For over a decade, the northern Indian River Lagoon (IRL) and its sub-regions, the north IRL, Banana River Lagoon, and Mosquito Lagoon have suffered marked ecological impacts as a result of recurring harmful algal blooms. Coincident with repeated blooms of the pelagophyte Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson (DeYoe et al., 1997), first observed in Mosquito Lagoon and the north IRL in 2012 (Gobler et al., 2013), have been widespread loss of seagrasses and drift macroalgae (Phlips et al., 2021), reduced larval recruitment of oysters (Walters et al., 2021), bivalve (Gobler and Sunda, 2012) and fish (Gobler et al., 2013) kills, and unusual mortality events of marine mammals (Florida Fish & Wildlife Conservation Commission, 2021) and birds (Lapointe et al., 2015).

This brown-tide forming pelagophyte is now considered a dominant constituent of the IRL phytoplankton community despite its relatively recent introduction to Florida coastal waters, with significant blooms observed in 2012, 2013, 2015, 2016, and 2018 (Phlips et al., 2019).

In response to eutrophication and loss of filter-feeders world-wide, numerous studies have investigated the ability of oysters, tunicates, and other grazers to attenuate nutrients (e.g., Cerco and Noel, 2007; Buzzelli et al., 2013; Kellogg et al., 2013; Bricker et al., 2018), bacteria (e.g., Draughon et al., 2008) and affect top-down control on bloom-forming phytoplankton (including brown tide, e.g., Galimany et al., 2017; Galimany et al., 2020; Galimany et al., 2021). While considered non-toxic, brown tides formed by Aureoumbra lagunensis, and another pelagophyte Aureococcus anophagefferens Hargraves et Sieburth (DeYoe et al., 1997), affect bivalve and zooplankton feeding due to their cellular structure (e.g., mucilaginous secretions), accumulated biomass, or a combination of these factors (Anderson et al., 2002). The exopolymeric secretions (EPS) of these brown tide species interfered with the ciliary beating required for movement of captured particles to the gut in bivalves (Gainey and Shumway, 1991; Robbins et al., 2010) and adhered to cilia on the surface of protozoa, affecting both motility and feeding (Liu and Buskey, 2000). Even when ingested, the EPS may shield cells from digestion (Bersano et al., 2002). Evidence for feeding and growth inhibition in the hard clam (Mercenaria mercenaria) and the blue mussel (Mytilus edulis) to different strains of Aureococcus anophagefferens was observed by Bricelj et al. (2001).

Several studies suggest top-down control of brown tide may be possible in the IRL. Bricelj et al. (2001) found that juvenile hard clams had normal feeding exposed to Aureococcus anophagefferens at cell densities < 3.5 x 10⁴ cells mL^-1, although clearance rates were depressed above this threshold. Eastern oysters (Crassostrea virginica) from the IRL had decreased clearance rates (from 2.19 to ≤ 0.12 L hr⁻¹ g⁻¹) when exposed to A. lagunensis (mean 3.26 × 10⁶ ± 0.20 × 10⁶ (SE) cells ml⁻¹), but absorption and efficiency was comparable to non-bloom conditions suggesting oysters could maintain their feeding requirements on brown tide (Galimany et al., 2017). Feeding experiments conducted on bivalve species widely distributed in the IRL include Eastern oysters, hooked mussels (Ischadium recurvum), charru mussels (Mytella charruana), green mussels (Perna viridis), Atlantic rangia (Rangia cuneata), and hard clams. These species were exposed to isotopically (¹⁵N) labeled microalgae, including Aureoumbra lagunensis and other picoplanktonic bloom-forming species isolated from the 2011 IRL “superbloom” (Galimany et al., 2020). While clearance rates differed among bivalves and microalgal treatments, bivalve tissues indicated assimilation of ¹⁵N-enriched bloom-forming microalgae, indicating the importance of a biodiverse community of filter-feeders and their potential to force top-down control of phytoplankton in the IRL (Galimany et al., 2020).

Another filter-feeder found ubiquitously in the IRL is the pleated tunicate, Styela plicata (Lesueur). Styela plicata is a solitary ascidian and one of the most introduced tunicate species worldwide (Baker et al., 2004; Pineda et al., 2013). There is no consensus regarding its native range although it is generally thought to be from the northwest Pacific (Carlton and Ruckelshaus, 1997; Lambert and Lambert, 1998; Lambert, 2001; Pineda et al., 2011; Pineda et al., 2013). Bingham (1992) and Carlton and Ruckelshaus (1997) list S. plicata as an introduced species in Florida. It is widespread and abundant in the IRL near inlets and the interior of the lagoon (Mook, 1983).

