Journal of Photochemistry and Photobiology B: Biology 143 (2015) 107–119

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Comparing the diel vertical migration of Karlodinium veneficum (dinophyceae) and Chattonella subsalsa (Raphidophyceae): PSII photochemistry, circadian control, and carbon assimilation Charles L. Tilney ⇑, Kenneth D. Hoadley, Mark E. Warner University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA

a r t i c l e

i n f o

Article history: Received 25 September 2014 Received in revised form 6 December 2014 Accepted 18 December 2014 Available online 3 January 2015

a b s t r a c t Diel vertical migration (DVM) is thought to provide an adaptive advantage to some phytoplankton, and may help determine the ecological niche of certain harmful algae. Here we separately compared DVM patterns between two species of harmful algae isolated from the Delaware Inland Bays, Karlodinium veneficum and Chattonella subsalsa, in laboratory columns. We interpreted the DVM patterns of each species with Photosystem II (PSII) photochemistry, rates of carbon assimilation, and specific growth rates. Each species migrated differently, wherein K. veneficum migrated closer to the surface each day with high population synchrony, while C. subsalsa migrated near to the surface from the first day of measurements with low population synchrony. Both species appeared to downregulate PSII in high light at the surface, but by different mechanisms. C. subsalsa grew slower than K. veneficum in low light intensities (bottom of columns), and exhibited maximal rates of C-assimilation (Pmax) at surface light intensities, suggesting this species may prefer high light, potentially explaining this species’ rapid surface migration. Contrastingly, K. veneficum showed declines in carbon assimilation at surface light intensities, and exhibited a smaller reduction in growth at low (bottom) light intensities (compared to C. subsalsa), suggesting that this species’ step-wise migration was photoacclimative and determined daily migration depth. DVM was found to be under circadian control in C. subsalsa, but not in K. veneficum. However, there was little evidence for circadian regulation of PSII photochemistry in either species. Migration conformed to each species’ physiology, and the results contribute to our understanding each alga’s realized environmental niche. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction It is widely accepted that the frequency of harmful algal blooms (HABs) is increasing and that some of these species are expanding their biogeographic ranges [36,92,63]. In seeking to understand why this is occurring, a thorough understanding of the biotic and abiotic controls on HAB dynamics is required. This is daunting given the sheer number and diversity of species responsible for HABs and the variety of environments in which they occur, but nevertheless much progress has been made [4,39,2,59]. One particularly complex factor that has long been studied and implicated in bloom initiation and persistence is diel vertical migration (DVM). DVM is a cyclical behavior in which the vertical distribution of organisms inverts between day and night, such that the

⇑ Corresponding author at: Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St Petersburg, FL 33701, USA. Tel.: +1 (302) 249 8818. E-mail address: [email protected] (C.L. Tilney). http://dx.doi.org/10.1016/j.jphotobiol.2014.12.023 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

distribution cycle (starting and ending in the same place), happens over approximately 24-h intervals (i.e. has a circadian period). DVM is particularly complex because it integrates a wide variety of cellular physiologies into a plastic behavior [17], via the use of a variety of sensory perceptions [51], which operate by incompletely characterized signal transduction pathways [1]. In this regard, understanding how DVM is employed by different algal species could be useful to better characterize their ecological niches [17]. Two hypotheses have been postulated to explain how DVM confers an adaptive advantage to phytoplankton. The first is that DVM provides access to deep nutrients when surface nutrients become depleted [23,16,61,18,69]. The second is that DVM provides relief from grazers, many of which show DVM patterns in an opposite phase to phytoplankton [71,9]. These hypotheses are not mutually exclusive and both factors were used to explain the dominance of a raphidophyte in a stratified lake [81]. Other advantages conferred by DVM include avoidance of flushing into unfavourable waters [3,14], and photosynthetic optimization [6]. There are a variety of

