Ecological Responses to Eutrophication and Restoration in a Shallow Subtropical Estuary

XIAO MA

B.S. Marine Biology Xiamen University 2012

M.S. Biological Oceanography Florida institute of Technology 2014

A dissertation submitted to the College of Engineering and Science at Florida Institute of Technology in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Oceanography

Melbourne, Florida May 2019

We the undersigned committee hereby approve the attached dissertation, “Ecological Responses to Eutrophication and Restoration in a Shallow Subtropical Estuary”, by Xiao Ma.

______Kevin B. Johnson, Ph.D., Major Advisor Professor of Oceanography

______John H. Trefry, Ph.D. Professor of Ocean Engineering and Marine Sciences

______Kelli Hunsucker, Ph.D. Assistant Professor of Oceanography

______Spencer Fire, Ph.D. Assistant Professor of Biological Sciences

______Richard Aronson, Ph.D., Professor and Head Department of Ocean Engineering and Marine Sciences

Melbourne, Florida May 2019

Abstract

Ecological Responses to Eutrophication and Restoration in a Shallow Subtropical Estuary

Xiao Ma

Major Advisor: Kevin B. Johnson, Ph.D.

Like many other estuaries in the world, the Indian River Lagoon (IRL) is experiencing serious environmental health challenges in recent years. Ecosystem challenges include the connected phenomena of Harmful Algal Blooms (HABs) due to eutrophication and accumulation of fine-grained organic-rich sediments on the benthos. Fine-grained organic-rich sediments, referred to as “muck”, is a source of nutrients and fuels algal blooms in the water column, and excess particles associated with algal blooms sink to bottom and contribute to muck. This dissertation aims to better understand these problems by addressing two main objectives: first, to determine whether key herbivorous mesozooplankton have the capacity to exert top-down control on the HAB species, and second, whether to explore alternative approaches of muck removal and ecosystem restoration to disrupt the vicious cycle of eutrophication perpetuated by organic sediments. For the first objective, grazing experiments revealed that Parvocalanus crassirostris, the dominant herbivorous copepod in the IRL, exerted some top-down control on the HAB species under laboratory conditions (Chapter 1). Additional laboratory grazing experiments on cell size and mucus effects were examined to better understand the low grazing rates (Chapter 2). For the second objective, estuarine

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ecosystems were monitored throughout a pilot aeration experiment to evaluate the feasibility and effectiveness of aeration for muck mitigation (Chapter 3). It was found that aeration facilitated benthic animal life, but only in the cold period when bottom waters held the most dissolved oxygen. Planktonic communities, on the other hand, displayed no significant responses to aeration.

Table of Contents Background ...... 1 Chapter 1: Differential and selective grazing on a novel algal bloom ...... 3 Abstract ...... 3 1.1 Introduction ...... 5 1.2 Materials and Methods ...... 8 1.3 Results ...... 10 1.4 Discussion ...... 18 References ...... 25 Chapter 2: The effect of algal cell size and mucilage on copepod grazing rates ...... 33 Abstract ...... 33 2.1 Introduction ...... 34 2.2 Materials and Methods ...... 37 2.3 Results ...... 40 2.4 Discussion ...... 44 References ...... 47 Chapter 3: Biological responses to aeration in an estuarine canal with fine-grained organic- rich sediments ...... 63 Abstract ...... 63 3.1 Introduction ...... 64 3.2 Materials and Methods ...... 67 3.3 Results ...... 71 3.4 Discussion ...... 98 References ...... 105 Conclusion ...... 112

List of Figures

Figure 1.1 Numbers of cells consumed per hour by Parvocalanus crassirostris grazing on different densities of Isochrysis galbana and an unidentified chlorophyte ...... 11 Figure 1.2 Mortality rates for Parvocalanus crassirostris exposed to different densities of Synechococcus sp...... 13 Figure 1.3 Numbers of cells consumed per hour by Parvocalanus crassirostris grazing on different densities of Isochrysis galbana and Synechococcus sp. .. 15 Figure 2.1 Grazing rates and mortality rates of Parvocalanus crassirostris exposed to different viscosities ...... 41 Figure 2.2 Grazing rates of Parvocalanus crassirostris on different single-sized beads and mix-sized beads ...... 43 Figure 3.1: Redwood Canal and the placement of 8 aeration diffusers for ecosystem testing and evaluation ...... 68 Figure 3.2 Study locations Redwood Canal and Anderson Canal, adjacent to the Grand Canal in Satellite Beach ...... 69 Figure 3.3 Two-dimensional nMDS ordination plots of planktonic assemblages in aerated and control canals in the months August 2017-June 2018...... 74 Figure 3.4 Two-dimensional nMDS ordination plots of planktonic assemblages in surface water over the year-long study period...... 77 Figure 3.5 Two-dimensional nMDS ordination plots of planktonic assemblages in Bottom water over the year-long study period...... 81 Figure 3.6 Microphytoplankton and Nanophytoplankton densities from surface vs. bottom waters from each canal in different months ...... 86 Figure 3.7 Surface water micro-phytoplankton composition in the Grand Canal, the Control Canal and the Aeration Canal in different months ...... 88 Figure 3.8 Bottom water micro-phytoplankton composition in the Grand Canal, the Control Canal and the Aeration Canal in different months ...... 89 Figure 3.9 Surface water nanophytoplankton composition in the Grand Canal, the Control Canal and the Aeration Canal in different months ...... 91 Figure 3.10 Bottom water Nano-phytoplankton composition in the Grand Canal, the Control Canal and the Aeration Canal in different months ...... 92 Figure 3.11 Aureoumbra lagunensis cell densities in surface vs. bottom waters at different sampling sites in February 2018, April 2018 and June 2018 ...... 94 Figure 3.12 Ciliates and phytoplankton density in surface water and bottom water in different months ...... 95

List of Tables

Table 1.1 Comparison of Parvocalanus crassirostris grazing rates on Isochrysis galbana in different incubation volumes...... 17 Table 3.1 List of plankton species found in the canals...... 72 Table 3.2 One-way ANOSIM and SIMPER test results for planktonic communities from surface Vs. bottom waters in different months...... 75 Table 3.3 One-way ANOSIM and SIMPER test results for planktonic communities from surface waters among different months ...... 78 Table 3.4 One-way ANOSIM and SIMPER test results for planktonic communities from bottom waters among different months ...... 82 Table 3.5 Benthic infauna species composition and abundance at each sampling location in different months...... 97

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Acknowledgements

I would like to give my greatest appreciation to my major advisor Dr. Kevin B.

Johnson for all of his patient instructions and valuable suggestions. His intelligence, great enthusiasm and continuous guidance have expanded my knowledge in oceanography and made this dissertation a reality. I am also grateful for the constructive comments from my committee members, Dr. John Trefry, Dr.

Kelli Hunsucker and Dr. Spencer Fire. Many thanks to my lab mates, Dr. L. Holly

Sweat, Hannah Kolb, Daniel Hope, Tony Cox, Angelica Zamora-Duran, Nayan

Mallick, Danielle Juzwick, Sean Crowley, Linda Roehrborn, Melissa Rivera and

Connor Wong for assistance with laboratory and field work. Thanks to Dr. John

Trefry, Dr. Austin Fox and Stacey Fox for the environmental data collection and analysis for the Muck Aeration Project. I am also thankful for the research funding support and scholarships offered by the St. Johns River Water Management

District, Brevard County Florida, Indian River Lagoon National Estuary Program and the Florida Institute of Technology. Thanks to Erik Stenn and AlgaGen LLC for culturing the copepods and algae.

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Dedication

I would like to dedicate this dissertation to my parents, Dr. Haizhou Ma and

Prof. Lingqin Li, whose love, unselfish support and example over many years laid the foundations for the discipline and application necessary to complete this work. I am eternally grateful and promise to keep making you both proud while living out my dreams.

Background

The Indian River lagoon (IRL), a complex shallow subtropical estuarine system, spans 156 miles (approximately 40%) of Florida's east coast. It associates with riverine, wetland and terrestrial systems, combining to create diverse habitats. Like many estuarine systems throughout the world, the IRL is experiencing serious environmental health challenges in recent years. The challenges include the connected phenomena of Harmful Algal Blooms (HABs) due to eutrophication and accumulation of fine-grained organic-rich sediments (“muck”) on the benthos. The fine-grained organic-rich sediments accumulated from various sources such as runoff and sewage (Trefry et al. 1990), terrestrial litter (Hedges et al. 1997; Trefry et al. 2007) and oil and industrial waste (Gray and Mirza 1979). Sedimented organic matter fuels estuarine benthic productivity, but unnaturally high levels can unbalance an ecosystem and stress native populations (Nixon 1995; Diaz 2001;

Schindler and Vallentyne 2008). Muck contributes >50% of total nitrogen flux to the IRL water column. Nutrient fluxes from muck aggravate eutrophication and contribute to harmful algal blooms (Lapointe et al. 2015). The IRL has lost tens of thousands of acres of seagrass because of algal blooms and is becoming a more phytoplankton-dominated system (Indian River Lagoon 2011 Consortium 2015).

When algal blooms die off, the cells are decomposed by bacteria and oxygen levels fall, potentially suffocating aerobic aquatic life (Vaquer-Sunyer and Duarte 2008).

Sinking dead materials accumulate on the bottom and contribute to muck, completing a vicious cycle of harm. This dissertation aims to better understand the

problems associated with muck sediments and HABs by addressing two main objectives: first, to determine whether key herbivorous mesozooplankton have the capacity to exert top-down control on the HAB species; second, to explore an alternative approach of muck removal and ecosystem restoration while monitoring the associated biological responses.

