tester et al. (2010) noted the isotherm for the observed ideal temperature for maximum Gambierdiscus species production (25°C) was above the 35°N latitude range for

Gambierdiscus species, suggesting Gambierdiscus species could be expanding past the current 35°N and 35°S ranges previously mentioned.

Figure 1.4: The geographical distribution of ciguatera (light gray band: range from 35°N to 35°S latitudes). The medium gray markings indicate coral reef regions within the range where ciguatera is found. The darkest gray markings indicate disease- endemic areas of ciguatera. (Figure adapted from Perez-Arellano et al., 2005)

Studies have addressed that temperature may play a role in the grazing rates of herbivores (Bruno et at., 2015; Carpenter, 1986; Ferriera et al., 1998; Polunin & Klumpp,

1992; Smith, 2008). Additionally, Carpenter (1968) and Klumpp and McKinnon (1989,

1992) address the potential for changes in the availability of food sources such as algae or seagrasses with changing temperatures. If food sources such as macroalgae and seagrasses are increasing with increasing temperature as suggested by Carpenter (1986) and Klumpp and McKinnon (1989, 1992) that would mean that there would be a larger availability of substrates for Gambierdiscus cells to grow on (Tester et al., 2010). Furthermore, if grazing rates by herbivorous fishes are influenced by temperature, they may also correlate with the rate of ciguatoxins being introduced into marine environments where toxic Gambierdiscus

15 species are found. Gambierdiscus cells live in the epiphytic communities of organisms such as seagrasses and macroalgae (Friedman et al., 2017; Hossen et al., 2015; Parsons et al., 2011; Tester et al., 2010). Understanding the factors affecting epiphyte grazing and the direct consumption of different macrophytes by herbivores within these ecosystems is an important step in understanding the trophic transfer of ciguatoxins from epiphytic

Gambierdiscus species into herbivores in these systems.

The culprit of CFP, Gambierdiscus species, can be found in tropical and subtropical regions worldwide primarily in the epiphytic community of coral, algae, and seagrass, but they have also been observed in sand and in the water column (Dickey & Plakas, 2010;

Erdner et al., 2008; Friedman et al., 2017; Hossen et al., 2015; Parsons et al., 2011; Tester et al., 2010; Yasumoto et al., 1977b, 1980). Gambierdiscus cells thrive in shallow coastal environments and reside on a variety of host species (including seagrasses and algae;

Hossen et al., 2015; Parsons et al., 2017; Friedman et al., 2017). Thalassia testudinum and

Halimeda incrassata were found to harbor high cell densities of Gambierdiscus (per gram wet weight macrophyte), including values within the top 20 highest cell densities ever recorded (maximum densities of Gambierdiscus cells per algal gram: Thalassia testudinum had 1463 and Halimeda incrassata had 4774; Cruz-Rivera & Villareal, 2005). Fish that frequent shallow coastal environments where Thalassia testudinum, Halimeda incrassata, and Gambierdiscus cells thrive may be highly susceptible to the accumulation of ciguatoxins in their tissues and thus likely to cause CFP outbreaks in humans who consume these fishes (Friedman et al., 2017).

Ciguatoxins are introduced into the marine food web via grazing by herbivorous or omnivorous fish (Clausing et al., 2018; Erdner et al., 2008; Friedman et al., 2017; Hossen

16 et al., 2015; Legrand, 1998; Lewis & Holmes, 1993; Loffler et al., 2015). When those lower trophic level fish are then consumed by carnivorous fish, the toxins transfer to the consumer and can biomagnify through the food web (Figure 1.5; Catasus 2019; Clausing et al., 2018;

Erdner et al., 2008; Friedman et al., 2017; Legrand, 1998; Lewis & Holmes, 1993).

Herbivorous and omnivorous fish that target food sources such as algae, detritus or seagrass in areas known to harbor Gambierdiscus cells are therefore at higher risk of becoming ciguatoxic and passing ciguatoxins to higher trophic levels. As grazing by these herbivorous and omnivorous fish is the first major step in introducing ciguatoxins into the marine food web (and eventually onto people’s plates), it is critical to study this process to better understand how ciguatoxins move through the food web and into fish such as grouper and snapper that people eat, causing CFP.

Figure 1.5: Depicts how ciguatoxins are introduced to marine food webs via grazing by herbivorous fish and subsequently how the toxins move up the food web to larger carnivorous fish that people consume thus causing CFP outbreaks. (Figure adapted from https://www.nbc-2.com/story/39239441/poisonous-toxins-could-be-in-local-seafood)

Dawson et al. (1955) first addressed the importance of grazing in understanding the geographical distribution of ciguatoxic fish. These researchers did a study in the Line

Islands and found that fish of the same species were toxic in one location and non-toxic in another location, leading them to conclude that this difference was most likely related to differences in food resources the fish were consuming in each location. Additionally, this study noted the presence of algae in the stomach content of many of the toxic fish that were collected, suggesting an algal source to the toxins in the fish (also noted in Randall, 1958).

However, it wasn’t until the 1970s that the source of this toxin in the algae was discovered to be the benthic (epiphytic) dinoflagellate, Gambierdiscus (Yasumoto et al., 1977b), then an unknown species later described as Gambierdiscus toxicus (Adachi and Fukuyo, 1979).

Currently, 16 species of Gambierdiscus have been described worldwide, some more toxic than others with new species continually being identified (Bravo et al., 2019; Díaz-Asencio et al., 2019; Fraga & Rodríguez, 2014; Jang et al., 2018). In a study done by Tester et al.

(2010), it was found that the different Gambierdiscus species thrive at different temperatures. Additionally, Tester et al. (2010) found that some species such as

Gambierdiscus carolinianus experienced its highest growth rates at 25°C in isolated laboratory settings while other species such as Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus pacificus, Gambierdiscus (now Fukuyoa) ruetzleri, and

Gambierdiscus ribotype 2 experienced increased growth rates or their maximum growth rates at 29°C or higher. Gambierdiscus species have also been found to have differing toxicity levels (Holland et al., 2013; Xu et al., 2016). Understanding the grazing rates of herbivores on Gambierdiscus species as a whole is important, however as more studies are published on the toxicity and growth rates of individual species of Gambierdiscus it will

18 become arguably more important to identify where the most toxic strains of Gambierdiscus grow and study the grazing dynamics of herbivores in those locations.

Although Gambierdiscus species were identified as the source of the toxins leading to CFP, little work has been done on the grazing of Gambierdiscus species and the transfer of the toxin into the food web until very recently (Clausing et al., 2018; Loeffler et al.,

2015). Loeffler et al. (2015) conducted the first known study addressing the impacts of herbivory on Gambierdiscus cell densities. In this study, researchers used both caged and partially caged artificial substrates, that supported the growth of Gambierdiscus cells, to address the effects of herbivory on Gambierdiscus cell densities. From this, they found that

Gambierdiscus cell densities were up to 374% higher on caged samples than on partially caged samples suggesting that grazing intensity is likely a controlling factor in the amount of Gambierdiscus cells present in natural environments. Additionally, Clausing et al.

(2018) conducted a grazing experiment addressing how ciguatoxins were directly taken up by grazers. They used the herbivorous fish Naso brevirostris (Acanthuridae; common name unicorn fish) and fed them gelatin pellets infused with Gambierdiscus polynesiensis for an extended period of time. They then extracted the tissues from those fish for toxin analysis.

Outside of the two above listed studies, there has been little work done on understanding the relationship between grazing dynamics by herbivores in relation to the consumption and abundance of Gambierdiscus cells. In addition to observing the grazing dynamics of pinfish between Thalassia testudinum and Halimeda incrassata across a temperature gradient, this study also aims to tie these findings into implications for CFP and help with modeling efforts for CFP outbreaks.

1.5. Study Overview

The purpose of this study it to address the grazing dynamics of pinfish on Thalassia testudinum and Halimeda incrassata across a temperature gradient. In addition, this study will also address the implications Ciguatera Fish Poisoning has in this area. Thalassia testudinum and Halimeda incrassata were chosen as they are all common in seagrass communities in the Florida Keys (Chacin, 2014; Lirman et al., 2019; Russell et al., 2014).

In addition, Thalassia testudinum and Halimeda incrassata are known to harbor toxic

Gambierdiscus species within the associated epiphytic community living on both macrophytes (Crus-Rivera & Villareal, 2005; Hossen et al., 2015; Parsons et al., 2017;

Friedman et al., 2017). Pinfish were chosen for this study as they are a common prey item for many commercially important fish species in the Florida Keys (Chacin, 2014).

Additionally, they are the most abundant vertebrate species in the northeastern Gulf of

Mexico which could make them an important link to Ciguatera Fish Poisoning (Chacin,

2014).

Heine Grass Bed (HGB; Figure 1.6 is a representative seagrass bed in the middle

Florida Keys where Gambierdiscus cells are present and abundant (as are pinfish,

Thalassia testudinum and Halimeda incrassata). HGB has also been studied on a monthly basis for many years and was found to have the highest average Gambierdiscus cell abundances of the four sites that were studied in the middle Florida Keys (Parsons et al.,

2017). These characteristics make HGB an ideal site to study the grazing dynamics of pinfish between both macrophyte species in addition to addressing how these three species might be associated with the introduction of ciguatoxins into the marine environment and food web in this location.

Figure 1.6: HGB Coordinates: N. 24°51’38.4”, W. 80°44’17.4” a) Identifies the location of the study site, HGB (marked with red circle), in relation to Florida and the Florida Keys. The largest image (colorful image) depicts the site at a closer angle, on the Florida Bay side of Lower Matecumbe Key in the middle Florida Keys. (Figure adapted from Google Earth)

2. RESEARCH PURPOSE

The overall objective of this study is to examine the grazing dynamics of the pinfish on Thalassia testudinum and Halimeda incrassata under varying temperatures within a seagrass bed environment (study site HGB) in the middle Florida Keys. This study will also aid in determining how ciguatoxins are being introduced into the marine food web through grazing processes occurring within seagrass beds in the middle Florida Keys and assist modeling efforts for CFP outbreaks. Additionally, it will identify if specific temperatures influence the grazing behaviors of pinfish. This will lend insight into how grazing pressures change seasonally and how they may change as the climate warms.

