Effects of Salinity on Diel Vertical Migration Behavior in Two Red-Tide Algae, Chattonella Antiqua and Karenia Mikimotoi

Plankton Benthos Res 9(1): 42–50, 2014 Plankton & Benthos Research © The Plankton Society of Japan

Effects of salinity on diel vertical migration behavior in two red-tide algae, Chattonella antiqua and Karenia mikimotoi

1, 1 1 2 TOMOYUKI SHIKATA *, SETSUKO SAKAMOTO , GOH ONITSUKA , KAZUHIRO AOKI & 1 MINEO YAMAGUICHI

1 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi 2–17–5, Hatsukaichi, Hiroshima 739–0452, Japan. 2 National Research Institute of Fisheries Science, Fisheries Research Agency, 2–12–4 Fukuura, Kanazawa, Yokohama, Kanagawa 236–8648, Japan. Received 17 May 2013; Accepted 4 December 2013

Abstract: We examined the effects of salinity on diel vertical migration (DVM) of two coastal flagellates, Chattonella antiqua and Karenia mikimotoi, in 90-cm-high columnar aquariums. Experiments were performed with surface salinities from 5 to 32 and bottom salinity constant at 32. Cells of each flagellate were injected into the bottom of the aquarium at night and the vertical distribution of cells was monitored every 4 h for 36 h in one series of experiments, or twice daily (day and night) for 5 days in another. Ascent and descent started at approximately the same time in all water columns, indicating that difference in surface salinity does not substantially affect DVM rhythm in the two flagellates. During daytime, C. antiqua and K. mikimotoi transited haloclines with surface salinities ≥15 and accumulated at the surface, although K. mikimotoi required 2 days to transit the halocline with surface salinity of 20 and 4 days for surface salinity of 15. However, neither flagellate could transit haloclines with surface salinities of 5 or 10; instead they accumulated in the halocline during daytime. At night, most cells of both species accumulated at the bottom in all water columns, although the distribution of K. mikimotoi gradually expanded to the upper layers on succes- sive days when surface salinity was 5 or 10. We demonstrated that low salinity in the surface layer blocks upward migration in two flagellate species and delays surface accumulation and weakens population synchrony of DVM at night in K. mikimotoi.