Chapter 1: Differential and selective grazing on a novel algal bloom

Abstract

In laboratory experiments, three species of phytoplankton were fed to

Parvocalanus crassirostris, an abundant and globally distributed herbivorous, calanoid copepod. The goal was to evaluate the potential for top-down control of

Synechococcus sp. and an unidentified chlorophyte, which co-dominated a long- lasting in situ harmful algal bloom (HAB) with extremely high field cell densities in the Indian River Lagoon (IRL). Grazing on Isochrysis galbana, a palatable microalga presented at the same densities, provided a baseline for comparison.

When presented individually, grazing rates on both HAB species were lower than grazing rates on the palatable alternative. Grazing on I. galbana and the chlorophyte increased with increasing concentrations of algae but grazing on

Synechococcus sp. decreased or ceased at densities over 4.8  105 cells ml-1.

Copepods did not consume the chlorophyte at any density when it was paired with the palatable alternative. When Synechococcus sp. was paired with I. galbana, both species were consumed, but grazing on I. galbana decreased, especially at higher densities of cells. Observations indicated that Synechococcus sp. produced mucilage, which potentially hampered grazing. Copepod mortality was higher whenever Synechococcus sp. was present in densities equal to or greater than 4.8 ×

105 cells ml-1, another phenomenon potentially connected to the presence of mucilage. Overall, the evidence suggests some top-down control of the co- dominant microalgae during blooms.

Keywords: Top-down control, Grazing, Cyanobacteria, Chlorophyte,

Parvocalanus crassirostris, Synechococcus

1.1 Introduction

Harmful algal blooms (HABs) are increasing in estuaries around the world

(Smayda 2008; Berry et al. 2015; Phlips et al. 2015; Cao et al. 2017). Synchronicity at the global scale has been attributed to climate change and eutrophication brought on by increased nutrient loading (Smayda 2008), whereas important local influences on the magnitude and composition of HABs include residence times, toxicity, and differential losses due to grazing (Turner 2006; Smayda 2008; Phlips et al. 2010; Turner 2010; Berry et al. 2015; Phlips et al. 2015; Cao et al. 2017).

In keeping with the global trend, the frequency and intensity of HABs in the

Indian River Lagoon (IRL) has increased in the last seven years, with increased availability of nutrients suspected as a key bottom-up influence (Phlips et al. 2010,

2011; Lapointe et al. 2015; Phlips et al. 2015). An unprecedented sequence of intense and long-lasting algal blooms started in 2011 (Phlips et al. 2015), with the initial bloom being dubbed the “superbloom” because of its ~12-month duration and concentrations of chlorophyll a that reached 136 μg L-1. In contrast to blooms dominated by dinoflagellates or diatoms (Phlips et al. 2015), the superbloom was co-dominated by a species of picocyanobacteria and a nanoplanktonic chlorophyte, which remains unidentified and was documented at > 1 million cells ml-1 during the event (Indian River Lagoon 2011 Consortium 2015). The bloom caused harm by decreasing light penetration to an extent and for a duration that led to the loss of tens of thousands of acres of seagrass, and the resulting decreased competition for nutrients may have promoted subsequent blooms of phytoplankton (Phlips et al.

2015). In addition to bottom-up stimulation by nutrients, the relatively small size and unusually high algal densities of the species in the superbloom led to questions about the possible failure of top-down control by zooplankton.

The algae targeted in this study were Isochrysis galbana and two strains isolated from the “superbloom,” an unidentified, unicellular, nanoplanktonic chlorophyte and the prokaryotic Synechococcus cf. elongatus. The haptophyte, I. galbana, is roughly spherical with a diameter of 4–6 µm, and it has been used widely as a palatable food when culturing copepods (Shields et al. 2005). The chlorophyte was a non-flagellated alga, with a diameter of 1–4 µm, which was present in the lagoon but never dominant until the 2011 superbloom (Phlips et al. 2015). The taxonomy, physiology, and ecology of this chlorophyte have not been documented. The other

HAB species, Synechococcus sp., typically has been characterized as a rod-shaped unicellular cyanobacterium, but its morphology has been shown to change substantially in response to environmental cues (Goclaw‐Binder et al. 2012). The genus has been shown to be ubiquitous in marine, estuarine, and freshwater environments (Partensky et al. 1999), and species have contributed significantly to algal blooms in coastal and estuarine systems with high nutrient loads (Badylak and

Phlips 2004), including Florida bay (Phlips et al. 1999; Berry et al. 2015), San

Francisco Bay (Ning et al. 2000), and the Black Sea (Uysal 2000). During the superbloom, concentrations of Synechococcus sp. exceeded 5 × 106 cells ml-1

(Phlips et al. 1999). This cyanobacterium exhibits broad salinity and temperature tolerances (Phlips et al. 1999), which easily encompass typical salinities and

temperatures in the IRL (Phlips et al. 2015). The strain used in this study was in the nanoplanktonic size range, ~5 µm in diameter, and the culture was observed to produce mucilage.

The grazer used in this study was Parvocalanus crassirostris, a small (~2 mm long), holoplanktonic, calanoid copepod that has been shown to dwell in the upper

6–20 m of tropical and subtropical estuarine, coastal, and oceanic waters (Milstein

1979; Turner and Dagg 1983; Wong et al. 1993; Tang et al. 1994; Almeida et al.

2012; Sun et al. 2012; Liu et al. 2013). These cosmopolitan copepods have been shown to tolerate a wide range of temperatures (15–31 ºC), salinities (20–37), and eutrophic or turbid conditions (Milstein 1979; Wong et al. 1993; Almeida et al.

2012). P. crassirostris mainly grazes on nanophytoplankton (Calbet et al. 2000), and according to a three-year record of mesozooplanktonic abundances, it was the dominant herbivorous copepod in the region of the superbloom (Johnson, unpublished data).

This study determined relative grazing rates at cell densities mimicking an unusual, but actual algal bloom. To this end, P. crassirostris was fed monocultures of i) the nanoplanktonic chlorophyte, ii) a cyanobacterium in the genus

Synechococcus, and iii) I. galbana at various densities based on field observations, as well as mixed cultures containing iv) the chlorophyte and I. galbana or v)

Synechococcus sp. and I. galbana at equal densities that summed to the targeted total density. Thus, the study contrasts mesozooplanktonic grazing on the HAB species isolated from the superbloom, at observed superbloom densities, with

grazing on a palatable species to elucidate the role of herbivory in the initiation, maintenance, and termination of blooms. This study addressed the following hypotheses:

H1: Copepod (Parvocalanus crassirostris) will graze on HAB species

(Synechococcus sp. and an unidentified chlorophyte).

H2: The grazing rates of Copepod (Parvocalanus crassirostris) on HAB species

(Synechococcus sp. and an unidentified chlorophyte) will be lower than its grazing rates on a palatable alternative (Isochrysis galbana).

H3: Copepod (Parvocalanus crassirostris) will actively graze on the palatable food

(Isochrysis galbana) rather than HAB species (Synechococcus sp. and an unidentified chlorophyte) when they are served together in same concentrations.

1.2 Materials and Methods

Copepods (Parvocalanus crassirostris) and algae (harmful algae and Isochrysis galbana) were cultured by Algagen, LLC (Vero Beach, FL 32961, USA). Starter cultures of the harmful algae were isolated from IRL by the Phlips Laboratory at the University of Florida. Cultures were grown in seawater filtered through 0.45-

µm filters. All algae were cultured in 2-L glass jars at 26 °C, with continuous aeration and 1,100 lux of light provided continuously by fluorescent bulbs.

Guillard’s (F/2) marine water enrichment solution was used as culture medium. For best yield, chlorophytes were cultured at a salinity of 25 and Synechococcus sp. at a salinity of 32. Copepods were cultured at 26 °C and a salinity of 25 in 2-L glass

jars that were aerated gently, and they were fed I. galbana at 15,000 cells ml-1 daily. For 24 h immediately preceding experiments, copepods were maintained without food in gently aerated, 2-L glass jars under a 12/12 light/dark cycle. To reduce stress in the experiments involving Synechococcus sp., P. crassirostris, and

I. galbana were gently acclimated to a salinity of 32 in advance of Synechococcus experiments.

Experiments were carried out in filtered seawater and held at 26 °C for 20 h under 1,100 lux of artificial light. Along with algae at specified treatment densities, adult copepods were placed in each replicate container, which was 3-ml capacity.

Controls to estimate algal growth and non-grazing mortality had the same algal densities, but without copepods. Five replicates were prepared for each treatment and control replicate (n = 5). In such laboratory experiments, behavioral artifacts due to containment have always been a concern (Elmore 1982), but the extreme algal densities required, near and exceeding 106 cells ml-1 and mimicking observed field densities, dictated final experimental volumes. Standard phytoplankton culture procedures (e.g., 1×104 cells ml-1) do not produce cell densities required to test superbloom conditions (Hoff and Snell 2007), a testament to the extreme nature of the IRL superbloom. Algagen, an algal culture company specializing in high density production, was contracted specifically to produce densities on par with the superbloom. Proprietary culture procedures at Algagen limit the volume produced for extreme cell densities. With a limited supply of the highest density cultures, we could only test the highest densities by separating them into small volumes.

According to our preliminary experiments, grazer densities of 2.5 individuals ml-1 were necessary in order to observe measurable grazing impacts in a reasonable experimental time frame for the bloom density treatments. The goal was to conduct replicated trials using monocultures and 50:50 mixtures of the appropriate algae at densities of 5.8×104, 1.2×105, 2.3×105, 4.8×105, 9.6×105, and 1.9×106 cells ml-1, but cultures only yielded 9.5×105 cells ml-1 for the trials involving mixtures of the chlorophyte and I. galbana. To our knowledge, copepod grazing rates with cell densities on par with the superbloom have not been tested. Exploring this unique phenomenon necessitated a compromise with the experimental design and it will, therefore, be important to discuss results with consideration of possible container artifacts.