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mechanisms that algae use to sense their immediate environment for perception and orientation, and a number of these are used to direct movement and DVM. These include phototaxis (movement in respect to light; e.g. [65], chemotaxis (movement in respect to chemicals; e.g. [12,91] and geotaxis/gravitaxis (movement in respect to gravity; e.g. [51,34]. Furthermore, DVM appears to be driven by an endogenous circadian clock in some species [99,80,85], which is thought to operate (in part) by controlling the sensitivity of the alga to the above taxes (e.g. sensitivity of phototaxis depends on the endogenous clock’s rhythm; [26]. The clock itself must be regulated however, and is most often controlled primarily by light, but can also receive inputs from pH and certain chemicals [79,21]. The role of the circadian clock has been studied extensively in one particular dinoflagellate, Lingulodinium polyedrum, which shows clock controlled rhythms in movement as well as bioluminescence, photosynthesis, cell division, protein synthesis, and ultrastructure [27,64,66]. It is important to determine whether a biological rhythm such as DVM and photosynthesis in microalgae is under clock control because clock-controlled rhythms provide an adaptive advantage by synchronizing specific cellular physiologies to the optimal time of day, without reliance on external factors [68,100,19]. Most studies that have noted the existence of endogenous control of algal DVM have cited movement prior to the light–dark or dark-light transitions (e.g. [23,18]. However, observation of a free-running rhythm in constant conditions should help to validate that these rhythms are indeed circadian [47]), but such measures have rarely been assessed [37,85]). Both the proximate (i.e. mechanistic) and ultimate (i.e. adaptive significance) drivers regulating DVM appear to vary among different species [51,18]), and even within a species under different environmental conditions (e.g. [23]). Despite an imperfect understanding of the DVM behavior, empirical testing among species could generate ecologically relevant knowledge about certain key species, such as those responsible for harmful algal blooms. In the current study we describe and compare the DVM patterns of two locally isolated HAB forming species from the Delaware Inland Bays in the Mid Atlantic region of the United States, a dinoflagellate, Karlodinium veneficum, and a raphidophyte, Chattonella subsalsa. Previous studies have assessed some aspects of DVM for Chattonella spp., including Chattonella antiqua [97,98]), and the same strain of C. subsalsa used in the current study [37]). In contrast, almost nothing is known of the vertical migrations of K. veneficum, with only one study reporting DVM in this species from a field study assessing DVM in phytoflagellates in response to water column structure [35]). Here, physiological measurements, including photochemistry, specific growth rates, and carbon assimilation rates, were used to help explain different DVM behaviors. Additionally, the potential role of a circadian clock was assessed by testing if rhythms in DVM and PSII photochemistry would free-run in constant light conditions.

2. Methods 2.1. Stock algal culture, column design, and lighting The dinoflagellate, K. veneficum (CCMP 2936) and the raphidophyte, C. subsalsa (CCMP 2191), were both isolated from the Delaware Inland Bays approximately 8 years before this study. Prior to this study, these cultures were grown in f/2 seawater medium under static 12:12 light:dark (LD) cycles. Two months before this study began, stocks were grown in filtered sea water (salinity 20) supplemented with f/2 nutrient concentrations (‘‘f/2 medium’’, [32,31]), at 24 °C, and grown under static 14:10 light:dark (LD) cycles at 50 lmol photons m2 s1 from above with cool white

fluorescence lights. In stock cultures and column experiments detailed below, the light period commenced at 6 a.m. EST (06:00), and ended at 8 p.m. (20:00). Three columns were constructed from extruded transparent acrylic piping, which was bonded to square acrylic bases. Columns were 1.82 m tall, had an internal diameter of 14.6 cm and held approximately 31 L of culture. Thirty nylon barbed-style sampling ports were threaded into the walls of the column, 3 each at 10 depths. The bottom sampling port was 2.8 cm above the bottom of the column, and ports above this were placed every 19 cm, with the final port placed at a depth of 9 cm from the top of the column. The tops of the columns were covered with a square of thin acrylic, which was removed for surface sampling (1 cm depth). Ports were sampled via 5 mL syringes, which were attached to the barbed ports by 3 cm of silicone tubing that remained clamped while not in use. The columns were kept in a temperature controlled room at 24 °C, and each illuminated from above by a set of 9 fan-cooled white LEDs (Cool White XLamp XP-G, Cree Inc., CA, USA), which were soldered to large aluminum heat sinks and attached to a digital controller (AquaController-Apex Jr., Neptune Systems, CA). Spectral output of the LEDs is presented in Supplemental Fig. 1, and showed peak output at 445 nm and between 520 nm and 580 nm, with a dip in output around 485 nm. The standard lighting regime in the columns was a 14:10 LD cycle with a surface light intensity at dawn and dusk of 230 l mol photons m2 s1, which ramped up to 1000 l mol photons m2 s1 at the mid day peak. Irradiance (PAR, 400–700 nm) at the top and bottom of the columns was measured in air, with a Li-Cor 4p quantum sensor. A two-point light attenuation coefficient (Kd) was calculated using the equation from Kirk [53]:

