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Effects of temperature, salinity and irradiance on growth of the novel red tide flagellate Chattonella ovata (Raphidophyceae)
Harmful Algae 9 (2010) 398–401
Contents lists available at ScienceDirect
Harmful Algae journal homepage: www.elsevier.com/locate/hal
Effects of temperature, salinity and irradiance on growth of the novel red tide flagellate Chattonella ovata (Raphidophyceae) Haruo Yamaguchi a, Koichiro Mizushima b,1, Setsuko Sakamoto b, Mineo Yamaguchi b,* a
Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan Harmful Algal Bloom Division, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 July 2009 Accepted 5 February 2010
To elucidate the mechanism of bloom outbreaks of the ‘novel’ noxious flagellate Chattonella ovata (Raphidophyceae), we examined the growth responses of the organism with 42 different combinations of temperature (10–35 8C) and salinity (10–35), and under various light intensities (0–381 mmol photons m2 s1). The three strains, CO2, CO3, and CO8 of C. ovata isolated from Hiroshima Bay, tolerated a wide range of temperature (15–32.5 8C) and salinity (10–35). The organism could grow rapidly at over 20 8C and salinity of 20, and the maximal growth rates of 1.11–1.47 divisions day1 were found in the combination 25–30 8C and salinity of 25–30. These maximum growth rates and optimal temperatures and salinities of the C. ovata strains were clearly higher than those of Chattonella antiqua and Chattonella marina. A statistical analysis showed that growth rates of C. ovata were significantly influenced by temperature–salinity interaction. Furthermore, growth of the strain CO2 and CO8 was observed at the irradiance 15–45 mmol photons m2 s1 or more and was saturated at over 300 mmol photons m2 s1. The half-saturation constants of irradiance for the growth of C. ovata were much higher than those of C. antiqua and C. marina. These results showed that C. ovata is better adapted to higher temperature, salinity and light environments than C. antiqua and C. marina. These physiological features of C. ovata would be an ecological advantage to development of the bloom during summer seasons. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Chattonella ovata Growth rate Irradiance Red tide Salinity Temperature
1. Introduction Species of the genus Chattonella (Raphidophyceae) are harmful flagellates which cause mass mortality of cultured fishes in coastal waters (Hallegraeff, 1993; Imai et al., 1998; Marshall and Hallegraeff, 1999). So far in Japanese waters, the most representative and harmful species recorded are Chattonella antiqua and Chattonella marina. The first bloom of C. antiqua occurred in Hiroshima Bay in 1969. Since then these species have caused severe damage to fish aquaculture in this area (Imai et al., 1989; Imai et al., 2006). From July through September in 2004, red tides repeatedly occurred in the Seto Inland Sea, Japan and caused severe damage to cultured fish (Hiroishi et al., 2005; H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008). The main causative species of the red tides was identified to be Chattonella ovata (Raphidophyceae) Y. Hara et Chihara (Hara et al., 1994). This is the first record of a bloom and
* Corresponding author. Tel.: +81 829 55 0666; fax: +81 829 54 1216. E-mail address: [email protected] (M. Yamaguchi). 1 Present address: Sanyo Techno Marine, Inc., 1-3-17 Nihonbashihoridome, Chuo-ku, Tokyo 103-0012, Japan. 1568-9883/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2010.02.001
damage to fisheries caused by C. ovata in Japan (Hiroishi et al., 2005), although vegetative cells have been observed since the mid 1980s (Yoshimatsu and Ono, 1986). Since 2004 C. ovata blooms have been conspicuous in coastal environments around Western Japan (Yamatogi et al., 2006). Blooms of C. ovata have been observed not only in Japan, but also in China and Mexico since 2001 (Lu and Hodgkiss, 2001; Barraza-Guardado et al., 2004; Corte´sAltamirano et al., 2006). Therefore it is critical to clarify the ecophysiological characteristics of the organism, for understanding the environmental factors controlling the bloom dynamics of C. ovata. We have already reported the nutrition and growth kinetics and morphology, distribution and germination of the cysts of C. ovata (H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008) in previous laboratory and field works. However, physical factors, especially temperature, salinity and irradiance, influencing bloom dynamics of C. ovata have not been fully clarified yet (Yamatogi et al., 2006). In the present study, we examined the effects of temperature, salinity and irradiance on the growth rate of C. ovata isolated from Hiroshima Bay. On the basis of the results the contribution of these physical factors to the bloom dynamics of C. ovata is discussed.
