Fine scale physical-biological interactions during a shift from relaxation to upwelling with a focus on Dinophysis acuminata and its potential ciliate prey

Contents lists available at ScienceDirect Progress in Oceanography journal homepage: www.elsevier.com/locate/pocean Fine scale physical-biological interactions during a shift from relaxation to upwelling with a focus on Dinophysis acuminata and its potential ciliate prey Patricio A. Díaz a,b,⁎ , Manuel Ruiz-Villarreal c , Beatriz Mouriño-Carballido d , Concepción Fernández-Pena c , Pilar Riobó e , Beatriz Reguera a a Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Vigo, Subida a Radio Faro 50, 36390 Vigo, Spain b Centro i∼mar & CeBiB, Universidad de Los Lagos, Casilla 557, Puerto Montt, Chile c Instituto Español de Oceanografía (IEO), Centro Oceanográfico de A Coruña, Paseo Marítimo Alcalde Francisco Vázquez 10, 15001 A Coruña, Spain d Departamento de Ecología y Biología Animal, Universidad de Vigo, Campus Universitario As Lagoas-Marcosende, E-36310 Vigo, Spain e Instituto de Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, 32208 Vigo, Spain ARTICLE INFO Keywords: Dinophysis acuminata Potential ciliate prey Physical-biological interactions Fine scale structure Turbulence, division rates Circadian variability, Galician Rías ABSTRACT Wind reversals and quick transitions from relaxation to upwelling in coastal areas cause major changes in water column structure, phytoplankton distribution and dominance, and rates of physiological processes. The cruise “ASIMUTH-Rías” (17–21 June 2013) was carried out in the Galician Rías and adjacent shelf, at the time of a DSP outbreak, to study small-scale physical processes associated with late spring blooms of D. acuminata and accompanying microzooplanktonic ciliates with the overall objective of improving predictive models of their occurrence.The cruise coincided with the initiation of an upwelling pulse following relaxation and deepening of a previously formed thin layer of diatoms. A 36-h cell cycle study carried on 18–20 June showed the vertical excursions of the thin layer, mainly delimited by the 13.5–14 °C isotherms and turbulence levels (ε) of 10 −8 –10 −6 m 2 s −3 , as well as marked changes in phytoplankton composition (increased density and dominance of diatoms). There was no evidence of daily vertical migration of D. acuminata, which remained in the top layer during the cycle study, but the opposite was observed in the ciliate populations. Dinophysis and its potential prey (Mesodinium species) cell maxima overlapped after midday, when the ciliate moved to the surface, suggesting an “ambush” strategy of Dinophysis to catch prey. A remarkable decline (from 0.65 to 0.33 d −1 ) in division rates (µ) of D. acuminata was associated with increased turbulence (ε< 10 −4 m 2 s −3 ) near the surface and a sharp drop of temperature (> 2°C in about 8 h). In contrast, high division rates (µ min ∼ 0.69 d −1 ) persisted at a mid-shelf station where environmental conditions below the mixed layer were more stable. The onset of upwelling pulses appears to have a double negative effect on the net growth of Dinophysis populations: a direct physical effect due to advective dispersion and an indirect effect, decreased division rates. The latter would be caused by the rapid cooling of the mixed layer, and the increased turbulence at the surface resulting in shear stress to the cells. The short-term impact of upwelling pulses (and the winds promoting it) on the physiology of Dinophysis and its ciliate prey, and the role of mid-shelf populations of Dinophysis as a relatively undisturbed reservoir for the inoculation of subsequent blooms are discussed. 1. Introduction Low density (10 2 –10 4 cells L −1 ) populations, i.e. low biomass harmful algal blooms, of Dinophysis acuminata, producer of diarrhetic shellfish poisoning (DSP) toxins, are the main cause of endemic shellfish harvesting bans in the Galician Rías Baixas, Northwest Iberian Peninsula (Blanco et al., 2005; Reguera et al., 2014). These rías are located in the northern limit of the Canary Current-Iberian Peninsula upwelling system (Fig. 1A). Winds, which vary considerably at a variety of temporal scales related to atmospheric oscillations, are the main drivers of hydrodynamics in upwelling systems (GEOHAB, 2005). Upwelling in northwestern Iberia is seasonal. On an annual basis, variability results from the seasonal evolution of the Azores high and the Iceland low which determine the wind regime and the onset of an upwelling (spring-summer) and a downwelling season (autumn-winter), separated by transition periods in spring and autumn (Bakun and Nelson, 1991). Variations in the wind regime on a time scale of days, due to changes in the position and strength of the high-low pressure https://doi.org/10.1016/j.pocean.2019.04.009 Received 6 October 2017; Received in revised form 18 February 2019; Accepted 29 April 2019 ⁎ Corresponding author at: Centro i∼mar & CeBiB, Universidad de Los Lagos, Casilla 557, Puerto Montt, Chile. E-mail address: [email protected] (P.A. Díaz). Progress in Oceanography 175 (2019) 309–327 Available online 29 April 2019 0079-6611/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T systems, cause short-term hydrodynamic variability resulting in upwelling-downwelling cycles (Álvarez-Salgado et al., 2003; Gilcoto et al., 2017). Turbulence plays a fundamental role in the structure and distribution of phytoplankton communities (Margalef, 1978; Smayda and Reynolds, 2001). Thus, diatoms dominate during mixing periods and dinoflagellates are more abundant during stable conditions (Margalef, 1978; Wyatt, 2014). Upwelling, an important source of turbulence in coastal systems, and wind stress controlling the upwelling-downwelling cycles, have been identified as the main drivers affecting the dominance of phytoplankton populations in the Galician Rías Baixas (Tilstone et al., 2000). In addition, short-term succession of these populations is modulated by semidiurnal and spring-neap tidal signals and its coupling to upwelling-downwelling events (Díaz et al., 2014). Recently, Villamaña et al. (2017) used a microstructure turbulence profiler during spring and neap tides to characterize internal wave activity in the outer reaches of the Ría de Vigo. These authors showed that enhanced mixing, partially linked to intense internal wave activity during spring tides, in addition to the constructive interference