Effects of small-scale turbulence on two species of Dinophysis

Harmful Algae 89 (2019) 101654

Contents lists available at ScienceDirect

Harmful Algae journal homepage: www.elsevier.com/locate/hal

Effects of small-scale turbulence on two species of Dinophysis a,⁎

a

b

a

María García-Portela , Beatriz Reguera , Maurizio Ribera d’Alcalà , Francisco Rodríguez , Marina Montresorb a b

T

Harmful Microalgae Group (VGOHAB), Centro Oceanográfico de Vigo, IEO, Vigo, Spain Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, Italy

ARTICLE INFO

ABSTRACT

Keywords: Dinophysis acuminata Dinophysis acuta Mesodinium rubrum Turbulence Growth rates Cell size Turbulent kinetic energy dissipation rate

Dinoflagellate species of Dinophysis, in particular D. acuminata and D. acuta, produce lipophilic toxins that pose a threat to human health when concentrated in shellfish and jeopardize shellfish exploitations in western Europe. In northwestern Iberia, D. acuminata has a long growing season, from spring to early autumn, and populations develop as soon as shallow stratification forms when the upwelling season begins. In contrast, D. acuta blooms in late summer, when the depth of the pycnocline is maximal and upwelling pulses are moderate. In situ observations on the hydrodynamic regimes during the two windows of opportunity for Dinophysis species led us to hypothesize that D. acuta should be more sensitive to turbulence than D. acuminata. To test this hypothesis, we studied the response of D. acuminata and D. acuta to three realistic turbulence levels elow (LT), ε ≈ 10−6 m2 s-3; medium (MT), ε ≈ 10-5 m2 s-3 and high (HT), ε ≈ 10-4 m2 s-3 egenerated by Turbogen, a highly reproducible, computer-controlled system. Cells of both species exposed to LT and MT grew at rates similar to the controls. Marked differences were found in the response to HT: D. acuminata grew slowly after an initial lag phase, whereas D. acuta cell numbers declined. Results from this study support the hypothesis that turbulence may play a role in shaping the spatio-temporal distribution of individual species of Dinophysis. We also hypothesize that, in addition to cell disturbance affecting division, sustained high shear generated by microturbulence may cause a decline in Dinophysis numbers due to decreased densities of ciliate prey.

1. Introduction Dinophysis species produce diarrhetic shellfish poisoning (DSP) toxins and/or pectenotoxins (PTXs). Filter-feeding molluscs exposed to Dinophysis blooms accumulate these toxins and become a threat to public health (Yasumoto and Murata, 1990). Harvest closures are enforced when toxin levels exceed local regulatory limits, causing considerable losses to bivalve exploitations, in particular in European Atlantic coastal waters (Blanco et al., 2005; Vale et al., 2008; Reguera et al., 2014). In western Iberia, northern limit of the Canary Current upwelling system, Dinophysis acuminata and D. acuta form low biomass (103- 105 cell L−1) blooms often associated with stratified conditions, and their maxima related to density gradients (Maestrini, 1998; Reguera et al., 2012; Velo-Suárez et al., 2008). The two Dinophysis species exhibit distinct seasonal patterns (Reguera et al., 1993; Díaz et al., 2016). Dinophysis acuminata has a long spring-summer growing season and is often recorded in surface waters where wind-generated turbulence is maximal (Velo-Suárez et al., 2008; Díaz et al., 2019a). In

contrast, D. acuta blooms in late summer, when thermal stratification is more developed, and its cell maxima are often found in or near the pycnocline (Reguera et al., 1995; Díaz et al., 2016). The distinct conditions associated with high cell densities of D. acuminata and D. acuta (Reguera et al., 1995; Palma et al., 1998; Escalera et al., 2006; Moita et al., 2006), and their vertical segregation in the water column when they co-occur (Escalera et al., 2012; Moita and Silva, 2001; Díaz et al., 2018), prompted the hypothesis that D. acuta is more sensitive to turbulence than D. acuminata. Forty years ago, partly influenced by his observations of phytoplankton succession in Ría de Vigo, Margalef (1978) identified turbulent mixing and nutrient concentrations as the key factors controlling microphytoplankton succession. In his famous “Mandala”, Margalef ordinated phytoplankton groups along two axes eturbulence and nutrientse and highlighted the distinction between organisms that thrived in turbulent, nutrient-rich conditions (diatoms and cocolithophorids), and those that tended to thrive in low turbulence and nutrient-poor waters (dinoflagellates). In this simple conceptual model, if turbulence

Corresponding author. E-mail addresses: [email protected] (M. García-Portela), [email protected] (B. Reguera), [email protected] (M. Ribera d’Alcalà), [email protected] (F. Rodríguez), [email protected] (M. Montresor). ⁎

https://doi.org/10.1016/j.hal.2019.101654 Received 25 February 2019; Received in revised form 21 June 2019; Accepted 31 July 2019 Available online 03 October 2019 1568-9883/ © 2019 Elsevier B.V. All rights reserved.

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

decayed but nutrients remained abundant, an alternative successional route led to dinoflagellate species forming high biomass blooms, i.e. “red tides”. In further explorations of his Mandala, focusing explicitly on the importance of morphology as an adaptive strategy to the hydrodynamic regime, Margalef et al. (1979) speculated that the succession from fast growing diatoms (“r strategists”) to slow dividing dinoflagellates equipped with defence mechanisms, swimming capabilities and other competitive strategies (“K strategists”), resulted from adaptations to decaying turbulence. Margalef’s Mandala, proposed when methodologies to quantify turbulence in the water column had not been developed, continues inspiring many researchers (see Wyatt, 2014) and several refinements to the theory were applied in the subsequent years (e.g., Smayda and Reynolds, 2001; Glibert, 2016). Plankton dynamics are affected by turbulence in many direct and indirect ways and at different scales (Mann and Lazier, 1991; Estrada and Berdalet, 1998). Macro- and mesoscale turbulence affects the horizontal transport of water masses containing planktonic organisms. At a finer scale, advective and turbulent flows with typical dimensions of a few meters to tens of meters affect vertical transport of organisms in the water column changing their light regime, nutrient supplies and potential for biological interactions (Mann and Lazier, 1991). The smallest-scale turbulence events are eddies in the range of millimeters to less than one meter (microscale). This microscale which includes molecular processes and individual cell movements, is the scale that more directly affects phytoplankton physiological processes. This is due to the fact that the 10–100 μm sized phytoplankton cells are below the Kolmogorov scale, and thus, phytoplankton organisms perceive turbulence in the ocean as laminar shear (reviewed by Estrada and Berdalet, 1998; Thomas et al., 1995). Measurements of microscale turbulence and the shear forces associated with it are essential to understand the effects of turbulence on planktonic organisms and to compare results from experiments where different devices were used to generate turbulence (Thomas and Gibson, 1990a,b; Juhl et al., 2000; Juhl and Latz, 2002). Advances in microelectronic technology and high vertical-resolution observational tools, such as microstructure turbulence profilers, have now made it possible to quantify microturbulence (GEOHAB, 2008). Turbulence intensity is quantified by ε (m2 s−3), the turbulent kinetic energy dissipation rate. Typical values of ε in coastal waters range from 10-7 to 10-2 m2 s−3 (reviewed by Kiørboe and Saiz, 1995) and in broad terms, dinoflagellates have been found to be more sensitive to turbulence than diatoms (Margalef et al., 1979; Thomas et al., 1995). Microscale turbulence studies have shown its effects on a range of biological processes in planktonic populations. These include predator–prey encounter rates (Rothschild and Osborn, 1988), nutrients diffusion and hence their uptake by planktonic osmotrophs (Karp-Boss et al., 1996; Barton et al., 2014), cell division rates (Berdalet, 1992), swimming behaviour (Karp-Boss et al., 2000), gamete encounter rates (Persson et al., 2008), infection rates by dinoflagellate parasites (Llavería et al., 2010) and gene expression and chain length in diatoms (Amato et al., 2017). Shear may also affect cell integrity (Thomas et al., 1997), morphology (Zirbel et al., 2000) and motility (Durham et al., 2013). Following the first establishment of Dinophysis cultures fed the ciliate Mesodinium rubrum (Park et al., 2006), laboratory studies addressing different aspects of the physiology of Dinophysis species have been carried out (Reguera et al., 2012; Hansen et al., 2016). In most cases, these experiments were performed in culture plates or in small (100–250 mL) containers, and without any stirring or aeration. In consequence, the effect of microscale turbulence on Dinophysis species has never been examined. Indeed, to scale up cultures of mixotrophic species of Dinophysis and its ciliate prey Mesodinium rubrum, itself a kleptoplastic mixotroph that feeds on cryptophytes (Hernández-Urcera et al., 2018), to the volume scales needed to assess the effects of turbulence is a cumbersome task. This work represents the first laboratory study testing the response of two Dinophysis species to different levels of turbulence generated