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
2. Materials and methods 2.1. Cultures The three strains of C. ovata (strains CO2, CO3 and CO8) used in the present experiment were isolated from Hiroshima Bay in 2004. These strains are clonal but not axenic. The stock cultures were maintained in 50-mL flasks containing 25 mL of modified SWM-3 medium at 25 8C under 150 mmol photons m2 s1 of cool-white fluorescent illumination on a 12:12 h L:D cycle (light period 06:00–18:00 h). The medium contained 2 mM NaNO3, 0.1 mM NaH2PO4, 0.2 mM Na2SiO3, 30 mM Na2EDTA, 2 mM Fe-EDTA, 2 nM Na2SeO3, 10 mL P-I metals, 2 mL S3 vitamins and 0.5 g L1 Tris, pH 7.8 (Imai et al., 1996). 2.2. Effects of temperature and salinity on growth rate The culture experiment was carried out at seven temperatures (10, 15, 20, 25, 30, 32.5, 35 8C) in combination with six salinities (10, 15, 20, 25, 30, 35) using a temperature gradient growth chamber (TG-100-AD, Nippon Medical & Chemical Instrument Co. Ltd, Japan) and growth cabinet (MLR-350, Sanyo Co. Ltd, Japan). The salinities of below 30 were obtained by diluting aged seawater from Hiroshima Bay with distilled water. A salinity of 35 was obtained by concentrating the natural seawater with a drying oven at 50 8C. Cultures of strain CO2, CO3 and CO8 were pre-adapted to experimental conditions through stepwise transfer of stock cultures to each temperature and salinity regime (Yamaguchi et al., 1997). Acclimated stock cultures were inoculated into triplicate PP (polypropylene)-capped test tubes (13 150 mm) for each experimental regime. Inoculum size was adjusted giving ca. 1/100 (v/v) of pre-cultures. Growth rates in each experimental regime were determined by measuring the in vivo chlorophyll (chl.) a fluorescence, because significant correlations (r > 0.912, p < 0.001) were found between the cell density and the chl. a fluorescence in each strain of C. ovata (data not shown). The in vivo chl. a fluorescence was measured every 2 days using a Turner Designs Model 10-100 R fluorometer (Brand et al., 1981), and growth rate (m: divisions day1) was calculated using data from the exponential portion of the growth curve by least squares regressions of the natural logarithm of fluorescence on day number (Yamaguchi and Honjo, 1989). The mean of growth rates was calculated using the three independent estimates of m. The fluorescence decline from the initial value at extreme temperatures and salinities was not quantified, and was evaluated to
399
represent a zero growth rate. Effect of temperature and salinity on the growth rates was tested by analysis of variance (two-way ANOVA). On the basis of the ANOVA results, cubic equations of the form,
m ¼ b00 þ b10 T þ b20 T 2 þ b30 T 3 þ b01 S þ b02 S2 þ b03 S3 þ b11 TS þ b12 TS2 þ b21 T 2 S; where m = growth rate, T = temperature, S = salinity and bnn = regression coefficients, were fitted by the multiple regression analysis. 2.3. Effect of irradiance on growth rate Effect of irradiance on the growth rate of C. ovata was examined using two strains CO2 and CO8. The culture experiment was carried out under the range of irradiance 0–381 mmol photons m2 s1 at 27.5 8C. Stock cultures of CO2 and CO8 were inoculated into PP-capped test tubes (37 130 mm) containing 50 mL of the SWM-3 medium (salinity was adjusted to 30). The inoculum size was adjusted giving ca. 1/100 (v/v) of pre-cultures to obtain ca.100 cells mL1 as an initial cell density. Growth rates at each irradiance were determined by measuring cell densities every day and were calculated as described above. Modified hyperbolic function (Eq. (1)) was fitted to the irradiance and growth rate data, which was originally proposed for the photosynthesis-irradiance curve without photo-inhibition (Lederman and Tett, 1981).
m ¼ mmax
I I0 ðK s I0 Þ þ ðI I0 Þ
(1)
where mmax, I0, and Ks are the maximum growth rate (divisions day1), threshold of light intensity for growth (mmol photons m2 s1) and half-saturation constant for growth rate mmol photons m2 s1), respectively. The equation was fitted by the least squares method using the software Deltagraph (Red Rock Software, Inc., USA). 3. Results and discussion Response surface contours of the growth rates of the three strains CO2, CO3, and CO8 of C. ovata isolated from Hiroshima Bay for the temperature and salinity combinations are shown in Fig. 1. All three strains tested in the present experiments showed similar responses to temperature and salinity regime. The strains tolerated a wide range of temperature (15–32.5 8C) and salinity (10–35). The strains could grow rapidly at over 20 8C and salinity of 20, and the
Fig. 1. Response surface contours of the growth rates of three strains of Chattonella ovata as functions of temperature and salinity.