Key words: acclimation, accumulation, ﬂagellate, halocline

(Watanabe et al. 1995, Smayda 1997, Kamykowski et al. Introduction 1998, Salonen & Rosenberg 2000). Diel vertical migration Some flagellate algae display diel vertical migration is therefore one of the most important physiological adap- (DVM) behavior. They start to swim toward the surface tations for survival and growth of flagellate algae. before dawn and to deeper layers at dusk (Yamochi & Abe Diel vertical migration behavior should be less subject to 1984, Olsson & Granéli 1991, Koizumi et al. 1996, Park et the external environment because its rhythm is controlled al. 2001). This DVM enables flagellates to optimize photo- by an endogenous clock (Roenneberg et al. 1989, Shikata synthesis regardless of weather or water clarity (Ault et al. 2013). However, some environmental conditions such 2000), to acquire nutrients over a wide depth-range (Wata- as nutrient concentrations and light levels can sometimes nabe et al. 1991, Hall & Paerl 2011), and to avoid predation affect this physiological behavior (Heaney & Eppley 1980, by zooplankton, which swim to the surface at night and re- Richter et al. 2002). Salinity is also an important environ- turn to deeper layers in the daytime (Lampert 1989). Diel mental condition for completion of DVM in coastal flagel- vertical migration thus aids in competition with other mi- lates. In coastal areas, freshwater inflow from rivers causes croalgae such as diatoms, which do not have this ability dramatic decreases in surface salinity, thereby forming strong haloclines (Shikata et al. 2008b). Therefore, flagel- * Corresponding author: Tomoyuki Shikata; E-mail, [email protected] lates performing DVM there often have to transit the halo- Migration of red-tide algae in halocline 43 cline. However, the rapid salinity changes that migrating flagellates must experience in the halocline can inhibit their swimming ability (Shikata et al. 2008a) and their survival (Mahoney & McLaughlin 1979, Shimura et al. 1979). In fact, the halocline can block the DVM ascent and descent of some flagellates (Legović et al. 1991, Bearon et al. 2006, Jephson & Carlsson 2009, Jephson et al. 2011). Because there have been few studies on the effects of environmental factors on the DVM behavior of flagellate algae, we examined the effects of salinity on DVM in the raphidophyte Chattonella antiqua (Hada) Ono and the dinoflagellate Karenia mikimotoi (Miyake & Kominami) Hansen & Moestrup. These two flagellates form harmful red tides that cause tremendous damage to aquaculture in- dustries along the Japanese coast (Okaichi 2004, Imai & Yamaguchi 2012). In both species, DVM facilitates the growth of individual cells and increases in population size and can lead to fish kills through surface accumulation of cells (Koizumi et al. 1996, Amano et al. 1998, Honjo 2004). Fig. 1. Columnar aquarium used as a culture vessel to observe diel vertical migration. The aquarium had nine sampling ports Materials and Methods for measurement of cell density and was capped with a daylight colored-LED lamp. Culture We isolated clonal strains of Chattonella antiqua (strain to the bottom of the aquarium via a long glass tube (length, CHA3) from the Yatsushiro Sea, Japan, in June 2010 and 1 m; inner diameter, 5 mm) and a funnel. After allowing Karenia mikimotoi (strain KM02) from near the North Ku- the water column to stabilize for 3–4 h, we used a syringe juku Islands, Japan, in July 2005. The C. antiqua strain and needle (gauge, 0.7 mm) to inject 25–50 mL of a cell was axenic, but that of K. mikimotoi was not. Cultures suspension containing about 10,000–20,000 cells mL－1 of were maintained in 50-mL Erlenmeyer flasks containing Chattonella antiqua or Karenia mikimotoi through the 25 mL of modified SWM-3 medium (Shikata et al. 2011) sampling port at the bottom of the aquarium; the injection with a salinity of 32 at 25°C under 300 μmol photons was done at night (2000 LT, Day 0). The final water depth m–2 sec–1 of white fluorescent irradiation on a 12-h : 12-h in the aquarium was about 85 cm. Thereafter, at fixed time light:dark cycle [light period, 0600–1800 local time (LT)]. intervals, 3.5 mL of the cell suspension was sampled from Irradiance in the incubator was measured with a Quantum the water surface or from each sampling port, and then the Scalar Laboratory Irradiance Sensor (QSL-2101, Biospher- aquarium was refilled from the surface with 17.5 mL of ical Instruments Inc., San Diego, CA, USA). fresh medium matching the surface salinity (5–32) and from the bottom sampling port with 17.5 mL of fresh me- Monitoring of DVM with different surface salinities dium matching the bottom salinity (32). We used a columnar aquarium as a culture vessel to To determine the vertical distribution of cells, we mea- monitor DVM (Fig. 1). The aquarium (inner diameter, sured the in vivo fluorescence (Brand et al. 1981) of the 5 cm; height, 90 cm) was made of acrylic, with nine sample from each depth by using a fluorometer (Model sampling ports (diameter, 13 mm) at intervals of 10 cm, 10–005R; Turner Designs, Sunnyvale, California, USA). starting from the bottom. Each port was plugged with a Previous studies (Yamaguichi & Honjo 1989, Yamaguchi silicone-rubber stopper. The aquarium was placed in a et al. 1991) showed that the cell densities of C. antiqua and temperature-controlled room (25°C) and capped with a K. mikimotoi were positively correlated with in vivo fluo- disc-shaped LED lamp (daylight color; INREDA, Spot- rescence. The salinity of each sample was measured at the light LED, Inter IKEA Systems B.V., Netherland). The light same time with an immersion refractometer (IS/Mill-E, intensity at the surface was 250 μmol photons m－2 sec－1. AS ONE Corporation, Japan) to observe the vertical distri- The experiments were performed with surface salinities bution of salinity. For sample collection during the dark varying from 5 to 32 and a bottom salinity kept constant at period, we used a red LED flashlight (peak wavelength, 32. To establish a salinity gradient, we first added 810– 640 nm) for illumination, because red light has little effect 820 mL of modified SWM-3 medium with a salinity of on the DVM rhythm of C. antiqua (Shikata et al. 2013) or 5–32, depending on the desired halocline. Then the same K. mikimotoi (Shikata et al. unpublished). volume of medium with a salinity of 32 was slowly added 44 T. Shikata et al.