with Turbogen, a highly reproducible computer-controlled device of oscillating grids (Amato et al., 2016), which generates realistic turbulence conditions comparable to those experienced by planktonic organisms in the field. 2. Material and methods 2.1. Cultures Dinophysis acuminata (strain VGO1349) was isolated from Ría de Vigo in July 2016 and Dinophysis acuta (VGO1065) from Ría de Pontevedra in October 2010. Culture procedures followed Park et al. (2006) with minor modifications (Hernández-Urcera et al., 2018). In short, a strain of the ciliate Mesodinium rubrum (AND-A0711), isolated from samples collected in Huelva (SW Spain), was fed the cryptophyte Plagioselmis prolonga (CR10EHU) from the Bay of Biscay (Northern Spain). These cultures were periodically given to Dinophysis as prey. The four species, grown in autoclaved seawater (salinity 32) enriched with K(-Si) medium (Keller et al., 1987) were acclimated to a temperature of 19 °C, an irradiance of 160–180 μmol photons m−2 s-1, and a 16:8 L:D photoperiod. 2.2. The turbulence-generating system Turbogen is composed of a polygonal aluminum frame, which can hold six Plexiglas cylinders containing up to 12 L in total (Amato et al., 2016). Turbulence is generated by oscillating grids attached via aluminum arms to a central pillar whose motion is driven by a computercontrolled motor. For the experiments illustrated in this study, light was provided by 12 LED lamps disposed in panels of 60 x 60 cm, and the volume of cultures in each cylinder was 4 litres. Turbulence levels were assessed using the equation suggested by Peters and Gross (1994): P/Cd = ½ SA·S−3 ·V-1 · f (t1-2 + t2-2)

(1)

where P is the theoretical rate of kinetic energy production, Cd is the drag coefficient set at 0.018, SA is the solid area of the grid set at 0.24%, S is the stroke length, V is the volume of the water, f is the oscillation frequency and t1, t2 the time the grid takes on each (upwards and downwards) vertical displacement. The ε values were estimated from the P/Cd values using the relationships between the two parameters obtained by Amato et al. (2016) used different methods for computing ε from particle image velocimetry data. In brief, the reported ε values may be affected by one order of magnitude error due to discrepancies among the different methods to asses it during the performance test. Since our study is comparative, the above uncertainty does not affect our inferences. Likewise, the turbulence regimes we explored mimic, to a very good extent, the range of in situ turbulence experienced by dinoflagellates in surface waters in the Galician upwelling system, from calm weather to strong northerly wind events, at the time of Dinophysis blooms (Villamaña et al., 2017; Díaz et al., 2019a). 2.3. Dinophysis experiments with three turbulence levels The set up for each treatment and species included one control (without turbulence) and two replicates in which cultures were exposed to different turbulence intensities. Three levels of turbulent kinetic energy dissipation rate (ε) were tested: low (0.5–8 × 10−6 m2 s-3), medium (0.3–4 × 10-5 m2 s-3) and high (0.5–4 × 10-4 m2 s-3). The low and medium turbulence (LT, MT) levels were in the range of those measured in the Galician Rías in the mixed layer, using a microstructure turbulence profiler (MSS), during spring-summer stratification combined with moderate upwelling (Villamaña et al., 2017), and the high (HT) values, when there are strong (> 10 m s-1) upwelling-promoting northerly winds (Diaz et al., 2019a). Preliminary tests were carried out with Mesodinium cultures to 2

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

confirm that the ciliate remained alive and available to its Dinophysis predator during the turbulence treatments for at least 3 days, i.e., the time between two consecutive re-feedings of Dinophysis. Mesodinium cultures grown in 2-L beakers (1.7 L culture volume) with an initial density of ≈1500 cells ml−1 and no cryptophyte prey added, were grown either in still conditions (control) or exposed to medium (ε≈106 -10-5 m2 s-3) and high (ε≈10-4 m2 s-3) turbulence levels. Samples of 5 mL taken at different time intervals were fixed with acid Lugol’s solution (1%) and cells counted three times using a Sedgewick-Rafter chamber with a Zeiss Axioskop 2 plus microscope (Zeiss, Oberkochen, Germany) with phase-contrast at a magnification of 100X and 200X. Growth rates (r) of Mesodinium in the control and treatments (MT and HT) during the preliminary tests were calculated as:

(in absolute value) than the treatments (kLT, kMT, kHT), and the difference would reflect ciliate mortality due to cell lysis:

C2 = C1 * e(r−g)

(6)

r = (ln Ct – ln Co) / (tt – t0)

g = r - ln (C2 / C1) = r + ln C1 / C2

(7)

m = kcontrol - ktreat

Growth/loss rates of Mesodinium in the preliminary tests in 1.7 L were assumed to be very similar in the 4 L cultures with Dinophysis in the Turbogen set up. These rates were used later as controls in simulations, for time intervals (t1-t2) of 1 day, to estimate the proportion of Mesodinium losses due to grazing. Mesodinium losses were considered to be equivalent to the difference between the ciliate growth rate (r) and the grazing rate, as follows:

(2)

where C1 and C2 are Mesodinium cell concentrations on day 1 and 2 respectively, r is the ciliate growth and g the grazing rate of Dinophysis upon Mesodinium. Ingestion rate (I) simulations for Dinophysis species (ciliates · Dinophysis−1 · day−1) were calculated adapting Frost (1972) as follows:

where C0 is the initial and Ct the final density of Mesodinium in time interval tt–t0. After that, Dinophysis cultures were started with an initial density of 200 cells mL−1 and M. rubrum provided as prey with a 1:10 ratio (Dinophysis:Mesodinium), to a final volume of 4 L. Cultures were resupplied with prey with a 1:5 ratio whenever Mesodinium cell numbers dropped below 50 cells mL−1 to avoid prey-limitation effects which may interfere with the response to turbulence. A volume of culture medium equivalent to the volume of Mesodinium culture to be added was removed by gentle reverse filtration (Dodson and Thomas, 1978) before re-feeding to keep constant the experimental volume at 4 L. pH was measured on day 0 and 6 with a Corning 240 pHmeter, Beckman electrode. Observed pH values were between 8 and 8.1 throughout the three sets of experiments. Gentle mixing was applied to each container to homogenize cell distribution before sampling. Three subsamples of 5 mL were collected every day from each culture container (a total of 90 mL per container from day 0 to day 6) and fixed with Lugol’s acid solution (1%). The concentration of Dinophysis and Mesodinium was estimated daily from triplicate counts of each sample using a Sedgewick-Rafter chamber with phase contrast and a Zeiss Axiophot light microscope. Each turbulence test lasted 6 days. Cultures of both species were subsequently kept in the same vessel in still conditions for 2 additional days to follow potential recovery from any possible negative impacts of turbulence. At the end of each experiment, control (undisturbed) cultures were saved to be used as inocula for the subsequent assay following concentration by reverse filtration through a 20-μm mesh. This concentrated culture was added as inoculum to fresh culture medium (4 L final volume) in 10 L Nalgene bottles so as the initial Dinophysis:Mesodinium ratio was 1:10. Intrinsic growth rates (r) of D. acuminata and D. acuta were calculated, following Wood et al. (2005), using the slope of the regression for the logarithmic (ln)-tranformed values of N (number of cells) versus time. Values of r (d−1) were estimated for three different time intervals: i) r0–6, for the whole turbulence treatment; ii) r6-8 for the still conditions period following and iii) rmax, for the interval where the maximal slope for each species and treatment was observed. One-way ANOVA (p < 0.01) and Tukey Honest Significant Differences (Tukey HSD) tests were carried out to test for differences in r (d−1) between controls (d0 d6) vs treatments (d0 - d6), with R software, version 3.1.3. Daily net growth rates (k) of Mesodinium co-cultured with Dinophysis, calculated according to Wood et al. (2005), were assumed to be the balance between growth and grazing (r-g) in the controls, plus turbulence-driven cell lysis, m, (r-g-m) in the treatments. Therefore, if the grazing capacity of Dinophysis were not affected by turbulence in the treatments: k = r - g (in the controls)