Kd ¼

  1 Ez1  Ln ðz2  z1Þ Ez2

where z is the depth and E is the light intensity. The resultant Kd was used to model the light field throughout the columns, which was used for the carbon assimilation experiments (see below). Small column diameters led to difficulties in measuring light intensities in the columns, and precluded our ability to make measurements in media-filled columns. Consequently, the light levels measured here are potentially overestimated. 2.2. Common experimental procedures As with the stock cultures, all experiments were conducted in seawater (salinity 20) amended with f/2 nutrients. Although this nutrient amendment is ecologically irrelevant, it served to control for nutrient conditions over the incubation periods tested. Initially, 30 L of f/2 medium was added to each column, and then 1 L of culture was added from the top at approximately 19:00 to avoid a high light exposure. Algae were given P24 h for acclimation before commencing sampling. Columns were cleaned between inoculations with liquid detergent and sponge, followed by bleach and multiple rinses with tap water and a final rinse with DI water. Sampling under darkness was always performed under dim green light (a head-torch with green filter, intensity 0.05, RM-ANOVA, n = 3). At the top and bottom of the column, maximal rPSII (1.2 nm2) occurred just before dawn, and decreased significantly throughout the day (P < 0.05, RM-ANOVA, p = 3), before rising back to the maxima through the second half of the night. Photosystem II reoxidation time (s) in K. veneficum at the top and bottom of the columns was not significantly different (Fig. 1G; 05:00–20:00; P > 0.05, RM-ANOVA, n = 4), and remained constant throughout the day at 490 ls, before rising gradually throughout the night to a maximum of 600–670 ls toward the end of the night. Tau (s) in C. subsalsa was similar at the top and bottom throughout the experiment (Fig. 1H), showing a midday minima of 600 ls, which increased steadily toward a maxima during the early night (700 ls), and then fell steadily back toward the midday minima. PSII connectivity (q) differed significantly between the top and bottom of the columns in K. veneficum (Fig. 1I; 05:00–20:00; P < 0.05, RM-ANOVA, n = 4). At the bottom, q was constant during the day and night, but remained at a slightly higher level during the day (0.41) than during the night (0.37). At the surface, q dropped significantly by 0.15 (P < 0.05, RM-ANOVA, n = 4) to a minimum at midday under peak irradiance, then returned to the level of the bottom cells by the end of the day, and rose slightly to a maxima in the early morning. PSII connectivity (q) in C. subsalsa also exhibited differences between the top and bottom of the column (Fig. 1J; P < 0.05, RM-ANOVA, n = 3). At the bottom, q dropped significantly by 0.06 (05:00) from the morning to the end of the day (P < 0.05, RM-ANOVA, n = 3) before rising back to the morning maxima through the night. At the top of the column, the drop in q from the morning maxima to the end of the day was also significant, and more abrupt than at the bottom (P < 0.05, RMANOVA, n = 3). In summary, the main differences between the diel photochemical patterns between K. veneficum and C. subsalsa were (1) the reduction of PSII in the dark in K. veneficum (decrease in Fv/Fm and increased s), and (2) the larger drop in PSII connectivity at the top (compared to the bottom) in K. veneficum, and (3) the inverted timing of maximal rPSII value (late day phase in K. veneficum and early day phase in C. subsalsa). When assessed over 6 days, the migratory patterns of K. veneficum and C. subsalsa were broadly similar (in terms of inter-quartile range, and migration amplitude) to that observed in Fig. 1 (Fig. 2A and B). Specifically, both maintained similar inter-quartile range and migration amplitude relative to those in Fig. 1. However, for K. veneficum, the population migrated closer to the surface with each proceeding day. Specifically, median migration amplitude, calculated as the difference between 13:00 and the following morning (05:00), increased significantly in K. veneficum when comparing day 2 to day 5 (+99 cm ± 5 SEM; P < 0.001, 1way-ANOVA, n = 3). Conversely, in C. subsalsa, the population immediately distributed with an upper quartile close to the surface, but as growth progressed, C. subsalsa began to migrate with greater synchrony toward the bottom, although median migration amplitude was not significantly different when comparing day 2 to day 6 (+63 cm ± 28 SEM; not significant, 1way-ANOVA, n = 3). Similarities and differences in photochemistry and photo-acclimation between K. veneficum and C. subsalsa were evident after monitoring the columns for 6 days. Prominent differences were highlighted in the measurements of light acclimated surface samples compared to dark acclimated samples. The operating quantum yield Fq0 /Fm0 was significantly lower at the surface than at the bottom every day in K. veneficum (Fig. 2C; P < 0.05; see legend) and C. subsalsa (Fig. 2D; P < 0.05; see legend). Midday surface Fq0 /Fm0 followed a similar trend in both species, increasing for the first 3 days