of the shear associated with the upwelling and tidal currents (FernándezCastro et al., 2018), caused a significant increase in nitrate diffusive fluxes. This nitrate supply could contribute to the dominance of largesized diatoms during the upwelling favourable season (April-September) (Figueiras and Ríos, 1993; Moita and Silva, 2001). Nevertheless, turbulent mixing may also have disruptive effects on phytoplankton cells and in broad terms, dinoflagellates have been found to be more sensitive to turbulence than diatoms (Margalef et al., 1979; Thomas et al., 1995). Deleterious effects include changes in morphology (Berdalet and Estrada, 1995; Zirbel et al., 2000) and decline of cell division rates (Thomas and Gibson, 1990b; Thomas and Gibson, 1990a; Gibson and Thomas, 1995; Thomas et al., 1995). The D. acuminata growth season, i.e. the time since a numerical increase in cell densities starts until the population declines to winter levels (< 40 cells L −1 ), has been found to be tightly coupled to the local spring-summer upwelling season (Díaz et al., 2013; Velo-Suárez et al., 2014). Therefore, interactions between biological processes in Dinophysis populations (growth, vertical migration, toxin production) and quick changes of water-column structure associated with upwellingdownwelling cycles need to be scrutinized for the development of realistic prediction models (Ruiz-Villarreal et al., 2016). For a given population, growth results from the positive balance between gains and losses and is governed by the fundamental equation: =dN t dt rN t( )/ ( ) Fig. 1. Map of the study area showing the (A) Iberian Peninsula; (B) Northwest coast of Spain (the box delimits the Galician Rías Baixas) and the northern half of Portugal; (C) Map of the survey’s sampling grid in the rías of Vigo (V0-V8) and Pontevedra (P1-P8). Red asterisks indicate the fixed station for the 36-h study (P2) and the shelf (100 m) station (V6) for estimates of vertical distribution of division rates; red square and black triangle, the mooring position of the ADCP and Cabo Silleiro Seawatch buoy, respectively. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article. P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 310 = +r µ I E g m n m where ris the net growth rate and µthe specific growth rate; I n and E m are immigration (advection, cyst germination) and emigration (dispersion, encystment), ggrazing and m, natural mortality rates. The in situ specific growth rate, µ , estimates the potential for intrinsic division without the interference of losses due to grazing, mortality and physical dispersion (Carpenter and Chang, 1988). Therefore, estimates of µhelp to identify oceanographic conditions that promote either active in situ division or accumulation resulting from physical/biological interactions (Reguera et al., 2003). Even more important for modeling purposes is to estimate μ max , i.e. the maximum potential division rate of the species under optimal conditions when resources are not limiting (VeloSuárez et al., 2009). Models tend to use constant division rates, derived from laboratory experiments, for short (week) time-scale forecasts. Nevertheless, fast day-scale shifts in coastal wind-driven circulation, modulated by tides, may have dramatic effects on the net growth rate of a species population. The model of Carpenter and Chang (1988), based on the mitotic index approach (McDuff and Chisholm, 1982), has been successfully used to estimate in situ division rates (µ avg ) of Dinophysis populations in different coastal environments (Chang and Carpenter, 1991; Garcés et al., 1997; Reguera et al., 2003; González-Gil et al., 2010; Aissaoui et al., 2014; Farrell et al., 2014). In most cases, an integrated or average value of µ(µ avg ) for the whole water column, estimated from vertical net haul samples, was obtained. Nevertheless, in two cases when estimates of division rates at different depths µ z were carried out, important vertical heterogeneities were observed (VeloSuárez et al., 2009; Farrell et al., 2014). Dinophysis acuminata, the target dinoflagellate species of this study, is an obligate kleptoplastidic mixotroph, i.e., it requires light, nutrients and plastids stolen from its ciliate prey for sustained growth (Park et al., 2006, Kim et al., 2008). To date, the phototrophic ciliate Mesodinium rubrum, which in turn requires cryptophyte prey belonging to the Teleaulax/Plagioselmis/Geminigera (TPG) clade, is the only known prey for Dinophysis species established in culture. This ciliate acts as a vector of cryptohyte plastids for the dinoflagellate (Park et al., 2006). The possibility of other plastid-retaining phototrophic ciliates being used as alternative prey to Mesodinium by Dinophysis species needs to be explored. For all these reasons, special attention was focused during the 36-h cycle study, on the distribution of Mesodinium and other plastidbearing microzooplanktonic ciliates in relation to their potential predator, Dinophysis. The cruise ASIMUTH-Rías was carried out in June 2013 in the Galician Rías of Vigo and Pontevedra and their adjacent shelf (Fig. 1B) to study small-scale physical-biological interactions in late spring blooms of D. acuminata during the upwelling season. These included the circadian variability of Dinophysis physiology (division rate, toxin content), daily vertical migration and biological interactions with their potential ciliate prey. Our main objective was to examine, with the support of high vertical resolution measurements of turbulence and current velocities, the short-term response of D. acuminata (distribution and physiology) and accompanying ciliate populations to rapid shifts from upwelling relaxation to new upwelling pulses in the Galician Rías Baixas. 2. Material and methods This work was part of the cruise “ASIMUTH-Rías 2013”, carried out on board R.V. Ramón Margalef from 17 to 21 June 2013 in the rías of Vigo and Pontevedra and their adjacent shelf. The cruise took place during a long-lasting DSP outbreak which led to shellfish harvesting closures from mid March to mid September in aquaculture sites in Ría de Pontevedra (Ruiz-Villarreal et al., 2016). Longitudinal transects from Ría de Pontevedra and Ria de Vigo (this one incomplete due to a ship breakdown on June 21) to the adjacent shelf were sampled on June 18 and 21 (Fig. 1C). In addition, a 36 h sampling, from 18 to 20 June, was carried out at a fixed station (P2, Bueu) from Ría de Pontevedra. This station is a hot-spot for diarrhetic shellfish poisoning (DSP) events, i.e. harvesting bans when toxins in shellfish meat exceed regulatory limits, in the Galician Rías Baixas (Blanco et al., 2013). 