(3)

k = r – g - m (in the treatments)

(4)

(5)

C = C1 [e(r

− g) · (t2−t1)

− 1] / (t2− t1) (r − g) = (C2 − C1) / (r − g) (8)

I = C · g / (D1 + D2) / 2

(9)

where C1, D1, and C2 D2 are the ciliate prey (C) and Dinophysis (D) cell counts at times 1 and 2 respectively, for time intervals (t2–t1) of 1 day; C is the average ciliate concentration available for the grazers and g the grazing rate during the time interval t2− t1. Values of r estimated during the same time interval and culture conditions during the preliminary tests (Table S1) were used as controls for the experiments with different turbulence treatments in the simulations to estimate grazing. 2.4. Cell size (biovolume) To compare possible changes in cell size and morphology of the two Dinophysis species due to the treatments, biovolume was estimated on 50 cells of each species from additional 15 mL Lugol-fixed subsamples collected in each turbulence treatment and in the control on days 2 and 6. Measurements were carried out with an Axiocam HRC (Zeiss, Oberkochen, Germany) digital camera coupled to a Leica DMR (Leica, Germany) microscope at a 630x magnification. Dinophysis acuminata geometric shape was considered equivalent to a flattened ellipsoid and that of D. acuta to a cone plus a truncated cone (Olenina, 2006). Regression analysis, two-way ANOVA (p < 0.01) and Tukey Honest Significant Differences (Tukey HSD) test were carried out on biovolume estimates with R software. 3. Results 3.1. Effect of turbulence on Mesodinium cultures Average growth rate of Mesodinium rubrum during 3 days of preliminary tests were 0.18 ± 0.03 d1 with still conditions, and 0.09 ± 0.06 d−1 with MT, while cell concentrations decreased moderately (r = -0.18 ± 0.05 d−1) with HT. Estimated daily growth rates varied between treatments. Compared with the still control, they were similar to (with MT) or higher (with HT) than on day 1, near zero (but positive) on day 2, and with high negative values on day 3 Cultures had a healthy appearance and Mesodinium cells were dispersed in the water column with no visible signs of concentration in the bottom of the culture container. Cultures of D. acuminata were re-supplied with Mesodinium on days 2 and 5 (Fig. 1A–C) and cultures of D. acuta on days 3 and 5 (Fig. 1D, E) except those with the HT treatment (Fig. 1F) that did not need refeeding until day 5.

and negative growth estimates in the control (kcont) should be lower 3

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

Fig. 1. Growth curves of Dinophysis acuminata (A, C, E) and D. acuta (B, D, F) and cell concentration of Mesodinium rubrum used as prey exposed to different turbulence levels (LT: low turbulence, MT: medium turbulence and HT: high turbulence) betweeen day 0 and day 6 and recovery time (shaded grey area) between day 6 and day 8 in still conditions. Black circles, controls (n = 3); white circles, treatments (n = 6); small bars represent std values. Notice that some of them can be smaller than the symbol. Cell concentration was estimated on triplicate cell counts; treatment data are the average value of two replicates.

3.2. Dinophysis growth response to different turbulence levels

3.2.2. Medium turbulence (MT) treatment Dinophysis acuminata exposed to MT, and re-supplied with prey on days 2 and 5, grew faster (0.37 d−1) than in the control (0.31 d−1) between day 0 and day 4, reached an earlier stationary phase on day 4, and began to decline after day 5 (Fig. 1B; Table 1). There were no significant differences between growth rates in controls and treatments from day 0 to day 6 (Table 2). Dinophysis acuta cultures exposed to MT (Fig. 1E), re-supplied with prey on days 3 and 5, showed a lag phase from day 0 to 1 and an intrinsic growth rate from day 1 to 6 (0.17 d−1) significantly lower (p < 0.01) than the control (0.23 d−1) (Tables 1,2). Cultures of D. acuminata with MT resumed moderate (0.13 d−1) growth when turbulence stopped, and reached a final yield lower than the control. Growth rates of D. acuta increased when turbulence ceased, from day 6 to 8 (0.31 d−1), reaching a final yield close to that of the control. Ingestion rate simulations of Mesodinium by D. acuminata were much higher in the treatment (4.94 ± 0.38 cells d−1) than in the control (2.31 ± 0.72 cells d−1) the first day, but declining in parallel with the control the following days (Fig. 2A). A similar pattern was observed with D. acuta cultures, with an ingestion of 6.69 ± 0.47 d−1in the treatment versus 3.99 ± 0.92 cells d−1 in the control the first day (Fig. 2B).

Dinophysis cultures with different turbulence levels and still controls were started with an initial 1:10 Dinophysis:Mesodinium ratio. Net growth rates of Mesodinium in all the treatments and controls were negative. 3.2.1. Low turbulence (LT) treatment Growth curves of D. acuminata and D. acuta cultures exposed to LT (Fig. 1A, D) were similar to those observed in the controls and no significant differences (p > 0.05) were observed in their intrinsic growth rates, from day 0 to day 6, in relation to the controls: 0.34 vs 0.35 d−1 for D. acuminata and 0.22 vs 0.20 d-1 for D. acuta (Tables 1, 2). Growth of the two species continued and final yields were very similar to the controls when turbulence stopped from day 6 to 8 (Tables 1, 2). Mesodinium loss rates were always higher in the treaments (except between days 2 and 3 just after first re-feeding) than in the control in cultures with D. acuminata. The opposite was observed in cultures with D. acuta (Supplementary Table S3). Daily ingestion of Mesodinium cells by both species of Dinophysis were maximal in the controls of both species (5.00 ± 0.07 and 6.06 ± 0.62) the first day, and about half these values subsequently, even after refeeding (Fig. 2). 4

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

Table 1 Average intrinsic growth rate (r) of Dinophysis acuminata and D. acuta in the controls and treatments (low = LT; medium = MT and high = HT turbulence) from day 0 to day 6 (d0-d6); intrinsic maximum growth (rmax, d−1) during the exponential growth phase and r (d−1) during recovery time from day 6 to day 8 (d6-d8). Cell concentration was estimated on triplicate cell counts; treatment data are the average value of two replicates. Time

d0-d6 d6-d8 (Recovery)

D. acuminata CLT

LT

CMT

MT

CHT

HT

r (d ) rmax (d−1)

0.34 ± 0.01

0.31 ± 0.01

0.21 ± 0.01

0.33 ± 0.01 0.37 ± 0.01 (r1-4) 0.13 ± 0.02

0.34 ± 0.01

r (d−1)

0.35 ± 0.01 0.37 ± 0.02 (r3-5) 0.07 ± 0.02

0.20 ± 0.01 0.23 ± 0.01 (r3-5) 0.07 ± 0.00

−1

0.22 ± 0.01

Time

0.23 ± 0.01

D. acuta

d0-d6 d6-d8 (Recovery)

CLT

LT

CMT

MT

CHT

HT

r (d−1) rmax (d−1)

0.22 ± 0.00

0.23 ± 0.01

−0.08 ± 0.01

0.13 ± 0.01

0.17 ± 0.01 0.31 ± 0.01 (r1-4) 0.31 ± 0.02

0.20 ± 0.01

r (d−1)

0.20 ± 0.01 0.23 ± 0.01 (r3-6) 0.09 ± 0.01

0.09 ± 0.00

0.04 ± 0.02

0.18 ± 0.01

Dinophysis cells were visibly concentrated in the bottom of the containers, in both the controls and the turbulence treatments, but were easily resuspended by gentle mixing carried out every day before sampling. In our experience, this is a common observation in the course of experiments and routine maintenance under laboratory conditions (see Hernández-Urcera et al., 2018). Microscopic examination at the end of the experiment confirmed that cells on the bottom had the bright red pigmentation and rotating movement characteristic of healthy, well-fed individuals. Some of them were seen to have Mesodinium prey still trapped by the dinoflagellate feeding peduncle.

