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 loads of nutrients (Smayda, 2008), whereas important local influences on the magnitude and composition of HABs include longevity, toxicity, and differential losses due to grazing (Turner, 2006, 2010; Smayda, 2008; Phlips et al., 2010, 2015; Berry et al., 2015; Cao et al., 2017).

An unprecedented sequence of intense and long-lasting algal blooms have afflicted the Indian River Lagoon (IRL) in Florida, starting in 2011 (Phlips et al., 2010, 2011, 2015; Lapointe 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 (2–4 × the concentrations recorded from 1996 to 2010). In contrast to blooms dominated by dinoflagellates or diatoms (Phlips et al., 2015), the superbloom was co-dominated by cyanobacteria and a nanoplanktonic chlorophyte. Both taxa were documented at > 1 million cells ml^–1 during the event (IRL Consortium, 2015). The bloom caused harm by decreasing light penetration 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 densities of the species in the superbloom led to questions about top-down control by herbivores like zooplankton.

The alga targeted in this study was an unresolved prochlorophyte in the Family Prochlorothricaceae, which was isolated from the 2011 superbloom. The prochlorophyta are a group of cyanobacteria abundant in nutrient-poor tropical waters and possessing a light-harvesting apparatus composed of the higher plant pigments chlorophylls a and b (Partensky et al., 1993; Post and Bullerjahn, 1994; Lewin, 2002). Studies of prochlorophytes are sparse, but no toxic species have been reported. The strain used in this study was an oval-shaped unicellular cyanobacterium, with cells approximately ∼5 μm in diameter. Heavy mucus production was observed during the bloom and in monocultures of the prochlorophyte. During the superbloom, concentrations of the prochlorophyte exceeded 5 × 10⁶ cells ml^–1. This exceptionally high density is unusual for a natural bloom, even a HAB, and it raises concerns about how such densities might impact grazers. Grazing on the prochlorophyte was evaluated to determine the potential for a key herbivore to exert top-down control. Grazing on Isochrysis galbana, a palatable microalga, was examined to contrast with grazing on the prochlorophyte. I. galbana is roughly spherical, with a diameter of 4–6 μm, and it is commonly used as a food for culturing copepods (Shields et al., 2005).

The grazer selected for this study is Parvocalanus crassirostris, a small (∼2 mm long), holoplanktonic, calanoid copepod that dwells 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). It is common for calanoid copepods to reside permanently in the mid- to upper water column (Ma and Johnson, 2017), where they feed on microalgae and nanoplankton. P. crassirostris is a cosmopolitan copepod and tolerates a wide range of temperatures (15–31°C), 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 3-year record of mesozooplanktonic abundances, it was the dominant herbivorous copepod in the region of the aforementioned superbloom (Sweat unpublished data).

This study aims to (1) determine the grazing rates of P. crassirostris on the prochlorophyte, (2) compare grazing rates on the HAB species with those on a palatable alternative (I. galbana), and (3) examine selective grazing when the HAB species is presented together with the palatable alternative at equal densities. Additionally, when it was discovered that the prochlorophyte produced heavy mucilage, altering culture media, this study investigated potential impact of that mucilage on copepod grazing rates and mortality.

When copepods were fed the prochlorophyte, some mortality was observed (Figure 1). Copepod mortality rates (less than 20%) were similar between less dense monocultures of the prochlorophyte (less than 2.3 × 10⁵ cells ml^–1) and mixed treatments (Figure 1). However, mortality rates were over 40% in monocultures containing the prochlorophyte at high densities (≥4.8 × 10⁵ cells ml^–1), and the rates increased substantially with densities ≥ 4.8 × 10⁵ cells ml^–1 (Figure 1A). Copepod mortality was not observed in monocultures of I. galbana.