ing the daytime (35 cm water depth; salinity, 9–11) in Design of the two experiments water columns with surface salinity of 5 or 10 (Fig. 2b, c). We conducted two experiments. In the first experiment The results were more complicated for K. mikimotoi (Fig. (hereafter, the short-term experiment), variations in the 3b–g). From daytime on Day 1, most cells accumulated at vertical distribution of cells were monitored in water col- the surface (0 cm water depth) in water columns with sur- umns with six different surface salinities (5, 10, 15, 20, 25 face salinities of 25 and 32. In the water column with a or 32). Samples were collected every 4 h for 36 h starting surface salinity of 20, most K. mikimotoi cells accumulated from 0000 LT on Day 1. In the second experiment (hereaf- in the upper part of the halocline (35 cm water depth; sa- ter, the long-term experiment), variations in the vertical linity, 21) on Day 1 but reached the surface on the follow- distribution of cells were monitored in water columns of ing day. When the surface salinity was 15, the maximum- two or three different surface salinities (for Chattonella density layer for K. mikimotoi during the daytime shifted antiqua: surface salinity 5 or 10, bottom salinity 32; for from the middle of the halocline (45 cm water depth; salin- Karenia mikimotoi: surface salinity 5, 10, or 15, bottom sa- ity, 25) on Day 1 to the upper halocline (35 cm water linity 32). Samples were collected twice a day (daytime, depth; salinity, 15) on Day 2. When the surface salinity 1230–1330 LT; nighttime, 1900–2000 LT) every day, from was 5 and 10, most K. mikimotoi cells accumulated in the daytime on Day 1 to daytime on Day 5. Both experiments middle of the halocline (45 cm water depth; salinity, 20– were performed in triplicate. Water temperature at the sur- 22) during the daytime on both days. At night, most cells face (1 cm water depth) measured with a temperature data of both species accumulated at or near the bottom regard- logger (RTR-52A, T&D Corporation, Japan) remained al- less of halocline strength (Figs. 2b, 3b). most constant (25±0.3°C). The halocline is defined as the Long-term experiment layer in which the difference in average salinity between neighboring sampling ports is ≥2. There was minimal dis- We followed the DVM behaviors of Chattonella antiqua turbance of salinity gradients in all water columns and Karenia mikimotoi for 5 days in the water columns throughout the experimental periods. For example, the sur- with quite low salinity in the upper layers, in which they face and bottom salinities in the most stratified column could not reach the surface in the short-term experiment, ranged from 4 to 7 and from 30 to 34, respectively, in the to see whether they could transit the haloclines and reach two experiments. the surface given more time. Average in vivo fluorescence of C. antiqua and K. mikimotoi at 10 sampling depths in the water column fluctuated in the range of 101–102 over 5 Results days, and increased only little by little at any of the surface salinities (Figs. 4a, 5a), indicating that the two species Short-term experiment grew only slightly over the experimental period at any of The vertical distributions of Chattonella antiqua and the surface salinities. Most C. antiqua cells could not tran- Karenia mikimotoi in water columns with different surface sit the haloclines and instead accumulated in the upper and salinities were monitored every 4 h for 36 h to examine middle parts of the haloclines (35–45 cm water depth; sa- how low salinity in the surface layer affected the DVM be- linity, 7–22) in the daytime and at the bottom at night for havior of the two flagellates over a short time-period. Av- all 5 days (Fig. 4b, c). Karenia mikimotoi also could not erage in vivo fluorescence of C. antiqua and K. mikimotoi transit the haloclines and accumulated in the upper or mid- at the 10 sampling depths in the water column generally dle part of the haloclines (35–45 cm water depth; salinity, fluctuated in the range of 101–102 over the experimental pe- 12–21) in the daytime when the surface salinity was 5 or riod, but there was no clear increasing or decreasing trend 10 (Fig. 5b, c). When the surface salinity was 15, however, under any experimental conditions (Figs. 2a, 3a). This most K. mikimotoi cells were found in the upper part of the means that total cell numbers in the water column were halocline in the daytime until Day 2, but thereafter the per- roughly constant, indicating that noticeable death and centage of cells at the surface in the daytime increased growth did not occur over the experimental period in any daily and exceeded 50% on Day 4 (Fig. 5d). At night, most of the haloclines. In all salinity gradients, C. antiqua K. mikimotoi cells accumulated at the bottom for all sur- started to ascend at 0000–0400 and to descend at 1600– face salinities over 5 days, but the percentage of cells at the 2000 (Fig. 2b–g); K. mikimotoi started to ascend at 0400– bottom gradually decreased and those in the upper layers 0800 and to descend at 1200–1600 (Fig. 3b–g). This indi- increased when the surface salinity was 5 or 10 (Fig. 5b, c). cated that the DVM rhythms of both species were approximately constant regardless of surface salinity. However, Discussion the layers of maximum in vivo fluorescence during the daytime differed for different surface salinities. For C. Diel vertical migration behavior should not only provide antiqua, most cells accumulated at the surface (0 cm water advantages to flagellates by enabling efficient acquisition depth) in water columns with surface salinities greater of nutrients and light, but would also expose them to the than 15 but remained in the upper part of the halocline dur- risk of down- and up-shocks due to salinity changes in Migration of red-tide algae in halocline 45