2.1. Meteorology and hydrology Measurements of wind speed and direction were recorded from a buoy (Seawatch, Oceanor), belonging to the national port system (Puertos del Estado) network, deployed in Cabo Silleiro (42°7.89N − 9°23.49W) (Fig. 1C), a representative station for the study area. Additionally, high frequency (every 1 min) wind speed and direction data were obtained from the meteorological station located on board R/V Ramón Margalef. Estimates of Ekman’s transport (m 3 s −1 km −1 ) according to Bakun (1973), every 6 h, were obtained from the Spanish Institute of Oceanography (IEO) (www.indicedeafloramiento.ieo.es) using geostrophic wind data from the buoy station. Tides and sea level measurements were taken with a tidal gauge from the IEO network deployed on Ría de Vigo harbour (http://indamar.ieo.es/). Differences in amplitude (< 10 cm) and tidal phase (1–2 min) in the tides semidiurnal component in both rías are negligible. 2.2. Microstructure turbulence profiler and ADCP measurements Measurements of turbulence microstructure were obtained with a MSS (Prandke and Stips, 1998) profiler during the 36 h cell-cycle study at the fixed station (P2), and at the V5 shelf station (∼100 m depth) on June 18 and 21. The profiler was equipped with 2 velocity microstructure shear sensors (type PSN06), a microstructure temperature sensor (FP07), a sensor to measure horizontal acceleration of the profiler and a high-precision CTD probe including a fluorescence sensor. The acquisition and processing of the shear data were performed with the commercial software SST-SDA (Standard Data Acquisition) and ProDat Sea & Sun Technology (www.sea-sun-tech.com/technology. html). The turbulent kinetic energy dissipation rate (ε) was estimated from the shear data following Fernández-Castro et al. (2014). In short, ε was computed in 512 data point segments, with 50% overlap, from the shear variance, under the assumption of isotropic turbulence, using the following equation: = u z 7.5 (W kg ) 2 1 (1) where νis the kinematic viscosity of seawater, ∂u=∂z the vertical shear and 〈·〉 represents the ensemble average. The shear variance was computed by integrating the shear power spectrum. The lower integration limit was determined considering the size of the bins, and set to 2 cpm. The upper cut-off wave number for the integration of the shear spectrum was set as the Kolmogoroff number =k( ·( / ) cpm) c 1 2 3 1/4 . An iterative procedure was applied to determine k c . The maximum uppercut-off was not allowed to exceed 30 cpm to avoid the noisy part of the spectrum. Assuming a universal form of the shear spectrum, εwas corrected for the loss of variance below and above the integration limits, using the polynomial functions reported by Prandke et al. (2000). Values of εwere then averaged in 1 m bins. Peaks due to particle collisions were removed by comparing the dissipation rates computed simultaneously from the two shear sensors. An RDI 600 Hz Sentinel Workhorse ADCP was moored, from June 14 to July 4, 2013, near the centre of the main navigation channel, between stations P2 and P3 (42° 21.41′ N; 08° 50.02′ W, Fig. 1C). Nominal depth at the mooring was 37 m. The ADCP was installed in a low drag float 2 m above the sea floor. Data were acquired through 3 min temporal ensembles and 1 m vertical bins. Raw data were postprocessed with the IMOS ADCP toolbox and several quality control tests carried out ─including tilting, echo intensity, correlation and side lobe P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 311 effects─to produce QC (quality controlled) current data at fixed depths above the sensor using tilt information. 2.3. Model simulations Surface current fields were obtained from a Regional Ocean Model System (ROMS) configuration developed at IEO in the framework of the Iberian Margin Ocean Observatory project RAIA (Otero et al., 2013). This configuration was the same as that used by Ruiz-Villarreal et al. (2016) to investigate the effects of hydrodynamical conditions on toxic dinoflagellate blooms in Galicia. A high resolution 1.3 km grid centred in the Galician coast is nested in a 4 km grid of the Iberian shelf and slope. Realistic forcing includes tides, run-off from several rivers and atmospheric fluxes from the operational configuration of the Weather Research and Forecasting (WRF) mesoscale model run by Meteogalicia (www.meteogalicia.es). This forecast model configuration allows describing the response of the shelf and rias circulation to upwellingdownwelling pulses and its interplay with different physical forcings (Ruiz-Villarreal et al., 2016). 2.4. Field sampling Data on Dinophysis cell densities in Ría de Pontevedra before and after the cruise were taken from weekly reports of phytoplankton distribution from the Galician Monitoring Programme at INTECMAR (www.intecmar.gal). In this programme, weekly plankton samples for quantitative analyses are collected with a dividable hose (tube-sampler) that samples the whole water column from 0 to 15 or 20 m, and immediately fixed on board with acidic Lugol’s iodine solution. During the survey, vertical profiles of temperature, salinity and fluorescence were obtained with a Sea-Bird SBE-25 conductivity-temperature-depth (CTD) profiler combined with a 12-bottle (2.5 L each) rosette (General Oceanics, USA). Water samples for quantitative analysis of microphytoplankton (Dinophysis) and ciliates (every 2 h during the 36 h study), and Dinophysis division rate estimates (during the 36 h study and one vertical profile on a shelf station, see Section 2.7) were taken from 6 different depths selected after reading the CTD profile. These depths included surface (3 m), near the seabed, at the chlorophyll (chl) maximum and wherever any relevant discontinuity of physical properties was observed. For Dinophysis studies, two kinds of samples were collected every 2 h from discrete depths: (1) unconcentrated seawater samples, immediately fixed with acidic Lugol’s iodine solution, for quantitative analysis and (2) concentrated water samples (2.5 L), filtered through 20-µm nytex filters and resuspended in 50 mL of seawater fixed with formalin, to enumerate cell-cycle phases of D. acuminata (concentration factor = 