Copepods grazed on the prochlorophyte in treatments with ≤ 4.8 × 10⁵ cells ml^–1; grazing rates on the denser prochlorophyte at treatments of 9.6 × 10⁵ and 1.9 × 10⁶ cells ml^–1 were indistinguishable from zero (Figure 2A). Compared to grazing rates on monocultures of I. galbana, rates on the prochlorophyte were significantly lower in treatments with ≥ 2.3 × 10⁵ cells ml^–1 (p < 0.05) (Figure 2A). Again, water that contained the prochlorophyte was more viscous than filtered seawater alone due to the production of mucilage.

In experiments with mixed cultures, grazing on I. galbana was markedly reduced compared to grazing recorded in monocultures (Figure 2), with grazing rates on I. galbana decreasing by 80% in the highest density mixed treatment as compared to the monoculture of I. galbana at the same density. In mixed cultures, the grazing rates on I. galbana were statistically indistinguishable from those on the prochlorophyte (Figure 2B). Conversely, grazing rates on the prochlorophyte in mixed cultures were equal to or higher than rates on the same species in monoculture (Figure 2). This was especially true for the highest algal density treatments (≥9.6 × 10⁵ cells ml^–1).

When exposed to I. galbana in PVP treated water, copepod mortality increased at higher viscosities (Figure 3A). Copepods grazed on I. galbana in low viscosity treatments, but grazing rates declined significantly with increasing viscosity, dropping from 6.8 × 10³ cells copepod^–1 h^–1 to zero (Figure 3B).

Top-down control via grazing has been shown to vary with the characteristics of grazers, their algal food, and environmental conditions. Our experiments involved consistent environmental conditions and life stage (adult) 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). Our focus was on characteristics of phytoplankton, including cell density, size, and apparent palatability (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 grazing by P. crassirostris, regardless of the species of algae being offered or whether they were offered as monocultures or mixed cultures containing the prochlorophyte. Mean grazing rates on I. galbana in monocultures ranged from 1.1 × 10³ to 1.8 × 10⁴ cells copepod^–1 h^–1, and are similar to rates reported for P. crassirostris feeding on other 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 typically generate small-scale currents to entrain particles, which subsequently are trapped by feeding appendages (Koehl and Strickler, 1981). It follows that encounter rates and the resulting grazing rates would, to some degree, depend on algal densities, provided there were no other constraints. Given that other studies have also shown correlations of grazing rates with higher algal densities (Frost, 1972), that general pattern does not appear to have been altered by experimental conditions, neither for I. galbana nor the prochlorophyte.

Consumption of the prochlorophyte presented as a monoculture or mixed with I. galbana varied from 0 to 3.0 × 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 in monocultures of I. galbana, especially at algal densities of 9.6 × 10⁵–1.9 × 10⁶ cells ml^–1. Grazing on I. galbana in mixed cultures also was depressed, which in combination with the relatively consistent rate of grazing on the prochlorophyte in all cultures, suggests that copepods did not select prey based on cell taste.

Copepod feeding mechanics are predominantly governed by viscous forces (Koehl and Strickler, 1981; 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.” Mucilage produced by the prochlorophyte in this study may have reduced grazing rates. Mucilage was unlikely to have been an artifact of containment, because cyanobacteria have been shown to produce it in natural assemblages found in Florida Bay (Berry et al., 2015) and off the coast of Venezuela (Ramos and Reyes-Vazquez, 1990). In previous work, polymeric exudates (a.k.a. mucilage) reduced copepod grazing rates, either via chemical inhibition or mechanical interference with filtration or particle capture (Malej and Harris, 1993; Dilling et al., 1998; Nejstgaard et al., 2007). The effects of mucilage were most obvious above a threshold cell density, because copepods grazed on low densities of the prochlorophyte at rates that were not significantly different from grazing on 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 the prochlorophyte in higher density mixed cultures could have been due to actual densities of cyanobacteria cells being half of those in monocultures. In general, mucilage may have interfered with handling of cells, with the increased viscosity rendering the delicate work of particle sorting and selection more difficult. The diameters of I. galbana and the prochlorophyte were similar (∼ 5 μm), making sorting based solely on size challenging. In addition, the experiments involving PVP indicated that viscosity had a substantial influence on P. crassirostris grazing rates. Copepods grazed effectively on I. galbana in low viscosity treatments, but grazing rates fell with increasing viscosity (Figure 3B). Feeding behaviors of suspension feeders have been shown to be influenced by water viscosity (Podolsky, 1994; Bolton and Havenhand, 1998). In fact, Podolsky (1994) found that high viscosities cut grazing by sand dollar larvae in half, and higher viscosities shifted ingestion to larger particles. “Brown tide,” Aureoumbra lagunensis, is another harmful algal bloom species that secretes extracellular polymeric substances (EPS), doing so mainly under hypersaline conditions (Liu and Buskey, 2000). Some species of protozoa had difficulty feeding on high densities of A. lagunensis, but they grew faster on cells separated from their EPS exudates via gentle centrifugation (Liu and Buskey, 2000). Given these reports and our results, it seems likely that depressed grazing reflects a natural consequence of increased viscosity.