and gradually decreasing for the last 3 days. Recovery in Fq0 /Fm0 from midday (13:00) to the end of the day (19:00) decreased in both species by day 6. Surface Fv/Fm values were significantly different from bottom Fv/Fm values in both K. veneficum (Fig. 2B; P < 0.05), and C. subsalsa (Fig. 2D; P < 0.05), although C. subsalsa showed larger, and gradually increasing declines. Non-photochemical quenching from the PSII antenna bed was significantly higher in the light acclimated surface cells of K. veneficum (0.5; Fig. 2E) compared to C. subsalsa (0.2; Fig. 2F) (P < 0.05, RMANOVA, n = 3). Compared to K. veneficum, there appeared to be greater diel variability in light-acclimated s in C. subsalsa at 19:00 in surface samples in the final 3 days (Fig. 2H) though this was not significant (P > 0.05, RM-ANOVA, n = 3). At midday at the surface, PSII connectivity in the light activated state (q0 ) dropped to a larger extent compared to dark-acclimated q in K. veneficum as compared to C. subsalsa (Fig. 2I and J; P < 0.05, RM-ANOVA, n = 3). However, a downward trend in q0 in C. subsalsa led to q0 values that were equivalent to those in K. veneficum by the final day. 3.2. Circadian control experiments Before shifting K. veneficum into continuous light (LL), it migrated in the same manner as observed in Fig. 2A, including the trend to shallower midday depth distributions over time. After shifting K. veneficum into LL, the depth distribution continued to rise almost linearly, without any evidence of DVM (Fig. 3A; P > 0.05, CircWave, n = 3). After returning K. veneficum back to LD, DVM returned immediately with similar phase and amplitude. Before shifting C. subsalsa into continuous light (LL), cells migrated in the same manner as observed in Fig. 2B, showing the same migration amplitude and characteristically wide inter quartile range observed in Figs. 1B and 2B. After shifting C. subsalsa into LL, it continued to migrate with significant diel periodicity (P < 0.01, CircWave, n = 3), with the same apparent phase as before the shift, but with diminished amplitude. After returning C. subsalsa back to LD, the amplitude of DVM was immediately regained. Before shifting K. veneficum into LL, Fv/Fm, s, and rPSII showed distinct diel fluctuations consistent with that depicted in Figs. 2C, G, and E respectively. Fv/Fm fluctuated by 0.1, driven predominantly by a fall during the night (at the bottom) and to midday declines (at the top) (Fig. 3C). Tau (s) decreased by 150 ls during the day before rising again at night (Fig. 3E), and rPSII tended to peak at midday and fell from midday through to the next morning (Fig. 3G). After shifting to LL, K. veneficum lost the diel fluctuations in Fv/Fm, s, and rPSII. Fv/Fm remained at day-time levels at the bottom, while cells at the surface had significantly lower Fv/Fm values at 13:00 compared to values at 13:00 during the first 2 days under LD (Fig. 3C; P < 0.05, RM-ANOVA, n = 3); s remained at 590 ls at the bottom, while steadily increasing from 530 ls to 670 ls at the surface; and there was no daytime increase in rPSII at the bottom or the surface, and remained significantly lower (Fig. 3G; P < 0.05, RM-ANOVA, n = 3) at the surface compared to midday values under LD. Similar to Fv/Fm and sigma, q remained low and had no continued diel periodicity (Fig. 3I). Upon returning K. veneficum to LD, Fv/Fm, s, rPSII, and q returned to pre-LL patterns. Before shifting C. subsalsa into LL, Fv/Fm, s, and rPSII showed diel fluctuations consistent with Fig. 2D, H, and F respectively; Fv/Fm at the surface was reduced at midday (13:00) and before dark (19:00) (Fig. 3D). Tau (s) was highest before dark (19:00) and lowest around midday (13:00) (Fig. 3F), and sigma was highest just before dawn and decreased until the end of the day (Fig. 3H). After shifting C. subsalsa into LL, Fv/Fm at the bottom was similar to the trends noted under LD conditions until the second night, while shifts in Fv/Fm at the surface did not continue as under LD conditions and generally remained low (Fig. 3D). Tau (s) at the bottom and top maintained a LD pattern only for 1 day (Fig. 3F), and rPSII at

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C. subsalsa

Light Int .

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Fv /Fm & Fq'/Fm'

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Fig. 2. DVM and PSII photochemistry in K. veneficum (A, C, E, G, I) and C. subsalsa (B, D, F, H, J) measured 1 h before dawn (05:00), at midday (13:00), and 1 h before dark (19:00) every day for 6 days, with columns kept under 14:10 LD cycles. Measurement began on the second day after inoculation. Filled gray bars underneath the data represent the dark period, and white areas represent the light period. Light intensity is presented above panels A, B. Presented for each species are: DVM (A and B); Fv/Fm and Fq0 /Fm0 (C and D); PSII light harvesting center quenching (E and F); s and s0 (ls, G and H); q and q0 (I and J). DVM is presented as the median (filled squares), upper (open triangles), and lower (open inverted triangles) quartiles. In photochemistry figures (C–J), dark acclimated measurements at the bottom (filled circles), at the surface (open circles), and light acclimated measurements at the surface (open inverted triangles) are presented. Light acclimated measurements at the bottom were obscuring dark measurements, and so were excluded for clarity. Asterisks in C and D represent significant differences (p < 0.05) between dark acclimated measurements at the surface and bottom samples as determined by RM-ANOVA and Bonferroni post tests (n = 3). Error bars represent ±1 SEM.

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C. subsalsa

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Fig. 3. DVM and PSII photochemistry in K. veneficum (A, C, E, G, I) and C. subsalsa (B, D, F, H, J) measured at 1 h before dawn (05:00), midday (13:00), and 1 h before dark (19:00) every day for 6 days, in the continuous light experiment (LL). Columns were kept under 14:10 LD cycles until day 3, when columns were shifted at midday (13:00) into continuous midday light for 2 days, before being returned to 14:10 LD cycles at midday (13:00) for a further 24 h. Light intensity during the incubations is presented above panels A and B. Dark filled gray bars underneath the data represent dark periods, white areas represent light periods, and pale filled gray bars represent ‘night-time’ during the constant light period. Light intensity is presented above panels A and B. Presented for each species are: DVM (A and B); Fv/Fm (C and D); s (ls, E and F); rPSII (nm2, G and H); q (I and J). DVM (A and B) is presented as the median (filled squares), upper (open triangles), and lower (open inverted triangles) quartiles. In photochemistry figures (C–J), dark acclimated measurements at the bottom (filled circles), and at the surface (open circles) are presented. Note the difference in scale between panels G and H. Error bars represent ±1 SEM.