50). Additional water samples of 250 mL, fixed with Lugol’s solution, were taken for ciliate analyses. Sampling for division rate estimates, during the cell-cycle study, was carried out every 2 h from 08:00 h to 02:00 h the next day (GMT), and every hour from 02:00 h to 08:00 h, which is the time window before dawn when phased division of Dinophysis acuminata. is observed, and proportion of dividing and recently-divided cells change very rapidly (Reguera et al., 2003). For toxin analyses, vertical net hauls (one every 2 h) with a 20-μm mesh net were collected and passed through a 150-μm mesh to eliminate large microzooplanktonic organisms; an aliquot (∼15 mL) from each haul sample, immediately fixed with acidic Lugol’s solution, kept for cell counts; 200 mL of the hauled material filtered through Whatman (Whatman, Maidstone, England) GF/F fiberglass filters (47 mm Ø, 0.7-µm pore size), the filter and filtered material placed in a centrifuge tube and covered with analysis grade methanol; 50 mL of the filtered haul water collected for extracellular toxin analyses in a Falcon tube and all tubes placed in the vessel’s deep-freeze and later transported in a portable fridge before final storage in the laboratory at −20 °C until analysis. 2.5. Toxin analyses Toxin analyses by LC-HRMS were carried out in positive mode with a Thermo Scientific Dionex High-Speed LC coupled to an Exactive mass spectrometer equipped with an Orbitrap mass analyzer and a HESI-II probe for electrospray ionization. Toxins were separated using an Aquity C18 column (2.1 × 150 mm, 3 µm particle size) maintained at 35 °C with a flow rate of 400 µL min −1 . The mobile phase consisted of 5 mM ammonium acetate pH 6.8 (A) and 95% MeOH: 5% mobile phase A (B). A linear gradient elution from 60% B to 100% B was run for 20 min. 100% B was held for 2 min before returning to the initial conditions of 60% B in 3 min. This percentage was held until min 30. Certified reference standard solutions of okadaic acid (OA), dinophysistoxin-2 (DTX2) and pectenotoxin-2 (PTX2) were purchased from the National Research Council (Canada). Calibration curves were obtained in triplicate for each toxin standard (variation coefficient 14%). The toxin concentration in sample extracts was quantified by comparing the area or the peaks obtained in the chromatograms with those of the certified reference material solutions. 2.6. Plankton analyses For quantitative analyses of Dinophysis species, 25 mL of unconcentrated acidic Lugol’s-fixed samples were left to sediment for 24 h and analysed under an inverted microscope (Nikon Eclipse 2000) using the method described in Utermöhl (1958). The whole surface of the chamber was scanned at a magnification of ×100, so that the detection limit was 40 cells L −1 . For quantitative analyses of ciliates, 100 mL of unconcentrated Lugol’s-fixed samples were left to sediment for 48 h. Taxonomic classification was based on Lynn and Small (2002) criteria. Counts were performed at a 200× magnification under a Nikon Eclipse TE 300 inverted microscope according to Utermöhl (1958). 2.7. Estimates of division rates To enumerate Dinophysis cell-cycle stages, the concentrated water samples (2.5 L filtered and resuspended in 50 mL, concentration factor 1/50) were left to sediment over 12 h and the whole surface of the chamber scanned under the inverted microscope at ×100, so the detection level ranged from 0.8 to 2 cells L −1 . In situ division rates were estimated, with a post-mitotic index approach, from the frequency of dividing (paired) and recently divided (incomplete development of the left sulcal list) cells, which were recognized by their distinct morphology as described in Reguera et al. (2003), following the model of Carpenter and Chang (1988): = + + + = µn T T t s ln f t f t 1 ( ) ( | ) [1 ( ) ( )] c r i n iciri 1 (2) where µis the daily average specific division rate, f c (t i ) is the frequency of cells in the cytokinetic (or paired cells) phase (c) and f r (t i ) is the half frequency of cells in the recently divided (incomplete development of the left sulcal list) (r) phase in the i th sample. T c and T r are the duration of the cand rphases, considered as “terminal events” (sensu Carpenter and Chang, 1988) in this work; nis the number of samples taken in a 24-h cycle, and t s is the sampling interval in hours. The duration of the selected terminal events, T c +T r , was estimated as the interval of time necessary for a cohort of cells to pass from one phase to the next; in this case, the time interval between the time t 0 —when the frequency of cells undergoing cytokinesis, ƒ c ,is maximum—and the time t 1 when the fraction of recently divided cells ƒ r is maximum: + =T T t t 1 2( ) ( ) c r 0 1 (3) where T c ,T r ,t 1 and t 0 are calculated after fitting a 5th degree Gaussian P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 312 function to the frequency data. Frequency of different cell-cycle stages was quantified only in samples providing a minimum number of specimens (n > 200) to have statistically sound results. For comparative purposes, the vertical distribution of the lower bound of division rates (µ min ) at peak-division time (06.00 GMT) was estimated from a single vertical profile at one shelf station (100 m deep) off Ría de Vigo on June 21. The ‘maximum frequency approach’ (McDuff and Chisholm, 1982) was used to estimate µ min at each depth z, (µ min ) z : = +µ ln f(1 ) min max (4) where f max is the frequency of dividing cells at each depth. This approach assumes that all cells which divide in a given day can be recognized as undergoing or just completed mitosis, in one single sample collected at the time-window when maximal division is expected. 