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2018 A Day in the Life of Picoplankton in Dickerson Bay, FL Isabelle G Basden

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COLLEGE OF ARTS & SCIENCES

A DAY IN THE LIFE OF PICOPLANKTON IN DICKERSON BAY, FL

By

ISABELLE BASDEN

A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded: Fall, 2018

The members of the Defense Committee approve the thesis of Isabelle Basden defended on November 28, 2018

______Dr. Janie Wulff Thesis Director

______Dr. Markus Huettel Outside Committee Member

______Dr. Sophie McCoy Committee Member

Acknowledgements

I would like to thank Dr. Janie Wulff for all of her guidance through out the planning and execution of this project. I would also like to thank my committee members, Dr. Sophie

McCoy and Dr. Markus Huettel, for their guidance on project design, data analysis, and writing. I would like to thank Kathleen Kaiser, Kate Hill, Alex Strawhand, Bobbie Renfro,

Ashley Dawdy, Samantha Politano, Kristie Dick, Connor O’Halloran, and Ariel Basden for field assistance and data collection. I thank Ruth Didier for her assistance and patience with the flow cytometer at the Florida State University College of Medicine

Facility. I thank Dr. Markus Huettel for analyzing DOC and TN in the Oceanography

Department. I would like to thank Jack Rudloe and Gulf Specimen Marine Lab for allowing me to use their dock space for the picoplankton fluctuation project in Dickerson Bay. I would also like to thank Dr. Gregg Hoffman for allowing me to use his dock space to conduct the sponge feeding trials at Shell Point. This research was funded by the FSU IDEA

Grant and the Bess H. Ward Honors Thesis Award. Finally I would like to thank my friends and family for their ongoing support and encouragement through out this project.

Abstract

Pelagic ecosystems play an important role in regulating the Earth’s biogeochemical processes. Picoplankton, cells > 2µm, are some of the most abundant in the pelagic community and responsible for 44‐90% of in tropical oceans.

This study attempts to understand the daily fluctuations of picoplanktonic organisms in relation to the abiotic and biotic factors that may influence their dynamics by 1) analyzing the fluctuations in picoplankton community structure and density from 8am to 8pm 2) analyzing the relationship between picoplankton fluctuations and abiotic factors including light, temperature, and the tidal cycle and 3) analyzing the impact of a sponge filter feeder on picoplankton . Picoplankton densities fluctuated significantly through out the day and tidal cycles and vertical mixing could play a large role in these daily dynamics.

Sponge feeding can significantly decrease the density of autotrophic picoplankton and could be a form of control for picoplankton communities.

Introduction

Pelagic ecosystems play an important role in regulating the Earth’s biogeochemical processes. Life in marine benthic ecosystems relies on the primary production of plankton that occurs in pelagic zones. Therefore, many of the organisms found in these environments have a large ecological impact. Pelagic environments are composed of planktonic, free floating, and nektontic, swimming, organisms. Plankton can be extremely diverse and can range from microscopic bacteria or protists to larger , such as or even . There are two size classes that compose the majority of the planktonic community in most environments; nanoplankton, size from 2µm‐20µm, and picoplankton, cell size <

2µm. Picoplankton are the most abundant and productive in the pelagic environment

(Campbell & Vaulot, 1993; Chavez, 1989; Platt et al., 1983). Organisms that fall into the picoplankton size class include both heterotrophic and autotrophic organisms. The two main groups of autotrophic picoplankton are picoeukaryotic protists and ;

Prochlorococcus‐like prochlorophytes and ‐like .

Prochlorococcus (0.5‐0.7 µm) are the smallest photosynthesizers and are typically the most abundant plankton in warm, oligotrophic waters (Partensky et al., 1999).

Synechococcus phytoplankton (0.6‐1.7µm) are slightly larger and can be found in cooler, more nutrient rich waters. The picoeukaryotic phytoplankton are much more diverse but less abundant than their prokaryotic counter parts (Otero‐Ferrer et al., 2018; Vaulot et al.,

2008).

The picoplanktonic are the base of the marine food web and it is estimated that these phytoplankton make up 60‐80% of the planktonic and 44‐90% of primary production in tropical oceans (Sherr & Sherr, 1991; Stockner & Anita, 1986).

Phytoplankton capture energy from the sun to convert inorganic CO2 into organic carbon for synthesis of cellular materials. Some of the converted organic carbon is released as dissolved organic carbon (DOC) via exudation (Biddanda & Benner, 1997; Fogg, Nalewajko,

& Watt, 1964). This is the beginning of the . The next step involves the heterotrophic picoplankton. Heterotrophic bacteria utilize approximately 50% of the DOC released by the autotrophic picoplankton and convert it to biomass with 60% efficiency (Fogg et al., 1964; Larsson & Hagström, 1982). This biomass can now be transferred to higher trophic levels via predation by filter feeders.