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(statistically at 28 and 66 cm; Fig. 5A). For C. subsalsa Pmax was 88 pg C cell1 h1 and was not significantly different (P > 0.05, 1way ANOVA, n = 3) between samples collected at shallow to intermediate column depths (1, 28, 66 cm; Fig. 5B). Declines in carbon uptake were observed in K. veneficum at 28 cm (P > 0.05) and 1 cm depth (P < 0.05), while no similar trend was noted for C. subsalsa, which instead maintained Pmax at the surface.

0.6 A

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4. Discussion

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Low

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4.1. DVM, photobiology, and growth

C. subsalsa

Fig. 4. Specific growth rate l (d1) in cells forced to grow in surface light intensities (High), bottom light intensities (Low), or in cells free to move within the column (Col.). In each case growth was calculated by regression over 6–9 days (see methods). Error bars represent ±1 SEM, and statistical differences (2-way and 1way ANOVA followed by Bonferroni post-tests, P < 0.05, n = 3), are denoted by capital letters at the top of each bar.

the bottom maintained a similar pattern to LD at reduced amplitude and with a general trend to lower values, but surface rPSII did not maintain the pattern observed under LD conditions (Fig. 3H). PSII connectivity (q) at the bottom continued as in LD until the end of the first day, increasing at 13:00 and decreasing to 19:00, and the amplitude was larger than under LD. At the surface, connectivity remained low and showed no rhythmicity (Fig. 3J). 3.3. Comparing growth between DVM and static cultures Growth rates of each species held under ramping 14:10 LD cycles at either surface or bottom light intensities, and in the columns, are presented in Fig. 4. In surface high-light flasks, K. veneficum and C. subsalsa both grew at approximately 0.5 d1, and were not significantly different from each other (P > 0.05, 2-way ANOVA, n = 3). However, in bottom low-light flasks K. veneficum growth was 0.36 d1 while C. subsalsa grew significantly slower at 0.22 d1 (P < 0.01, 2-way ANOVA, n = 3). C. subsalsa growth in the columns was 0.47 d1 and was significantly faster than low-light acclimated cells (P < 0.05, 1-way ANOVA, n = 3), but not significantly different from high-light acclimated cells (P > 0.05, 1-way ANOVA, n = 3). K. veneficum growth in the columns (0.17 d1) was significantly slower than high-light acclimated cells (P < 0.05, 1-way ANOVA, n = 3) but not significantly different from low-light acclimated cells (P > 0.05, 1-way ANOVA, n = 3). 3.4. Carbon assimilation rates Maximal carbon fixation (Pmax) in K. veneficum was 9.0 pg C cell1 h1 and occurred at intermediate column depths

10

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K. veneficum and C. subsalsa both showed clear patterns of DVM, but migrated differently within each day. The main differences in DVM patterns between the two species were (1) a rapid descent in K. veneficum compared to a slow descent in C. subsalsa, and (2) less synchrony in C. subsalsa’s migration compared to K. veneficum. Regarding the descent, it may be that K. veneficum descends actively (e.g. via negative phototaxis and/or positive geotaxis) [20,51], whereas C. subsalsa descends passively via a ‘dispersal’ mechanism like that of Karenia brevis, as noted by Van Dolah et al. [93]. The lower migration synchrony in C. subsalsa compared to K. veneficum is difficult to explain, but could be due to individual-specific migrations (to different depths) resulting from phased/asynchronous cell division, where biochemically unequal cell division and/or cell division gating, limits each cell’s timing and extent of migration [50,93]. A similar pattern has been noted in the dinoflagellate K. brevis that divides unevenly, such that the ‘poorer’ daughter cells migrate higher and earlier than the parent cells [84]). Alternatively, the wide C. subsalsa distribution may be due to observed bioconvection streams (i.e. ‘clumps’ of cells moving rapidly downwards creating a stream of cells). Bioconvection streams are initiated by dense swarms of cells at the surface, which causes the surface water to become denser than the subsurface water, creating an overturning instability [70]). However, it is not clear how this affects C. subsalsa specifically because K. veneficum also forms such ‘streams’, but contrasting cell size and density could mediate such differences. Although we have discussed this difference from the perspective of C. subsalsa, the converse effects from K. veneficum may be equally important (e.g. more synchronous division, less efficient streaming). Regardless of the mechanisms, a wide vertical distribution in-situ may be advantageous by limiting the probability of catastrophic population loss from an area due to currents or grazing [3,14,9]), or by increasing the probability of partial transfer to a better environment and hedging survival through wider distributions [90]). The appearance of cells at the surface despite evidence for the majority of cells occurring at lower depths, highlights that sub-populations can migrate differently from the population as a whole. Individual level assessments

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Fig. 5. Carbon assimilation rates as pg C cell1 h1, in K. veneficum (A), and C. subsalsa (B) calculated from 55 min incubations using sub-samples from 5 depths in the columns. Statistical differences (1-way ANOVA followed by Bonferroni post-tests, P < 0.05, n = 3) between rates at each depth are denoted by capital letters at the top of each bar. Error bars represent ±1 SEM.