3. Results 3.1. Hydrographic conditions before and during the cruise June 2013 showed a succession of short-term (5–14 days) upwelling-downwelling cycles which is a common feature during the upwelling season in the study area (Alvarez-Salgado et al, 2003). Nevertheless, daily average Ekman’s transport estimate (751 ± 1201 m 3 s −1 km −1 ), with a maximal value of 2587 m 3 s −1 km −1 on June 23, showed a strong positive anomaly in relation to the historic mean (1985–2013) for June (373 ± 363 m 3 s −1 km −1 ) (Fig. 2A) (Díaz et al., 2016). Northerly winds with a mean velocity of 8.1 m s −1 and a maximum of 10.5 m s −1 on June 19 (Fig. 2B), were predominant during the cruise, which started with neap tides (Fig. 2C). Hydrographic conditions in two longitudinal Ría de Vigo and Ría de Pontevedra-shelf transects showed common patterns in their response to a transition, that had occurred 2 days before (June 16), from relaxation to upwelling. These included shoaling of the isotherms (not so clear in Fig. 3A because of the missing stations in the outer reaches of Ría de Vigo) and a quick drop of temperature (> 2 °C) in the top 10 m of the two rías (Fig. 3). A higher resolution examination during the 36-h study showed that maximal gradients of temperature and salinity were found at 18–19 m at the beginning of the cruise (Fig. 4B–C), and at 10 m 26 h later. A prominent thin fluorescence layer was formed and followed the excursions of the pycnocline (Fig. 4D). Wind velocities, obtained from the meteorological station on board R/V Ramón Margalef, were very variable, with a mean velocity of 4.3 m s −1 . From 08:00 h to 20:00 h on June 19, a significant increase in northerly winds velocity was observed, with a maximum of 12.6 m s −1 at 15:00 h (Fig. 4A). Nevertheless, from 20:00 h onwards there was a progressive decline, with values lower than 1 m s −1 at the end of the 36-h cycle study. At the same time, high (∼10 −4 m 2 s −3 ) near surface values of turbulence (ε), with maxima from 00:00 h to 02:00 h and 10:00 h to 14:00 h on June 19 and from 20:00 h (June 19) to 02:00 h on June 20, were observed, followed by a progressive decline the last day (June 20) from 04:00 h onwards. Values of εat the depth of the thin layer, delimited by the 13.5–14 °C isotherms, increased from 10 −8 m 2 s −3 at the beginning of the cycle study (20:00 h on June 18) to 10 −6 m 2 s −3 (00:00 h on June 19). These changes coincided with the progressive shoaling of the thin layer that reached a 10 m depth between 00:00 h and 02:00 h on June 20. Thus, the uprising velocity of the inflowing water was around 0.34 m h −1 , and the thin layer was always associated with the pycnocline (Fig. 4F–I). Fig 2. Time series of (A) Estimates of daily upwelling indices, Q x (m 3 s −1 km −1 ) (positive values indicate upwelling), estimated from Silleiro buoy (Western Galicia) data, during June 2013, (B) vector diagram of wind direction and velocity (m s −1 ) recorded hourly at the Cabo Silleiro buoy (positive values correspond to northerly winds) and C) sea level (m) recorded hourly. Shaded area indicates the days of the cruise. P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 313 Currents from the ADCP moored at the centre of the navigation channel (red square in Fig. 1, close to the position of the 36 h study) showed the currents variability in response to tides and to the northerly (upwelling) and southerly (downwelling) winds (Fig. 5). Before the cruise, a downwelling event on June 16 was associated with surface inflow and also with northward cross-channel flow (Fig. 5A, B). Tidal modulation of the currents was observed although the downwelling event occurred near neap tides. After this downwelling event, weak upwelling was associated on June 18 midday with inflow in the bottom layers and a near surface outflow, modulated by tides, of the water previously retained in the inner parts of the ria during downwelling. The outflowing layer became progressively thinner while the inflow layer increased (Fig. 5A). The tidal signal was clearer when the cycle study began (June 18 night). During the rest of the cycle, inflow and outflow occurred during ebb and flood (tidal amplitude was increasing), although upwelling conditions until relaxation on June 20 modulated the currents due to the upwelling-induced surface outflow and bottom inflow. After the cycle, the tidal signal was clear too, and again modulated by the peak of the upwelling-induced surface outflow on June 22. Results of model simulations for sea surface temperature (SST) and current velocities confirmed changes from weak inflowing currents favouring retention inside the rías on June 16 to strong outflowing currents from June 18 (the day of initiation of the 36-h study) onwards (Fig. 6). From June 19, these simulations revealed the presence of cold (∼14 °C) upwelled surface water along the Galician coast, to the south of Ría de Vigo. Although the model did not resolve in detail the circulation inside the Ria due to the coarse resolution (1 km), it was possible to assess variability of the inflow-outflow into the Ría, and a picture compatible with that depicted by the ADCP was obtained. 3.2. Distribution of Dinophysis and other dinoflagellate species and ciliates During relaxation, longitudinal ría-shelf transects showed that maximal densities of Dinophysis (1 × 10 3 cells L −1 ) near the surface (3–5 m) in the inner reaches of the rías were located in the warm (> 16 °C) water layer, most likely advected from the shelf the previous days (Fig. 3A–B). After the upwelling pulse, mixed surface waters cooled down (< 15 °C) and Dinophysis maxima (2.2 × 10 3 cells L −1 ), located in the top 5 m of the water column, moved off to the outer reaches of the rías and shelf waters (Fig. 3C–D). Data from the Galician Monitoring Centre on the distribution of Dinophysis (integrated tube-samples) in Ría de Pontevedra (10 stations) the weeks before, during and after the cruise (Fig. 7) support the idea of Fig. 3. Vertical distribution of temperature (°C) and D. acuminata (cells L −1 ) in ría-shelf transects from Ría de Vigo (A, C) and Ría de Pontevedra (B, D) on June 18 and June 21. P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 314 Fig. 4. Vertical distribution of (A) Wind velocities recorded every 1-minute on board R/V Ramón Margalef; (B) Temperature; (C) Salinity, (D) In vivo fluorescence and E) Turbulent kinetic energy dissipation rate (ε) derived from the microstructure profiler deployed at a fixed station (P2) during the 36-h cycle study, 18–20 June 2013. Arrows in the top panel (sea level) indicate the hours corresponding to the vertical profiles of the temperature, salinity and fluorescence shown below (F-E). P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 315 Fig. 5. Eastwards (top panel) and northwards (bottom panel) current velocity components (instantaneous flow variability) from the bottom mounted ADCP at station P3 in Ría de Pontevedra between 14 and 26 June. ADCP pressure sensor is plotted as a reference. Fig. 6. Model generated distribution of daily sea surface temperature (SST) (°C) and surface currents from June 16 to 21. P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 