Filter feeding, the process of capturing plankton and dissolved nutrients suspended in the water column, is a widely used method for acquiring food in marine environments.

Filter feeding animals are crucial in marine environments because they transfer energy from pelagic to benthic environments, improve water quality preventing phytoplankton blooms, and improve water clarity, which allows for more efficient for corals and algae (Gili & Coma, 1998; Lesser, 2006; Peterson et al., 2006; Wulff, 2013). Filter feeders have the ability to greatly impact the planktonic community depending on their abundance, clearance rate, filtering efficiency, and grazing selectivity.

Do to their small size, very few organisms can efficiency feed on picoplankton. One of the groups of picoplanktivores includes the heterotrophic microflagellates (3‐7µm). These organisms feed by creating a current with their flagella to push bacteria prey towards the cell membrane where it is phagocytized. Mucus mesh feeders, such as the Appendicularians and Thalicean salps utilize mucus nets to capture picoplanktonic prey. The only major group of benthic picoplanktivores are the sponges (Phylum Porifera). Sponges are able to filter large amounts of water efficiently due to their aquiferous system made of extensive canals. Flagellated cells, called choanocytes, pull water through incurrent pores, ostia, and into the canal system by beating the flagella. Food particles are sieved through choanocyte villi and the water exits through the excurrent pore, called the osculum. Sponges play an important role in transferring pelagic biomass to benthic ecosystems.

The Earth is changing do to anthropogenic influences. As CO2 continues to be released into the atmosphere, sea surface temperatures increase and the ocean becomes more acidic. Picoplankton are directly linked to these abiotic changes in many ways.

Autotrophic picoplankton are needed to convert atmospheric CO2 into DOC. Increased sea surface temperatures can cause increased prevalence of increased picoplankton growth and harmful algal blooms. Biotic interactions in the oceans are also altered by human impact; such as overfishing, introduction of invasive species, and increased disease prevalence. It has been recorded that consumers can be a major form of control for primary productivity in Lakes via trophic cascades (S R Carpenter et al., 2001; Stephen R. Carpenter

& Kitchell, 1988; Vanni & Layne, 1997). Overfishing of tertiary consumers in the ocean could cause picoplankton blooms via increases in secondary consumers and therefore decreases in populations. It is important to study the fine scale fluctuations of picoplankton to better understand how these populations will be affect by future global changes.

This study attempts to understand the daily fluctuations of picoplanktonic organisms in relation to the abiotic and biotic factors that may influence their dynamics by

1) analyzing the fluctuations in picoplankton community structure and density from 8am to 8pm 2) analyzing the relationship between picoplankton fluctuations and abiotic factors including light, temperature, and the tidal cycle and 3) analyzing the impact of a sponge filter feeder on picoplankton abundance.

Methods:

Study Site

This study was conducted in the Northern Gulf of Mexico in Dickerson Bay, Panacea FL.

Samples were collected from Gulf Specimen Marine Lab’s a floating dock.

Water Sample Collections

A HOBO data logger was deployed off of the dock to measure light intensity and temperature through out the day. Three water samples were collected six inches from the surface of the water every hour from 8am to 8pm. samples were fixed for preservation immediately after being taken. For picoplankton analysis, 1.7mL were filtered through a

100µm mesh into a 2mL cryovial and fixed with formaldehyde to a concentration of 0.5%.

Samples were put on dry ice until returned to FSU in a ‐80*C freezer, where they were kept until analysis. For nutrient analysis ~25mL were filtered through G/F glass fiber filters and fixed with HCl to a pH of 2. Samples were kept in a dark refrigerator until analysis.

Picoplankton Analysis

Picoplankton samples were analyzed with a FACSCanto Flow Cytometer in the FSU

College of Medicine. Flow cytometry is laser technology that measures the physical and chemical properties of particles suspended in a fluid. Autotrophic groups were distinguished with flow cytometry by their distinct pigment concentrations. When excited by the Blue 670LP laser Prochlorococcus only emits red fluorescence (chlorophyll a) and

Synechococcus emits red and orange fluorescence (from phycoerythrin). 500µL of the sample was run through the flow cytometer for five minutes at a high flow rate. To determine heterotrophic density, 10µL of SYBR1 Green dye(a nucleic acid gel stain) was added to 250µL of the sample to stain all DNA containing particles. The stained SYBR1

Green cells are triggered by the FITC laser. The sample was run for 3 minutes at a low flow rate. Autotrophic populations were distinguished and subtracted from the total DNA population to get the heterotrophic population (adapted from Stramaitis, 2012 unpublished).