Specific objectives and null hypotheses are listed below:

Objective 1: Assess pinfish grazing for Thalassia testudinum versus Halimeda

incrassata at ambient temperature.

Null Hypothesis 1: Pinfish do not consume either macrophyte at ambient

temperature.

Null Hypothesis 2: Pinfish consume both macrophytes equally at ambient

temperature.

Objective 2: Determine the effect of water temperature on pinfish grazing and on

preference for Thalassia testudinum versus Halimeda incrassata.

Null Hypothesis 3: Pinfish grazing is not affected by temperature.

Null Hypothesis 4: Pinfish do not consume one macrophyte over the other

consistently across all temperatures.

3. METHODS

3.1. Study Site

Heine Grass Bed (HGB) is a shallow seagrass bed with an average depth of around

2 meters. It is located on the western side of the middle Florida Keys (Coordinates: N.

24°51’38.4”, W. 80°44’17.4”; for image see Figure 1.6 from Introduction). The species composition at HGB is predominantly Thalassia testudinum and Halimeda incrassata with other species considered minor background species in this experiment (Parsons, 2017).

According to Parsons (2017), temperatures at HGB averaged 31.28°C (range 26.98°C to

34.37°C) in the summer (June 21st – September 22nd) and 22.96°C (range 15.71°C to

28.55°C) in the winter (December 21st – March 20th). During the spring and fall the temperatures averaged at 27.94°C (range 19.46°C to 32.18°C) and 26.00°C (range 18.83°C

22 to 31.90°C) respectively (Equinox and Solstice 2010 – 2019; Parsons, 2017). Salinities from that study averaged 36 parts per thousand (ppt) year-round (Parsons, 2017).

3.2. Field Collection Procedures

Prior to the start of each experimental treatment, Thalassia testudinum and

Halimeda incrassata were collected from HGB. Pinfish were obtained locally in the

Florida Keys from the World Wide Sportsman bait shop. A YSI Pro Plus Multiparameter

Meter was used to record water temperature and salinity at the time of collection. Within

HGB, healthy Thalassia testudinum and Halimeda incrassata were targeted for collection.

Thalassia testudinum and Halimeda incrassata samples were collected separately using

SCUBA equipment.

Initial tests utilizing living macrophyte samples that were cleaned of epiphyte with intact root systems were attempted, but there was little interest in grazing by the pinfish.

This was most likely a result of the toughness of the plants structure and/or the lack of epiphytes present on the macrophytes (Hemminga & Duarte, 2000; Klumpp et al., 1992;

Nakaoka, 2005; Prado & Heck, 2011; Valentine & Heck, 1999; Zapata & McMillan, 1979).

Because of this, both macrophyte species were made into agar following the methods and standards set forth by Prado and Heck (2011). This removed any structural deterrents that may have been present when the macrophytes were in their natural form (Prado & Heck,

2011). Additionally, it allowed for the epiphytic communities found on each macrophyte to remain intact once they were reconstituted into agar.

Both macrophytes were collected by cutting the blades or thalli at the base of the plant; they were then separated into plastic containers by species and the containers were

23 capped underwater to minimize disturbance to the epiphytic community growing on them.

Following the guidelines set by the Special Activities License issued to Dr. Parsons from the Florida Fish and Wildlife Conservation Commission (SAL_17_1262), the number of

Thalassia testudinum blades and Halimeda incrassata thalli collected were monitored making sure not to remove more than allowed from the site so as to minimally impact and preserve the seagrass bed. Additionally, samples were collected equally throughout the seagrass bed to accurately portray the entire sample site. Collected samples of Thalassia testudinum and Halimeda incrassata were placed into separate one-gallon Ziploc bags corresponding to the species type. The samples were given time to rest allowing suspended particles in the water to settle. Excess water was then poured off, taking care to not pour out any of the epiphytes that had become dislodged during the transfer. Following this step, the Ziploc bags were placed on ice in Yeti coolers until returning to the field lab (the Florida

Institute of Oceanography’s Florida Keys Marine Laboratory in Layton, FL) where they were placed into freezers set at -20°C.

Initial attempts to collect pinfish using seine nets, cast netting, sabiki rigs, otter trawls, and bait and trap techniques were all unsuccessful. Thus, pinfish used for experimentation were purchased from the World Wide Sportsman bait shop in Islamorada

Florida. The World Wide Sportsman bait shop in Islamorada receives pinfish regularly

(daily to weekly) from fisherman that collect the fish locally. The fish were transported in an Igloo cooler with 60 liters of seawater. An assortment of medium sized limestone rocks were places into the cooler providing shelter for the pinfish along with two Marine Metal

Bubbles aerators while being transported back to Vester Marine Field Station. The pinfish purchased from the World Wide Sportsman bait shop were all greater than 120mm in

24 length and were all within 10mm of each other. This size range was selected to target herbivorous individuals. Pinfish that are 120mm or larger have been observed to be herbivorous (Darnell, 1958; Stoner, 1980). At the start of each experimental trial all pinfish used were between the lengths of 130mm to 140mm.

The following chart (Figure 3.1) represents a timeline of the study highlighting when samples were collected, when agar was made, when control and experimental trials were run, and what days wet weight measurements were recorded.

3.3. Experimental Set Up

Macrophyte samples (on ice in Yeti coolers) and pinfish (in Igloo cooler with saltwater and bubblers) were transported back to Florida Gulf Coast University’s Vester

Marine Field Station in Bonita Springs, FL within 24 hours of collection. Macrophyte samples were stored in a freezer at -20°C until they were made into agar. Pinfish were acclimated to a 473-liter holding tank via drip acclimation over a 4-hour period that was held at the ambient temperature set for this project (26°C). Once pinfish were acclimated, they were placed into the holding tank until experimentation following standards set forth by the Institutional Animal Care and Use Committee protocols. Following feeding methods used by Prado and Heck (2011), pinfish were fed a diet of frozen shrimp and the macrophytes that were used for this study (Thalassia testudinum and Halimeda incrassata agar cubes) while in the holding tank. The holding tank for the pinfish utilized 36 ppt artificial seawater that was made by combining Instant Ocean Sea Salt with reverse osmosis water. A filtration system was set up on the holding tank that would pump dirty water out of the main 473-liter compartment of the tank into a carbon filter, through a UV sterilizer, into a large plastic container filled with Bio Balls, and then through a PVC tube fitted with a number of small openings for aeration before flowing back into the main holding tank

(Figure 3.2). This process allowed for the excess nutrients to be filtered out of the water and the UV sterilizer prevented the pinfish from getting fin rot.

Figure 3.2: Pinfish Holding tank set up a) Shows where water is removed from the tank. b) Carbon filter that dirty water passes through c) UV sterilizer d) Bio Ball filtration tank e) & f) Show the entire tank set up from the front and side angles. Looking at (f) water is removed from the left side of the tank filtered and sterilized then flows back into the right side of the tank. Figures (e) and (f) also show the mesh barrier that was put into the holding tank creating a separation device for pinfish once they had undergone a feeding trial (discussed further in section 3.3.2).

One control trial and three experimental trials were run for this study. Each trial was performed in a temperature-controlled room at Vester Marine Field Station. For all trials (control and experimental) eight 19-liter clear aquarium mesocosm tanks were set up and filled with water taken from the pinfish holding tank. Tanks were fitted with Elemental

Solutions H2O Titanium Heaters and set to the following temperatures 30°C, 29°C, 28°C,

27°C, 26°C, 25°C, 24°C, and 23°C for tanks 1 through 8 respectively, thus simulating the average range of temperatures observed at HGB across all seasons (Parsons, 2017). Each tank was fitted with an Onset HOBO Data Logger that recorded light and temperature measurements every 15 minutes throughout the duration of the experiment. The tank heaters were set at the above listed temperatures but the actual temperature averages recorded by the HOBO Data Loggers were as follows plus or minus the range between all trials: 30.3°C ±0.3, 28.6°C ±0.1, 27.6°C ±0.5, 26.8°C ±0.1, 25.5°C ±0.1, 24.3°C, 23.9°C

±0.5, and 22.2°C ±0.2 for tanks 1 through 8 respectively. For the remainder of the experiment the tank temperatures will be referred to as 30°C, 28°C, 27°C, 26°C, 25°C,

24°C, 23°C and 22°C for tanks 1 through 8 respectively (Figure 3.3). The tanks were set up in groups of 4 under Sun Blaze T5 HO 48-inch fluorescent lights. The lights were fitted with timers set to a 12:12 hour light to dark ratio throughout the length of the experiment; these conditions simulate the average light exposure for all species over a yearly period.

At the beginning of each trial tanks were cleaned, filled with salt water from the pinfish holding tank, and heaters and lights were turned on.

By putting the tanks in groups of 4 it ensured each tank would receive an equal amount of light. Additionally, it standardized the fish’s feeding behaviors by allowing each fish to be in a similar environment where they could see 2 or 3 other fish at any time during the experiment (Figure 3.3). According to Darnell and Wissing (1975), the rate at which pinfish consume food varies depending on the level of isolation that the fish feels. In complete isolation pinfish will not consume enough food to survive, but when pinfish were able to see other pinfish while in captivity, they are more likely to have similar feeding

29 rates to those observed in the wild (Darnell & Wissing, 1975). Additionally, cardboard was placed on the outside walls of the tanks so that the feeding behaviors of the pinfish would not be influenced by the presence of people in the lab.

Agar cubes were made for each macrophyte species using the steps outlined by

Prado and Heck (2011) as a guide. First, the frozen macrophyte samples, with epiphytes still attached, were thawed at room temperature then dried in a drying oven at 70°C for 24 hours (Prado & Heck, 2011). Once the samples were dried, they were ground into a fine powder using a high-powered blender. The agar cubes were made by combining 4 grams of a single macrophyte species with a heated mixture of 100 milliliters of distilled water and 2 grams of agar (Fisher Scientific agar powder; Prado & Heck, 2011). The agar and water mixture was made by putting the two components together in a beaker and placing it in an autoclave on the liquid cycle to incorporate. Once the new homogenous mixture

(agar and water) was made it was cooled for one hour at room temperature before the ground macrophyte powder was added. By allowing the agar and water mixture to cool for

30 an hour the consistency became very viscous allowing the macrophyte powder to stay uniformly distributed throughout the agar cube, rather than settle at the bottom of the cube.