Table 2 Growth rates: comparative statistical results carried out using i) treatments exposed to turbulence from days 0–6 and during the recovery (d6-d8), ii) controls in still conditions and treatments between days 0 and 6, and iii) controls in still conditions and treatments during the recovery period between days 6 and 8. Comparison

Species

Treatments LT

MT

HT

Controls vs treatment between d0-d6

D. acuminata D. acuta

– –

– 2.76 × 10−7

10−10 5.5 × 10−13

Controls vs treatment between d6-d8

D. acuminata D. acuta

7.88 × 10−7 2.70 × 10−4

2.09 × 10−4 5.41 × 10−9

3.55 × 10−7 0.01

3.2.4. Comparison of growth response to turbulence by the two Dinophysis species Growth rate differences between the two species were greater within the HT treatment than within the MT and LT treatments. Overall, D. acuminata grew moderately well (rmax:: 0.20 d−1) under HT, but at a lower rate than in the control flasks without turbulence (0.34 d−1) (p < 0.01). Under HT, D. acuta ceased growth (-0.08 d−1) and it did not recover when it was returned to still conditions, i.e. no growth was observed on days 6–8 (Tables 1,2). With the LT and MT treatments. Dinophysis acuminata exhibited higher growth rates (rmax: 0.35 d−1, 0.33 d−1) and reached higher yields than D. acuta (rmax: 0.20 d−1, 0.17 d−1).

3.2.3. High turbulence (HT) treatment Growth curves of both, D. acuminata and D. acuta showed an initial lag phase when exposed to the highest turbulence, followed by distinct responses (Fig. 1C, F). Dinophysis acuminata lag phase lasted from day 0 to day 1 and a positive, though moderate (rmax = 0.23 ± 0.01 d−1), growth followed from day 1 to day 5 (Fig. 1C, Table 1). Prey was resupplied on days 2 and 5. Growth rates in the treatments were significantly lower (p < 0.01) than in the controls for this species between day 0 and 6 (Table 2). Dinophysis acuta lag phase lasted from day 0 to day 2, followed by a decline in cell numbers from day 2 to day 4 and a stationary phase from day 4 to day 6, with little change when turbulence ceased (Fig. 1F). Thus, growth rates in the treatments (negative growth), were significantly lower (p < 0.01) than in the control for this species between day 0 and 6. Ciliate prey was above the 1:5 Dinophysis:Mesodinium ratio on day 3, so new prey was added on day 5 only (Fig. 1F). When turbulence stopped, growth rate (0.07 d−1) was negligible whereas a late exponential phase was observed in the control growth curves. (Tables 1,2). Ingestion rates of Mesodinium by D. acuminata with the HT treatment were again much higher (6.36 ± 0.26 cells d−1) than in the control (3.81 ± 0.25 cells d−1) the first day, but declined in parallel with the control the following days (Fig. 2A). A different pattern was observed with D. acuta cultures, where ingestion rates were very similar in control and treament during the first day (5.85 ± 0.37 in the treatment versus 5.13 ± 0.05 cells d−1 in the control); a minimum was reached between days 3 and 4 when no feeding occurred (Fig. 2B). Mesodinium losses were considerably lower in the treatment than in the control in the case of D. acuta, but differences were not so consistent in the case of D. acuminata (Supplementary Table S3).

3.3. Changes in cell size (biovolume) Mean values of D. acuminata and D. acuta biovolumes did not show significant differences (Supplementary Table S2) between control and treatments (Fig. 3). A few (< 1% of the population) small-sized cells were observed in both controls and treatments. These cells did not correspond to the description of “gamete-like” cells in the life cycle of Dinophysis species, with smaller size and different shape than the normal vegetative cells (Reguera and González-Gil, 2001). Average measurements of these small-sized cells were: L = 37.28 ± 2.30; D = 23.51 ± 3.38 μm, for D. acuminata (n = 9) and L = 55.68 ± 3.34 and D = 35.36 ± 4.29 μm for D. acuta (n = 11). 4. Discussion 4.1. Effect of turbulence on D. acuminata and D. acuta growth rates This study focused on the behaviour of well-fed cells of Dinophysis acuminata and D. acuta at different turbulence (ε) levels between ε ≈ 10−6 m2 s-3 and 10-4 m2 s-3. These are realistic values equivalent to those measured with microstructure profilers in the Galician Rías under 5

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

Fig. 2. Ingestion rate (ciliates·Dinophysis−1·day−1) simulations for Dinophysis acuminata (A) and D. acuta (B) exposed to different turbulence levels (LT: low turbulence, MT: medium turbulence and HT: high turbulence) and their controls, betweeen day 0 and day 6.

different wind intensities in spring-summer (Villamaña et al., 2017; Díaz et al., 2019a). The intrinsic growth rates (Table 1) of the two Dinophysis species were unaffected by the LT levels assayed, and D. acuta was demonstrated to be less tolerant than D. acuminata to the MT and especially HT treatments. In contrast, D. acuminata was able to grow with HT (rmax = 0.23 d-1), although at a signicantly (p < 0.01) lower rate than the control, and the LT and MT treatments (rmax = 0.37 d-1). Dinophysis acuminata growth rates during recovery time were always significantly lower in the treatments than in the control. A similar response was detected in D. acuta with LT, while almost no growth was detected in the HT treatment when turbulence was stopped. Low growth during the recovery period in D. acuminata cultures can be interpreted as a short term response to the change, a sort of ‘lag-phase’ that might have been overcome if the still conditions had been tested for longer. The change to still conditions in the case of D. acuta following medium turbulence may represent a relief from stress, and would explain the increased growth from day 6 to 8 and a final yield matching the control. In contrast, in the HT treatment, cells were heavily stressed –cell numbers declined during the treatment – and could not recover over the two days in which turbulence was stopped (Table 2). In addition to the effect on Dinophysis, MT and HT also affected the ciliate prey, causing a moderate (with MT) to sharp (with HT) decline in

cell densities after three days of exposure (Table S1). We did not observe cells of Mesodinium in poor condition, so it may be assumed that the ciliate individuals damaged by excessive turbulence probably lyzed and disintegrated. 4.2. Effect of turbulence on cell size Cell size differences between Dinophysis cells under different treatments and the controls were not significant, even in the case of D. acuta cultures exposed to HT. In field populations of D. acuminata in the Galician Rías, important differences in biovolume (33% increase) were observed between prey limited (starving) and recently fed (full of food vacuoles) cells (González-Gil et al., 2010). This situation was not observed in our experiments. Dinophysis size did not exhibit significant changes, and this was considered proof of cells being well fed. Most previous studies of phytoplankton cultures have shown inverse relationships between division rate and under high turbulence (Thomas and Gibson, 1990a,b; Thomas et al., 1995; Sullivan and Swift, 2003; Berdalet, 1992). However, a few studies reached similar conclusions to the present one. For instance, the cross-sectional area of Gonyaulax polyedra, a proxy for cell volume, did not change at high turbulence (ε≈10−3 m2 s-3) applied for 10 days (Sullivan et al., 2003), nor did the cell volume of Heterocapsa triquetra during a treatment of the same 6

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

Fig. 3. Boxplot of biovolumen estimates (μm3) under still conditions (controls, n = 50), in each turbulent treatment (LT, low turbulence; MT, medium turbulence and HT, high turbulence; n = 50 each) of A) D. acuminata and B) D. acuta for sampling days 2 and 6.

duration at turbulence levels of ε≈10-4 m2 s-3 (Havskum and Hansen, 2006). Some studies have shown parallel trends between increased biovolume and turbulence. A 20% increase in the cross-sectional area of Alexandrium catenella was observed after 6 days with values higher than 10-5 m2 s-3, and an even more remarkable increase in cell volume was detected in Alexandrium minutum and Prorocentrum triestinum at levels of ε≈2×10-4 m2 s−3 (Sullivan et al., 2003). These contrasting results suggest that changes in cell size in response to turbulence must be considered at a species level.