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
400
Table 1 Results of two-way ANOVA of the growth rate of Chattonella ovata as a function of temperature, salinity and temperature–salinity interaction. Strain
Source of variation
DF
SS
MS
F
CO2
Temperature Salinity Interaction Error Total
6 5 30 84 125
16.78 3.64 6.17 0.56 27.15
2.797 0.727 0.206 0.007
416.8*** 108.3*** 30.7***
CO3
Temperature Salinity Interaction Error Total
6 5 30 84 125
16.13 1.21 6.15 0.23 23.73
2.689 0.242 0.205 0.003
967.3*** 87.1*** 73.7***
CO8
Temperature Salinity Interaction Error Total
6 5 30 84 125
21.42 3.10 6.25 0.43 31.19
3.569 0.621 0.208 0.005
702.6*** 122.2*** 40.9***
***
p < 0.001.
maximal growth rates of 1.21, 1.11, and 1.47 divisions day1 were obtained in the combination of 30 8C and salinity of 25 for CO2, 25 8C and 25 for CO3, and 30 8C and 30 for CO8, respectively. Furthermore, higher growth rates (>1.0 divisions day1) were observed even at 32.5 8C. The result was almost consistent with that obtained in the C. ovata strain isolated from the coastal water in western Kyushu, Japan (Yamatogi et al., 2006). These results indicate that C. ovata prefers high temperature and salinity environments. In contrast, previous studies have shown that maximum growth rates of other species of Chattonella (C. antiqua and C. marina) were obtained at 25 8C and salinity of 20–25 (Yamaguchi et al., 1991). In addition the growth remarkably dropped at 30 8C and maximal growth rates of these two species were less than 1.1 divisions day1 (Yamaguchi et al., 1991; Nakamura and Watanabe, 1983; Marshall and Hallegraeff, 1999). Therefore C. ovata presumably predominates over other Chattonella species in the environments of higher temperature and salinity. A two-way ANOVA result shows that temperature, salinity, and temperature–salinity interaction significantly affect the growth rate of C. ovata at the 0.1% level (Table 1). Especially over 61% of the total sum squares was accounted for by the sum of temperature, which was higher than the values for salinity (5.1–13%). Thus temperature is the most important factor contributing to the observed variation in growth rates. On the basis of the ANOVA results, the obtained multiple regression equations of the growth rate of each strain on temperature and salinity were as follows:
formulae it is now possible to estimate the in situ growth rates of the organisms using temperature and salinity data from field observations (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Toda et al., 1994). In the present experiments, growth rates of the C. ovata strains dropped sharply to less than 0.2 divisions day1 at below 15 8C and growth was not observed at 10 8C (Fig. 1). Thus vegetative cells of C. ovata cannot over-winter in temperate waters such as the Seto Inland Sea, because the water temperature decreases to 10 8C or below during the winter season in these waters (Imai et al., 1998; Oda, 2007; Seiki et al., 2008). We have previously found the resting cysts of C. ovata from the natural sediments in Hiroshima Bay (M. Yamaguchi et al., 2008). This indicates that C. ovata has a dormant cyst stage in its life cycle like the two other species of Chattonella and the over-wintered cysts play an important role in initiating summer blooms C. ovata (Imai and Itoh, 1987; Imai, 1990; Yamaguchi and Imai, 1994). Furthermore the previous works have shown that the C. ovata cysts actively germinated at 27.5–30 8C but not at 15 8C or below in the laboratory conditions (M. Yamaguchi et al., 2008). Thus the lower limit and the optimum temperatures for the germination of C. ovata cysts was estimated to be 2.5 and 7.5 8C higher than that of C. antiqua and C. marina, respectively (Imai, 1990; M. Yamaguchi et al., 2008). These results suggest that C. ovata prefers warmer waters not only for the germination of the cysts but also for growth of the vegetative cells than other Chattonella species. Therefore a high water temperature is concluded to be an important factor for the initiation and development of C. ovata bloom. The relationships between growth rates and irradiances and the fitted rectangular hyperbola for the two strains (CO2 and CO8) of C. ovata are shown in Fig. 2. The growth of the strain CO2 and CO8 was observed at the irradiance 15–45 mmol photons m2 s1 or more and saturated at over 300 mmol photons m2 s1. As shown in Fig. 2, the observed values of the growth rate closely fit the
CO2;
m ¼ 0:58034 0:00894TS 0:00223S2 þ 0:00004TS2 0:00021T 2 S; CO3;
m ¼ 0:947347 0:23914T þ 0:01747T 2 0:00034T 3 0:00001S3 þ 0:00003TS2 ; CO8;
m ¼ 2:6997 0:56579T þ 0:03105T 2 0:00059T 3 0:00121S2 þ 0:00555TS 0:0001T 2 S: The regression model fits the data well with R2 values of 0.61, 0.69 and 0.78 for CO2, CO3 and CO8, respectively. Based on these
Fig. 2. Effect of irradiance on growth of Chattonella ovata (strains CO2 and CO8).