The initial and final densities of microalgae were measured via flow cytometry

(BD Accuri C6), and copepod survival was scored via stereo microscopy. Grazing rates (cells copepod-1 h-1) were determined using equations from Frost (1972), with the mean of replicate controls used for comparison (Frost 1972). Statistically significant differences in grazing rates among different densities of algae were determined via analysis of variance (ANOVA) and post-hoc Tukey pairwise comparisons (α = 0.05).

1.3 Results

Figure 1.1 Numbers of cells consumed per hour by Parvocalanus crassirostris grazing on different densities of Isochrysis galbana and an unidentified chlorophyte presented as (a) monocultures and (b) mixtures of 50% I. galbana and 50% chlorophyte. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05).

Parvocalanus crassirostris grazed on monocultures of Isochrysis galbana and the chlorophyte, without suffering any mortality. Grazing rates increased with increasing densities of both I. galbana and the chlorophyte (Fig 1.1a). However, grazing pressure on the chlorophyte was significantly less than that exerted on the more palatable I. galbana at all densities (Fig 1.1a).

There also was no copepod mortality in experiments employing mixed cultures of I. galbana and the chlorophyte. In these treatments, the pattern of increased grazing on denser cultures of I. galbana was similar to that observed in experiments with monocultures. However, the grazing rates on I. galbana were lower compared to the grazing rates on the monocultures. In addition, grazing on the chlorophyte in the mixed culture decreased or ceased altogether (Fig 1.1b).

Figure 1.2 Mortality rates for Parvocalanus crassirostris exposed to different densities of Synechococcus sp. presented as (a) a monoculture and (b) a mixture of 50% Isochrysis galbana and 50% Synechococcus sp.. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05). Note: mean mortality rates in the mixed culture (b) were statistically equivalent and not significantly different from zero.

When exposed to Synechococcus sp. in monoculture, some copepods died in treatments with densities  1.2×105 cells ml-1, and mortality increased substantially in treatments employing densities  4.8×105 cells ml-1 (Fig 1.2b). Without knowledge of when ailing copepods ceased grazing or died, grazing rates for all densities were calculated using the assumption that they had lived for half of the experiment (Turner et al. 2012); therefore, they likely represented underestimates of actual grazing rates. Copepods grazed on Synechococcus sp. primarily in treatments with  4.8105 cells ml-1, and these grazing rates generally were lower than those observed in experiments employing monocultures of I. galbana (Fig

1.3a). Water that contained Synechococcus sp. was more viscous than filtered seawater due to mucilage production.

Figure 1.3 Numbers of cells consumed per hour by Parvocalanus crassirostris grazing on different densities of Isochrysis galbana and Synechococcus sp. presented as (a) monocultures and (b) mixtures of 50% I. galbana and 50% Synechococcus sp.. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05).

As in experiments with Synechococcus sp. monocultures, there was some copepod mortality in the mixed treatments (50% I. galbana + 50% Synechococcus sp.; Fig 1.2b). However, mortality rates in mixed cultures were lower than those in monocultures with similar densities (Fig 1.2). In these mixed culture experiments, grazing rates on I. galbana and Synechococcus sp. were statistically indistinguishable (Fig 1.3b) because grazing rates on I. galbana were markedly lower relative to those observed in monocultures (Fig 1.3). In fact, grazing rates on

Synechococcus sp. in mixed cultures were equal to or higher than rates observed for monocultures (Fig 1.3).

Table 1.1 Comparison of Parvocalanus crassirostris grazing rates on Isochrysis galbana in different incubation volumes.

1.4 Discussion

Crowding and container effects are of common concern to researchers doing laboratory feeding/grazing rate studies (Folt and Goldman 1981; Peters 1984;

Helgen 1987; Burns 1995, 2000; Preuss et al. 2009; Lee et al. 2012). Potential artifacts could arise from physical interference among zooplankton, interactions between zooplankton and vessel walls (Peters 1984), or chemical mediated interactions among competitors (Folt and Goldman 1981). Some studies demonstrate that crowding or container effects may lead to depression of zooplankton feeding rates (Folt and Goldman 1981; Helgen 1987; Burns 1995,

2000; Preuss et al. 2009; Lee et al. 2012). However, (Mullin 1963) found that container size was not a significant factor on feeding rates when testing copepods.

Small containers can also crowd grazers, and we must acknowledge the effects of such conditions in this study cannot be known with certainty. However, (Lee et al.

2012) demonstrated that some copepod species could be raised at densities as high as 20 individuals ml-1(Lee et al. 2012). In fact, many copepod grazing studies have been done with small containers or high grazer densities similar to our experimental settings (Hong et al. 2013; Motwani and Gorokhova 2013; Boersma et al. 2016; Ger et al. 2016; Rangel et al. 2016; Svensen and Vernet 2016; Mathews et al. 2018). Most importantly, we conducted a small-scale trial with a range of volumes and grazer densities which showed that the grazing rates of Parvocalanus crassirostris reported here, when feeding on Isochrysis galbana, did not change substantially under less unnatural conditions (Table 1.1).

Top-down control via grazing has been shown to vary with the characteristics of grazers, algal food, and environmental characteristics. Our experiments involved adults of one grazer, which limited the influence of body size, foraging speed, feeding efficiency, and life history stage (Peters 1984; Hansen et al. 1997; Atkinson

1998; Nejstgaard et al. 2007; Seuront and Vincent 2008). In fact, our focus was on characteristics of phytoplankton, in particular, their density, size, swimming speed, movement pattern palatability, and chemical constituents (Peters 1984; Hansen et al. 1994; Kiørboe et al. 1996; Norberg and DeAngelis 1997; Calbet 2001; Seuront and Vincent 2008).

Algal density had a substantial influence on P. crassirostris grazing rates, regardless of the species of algae being offered or whether they were offered as monocultures or mixed cultures containing HAB species. Mean grazing rates on I. galbana in monocultures ranged from 1.1×103 to 1.8×104 cells copepod-1 h-1, which were similar to rates reported for P. crassirostris feeding on nanoplankton (Calbet et al. 2000), Tisbe furcata feeding on a flagellated haptophyte and a flagellated cryptophyte of similar size (Abu-Rezq et al. 1997), and Pseudodiaptomus euryhalinus feeding on an Isochrysis sp. (Anzueto-Sánchez et al. 2014). P. crassirostris has been documented to be a suspension feeder, with no evidence of raptorial behavior (McKinnon and Klumpp 1998). Such copepods have been observed to generate small-scale currents that entrained particles, which subsequently were caught by feeding appendages (Koehl and Strickler 1981). It follows that encounter rates and the resulting grazing rates would, to some degree,

have depended on algal densities, provided there were no other constraints. Given that other studies have shown positive density-dependent grazing patterns (e.g.,

Frost 1972), artifacts due to containment were not expected to affect grazing on

HABs substantially or differentially.

Consumption of the chlorophyte and Synechococcus sp. varied from 0 to 3×103 cells copepod-1 h-1 at densities from 5.8×104 to 1.9×106 cells ml-1. These rates were low compared to consumption of I. galbana, especially at algal densities of

9.5×105–1.9×106 cells ml-1. In fact, P. crassirostris effectively stopped grazing on chlorophytes when they were mixed with I. galbana at any density. In contrast, grazing on Synechococcus sp. persisted at low rates in mixed cultures.

Potentially related chlorophytes have not been reported to produce toxins or inhibit grazing by zooplankton (e.g. DeBiase et al. 1990). When feeding on the chlorophyte from the IRL, P. crassirostris always survived so any negative effects were not lethal in the short term. Phytoplankton have been reported to produce secondary metabolites and structural defenses that render them less palatable to grazers (Mitra and Flynn 2006). For example, Leising et al. (2005) found that

Calanus pacificus rejected certain Thalassiosira spp. even though they were abundant, the right size, and congeneric with preferred prey, because they were producing chemicals that had deleterious effects on reproduction. Selective avoidance of harmful algal species also has been shown to be a complex process influenced by the density of toxic species, presence of other sources of food, and consequences of sublethal effects (Teegarden et al. 2001; Turner and Borkman

2005; Cohen et al. 2007). Alternatively, copepods have exhibited size-selective feeding (Wilson 1973; Berggreen et al. 1988), and this chlorophyte was 1–4 µm in diameter or roughly half the diameter of I. galbana (4–6 µm). P. crassirostris has been observed to graze on nanophytoplankton with diameters of 2–20 µm (Calbet et al. 2000), but this chlorophyte and other algae at the lower edge of this range may be more difficult for P. crassirostris to detect, capture, or consume. The potential for a grazer to control a harmful bloom may be dependent on whether the algae matches the grazer’s optimal prey size.

Regarding detection, it has been shown that background plankton, meaning other particles that may obscure targets, can reduce grazing or predation (Johnson and

Shanks 1997). When the chlorophyte and I. galbana were offered together in mixed cultures, grazing rates on the chlorophyte dropped to nearly zero at most densities, while grazing on I. galbana persisted but the rates were lower than in monocultures

(Fig 1.1b). Thus, P. crassirostris may be deliberately selecting I. galbana cells or deliberately avoiding the chlorophyte. Selective avoidance of harmful algal species has been observed in previous studies of zooplankton grazing (Teegarden et al.

2001; Turner and Borkman 2005; Cohen et al. 2007). Another possibility is that the larger I. galbana cells may have functioned as background plankton that obscured chlorophytes and hindered detection or capture (Johnson and Shanks 2003). In summary, results indicated that P. crassirostris was unlikely to exert effective top- down control on the chlorophyte that bloomed in the IRL, but further work is needed to elucidate the exact mechanism that reduced grazing pressure.