Nutrient Analysis

DOC and TN concentrations were analyzed with a Shimadzu TOC‐V analyzer with

ASI autosampler in the Oceanography Department. DOC is measured using “oxidative combustion‐infrared analysis” TN is measured using “oxidative combustion‐ chemiluminescence.”

Sponge Filter Feeding Trials

Halichondria corrugata, a possible endemic sponge to the Northern Gulf of Mexico, was the species used in the feeding trials. This species is found in seagrass bed habitats and dock fouling communities and harbors photosynthetic symbionts.

H. corrugata fragments of relatively equal size were cut from 10 healthy sponges from a dock community in Shell Point, FL. The fragments were attached to a 2‐inch PVC pipe using cable ties. Placing the sponge on PVC prevented any human contact, which can disturb the sponge, after attachment. All samples were left for at least one week to heal and regrow before the experiment began.

One sponge fragment at a time was enclosed in a watertight chamber. The chambers were made from 6 L, food safe Rubbermaid containers with snap on lids. All chambers were conditioned by scrubbing and soaking with ambient seawater prior to experimental use. The change in prey cells, nitrogen, and carbon in the chambers was measured by taking water samples with a 30mL syringe at minute 0, 10, and 20, and 30. The chambers were gently circulated by hand throughout the trial to prevent particles from settling. A trial without a sponge in the chamber was conducted simultaneously with three other sponge feeding trials. Water samples were prepared for analysis with the same methods as described previously but only autotrophic plankton were analyzed (adapted from

Stramaitis, 2012 unpublished).

Results

Picoplankton Dynamics

There was a significant difference in abundance of the different types of picoplankton in Dickerson Bay, FL (One‐way ANOVA df=3, F=282.9, p<0.001). The heterotrophic bacteria were the most abundant, followed by Synechococcus, with

Prochlorococcus and being the least abundant (Figure 1). Picoplankton densities fluctuated significantly through out a 13hour period. Autotrophic plankton gradually decreased during the first half of the day then increased in the afternoon before beginning to decrease again as the sunset. Figure 3 shows the broken down trend of the autotrophic densities. There is a less clear trend for the heterotrophic bacteria densities

(Figure 2).

Fig 1. represents the mean density of each picoplankton group at each hour of the day

Fig 2. represents the density fluctuations of heterotrophic and autotrophic picoplankton through out the day

Fig 3. represents the density fluctuations of each of the autotrophic picoplankton groups through out the day

Abiotic Conditions

Mean light intensity at each hour started around 1000lux at 9am and rapidly decreased after the first hour (Fig. 4). It is likely that the HOBO logger sensor was obstructed after the first readings, therefore light intensity will not be used in further analysis. Mean temperature followed an expected change through out the day with increasing in the morning and early afternoon and then decrease as the sun started to set

(Fig. 5). Peak temperature was 75.6•F.

The relation between picoplankton fluctuations and average temperature is represented in figure 6. There does not seem to be a distinct relationship between in the fluctuations in or autotrophs and temperature. Lastly, figure 7 represents the relationship of picoplankton fluctuations and the tidal cycle. This graph shows that the tide and autotrophic picoplankton could be correlated.

Fig 4. represents the mean light intensity (lux) for each hour

Fig 5. represents the mean temperature (•F) for each hour

Fig. 6 shows the fluctuations in picoplankton density in relation to the mean temperature at each hour.

Fig. 7 shows the fluctuations in picoplankton density in relation to the tidal depth at each hour

Nutrient Concentration Fluctuations

There was not a significant change in DOC concentrations at each hour (Fig. 8). The concentrations could be highly variable between each replicate. Mean DOC concentration ranged from 34.9‐91.9µmol/L. There also was not significant a change in mean TN at each hour (Fig. 9). Mean TN concentration ranged from 2.0‐4.9µmol/L.

Figures 10 and 11 represent the relationship between autotrophic density fluctuations and nutrient concentrations at each hour. There is a possible negative correlation between autotrophs and DOC and a possible positive correlation between autotrophs and TN.

Figures 12 and 13 represent the relationship between heterotrophic density fluctuations and nutrient concentrations at each hour. There a possible positive correlation between heterotrophic density fluctuations and both DOC and TN concentration with a lag time.