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
hyperbolic function and can be expressed as the following formulas: ðI 20:1Þ ðR2 ¼ 0:94; n ¼ 20; p < 0:01Þ; CO2 : m ¼ 2:09 ðI þ 138Þ CO8 : m ¼ 1:49
ðI 15:5Þ ðI þ 56:5Þ
ðR2 ¼ 0:92; n ¼ 20; p < 0:01Þ:
Obtained parameters mmax, I0 and Ks were 2.09 divisions day1, 20.1 mmol photons m2 s1 and 178 mmol photons m2 s1 for CO2 and 1.49 divisions day1, 15.5 mmol photons m2 s1 and 87.5 mmol photons m2 s1 for CO8, respectively. These parameters for the growth of C. ovata are larger than those of C. antiqua and C. marina (Yamaguchi et al., 1991). Especially half-saturation constants for the growth (Ks) of C. ovata are two to three times larger than those of the other two species. This suggests that C. ovata requires considerably higher irradiances for its growth than C. antiqua and C. marina. C. ovata was thought to be a hidden flora member in the Seto Inland Sea, Japan because its vegetative cells had been observed since the 1980s (Yoshimatsu and Ono, 1986). However, a bloom and damage to fisheries caused by C. ovata occurred in the summer of 2004 for the first time (H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008). Thus it is critical to determine the key factors causing the outbreaks of C. ovata in the summer for understanding the bloom dynamics of the organism. From the results obtained from the present experiments, we can demonstrate the importance of high water temperature, salinity and irradiance for the growth of C. ovata. Actually, the summer in 2004 has been severer than usual. The surface water temperature from February to July in 2001 in the eastern Seto Inland Sea was more than 1 8C higher than the normal year (Oda, 2007). In addition, referring to online data provided from Japan Meteorological Agency (http://www.jma.go.jp/jma/), we summarized and compared the hours of sunlight (h) and the mean values of the daily solar radiation (MJ m2) in Hiroshima City during June–August for 2004 with that of the last 5 years (1999– 2003). These values of the summer of 2004 were 623.6 h and 19.2 MJ m2, respectively and clearly higher than those of the last 5 years (538.2 h and 17.4 MJ m2). Therefore the water temperature and irradiance in 2004 were suitable for the growth of C. ovata. Thus we can conclude that high water temperature and irradiance are critical physical factors contributing to the outbreaks of C. ovata blooms in the summer of 2004. The physiological features obtained in the present study suggest that C. ovata is well adapted to warmer water and higher irradiance environment and can predominate over C. antiqua and C. marina. Since there is a possibility for future outbreaks of C. ovata in temperate and subtropical waters, careful monitoring of this species is necessary to prevent damages to fisheries caused by the organism. For that purpose the growth parameters of temperature, salinity and irradiance obtained in the present study is useful to establish a prediction model of the organism. References Brand, L.E., Guillard, R.R.L., Murphy, L.S., 1981. A method for the rapid and precise determination of acclimated phytoplankton reproduction rates. J. Plankton Res. 3, 193–201. Barraza-Guardado, R., Corte´s-Altamirano, R., Sierra-Beltra´n, A., 2004. Marine dieoffs from Chattonella marina and Ch. cf. ovata in Kun Kaak Bay, Sonora in the Gulf of California. In: Wyatt, T. (Ed.), Harmful Algae News. IOC of UNESCO, No. 25, pp. 7–8.
