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 sa- linities 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 accumu- lated 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 bot- tom 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 mi- gration 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 sur- vival (Mahoney & McLaughlin 1979, Shimura et al. 1979). In fact, the halocline can block the DVM ascent and de- scent 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 di- noflagellate 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.