Fig. 8 shows mean DOC concentrations at each hour

Fig. 9 shows the mean TN concentrations at each hour

Fig. 10 shows the fluctuations in autotrophic picoplankton density in relation to DOC concentration fluctuations

Fig. 11 shows the fluctuations in autotrophic picoplankton density in relation to TN concentration fluctuations

Fig. 12 shows the fluctuations in heterotrophic picoplankton density in relation to DOC concentration fluctuations

Fig. 13 shows the fluctuations in heterotrophic picoplankton density in relation to TN concentration fluctuations

Sponge Filter Feeding Trials

There was a significant difference in autotrophic mean cell density at each time interval for the water samples taken from the sponge feeding chambers (One‐way ANOVA df=3, F=15.18, p<0.001) (Fig. 14). Mean cell density was significantly greater for sponge feeding at time 0 (mean = 274416.67, SD = 21416.88, p<0.05) than at time 10, 20, or 30 minutes. After 30 minutes in the sponge chamber the autotrophic mean cell density was

142733.33cells/mL with a SD of 39378.85cells/mL. Statistical analysis could not be completed for the control because there is only one replicate. The autotrophic water sample for the control chamber started with a cell density of 224922.22cells/mL at time 0.

At time 30 the cell density was 206822.2222cells/mL. Figure 14 shows the break down of change in cell density for each autotrophic group. The total change in autotrophic mean cell density for the sponge feeding chambers was ‐131683cells/mL with a SD of 29762.74cells/mL. The total change in cell density for the control chamber was ‐

181000cells/mL (Fig. 15). Figure 16 represents the total change in cell density for each autotrophic group in the sponge feeding chambers and the control chamber.

Fig. 14 shows the density of autotrophic cells at 10 minute intervals in the sponge feeding chamber (n=3) and the control chamber (n=1). Letters show significance.

Fig. 15 shows the density of each group of autotrophic cells at 10 minute intervals in the sponge feeding chamber (n=3) and the control chamber(n=1).

Fig. 16 shows the total change in autotrophic cells in 30 minutes for both the sponge feeding chamber and the control chamber

Fig. 16 shows the total change in each group of autotrophic cells in 30 minutes for both the sponge feeding chamber and the control chamber.

Discussion

Picoplankton community structure and abundance in Dickerson Bay, FL are extremely dynamic and both heterotrophic and autotrophic picoplankton densities fluctuate significantly through out the day. Heterotrophs are the most abundant followed by the Synechococcus with Prochlorococcus and eukaryotic picoplankton being the least abundant at this site. Many studies have found that the abundance of Prochlorococcus decreases at high light irradiance and therefore is not as abundant at the water surface. All of my samples were collected six inches from the surface of the water, which could explain the lower densities of Prochlorococcus.

These dynamics can be driven by both biotic and abiotic factors. In Dickerson Bay, the tidal cycle and vertical mixing could play a large role in the picoplankton fluctuations seen throughout the day. In other studies, hydrodynamic processes have been found to alter picoplankton abundance over a period of hours (Koseff et al. 1993; Legendre &

Rassoulzadegan, 1995). There are also possible correlations between picoplankton densities and nutrient concentrations but more research needs to be conducted to understand the lag time between nutrient fluctuations and picoplankton response. It is also possible that nutrient levels may not be a driving factor of picoplankton dynamics on the hourly period because picoplankton are very efficient at scavenging for nutrients do to their high surface area to volume ratio. This advantage allows picoplankton to survive in oligotrophic waters (Partensky, Garczarek, Hess, & Vaulot, 1999). Because there is high salinity variability in shallow waters, it is likely that salinity levels also played a role in the picoplankton fluctuations. In addition to abiotic drivers, predation can also alter picoplankton abundances.

Sponge feeding can significantly decrease the density of autotrophic picoplankton in just 10 minutes. Halichondria corrugata consumed a mean of 130,000cells/mL within 30 minutes compared to the control chamber in which the picoplankton density decreased by less than

20,000cells/mL in 30 minutes. H. corrugata fed selectively with a preference of

Prochloroccus followed by Synechococcus, and the picoeukaryotes. This trend aligns with the abundance of each autotrophic group at the site. Most of the sponge feeding occurred with in the first 10 minutes of closure within the chamber. This may mean that it is too energetically costly for the sponge to continue to pump in low food environments. Sponges are the dominant benthic filter‐feeders and therefore have the ability to control primary productivity in pelagic oceans. Peterson et al., 2006 found that sponges have the potential to prevent phytoplankton blooms in Florida Bay.

There are a multitude of harmful changes being made in oceanic environments from climate change and to overfishing and disease outbreak. Many of these changes can be linked to picoplankton due to their high abundance and ecological importance. Understanding the pelagic ecosystem and the dynamics of organisms at the base of this food web is essential determining the health of our oceans.

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