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Contents lists available at ScienceDirect
Harmful Algae journal homepage: www.elsevier.com/locate/hal
Effects of temperature, salinity and irradiance on growth of the novel red tide flagellate Chattonella ovata (Raphidophyceae) Haruo Yamaguchi a, Koichiro Mizushima b,1, Setsuko Sakamoto b, Mineo Yamaguchi b,* a
Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan Harmful Algal Bloom Division, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 July 2009 Accepted 5 February 2010
To elucidate the mechanism of bloom outbreaks of the ‘novel’ noxious flagellate Chattonella ovata (Raphidophyceae), we examined the growth responses of the organism with 42 different combinations of temperature (10–35 8C) and salinity (10–35), and under various light intensities (0–381 mmol photons m2 s1). The three strains, CO2, CO3, and CO8 of C. ovata isolated from Hiroshima Bay, tolerated a wide range of temperature (15–32.5 8C) and salinity (10–35). The organism could grow rapidly at over 20 8C and salinity of 20, and the maximal growth rates of 1.11–1.47 divisions day1 were found in the combination 25–30 8C and salinity of 25–30. These maximum growth rates and optimal temperatures and salinities of the C. ovata strains were clearly higher than those of Chattonella antiqua and Chattonella marina. A statistical analysis showed that growth rates of C. ovata were significantly influenced by temperature–salinity interaction. Furthermore, growth of the strain CO2 and CO8 was observed at the irradiance 15–45 mmol photons m2 s1 or more and was saturated at over 300 mmol photons m2 s1. The half-saturation constants of irradiance for the growth of C. ovata were much higher than those of C. antiqua and C. marina. These results showed that C. ovata is better adapted to higher temperature, salinity and light environments than C. antiqua and C. marina. These physiological features of C. ovata would be an ecological advantage to development of the bloom during summer seasons. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Chattonella ovata Growth rate Irradiance Red tide Salinity Temperature
1. Introduction Species of the genus Chattonella (Raphidophyceae) are harmful flagellates which cause mass mortality of cultured fishes in coastal waters (Hallegraeff, 1993; Imai et al., 1998; Marshall and Hallegraeff, 1999). So far in Japanese waters, the most representative and harmful species recorded are Chattonella antiqua and Chattonella marina. The first bloom of C. antiqua occurred in Hiroshima Bay in 1969. Since then these species have caused severe damage to fish aquaculture in this area (Imai et al., 1989; Imai et al., 2006). From July through September in 2004, red tides repeatedly occurred in the Seto Inland Sea, Japan and caused severe damage to cultured fish (Hiroishi et al., 2005; H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008). The main causative species of the red tides was identified to be Chattonella ovata (Raphidophyceae) Y. Hara et Chihara (Hara et al., 1994). This is the first record of a bloom and
* Corresponding author. Tel.: +81 829 55 0666; fax: +81 829 54 1216. E-mail address: [email protected] (M. Yamaguchi). 1 Present address: Sanyo Techno Marine, Inc., 1-3-17 Nihonbashihoridome, Chuo-ku, Tokyo 103-0012, Japan. 1568-9883/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2010.02.001
damage to fisheries caused by C. ovata in Japan (Hiroishi et al., 2005), although vegetative cells have been observed since the mid 1980s (Yoshimatsu and Ono, 1986). Since 2004 C. ovata blooms have been conspicuous in coastal environments around Western Japan (Yamatogi et al., 2006). Blooms of C. ovata have been observed not only in Japan, but also in China and Mexico since 2001 (Lu and Hodgkiss, 2001; Barraza-Guardado et al., 2004; Corte´sAltamirano et al., 2006). Therefore it is critical to clarify the ecophysiological characteristics of the organism, for understanding the environmental factors controlling the bloom dynamics of C. ovata. We have already reported the nutrition and growth kinetics and morphology, distribution and germination of the cysts of C. ovata (H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008) in previous laboratory and field works. However, physical factors, especially temperature, salinity and irradiance, influencing bloom dynamics of C. ovata have not been fully clarified yet (Yamatogi et al., 2006). In the present study, we examined the effects of temperature, salinity and irradiance on the growth rate of C. ovata isolated from Hiroshima Bay. On the basis of the results the contribution of these physical factors to the bloom dynamics of C. ovata is discussed.