Tunicate suspension feeding is characterized by pumping water via movement of cilia along the stigmatal openings of a branchial basket lining the pharynx, through the inhalant siphon into the pharynx (Petersen, 2007). An endostyle continuously produces a mucus net, whereby captured particles are rolled into a food string and carried to the oesophagus (Petersen, 2007). There is no evidence of particle selection once taken into the pharynx: any particles entering the pharynx can be captured by the mucus net, and experimental studies indicate no difference between particles in suspension and those ingested, except for very large particles or their own eggs and larvae (Randløv and Riisgård, 1979; Robbins, 1983; Klumpp, 1984; Stuart and Klumpp, 1984; Bingham and Walters, 1989; Ribes et al., 1998). Particle selection can occur at the inhalant siphon aperture where specially arranged tentacles prevent larger particles from entering the pharynx. Regulation of suspension feeding in ascidians is mediated by temperature as well as gut fullness (Petersen et al., 1999; Petersen, 2007).

The present study aimed to determine if the pleated tunicate Styela plicata contributes to top-down control of brown tide in the IRL. Feeding experiments were conducted at varying cell densities of Aureoumbra lagunensis compared to a control microalga. Additionally, a feeding experiment using Aureococcus anophagefferens was conducted. Although not yet observed in Florida, Aureococcus anophagefferens blooms may be a future threat to the IRL, as it has spread to other estuaries along the Atlantic coast of the United States by similar transport mechanisms as Aureoumbra lagunensis (e.g., ship bilge water).

The present study examined the filtration rate of the pleated tunicate Styela plicata fed varying cell densities of either Aureoumbra lagunensis or Aureococcus anophagefferens, brown tide-forming pelagophytes. All experiments included a control in which tunicates were fed Tisochrysis lutea Bendif et Probert (previously classified as Isochrysis sp. [clone T-Iso]; Bendif et al., 2013), a microalga regularly used as bivalve feed in aquaculture facilities and feeding experiments, which is of comparable cell size (4–6 μm); and a negative control that included replicate treatment beakers with the microalgae, but no tunicate, which accounted for sedimentation or adherence of cells to the beaker walls. Experiments were conducted in the aquaculture laboratory at Harbor Branch Oceanographic Institute (HBOI), Florida Atlantic University, Fort Pierce, Florida between July 2014 and July 2015.

Although the pleated tunicate Styela plicata had a significant decline in filtration rate (FR) when exposed to Aureoumbra lagunensis for five hours, these filter-feeders had comparable FR to the control when exposed for 24 hrs, effectively filtering brown tide cells across several cell densities. Tunicate size did not affect FR across all experiments. For example, in Experiment 1 the tunicate with the greatest mean FR exposed to A. lagunensis (542 mL hr^-1) weighed 33 grams (g) whereas the largest tunicate (45.4 g) had a mean FR of only 52 mL hr^-1. The tunicate with the greatest mean FR exposed to Tisochrysis lutea (2975 mL hr^-1) weighed 16.8 g whereas the largest tunicate (51.2 g) had a mean FR of 2094 mL hr^-1. These results are consistent with Draughon et al. (2010) who found there was no relationship between average hourly filtration rate and whole animal weight (R² = 0.0001) or dry organ weight (R² = 0.0067) of S. plicata fed either the bacteria Escherichia coli or the microalgae Nannochloropsis sp. They recommended that FR for the pleated tunicate not be reported based on live or dry weight. In the present study, mean FR of S. plicata fed T. lutea (the control) at 5.0 x 10⁵ cells mL^-1 across the three experiments often exceeded 2000 mL hr^-1 animal^-1. Laboratory measurements by Draughon et al. (2010) of S. plicata FR exposed to Nannochloropsis sp. or E. coli at 10⁵ cells mL⁻¹ filtered 3065 mL hr⁻¹ animal⁻¹ ( ± 1284 mL hr⁻¹ s.d.) and 4654 mL hr⁻¹ animal⁻¹ (± 810 mL hr⁻¹ s.d.), respectively.

The present study did not employ the use of stable isotopes to determine whether tunicates were assimilating nutrients of consumed particles, although feces were produced by tunicates which indicated digestion of material. It is expected that tunicates were not feeding at a consistent rate throughout the 24 hour exposure period, as suggested by the standard deviation of mean FR, especially at low concentrations of A. lagunensis (10³ cells mL^-1) ( Figure 5 ). Slowing of ciliary beating and lower filtration rate in tunicates could be a result of cellular characteristics of A. lagunensis (i.e., the EPS). Some pico- and nanoplankton develop surface features to increase their mobility through mucus, effectively reducing capture efficiency by mucus-based filters (Jacobi et al., 2020) such as those in tunicates; however, both pelagophytes used in this study are non-motile so this is not assumed to be an evasive mechanism in Experiment 1 or 2. To strengthen Experiment 3, it should be replicated with hourly water samples and calculations of FR in order to determine treatment-specific feeding behavior. The samples taken only at Hour 0 and Hour 24 obscures feeding behavior and averages out the estimated hourly FR. Further, while the experimental treatments exposing tunicates to pelagophytes at 10⁵ cells mL^-1 is representative of median cell densities observed during the 2012 brown tide bloom in the IRL (ranging 10⁴ and 10⁶ cells mL^-1; Gobler et al., 2013) additional treatments representing peak cell densities of IRL brown tides (> 10⁶ cells mL^-1; Gobler et al., 2013; Phlips et al., 2021) would have contributed valuable information to this study.