nuclear division. Berdalet (1982) exposed Gymnodinium nelsonii (=Akashiwo sanguinea) to high (ε = 10−4 m2 s-3) turbulence generated with an oscillating grid for 10 days. Cell division was inhibited, but there was a 50% increase in cell volume and a 10-fold increase in the RNA and DNA cell quota. Division rates and the normal cell parameters were quickly restored 2 days after turbulence stopped. Therefore, the turbulence levels used did not seem to affect DNA synthesis (phase S of cell cycle), but, in agreement with results of Pollingher and Zemel (1981), inhibited the phase preparing for mitosis (phase G2). These observations led Berdalet (1982) to hypothesize that failure of the cells to divide resulted from physical disturbance of the microtubule assemblage (the spindle) asociated with chromosome separation during mitosis. Agitation in most of the early studies was described as rotation speed or oscillations per unit time, and only in very few cases expressed with the appropriate fluid mechanic terms, such as rate of turbulent energy dissipation (ε) (Thomas and Gibson, 1990a). A data set with contrasting results for the same species by different authors and or different turbulence generating devices was critically revised by Berdalet et al. (2007). Gonyaulax polyedra (=Lingulodinium polyedra) showed negative effects on growth with turbulence (ε) values between 164×10−4 and 0.18×10-4 m2 s-3 when using a Couette cylinder (Thomas and Gibson, 1990b), while Sullivan and Swift (2003) obtained significantly higher growth rates (compared to still conditions) for the same species using higher turbulence (ε≈10-4 m2 s-3) generated by vertically oscillating rods. In another study, where the same device was used to generate even higher turbulence (ε≈10-3 m2 s3 ), growth rates of Gonyaulax polyedra diminished compared with still conditions during a 10-d experiment (Sullivan et al., 2003). These differences document the difficulties met when comparing results obtained with the same species/strain where turbulence was generated with different devices (Peters and Redondo, 1997).

4.3. Effect of turbulence on dinoflagellates physiology Early studies related the development of high density dinoflagellate populations (red tides) to calm weather and water column stratification (Wyatt and Horwood, 1973; Margalef et al., 1979; Estrada et al., 1987). These low turbulence conditions would favour the development of “K strategists” or “stress tolerant” species, sensu Smayda and Reynolds (2001) with acquisitive strategies (e.g. swimming ability, storage of resources, production of allelopathic substances) to exploit stratified environments. In situ observations combined with laboratory experiments (White, 1976; Pollingher and Zemel, 1981) showed that, in contrast with the calm conditions favouring cells aggregation and in situ growth, small-scale turbulence could disrupt and/or cease cellular division in dinoflagellates. Thus, continuous rotary shaking of cultures of the toxic marine dinoflagellate Gonyaulax excavata (=Alexandrium catenella) at speeds of 125 rpm and greater caused rapid death and disintegration of cells, while shaking at lower speeds caused decline in growth rates compared to cultures in still conditions (White, 1976). Pollingher and Zemel (1981) found that disturbances of cell division rates of Peridinium cinctum in lake Kinneret (Israel) were observed only if strong wind episodes occurred during the few hours preceding 7

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

Sullivan and Swift (2003) tested the effect of small-scale turbulence on 10 bloom-forming dinoflagellate species using the same experimental design. Turbulence was generated by vertically oscillating cylindrical rods. The species responses to HT (ε≈10−4 m2 s-3) were diverse, ranging from growth inhibition or decline to no apparent effects or even incresed growth rates. It is worth pointing out that two species of thecate dinoflagellates with similar shape and size – Lingulodinium polyedra and Alexandrium catenella - reacted in different ways to similar levels of turbulence (Sullivan et al., 2003). Nevertheless, A. catenella is a chain-forming dinoflagellate. Field observations by the same authors showed that under high turbulence, A. catenella grew more slowly but formed longer chains, providing increased swimming velocity and a capacity to migrate to layers where shear stress was lower. Chain formation interacts with the turbulence dissipation rates, i.e. has rheological properties (Wyatt and Ribera d’Alcalà, 2006). These results highlight the importance of species-specific differences in the response to turbulence, and confirmed with laboratory and field measurements the importance of morphology as an adaptive strategy to the hydrodynamic regime proposed by Margalef et al. (1979). Little is known about the effect of turbulence on dinoflagellates toxin production and content. Contradictory results observed in cultures of several Alexandrium species are difficult to compare because the turbulence generating systems applied were different, and some of the turbulence values employed (ε = 2.7×10−3), unrealistically high (Juhl et al., 2001; Bolli et al., 2007). High turbulence causing a decline in growth rates may have an indirect effect in toxin accumulation (celltoxin content). Uncoupling between division and toxin production ratess was the accepted explanation for increased toxin per cell in field populations (Pizarro et al., 2009) and laboratory cultures of Dinophysis (Nielsen et al., 2013) when they entered the stationary phase. Another potential effect of turbulence on toxin producing dinoflagellates might be increased release of extracellular toxins by mechanical stress inflicted on cells. It is well known that shear stress due to sampling and filtration contribute to high levels of toxin release, a process that has been taken advantage of to adsorb lipophilic toxins with passive samplers (e.g. SPATT) (MacKenzie et al., 2004; Rundberget et al., 2007). But studies of toxin dynamics need to measure both intra and extracellular toxin production, a task rarely undertaken in studies of Dinophysis

populations (Rothschild and Osborn, 1988; Peters and Gross, 1994). The Dinophysis feeding mechanism, involving the use of a feeding tube to pierce prey (myzocytosis), is quite a complex process (Park et al., 2006). In addition, experiments with high density cultures of Mesodinium, D. acuminata (Mafra et al., 2016; Ojamäe et al., 2016) and D. acuta (Papiol et al., 2016) have shown that Dinophysis can secrete a mucous trap to capture ciliate prey. Jiang et al. (2018) used a highspeed microscale imaging system to detail D. acuminata detection of its M. rubrum prey by chemoreception, the ciliate’s escape mechanisms, and the use of mucus filaments by Dinophysis to immobilize it. It could well be that turbulence beyond a threshold value may lower the dinoflagellate’s ability to detect, capture and handle its prey. Dinophysis species studied in this paper have a cell size (< 80 μm) that falls below the Kolmogorov scale, where shear forces are the main effect of turbulence. Shear plays a relevant role in determining prey capture success (Shimeta and Jumars, 1991; Kiϕrboe and Titelman, 1998). The estimated values of shear rates to which Dinophysis cells were exposed in our experiments were: δ≈1.4 s−1at LT conditions; ≈2.4 s-1 at MT and ≈15 s−1 at HT. This implies that at a distance equivalent to the cell diameter (e.g., 50 μm), the water would move with a speed of 75, 125 and 750 μm s−1 for LT, MT and HT, respectively. In Jiang et al. (2018) videograms, it was estimated that Dinophysis feels its prey by chemoreception at a distance of 89 ± 39 μm. It can be hypothesized that at HT, the water speed generated by shear would reduce the ability of Dinophysis to detect its prey and capture it. This view is supported by the lower consumption rates of Mesodinium in the HT treatment of D. acuta, and its decline in numbers under this treatment (Fig. 1F). Thus, D. acuta cells were probably in poorer condition than D. acuminata under the HT treatment, and this would have had a negative impact on their growth rates and ability to capture prey. However, given the short duration of the experiment, it is difficult to assess whether the decline in Dinophysis numbers was a direct consequence of cellular stress or a shortage of Mesodinium prey, or a combination of both processes. This view is supported by the observation during the present study that ingestion rates of the two species, in all controls and treatments, were much higher the first day of the experiment, and showed a progressive decline even when ciliate concentrations were increased by refeeding. Considering the intermittent feeding behaviour of Dinophysis, its capacity to overfeed until deformed, and its ability to fast for 2 months without prey, it is possible that the heavy feeding of the first day (treatment and controls) was sufficient to support growth and division throughout the experiment. Growth differences should be then more likely be attributed to the turbulence effects.