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
2. Materials and methods 2.1. Cultures The three strains of C. ovata (strains CO2, CO3 and CO8) used in the present experiment were isolated from Hiroshima Bay in 2004. These strains are clonal but not axenic. The stock cultures were maintained in 50-mL flasks containing 25 mL of modified SWM-3 medium at 25 8C under 150 mmol photons m2 s1 of cool-white fluorescent illumination on a 12:12 h L:D cycle (light period 06:00–18:00 h). The medium contained 2 mM NaNO3, 0.1 mM NaH2PO4, 0.2 mM Na2SiO3, 30 mM Na2EDTA, 2 mM Fe-EDTA, 2 nM Na2SeO3, 10 mL P-I metals, 2 mL S3 vitamins and 0.5 g L1 Tris, pH 7.8 (Imai et al., 1996). 2.2. Effects of temperature and salinity on growth rate The culture experiment was carried out at seven temperatures (10, 15, 20, 25, 30, 32.5, 35 8C) in combination with six salinities (10, 15, 20, 25, 30, 35) using a temperature gradient growth chamber (TG-100-AD, Nippon Medical & Chemical Instrument Co. Ltd, Japan) and growth cabinet (MLR-350, Sanyo Co. Ltd, Japan). The salinities of below 30 were obtained by diluting aged seawater from Hiroshima Bay with distilled water. A salinity of 35 was obtained by concentrating the natural seawater with a drying oven at 50 8C. Cultures of strain CO2, CO3 and CO8 were pre-adapted to experimental conditions through stepwise transfer of stock cultures to each temperature and salinity regime (Yamaguchi et al., 1997). Acclimated stock cultures were inoculated into triplicate PP (polypropylene)-capped test tubes (13 150 mm) for each experimental regime. Inoculum size was adjusted giving ca. 1/100 (v/v) of pre-cultures. Growth rates in each experimental regime were determined by measuring the in vivo chlorophyll (chl.) a fluorescence, because significant correlations (r > 0.912, p < 0.001) were found between the cell density and the chl. a fluorescence in each strain of C. ovata (data not shown). The in vivo chl. a fluorescence was measured every 2 days using a Turner Designs Model 10-100 R fluorometer (Brand et al., 1981), and growth rate (m: divisions day1) was calculated using data from the exponential portion of the growth curve by least squares regressions of the natural logarithm of fluorescence on day number (Yamaguchi and Honjo, 1989). The mean of growth rates was calculated using the three independent estimates of m. The fluorescence decline from the initial value at extreme temperatures and salinities was not quantified, and was evaluated to
399
represent a zero growth rate. Effect of temperature and salinity on the growth rates was tested by analysis of variance (two-way ANOVA). On the basis of the ANOVA results, cubic equations of the form,
m ¼ b00 þ b10 T þ b20 T 2 þ b30 T 3 þ b01 S þ b02 S2 þ b03 S3 þ b11 TS þ b12 TS2 þ b21 T 2 S; where m = growth rate, T = temperature, S = salinity and bnn = regression coefficients, were fitted by the multiple regression analysis. 2.3. Effect of irradiance on growth rate Effect of irradiance on the growth rate of C. ovata was examined using two strains CO2 and CO8. The culture experiment was carried out under the range of irradiance 0–381 mmol photons m2 s1 at 27.5 8C. Stock cultures of CO2 and CO8 were inoculated into PP-capped test tubes (37 130 mm) containing 50 mL of the SWM-3 medium (salinity was adjusted to 30). The inoculum size was adjusted giving ca. 1/100 (v/v) of pre-cultures to obtain ca.100 cells mL1 as an initial cell density. Growth rates at each irradiance were determined by measuring cell densities every day and were calculated as described above. Modified hyperbolic function (Eq. (1)) was fitted to the irradiance and growth rate data, which was originally proposed for the photosynthesis-irradiance curve without photo-inhibition (Lederman and Tett, 1981).
m ¼ mmax
I I0 ðK s I0 Þ þ ðI I0 Þ
(1)
where mmax, I0, and Ks are the maximum growth rate (divisions day1), threshold of light intensity for growth (mmol photons m2 s1) and half-saturation constant for growth rate mmol photons m2 s1), respectively. The equation was fitted by the least squares method using the software Deltagraph (Red Rock Software, Inc., USA). 3. Results and discussion Response surface contours of the growth rates of the three strains CO2, CO3, and CO8 of C. ovata isolated from Hiroshima Bay for the temperature and salinity combinations are shown in Fig. 1. All three strains tested in the present experiments showed similar responses to temperature and salinity regime. The strains tolerated a wide range of temperature (15–32.5 8C) and salinity (10–35). The strains could grow rapidly at over 20 8C and salinity of 20, and the
Fig. 1. Response surface contours of the growth rates of three strains of Chattonella ovata as functions of temperature and salinity.