The mortality observed when P. crassirostris was fed the cyanobacterium

Synechococcus sp. (Fig 1.3a), especially in high-density monocultures, may have been due to toxicity or the effects of mucilage. Although the genus has been reported as a potential food for some copepods (Koski and Klein-Breteler 2003; Kâ et al. 2012), its members have exhibited genetic diversity and several strains are capable of producing hepatotoxins or neurotoxins that affect zooplankton (Martins et al. 2005, 2007). Lower mortality in mixed cultures of Synechococcus sp. and I. galbana, even at higher total densities, indicated that acute toxicity was unlikely.

Aside from possible toxicity, the mucilage produced by Synechococcus sp. in this study may have killed some P. crassirostris or at least reduced their grazing rates

(Ma and Johnson unpublished data). Production of mucilage was unlikely to have been simply an artifact of containment, because Synechococcus spp. have been shown to produce mucilage in natural assemblages found in Florida Bay (Berry et al. 2015) and off Venezuela (Ramos and Reyes-Vazquez 1990). Polymeric exudates (a.k.a. mucilage) have reduced copepod grazing rates via chemical inhibition or mechanical interference with filtering or capture due to increased viscosity or aggregation of cells (Malej and Harris 1993; Dilling et al. 1998;

Nejstgaard et al. 2007). Eventually, an inability to capture food could have led to starvation and death of copepods, but such an effect was unlikely in these 20-h experiments because copepods survived without food in filtered seawater for 24 h.

The effect of either a toxin or mucilage appeared to occur above a threshold density because copepods grazed on low-density cultures of Synechococcus sp. at rates that

were not significantly different from consumption of palatable I. galbana. The presence of a threshold has been shown for Acartia clausi grazing on the toxic dinoflagellate Alexandrium minutum, with consumption decreasing dramatically when A. minutum was abundant and producing more toxin (Guisande et al. 2002).

In fact, the apparent increase in grazing on Synechococcus sp. in higher density mixed cultures may have been due to the actual densities of these cells being half of those in monocultures. Interestingly, grazing on I. galbana in mixed cultures was depressed. In combination with the relatively consistent rate of grazing on

Synechococcus sp. in all cultures, this result indicated there was little selectivity and no obvious background effects as seen in experiments with the chlorophyte.

One possible explanation was interference from mucilage, with the increased viscosity of the water making the delicate work of sorting particles and selecting I. galbana more difficult. Additionally, the diameters of I. galbana and

Synechococcus sp. were similar (~ 5 µm), which would have made sorting based on size difficult. Overall, the results indicated that P. crassirostris would struggle to exert effective top-down control on blooms of Synechococcus sp., although further work is needed to identify the mechanism and any threshold.

Population dynamics during algal blooms, including HABs, have been shown to be regulated both by bottom-up controls affecting growth rates and top-down controls affecting loss rates (Buskey 2008). Top-down controls include pathogens, physical advection, and, importantly, grazing (Buskey 2008; Smayda 2008). Some studies have shown that copepods retarded or helped terminate harmful blooms

(Kozlowsky-Suzuki et al. 2003; Campbell et al. 2005; Jansen et al. 2006), with grazing rates on particular phytoplankton for dominant grazers being key factors that determined the degree of control (Campbell et al. 2005, 2009; Olson et al.

2006; Turner 2006). For example, Calanus finmarchicus fed selectively on diatoms and exerted only half of the grazing pressure on toxic Alexandrium spp. that was documented for a nonselective generalist, Acartia hudsonica, and in combination, reduced bottom-up, competition for nutrients and top-down control facilitated a bloom of the toxic dinoflagellate (Teegarden et al. 2001; Campbell et al. 2005).

The relatively low grazing rates for P. crassirostris feeding on the HABs, especially on the chlorophyte in the presence of alternative prey, indicated that mesozooplankton were unlikely to prevent or terminate blooms of these phytoplankton, with chemical inhibition, mechanical inhibition, or size selectivity suggested as possible reasons for limited consumption. While some grazing persisted on Synechococcus sp. in mixed cultures, grazing rates were still relatively low and top-down control by mesozooplankton seems unlikely. In addition to further work on the causes of reduced grazing, an improved understanding of grazing by microzooplankton would be valuable because this trophic link represents an important expansion of the original phytoplankton-mesozooplankton- fish trophic web (e.g., Calbet et al. 2003; Liu and Dagg 2003; Calbet and Landry

2004).

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Chapter 2: The effect of algal cell size and mucilage on copepod grazing rates

Abstract

Some recent Harmful Algal Blooms (HABs) have been composed largely of small-sized algal cells and mucilage-producing species which can alter water viscosity. In spite of the presence of potential algal grazers, HABs comprised of such species have persisted, and are becoming more common in, many locations.

Lab grazing experiments were conducted with one of the most cosmopolitan herbivorous copepods (Parvocalanus crassirostris) to examine the effects of small cell sizes and mucus on grazing rates. Copepods showed limited grazing rates on small-sized beads and in high viscosity treatments. Copepod mortalities were also observed in high viscosity treatments. Results reveal potential limitations of top- down HAB controls.

Keywords: Grazing, Size Selection, Mucilage effects, Parvocalanus crassirostris

2.1 Introduction

Harmful Algal Blooms (HABs) are an increasing problem in estuaries around the world (Berry et al. 2015; Phlips et al. 2015; Cao et al. 2017). Two primary HAB- drivers, eutrophication and climate change, have been proposed (Smayda 2008).

Recent studies have heightened attention on the influence of those drivers on phytoplankton structure and dynamics (Zingone et al. 2010; O’neil et al. 2012;

Phlips et al. 2015). One example is the change of the HAB species in Indian River

Lagoon (IRL) system. Before 2007, algal blooms in the IRL were dominated by large-sized dinoflagellates (Howell 1953; Steidinger et al. 1998; Badylak et al.

2004; Hsia et al. 2006; Phlips et al. 2011). However, since 2011, the blooms are primarily composed of small cyanobacteria and eukaryotes (Chlorophytes and

Ochrophytes) (Indian River Lagoon 2011 Consortium 2015). Some HABs in the

IRL, such as 2011 “superbloom” component Synechococcus sp. (Indian River

Lagoon 2011 Consortium 2015) and “brown tide” Aureoumbra lagunensis bloomed in 2012 and 2016 (Phlips et al. 2015) were observed to produce extracellular polymeric substances (mucilage), which could potentially increase the viscosity of the water. A question that arises is whether the small cell size or mucilage produced by these HAB species affect their potential top-down controls. This study aims to

1) evaluate the effect of mucus/viscosity on copepod grazing rates independent of temperature metabolic effects and 2) test the effect of different prey particle sizes on copepod grazing rates.

Copepod feeding mechanics are predominantly governed by viscous forces (Yen

2000; van Duren and Videler 2003). Viscosity indicates how well a fluid can resist deformation under stress and is sometimes equated with fluid “thickness”. The degree to which an organism is prone to influence by viscosity can be evaluated by its Reynolds number (Re). With small feeding appendage size and slow moving speed, the act of copepod grazing has relatively low Re, often <1, and is viscosity- dominated (Yen 2000). Copepods have evolved special feeding adaptations to enable food capture at low Reynolds numbers. For example, most calanoid copepods are suspension feeders who draw water past mouthparts and use their second maxillae to capture parcels containing food particles (Koehl and Strickler

1981).

The viscosity of seawater changes with temperature, salinity and chemical composition. Some phytoplankton species secrete extracellular polymeric substances (EPS) into surrounding water and change the viscosity (Seuront et al.

2006; Kesaulya et al. 2008; Seuront and Vincent 2008). In an extreme example, the haptophyte flagellate Phaeocystis sp. forms mucus-producing colonies which can increase seawater viscosity by 274% (Schoemann et al. 2005; Seuront et al. 2006).

Phytoplankton polymeric exudates have been repeatedly reported as reducing grazing rates of copepods (Malej and Harris 1993; Dilling et al. 1998; Nejstgaard et al. 2007) and also other filter feeders such as Euphausiids (Dilling et al. 1998) and protozoa (Liu and Buskey 2000). Potentially increase the viscosity of the water, and likely decrease top-down control on those HABs.

Size-selective feeding is another common factor influencing zooplankton grazing

(Wilson 1973; Berggreen et al. 1988; Hansen et al. 1994). Prey size selection by zooplankton is generally taxonomy and species dependent (Wilson 1973;

Berggreen et al. 1988; Hansen et al. 1994, 1997). For example, B. Hansen et al.

(1994) found that the length ratio between predator and their optimal prey is around

18:1 for copepods, 8:1 for ciliates and 1:1 for dinoflagellates. While feeding on microplankton of a consistent size range, omnivorous copepods are capable of switching feeding strategies between suspension feeding and ambush feeding

(Kiørboe et al. 1996; Atkinson 1998). In comparison, Parvocalanus crassirostris feeds only via suspension feeding and primarily grazes on nanoplankton (Calbet et al. 2000).

The grazer used in this study was Parvocalanus crassirostris, a small (~2mm) holoplanktonic calanoid copepod, common in estuaries, and with a broad geographic range including tropical and subtropical coasts and oceans (Milstein

1979; Turner and Dagg 1983; Wong et al. 1993; Almeida et al. 2012; Sun et al.

2012; Liu et al. 2013). According to a three-year record of mesozooplankton abundance in the Northern Indian River Lagoon, P. crassirostris was the most abundant herbivorous copepod in this area (Johnson, unpublished data). These cosmopolitan copepods tolerate a wide range of temperatures (15 to 31 ºC)

(Milstein 1979; Wong et al. 1993) and salinities (20 to 37) (Milstein 1979; Wong et al. 1993; Chew and Chong 2011). Isochrysis galbana was used in this experiment as a palatability control for comparison to mucus producing HAB species of

undetermined palatability. I. galbana is a haptophyte with cell size (4-6 µm) and shape (unicellular) comparable to those mucilage producing species. I. galbana is a palatable and advantageous food for copepods and widely used in the aquaculture industry (Shields et al. 2005). This study addresses the following hypotheses:

H1: Copepod (Parvocalanus crassirostris) grazing rates will be lower on small (< 5

µm) relative to large (> 5 µm) beads.