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of swimming behavior (e.g. via video analysis; [88]) and physiology would be highly informative to further characterize these patterns in DVM. In the longer duration experiment (Fig. 2), K. veneficum increased its migration amplitude significantly by migrating closer to the surface each day. This is surprising, given that Pmax was recorded at a light level equivalent to an intermediate depth (Fig. 5A). Importantly, cell growth in the columns by day 6 would have increased light attenuation, so the light levels used to determine Pmax were probably overestimated, and the true (day 6) Pmax may have been shallower. Consequently, K. veneficum appears to migrate to a depth optimal for photosynthesis, similar to what was described for the dinoflagellate Prorocentrum triestinum [6]. The high growth (Fig. 4) and maximal photosynthesis (Fig. 5) at low to intermediate light intensities may facilitate the migration strategy of K. veneficum. Although the majority of K. veneficum cells did not require an immediate use of fast photoprotective mechanisms (see below), a small population of cells at the surface did use these mechanisms. Such photoprotective mechanisms were evidenced by the midday reductions in Fq0 /Fm0 at the surface, which were not evident in dark acclimated samples, and recovered toward the end of the day. These photoprotective mechanisms included the use of both PSII connectivity and non-photochemical quenching in the antennae complex. Such photoprotective mechanisms at the surface did not change over time in K. veneficum. Although not directly measured, antennae-based non-photochemical quenching is consistent with the operation of a xanthophyll cycle [60,11,29,40]), as previous reported in both dinoflagellates [11]) and raphidophytes [41]. Reports of PSII connectivity as it relates to photoprotection are rare [29,43,89]). Trimborn et al. [89] suggest that high q at the start of the day can also provide photoprotection by enhancing electron transport rates to rapidly set-up a thylakoid pH gradient and initiate NPQ mechanisms. Correspondingly, Ihnken et al. [43] showed a positive relationship between NPQ and q in the microalga Dunaliella tertiolecta, in the first few minutes of illumination from darkness (high q correlating with high NPQ). Our results also show morning maxima in reaction center connectivity, such that NPQ could be initiated quickly in both species. However q, and q0 particularly, decrease during exposure to high-light throughout the day, which would not enhance photochemical e sinks during this period. Instead q0 may correspond to other NPQ mechanisms, including LHC-quenching and RCII downregulation. In particular, by preventing absorbed excitation energy from entering closed RCIIs, LHCquenching should by definition reduce q, by increasing the likelihood that excitation energy transiting between PSII units via LHCIIs would be quenched. Indeed, Gorbunov et al. [29] also showed a positive correlation with q and r0PSII in Symbiodinium spp. in agreement with our data. Consequently, reductions in q and q0 may simply indicate specific photoprotective mechanisms, rather than being an active mechanism itself. Although unlikely to be the case here, reductions in q may contribute to photoprotection by allowing continued quenching by photoinactivated RCIIs, rather than adding excitation pressure to remaining functional RCIIs. Similar to K. veneficum, C. subsalsa exhibited similar reductions in surface Fq0 /Fm0 that recovered slightly by the end of the day and were not evident in surface Fv/Fm values during the first 2 days, suggesting non-photochemical photoprotective mechanisms were implemented and effective at preventing photoinhibtion. From the first day onwards, >25% of the C. subsalsa population migrated near to the surface each day, thus a larger proportion of the population (compared to K. veneficum) required rapid photoprotection. Mechanistically, C. subsalsa differed from K. veneficum, in being much less reliant on LHC-quenching and less reliant on PSII connectivity. The lower reliance on LHC-quenching, along with the smaller drop in q (Figs. 1J and 2J) and larger increases in s and s0 (time for electron flow out of PSII) toward the end of the day in C. subsalsa