After all the components were mixed together the solution was poured into 1-inch silicone cube molds and placed into a refrigerator for an hour to set (Prado & Heck, 2011). Once cooled, samples were removed from the mold and placed into a bucket of water taken from the pinfish holding tank for 24 hours. This occurred during the food deprivation period the pinfish underwent leading up to each experiment (this will be discussed in more detail in section 3.3.2; Figure 3.1). Initial experimental attempts found that within the first 24 hours the agar cubes absorbed water and some floated. Allowing the agar to acclimate for 24 hours minimized this from occurring for the remainder of the experiment (some agar blocks still absorbed a small amount of water during experimentation, but no agar blocks were observed floating in any of the trials. Agar cubes that absorbed water during experimentation had negative wet weights at the end of the trial periods). After the 24-hour acclimation period each agar cube was weighed on a Baoshishan (500/0.001) Analytical

Electronic Balance to the nearest 0.001 gram and placed into trial tanks. A wet weight was used to measure the change in weight of the agar cubes. To standardize the weighing process, one agar cube was removed from the water at a time and placed on a dry paper towel for 20 seconds before weighing. After the weight was recorded the agar cube was placed back into the tank it was removed from.

3.3.1. Control Trial

A control trial without fish was done prior to the start of the experimental trials to monitor the decomposition rate of the agar cubes under each temperature treatment that

31 would be used during the experiment (Figure 3.1). For this trial, 3 replicates of each macrophyte agar cube were placed into each tank to account for any anomalies that might occur during the trial (48 total cubes: 24 Thalassia testudinum, 24 Halimeda incrassata;

Figure 3.4). Each replicate was tagged prior to weighing to ensure they were not mixed up during the weighing process. Tanks were monitored daily and every 48 hours the wet weight of each agar cube was recorded (Day 1, Day 3, Day 5, Day 7, and Day 9; Figure

3.1). The control trial went on for a total of 9 days (2 days longer than the experimental trials). From the results of the control trial and lengths of other previous grazing experiments it was determined that 7 days was an appropriate amount of time to run the experimental trials to see grazing rates and maintain the structural integrity of the agar cubes (Clausing et al., 2018; Darcy, 1985; Montgomery & Targett, 1992; Prado & Heck,

2011).

Figure 3.4: Schematic of the control trial. The experimental tanks are represented by the blue rectangles and labeled with the tank number and temperature. The larger white rectangles that encompass the tanks represent the lights that the tanks were placed under during the experiment. Dark green squares represent the Thalassia testudinum agar cubes and light green squares represent the Halimeda incrassata agar cubes.

3.3.2. Experimental Trials

Three experimental trials were conducted for this experiment each following the same procedures. Pinfish were fed prior to transfer to experimental tanks; this ensured all

32 pinfish would begin their 24-hour food deprivation period at the same time. They were then collected, measured to the nearest 0.5 centimeter, and weighted to the nearest 0.1 gram.

Once the tanks were set up and had reached the appropriate temperatures, pinfish were placed into the tanks and underwent a 24-hour food deprivation period (Figure 3.1; Prado

& Heck, 2011). After 24 hours, one Thalassia testudinum and one Halimeda incrassata agar cube (that had been soaking for the entire food deprivation period as explained above) were weighed and placed into each tank (Figure 3.5). All tanks were monitored daily and agar cubes were weighed every 48 hours (Day 1, Day 3, Day 5, and Day 7) to allow for minimal disturbance to the fish (Figure 3.1). At the end of the 7-day experimental period final fish length to the nearest 0.5 centimeter and weight to the nearest 0.1 gram was recorded and fish were put back into the holding tank. A mesh divider was made out of oyster netting and PVC separating the holding tank into two halves while still allowing water exchange throughout the tank. One side of the divider was for fish that had not been used for experimentation yet and the other side was for fish that had already been used for experimentation. This ensured that pinfish were not being used for multiple experiments.

At the end of the experiment tanks were drained, cleaned, and set back up for the next trial.

Figure 3.5: Schematic of the experimental trials. The experimental tanks are represented by the blue rectangles and labeled with the tank number and temperature. The larger white rectangles that encompass the tanks represent the lights that the tanks were placed under during the experiment. The dark green squares represent the Thalassia testudinum agar cubes and the light green squares represent the Halimeda incrassata agar cubes. Pinfish are represented by the gray fish shape.

3.4. Data Analysis

All data collected for this experiment were recorded and organized in Microsoft

Excel then uploaded into SPSS (version 26) for analysis. Before testing each hypothesis, the change in control trial agar wet weights were first analyzed in SPSS to determine if they were significantly different from zero for each macrophyte. The results of these analyses were then used to determine if the data needed to be standardized for the degradation of the agar at each temperature. Different statistical analyses were used to answer each hypothesis (Table 1). The normality of the data and the residuals were tested to determine if parametric or non-parametric tests should be used. The data were normal for Hypothesis 1, so two One Sample T-Tests were run to determine if pinfish ate a significant amount of either macrophyte agar cube at ambient temperature. Similarly, the data were normal for Hypothesis 2, so an Independent Samples T-Test was used to identify if one macrophyte was more palatable than the other to pinfish at ambient temperature. The data were not normally distributed for Hypothesis 3, so a linear regression was used to test the normality of the residuals which were also found to not be normally distributed.

Therefore, in this case, two Linear Regression analyses were run using ranked data to determine if there was a linear change in the amount of Thalassia testudinum agar or

Halimeda incrassata agar eaten with changing temperature (i.e., more or less as temperature increased or decreased; Iman and Conover, 1979). Two Non-Parametric

Kruskal-Wallis analyses were also used for Hypothesis 3 to determine if the consumption of each macrophyte was higher or lower at a particular temperature (i.e., not linear). The data were not normal for Hypothesis 4, so the consumption data were ranked and input into

34 a Linear Mixed Model to determine if one macrophyte was consistently consumed over the other across temperatures.

Table 1: A table of all statistical tests being run by hypothesis. The table includes the hypothesis number and hypothesis, the variables that were compared, the statistical test that was used for each hypothesis, and the purpose of each statistical test being run.

Variables Statistical Hypothesis Purpose of Test Compared Tests H1: Pinfish do Amount eaten of Determine if pinfish are not consume each macrophyte 2 – One Sample eating either macrophyte either macrophyte at ambient T-Tests agar cubes at ambient at ambient temperature temperature temperature (26.8°C) H2: Pinfish Amount eaten of Determine if one consume both each macrophyte macrophyte agar cube is macrophytes 1 – Independent at ambient more palatable than the equally at Samples T-Test temperature other to pinfish at ambient (26.8°C) ambient temperature temperature Determine if there is a linear relationship between the 2 – Linear consumption of either Amount of Regressions macrophyte agar by H3: Pinfish Thalassia pinfish across grazing is not testudinum and temperatures affected by Halimeda Determine if the temperature incrassata eaten at 2 – Non- consumption of either all temperatures Parametric macrophyte agar by Kruskal-Wallis pinfish is higher at a tests specific temperature (i.e. non-linear) H4: Pinfish do Determine if pinfish Amount of not consume one consume Thalassia Thalassia macrophyte over testudinum agar or testudinum and 1 – Linear the other Halimeda incrassata Halimeda Mixed Model consistently agar over the other incrassata eaten at across all consistently across all all temperatures temperatures temperatures

4. RESULTS

4.1. Overview

Overall, every pinfish that was used in this experiment grazed on the Thalassia testudinum agar or Halimeda incrassata agar across various temperatures over the 7-day experimental periods with the exception of one pinfish (experimental trial 1, tank 7, 23°C).

It is worth noting that all pinfish from experimental trial 1 exclusively ate the Halimeda incrassata agar and not the Thalassia testudinum agar with the exception of the pinfish in tank 2 (28°C). This pinfish (experimental trial 1, tank 2, 28°C) ate both the Thalassia testudinum agar and the Halimeda incrassata agar during the study. The wet weights of any agar cubes from this study that had negative values (i.e. the agar gained weight and had no visible observation of bite attempts from pinfish) were given a value of zero since there were no signs (through wet weight recordings or visual observation) that the pinfish were eating those cubes. Additionally, as mentioned above, the pinfish from experimental trial 1 in tank 7 (23°C) did not consume any amount of either the Thalassia testudinum agar or the Halimeda incrassata agar during the study period. Seven days was a sufficient time for all but one pinfish (experimental trial 1, tank 7, 23 °C) to consume at least some amount of the macrophyte agar cubes while leaving some agar behind at the end of the experiment for post-grazing weight measurements. The pinfish used in the experimental trials all appeared to be well adjusted to the experimental tanks before grazing trials began as well.

4.1.1. Control Trial

During the control trial both the Thalassia testudinum and Halimeda incrassata agar cubes maintained a relatively uniform weight across all temperatures (average change in weight of agar cubes from initial wet weight recordings: Thalassia testudinum = 1.7% and Halimeda incrassata = 1.5%) and showed no signs of deterioration over the first 7 days of the trial. However, by Day 8, the agar cubes began deteriorating, and by Day 9 approximately one-third of the cubes began to break apart. As discussed in the methods, this was one of the determining factors for running the experimental trials for 7 days.

4.1.2. Experimental Trial 1

During the first experimental trial, the pinfish showed little interest in the agar cubes for the first 2 days. On Day 3, there were visible bite marks on the Halimeda incrassata agar cube from tank 2 (28°C; Figures 4.1a and 4.1b). The other tanks showed little change in the wet weights of the agar. There was little change in any agar cubes on

Day 4. On Day 5, it appeared that pinfish from all tanks, with the exception of the pinfish in tank 7 (23°C), showed some interest in at least one of the agar cubes in each tank with small recorded changes in wet weights and a few visible bite marks in some of the agar cubes. Between Day 5 and Day 6, the pinfish from tank 2 (28°C) had consumed nearly half of the Thalassia testudinum agar cube and roughly 5/6 of the Halimeda incrassata cube

(Figure 4.1c). The rest of the experimental tanks showed similar grazing amounts on agar as the previous day (Day 5). On Day 7, the pinfish from tank 2 (28°C) had consumed another gram in wet weight from the Thalassia testudinum agar cube and the majority of the remaining portion of the Halimeda incrassata agar cube. Additionally, on Day 7 there

37 appeared to be interest from all fish on at least one macrophyte agar cube in every tank, with the exception on the pinfish in tank 7 (23°C), according to the wet weight recordings and visual assessment. All of the total change in wet weights recorded for the Thalassia testudinum agar cubes (with the exception of the agar cube in tank 2, 28°C) and one of the

Halimeda incrassata agar cubes (tank 7, 23°C) were slightly negative in wet weight (the agar cubes had slightly gained weight during the experimental period). This was determined to be a result of these agar cubes absorbing a small amount of water during this trial in addition to not being grazed upon at all by pinfish (wet weights and visual assessments were used to come to this conclusion).