The sedimentation controls in Experiment 3 indicate that removal of cells was a result of the presence of tunicate filtration rather than sedimentation; the moderate aeration provided in all beakers allowed the unflagellated, non-motile brown tide cells (DeYoe et al., 1997) to remain in suspension rather than settling. Tunicates may also spontaneously squirt as a method of clearing the branchial basket of waste material or in the case of overloading (Petersen 2007); while not observed, periodic squirting may have contributed to water movement throughout the experimental beakers to prevent settling, or may explain hour-to-hour fluctuations in cell densities, highly variable hourly FR, or instances of calculated negative FR.

There are various methods for measuring and calculating the filter feeding capacity of invertebrate suspension feeders in the lab and in the field which can confound direct comparisons between studies. For example, Fiala-Médioni (1978a) calculated the “pumping rate” (speed of water flow through siphons) of S. plicata as 3.30 cm sec^-1 and filtration rate as 4.9 L h^-1 gm^-1 total dry weight at a of 19-22°C (Fiala-Médioni, 1978a) and between 5.3 and 10.7 cm sec^-1 and 10.5 and 11.5 L h^-1 gm^-1 total dry weight at temperatures 15°C (Fiala-Médioni, 1978b). Sumerel and Finelli (2014) measured both “flow speed” (cm s^-1) and clearance rate (L hr^-1) for S. plicata. Galimany et al. (2017) employed the biodeposition method (Iglesias et al., 1998), a method used in studying bivalve feeding, which uses inorganic matter of water as a tracer of ingestion, egestion, and rejection feeding processes. Use of bivalve clearance of A. lagunensis in their study (in L hr^-1), defined by the volume of seawater passing through the gills per unit of time, may be used for rough comparison with the present study, acknowledging the methodological differences: the oyster Crassostrea virginica had a mean FR measured in situ at sites in the Indian River Lagoon between 2 and 3 L hr^-1; the hard clam Mercenaria mercenaria was less efficient at filtering high seston loads, with mean FR lower at <1 L hr^-1.

The reduction in FR observed in Experiment 1 of the present study was consistent with reductions of FR by bivalves important to the IRL (Bricelj et al., 2001; Gobler et al., 2013). The ecological function of suspension feeding invertebrates is important for maintaining water quality, and thus feeding behavior when exposed to algal blooms is of critical importance. Oysters serve an important role in reef formation and provide heterogeneous habitat for other organisms (Boudreaux et al., 2006). Aureoumbra lagunensis blooms have caused declines in oyster spat recruitment in Mosquito Lagoon, the northern-most segment of the IRL and one of the main subregions most affected by brown tide (Makris, 2016; Walters et al., 2021). Buskey et al. (1997) and Gobler et al. (2013) implicate the role of an extra-cellular polysaccharide sheath surrounding cells of A. lagunensis as cause for the disruption of grazing by filter-feeders. Liu and Buskey (2000) demonstrated that A. lagunensis with thick EPS was not suitable food for the protist Euplotes sp. and other hypotrichous ciliates; the protist Aspidisca sp. had reduced filtration rates and growth rates when exposed to the thick-EPS. Gainey and Shumway (1991) and Bricelj et al. (2001) implicate a dopamine-mimetic bioactive compound associated with the extra-cellular polysaccharide sheath of Aureococcus anophagefferens with the reduction of activity of gill lateral cilia in bivalves necessary to generate movement of water through the gills. In tunicates, cilia are responsible for the movement of trapped particles in a mucus net toward the oesophagus (MacGinitie, 1939; Petersen 1997), so while a similar mechanism may have been operative in the short-term experiments of the present study, identifying the mechanism for feeding inhibition and what explains the comparable (not statistically significant difference of) FR between treatments at 24 hours in Experiment 3 is only hypothesized in the present study and warrants further investigation. While a 24 hour experiment exposing tunicates to different starting cell densities of A. anophagefferens was not conducted, we hypothesize that similar results would be observed.