(Figs. 1H and 2H) compared to K. veneficum, strongly suggest that different mechanisms of photoprotection are used in this species. C. subsalsa may instead be more reliant on one or more other mechanisms such as; cyclic electron flow around PSII [24]) or PSI [5,22]); the use of alternative electron sinks such as O2 at RuBisCo (i.e. photorespiration, [55]), and nitrate by nitrate-reductases via NADPH [30,60]); the use of constitutive RCII NPQ mechanisms (so-called ‘‘RCII downregulation’’, [57,44]); or enhanced PSII repair cycles [45]). Moreover, differences in cell size may also play a role in the relative light harvesting capabilities of each alga, as C. subsalsa is approximately 3 times larger than K. veneficum. It is not possible to determine which mechanisms are primarily employed by C. subsalsa here, but given limited LHC-quenching, and the coincident reductions in surface Fv/Fm in tandem with increases in s late in the day, RCII downregulation appears likely [44]). An even larger difference in s was observed between the species in LL at the surface (constant high light, Fig. 3). After 3 days, Fv/Fm in C. subsalsa at the surface declined further (Fig. 3D), and this may be due to longerterm photoprotective mechanisms that were not relaxed within 45 min of dark acclimation. This loss in Fv/Fm is consistent with the finding of Warner and Madden [96] that C. subsalsa cannot maintain high maximal rates of electron transport for prolonged periods (>1 week) when shifted into high light. However, a decrease in Fv/Fm does not always imply reductions in overall electron transport or photosynthesis [7], and indeed, Pmax still occurred at the surface after 7 days (Fig. 4B). Ultimately, the low growth (Fig. 4B) and C-assimilation (Fig. 5B) rates in low-light grown C. subsalsa, provides further evidence that this alga maximizes photosynthesis and growth under high light. Thus, C. subsalsa and K. veneficum migrate, photoacclimate, and photoprotect in different ways, which will affect their ecological niche in the Delaware Inland Bays and elsewhere, but the importance of these differences will depend on stratification and mixing conditions. C. subsalsa relies on rapid photoprotective mechanisms, which over 3–4 days in a static column, resulted in reduced Fv/Fm but did not immediately affectPmax (Fig. 5B). C. subsalsa is poised to exploit periods (or seasons) with high light, and can acclimate quickly to rapid changes in light intensity. Interestingly, this is consistent with another harmful alga, Cochlodinium cf. polykrikoides, which is also a vertical migrator, with strong phototactic responses, that is minimally photoinhibited in high light (1600 lmol photons m2 s1), perhaps suggesting a common niche of these species [56]. K. veneficum migrated to an optimal light intensity in the column (directed by its prior photoacclimation state; and potentially self shading in uni-algal culture, [78] which enabled this species to maintain high Fv/Fm values and C-assimilation rates near to Pmax. As a result, K. veneficum uses migration to photoacclimate and prevent photoinhibition, and is better suited to stable light conditions, and may outcompete C. subsalsa in low light conditions. Although our results may not be representative of the two species tested as a whole due to intraspecific differences in photobiology and physiology, we suggest the coarse differences we observed between the two cultures are likely to remain relevant even in the context of intraspecific variability. 4.2. Circadian rhythms DVM continued in continuous light in C. subsalsa (Fig. 3B) but not in K. veneficum (Fig. 3A), suggesting that DVM may be under clock control in C. subsalsa but not in K. veneficum. It is not clear why K. veneficum did not appear to entrain its migratory behavior to a circadian oscillator like C. subsalsa (this study) and other dinoflagellates [26]. Perhaps entraining DVM to the circadian clock is inhibitory to an opportunistic predator like K. veneficum [74], where the optimal depth may be independent of light since K. veneficum may migrate toward its prey (e.g. see [8]). In contrast, although C. subsalsa is likely bactivorous [46], a greater reliance

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on photosynthesis may make circadian rhythms in DVM more advantageous. Previous reports of DVM in constant light in other dinoflagellates and in Chattonella antiqua show rhythms with high amplitude lasting >3 days [99,80,85]. Thus, the lack of any robust circadian rhythm in K. veneficum, and the low-amplitude rhythm in C. subsalsa, was surprising. Although these results may correctly reflect the true extent of circadian rhythmicity in these species, i.e. zero entrainment (K. veneficum) or low levels of entrainment (C. subsalsa), a number of factors may have contributed to the lower than expected rhythmicity. Firstly, light spectra used for entrainment may not have been optimal, since entrainment to white light yields weaker DVM rhythms in C. antiqua compared to blue light [85]). Spectral changes experienced in the environment may entrain stronger rhythms of DVM (see also [25]). Secondly, culture artefacts may have developed over time, since circadian rhythms in photosynthesis have been lost in older cultures of G. polyedra [87]), and phototaxis has been lost in older cultures of Kryptoperidinium foliaceum (linked to eyespot degeneration; [65]). However, the cultures used in this study have only been in culture for 8 years, hence mutation may be unlikely, but cannot be ruled out. Lastly, deprivation of non-photic clock inputs (e.g. nitrate and pH) may have repressed circadian rhythmicity of DVM [79,21]). Photosynthesis and PSII activity are known to show circadian rhythms in a number of phytoplankton [10]) including many dinoflagellates [38,75,76,86] and can continue with high amplitude in excess of 3 days in constant conditions [10,76]. In particular, the maximum quantum yield of PSII follows a circadian rhythm (e.g. [82,62,86], and was initially thought to stem from PSII rather than downstream from the plastoquinone (PQ) pool or PSI [82]). Additionally, Sorek et al. [86] noted that along with rhythms in Fv/Fm, transcription of the oxygen-evolving enhancer gene showed circadian rhythmicity in the dinoflagellate Symbiodinium sp., implying that PSII rhythmicity may be linked to the donor side of PSII. Consequently it was surprising to observe such low circadian variability in PSII fluorescence measures in both species in constant conditions here. Most prior results showing circadian regulation of Fv/Fm were determined with multiple turnover fluorescence induction, whereby the PQ pool becomes fully reduced, but this had no effect on the circadian rhythm of Fv/Fm in C. subsalsa here (data not shown). MacKenzie and Morse [62] found no circadian rhythm in oxygen evolution or Fv/Fm in extracted chloroplasts from L. polyedrum, and proposed that CO2 availability drives the circadian rhythms of photosynthesis and electron transport in L. polyedrum in vivo. However, components localized at PSII may affect Fv/Fm more than oscillations mediated by the dark reactions. For example, psbA synthesis and degradation is controlled by light rather than a circadian oscillator [95]b) and could severely affect PSII function and Fv/Fm. In K. veneficum, the majority of the diel periodicity in Fv/Fm stems from the night-time drop which may be related to chlororespiratory reduction of the PQ pool [49,72]. Large decreases in Fv/Fm by chlororespiration do not occur in the light because accumulation of PQH2 is prevented by linear electron transport through the Z-scheme and a supply of oxygen. Thus, a large proportion of the diel variability in Fv/Fm in K. veneficum cannot occur in constant conditions, as no shift between chlororespiration and photosynthesis occurs. If the majority of the diel periodicity were due to light-dependent downregulation at PSII in K. veneficum and C. subsalsa at the surface, then diel periodicity is unlikely to continue in the absence of changes in light intensity. 4.3. Value of DVM In K. veneficum, a large reproductive cost was associated with DVM, but due to the low coefficient of determination of the regression to calculate growth (0.38 ± 0.27 S.D.), this result was

