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Effects of temperature, salinity and irradiance on the growth of the harmful dinoflagellate Prorocentrum donghaiense Lu
Harmful Algae 9 (2010) 13–17
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Harmful Algae journal homepage: www.elsevier.com/locate/hal
Effects of temperature, salinity and irradiance on the growth of the harmful dinoflagellate Prorocentrum donghaiense Lu Ning Xu *, Shunshan Duan, Aifen Li, Chengwu Zhang, Zhuoping Cai, Zhangxi Hu Institute of Hydrobiology, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 September 2008 Received in revised form 19 June 2009 Accepted 19 June 2009
The effects of temperature, salinity and irradiance on the growth of the causative organism of large-scale red tide along the east Chinese coast Prorocentrum donghaiense were examined in the laboratory. The optimum irradiance for growth was >30 mmol m2 s1. A moderate specific growth rate of 0.33 d1 was observed at 2 mmol m2 s1, the minimum irradiance in the experiments, and photoinhibition did not occur even at 230 mmol m2 s1, the maximum irradiance in the experiments. From 42 different combinations of temperature (10–31 8C) and salinity (10–40 psu) under saturated irradiance, P. donghaiense exhibited its maximum specific growth rate of 0.77 d1 at a combination of 27 8C and salinity of 30 psu. Optimum growth rates of >0.60 d1 were observed at temperatures ranging from 20 to 27 8C and at salinities from 25 to 35 psu. The organism was able to grow at temperatures ranging from 10 to 27 8C and at salinities from 20 to 40 psu, but it could not grow at temperature 31 8C or at salinities 10 psu. Temperature had the greatest influence on the growth rate, followed by salinity and the interaction between temperature and salinity. It is noteworthy that P. donghaiense adapts to low irradiance, low temperature ranging from 10 to 20 8C and a broad range of salinities, demonstrating its ability to dominate in estuaries, and bloom in early spring. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Growth rate Irradiance Prorocentrum donghaiense Salinity Temperature
1. Introduction The East China Sea is an important fishery area; meanwhile it contains a number of natural reserves, and hence plays an important role in the Chinese coastal ecosystems. Changjiang River, which is the largest river in China and the third largest in the world, is the primary source of freshwater to the East China Sea. Historical data showed that during the past 20 years, the Changjiang River estuary and its adjacent sea has changed into an area where red tides frequently occur, since about 1/4 of red tide in China occurred in this area (Zhou et al., 2003). In the 1980s and early 1990s the red tide-causative species were mainly Skeletonema costatum, Pseudo-nitzschia pungens and Noctiluca scientillans, which bloomed after June every year. However, this species have been displaced by extensive Prorocentrum blooms along the east Chinese coast, near Changjiang River estuary and Zhejiang coast have been found in early summer in recent years (2000–2006). Such blooms extended to 10,000 km, lasted for one month, and had severe impacts on the aquaculture and the marine ecosystem. Although there are some lingering controversies regarding its taxonomy, the causative organism has been affirmed as a new
species Prorocentrum donghaiense Lu (Lu and Goebel, 2001; Lu et al., 2002, 2003, 2005). It has been reported that blooms of the same species have also occurred in Japan, South Korea and Turkey. The outbreaks of red tides are associated with some complex ecological and oceanographic processes, and can be affected by a variety of environmental factors (Sunda et al., 2006). Among them, water temperature, salinity and light are believed to be the most basic factors for the survival and reproduction of red tide organisms. Multiple laboratory studies have confirmed that environmental factors such as water temperature, salinity and light can significantly influence the growth rate of harmful algal species, and thus play a crucial role in the formation and demise of blooms (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Yamamoto et al., 2002; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). Since the physiology of P. donghaiense Lu is yet to be investigated, in the present study, we examined the effects of water temperature, salinity and irradiance on the growth of this described affirmed species under nutrient-replete laboratory conditions. 2. Methods 2.1. Organism and culture conditions
* Corresponding author. Tel.: +86 20 85224366; fax: +86 20 85220239. E-mail address: [email protected] (N. Xu). 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.06.002
A strain of P. donghaiense, provided by Dr. Songhui Lu, Jinan University, Guangzhou, China, was previously isolated from the
N. Xu et al. / Harmful Algae 9 (2010) 13–17
14