Mortality was observed when P. crassirostris was fed the prochlorophyte, especially in high-density monocultures (Figure 1). This result may have been due to toxicity or physical effects associated with mucilage. Although toxic cyanobacteria species are common (Martins et al., 2005, 2007; Valério et al., 2010), many other cyanobacteria may serve as food for copepods (Koski and Klein-Breteler, 2003; Kâ et al., 2012). Lower mortality in mixed cultures of the prochlorophyte and I. galbana, even at higher total densities, suggested that acute toxicity is unlikely. High mortality rates also were observed when P. crassirostris was feeding on palatable algae in water with high viscosity (Figure 3B), and PVP has been used in analogous experiments with no evidence of toxicity to delicate zooplankton (Podolsky and Emlet, 1993; Larsen et al., 2008). One possible explanation for copepod mortality is that oxygen diffuses more slowly in viscous water creating a risk of suffocation. Copepods have no gills, and oxygen uptake and carbon dioxide release take place through the integument and in the hindgut (Blaxter et al., 1998). Mucilage and PVP may have interfered with effective exchange of gases. In addition, I. galbana occasionally formed aggregates when PVP was added, and aggregates may have been difficult for copepods to grasp or masticate, which may cause stress, or occupy the copepod’s time examining non-consumable aggregate particles. Mortality from starvation was unlikely, however, due to the short duration of the experiments. The presence of non-consumable particles, however, could be considered a form of background plankton that reduces overall grazing rates (Johnson and Shanks, 1997, 2003).

Crowding and container effects have been a common concern when conducting laboratory grazing studies (Folt and Goldman, 1981; Peters, 1984; Helgen, 1987; Burns, 1995, 2000; Preuss et al., 2009; Lee et al., 2012). Potential artifacts can arise from physical interference among zooplankton, interactions between zooplankton and vessel walls or boundary layers (Peters, 1984), or chemically-mediated interactions among competitors (Folt and Goldman, 1981). Some studies demonstrate that crowding or container effects can 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 influence on feeding rates when specifically testing copepods. Small containers also can crowd grazers, and we must acknowledge that the influence of such effects 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. In fact, numerous copepod grazing studies have utilized small containers or high densities of grazers (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). Overall, evidence suggests the present experiments yielded useful insights into HAB grazing by copepods.

In conclusion, grazing on the prochlorophyte by P. crassirostris declined when algal densities were high and the water was viscous, whereas grazing on palatable algae increased with algal density. Copepod mortality increased with increasing viscosity, whether created by mucilage or a polymer. Copepod mortality and lower grazing rates in the presence of cultured prochlorophyte densities, meant to reflect in situ superbloom densities, suggest that P. crassirostris, a dominant herbivorous copepod, could be prevented from exerting top-down control in bloom conditions.

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

XM and KJ conceived the study. XM carried out data collection. XM and CJ analyzed data. All authors contributed to interpretation of the results, preparation and revision of the manuscript, and approved the submission.

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.

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