Figure 4.1: Bite marks on agar cubes made by pinfish. a) Shows the Halimeda incrassata agar cube from tank 2 (28°C) on Day 3 before the wet weight was recorded. b) Shows the same Halimeda incrassata agar cube from Tank 2 (28°C) after the wet weight was recorded on Day 3. c) Shows both the Thalassia testudinum and Halimeda incrassata agar cubes from tank 2 (28°C) on Day 6.

4.1.3. Experimental Trial 2

During the second experimental trial, the pinfish showed little interest in the agar cubes on Day 1. On Day 2 the pinfish in tank 6 (24°C) consumed about 1/10 of the

Thalassia testudinum agar cube (Figure 4.2a); the other experimental tanks showed little change in the agar cubes. There appeared to be some interest in the agar cubes by pinfish on Day 3 in all tanks with the exception of tank 4 (26°C). The pinfish in tank 2 (28°C), tank 5 (25°C; Figure 4.2b), tank 6 (24°C), and tank 7 (23°C; Figure 4.2c) all showed visual signs of grazing as well. There was little change on Day 4. On Day 5 it appears that all the pinfish showed some interest in the agar cubes in all tanks. The pinfish in tank 2 (28°C) showed the most interest of the 8 tanks on Day 5. Day 6 had similar results as Day 5 where all pinfish showed some interest in the agar cubes. On Day 7 the pinfish in tank 2 (28°C) had consumed roughly 20% of each macrophyte agar cube (Figure 4.2d). Additionally, the pinfish in tank 4 (26°C), tank 5 (25°C), tank 6 (24°C), and tank 7 (23°C) all showed an increase in the amount of agar they consumed. Over the course of the seven-day experimental period the data (along with visual observation) showed some signs of grazing in all tanks and on all agar cubes by pinfish.

Figure 4.2: Bite marks on agar cubes made by pinfish. a.) Shows a comparison of the bite marks on the Thalassia testudinum agar cube (left) compared to the Halimeda incrassata agar cube (right) from tank 6 (24°C) on Day 2. b.) Shows a bite mark in the Thalassia testudinum agar cube from tank 5 (25°C) on Day 3. c.) Shows multiple bite marks in the Thalassia testudinum agar cube from tank 7 (23°C) on Day 3. d.) Shows multiple bite marks on both the Thalassia testudinum (back) and Halimeda incrassata (front) agar cubes from tank 2 (28°C) on Day 7.

4.1.4. Experimental Trial 3

During the third experimental trial, pinfish showed little interest in the agar cubes for the first 2 days. On Day 3 there was some interest in the Halimeda incrassata agar cubes by pinfish in tank 1 (30°C), tank 2 (28°C), tank 3 (27°C), and tank 4 (26°C); the remaining tanks (tank 5, 25°C; tank 6, 24°C; tank 7, 23°C; and tank 8, 22°C) showed little change in wet weights of agar cubes. There was little change on Day 4. On Day 5 it appeared that all the pinfish had shown interest in the agar cubes. The fish in tank 3 (27°C) took visible bite marks out of the Halimeda incrassata agar cube (Figure 4.3a) and the fish in tank 8 (22°C) took visible bite marks out of both the Thalassia testudinum and Halimeda

40 incrassata agar cubes (Figure 4.3b). On Day 6 there was increased interest in the agar cubes by pinfish in tank 2 (28°C) and tank 5 (25°C) as well as the pinfish in tank 8 continually eating both agar cubes. On the final day (Day 7) the pinfish in tank 1 also showed increased interest in the agar cubes. Over the course of the seven-day experimental period the data

(along with visual observation) showed some signs of grazing in all tanks and on all agar cubes by pinfish.

Figure 4.3: Bite marks in agar cubes made by pinfish. a) Shows bite marks in Halimeda incrassata agar cube (right) made by the pinfish in tank 3 (27°C) on Day 5. b) Shows the bite marks in both the Thalassia testudinum agar cube (front) and Halimeda incrassata agar cube (back) made by the pinfish in tank 8 (22°C) on Day 5.

4.2. Results of Statistical Analyses

4.2.1. Control Trial

The total percent change in wet weight from each of the three agar cubes per macrophyte were averaged for each temperature. That data was then tested in SPSS to determine if the average percent change in wet weight of each macrophyte agar cube at each temperature differed significantly from zero. The results of the Linear Regression analyses showed that there was not a significant difference in the average percent change in wet weight of agar cubes from either macrophyte from zero across temperatures

(Thalassia testudinum p = 0.637 and Halimeda incrassata p = 0.387). Because there was not a significant difference in the average percent change in wet weight of either macrophyte cube across temperatures, the weight data from each experimental trial (1, 2, and 3) were not corrected for control cube weight loss.

4.2.2. Experimental Trials

The agar cube consumption data were tested below three ways: 1) raw amount of agar consumed per pinfish (g agar fish-1); agar consumed per gram fish weight (g agar g fish-1) and agar consumed per centimeter fish length (g agar cm fish-1). All subsequent statistical analyses gave similar statistical results so the analyses using the raw data are presented below.

Thalassia Halimeda Linear (Thalassia) Linear (Halimeda) y = 0.0784x - 3.6805 y = 0.2436x - 6.6293 R² = 0.0394 R² = 0.1267 p = 0.637 p = 0.387 4 3 2 1 0 22 23 24 25 26 27 28 29 30 -1 -2 -3 -4

Average Change in Wet Weight (%) Weight Wet in Change Average -5 Temperature (°C) Figure 4.4: The average percent change in wet weight for the control agar cubes at each temperature tested in this study for both macrophytes (Thalassia testudinum in dark green and Halimeda incrassata in light green). Linear equations, r squared values, and p values are listed for both species under the corresponding macrophyte they are for.

4.2.3. Hypothesis 1: Do pinfish consume either macrophyte at ambient temperature?

Ambient temperature for this experiment was determined to be 26.8°C (This is the actual temperature recording from tank 4 in all trials; the labeled temperature for reference is 26°C). The amount of Thalassia testudinum and Halimeda incrassata agar cubes that each pinfish consumed at ambient temperature was recorded. When the consumption amounts were graphed (Figure 4.5), the data showed that the pinfish from experimental trial 1 did not consume any of the Thalassia testudinum agar cube but did consume some of the Halimeda incrassata agar cube. The pinfish from experimental trial 2 consumed both the Thalassia testudinum and Halimeda incrassata agar cubes. A similar result was seen by the pinfish from experimental trial 3 with both the Thalassia testudinum and

Halimeda incrassata agar cubes being grazed on. Additionally, the pinfish from

43 experimental trial 2 consumed the largest amount of both macrophyte agar cubes while the pinfish from experimental trial 1 consumed the smallest amount of both macrophyte agar cubes.

2.5 Thalassia Halimeda 2

1.5

0.5 Total Consumption (g (g ww) Consumption Total

0 1 2 3 Experimental Trial

Figure 4.5: The total amount of Thalassia testudinum agar and Halimeda incrassata agar consumed by the individual pinfish in each experimental trial at ambient temperature (26.8°C, tank 4). This figure represents the data used for both Hypothesis 1 and Hypothesis 2.

Two, One Sample T-Tests were run to test the consumption of Thalassia testudinum and Halimeda incrassata to zero consumption at ambient temperature (26.8°C). The results showed there were not statistically significant differences in the consumption of either macrophyte from zero consumption (the amount of Thalassia testudinum eaten p = 0.292 and the amount of Halimeda incrassata eaten p = 0.155) at ambient temperature (Table 2).

However, as indicated in Figure 4.5, the pinfish in all three experimental trials (with the exception of the pinfish in experimental trial 1 not consuming any Thalassia testudinum agar) did consume some amount of each agar cube placed into the experimental tanks. The

44 data from experimental trial 2 were much larger than the data from experimental trial 1 or experimental trial 3 causing variability in the data.

Table 2: Results of both One Sample T-Tests comparing the actual amount consumed of each macrophyte agar by pinfish to zero consumption, individually, at ambient temperature (26.8°C). One-Sample Test Test Value = 0 95% Confidence Interval Sig. (2- df of the Difference tailed) Lower Upper Amount 2 0.292 -1.12979 2.23979 Thalassia Eaten Amount 2 0.155 -0.99411 3.14544 Halimeda Eaten *df refers to the degrees of freedom that are associated with the sources of variance for each analysis.

4.2.4. Hypothesis 2: Do pinfish consume both macrophytes equally at ambient temperature?

The amounts of Thalassia testudinum and Halimeda incrassata agar consumed by pinfish were recorded individually at ambient temperature (26.8°C). The results were graphed (Figure 4.5), the graph shows that pinfish consumed higher amounts of Halimeda incrassata agar than Thalassia testudinum agar in all three experimental trials 1 however, the variability between the three experimental trials could influence the significance of the results.

An Independent Samples T-Test was run comparing the amount of each macrophyte that was eaten by pinfish at ambient temperature. The results showed upon

45 further statistical testing that there was not a significant preference by pinfish between the consumption of either macrophyte at ambient temperature (p = 0.448; Table 3).

Table 3: Results of the Independent Samples T-Test comparing the actual amount consumed of each macrophyte by pinfish to identify if there was a preference between the two macrophytes by pinfish at ambient temperature. Independent Samples Test Levene's Test for Equality t-test for Equality of Means of Variances 95% Confidence Interval Sig. (2- Sig. df of the Difference tailed) Lower Upper Equal Amount variances 0.670 4 0.448 -2.242808 1.201474 Eaten assumed *Equal variance is assumed since the significance of the Lavene’s Test for Equality of Variance is > 0.05. df refers to the degrees of freedom that are associated with the sources of variance.