East China Sea, where red tides occurred. An axenic culture was obtained through repeated washing using capillary pipettes. Silicate was eliminated from an f/2 culture medium (Guillard, 1975) and artificial seawater (salinity of 30.5 psu, Harrison et al., 1980) was used as the culture medium. The culture was maintained at constant temperature (24 8C) and irradiance (120 mmol m2 s1) in a 12 h:12 h (light:dark) photoperiod cycle. 2.2. Irradiance experiments The irradiance experiments were carried out in an artificial climate incubator (CC275TL2H, Hangzhou, China) following the method of Kim et al. (2004). The pre-culture lasted for 1 week at 24 8C and 140 mmol m2 s1 irradiance from cool-white fluorescent bulbs with a 12 h light:12 h dark cycle. The cultures were inoculated with 500 cells ml1 in three identical 50 ml capped test tubes (25 mm 150 mm) containing 35 ml of the modified f/2 medium and were gently mixed twice daily. Using this density of inoculum, the complete growth cycle lasted for 12–15 days. Eleven different irradiance levels (2, 4, 7, 15, 30, 60, 90, 110, 140, 170, 230 mmol m2 s1) were established by wrapping the tubes with UV-absorbing vinyl screens. A Li-190SA quantum light meter (LICOR Biosciences) was used to confirm the irradiance levels by wrapping the receiver sensor with UV-absorbing vinyl screens. Every 1–2 days, 100 ml sample was taken from each test tube. A light microscope was used to determine the growth phase by counting the cells using a Sedgwick–Rafter counting chamber. The specific growth rates (m, d1) of samples in the exponential growth phase were calculated according to the method of Guillard (1973) by a least squares fit of a straight line to the data after logarithmic transformation. Eq. (1), modified from Lederman and Tett (1981), was used to describe the relationship between growth rate and irradiance:
m¼
mm ðI I0 Þ ðI þ K s 2I0 Þ
(1)
where m is the specific growth rate (day1), mm is the maximum specific growth rate (day1), I is the irradiance (mmol m2 s1), I0 is the compensation irradiance (mmol m2 s1) and Ks is the irradiance at mm/2 (half-saturation light intensity).
Fig. 1. Specific growth rate (d1) of P. donghaiense as a function of irradiance level.
during the exponential growth phase were calculated as described above. 2.4. Statistical analysis Analysis of variance (ANOVA) was used to determine the effects of temperature and salinity on the growth rate. Cubic polynomial equations were determined based on the ANOVA results (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Ellegaard et al., 1993; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). The computer application SPSS 11.5 for windows (SPSS, Chicago, IL, USA) was used for the statistical analysis. 3. Results 3.1. Effect of irradiance on growth The specific growth rate of P. donghaiense was 0.33 d1 at 2 mmol m2 s1, the minimum irradiance set in this study (Fig. 1). Growth of the organism saturated at 30 mmol m2 s1 and that the optimum irradiance for growth was >30 mmol m2 s1. Photoinhibition did not occur even at the maximum irradiance of 230 mmol m2 s1. The following hyperbolic equation described the exponential growth phase:
2.3. Temperature and salinity experiments Additional growth experiments were conducted using a crossed factorial design with 42 different combinations of six temperatures (10, 15, 20, 24, 27, 31 8C) and seven salinities (10, 15, 20, 25, 30, 35, 40 psu) under the same light irradiance condition (about 120 mmol m2 s1). Salinities <30.5 psu were prepared by diluting artificial seawater with de-ionized water, and salinities >30.5 psu were prepared by evaporating artificial seawater in a drying oven at 70–80 8C. To minimize shock to the inoculum due to rapid changes in temperature and salinity, cultures were pre-acclimated to the desired experimental conditions by stepwise transfer over a period of 1–4 weeks according to the method outlined by Yamaguchi and Honjo (1989) and Kim et al. (2004). If the transferred cells did not grow under the experimental regime, the growth experiment was not carried out, and the growth rate at that particular combination of temperature and salinity was regarded as zero. Acclimated stock cultures were inoculated into three identical capped test tubes (25 mm 150 mm) containing 35 ml of modified f/2 medium (Guillard, 1975) without silicate for each experimental regime. All test tubes were inoculated with 500 cells ml1 and were gently mixed twice daily. Every 1–2 days, 100 ml samples were removed for microscopic examination. Growth rates
m¼
0:67ðI 0:1Þ ðI þ 2:41Þ
(2)
The compensation irradiance (I0) was 0.1 mmol m2 s1. The maximum growth rate (mm) and half-saturating irradiance (Ks) were 0.67 d1 and 2.61 mmol m2 s1, respectively. 3.2. Effects of temperature and salinity on growth Growth of P. donghaiense was observed at 10–15 8C, with the maximum growth rate of 0.2 d1 within this range of temperatures. The specific growth rates increased as temperatures were increased from 15 to 20 8C. Additionally, the growth rate appeared to increase slightly between 20 and 27 8C, attaining the maximum growth rate of 0.77 d1 under the combination of temperature 27 8C and salinity 30 psu. However, P. donghaiense could not grow when the temperature exceeded 31 8C (Fig. 2). The impact of salinity on P. donghaiense growth exhibited as a bell-shaped curve. P. donghaiense displayed optimal growth at the salinities of 25–35 psu, while the growth was prohibited as the salinity was up to 40 psu. Growth limitation was apparent at 20 psu, and the organism could not survive at salinity <15 psu (Fig. 3).