Despite pelagophyte impacts on suspension feeding behavior, studies indicate that bivalves can feed on A. lagunensis and may be a mechanism for top-down control of blooms in the IRL. Galimany et al. (2020) exposed several IRL bivalves including eastern oysters (C. virginica), hooked mussels (Ischadium recurvum), charru mussels (Mytella charruana), green mussels (Perna viridis), Atlantic rangia (Rangia cuneata), and hard clams (M. mercenaria) to a mixed culture at 3.0 x 10⁴ cells mL^-1 that included ¹⁵N-enriched A. lagunensis. Clearance rates did vary between species, but isotopic labelling of bivalve tissue indicated that all species assimilated brown tide cells. In a follow-on study, eastern oysters, hooked mussels, and hard clams were exposed to isotopically labeled (¹³C and ¹⁵N) A. lagunensis at cell densities of 10⁴ and 10⁶ cells mL^-1. Clearance rates declined with increasing algal density, but bivalves assimilated the greatest number of cells at bloom concentrations (10⁶ cells mL^-1) (Galimany et al., 2021). Interspecific variation in filtration rate indicates the importance of a biodiverse suspension feeding community in the IRL.

A conservative estimate of filtration capacity at lagoon-scale using their laboratory-based measures of filtration rate of the Eastern oyster and hard clam (the most common species targeted for restoration efforts) estimate filtration of 0.002% the volume of the IRL at brown tide concentrations of 10⁴ cells mL^-1. An additional 1% is estimated based on restored oyster reefs found in Mosquito Lagoon only but doesn’t consider the suite of filter feeding organisms throughout the ecosystem (Galimany et al., 2021). The authors caution that laboratory-based estimates are merely a proxy for conditions in the field, but still provide evidence for these nature-based solutions of improving water quality. Draughon et al. (2008) investigated the use of S. plicata as a bioremediator and estimated a population of 200 S. plicata could filter >600 L hr^-1 of algae or bacteria. Styela plicata not only removed E. coli at cell densities of 10⁶ cells mL^-1 but killed all cells, rendering them unable to replicate (Draughon et al., 2010). No published estimates of S. plicata densities in the IRL are available from which extrapolations of total volume of the IRL that could be filtered during a brown tide can be made; however, a simple calculation using mean FR from the present study estimates that 200 S. plicata could filter 117.4 L hr^-1 (or approximately 2,818 L day^-1) of Aureoumbra lagunensis at cell densities exceeding 10⁵ cells mL^-1.

Despite various methodological approaches, numerous studies measuring suspension feeding of invertebrate organisms found in the IRL demonstrate the capacity to exert top-down control of phytoplankton. Suspension-feeding bivalves are capable of efficiently capturing particles as small as 1 μm (Møhlenberg and Riisgård, 1978; Galimany et al., 2020), as can ascidians (Jacobi et al., 2020). Further, Jacobi et al. (2020) found that ascidians could retain particles at submicron sizes (using microspheres and planktonic cells measuring as small as 0.3 μm); although the microspheres were captured at greater efficiencies than phytoplankton of comparable size, possibly due to evasive characteristics of phytoplankton. In their study, S. plicata had a capture efficiency rate of >25% of Prochlorococcus sp., which has a cell diameter of ~0.6 μm; and a capture efficiency rate of >50% of Synechococcus sp. and another eukaryotic microalgae “NanoEuk,” which had median cell diameters of ~1.05 μm, and ~6 μm, respectively (Jacobi et al., 2020). Galimany et al. (2020) found that bivalves in their study could also consume and assimilate Synechococcus sp. (although their strain was larger at 5.74 ± 07 μm diameter) but resulted in lower clearance rates. Other physical or chemical surface properties of Synechococcus sp. may explain its low preference by bivalves (Rosa et al., 2017).

The lack of any known particle selection mechanism in ascidians, and their ability to capture and retain sub-micron sized particles, warrants additional investigation and direct comparisons of filtration and assimilation of bloom-forming phytoplankton with that of bivalves present in the IRL. Styela plicata and other tunicates are ubiquitous in the IRL and likely contribute to improvements in water quality. The present study adds additional support that top-down control by extant or restored populations of a diverse suite of suspension feeding organisms could be used as one tool in the “restoration tool box.”

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

PK conceived of and conducted experiments as part of her Master of Science Thesis at Florida Atlantic University. JS and JH served as committee members and HBOI Summer Internship advisor and major professor, respectively. JS provided key training and guidance on use of lab and aquaculture facilities, as well as procurement of algal specimens and manuscript review. PK took the lead on design and execution of the experiments, data analysis, and writing the manuscript. All authors contributed to the article and approved the submitted version.

This project was funded by the Link Foundation and Harbor Branch Foundation as part of the Harbor Branch Oceanographic Institute Summer Internship, and Harbor Branch Graduate Research Fellowship, respectively. The authors acknowledge and thank the IRL Council, host of the Indian River Lagoon National Estuary Program (IRLNEP), and the U.S. EPA for providing encouragement and funding to support publication of this manuscript. The funding was provided through a EPA Region 4 supplemental funding grant to the IRLNEP.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.