4.2.5. Hypothesis 3: Is pinfish grazing affected by temperature?

The individual amounts of Thalassia testudinum agar and Halimeda incrassata agar consumed by pinfish for each of the three experimental trials were recorded for testing.

Each macrophyte was tested individually for a change in the amount of consumption by pinfish across temperatures. When the data were graphed (Figure 4.6), it appeared that there was a slight increase in the consumption of both the Thalassia testudinum and

Halimeda incrassata agar cubes by pinfish with increasing temperature. However, the positive linear relationship recorded for the consumption of Thalassia testudinum was insignificant (p = 0.154; R2 = 0.0901), and significant but weak (p = 0.022; R2 = 0.2154) for Halimeda incrassata (Table 4).

30 y = 0.8321x - 8.8221 R² = 0.0901 p = 0.154 25

Ranked Ranked Thalassia Consumed 5

0 22 23 24 25 26 27 28 29 30 a. Temperature (°C) ______

30 y = 1.2865x - 20.468 R² = 0.2154 p = 0.022 25

Ranked Ranked Halimeda Consumed 5

0 22 23 24 25 26 27 28 29 30 b. Temperature (°C) Figure 4.6: Both graphs in this figure are looking at the ranked amount of Thalassia testudinum (a) or Halimeda incrassata (b) consumed by pinfish from all experimental trials at each temperature tested in this study. a) The ranked amount of Thalassia testudinum agar consumed by each pinfish in each experimental trial for all temperatures tested in this study. b) The ranked amount of Halimeda incrassata agar consumed by each pinfish in each experimental trial for all temperatures tested in this study. The linear equation, r squared value and p value from the linear regression analysis for each macrophyte are listed in the top right corner of each graph.

When looking at the graphed data (Figure 4.6), the results of these analyses, specifically the significance of the data for the Halimeda incrassata agar cubes, appear to be driven by the increased consumption amounts at 28°C. In experimental trial 1 the pinfish in tank 2 (28°C) consumed the majority of the Thalassia testudinum agar (12.268g) and nearly the entirety of the Halimeda incrassata agar (20.856g) while the rest of the pinfish in experimental tanks set at different temperatures did not have this same feeding reaction

(average consumption by pinfish in remaining tanks: Thalassia testudinum = 0.000g and

Halimeda incrassata = 0.186g). A similar result occurred in experimental trial 2 where the pinfish in tank 2 (28°C) consumed the largest portions of Thalassia testudinum and

Halimeda incrassata compared to the other pinfish in the trial (pinfish in tank 2, 28°C consumption: Thalassia testudinum = 3.384g and Halimeda incrassata = 4.388g; average consumption by pinfish in remaining tanks: Thalassia testudinum = 1.076g and Halimeda incrassata = 1.278g). However, in experimental trial 2, the pinfish consumed less of both agar cubes than the pinfish in the same tank from experimental trial 1. In experimental trial

3 the pinfish in tank 2 (28°C) consumed both the Thalassia testudinum and Halimeda incrassata agar at a similar quantity to the rest of the pinfish in the trial, and less than the pinfish from the same tank in previous trials (pinfish in tank 2, 28°C consumption:

Thalassia testudinum = 0.830g and Halimeda incrassata = 1.967g; average consumption by pinfish in remaining tanks: Thalassia testudinum = 0.606g and Halimeda incrassata =

1.314g. Due to the large range in grazing amounts by the individual pinfish in tank 2 (28°C) from each experimental trial, it was apparent that the grazing amount of the pinfish in this tank (tank 2, 28°C) from experimental trial 1 might be an outlier. To test this, the data from tank 2 (28°C) in experimental trial 1 was further tested and it was determined that the

48 grazing amounts for the Thalassia testudinum agar and the Halimeda incrassata agar were outliers. When the data were run again with these outlier points removed, the statistical outcome remained the same as before the outlier was removed. There was not a significant linear relationship in the consumption of Thalassia testudinum agar by pinfish over the temperature gradient tested (22°C - 30°C; p = 0.247) and there was a significant but weak linear relationship in the consumption of Halimeda incrassata agar over the temperature gradient tested (22°C - 30°C; p = 0.041; R2 = 0.1849).

Table 4: Results of the Linear Regression analyses (with all data points) identifying if a linear relationship was or was not present between the amount of Thalassia testudinum agar or Halimeda incrassata agar consumed by pinfish with temperature. ANOVAa Model Sum of Squares df Sig. Regression 103.594 1 0.154b Thalassia Residual 1046.406 22 Total 1150.000 23

Regression 247.661 1 0.022b Halimeda Residual 902.339 22 Total 1150.000 23 a. Dependent Variable: Rank Thalassia (top) Rank Halimeda (bottom) b. Predictors: (Constant), Temp. (°C)

*Regression, Residual, and Total refer to the sources of variance and the Sum of Squares that are associated with each source of variance. df refers to the degrees of freedom that are associated with the sources of variance for each analysis.

To further assess the effects of temperature on pinfish grazing, the data were set up to identify if either macrophyte agar was consumed at a specific temperature (i.e. in a non- linear fashion). The total amounts of Thalassia testudinum and Halimeda incrassata

49 consumed by pinfish were recorded individually at each temperature from all three experimental trials for testing. When the total consumption of each macrophyte was averaged from the three experimental trials for each temperature and graphed (Figure 4.7), the graph showed evidence that 28°C may be the preferred grazing temperature for pinfish on both Thalassia testudinum and Halimeda incrassata. However, there was some variability within the three experimental trials as shown by the error bars (particularly

28°C).

16 Halimeda 14 Thalassia 12 10 8

(g ww) (g 6 4 2

Average Amount of Agar Consumed Agar of Amount Average 0 22 23 24 25 26 27 28 30 Temperature (°C) Figure 4.7: The average amount of each macrophyte agar cube consumed by pinfish (in grams wet weight) from all three experimental trials preformed across the temperature gradient (22°C – 30°C) used in this study. Standard error bars represent the error associated with the average consumption amounts for each macrophyte at each temperature. The dark green bars show the average amount of agar consumption by pinfish for Thalassia testudinum and the light green bars show the average amount of agar consumption by pinfish for Halimeda incrassata.

The results of the Non-Parametric Kruskal-Wallis analyses (Table 5) showed that there were not statistically significant differences in the amount of Thalassia testudinum

50 agar or Halimeda incrassata agar consumed by pinfish at a specific temperature (Thalassia testudinum consumed p = 0.569 and Halimeda incrassata consumed p = 0.276). However, in tank 2 (28°C) the amount of both Thalassia testudinum agar and Halimeda incrassata agar that were consumed by pinfish in all three trials, when averaged, was much larger than the rest of the average consumption rates for both macrophytes (Figure 4.8). Due to the large standard error associated with this temperature (28°C), it weakens the significance of this temperature. As mentioned above, in experimental trial 1, the pinfish from tank 2

(28°C) consumed the most of each macrophyte agar cube for the entire experiment. In experimental trial 2, a different pinfish in the same tank (tank 2, 28°C) consumed the second most of each macrophyte agar cube for the entire experiment. These results were not observed by the pinfish in tank 2 (28°C) for experimental trial 3. Due to the changes in the grazing behaviors of the three pinfish tested at 28°C it caused a large standard error to be present when the consumption amounts of each macrophyte from the three experimental trials were averaged.

Additionally, as with the linear regression analyses, the data for the Non-Parametric

Kruskal-Wallis analyses were run again with the outlier points (Thalassia testudinum and

Halimeda incrassata data points from tank 2, 28°C, experimental trial 1) removed.

However, rerunning the analysis with these points removed did not change the statistical outcome of the results (Thalassia testudinum p = 0.829; Halimeda incrassata p = 0.476).

Table 5: Results of the Non-Parametric Kruskal-Wallis analyses identifying if there was a significant difference in the amount of consumption of either macrophyte by pinfish at a specific temperature. Top table (1) is for Thalassia testudinum and bottom table (2) is for Halimeda incrassata. Hypothesis Test Summary Null Hypothesis Test Sig. Decision The distribution of the Independent- amount of Thalassia Samples Retain the null 1 testudinum eaten is the 0.569 Kruskal-Wallis hypothesis. same across categories of Test Temp. (°C).

The distribution of the Independent- amount of Halimeda Samples Retain the null 2 incrassata eaten is the 0.276 Kruskal-Wallis hypothesis. same across categories of Test Temp. (°C). Asymptotic significances are displayed. The significance level is .050.

Figure 4.8: Boxplots from the Non-Parametric Kruskal-Wallis analysis for Thalassia testudinum (top) and Halimeda incrassata (bottom).

4.2.6. Hypothesis 4: Do pinfish consume one macrophyte over the other consistently across all temperatures?

The total amounts of Thalassia testudinum agar and Halimeda incrassata agar consumed by pinfish were recorded for each experimental trial. When the data from all three trials were ranked, averaged, and graphed (Figure 4.9), the average consumption of

Halimeda incrassata agar by pinfish appears to be greater than the average consumption of Thalassia testudinum agar for each temperature suggesting that Halimeda incrassata could be favored consistently across temperatures tested. However, in contrast, the error bars for both macrophytes overlap at all temperatures tested suggesting that there may not actually be a consistent preference.

50 Ranked Halimeda 45 Ranked Thalassia 40 35 30 25 20 15 10

5 Average Ranked Amount Average Ranked Amount Consumed 0 22 23 24 25 26 27 28 30 Temperature (°C) Figure 4.9: The average ranked amount of each macrophyte agar consumed by pinfish from all three experimental trials preformed across the temperature gradient (22°C – 30°C) used in this study. Standard error bars represent the error associated with the ranked average amount of agar consumed by pinfish for each macrophyte at each temperature. The dark green bars show the ranked average consumption by pinfish for Thalassia testudinum agar and the light green bars show the ranked average consumption by pinfish for Halimeda incrassata agar.

A Linear Mixed Model analysis was run to determine if pinfish consumed one macrophyte consistently more than the other across all temperatures. The results of the

Linear Mixed Model (Table 6) using ranked data indicated that there was a marginally significant difference in the consumption of one macrophyte over the other consistently across all temperatures (p = 0.052). Pinfish consistently consumed more Halimeda incrassata agar than Thalassia testudinum agar across the temperature gradient tested

(22°C – 30°C).