H2: Copepods (Parvocalanus crassirostris) grazing rates will be lower in high viscosity water.

2.2 Materials and Methods

Copepods (Parvocalanus crassirostris) and algae (Isochrysis galbana) were cultured by Algagen, LLC (Vero Beach, FL 32961, USA). Copepod and algae were cultured in 2-L glass jars held at 25 °C and a salinity of 25, with 12/12 light/dark cycle under 1100 lux of artificial light. Copepods were fed I. galbana at 1 to 5 ×104 cells ml-1 daily (Hoff and Snell 2007). For 12 h immediately preceding experiments, copepods were maintained without food in gently aerated.

Mucilage effect experiments

Copepods (P. crassirostris) were fed with algal cultures (I. galbana) at different viscosities to test a potential effect of mucilage on copepod grazing rates. Algal density (1.8×104 cells ml-1) was held constant for the viscosity experiments. In order to manipulate the viscosity of I. galbana culture, the heavy molecule polymer polyvinyl pyrrolidone (PVP, average MW 360,000) was used as a thickening agent.

PVP was chosen because it has no detectable toxic effect on delicate planktonic larvae and has been used in analogous experiments (Podolsky and Emlet 1993).

Kinematic viscosity was measured using a falling-ball viscometer (model # GV-

2100 by Gilmont) while adding PVP until the algal cultures reach the desired viscosity. The viscosity treatments were 0.92, 1.45, 1.98, 2.50, 3.03 and 3.56 ×10-6 m2 s-1, where the viscosity of 0.92 ×10-6 m2 s-1 was non-PVP treatment (seawater only at the same temperature and salinity).

Mucilage effect experiments were carried out in beakers filled with 500-ml filtered seawater and held at 25±0.1 °C (controlled by micro temperature controller) and a salinity of 25 for 11 h under 1100 lux of artificial light with gentle aeration. Preliminary experiments showed that treatment effects on algal densities could be detected after 11 hours, and this knowledge determined the length of experimental runs. Ten adult copepods were placed in each container with algal density of 1.8×104 cells ml-1, and grazing control beakers had the same algal density and viscosity as these treatments but lacked copepods. Three replicate treatments and controls were prepared for each experiment (n = 3). The initial and final densities of microalgae were measured via flow cytometry (BD Accuri C6), and copepod survival was scored via stereo microscopy.

Statistically significant differences in grazing rates among different treatments were determined via Student's t-test (α = 0.05) or analysis of variance (ANOVA) and post-hoc Tukey pairwise comparisons (α = 0.05). Grazing rates (cells copepod-

1 h-1) were determined using the equation described in (Frost 1972).

Size selection experiments

Copepods were fed six different sizes of micro-polystyrene beads (2.0-2.4 µm,

3.0-3.4 µm, 5.0-5.9 µm, 7.0-7.9 µm, 8.0-12.9 µm, 13.0-17.9 µm) (Spherotech, Inc.) separately to examine the effect of prey size on copepod grazing rates. Copepods were also fed combinations of different size beads to test size selectivity. The concentration of the beads was 500 beads ml-1 for both basic grazing rate and size selection experiments. Grazing control treatments (n=3) had the same seawater volume and bead density, but lacked copepods. The initial and final bead densities were measured via flow cytometry (BD Accuri C6).

Size selection experiments were carried out in 500-ml beakers filled with 200-ml filtered seawater and held at 25±0.1 °C (controlled by micro temperature controller) and a salinity of 25 for 6 h under 1100 lux of artificial light. Preliminary experiments showed that copepods proactively grazed on beads for about 6 hours, with grazing rates in the same range as that for some algae. After 6 hours, however, grazing rates on beads decrease for an unknown reason. For this reason, size selection experiments were limited to 6 hour duration. Beakers were gently aerated to keep the beads suspended during the experiments. Ten adult copepods were placed in each container with beads density of 500 beads ml-1, and control beakers had the same bead densities as the treatments, but lacked copepods. Beads were flavored with I. galbana culture extraction (filtered with a 0.45 µm membrane) to encourage copepod grazing. Three replicate treatments and controls were prepared for each experiment (n = 3), and the control mean was used for comparison with

treatment replicates. The initial and final densities of beads were measured via flow cytometry (BD Accuri C6).

Statistically significant differences in grazing rates among different treatments were determined via analysis of variance (ANOVA) and post-hoc Tukey pairwise comparisons (α = 0.05). Since there was no need to consider beads density changes due to population growth during the experiment, grazing rates (beads copepod-1 h-1) were determined using an equation described in Abu-Rezq et al. (1997) and

Anzueto-Sánchez et al. (2014):

퐺푅 = (퐶2 − 퐶1) × 푉 ÷ (푛 × 푡)

퐺푅 = grazing rate (number of beads ingested copepod-1 h-1)

퐶1 = final beads density of treatment replicates (beads ml-1, n = 5)

퐶2 = final beads density of control mean (beads ml-1)

푉 = volume of the vials (ml)

푛 = number of copepods

푡 = duration of the experiment (h)

2.3 Results

Figure 2.1 A) Mean numbers of cells consumed per hour (± a standard deviation, SD) by Parvocalanus crassirostris grazing on Isochrysis galbana at the same algal density (1.8×104 cells ml-1) but in different viscosities. B) Mean mortality rates (± a standard deviation, SD) for Parvocalanus crassirostris exposed to different viscosities. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05).

Copepods grazed on Isochrysis galbana in low viscosity treatments, and the grazing rates significantly declined with increasing viscosity (Fig. 2.1A). When exposed to I. galbana with PVP treatments, copepod mortalities were observed at the highest viscosities (Fig. 2.1B). Without knowledge of when impaired copepods ceased grazing or died, grazing rates for all treatments were calculated assuming half duration grazing (Turner et al. 2012) (Fig. 2.1A).

Figure 2.2 A) Mean numbers of beads consumed per hour (± a standard deviation, SD) by Parvocalanus crassirostris grazing on different single-sized beads B) Mean numbers of beads consumed per hour (± a standard deviation, SD) by Parvocalanus crassirostris grazing on mix-sized beads. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05).

There was no copepod mortality observed in size selection experiments.

Parvocalanus crassirostris grazed differentially on different size beads when fed separately (Fig. 2.2A). Copepod grazing rates on 7.0-7.9 µm beads were significantly higher than the smallest beads (2.0-2.4 µm) and the largest beads

(13.0-17.9 µm) (Fig. 2.2A). When fed mixed bead sizes, the grazing rates on the extreme-sized beads increased and were not significantly different from rates on mid-sized beads (Fig. 2.2B).

2.4 Discussion

Viscosity had a substantial influence on Parvocalanus crassirostris grazing rates. Copepods grazed on I. galbana in low PVP content/viscosity treatments, but grazing rates dropped significantly with increasing viscosity (Fig. 2.1A). Feeding behaviors of suspension feeders are highly influenced by water viscosity (Podolsky

1994; Bolton and Havenhand 1998). Polymetic exudates (mucus or mucilage) can reduce copepod grazing rates by chemically inhibiting grazing, mechanically increasing seawater viscosity or facilitating cellular aggregations via adhesion

(Malej and Harris 1993; Dilling et al. 1998; Nejstgaard et al. 2007). Podolsky

(1994) found that high viscosities reduced grazing of Dendraster excentricus larvae by half, and higher viscosities tend to shift ingestion toward larger particles.

“Brown tide”, Aureoumbra lagunensis, is another example of an alga which secretes extracellular polymeric substances (ESP), which it does under hypersaline conditions (Liu and Buskey 2000). Some species of protozoa have difficulties

feeding on high densities of A. lagunensis, but grow faster on A. lagunensis cells that have been separated from their EPS exudate via gentle centrifugation (Liu and

Buskey 2000). Given these analogous evidences from the literature, it seems likely that depressed grazing observed at higher viscosities in this study reflects possible natural effects on P. crassirostris when feeding on EPS-producing algae.

High mortality rates were observed when P. crassirostris was feeding on algae at high viscosities (Fig. 2.3B). PVP has been used in analogous experiments, and showed no detectable toxic effects on delicate zooplankton (Podolsky and Emlet

1993; Larsen et al. 2008). One possible explanation for copepod mortality is that this heavy molecular polymer blocked the respiratory passage. Copepods have no gills, and oxygen uptake and carbon dioxide release take place through the integument and in the hindgut (Blaxter et al. 1998). Therefore, it was possible that copepods suffocated due to polymer sticking on their bodies. I. galbana were observed to form aggregates when mixed with PVP during the experiments.

Another possibility is that copepods had a hard time grasping and holding I. galbana which could lead to starvation. Clumped algal cells might exacerbate any suffocation problems. These copepods may not be as well adapted as a population with a history of co-existing with harmful algal blooms. Studies have demonstrated that zooplankton from areas routinely exposed to HABs showed more resistance to toxin secretions compared to populations from non-HAB areas (Hairston Jr et al.

2001; Colin and Dam 2002). However, one of the reasons that mucus effects have been tested on this grazer was the possibility that the grazer was impaired by

mucus, contributing to the failure of top-down control. These experimental outcomes are consistent with that hypothesis.