4.4. Turbulence and feeding behaviour of mixotrophic dinoflagellates Our experimental setup was prompted by observations and results from all the previous authors. Nevertheless, this is the first experiment where turbulence is tested on obligate mixotrophs, species of the genus Dinophysis with a complex system to catch prey that may be affected by turbulence in a different manner. All the studies described in the previous section were carried out with auxotrophic dinoflagellates, i.e., requiring light and inorganic nutrients plus a few vitamins and trace compounds. Dinophysis species are obligate kleptoplastidic mixotrophs requiring light, nutrients and plastids stolen from their prey (the ciliate Mesodinium); the ciliate is eaten by myzoctosis, through a feeding tube that pierces the prey and sucks its contents (Park et al., 2006). There is a single study on the effects of turbulence on the mixotroph Fragilidium subglobosum fed high densities (> > 10 cells mL−1) of Ceratium tripos (=Tripos muelleri) (Havskum et al., 2005). In that work, low, medium and high turbulence values (ε≈10-7, 5×10-7, 5×10-6 and 10-4 m2 s-3) comparable with the ones used in the present investigation did not affect growth of the dinoflagellate. Nevertheless, Fragilidium subglobosum is a facultative mixotroph, i.e., a species containing its own permanent plastids and able to grow exclusively by photosynthesis (Park et al., 2010). When Fragilidium feeds, the cingular region opens to engulf its prey, and the theca are shed after feeding. The result is the formation of a Fragilidium pellicle cyst, with a shape adapted to that of the prey (Park et al., 2010; Rodríguez et al., 2014). Theoretically, turbulence increases grazing rates in microplankton

4.5. Implications for ecology and population dynamic studies of Dinophysis populations Defining functional relationships between small-scale turbulence and dinoflagellate physiology in the laboratory is a first step toward producing mathematical relationships to describe turbulence effects (Sullivan et al., 2003). These relationships should be integrated into population dynamics models (Donaghay and Osborn, 1997), especially where wind-driven circulation plays a key role in determining phytoplankton dominance. Observations in the Galician Rías and northern Portugal show that Dinophysis thrives in stratified conditions combined with moderate upwelling, and aggregates in thin layers often associated with density discontinuities (Reguera et al., 2012). However in the case of D. acuminata, the most persistent species in the region, cell maxima in latespring populations are often found in the top 5 m (Velo-Suárez et al., 2008; Díaz et al., 2019a). This is the mixed layer zone where turbulence values are highest, especially during strong northerly winds in the upwelling season. In contrast, D. acuta populations thrive in shelf waters in late summer, when thermal stratification and depth of the mixed layer is maximal, and combined with moderate upwelling. Under these 8

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

conditions, D. acuta is found in or near the pycnocline (Moita et al., 2006; Díaz et al., 2016). Turbulence levels in these layers are considerably lower, as confirmed by field measurements with turbulent profilers (Villamaña et al., 2017; Díaz et al., 2019a). This study confirms that D. acuminata can endure higher levels of turbulence than D. acuta. This is compatible with field observations of the vertical segregation of the two species when they co-occur (Díaz et al., 2019b; Escalera et al., 2012). The volume of D. acuta is three times that of D. acuminata, and it is more compressed dorsoventrally. Being larger renders D. acuta more vulnerable to shear stress, but the higher surface:volume ratio of flattened cells allows them to harvest nutrients and light more efficiently. Estimates from laboratory experiments and field observation suggest that D. acuta is a faster swimmer than D. acuminata (Smayda, 2010). Recent studies show that D. acuta is more susceptible to photodamage under high light intensities (370–650 μmol photons m−2s−1) than D. acuminata but survives better with low light (10 μmol photons m−2 s−1) and prolonged (28 d) darkness (García-Portela et al., 2018). These varying considerations indicate that phytoplankton species must adapt to a variety of changes in their environment, not just to turbulence. But we can conclude that the different responses of D. acuminata and D. acuta to turbulence add to other differences (use of light, swimming capabilities) and allow D. acuta to grow in deeper waters than D. acuminata. Smayda and Reynolds (2001), in a conceptual model based on Margalef’s Mandala and Reynolds’ Intaglio (Reynolds, 1987), grouped dinoflagellate bloom species characterized by distinct life-forms and adaptive strategies, into nine habitat preferences along an onshore–offshore turbulence-nutrient gradient. In this scheme, Dinophysis species were included in Type VII, as “they seem to represent a transitional life form (Type VII) (...) more attuned to less pronounced, smaller-scale convective currents than Type V (upwelling relaxation) bloom species (...) blooms of this slow growing HAB assemblage may often result primarily from physical accumulation, rather than active growth" (Smayda and Reynolds, 2001). However, Dinophysis species associated with toxic events comprise a list of species with diverse morphologies (from the small oval species of the Dinophysis acuminata complex to the extreme forms with elongated prolongations of Dinophysis miles) and adaptations to thrive in a wide range of environmental conditions from coastal to oceanic waters (Reguera et al., 2014). Both Dinophysis species considered in the present study reach their annual maxima during upwelling relaxation events in the Galician Rías, but have distinct niches (Díaz et al., 2019b). Further, the belief that Dinophysis species are slow growers has proved false in laboratory cultures where several species of the genus can exhibit up to one division per day provided they are not prey limited (Park et al., 2006; Nielsen et al., 2013; Hernández-Urcera et al., 2018).

assistance and Tim Wyatt for fruitful discussions and English revision. This study was funded by Spanish projects DINOMA (RETOS Programme, CGL2013-48861-R) and MARBioFEED (ERANET Marine Biotechnology, PCIN-2015-252) and by a mobility grant from the Spanish Ministry MINEICO to M. García Portela (grant EEBB-I-1712341) to carry out these experiments at SZN. This research article is part of M. García-Portela PhD thesis, which is appended to the “Marine Science, Technology and Management” (DO*MAR) doctoral programme at the University of Vigo.[CG] Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.hal.2019.101654. References Amato, A., Dell’Aquila, G., Musacchia, F., Annunziata, R., Ugarte, A., Maillet, N., Carbone, A., Ribera d’Alcala, M., Sanges, R., Iudicone, D., Ferrante, M.I., 2017. Marine diatoms change their gene expression profile when exposed to microscale turbulence under nutrient replete conditions. Sci. Rep. 7 (1), 3826. Amato, A., Fortini, S., Watteaux, R., Diano, M., Espa, S., Esposito, S., Ferrante, M.I., Peters, F., Iudicone, D., Ribera d’Alcalà, M., 2016. TURBOGEN: computer-controlled vertically oscillating grid system for small-scale turbulence studies on plankton. Rev. Sci. Instrum. 87 (3), 035119. Barton, A.D., Ward, B.A., Williams, R.G., Follows, M.J., 2014. The impact of fine-scale turbulence on phytoplankton community structure. LOFE 4 (1), 34–49. Berdalet, E., 1992. Effects of turbulence on the marine dinoflagellate Gymnodinium nelsonii. J. Phycol. 28 (3), 267–272. Berdalet, E., Peters, F., Koumandou, V.L., Roldán, C., Guadayol, O., Estrada, M., 2007. Species-specific physiological response of dinoflagellates to quantified small-scale turbulence. J. Phycol. 43 (5), 965–977. Blanco, J., Moroño, Á., Fernández, M.L., 2005. Toxic episodes in shellfish, produced by lipophilic phycotoxins: an overview. Galician J. Mar. Resour. 1, 1–70. Bolli, L., Llavería, G., Garcés, E., Guadayol, Ò., Lenning, K.V., Peters, F., Berdalet, E., 2007. Modulation of ecdysal cyst and toxin dynamics of two Alexandrium (Dinophyceae) species under small-scale turbulence. Biogeosciences 4 (4), 559–567. Díaz, P., Ruiz-Villarreal, M., Mouriño-Carballido, B., Fernández-Pena, C., Riobó, P., Reguera, B., 2019a. Fine scale physical-biological interactions during a shift from relaxation to upwelling with a focus on Dinophysis acuminata and its potential ciliate prey. Prog. Oceanogr. 175. https://doi.org/10.1016/j.pocean.2019.04.009. Díaz, P., Reguera, B., Moita, T., Bravo, I., Ruiz-Villareal, M., Fraga, S., 2019b. Mesoscale dynamics and niche segregation of two Dinophysis species in Galician-Portuguese coastal waters. Toxins 11 (1), 37. Díaz, P.A., Ruiz-Villarreal, M., Pazos, Y., Moita, T., Reguera, B., 2016. Climate variability and Dinophysis acuta blooms in an upwelling system. Harmful Algae 53, 145–159. Dodson, A.N., Thomas, W.H., 1978. Reverse filtration. In: Sournia, A. (Ed.), Phytoplankton Manual. Monographs on Oceanographic Methodology 6. UNESCO, Paris, pp. 104–107. Donaghay, P.L., Osborn, T.R., 1997. Toward a theory of biological-physical control of harmful algal bloom dynamics and impacts. Limnol. Oceanogr. 42, 1283–1296. Durham, W.M., Climent, E., Barry, M., De Lillo, F., Boffetta, G., Cencini, M., Stocker, R., 2013. Turbulence drives microscale patches of motile phytoplankton. Nat. Commun. 4, 2148. Escalera, L., Reguera, B., Pazos, Y., Moroño, A., Cabanas, J.M., 2006. Are different species of Dinophysis selected by climatological conditions? Afr. J. Mar. Sci. 28 (2), 283–288. Escalera, L., Pazos, Y., Doval, M.D., Reguera, B., 2012. A comparison of integrated and discrete depth sampling for monitoring toxic species of Dinophysis. Mar. Pollut. Bull. 64 (1), 106–113. Estrada, M., Berdalet, E., 1998. Effects of turbulence on phytoplankton. NATO ASI Ser. Ser. G 41, 601–618. García-Portela, M., Riobó, P., Reguera, B., Garrido, J., Blanco, J., Rodríguez, F., 2018. Comparative ecophysiology of Dinophysis acuminata and D. acuta: Effect of light intensity and quality on growth, cellular toxin content and photosynthesis. J. Phycol. 54, 899–917. GEOHAB, 2008. In: Gentien, P., Reguera, B., Yamazaki, H., L, Fernand L., Berdalet, E., Raine, R. (Eds.), Global Ecology and Oceanography of Harmful Algal Blooms, GEOHAB Core Research Project: HABs in Stratified Systems. IOC and SCOR, Paris, France, and Newark, Delaware, USA, pp. 59. Glibert, P.M., 2016. Margalef revisited: a new phytoplankton mandala incorporating twelve dimensions, including nutritional physiology. Harmful Algae 55, 25–30. González-Gil, S., Velo-Suárez, L., Gentien, P., Ramilo, I., Reguera, B., 2010. Phytoplankton assemblages and characterization of a Dinophysis acuminata population during an upwelling–downwelling cycle. Aquat. Microb. Ecol. 58 (3), 273–286. Hansen, P.J., Ojamäe, K., Berge, T., Trampe, E.C., Nielsen, L.T., Lips, I., Kühl, M., 2016. Photoregulation in a kleptochloroplastidic dinoflagellate, Dinophysis acuta. Front. Microbiol. 7, 785–796. Havskum, H., Hansen, P.J., 2006. Net growth of the bloom-forming dinoflagellate Heterocapsa triquetra and pH: why turbulence matters. Aquat. Microb. Ecol. 42 (1), 55–62.