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not considered further. Contrastingly, DVM in C. subsalsa, did not show a reproductive advantage, or cost, under nutrient replete LD conditions compared to cells held at surface light intensities over 6–9 days (Fig. 4; ‘col.’ vs. ‘high’). Although reliant upon estimated growth rates without mixing the column, the coefficients of determination for these rates were high (0.95 ± 0.04 S.D.), and provide support for our results. This suggests that the adaptive value of DVM may occur in conditions not tested here, such as nutrient depletion [15,61]), or high grazing pressure [9]). Only two studies in cyanobacteria have directly measured an adaptive advantage of circadian rhythms (i.e. reproductive fitness, [48,100,102], so the unexpected lack of a circadian rhythm in DVM in our K. veneficum isolate may present a unique strain to study the adaptive value of circadian rhythms if new isolates from the field do show high levels of circadian rhythmicity in DVM. It is worth noting however that continued rhythmicity under constant conditions, although critical in differentiating diurnal rhythms from circadian rhythms, is just one of at least four main features that define circadian rhythms [94]). Consequently, we cannot definitively rule out that DVM or photosynthesis were not under ‘circadian control’ in these algae, only that free running under constant conditions was not observable. 5. Conclusion We present the first detailed assessment of DVM in K. veneficum and found distinct patterns of DVM between K. veneficum and C. subsalsa. Each species’ photosynthetic physiology appeared to define their migration patterns in the conditions used in the current study. C. subsalsa appeared to be a high-light specialist, and should be competitive during peak midday light intensities in the field. C. subsalsa tended to have a wide vertical distribution, which may influence lateral population dispersal and losses in the field. K. veneficum’s migration was consistent with this species’ mixotrophic and generalist life style, and we suggest that its lack of circadian control of DVM is consistent with the potential for preydependent depth orientation. To more fully determine the migratory preferences of these algae, DVM will need to be assessed under more conditions, such as nutriclines and with predator/prey additions, which will further help to characterize the possible ecological and biogeochemical roles that such migratory patterns play in nature. Acknowledgements We wish to thank Gary Sterling for help building the columns, and Jonathan H. Cohen for advice and help with circadian analyses, and Wei-Jun Cai for use of the DIC analyser. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014. 12.023. References [1] C.Y. Allan, P.R. Fisher, Phototaxis: microbial, in: Encyclopedia of Life sCiences, 2011. doi: http://dx.doi.org/10.1002/9780470015902.a0000399.pub2. [2] D.M. Anderson, Approaches to monitoring, control and management of harmful algal blooms (HABs), Ocean Coast. Manag. 52 (7) (2009) 342–347. [3] D.M. Anderson, K.D. Stolzenbach, Selective retention of two dinoflagellates in a well-mixed estuarine embayment: the importance of diel vertical migration and surface avoidance, Mar. Ecol. Prog. Ser. 25 (1985) 39–50. [4] D.M. Anderson, J.M. Burkholder, W. Cochlan, P.M. Glibert, C.J. Gobler, C.A. Heil, R.M. Kudela, M.L. Parsons, J.E.J. Rensel, D.W. Townsend, V.L. Trainer, G.A. Vargo, Harmful algal blooms and eutrophication: examining linkages from selected coastal regions of the United States, Harm. Algae 8 (2008) 39–53.

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Comparing the diel vertical migration of Karlodinium veneficum (dinophyceae) and Chattonella subsalsa (Raphidophyceae): PSII photochemistry, circadian control, and carbon assimilation.

Diel vertical migration (DVM) is thought to provide an adaptive advantage to some phytoplankton, and may help determine the ecological niche of certai...
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