The potential for a grazer to control an algal bloom may depend, in part, on whether the algae match the grazer’s optimal prey size. Parvocalanus crassirostris grazed beads at distinctly different rates based upon size, with the greatest rate being on mid-sized beads from 7.0-7.9 µm. Mid-size bead grazing was significantly greater than grazing on both smaller and larger beads (2.0-2.4 µm and13.0-17.9

µm, respectively) (Fig. 2.2A). For a dominant herbivore like P. crassirostris, a bloom of smaller cells could present a challenge. One key harmful algal species in the Indian River Lagoon Superbloom was an unidentified chlorophyte (cell size 1-4

µm). In laboratory grazing experiments, P. crassirostris grazing rates were low on this chlorophyte relative to Isochrysis galbana (cell size 4-6 µm) presented at the same densities (Ma and Johnson, unpublished data). Other algal species comprising that same bloom were a picocyanobacteria (< 1 µm) (Indian River

Lagoon 2011 Consortium 2015) and Aureoumbra lagunensis (3-5 μm) (Phlips et al.

2015). All three of these harmful bloom species are smaller than the apparent optimal prey size for P. crassirostris, and may be difficult for the grazers to detect, capture or consume. This copepod species was the dominant mesozooplanktonic herbivore during most of the IRL Superbloom and its inability to exert clear top- down control on this mixed bloom of small-celled algae may be partially explained by its larger preferred prey size. When different sized beads were offered together at the same concentrations, the grazing rates on different sizes was less distinct

(Fig. 2.2B). P. crassirostris were observed to avoid small-sized chlorophytes (1-4

µm) when they are offered together with I. galbana (4-6 µm) at the same algal density (Ma and Johnson, unpublished data). Thus, P. crassirostris are able to tell the difference of different algal cells. Possible explanation of indiscriminate grazing on different beads is that both smaller and larger beads may have functioned as background plankton (Johnson and Shanks 1997, 2003) that obscured the preferred prey sizes and all beads were grazed at the same rate when they taste similar.

In conclusion, Parvocalanus crassirostris showed lower gazing rates on extreme sized beads compare to middle sized beads. Copepod mortalities were observed in high viscosity treatments and grazing rates decreased as increasing of viscosity.

Therefore, P. crassirostris, a dominant herbivorous copepod present during recent

HABs, could have limited grazing pressure on algal blooms with small cell sizes and mucilage production. This failure of top-down controls could contribute to the increasing prevalence of HABs resulting from smaller-celled, mucus-producing algae.

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Chapter 3: Biological responses to aeration in an estuarine canal with fine-

grained organic-rich sediments

Abstract

Fine-grained organic-rich sediments, usually referred to as “muck”, are detrimental to the overall quality of the Indian River Lagoon (IRL) ecosystem. One potential restoration method is water column aeration which generates fine bubbles, introducing oxygen to react with and neutralize organic material and purge toxic gasses from sediments. A pilot aeration experiment was carried out in a residential canal of the IRL estuary to examine ecosystem outcomes. Biological responses

(both phytoplankton and benthic infauna) were monitored throughout the aeration process, and compared against control conditions, to evaluate the feasibility and effectiveness of aeration for muck mitigation. Aeration facilitated benthic animal life only in the cold period when bottom waters held the most dissolved oxygen.

Planktonic communities displayed no significant responses to aeration.

Keywords: Aeration, fine-grained organic-rich sediments, Biological responses,

Phytoplankton community, Benthic infauna

3.1 Introduction

Estuaries are dynamic ecosystems often made eutrophic due to runoff and the accumulation of anthropogenic nutrients (Diaz 2001; Zhang et al. 2009). Nutrients drive estuarine productivity, but unnaturally high levels can unbalance an ecosystem and stress native populations(Nixon 1995; Diaz 2001; Schindler and

Vallentyne 2008). Due to coastal development and associated anthropogenic nutrient inputs, estuaries worldwide are experiencing the negative impacts of eutrophication, often including the accumulation of organic sediments (Puente and

Diaz 2015). Although organic sediments are potential food for benthic fauna

(Haines and Montague 1979), too much can cause hypoxia or anoxia via bacterial decomposition (Vaquer-Sunyer and Duarte 2008). Aquatic animals require oxygen, and changes to dissolved oxygen (DO) levels can rapidly alter the survivorship, behavior, abundance and composition of submerged fauna (Diaz and Rosenberg

1995; Vaquer-Sunyer and Duarte 2008). Sustained hypoxic conditions encourage reduced environments fostering sulfur-reducing bacteria and toxic hydrogen sulfide

(H2S) production (Vaquer-Sunyer and Duarte 2010). The presence of hydrogen sulfide can decrease survival times under hypoxia by 30% in some marine benthic communities (Vaquer-Sunyer and Duarte 2010), so these impairments often occur together and can enhance one another.

The Indian River lagoon (IRL), a complex shallow subtropical estuary associated with riverine, wetland and terrestrial systems, spans 156 miles (approximately

40%) of Florida's east coast. Like other estuaries, the IRL is highly impacted by the

buildup of fine-grained organic-rich sediments. The IRL is a low energy estuary, which contributes to the settlement and accumulation of fine-grained organic-rich sediments (Byers and Grabowski 2014). Extreme fine-grained organic-rich sediments have been referred to as “muck” (Trefry and Trocine 2011), operationally defined as being composed of > 10% organic matter, >60% silt and clay, and > 75% water by weight. Recent muck surveys have revealed IRL muck thickness to be 67% greater relative to three decades earlier (Trefry et al. 1990;

Trefry and Trocine 2011). Mitigating IRL muck pollution to restore healthy benthic habitats and remove a source of eutrophication will require a multi-pronged approach of IRL muck treatment, removal, and upstream input reduction.

Muck itself is a source of nutrients. It contributes more than 15 tons/km2/year nitrogen (>50% of total nitrogen flux) to the IRL water column, and the flux rate increases with temperature (Trefry et al. 2016). Nutrient fluxes from muck aggravate eutrophication and fuel harmful algal blooms (Brevard County 2016).

The IRL has lost tens of thousands of acres of seagrass because of algal blooms and is becoming a more phytoplankton-dominated system (Indian River Lagoon 2011

Consortium 2015). When algal blooms die off, the cells are decomposed by bacteria and oxygen levels fall, potentially killing aquatic life (Vaquer-Sunyer and

Duarte 2008). Sinking dead materials accumulate on the bottom and contribute to muck, completing a vicious cycle of harm.

One important component of the restoration process is environmental muck dredging (Brevard County 2016). It is apparent, however, that dredging alone will

not solve the problems. Other treatments, including flushing and water column aeration, are being considered to compliment removal and input regulation. These treatments are experimental, with little data available to confidently predict ecosystem outcomes. A pilot aeration experiment was funded by the IRL National

Estuary Program and carried out in Satellite Beach canals connected to the IRL estuary. This study follows the biological responses to aeration in an estuarine canal, simultaneously following a control canal for comparison.

Aeration was intended to purge gasses such as H2S from the sediments

(Jermalowicz‐Jones 2016) and introduce oxygen to react with and neutralize sediment organic content. Previous studies in other systems have showed four potential effects of aeration for improving water and sediment quality. First, it could increase dissolved oxygen by introducing bubbles and disrupting the stratification of water column (Jermalowicz‐Jones 2016). Second, the aeration could accelerate the decomposition of muck via oxidation of organic material.

Third, the aeration may promote the conversion of eutrophic nitrogen to inert N2 gas. (Lim et al. 2010; Harris et al. 2015). Finally, when H2S is oxidized during the aeration process, previously bound iron may precipitate phosphorus and reduce its bioavailability (Katsev and Dittrich 2013). Water column aeration has been attempted in Florida freshwater lakes and other locations (Katsev and Dittrich

2013; Harris et al. 2015; Jermalowicz‐Jones 2016). However, estuarine muck aeration has not been studied and a comprehensive evaluation of biological impacts is needed.

This project examined the impacts of aeration on benthic infauna and plankton.

Two hypotheses were posed and examined.

H1: The composition and density of phytoplankton community will be significantly changed as a result of canal aeration.

H2: The benthic infauna community, largely absent in anoxic canal sediments, will become reestablished following aeration.

3.2 Materials and Methods

Study sites

The aeration treatment was carried out at Redwood Canal (Satellite Beach,

Florida), and a non-aeration control were monitored 800 meters away at Anderson

Canal. Both canals were near the Grand Canal in Satellite Beach, all connected to the Indian River Lagoon (Fig 3.2). The aeration system installed by our collaborators, The Allied Group, consisted of five micro-porous diffusers wet on the surface of muck at 50-m spacing in Redwood canal (Fig 3.1). Three additional diffusers were installed at the open-end of the canal to establish a curtain to limit loss or gain of muck in the canal (Fig 3.1). The aeration was initiated on August

2017 and continued until September 2018. The samplings were carried out before the aeration on August 2017, and after the aeration on September 2017, November

2017, February 2018, April 2018 and June 2018. Both phytoplankton and benthic infauna samples were collected in each survey (details below).

Figure 3.1: Redwood Canal and the placement of 8 aeration diffusers for ecosystem testing and evaluation. Grey squares are aeration diffusers and green lines are sunken benthic aeration tubing.

Figure 3.2 Study locations in A) Redwood Canal (aeration) and Anderson Canal (control), adjacent to the Grand Canal in Satellite Beach. (B) Anderson Canal (control) sampling stations (CM: Control month/Grand Canal; C1: Control 1; C2: Control 2). (C) Redwood Canal (aeration) sampling stations (AM: Aeration month/Grand Canal; A1: Aeration 1; C2: Aeration 2).

Phytoplankton collection and laboratory processing

Phytoplankton samples were collected using a water pump that captures water from specified depths (0.5m below the surface and 0.5m above the bottom) at the 6 stations indicated in Figure 2. Three replicates (n=3) were taken at each sampling depth at each station. Each phytoplankton sample were split into two subsamples.