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
400
Table 1 Results of two-way ANOVA of the growth rate of Chattonella ovata as a function of temperature, salinity and temperature–salinity interaction. Strain
Source of variation
DF
SS
MS
F
CO2
Temperature Salinity Interaction Error Total
6 5 30 84 125
16.78 3.64 6.17 0.56 27.15
2.797 0.727 0.206 0.007
416.8*** 108.3*** 30.7***
CO3
Temperature Salinity Interaction Error Total
6 5 30 84 125
16.13 1.21 6.15 0.23 23.73
2.689 0.242 0.205 0.003
967.3*** 87.1*** 73.7***
CO8
Temperature Salinity Interaction Error Total
6 5 30 84 125
21.42 3.10 6.25 0.43 31.19
3.569 0.621 0.208 0.005
702.6*** 122.2*** 40.9***
***
p < 0.001.
maximal growth rates of 1.21, 1.11, and 1.47 divisions day1 were obtained in the combination of 30 8C and salinity of 25 for CO2, 25 8C and 25 for CO3, and 30 8C and 30 for CO8, respectively. Furthermore, higher growth rates (>1.0 divisions day1) were observed even at 32.5 8C. The result was almost consistent with that obtained in the C. ovata strain isolated from the coastal water in western Kyushu, Japan (Yamatogi et al., 2006). These results indicate that C. ovata prefers high temperature and salinity environments. In contrast, previous studies have shown that maximum growth rates of other species of Chattonella (C. antiqua and C. marina) were obtained at 25 8C and salinity of 20–25 (Yamaguchi et al., 1991). In addition the growth remarkably dropped at 30 8C and maximal growth rates of these two species were less than 1.1 divisions day1 (Yamaguchi et al., 1991; Nakamura and Watanabe, 1983; Marshall and Hallegraeff, 1999). Therefore C. ovata presumably predominates over other Chattonella species in the environments of higher temperature and salinity. A two-way ANOVA result shows that temperature, salinity, and temperature–salinity interaction significantly affect the growth rate of C. ovata at the 0.1% level (Table 1). Especially over 61% of the total sum squares was accounted for by the sum of temperature, which was higher than the values for salinity (5.1–13%). Thus temperature is the most important factor contributing to the observed variation in growth rates. On the basis of the ANOVA results, the obtained multiple regression equations of the growth rate of each strain on temperature and salinity were as follows:
formulae it is now possible to estimate the in situ growth rates of the organisms using temperature and salinity data from field observations (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Toda et al., 1994). In the present experiments, growth rates of the C. ovata strains dropped sharply to less than 0.2 divisions day1 at below 15 8C and growth was not observed at 10 8C (Fig. 1). Thus vegetative cells of C. ovata cannot over-winter in temperate waters such as the Seto Inland Sea, because the water temperature decreases to 10 8C or below during the winter season in these waters (Imai et al., 1998; Oda, 2007; Seiki et al., 2008). We have previously found the resting cysts of C. ovata from the natural sediments in Hiroshima Bay (M. Yamaguchi et al., 2008). This indicates that C. ovata has a dormant cyst stage in its life cycle like the two other species of Chattonella and the over-wintered cysts play an important role in initiating summer blooms C. ovata (Imai and Itoh, 1987; Imai, 1990; Yamaguchi and Imai, 1994). Furthermore the previous works have shown that the C. ovata cysts actively germinated at 27.5–30 8C but not at 15 8C or below in the laboratory conditions (M. Yamaguchi et al., 2008). Thus the lower limit and the optimum temperatures for the germination of C. ovata cysts was estimated to be 2.5 and 7.5 8C higher than that of C. antiqua and C. marina, respectively (Imai, 1990; M. Yamaguchi et al., 2008). These results suggest that C. ovata prefers warmer waters not only for the germination of the cysts but also for growth of the vegetative cells than other Chattonella species. Therefore a high water temperature is concluded to be an important factor for the initiation and development of C. ovata bloom. The relationships between growth rates and irradiances and the fitted rectangular hyperbola for the two strains (CO2 and CO8) of C. ovata are shown in Fig. 2. The growth of the strain CO2 and CO8 was observed at the irradiance 15–45 mmol photons m2 s1 or more and saturated at over 300 mmol photons m2 s1. As shown in Fig. 2, the observed values of the growth rate closely fit the
CO2;
m ¼ 0:58034 0:00894TS 0:00223S2 þ 0:00004TS2 0:00021T 2 S; CO3;
m ¼ 0:947347 0:23914T þ 0:01747T 2 0:00034T 3 0:00001S3 þ 0:00003TS2 ; CO8;
m ¼ 2:6997 0:56579T þ 0:03105T 2 0:00059T 3 0:00121S2 þ 0:00555TS 0:0001T 2 S: The regression model fits the data well with R2 values of 0.61, 0.69 and 0.78 for CO2, CO3 and CO8, respectively. Based on these
Fig. 2. Effect of irradiance on growth of Chattonella ovata (strains CO2 and CO8).