316 advection into and out of the ría suggested before (Fig. 5). Prior to the cruise (June 10), D. acuminata cell densities ranged between 280 and 1240 cells L −1 . The week of the cruise (June 18), relaxation-driven cross-shelf transport led to a Dinophysis cell maximum (> 3 × 10 3 cells L −1 ) in the innermost part of the ría. The ADCP records provided evidence of this transport between 15 and 17 of June (Fig. 5). The surface outflow during ebb tide coincided with the observation of a dense population of Dinophysis on June 18. The population decline observed during the cycle was depicted in the following week’s monitoring results (June 25) from Ría de Pontevedra (Fig. 7). During the 36-h cycle study, vertical haul-samples from the top 15 m showed the low-density microplankton community was dominated by large chain-forming diatoms (Detonula pumila,Thalassiosira rotula,Chaetoceros spp) with half-empty cells and a decaying appearance in addition to detritus. The most striking temporal change was the sudden inflow of Protoperidinium spp. (P. conicum,P. divergens,P. diabolum) between 12:00 h and 20:00 h on June 19 (Table 1). Remarkable changes were also observed in D. acuminata, the most abundant dinoflagellate species throughout the study. Densities in the vertical net hauls ranged from 7 to 8 cells mL −1 at 20:00 h on June 18 to 150–170 cells mL −1 at 16:00–18:00 h on June 19. These extreme values coincided with high and low tide respectively (Table 1). From all the analysed samples collected at discrete depths, there was no evidence of daily vertical migration of D. acuminata during the 36 h study, and cell maxima remained in the warmer (> 16 °C) and more brackish (< 35) surface layer, above the 25.4 σ t isopycnal (Fig. 8A). Cell densities increased during the outward tidal flow (ebb tide), and the cell maximum, 2.4 × 10 3 cell L −1 , at 5 m, was observed at 03.00 on June 19, coinciding—as in the haul samples—with low tide. In contrast Mesodinium cells with marked morphological variability, probably including at least two species (Mesodinium rubrum and M. major), showed maximal cell densities below the pycnocline at night, but aggregated at the surface between 14:00 h and 16:00 h, the only time window when they overlapped the Dinophysis cell maxima (Fig. 8A). As in the case of Dinophysis, the ciliate cell maximum (5 × 10 3 cell L −1 ), was observed at the surface (3 m), on June 19 at low tide. Mesodinium spp. were the main component of the microzooplanktonic ciliate populations at peak hours (14:00 to 16:00 h in the afternoon and 02:00–04:00 h at night (Fig. 8B). Other identified plastidretaining ciliate species, such as Laboea strobila, Strombidium spiralis, Strombidium acutum and Strombidium epidemum reached high densities, sometimes overtaking Mesodinium’s dominance. In addition, a relevant contribution of ciliates for which retained plastids have not been described, including Legaardiella sol,L. ovalis,Lomhaniella oviformis and the well known prey of the heterotrophic Phalacroma rotundatum, Tiarina fusus (Fig. 8B) was noted. Attention should be paid to a second cell maximum of Dinophysis near the bottom which appeared on three occasions just before low tide (Fig. 8A). 3.3. Estimates of µ and particulate and dissolved toxins during the 36 h study Monitoring for 36 h at the fixed station the fraction of cells undergoing mitosis showed that there were marked differences in frequencies of mitotic cells at different depths (Table 2). Maximal frequencies were observed at 3–7 m—above the 25.4 σ t isopycnal—where the cell maxima were found throughout the two cycles on June 19 and June 20. Frequencies observed in the cell-maximum layer were used to estimate Fig. 7. Weekly variation of Dinophysis acuminata cell densities (data from the Galician Monitoring Centre) in Ría de Pontevedra (mean of 10 monitoring stations) from February to September 2013 (top panel) and spatial distribution of D. acuminata cell maxima in Ría de Pontevedra before (June 10), during (June 18) and after (June 25) the cruise ASIMUTH-Rías 2013. Error bars in the top panel represent mean and standard deviation (n = 10). P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 317 the time. The two populations appeared to have distinct niches and distinct responses to physical forcing. In contrast, Villarino et al. (1995) reported that D. acuminata and Mesodinium performed a similar DVM pattern in Ría de Vigo. The fact that their sampling took place at the end of summer (19–20 September 1991) under very calm weather and strong thermal stratification (4.5 °C gradient between the surface and 9 m depth) may explain the different behaviour of the dinoflagellate and its ciliate prey. These observations support the view that Dinophysis behaviour varies depending on the nutritional status and phase of the population growth (Reguera et al., 2012). But our observations in previous studies and here suggest a possible “ambush” strategy of Dinophysis to catch its fast swimming ciliate prey: to remain the whole day Fig. 11. Vertical profiles on a shelf station (V6) off Ría de Vigo. (A-B) Vertical distribution of temperature, salinity and in vivo fluorescence on 18 and 21 June at 06:00 GMT. (C) Vertical distribution of Dinophysis acuminata (cells L −1 ) and µ min , and of (D) Total densities of diatoms and dinoflagellates (cells L −1 ) on June 21, 2013. P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 324 at a fixed depth and intercept the ciliates as they ascend. Physiological studies with D. acuminata and M. rubrum cultures shed new light on their behavioural differences. Both species are kleptoplastidic mixotrophs, but carbon uptake from live prey in Mesodinium represents only about 2% of its daily intake, whereas it is 50% in the case of Dinophysis acuminata (reviewed by Hansen et al., 2013). Thus, D. acuminata may grow in nutrient-poor surface waters provided it has ciliate prey, but Mesodinium will be more dependent on resources from nutrient-rich layers, in or below the pycnocline, visited during DVM at night, and light for photosynthesis at midday. Of special interest are other plastid-bearing ciliates which co-occured with D. acuminata in this study. Plastid-retaining oligotrichous ciliates (e.g. Cyrotostrombidium,Laboea,Strombidium and Tontonia) might provide alternative prey for growth of Dinophysis species provided they retain plastids of suitable cryptophyte species (Stoecker et al., 2009). So far, only cryptophyte plastids belonging to the Tetraselmis/Plagioselmis/Geminigera (TPG) clade have been regularly found in field specimens of Dinophysis species (Reguera et al., 2012). Over 90% of the plastids sequenced from Dinophysis, mainly D. acuminata, in the Galician Rías had a difference of just 1 bp from those of Teleaulax amphioxeia (Rial et al., 2015). Nevertheless, plastids from more species and strains of Dinophysis and from more mixotrophic ciliates need to be analyzed as potential alternative prey to Mesodinium. 