One subsample (30 ml) were filtered with 53-µm mesh, kept cool, and analyzed using flow cytometer (BD Accuri C6) within 12 hrs of collection. The other subsample (250 ml) were preserved in 4% formalin pending analysis and identification via compound microscopy. Those samples were concentrated using the sedimentation technique described in Standard Operating Procedure for

Phytoplankton Analysis (Grace Analytical Lab 1994). The sedimentation process was conducted in 24 cm graduated cylinders and took 6 days. After cells have settled, overlying water were carefully decanted using a syringe. Concentrated samples were counted using a graduated Sedgewick Rafter counting chamber until

≥ 300 cells were counted. Phytoplankton were identified to the lowest possible taxonomic level. Flow cytometer (Accuri C6) can differentiate nano-phytoplankton into groups according to their pigment contents and algal sizes, and thereby provide algal densities for each group. Counting via compound light microscopy was used to determine species present and algal densities of micro-phytoplankton.

Benthic infauna collection and laboratory processing

Benthic infauna were collected randomly around each station’s coordinates with a Wildco Petite Ponar Grab (grab area 225 cm2) (McGee et al. 1993; Poirrier et al.

2008). Three replicates (n=3) were taken at each sampling location. Total sediment grab volumes were measured to enable calculations of grab penetration depth.

Sediments were sieved through a 500 µm mesh sieve (Soares and Sobral 2009) and retained organisms were frozen for lab analysis. Aliquots of each infauna sample

(1/4 or 1/8) were identified and counted via stereomicroscopy (8-35× magnification). Organisms were identified to the lowest possible taxonomic level and benthic densities determined.

Statistical analysis

All statistical analyses were performed using RStudio. For key species, mean densities were compared temporally and spatially using ANOVA. A one-way analysis of similarities (ANOSIM, α=0.05) based on Bray-Curtis similarities were used to detect differences in assemblages among different sites and between before and after aerations. A similarity percentage analysis (SIMPER) was used to determine which species contributed most to those differences.

3.3 Results

Table 3.1 List of plankton species found in the canals.

52 species/groups of micro-phytoplankton and micro-zooplankton were found in the canals during the sampling year and most of the micro-phytoplankton species were dinoflagellates (Table 3.1). 21 species/groups of nanophytoplankton were found in the canals with 11 groups of cyanobacteria (Table 3.1). Aureoumbra lagunensis, notoriously referred to as “brown tide”, bloomed from February 2018 to June 2018. The rest of the nanophytoplankton groups were unidentified prokaryotes (Table 3.1).

Figure 3.3 Two-dimensional nMDS ordination plots of planktonic assemblages in aerated and control canals (both surface and bottom water) in the months August 2017-June 2018.

Canal water columns were highly stratified during the summer, fall and spring, as evidenced by the distinct assemblages near the surface vs. near the bottom (Fig

3.3, Table 3.2). The dissimilarities between surface and bottom waters mainly came from nano-sized cyanobacteria, nano-sized phytoplankton and micro-sized dinoflagellates (Table 3.2). Water columns were more mixed up during winter and spring (Fig 3.3, Table 3.2). There was no significant dissimilarity observed between surface and bottom water communities during the cold period (R < 0.25) (Table

3.2).

Figure 3.4 Two-dimensional nMDS ordination plots of planktonic assemblages in surface water over the year-long study period. Treatments are canal location, with aeration, control and Grand Canal represented by shape and dates represented by color.

Figure 3.5 Two-dimensional nMDS ordination plots of planktonic assemblages in Bottom water over the year-long study period. Treatments are canal location, with aeration, control and Grand Canal represented by shape and dates represented by color.

Surface water planktonic communities were different among different months.

Surface water communities were more spread out in summer and fall and converged in winter and spring (Fig 3.4). The dissimilarities among different months mainly came from nano-sized cyanobacteria and nano-sized phytoplankton

(Table 3.3). Same patterns were found in the bottom water communities (Fig 3.5 and Table 3.4).

Figure 3.6 A) Microphytoplankton B) Nanophytoplankton densities from surface vs. bottom waters from each canal in different months.

Nanophytoplankton dominated the water columns all the year round in all sampling sites (Fig 3.6B). During the non-bloom period, the densities of nanophytoplankton were around 2 magnitudes higher than the densities of microphytoplankton, and were 4-5 magnitudes higher during the bloom period (Fig

3.6). The densities of phytoplankton from surface and bottom waters were consistent in most of the sampling months, but more microphytoplankton were found in the surface waters in September 2017 (Fig 3.6A) and more nanophytoplankton were observed in the surface waters during the bloom fading period (April to June, 2018) (Fig 3.6B). The main difference between aeration canal and control canal was around 47% and 40% less cell counts for nanophytoplankton in surface aeration canal in April and June 2018, respectively

(Fig 3.6B).

Figure 3.7 Surface water micro-phytoplankton composition in the Grand Canal (CM, AM), the Control Canal (C1, C2) and the Aeration Canal (A1, A2) in different months.

Figure 3.8 Bottom water micro-phytoplankton composition in the Grand Canal (CM, AM), the Control Canal (C1, C2) and the Aeration Canal (A1, A2) in different months.

For microphytoplankton communities, Dinoflagellates were abundant both in the surface (Fig 3.7) and bottom (Fig 3.8) waters throughout the sampling year.

Diatoms grew better during the winter time (Fig 3.7 and Fig 3.8), and they took larger proportion in bottom waters compare to the surface waters at the same sampling location in the same month. Euglenoids appeared throughout the sampling year (Fig 3.7 and Fig 3.8). Microphytoplankton compositions in surface aeration canal were similar to surface Grand Canal and surface control canal (Fig

3.7), but bottom aeration canal was more consistent with bottom Grand Canal after the aeration (Fig 3.8).

Figure 3.9 Surface water nanophytoplankton composition in the Grand Canal (CM, AM), the Control Canal (C1, C2) and the Aeration Canal (A1, A2) in different months.

Figure 3.10 Bottom water Nano-phytoplankton composition in the Grand Canal (CM, AM), the Control Canal and the Aeration Canal in different months.

For nanophytoplankton communities, cyanobacteria dominated both the surface

(Fig 3.9) and bottom (Fig 3.10) waters from August 2017 to November 2017, then

Aureoumbra lagunensis bloomed and dominated the water column from February

2018 to June 2018 (Fig 3.9 and Fig 3.10). A. lagunensis abundance reached the maximum in February 2018 and decreased in April and June 2018, while nanocyanobacteria and other non-cyanobaterial nanophytoplankton increased during the brown tide fading period (Fig 3.9 and Fig 3.10). Nanophytoplankton compositions in surface aeration canal were not different from surface Grand Canal and surface control canal (Fig 3.9), but bottom aeration canal was more consistent with bottom Grand Canal after the aeration (Fig 3.10).

Figure 3.11 Aureoumbra lagunensis cell densities in surface vs. bottom waters at different sampling sites in A) February 2018, B) April 2018 and C) June 2018. Different letters identify significantly different means based on analysis of variance and Tukey’s post-hoc pairwise comparisons (α = 0.05).

Figure 3.12 Ciliates and phytoplankton density in A) surface water and B) bottom water in different months.

A “brown tide” (Aureoumbra lagunensis) was observed during the sampling year from February 2018 to June 2018. The cell counts for A. lagunensis reached extreme values of 3.5 × 106 cells ml-1 in February 2018, and continuously decreased in abundance by a factor of 10 through June 2018 (Fig 3.11). The densities of A. lagunensis from surface and bottom waters were consistent in February 2018 (Fig

3.11A), but more of A. lagunensis was found in the surface waters at C2, C1 and

CM in April 2018 (Fig 3.11B) and all sites in June 2018 (Fig 3.11C). The difference between the aeration canal and control canal was around 35% and 50% lower cell counts for A. lagunensis in surface aeration canal during April and June

2018, respectively (Fig 3.11B and C). During the bloom period, ciliates density increased dramatically from 10 to 100 individuals ml-1(Fig 3.12).

Table 3.5 Benthic infauna species composition and abundance (mean densities) at each sampling location in different months.

Benthic infauna only appeared from late fall to early spring (Table 3.5). Benthic infauna was more abundant and diverse in aeration canal than control canal in the winter time (Table 3.5).

3.4 Discussion

This study of biological responses to muck aeration was conducted in parallel to a geochemical evaluation of muck aeration (Trefry and Fox 2019). Environmental data/samples discussed in this chapter were collected simultaneously as the biological samples. Those environmental data included both water quality and sediment quality parameters (Trefry and Fox 2019).

The average algal density of phytoplankton in the canals was 5 × 104 cell ml-1 (20

µg L-1 chlorophyll a) during the non-bloom period and reached the maximum of

3.5 × 106 cell ml-1 (180 µg L-1 chlorophyll a) during the brown tide. The concentration of chlorophyll a and cell counts are comparable with other eutrophic estuary systems, especially in a bloom period (Glibert et al. 2010). The Grand

Canal system is a highly-polluted shallow brackish waterway with limited water exchange though the small outlets connected to the Indian River Lagoon (IRL).

With high water column concentrations of total dissolved nitrogen (40 - 400 µg N

L-1) and phosphorus (16 – 59 µg P L-1), high phytoplankton cell counts and chlorophyll a concentrations are to be expected.

Nanophytoplankton dominated the water columns all the year round in all sampling sites (Fig 3.3.6) and the densities were 2-5 orders of magnitude higher than the densities of microphytoplankton during the sampling year. Although, studies suggested that small phytoplankton (pico- and nano- size ranges) are more indicative of oligotrophic open ocean ecosystems than nutrient-enriched coastal waters (Malone 1980; Takahashi and Bienfang 1983; Marañón et al. 2001;

Anabalón et al. 2016), more and more nanophytoplankton-dominated eutrophic estuaries have also been reported in recent years (Gin et al. 2000; Li et al. 2013; Fu et al. 2016). In particular, nanophytoplankton have bloomed frequently in the IRL in the recent years (St. Johns River Water Management District 2012), and Phlips et al. (2015) suggested that extreme climatic conditions tend to promote the growth of those nanophytoplankton species.