5. Concluding remarks Dinophysis acuta is more sensitive than D. acuminata to high turbulence levels, a fact that combined with other ecophysiological traits, allows the former to thrive in thermally-stratifed waters with deeper pycnoclines, at depths where light intensity is low and blue light predominates. We hypothesize that, in addition to cell disturbance affecting cell division, high shear at cell surfaces generated by HT may disturb the prey capture process in mixotrophic species of Dinophysis and cause a decline of prey density. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We thank Alessandro Manfredonia, Cosimo Vestito and Gianluca Zazo from Stazione Zoologica Anton Dohrn (SZN) for technical 9

Harmful Algae 89 (2019) 101654

M. García-Portela, et al.

between picked cells and plankton concentrates. Harmful Algae 8, 926–937. Pollingher, U., Zemel, E., 1981. In situ and experimental evidence of the influence of turbulence on cell division processes of Peridinium cinctum forma westii (Lemm.) Lefèvre. Br. Phycol. J. 16 (3), 281–287. Reguera, B., Bravo, I., Mariño, J., Campos-Loriz, M.J., Fraga, S., Carbonell, A., 1993. Trends in the occurrence of Dinophysis spp in Galician coastal waters. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, pp. 559–564. Reguera, B., Bravo, I., Fraga, S., 1995. Autoecology and some life history stages of Dinophysis acuta Ehrenberg. J. Plankton Res. 17, 999–1015. Reguera, B., González-Gil, S., 2001. Small cell and intermediate cell formation in species of Dinophysis (Dinophyceae, Dinophysiales). J. Phycol. 37 (2), 318–333. Reguera, B., Riobó, P., Rodríguez, F., Díaz, P.A., Pizarro, G., Paz, B., Franco, J.M., Blanco, J., 2014. Dinophysis toxins: causative organisms, distribution and fate in shellfish. Mar. Drugs 12, 394–461. Reguera, B., Velo-Suárez, L., Raine, R., Park, M.G., 2012. Harmful Dinophysis species: a review. Harmful Algae 14, 87–106. Reynolds, C.S., 1987. The response of phytoplankton communities to changing lake environments. Swiss J. Hydrol. 49 (2), 220–236. Rodríguez, F., Riobó, P., Rial, P., Reguera, B., Franco, J.M., 2014. Feeding of Fragilidium cf. duplocampanaeforme and F. subglobosum on four Dinophysis species: prey specificity, local adaptation and fate of toxins. Aquat. Microb. Ecol. 72 (3), 241–253. Rothschild, B.J., Osborn, T.R., 1988. Small-scale turbulence and plankton contact rates. J. Plankton Res. 10, 465–474. Rundberget, T., Sandvik, M., Larsen, K., Pizarro, G.M., Reguera, B., Castberg, T., Gustad, E., Loader, J.I., Rise, F., Wilkins, A.L., Miles, C.O., 2007. Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2. Toxicon 50, 960–970. Shimeta, J.E.F.F., Jumars, P.A., 1991. Physical mechanisms and rates of particle capture by suspension-feeders. Oceanogr. Mar.Biol. Annu. Rev 29 (19), l-257. Smayda, T.J., 2010. Adaptations and selection of harmful and other dinoflagellate species in upwelling systems. 2. Motility and migratory behavior. Progr. Oceanogr. 85, 71–91. Smayda, T.J., Reynolds, C.S., 2001. Community assembly in marine phytoplankton: application of recent models to harmful dinoflagellate blooms. J. Plankton Res. 23, 447–461. Sullivan, J.M., Swift, E., 2003. Effects of small-scale turbulence on net growth rate and size of ten species of marine dinoflagellates. J. Phycol. 39 (1), 83–94. Sullivan, J.M., Swift, E., Donaghay, P.L., Rines, J.E., 2003. Small-scale turbulence affects the division rate and morphology of two red-tide dinoflagellates. Harmful Algae 2 (3), 183–199. Thomas, W.H., Gibson, C.H., 1990a. Effects of small-scale turbulence on microalgae. J. Appl. Phycol. 2, 71–77. Thomas, W.H., Gibson, C.H., 1990b. Quantified small-scale turbulence inhibits a red tide dinoflagellate, Gonyaulax polyedra Stein. Deep Sea Res. Part 1. Oceanogr. Res. Pap. 37 (10), 1583–1593. Thomas, W.H., Vernet, M., Gibson, C.H., 1995. Effects of small-scale turbulence on photosynthesis, pigmentation, cell division, and cell size in the marine dinoflagellate Gonyaulax polyedra (Dinophyceae). J. Phycol. 31, 50–59. Thomas, W.H., Tynan, C.T., Gibson, C.H., 1997. Turbulence phytoplankton interrelationships. In: In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research 12. Biopress, pp. 283–324. Vale, P., Botelho, M.J., Rodrigues, S.M., Gomes, S.S., Sampayo, M.A.M., 2008. Two decades of marine biotoxin monitoring in bivalves from Portugal (1986–2006): a review of exposure assessment. Harmful Algae 7, 11–25. Velo-Suárez, L., González-Gil, S., Gentien, P., Lunven, M., Bechemin, C., Fernand, L., Raine, R., Reguera, B., 2008. Thin layers of Pseudo-nitzschia spp. and the fate of Dinophysis acuminata during an upwelling‒downwelling cycle in a Galician Ría. Limnol. Oceanogr. 53 (5), 1816. Villamaña, M., Mouriño-Carballido, B., Marañón, E., Cermeño, P., Chouciño, P., da Silva, J.C.B., Díaz, P.A., Fernández-Castro, B., Gilcoto, M., Graña, R., Latasa, M., Magalhaes, J.M., Otero-Ferrer, L.J., Reguera, B., Scharek, R., 2017. Role of internal waves on mixing, nutrient supply and phytoplankton community structure during spring and neap tides in the upwelling ecosystem of Ría de Vigo (NW Iberian Peninsula). Limnol. Oceanogr. 62 (3), 1014–1030. White, A.W., 1976. Growth inhibition caused by turbulence in the toxic marine dinoflagellate Gonyaulax excavata. J. Fish. Res. Bd. Can. 33, 2598–2602. Wood, A.M., Everroad, R.C., Wingard, L.M., 2005. Measuring growth rates in microalgal cultures. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Phycological Society of America. Elsevier, Amsterdam, pp. 269–285. Wyatt, T., 2014. Margalef’s mandala and phytoplankton bloom strategies. Deep Sea Res. II 101, 32–49. Wyatt, T., Ribera d’Alcalà, M., 2006. Dissolved organic matter and planktonic engineering. Production and Fate of Dissolved Organic Matter in the Mediterranean Sea. CIESM Workshop Monograph 28. pp. 13–23. Yasumoto, T., Murata, M., 1990. Polyether toxins involved in seafood poisoning. In: Hall, S., Strichartz, G.R. (Eds.), Marine Toxins. American Chemical Society, Washington DC, pp. 120–132 ACS Symposium Series 418. Zirbel, M.J., Veron, F., Latz, M.I., 2000. The reversible effect of flow on the morphologyof Ceratocorys horrida (Peridiniales, Dinophyta). J. Phycol. 36, 46–58.