4.3. Changes in division-rates (µ) in D. acuminata populations during the transition from relaxation to upwelling The post-mitotic index method applied in this study is a simple and reliable way to estimate in situ division rates of Dinophysis species representing a small fraction of the microplankton community. This method has been applied in several previous studies in the Galician Rías, and has provided background information on the cell-cycle behaviour of different species of Dinophysis, including their specific timewindow for synchronized or in-phase division (Reguera et al., 2003; Pizarro et al., 2008; Velo-Suárez et al., 2008, 2009). Estimates of in situ division rates (µ avg ) of Dinophysis spp. in previous studies have shown large variability related to the population growth phase and to changing environmental conditions. Almost nil division rates (0.09 d −1 ) were observed during the stationary phase of a D. acuminata population in the same area and time of year (mid-June 1998) (Reguera et al., 2003) and in a D. acuta population with a sharp increase in net growth associated with physical accumulation of stationary-phase shelf populations (Escalera et al., 2010). In contrast, high rates close to one division per day (µ= 0.6 d −1 ), presumably triggered by a recent encounter and feeding on a Mesodinium patch, were observed in June 2005 (González-Gil et al., 2010; Velo-Suárez et al., 2014). Velo-Suárez et al. (2009) showed that during late spring relaxation, increased densities of D. acuminata inside the rías resulted from physical advection combined with high in situ growth. The opposite scenario is described in the present study at the onset of an upwelling pulse near neap tide. Decreased densities of a D. acuminata population resulted from a combination of physical dispersion and accompanying decline of in situ (µ avg )growth.Upwelling-induced transport of the toxic dinoflagellate Gymnodinium catenatum and other harmful microalgae off the Galician Rías Baixas (Fermin et al., 1996), and the reset of the phytoplankton succession and rapid development of diatom-dominated assemblages of colonizers following upwelling pulses (Nogueira and Figueiras, 2005; Tilstone et al., 2000) are well documented. Upwelling pulses lead to increased flushing rates of surface waters (Álvarez-Salgado et al., 1993). During the first stages of a relaxation-upwelling transition, increased outflows lead to a remarkable decrease of planktonic populations before a new diatom bloom is triggered (Varela et al., 2008). Increased shear stress in the pycnocline region and higher horizontal water velocities in the top layer have been recorded even during moderate upwelling pulses (Velo-Suárez et al., 2010). ADCP records in the present study provide strong evidence of increased surface outflow between 17 and 19 June (Fig. 5). A different matter is the direct negative effect (reduced division rate) that the onset of an upwelling pulse and increased turbulence may have on the physiology of D. acuminata. The effect of microscale turbulence in altering or even bringing to a halt cellular division (Berdalet, 1992; Juhl and Latz, 2002; Sullivan et al., 2003; Berdalet et al., 2007), motility, sexual processes, cell shape (Berdalet and Estrada, 1995; Zirbel et al., 2000) and the transport of substances in and out of the cells (Karp-Boss et al., 1996) in dinoflagellates is well documented. In the present study, the division rate of a very fit population of D. acuminata (µ avg = 0.65 d −1 , almost one doubling per day) was halved (to 0.33 d −1 ) in 24 h following the onset of upwelling 2 days before and the shoaling of colder, nutrient richer upwelling waters at an estimated rate of 0.33 m h −1 . Further, the circadian rhythm (maximum division at dawn) was altered, and the shape of the frequency distribution curves evolved from an acute-, indicating more synchronized division, to a wider and flatter dome. Upwelled waters may contribute to increase phytoplankton cell densities by shoaling pycnoclines and bringing cells closer to the well illuminated surface layer. This was not the case in the population studied here, because D. acuminata cell maxima were already in the top layer before the upwelling pulse, and they remained in this layer throughout the 36-h cycle. The onset of northerly winds could have at least two effects on the near-surface layer: increased turbulence and increased flushing, as well as the upwelling itself. ADCP and turbulence profiler data from the top 3 m are not reliable due to side lobe effects. Nevertheless, turbulence profiles provided evidence of changes of ɛover two orders of magnitude (from 10 −6 to 10 −4 m 2 s −3 ) at 5 m during several time windows in the cell cycle (Fig. 4E). It is true that turbulence increased before division time in the first cycle, on June 19 at dawn, but division one day tends to reflect the previous 24 h life history of the cells and in any case, northerly winds intensification started on June 18 (Fig. 2B). There is hardly any information available on the effect of microscale turbulence on Dinophysis division, but a recent study by García-Portela (2018) with D. acuminata cultures exposed to different levels of turbulence with an oscillating grid showed very poor growth under high turbulence (ɛ = 10 −4 –10 −3 m 2 s −3 ) compared with lower values (ɛ = 10 −6 –10 −5 m 2 s −3 ). In addition, upwelled waters cause cooling of the surface layer, which in the present study was from 16.84 °C to 14.67° at 5 m, from 20:00 h on June 19 to 04:00 h on June 20, i.e., more than 2 °C drop in only 8 h. Our results suggest that downwelling-upwelling transitions within the rías have a double effect, both contributing to decreased cell densities in established populations of D. acuminata: a direct physical effect of advective dispersion, and a physiological stress inflicted on the cells by the rapid rising of turbulence