Fig. 2. Short-term (36 h) experiments for diel vertical migration of Chattonella antiqua in water columns with various surface salinities (5, 10, 15, 20, 25, and 32) and bottom salinity of 32. (a): Temporal variations in in vivo fluorescence averaged over the entire water column (mean±SD, n=3). (b–g): Time-series sections of the vertical percentage distribution of cells (mean, n=3) in the columns and vertical profiles of the average salinity over the experimental period. White and black bars above each panel represent light and dark periods, respectively. Gray areas in (b)–(f) represent the halocline. coastal areas where there are frequent inflows of fresh (Raphidophyceae) and Heterocapsa triquetra (Ehrenberg) water to the surface layer. Therefore, coastal flagellates F. Stein (Dinophyceae), cessation of upward swimming should have the ability to quickly sense salinity changes and a decrease in swimming speed have been observed and to control swimming patterns. In some flagellates, when they transit a strong halocline in laboratory condi- such as Tetraselmis sp. (Prasinophyceae), Heterosigma tions (Erga et al. 2003, Bearon et al. 2006, Jephson et al. akashiwo (Y. Hada) Y. Hada ex Y. Hara & M. Chihara 2011). Some flagellates such as Gyrodinium aureolum 46 T. Shikata et al.

Fig. 3. Short-term (36 h) experiments for diel vertical migration of Karenia mikimotoi in water columns with various surface salinities (5, 10, 15, 20, 25, and 32) and bottom salinity of 32. (a): Temporal variations in in vivo ﬂuorescence averaged over the entire water column (mean±SD, n=3). (b–g): Time-series sections of the vertical percentage distribution of cells (mean, n=3) in the columns and vertical proﬁles of the average salinity over the experimental period. White and black bars above each panel represent light and dark periods, respectively. Gray areas in (b)–(f) represent the halocline.

Hulburt form a thick bloom in a strong halocline (Bjørnsen when salinity is low (＜10) after inﬂow from a large river. & Nielsen 1991). We found that Chattonella antiqua and Onitsuka et al. (2011) observed that a maximum layer of Karenia mikimotoi stopped ascent in the halocline when the cell density of C. antiqua was located in the halocline there is low salinity in the upper layer (Figs. 2b, 3b). only at sampling stations where the surface salinity was According to ﬁeld observations by Katano et al. (2012), quite low. Chattonella spp. does not migrate into the surface layer Some microalgae control intracellular concentrations of Migration of red-tide algae in halocline 47