Table 6: Results of the Linear Mixed Model analysis identifying if pinfish consume one macrophyte over the other consistently across all temperatures. Pairwise Comparisonsa 95% Confidence Interval for Differenceb (I) (J) Lower Upper Macrophyte Macrophyte Sig.b Bound Bound Halimeda Thalassia 0.052 -0.067 14.608 incrassata testudinum Thalassia Halimeda 0.052 -14.608 0.067 testudinum incrassata Based on estimated marginal means a. Dependent Variable: Ranked Amount Eaten. b. Adjustment for multiple comparisons: Least Significant Difference (equivalent to no adjustments). *Column I represents treatment 1 and column J represents treatment 2.

Table 7: An overview of the results from the statistical analyses that were run for this study. This table includes the hypotheses tested, the null hypothesis of each hypothesis, whether or not there was a significant preference identified, and the possible reasons for the significance or insignificance of each analysis.

Null Significant Analysis Possible Reasons Why Hypothesis Difference - The sample size used for this experiment H1: Pinfish do - The laboratory environment not consume that the pinfish were in either 2 – One Sample - Reconstituted Thalassia YES/NO macrophyte at T-Tests testudinum and Halimeda ambient incrassata into agar form temperature - The feeding preference of the individual or specific fish used in the experiment H2: Pinfish consume both - The sample size used for this macrophytes 1 – Independent experiment NO equally at Samples T-Test - Longevity of reconstituted ambient macrophytes in agar form temperature - Availability of the food source for pinfish - A change in metabolic demand 2 – Linear YES/NO with changing temperature H3: Pinfish Regressions - Reconstituted Thalassia grazing is not testudinum and Halimeda affected by incrassata into agar form temperature 2 – Non- Parametric - The sample size used in this NO Kruskal-Wallis experiment Tests H4: Pinfish do not consume - Reconstituted Thalassia one testudinum and Halimeda macrophyte 1 – Linear YES/ incrassata into agar form over the other Mixed Model Marginal - The sample size used in this consistently experiment across all temperatures

5. DISCUSSION The overall results of this study are summarized in Table 7. In summary, pinfish did not consume one macrophyte agar (Thalassia testudinum or Halimeda incrassata) at a significantly higher amount than the other at ambient temperature. Additionally, the results also suggest that pinfish are not significantly increasing their grazing intensity on Thalassia testudinum agar across a temperature gradient (22°C – 30°C). However, the grazing intensity of pinfish on Halimeda incrassata agar did significantly increase with increasing temperature but the strength of this linear correlation is fairly weak (R2 = 0.2154). Finally, pinfish ate a marginally significantly larger amount of the Halimeda incrassata agar cubes than the Thalassia testudinum agar cubes over the temperature gradient tested (22°C –

30°C; p = 0.052).

This study utilized Thalassia testudinum and Halimeda incrassata in a reconstituted agar form as a proxy for the living macrophytes which produced results that may differ from the way these results would present themselves in a natural environment.

Furthermore, the results of this study are based off of a relatively small experimental sample size, thus it may be that underlying trends were masked by the variability in the data. Finally, this was a controlled study that was performed in a laboratory and thus, was not able to factor in all environmental conditions that would be found in the field.

5.1. Significant Conclusions

5.1.1. Hypothesis 1: The consumption of either macrophyte by pinfish at ambient temperature was insignificant.

The null hypothesis tested was that pinfish would not consume either macrophyte at ambient temperature (26.8°C). While the data from Table 2 suggest that pinfish were not

57 consuming either macrophyte agar in significant proportions at ambient temperature

(Thalassia testudinum, p = 0.292; Halimeda incrassata, p = 0.155), there is evidence through both visual and wet weight measurements that indicate pinfish were consuming some portion of each macrophyte agar at ambient temperature during the experimental trials (Figure 4.5). The statistical insignificance of pinfish grazing on either macrophyte at ambient temperature could be due to the range in grazing rates by pinfish between experimental trials 1, 2, and 3 at ambient temperature causing variability in the data.

Having a larger sample size for a future study could help strengthen the results seen at ambient temperature.

Conversely, the statistical insignificance of these results could also be caused by a number of other reasons such as the environment the fish were in, the form the macrophytes were in, or the feeding preference of the fish. Darnell and Wissing (1975) found that pinfish exhibited several different grazing rates depending on their environment. They also found that pinfish needed to consume a minimum of 5.75% of their body weight per day to survive. When pinfish were in complete isolation (no other fish in the tank nor could they see fish in other tanks), they did not meet this minimum consumption level, inadvertently starving themselves. Conversely, the consumption rate of pinfish tripled (compared to their feeding rates in complete isolation) when they were isolated but were able to see pinfish in other tanks during experimentation; this feeding rate was determined to be the most realistic to feeding rates observed in the field. Finally, the feeding rates of pinfish were greater than feeding rates observed in the field when pinfish were placed together in one tank and able to feed in groups (Darnell & Wissing, 1975). In the current study, pinfish were in isolated tanks but were always able to see 2 – 3 other pinfish during the experiment

58 to try and simulate more realistic grazing amounts to those that would be observed in the field according to Darnell & Wissing’s (1975) study. However, there could have been other factors influencing the low consumption amounts by pinfish that were seen in this experiment as well.

The use of agar cubes could have influenced the feeding behavior of pinfish on the macrophytes. A number of studies have identified that pinfish use both visual and scent cues to identify food sources (Darcy, 1985; Luczkovich, 1988; Stoner & Livingston, 1984).

The macrophytes were presented to the pinfish in a form physically different from the natural morphological form pinfish would encounter in the wild, which may have reduced the ability of the pinfish to recognize the plants as food visually. However, reconstituting these species into agar created a substrate that could be more easily consumed and digested by pinfish (Prado & Heck, 2011). Chemical cues such as taste and smell were not altered, so combined with the increased digestibility of the agar, it presented an appealing meal for the pinfish, albeit visually different from natural forms (Darcy, 1985; Luczkovich, 1988;

Stoner & Livingston, 1984). There were no indications in previous studies suggesting that there were alarm pheromones produced by either Thalassia testudinum or Halimeda incrassata when they were ground into powder to make the agar (Hay, 1984; Prado &

Heck, 2011). While there is evidence that some Halimeda species produce chemical defense mechanisms to deter grazing pressures (Cruz-Rivera & Villareal, 2005; Hay, 1984;

Paul & Hay, 1986), Hay (1984) found that Halimeda incrassata did not appear to produce a chemical defense that was toxic to fish in its natural form.

Finally, the feeding preference of the pinfish could be a controlling factor on the amount of agar the pinfish consumed in this study. Many studies have identified pinfish as

59 omnivorous fish that undergo feeding habit transitions throughout their lives and that once they become larger than 120mm they become predominantly herbivorous (Chacin, 2014;

Darcy, 1985; Luczkovich, 1988; Montgomery & Targett, 1992; Stoner, 1980; Stoner &

Livingston, 1984). Pinfish have been observed to be generalist feeders but in certain environments they can become fairly selective feeders (Darcy, 1985; Luczkovich, 1988;

Stoner 1980). Luczkovich (1988) found that the availability of prey may have had a determining effect on how selective pinfish were in their feeding behaviors. Having food consistently available and obtainable throughout the current experiment could have influenced the selectivity of the pinfish causing them to be more selective and thus not consuming a large amount of either macrophyte in this study. Additionally, following the methods of Prado and Heck (2011), pinfish in this study were fed a combination of frozen shrimp and both macrophytes that would be used in this experiment (Thalassia testudinum agar cubes and Halimeda incrassata agar cubes) while in the holding tank before experimentation. All pinfish were 10 – 20 mm over the “predominantly herbivorous” body size (≥120mm; Darcy, 1985; Livingston, 1980; Luczkovich, 1988; Montgomery & Targett,

1992; Stoner, 1980; Stoner & Livingston, 1984). Pinfish used in this study were 130 –

140mm in length. While the pinfish in this experiment did consume both types of macrophyte agar cubes during this study, feeding them frozen shrimp in addition to

Thalassia testudinum agar and Halimeda incrassata agar while in the holding tank

(following Prado & Heck, 2011 procedures) may have influenced the herbivorous feeding behaviors of the pinfish used in this study.

A number of studies have identified adult pinfish feeding on plant materials such as seagrass and green algae extensively (seagrasses such as Thalassia testudinum,

Syringodium filiforme, and Zostera marina L. made up 18 – 90% of adult pinfish’s diets and unspecified green algae species made up to 50% of their diets; Adams, 1976; Darnell,

1958; Hansen, 1969; Montgomery & Targett, 1992; Stoner, 1980; Stoner & Livingston,

1984). Conversely, other studies have identified that it is an uncommon occurrence for many organisms living among seagrass beds to graze exclusively upon living seagrass leaves or algae and in turn are dependent on other food sources such as epiphytes as their target food source (Hemminga & Duarte, 2000; Klumpp et al., 1992; Nakaoka, 2005;

Valentine & Heck, 1999; Zapata & McMillan, 1979). For these reasons, there was great caution taken to ensure that the epiphytic community remained intact on the macrophytes used to make the agar cubes. This was done to represent a realistic epiphytic species composition on the Thalassia testudinum and Halimeda incrassata as they would be found in the wild.

5.1.2. Hypothesis 2: Pinfish do not show a significant preference between either macrophyte agar cubes at ambient temperature.

There was not a significant preference seen in the consumption of one macrophyte over the other by pinfish at ambient temperature (26.8°C). The data from Table 3 support the null hypothesis that pinfish consume both Thalassia testudinum agar and Halimeda incrassata agar equally at ambient temperature (p = 0.448). The insignificance in preference by pinfish could be a result of the variability in the data. As addressed in

Hypothesis 1, there was variability in the data collected on the grazing amounts of pinfish at ambient temperature. While the graphed data (Figure 4.5) shows that the Halimeda incrassata agar was consumed in larger amounts compared to Thalassia testudinum agar

61 per experimental trial, the consumption amounts of both macrophytes is very different from one another between experimental trials. For a future study, utilizing a larger sample size with more experimental trials could lend stronger results on the palatability of the macrophyte to pinfish at ambient temperature. For example, the significant results in

Hypothesis 4 argue a need for more data (power) as well.