and cooling of surface waters. Mid-shelf (100 m isobath) surface water conditions did not show such marked changes as those inside the rías, where shoaling of isoclines during upwelling and sinking during downwelling created contrasting short-lived vertical structures. Fig. 3 shows that on June 18, when the top 15–20 m were homogeneous, there was a conspicuous stratification at the mid-shelf stations that was not completely eroded after the upwelling pulse (Figs. 10C, 11B). Further, temperature in the top layer (∼15 °C) did not show significant changes, and division rates (µ min ) at the surface cell maximum at station V6 (0.36 d −1 ) were as high as those inside the ría before the upwelling event. But the most surprising observation was the extremely fit condition (µ min = 0.69) of the low density population in the subsurface layer (10–18 m) at this station on June 21. The value of µ min there indicates that the whole population had gone through mitosis, and all cells could be recognized as recentlydivided specimens in a single sample at peak division hour (06:00 h, dawn), i.e. they were perfectly synchronized, whereas populations inside the ría divided in-phase, i.e., all cells going through mitosis were doing so within a discrete time window. These observations suggest that when Dinophysis and other dinoflagellate species are transported out of the rías to mid-shelf waters during upwelling pulses, this region P.A. Díaz, et al. Progress in Oceanography 175 (2019) 309–327 325 (upwelling front) will act as the pelagic seed bank (sensu Smayda, 2002) where cells will aggregate and keep in good condition (high division rates) until the next relaxation period re-introduces them into the rías. Several facts support this view. First, upwelling fronts in shelf waters have been described as horizontal retention areas where good planktonic swimmers can aggregate (Smayda, 2010a; Smayda, 2010b). In addition, upwelling fronts have been identified as important horizontal limits for the distribution of Gymnodinium catenatum and Dinophysis species in Portuguese shelf waters (Moita, 1993; Moita et al., 1998), and as the “incubator” of inoculum populations of Dinophysis acuta before they are transported to shellfish cultivation areas in SW Ireland (Raine et al., 2016). Changes of toxin (OA) accumulation per cell throughout the two cell cycles did not show any clear circadian pattern. The “normal” expected result would have been a decline in toxin per cell at 06.00 on June 19 coinciding with the peak in recently divided cells. Nevertheless, toxin per cell estimates from net-haul samples may be noisy due to contamination with other species. Results are reliable when there is an overwhelming dominance of the target organism in the net-haul material, as is often the case during downwelling (Reguera et al., 2011, 2014). But in the present study, samples had variable amounts of the predominant diatoms. 4.4. Concluding remarks High resolution vertical sampling at a fixed station in Ría de Pontevedra (Galician Rías, NW Spain) showed physical processes (upwelling pulse, changes in turbulence) associated with the shoaling, erosion and renewal of a diatom thin layer. A co-occurring population of Dinophysis acuminata had its cell maxima at the surface throughout a 36 h cell cycle study, with no evidence of daily vertical migration. In contrast, its ciliate prey (Mesodinium spp.) maxima were below the pycnocline at night and near the surface in the afternoon, when they coincided with the dinoflagellate maximum. Other mixotrophic ciliates with similar behaviour to Mesodinium also co-occurred with D. acuminata. Their potential role as alternative prey for Dinophysis deserves investigation. The onset of upwelling pulses and associated increase of surface outflow caused advective dispersion of phytoplankton populations, in particular of Dinophysis, and a rapid cooling of the surface layer where the latter was distributed. In addition, the northerly winds increase turbulence directly, maximaly at the surface. Therefore, the onset of upwelling pulses and the intensified northerly winds promoting them will cause a decline in the population growth of Dinophysis due to: i) a direct physical effect of advective dispersion favouring the transport of cells in the surface layer of the rías to shelf waters, and ii) an indirect effect of physiological disturbance of the cells, mainly through enhanced turbulence and rapid temperature drop in the surface layer, leading to reduced division rates. At the same time, shelf conditions are more stable, with a moderate decline of surface temperature, and relatively similar levels of turbulence in the upper 30 m before and after the upwelling pulse. Shelf populations of Dinophysis seemed relatively undisturbed, showing division rates comparable to those of the populations in the ría before the onset of upwelling. These shelf populations may act as a reservoir for subsequent bloom formation. Acknowledgements We thank I. Ramilo, P. Rial and G. Fernández for technical assistance during the ASIMUTH-Rías 2013 cruise and phytoplankton counts. This work and the sampling cruise were funded by project ASIMUTH (EC FP7-SPACE-2010-1 grant agreement number 261860). Additional support came from Spanish project REMEDIOS (MINECO, Programa RETOS, CTM2016-75451-C2-2-R), and the EU Interreg Atlantic Area PRIMROSE (EAPA_182/2016), an IOC-SCOR GlobalHAB endorsed project. Patricio A. Díaz had a PhD student fellowship from BECAS–CHILE, National Commission for Scientific and Technological Research (CONICYT) and is now funded by projects PAI79160065 (The Attraction and Insertion of Advanced Human Capital Program) and REDES170101 (International Cooperation Programme), CONICYT, Chile. References Aissaoui, A., Dhib, A., Reguera, R., Ben Hassine, O.K., Turki, S., Aleya, L., 2014. First evidence of cell deformation occurrence during a Dinophysis bloom along the shores of the Gulf of Tunis (SW Mediterranean Sea). Harmful Algae 39, 191–201. Álvarez-Salgado, X.A., Figueiras, F.G., Pérez, F.F., Groom, S., Nogueira, E., Borges, A.V., Chou, L., Castro, C.G., Moncoiffé, G., Ríos, A.F., Miller, A.E., Frankignoulle, M., Savidge, G., Wollast, R., 2003. The Portugal coastal counter current off NW Spain: new insights on its biogeochemical variability. Prog. Oceanogr. 56, 281–321. Álvarez-Salgado, X.A., Rosón, G., Pérez, F.F., Pazos, Y., 1993. 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