Havskum, H., Hansen, P.J., Berdalet, E., 2005. Effect of turbulence on sedimentation and net population growth of the dinoflagellate Ceratium tripos and interactions with its predator, Fragilidium subglobosum. Limnol. Oceanogr. 50 (5), 1543–1551. Hernández-Urcera, J., Rial, P., García-Portela, M., Lourés, P., Kilcoyne, J., Rodríguez, F., Fernández-Villamarín, A., Reguera, B., 2018. Notes on the cultivation of two mixotrophic Dinophysis species and their ciliate prey Mesodinium rubrum. Toxins 10 (12), 505. Jiang, H., Kulis, D.M., Brosnahan, M.L., Anderson, D.M., 2018. Behavioral and mechanistic characteristics of the predator-prey interaction between the dinoflagellate Dinophysis acuminata and the ciliate Mesodinium rubrum. Harmful Algae 77, 43–54. Juhl, A.R., Velázquez, V., Latz, M.I., 2000. Effect of growth conditions on flow-induced inhibition of population growth of a red tide dinoflagellate. Limnol. Oceanogr. 45 (4), 905–915. Juhl, A.R., Trainer, V.L., Latz, M.I., 2001. Effect of fluid shear and irradiance on population growth and cellular toxin content of the dinoflagellate Alexandrium fundyense. Limnol. Oceanogr. 46, 758–764. Juhl, A.R., Latz, M.I., 2002. Mechanisms of fluid shear-induced inhibition of population growth in a red-tide dinoflagellate. J. Phycol. 38 (4), 683–694. Karp-Boss, L., Boss, E., Jumars, P.A., 1996. Nutrient fluxes to planktonic osmotrophs in the presence of fluid motion. Oceanogr. Mar. Biol. 34, 71–107. Karp-Boss, L., Boss, E., Jumars, P.A., 2000. Motion of dinoflagellates in a simple shear flow. Limnol. Oceanogr. 45, 1594–1602. Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R., 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23 (4), 633–638. Kiørboe, T., Saiz, E., 1995. Planktivorous feeding in calm and turbulent environments with emphasis on copepods. Mar. Ecol. Prog. Ser. 122, 135–145. Kiϕrboe, T., Titelman, J., 1998. Feeding, prey selection and prey encounter mechanisms in the heterotrophic dinoflagellate Noctiluca scintillans. J. Plankton Res. 20 (8), 1615–1636. Llavería, G., Garcés, E., Ross, O.N., Figueroa, R.I., Sampedro, N., Berdalet, E., 2010. Small-scale turbulence can reduce parasite infectivity to dinoflagellates. Mar. Ecol. Prog. Ser. 412, 45–56. MacKenzie, L., Beuzenberg, V., Holland, P., McNabb, P., Selwood, A., 2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44 (8), 901–918. Mafra Jr, L.L., Nagai, S., Uchida, H., Tavares, C.P., Escobar, B.P., Suzuki, T., 2016. Harmful effects of Dinophysis to the ciliate Mesodinium rubrum: implications for prey capture. Harmful Algae 59, 82–90. Mann, K.H., Lazier, J.R.N., 1991. Dynamics of Marine Ecosystems. Biological-physical Interactions in the Oceans. Blackwell Scientific Publications, Boston. Margalef, R.M., 1978. Life forms of phytoplankton as survival alternatives in an unstable environment. Oceanol. Acta 1, 493–509. Margalef, R., Estrada, M., Blasco, D., 1979. Functional morphology of organisms involved in red tides as adapted to decaying turbulence. In: Taylor, D.L., Seliger, H.H. (Eds.), Toxic Dinoflagellate Blooms. Elsevier, pp. 89–94. Moita, M.T., Silva, A.J., 2001. Dynamics of Dinophysis acuta, D. acuminata, D. tripos and Gymnodinium catenatum during an upwelling event off the Northwest coast of Portugal. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal Blooms 2000. IOC of UNESCO, pp. 169–172. Moita, M.T., Sobrinho-Gonc¸alves, L., Oliveira, P.B., Palma, S., Falca˜o, M., 2006. A bloom of Dinophysis acuta in a thin layer off NW Portugal. Afr. J. Mar. Sci. 28, 265–269. Nielsen, L.T., Krock, B., Hansen, P.J., 2013. Production and excretion of okadaic acid, pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta–effects of light, food availability and growth phase. Harmful Algae 23, 34–45. Ojamäe, K., Hansen, P.J., Lips, I., 2016. Mass entrapment and lysis of Mesodinium rubrum cells in mucus threads observed in cultures with Dinophysis. Harmful Algae 55, 77–84. Olenina, I., 2006. Biovolumes and Size-classes of Phytoplankton in the Baltic Sea, Helsinki, HELCOM Balt. Sea Environ. Proc. No. 106. pp. 144. Palma, A.S., Vilarinho, M.G., Moita, M.T., 1998. Interannual trends in the longshore variation of Dinophysis off the Portuguese coast. In: Reguera, B., Blanco, J., Fernández, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO. Santiago de Compostela, pp. 124–127. Papiol, G.G., Beuzenberg, V., Selwood, A.I., MacKenzie, L., Packer, M.A., 2016. The use of a mucus trap by Dinophysis acuta for the capture of Mesodinium rubrum prey under culture conditions. Harmful Algae 58, 1–7. Park, M.G., Kim, S., Kim, H.S., Myung, G., Kang, Y.G., Yih, W., 2006. First successful culture of the marine dinoflagellate Dinophysis acuminata. Aquat. Microb. Ecol. 45, 101–106. Park, M.G., Kim, M., Kim, S., Yih, W., 2010. Does Dinophysis caudata (Dinophyceae) have permanent plastids? J. Phycol. 46 (2), 236–242. Persson, A., Smith, B.C., Wikfors, G.H., Alix, J.H., 2008. Dinoflagellate gamete formation and environmental cues: observations, theory and synthesis. Harmful Algae 7 (6), 798–801. Peters, F., Gross, T., 1994. Increased grazing rates of microplankton in response to smallscale turbulence. Mar. Ecol. Prog. Ser. 115, 299–307. Peters, F., Redondo, J.M., 1997. Turbulence generation and measurement: application to studies on plankton. Sci. Mar. 61, 205–228. Pizarro, G., Paz, B., González-Gil, S., Franco, J.M., Reguera, B., 2009. Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: differences

10