Additionally, due to the structural longevity of the reconstituted macrophytes, pinfish were only able to graze upon each macrophyte for a total of 7 days. If the pinfish were able to graze on the agar for a longer period of time, without the agar breaking down, it could lead to better insight into whether or not there is a significant palatable difference here. For this experiment, grazing amounts were calculated over a 7-day period

(determined based off of the results of pilot trials and the control trial). It was determined that the agar would break down (with no outside influence) at an increased speed between

Day 7 and Day 9. Testing a variety of agar solutions or reconstituting the macrophytes in a different substrate could offer greater structural longevity of the agar cubes allowing grazing experiments to be done for a longer period of time.

5.1.3. Hypothesis 3: Temperature had both significant and insignificant effects on pinfish grazing.

It was hypothesized that a range of temperatures (22°C – 30°C) would not have a significant effect on how much agar (Thalassia testudinum versus Halimeda incrassata) pinfish were eating. However, the results from the Linear Regression analysis (Table 4) indicate that while this hypothesis was true for Thalassia testudinum agar cubes (pinfish did not significantly increase their consumption of Thalassia testudinum agar across a

62 temperature gradient, p = 0.154), pinfish did show a statistically significant increase in the consumption of Halimeda incrassata agar cubes with increasing temperature (p = 0.022).

The temperature-grazing relationship, however, is not a strong relationship (R2 = 0.2154).

As mentioned in the results section, the data points for 28°C are likely driving the significance of the linear regression for Halimeda incrassata agar consumption by pinfish

(Figure 4.6). Additionally, when these outlier points were removed there was not a change in the statistical outcome of either test (Thalassia testudinum p = 0.247; Halimeda incrassata p = 0.041, R2 = 0.1849). The following discussion of these results should be interpreted with this in mind.

Many studies have identified that the grazing rates of marine herbivores can fluctuate with varying water temperature (Bruno et at., 2015; Carpenter, 1986; Ferriera et al., 1998; Polunin & Klumpp, 1992; Smith, 2008). One of the possible explanations found in these studies was that the grazing rates of marine herbivores shifted as a result of the availability of food increasing or decreasing with changing temperatures (Carpenter, 1986;

Klumpp & McKinnon, 1989, 1992). In the current study, the availability of food offered to the pinfish remained constant throughout the study which could have influenced the results.

A study by Bruno et al., (2015) suggested that the metabolic demand of a marine organism may change with changing temperature thus causing a shift in the grazing rate of the organism with variable temperature. This could explain why there was a statistically significant linear relationship in the amount of Halimeda incrassata agar consumed by pinfish with increasing temperature. However, it doesn’t explain why there was not a significant relationship seen in the amount of Thalassia testudinum agar consumed by pinfish. From the literature, it appears that Thalassia testudinum would be a more palatable

63 option to pinfish in the wild when compared with Halimeda incrassata (Adams, 1976;

Montgomery & Targett, 1992; Stoner & Livingston, 1984). However, in this study (see

Hypothesis 4) this might not be the case. Additionally, as addressed in Hypotheses 1, the reconstitution of both macrophytes into agar form could have influenced the palatability and/or the amount of consumption pinfish had on both macrophytes in this study.

Two Non-Parametric Kruskal-Wallis analyses were done for each macrophyte to identify if pinfish were consuming a larger amount of either macrophyte at a specific temperature. The results from these Non-Parametric Kruskal-Wallis analyses (Table 5) indicated that there were not statistically significant differences in the consumption of either macrophyte at a specific temperature (i.e. identifying if pinfish were eating more at a specific temperature in a non-linear fashion; Thalassia testudinum, p = 0.569; Halimeda incrassata, p = 0.276). While there were not significant relationships here, the boxplots in

Figure 4.8 show that 28°C (tank 2) stands out as a potential preferred grazing temperature for both macrophytes compared to the rest of the data. As mentioned in the results section, the data points from tank 2 (28°C) in experimental trial 1 were determined to be outliers.

However, there was still a lack of significance in the consumption of Thalassia testudinum agar or Halimeda incrassata agar at a specific temperature when the data was run again with these outliers removed. This is likely because the consumption amounts of the

Thalassia testudinum agar and the Halimeda incrassata agar by the pinfish in tank 2 (28°C) in experimental trial 2 were also much higher than the rest of the recorded consumption amounts by pinfish from this study. As a result, this still caused a large variability in the data at 28°C. The Non-Parametric Kruskal-Wallis analysis is a very conservative statistical test so, having a large variability in the data, such as the one present in tank 2 (28°C) for

64 this experiment, can cause a lack of significance to be concluded when there may actually be a difference in the grazing amounts at this temperature.

Further testing with a larger sample size would need to be done to identify if there is an actual increase in the grazing amounts of pinfish at this temperature or if this was just an anomaly seen in the specific fish that were tested in this experiment. It should also be noted that each pinfish in tank 2 (28°C) for each experimental trial were not significantly larger in mass or in length than any of the other pinfish used for this study. While individual specialization is a phenomenon that occurs in nature (Bolnick et al., 2002; Svanbäck &

Persson, 2004; Woo et al., 2008) and could explain why the pinfish from experimental trial

1 and experimental trial 2 at 28°C (tank 2) consumed much larger amounts of both macrophyte agar cubes, it is also an unlikely occurrence that the only two pinfish experiencing this would be placed into the same temperature tank in two separate trials where new pinfish were used every time.

5.1.4. Hypothesis 4: The consumption of Halimeda incrassata agar cubes was marginally significantly greater that the consumption of Thalassia testudinum agar cubes over the temperature gradient tested (22°C – 30°C).

The results of the Linear Mixed Model analysis (Table 6) indicate that there was a marginally significant preference in the consumption of Halimeda incrassata agar over the

Thalassia testudinum agar by pinfish across the 22°C – 30°C temperature gradient used in this study (p = 0.052). The reconstituted agar form that the macrophytes were in may have caused pinfish to consume a marginally significantly larger amount of Halimeda incrassata agar compared to Thalassia testudinum agar across the 22°C – 30°C temperature gradient

65 tested. Thalassia testudinum and Halimeda incrassata have very different visual and structural components when they are in their natural form (Thalassia testudinum is a species of fibrous seagrass and categorized as a marine angiosperm while Halimeda incrassata is a species of macroalgae and categorized as a calcareous green algae). By reconstituting these macrophytes into agar form it allowed pinfish to digest each macrophyte more easily (Prado & Heck, 2011). By having the macrophytes presented in a reconstituted form, and because pinfish are classified as generalist feeders, this may have minimized or even eliminated the controlling factors and/or recognizability between the two macrophytes to pinfish therefore influencing the grazing preference by pinfish.

Utilizing reconstituted macrophyte samples instead of presenting the macrophytes as they would be in the wild had numerous benefits for this study such as the ability to quantify grazing amounts with more certainty and increasing the digestibility of each macrophyte for pinfish. However, there were also drawbacks to this approach including the removal of large-scale controlling factors that would have been present if the macrophytes were in their natural forms. For example, based on the structural components of both macrophytes, it appears that Thalassia testudinum would likely be a more palatable option than Halimeda incrassata for marine herbivores (Adams, 1976; Montgomery &

Targett, 1992; Stoner & Livingston, 1984). However, the results of the Linear Mixed

Model analysis suggest the opposite (pinfish preferred Halimeda incrassata in a marginally significantly higher amount than Thalassia testudinum agar across the 22°C – 30°C temperature gradient tested).

Halimeda species appear to be difficult to digest for many grazers due to their toughness and high calcium carbonate concentrations (sometime >90%per dry mass of

66 tissue; Castro-Sanquino et al., 2017; Cruz-Rivera & Villareal, 2005; Hay, 1997; Hay &

Fenical, 1988; Hay et al., 1994; Pennings & Paul, 1992). However, studies have observed

Halimeda species being grazed on by larger fish such as parrotfish and/or small invertebrates (Castro-Sanquino et al., 2017). In this study, by reconstituting the Halimeda incrassata into agar it most likely weakened the tough exterior structure of the algae that might otherwise deter many grazers in the wild. Furthermore, some Halimeda species have been observed to produce chemical defenses to deter grazing pressures on the species in the wild as addressed in Hypothesis 1 (Cruz-Rivera & Villareal, 2005; Hay, 1984; Paul &

Hay, 1986). Even though Hay (1984) suggests that Halimeda incrassata may not produce toxic chemical defense deterrents like other species of Halimeda, due to the similarity in appearance of Halimeda species, some fish may instinctively avoid it. Additionally, the results of the current study suggest these chemical defenses may not have been present after it was made into agar form since there was a marginally significant preference in the consumption of Halimeda incrassata agar over Thalassia testudinum agar by pinfish over the temperature gradient tested in this study (22°C - 30°C).

Lastly, it should be noted that there was variability in the data as seen from the size of the error bars from Figure 4.9. In a future study, having a data set with a larger sample size will lend more clarity to the results. Additionally, observing the results of grazing preference by pinfish across this temperature range (22°C – 30°C) with macrophytes that were presented in their natural form could lend better insight into the food preference of pinfish in the wild.

5.2. Implications for Ciguatera Fish Poisoning

Understanding the grazing dynamics of pinfish in CFP-prone environments lends insight into how ciguatoxins are entering and moving through these seagrass bed environments and into the marine food web. While this study suggests that pinfish are not consuming Thalassia testudinum agar or Halimeda incrassata agar in statistically significant quantities at ambient temperature in simulated environments that are representative of seagrass beds found in the middle Florida Keys, when looking at the raw data and from visual observation during experimentation there was some observed consumption of all of the agar cubes by pinfish at ambient temperature and at the rest of the temperatures tested (with the exception of some of the Thalassia testudinum agar cubes and one Halimeda incrassata agar cube from experimental trial 1). These results indicate that while there is variability in the data, pinfish are likely eating both macrophytes from this study in some quantity across temperatures. Or at least, there are no strong chemical deterrents in the Thalassia testudinum or Halimeda incrassata that would keep the pinfish from eating them or grazing the epiphytes off them.