H. Yamaguchi et al. / Harmful Algae 9 (2010) 398–401
hyperbolic function and can be expressed as the following formulas: ðI 20:1Þ ðR2 ¼ 0:94; n ¼ 20; p < 0:01Þ; CO2 : m ¼ 2:09 ðI þ 138Þ CO8 : m ¼ 1:49
ðI 15:5Þ ðI þ 56:5Þ
ðR2 ¼ 0:92; n ¼ 20; p < 0:01Þ:
Obtained parameters mmax, I0 and Ks were 2.09 divisions day1, 20.1 mmol photons m2 s1 and 178 mmol photons m2 s1 for CO2 and 1.49 divisions day1, 15.5 mmol photons m2 s1 and 87.5 mmol photons m2 s1 for CO8, respectively. These parameters for the growth of C. ovata are larger than those of C. antiqua and C. marina (Yamaguchi et al., 1991). Especially half-saturation constants for the growth (Ks) of C. ovata are two to three times larger than those of the other two species. This suggests that C. ovata requires considerably higher irradiances for its growth than C. antiqua and C. marina. C. ovata was thought to be a hidden flora member in the Seto Inland Sea, Japan because its vegetative cells had been observed since the 1980s (Yoshimatsu and Ono, 1986). However, a bloom and damage to fisheries caused by C. ovata occurred in the summer of 2004 for the first time (H. Yamaguchi et al., 2008; M. Yamaguchi et al., 2008). Thus it is critical to determine the key factors causing the outbreaks of C. ovata in the summer for understanding the bloom dynamics of the organism. From the results obtained from the present experiments, we can demonstrate the importance of high water temperature, salinity and irradiance for the growth of C. ovata. Actually, the summer in 2004 has been severer than usual. The surface water temperature from February to July in 2001 in the eastern Seto Inland Sea was more than 1 8C higher than the normal year (Oda, 2007). In addition, referring to online data provided from Japan Meteorological Agency (http://www.jma.go.jp/jma/), we summarized and compared the hours of sunlight (h) and the mean values of the daily solar radiation (MJ m2) in Hiroshima City during June–August for 2004 with that of the last 5 years (1999– 2003). These values of the summer of 2004 were 623.6 h and 19.2 MJ m2, respectively and clearly higher than those of the last 5 years (538.2 h and 17.4 MJ m2). Therefore the water temperature and irradiance in 2004 were suitable for the growth of C. ovata. Thus we can conclude that high water temperature and irradiance are critical physical factors contributing to the outbreaks of C. ovata blooms in the summer of 2004. The physiological features obtained in the present study suggest that C. ovata is well adapted to warmer water and higher irradiance environment and can predominate over C. antiqua and C. marina. Since there is a possibility for future outbreaks of C. ovata in temperate and subtropical waters, careful monitoring of this species is necessary to prevent damages to fisheries caused by the organism. For that purpose the growth parameters of temperature, salinity and irradiance obtained in the present study is useful to establish a prediction model of the organism. References Brand, L.E., Guillard, R.R.L., Murphy, L.S., 1981. A method for the rapid and precise determination of acclimated phytoplankton reproduction rates. J. Plankton Res. 3, 193–201. Barraza-Guardado, R., Corte´s-Altamirano, R., Sierra-Beltra´n, A., 2004. Marine dieoffs from Chattonella marina and Ch. cf. ovata in Kun Kaak Bay, Sonora in the Gulf of California. In: Wyatt, T. (Ed.), Harmful Algae News. IOC of UNESCO, No. 25, pp. 7–8.
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