During the non-bloom period, cyanobacteria and dinoflagellates dominated the nanophytoplankton (Fig 3.9 and Fig 3.10) and microphytoplankton (Fig 3.7 and Fig

3.8) communities, respectively. Cyanobacteria must compete for low concentrations of inorganic phosphate (DIP) and possess adaptations for acquiring organic phosphorus compounds (Davis et al. 2010; O’neil et al. 2012). They display flexibility in the nitrogen sources they can exploit to form blooms (Glibert et al. 2004; O’neil et al. 2012). The highest concentrations of cyanobacteria are usually associated with eutrophication (Glibert and Burkholder 2006; O’neil et al.

2012). Dinoflagellates and their cysts can also be abundant in eutrophic conditions

(Dale 2001; Anderson et al. 2002; Bužančić et al. 2016). A large fraction of

dinoflagellates are mixotrophic, combining photosynthesis with ingestion of organic particles (phagotrophy) (Stoecker 1999). By mixing sources of energy and carbon, switching their nutritional strategies between photosynthesis and phagotrophy according to light availability, some species can avoid light- dependence (Skovgaard 2000). The waters in residential canals contain high concentrations of suspended solids with Secchi depths as shallow as 1 m during the non-bloom period – and only 30 cm during a strong bloom. Dinoflagellates are competitive in this turbid environment. Aided by their relatively larger size, motility and thick cell-wall features, dinoflagellates can avoid many would-be grazers (Graham et al. 2008). Diatoms were more abundant in the bottom waters compare to surface. It is because most of diatoms found in the canal waters are pennate diatoms and many pennates are benthic species (Leynaert et al. 2018), it is possible that those diatoms attached to the particles in the bottom waters and were collected through the water pump. Another abundant group, the euglenoids, are often used as indicators of eutrophic or polluted conditions (Singh et al. 2013). Like dinoflagellates, euglenoids can be phagotrophic and have strong motile abilities

(Graham et al. 2008). Cyanobacteria, dinoflagellates and euglenoids were relatively more abundant during warm periods, while diatoms were more abundant during cold periods (Figure 7-10). This pattern is consistent with observed phytoplankton succession cycles in the IRL (Phlips et al. 2010), and other estuaries (Domingues et al. 2005; Barbosa et al. 2010).

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A “brown tide” (Aureoumbra lagunensis) was observed during the sampling year, with cell counts reaching extreme values of 3.5 × 106 cells ml-1 in February

2018, and decreasing in abundance by a factor of 10 through June 2018 (Fig 3.11).

A. lagunensis has bloomed in the IRL frequently in recent years (Sullivan 2017;

Walters et al. 2017). Studies suggested that A. lagunensis is well adapted to warm hypersaline environments with salinities over 40 and temperature between 25 and

30 ℃(Rhudy et al. 1999; Liu and Buskey 2000). However, salinity and temperature ranges recorded during this bloom were 15 to 20 and 20 to 25℃, respectively. A. lagunensis can form blooms in a wide range of environmental conditions. This bloom may have been triggered by an increase of dissolved inorganic nitrogen and phosphorus following Hurricane Irma and subsequent rainfalls in October and

November 2017. Concentration of ammonium combined with nitrite+nitrate peaked at > 200 µg N L-1 in November and December and phosphate reached its highest value around 50 - 60 µg P L-1 in December in both canals. Coincident with the bloom was near-complete removal of dissolved inorganic nitrogen and a sharp increase in dissolved organic nitrogen in May and June 2018 in both canals. Since

A. lagunensis are able to use organic nitrogen and phosphorus (Rhudy et al. 1999;

Liu and Buskey 2000), the bloom continued until June 2018. Ong et al. (2010) observed that A. lagunensis can maintain high growth rates under low light conditions. Therefore, the highest cell density was recorded in February 2018 when

Secchi depth was only 30 cm. Observed phytoplankton differences between the aeration and control canals were 35% and 50% lower cell counts for A. lagunensis

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in aeration canal surface water during April and June 2018, respectively (Fig 3.11B and C). This difference could be due to the oxygenated water in the aeration canal where organic nitrogen has been decomposed to inorganic forms. A. lagunensis grows preferentially, however, on reduced forms of nitrogen (Muhlstein and

Villareal 2007; Kang et al. 2015) and aeration could potentially help to weaken the brown tide.

Planktonic communities in surface and bottom waters were quite different during the summer, fall and spring (Fig 3.3, Table 3.2). Planktonic communities from surface waters were more similar to surface communities from other sites, even in the opposing canal, rather than bottom water communities from the same site. This was also true for the bottom water planktonic communities. Canal water columns were more mixed during winter and spring (Fig 3.3, Table 3.2). The canals are shallow waterways, with average depths of 2.5 m. Temperatures were uniform between the surface and bottom of all sampling stations, while salinities and ammonium concentrations were higher in bottom water, and light intensities and nitrite+nitrate concentrations decreased from surface to bottom. Thus, the dissimilarities of planktonic communities between the surface and bottom during the warm period could be due to these environmental factors. For example, more live cells were observed in the surface waters where maximum sunlight was available, but more dormant phytoplankton cysts were found in bottom waters.

Distinct planktonic communities developed through time (Fig 3.4 and Fig 3.5), and communities from aeration and control canals shifted together. These changes

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resulted from seasonal shifts, rather than the aeration. Temperature, salinity and nutrients concentrations changed month to month, but were generally indistinguishable when comparing between the aeration and control canals.

Dissolved organic nitrogen and ammonium concentrations in the aeration canal were lower compared to the control canal from April to June 2018, and less

Aureoumbra lagunensis were recorded in surface waters during that time.

However, this difference was relatively small when compared to the seasonal shifts of the whole planktonic community.

During the bloom period, ciliate densities increased dramatically (Fig 3.12), reaching a maximum right after the peak of the Aureoumbra lagunensis.

Nanophyotoplankton are generally grazed by microzooplankton (Hansen et al.

1994; Calbet et al. 2003; Liu and Dagg 2003; Calbet and Landry 2004) and ciliates could be an important top-down control on brown tide. However, a study has shown that the thick mucus layer of exopolymer secretions (EPS) produced by A. lagunensis during hypersaline conditions may impair microzooplankton grazing

(Liu and Buskey 2000). The large increase of ciliate density during the bloom could due to weak EPS production from A. lagunensis at lower salinities (15-20 during the bloom).

During the warm summer period, waters at depths >1.5 m in both canals were anoxic and no benthic infauna were observed (Table 3.5). During the colder months

(November 2017 to February 2018), however, benthic infauna were more abundant and diverse in the aeration canal compared to the control canal (Table 3.5). Studies

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have shown that sediment qualities (silt-clay content, organic matter and porosity) are key factors of benthic infauna communities (Howarth et al. 2011; Lawless and

Seitz 2014; Robertson et al. 2015). Muck depth and sediment qualities were similar regardless of season and aeration, making it unlikely that benthic infauna community differences in colder months were due to sediment conditions. The aerated treatment stations, however, did experience higher bottom water dissolved oxygen and lower sediment ammonium flux. This could have provided a more oxygenated, and less toxic, environment for benthic infauna. This finding is consistent with the study that oxygen level, rather than chemical pollutants, controls the abundance of benthic infauna (Guerra-Garcia and García-Gómez

2005). Aeration oxygenated the bottom waters and facilitated colonization by benthic infauna during the cold months, but it could not overcome warm water column hypoxia in the summer. Benthic infauna found in canals are the species have adapted to polluted or otherwise harsh environments. For instance, Capitella capitata tolerates sewage-impacted sites (Sánchez et al. 2013); polychaete annelids such as Ctenodrilus serratus and Capitella capitata usually have high tolerance of heavy metal pollutants (Reish and Carr 1987); Mulinia lateralis is an opportunistic bivalve species which is known to have invaded rapidly after anoxic events occurred in a polluted estuary (Oviatt et al. 1984). Leptochelia dubia and Capitella capitata were found in aliphatic hydrocarbon polluted area (Guerra-García et al.

2003).

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In conclusion, the aeration process facilitated benthic animal life during the cold period of the study. Water column mixing and the increased capacity of water to hold dissolved gasses would have assisted the aeration process in enhancing dissolved oxygen. In contrast, aeration did not significantly change the planktonic community relative to the control canal. There were marked differences in near- surface and near-bottom planktonic communities, and these were consistent between locations, regardless of aeration or lack thereof.

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Conclusion

The overall health of the Indian River Lagoon (IRL) has being challenged by frequent Harmful Algal Blooms (HABs) and accumulation of fine-grained organic- rich sediments (referred to as “muck”) on the benthos in recent years. This dissertation answered two questions: first, whether the key herbivorous mesozooplankton in the IRL have the capacity to exert top-down control on the

HAB species, and second, whether the aeration is an effective restoration approach to solve the muck problem in the IRL. For the first question, grazing experiments revealed that Parvocalanus crassirostris, the dominant herbivorous copepod in the

IRL, exerted some top-down control on the HAB species under laboratory conditions (Chapter 1). Additional laboratory grazing experiments on cell size and mucus effects were examined to better understand the low grazing rates. The results showed that small cell sizes and mucilage production are possible reasons of low grazing rates on HABs (Chapter 2). For the second question, estuarine ecosystems were monitored throughout a pilot aeration experiment to evaluate the feasibility and effectiveness of aeration for muck mitigation (Chapter 3). It was found that aeration facilitated benthic animal life, but only in the cold period when bottom waters held the most dissolved oxygen. Planktonic communities, on the other hand, displayed no significant responses to aeration.

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