Parsons et al. (2017) found that Thalassia testudinum and Halimeda incrassata samples collected from HGB had the highest Gambierdiscus cell abundances compared to other sites that were sampled in the middle Florida Keys. Additionally, studies have identified Thalassia testudinum and Halimeda incrassata being among the top 20 macrophytes observed with the highest cell densities of Gambierdiscus per algal gram living within their epiphytic community (maximum densities of Gambierdiscus cells per algal gram: Thalassia testudinum had 1463 and Halimeda incrassata had 4774; Cruz-

Rivera & Villareal, 2005). The results from Hypothesis 4 of this study suggest that pinfish

68 prefer to graze on Halimeda incrassata agar over Thalassia testudinum agar across the temperature gradient tested (22°C - 30°C). If these results are a realistic representation of the grazing amounts of pinfish both in the wild also and on these macrophyte in their natural forms, there is a chance that toxic Gambierdiscus cells are being consumed in larger quantities than if there were no preference or if Thalassia testudinum agar had been preferred by pinfish since Halimeda incrassata has been found to have just over three times the Gambierdiscus cells per algal gram living within its epiphytic community.

Furthermore, the findings from this study lead to the assumption that pinfish may be targeting the epiphytes living on both Thalassia testudinum and Halimeda incrassata more than the macrophytes themselves. This assumption exemplifies the importance of studies such as this one seeing as Gambierdiscus species make up part of the epiphytic communities living on these macrophytes. If the results of this study are a realistic representation of the grazing behaviors of pinfish in the wild, the results suggest that the consumption of these species (Thalassia testudinum and Halimeda incrassata) by pinfish may not be a major vector for ciguatoxin entering the food web in seagrass beds found in the middle Florida Keys or other similar environments. However, further testing, both in laboratory and in field settings, should be done before this conclusion can be drawn with confidence. Additionally, the consumption of these macrophytes by pinfish in any quantity still suggests that ciguatoxins (via the consumption of Gambierdiscus cells) are being introduced into the marine food web to some extent in environments similar to HGB, even if it appears to be a small extent as suggested in this study.

Parsons et al. (2017) also found that the general trend in abundance of

Gambierdiscus cells in the middle Florida Keys were highest in winter months suggesting

69 that colder temperatures are ideal for the growth of Gambierdiscus cells. The results of the present study identified that there was not a significant linear relationship in the amount of

Thalassia testudinum agar being consumed by pinfish with increasing temperature and that there was a significant but weak linear relationship in the amount of Halimeda incrassata agar being consumed by pinfish with increasing temperature. Additionally, the results also suggested that there was not a statistically significant relationship in the grazing amounts of pinfish at a specific temperature. The data also suggests that 28°C could be a possible ideal peak grazing temperature. However, further testing should be done with a larger sample size before that conclusion is drawn with confidence. These results in combination with the results found by Parsons et al. (2017) could indicate that while pinfish are likely not consuming higher amounts of Thalassia testudinum and that there is a significant but weak increase in consumption of Halimeda incrassata with changing temperature, they may be inadvertently consuming larger amounts of Gambierdiscus cells in cooler temperatures (i.e. winter months) as there are more Gambierdiscus cells recorded in the epiphytic community of macrophytes found in the middle Florida Keys during that time of year (see below). Furthermore, there is evidence that certain species of Gambierdiscus thrive at different temperatures within tropical and sub-tropical regions and that certain species of Gambierdiscus are more toxic than others (Holland et al., 2013; Tester et al.,

2010; Xu et al., 2016). While it is important to understanding how temperature influences the grazing dynamics of marine herbivores such as pinfish that can be affected by CFP, understanding how the abundance and toxicity of Gambierdiscus species is related to temperature is also equally important for understanding the implications of CFP in these

CFP endemic areas. The results of this study, along with others, aid in predicting the

70 transfer of ciguatoxins into marine food webs through modeling efforts. Nevertheless, further testing on both the grazing dynamics of herbivores in these areas and the effects of temperature on the abundance of different Gambierdiscus species should be done before these algorithms are created.

The results of this study suggest that as global temperatures rise and ocean warming occurs, pinfish may not consume larger amounts of food (although there is a significant but weak increase in consumption of Halimeda incrassata agar with temperature from this study). Additionally, Parsons et al, (2017) found that Gambierdiscus species as a whole thrived in colder winter months suggesting that the Gambierdiscus species may find the conditions in the middle Florida Keys unfavorable for increased reproduction with rising temperatures. This could lead to lower chances for ciguatoxins to be introduced into marine environment via pinfish consuming Thalassia testudinum or Halimeda incrassata in the middle Florida Keys. However, this presents the question of whether or not warming sea surface temperatures will increase the range of Gambierdiscus cells allowing them to thrive in higher latitudes thus affecting pinfish communities in different locations.

5.3. Implications for Pinfish Feeding Behaviors

5.3.1. Laboratory Environment Pinfish Were In

The feeding behaviors of pinfish in laboratory settings have been observed to have a wide range depending on the environment that they are in (Darnell & Wissing, 1975).

While the results of this study are believed to most closely resemble field studies compared to other laboratory settings and tank setups, there are many factors that were controlled and/or not present in this study as compared to a field study. This study can be used as a

71 proxy for the grazing amounts of pinfish in the wild, however, it should not be used as a direct representation of feeding behaviors for pinfish found in their natural environments.

5.3.2. Reconstituted Macrophytes Used for Feeding Trials

Preliminary attempts to utilize Thalassia testudinum and Halimeda incrassata in their natural forms were done. Initial attempts were done using living samples of Thalassia testudinum and Halimeda incrassata with their root systems intact and cleaned of epiphytes. However, pinfish appeared to have no interest in the macrophytes in this form; there was not a measurable change in weight of either macrophyte over an extended period of time (7 – 14 days) nor were there visible bite marks on either macrophyte in this time frame. Epiphyte free clippings of shoots and thalli from both macrophytes were also used in a separate attempt with similar results; pinfish did not consume either macrophyte in a measurable amount nor were there any visible bite marks present on the macrophyte before they began to decompose (~7 days).

Hemminga and Duarte (2000) note that marine herbivores target food sources such as seagrasses, macroalgae, and/or epiphytes within seagrass bed environments.

Additionally, within these environments, some herbivores may specifically target epiphytes as their main food source if they do not possess the ability (through mechanical or chemical means) to break down the complex structures of seagrasses and/or macroalgae

(Klumpp et al., 1992; Nakaoka, 2005; Valentine & Heck, 1999; Zapata & McMillan, 1979).

Pinfish have been observed to consume large amounts of seagrasses such as Thalassia testudinum, Syringodium filiforme, and Zostera marina L. and smaller amounts of green algae species (species unspecified; Adams, 1976; Darcy, 1985; Darnell, 1958; Hansen,

1969; Livingston, 1980; Montgomery & Targett, 1992; Stoner, 1980; Stoner & Livingston,

1984). However, in this experiment, as stated above, pinfish did not show much, if any, interest in either of the macrophytes (Thalassia testudinum or Halimeda incrassata) when they were cleaned of epiphytes. This suggests that they may be targeting the epiphytic communities living on these macrophytes more than the macrophytes themselves.

The next reasonable attempts for setting up this experiment would have been to try using living samples of each macrophyte still covered in epiphytes with their root systems intact or to use clippings of each macrophyte while still covered in epiphytes. However, there was not a reasonable way to measure the consumption of either macrophyte by pinfish with epiphytes without disturbing the epiphytic community living on the macrophyte and thus skewing the data. This led to the conclusion that reconstituting each macrophyte, with the epiphytic community still intact, into agar form was the best solution for this experiment. Reconstituting macrophytes into agar form has been used in other grazing experiments as a proxy for quantifying grazing amounts/rates in laboratory settings (Prado

& Heck, 2011).

5.4. Future Research

There are still many questions that need to be answered in relation to the grazing dynamics of pinfish. Further studies addressing the grazing dynamics of pinfish in relation to temperature will lend better insight into how temperature influences the grazing behaviors of pinfish. Additionally, readdressing the diet of pinfish to include a more extensive list of what organisms (fauna and/or flora) pinfish prey upon will benefit the overall understanding of what pinfish are targeting as a food source; it could also lend

73 insight into how large of a role pinfish play in CFP trophic transference. Lastly, understanding how location contributes to the grazing choices pinfish make could lend better insight into whether or not the diet of pinfish changes based off of the area they are found. By further addressing the grazing amounts of pinfish in the wild, it will more accurately represent what these fish are consuming at different temperatures and locations where they are found.

To further the results of this study, a field study could be done to observe the grazing dynamics of pinfish in seagrass beds located in the middle Florida Keys.

Furthermore, changing the epiphytic community of each macrophyte (having no epiphytic community in one sample, a natural epiphytic community in another sample, and having an epiphytic community only consisting of Gambierdiscus species in the final sample) could aid in identifying if pinfish are targeting certain macrophytes based on their epiphytic community. This could also aid in identifying if Gambierdiscus species are a driving factor in the palatability of certain macrophytes to pinfish.

5.5. Studies on Other Herbivorous Species and Macrophytes

This study was done to identify the grazing dynamics of pinfish between Thalassia testudinum and Halimeda incrassata under varying temperatures. However, there are many species of herbivores found in seagrass ecosystems that could be influencing the transfer of ciguatoxins into the marine food web. Additionally, there are many other macrophytes found within seagrass ecosystems that could be studied as well. Conducting studies on different herbivorous species, different macrophytes, and the amount and species of

Gambierdiscus found on those macrophytes will add to the overall knowledge on how ciguatoxins are being introduced into the marine food web.

5.6. Improved Experimental Design

There are areas where this study can be improved for future studies. When the experiment was set up, agar cubes were placed into the same location after being weighed on Day 1, Day 3, Day 5, and Day 7. By not randomizing the placement of agar cubes it could have influenced the feeding behaviors of the pinfish. Additionally, increasing the amount of artificial structures within the experimental tanks could simulate a more natural environment for pinfish which might also influence their feeding behaviors. There was also a lack of resources associated with this experiment. Finding and having access to pinfish that were the same size as one another was very difficult for this experiment due to the massive red tide blooms in south Florida from 2017 through 2019 that killed or displaced pinfish on the west coast of Florida; this caused a lack of availability of pinfish both in the wild and at many bait shops. As a result of this, only three experimental replicates were performed. Having a larger number of experimental replicates could potentially overcome the variability in the data, specifically the variability associated with tank 2 (28°C) and changed the significance of some of the statistical analyses.

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