Fig. 4. Long-term (5 days) experiments for diel vertical migration of Chattonella antiqua in water columns with surface salinities of 5 and 10, into which C. antiqua did not penetrate in the short-term experiment, and with a bottom salinity of 32. (a): Daily variations in the in vivo fluorescence (mean±SD, n=3) averaged over the entire water column; white and black bars represent light and dark periods, respectively. (b, c): Time-series sections of the vertical percentage distribution of cells (mean, n=3) in daytime and at night in the columns with surface salinities of 5 (b) or 10 (c), and vertical profiles of average salinities in the daytime over the experimental period; gray areas represent the halocline. organic osmolytes such as glycerol and ribitol, and ions such shown). These results indicate that both species could as K＋ and Cl－ so as to tolerate salinity changes (Hellebust avoid low salinities causing physiological damage. 1985, Kirst 1989, Gustavs et al. 2010). However, osmotic The lowest salinity into which a flagellate can enter by pressure moves water in or out of the cell across the cell DVM varies among species: H. akashiwo, 8 (Bearon et al. membrane before osmoregulation is accomplished; there- 2006); C. antiqua, 7 (this study); and K. mikimotoi, 12 (this fore the volume and shape of the cell change immediately study). These values are usually close to the threshold sa- after a salinity change (McMillan & Johansen 1988, Kirst linity for their growth: H. akashiwo, 2–5 (Tomas 1978, 1989). In consequence, salinity changes can cause tempo- Martínez et al. 2010); C. antiqua, 5–10 (Shikata et al. rary inactivation of motility and cause cell death (Helle- 2010); and K. mikimotoi, 10–15 (Yamaguchi & Honjo bust 1985, Kirst 1989, Shikata et al. 2008b). In the present 1989). This suggests that coastal flagellates can transit study, however, the average in vivo fluorescence (that is, haloclines as long as the salinity above the halocline is total cell numbers) of C. antiqua and K. mikimotoi in the above the threshold for their growth. From this perspec- water column did not decrease over the experimental peri- tive, C. antiqua can photosynthesize and grow more suc- ods, even when the surface salinity was extremely low. cessfully in shallower layers than K. mikimotoi when sur- Our microscopic observations revealed that most cells in face salinity is quite low. The present study found that, the halocline were motile and had a normal shape (data not compared with K. mikimotoi, C. antiqua requires a much 48 T. Shikata et al.

Fig. 5. Long-term (5 days) experiments for diel vertical migration of Karenia mikimotoi in water columns with surface salinities of 5, 10 and 15, into which K. mikimotoi did not penetrate during the short-term experiment, and with a bottom salinity of 32. (a): Daily variations in the in vivo fluorescence (mean±SD, n=3) averaged over the entire water column; white and black bars represent light and dark periods, respectively. (b–d): Time-series sections of the vertical percentage distribution of cells (mean, n=3) in daytime and at night in the columns with surface salinities of 5 (b), 10 (c) or 15 (d), and vertical profiles of average salinities in the daytime over the experimental period; gray areas represent the halocline. shorter period to reach surface layers with low salinities, rivers such as the Chikugo River and the Kuma River, re- and that C. antiqua can concurrently descend to the bot- spectively, and why C. antiqua blooms can develop when tom at night even when the surface salinity is extremely surface salinity is extremely low (10–15) in these estuaries low during the day, allowing secure nutrient-uptake in (Matsubara et al. 2009). On the other hand, K. mikimotoi deeper layers. These advantages due to DVM in C. antiqua rarely blooms in strongly enclosed bays around Japan, and may partly explain why C. antiqua frequently blooms in the salinity necessary for blooming of this species is rather the Ariake Sea and the Yatsushiro Sea (Matsubara et al. high (mainly 28–33) (Matsuoka et al. 1989). 2009, Aoki et al. 2012), where there are inflows from large The presence of low-salinity surface layers strengthens Migration of red-tide algae in halocline 49 vertical stability of the water column, which promotes cell Balzano S, Sarno D, Kooistra WHCF (2011) Effects of salinity accumulation by DVM and thereby increases the likeli- on the growth rate and morphology of ten Skeletonema strains. hood of red tide formation by flagellates (Hershberger et al. J Plankton Res 33: 937–945. 1997). 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