N. Xu et al. / Harmful Algae 9 (2010) 13–17
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Fig. 2. Specific growth rate (d1) of P. donghaiense as a function of temperature at salinity of 30 psu.
Fig. 4. Contour plots for specific growth rate (d1) of P. donghaiense as a function of temperature and salinity.
Table 1 Summary of two-way ANOVA of growth rate of P. donghaiense as a function of temperature, salinity and their interaction. Source of variation Temperature Salinity Temperature salinity Error Total Fig. 3. Specific growth rate (d1) of P. donghaiense as a function of salinity at temperature of 24 8C.
The interaction of temperature and salinity had significant effect on the growth rates of P. donghaiense. Temperature affected the range of salinity for optimal growth of P. donghaiense. The broadest range of salinities for optimal growth (from 15 to 40 psu) was observed at 24 8C, whereas the range of salinities for optimal growth tended to be narrow. The contour plots for the specific growth rate of P. donghaiense as a function of temperature and salinity are shown in Fig. 4. P. donghaiense had a relatively wide range of temperature and salinity for optimum growth. At temperatures of 20–27 8C and salinities of 20–40 psu, growth rates exceeded 0.60 d1, 80% of the maximum value. Therefore, P. donghaiense seems well adapted to conditions found in spring in the East China Sea. Two-way ANOVA indicated highly significant effects of temperature, salinity and the temperature–salinity interaction on the growth rates of P. donghaiense at the 0.1% level (Table 1). Of the total sum of squares, 49%, 31% and 19% were accounted for by the sum of squares for temperature, salinity and the temperature– salinity interaction, respectively. This indicates that the contribution of temperature was the highest. On the basis of the ANOVA results, cubic equations could be developed as the form shown in Eq. (3):
m ¼ b00 þ b10 T þ b01 S þ b11 TS þ . . . þ b30 T 3 þ b03 S3
(3)
where m is the specific growth rate, T the temperature, S the salinity and bnn are the regression coefficients, fitted by means of the stepwise forward regression method. The multiple regressions
*
d.f.
Sum of squares
5 6 30 84
5.010 3.118 1.944 0.098
125
10.169
Mean square
F
1.002 0.520 0.065 0.001
857.804* 444.901* 55.466*
p < 0.001
of the specific growth rate of P. donghaiense on temperature and salinity obtained were as follows:
m ¼ 1:98246 þ 0:121461T þ 0:0854S 0:00513TS 0:00012T 3 þ 0:000226T 2 S 0:000057TS2 0:0000047S3
ðR2 ¼ 0:906Þ
(4)
The regression model fits the observed data well, with the adjusted R2 value of 0.906. Although the in situ growth rate can be influenced by a series of environmental factors such as irradiance, nutrients and grazing pressure etc., this model provides a valuable reference to estimate the in situ growth rates as a function of temperature and salinity under saturating light and nutrients. 4. Discussion Four stages, namely initiating stage, developing stage, proliferating stage and dispersing stage, describing the process of largescale P. donghaiense blooms in the East China Sea in spring have been proposed by Zhou and Zhu (2006), who believed that the developing stage is the most important stage determining the timing and scale of subsequent outbreak of the blooms (Zhou and Zhu, 2006). Correspondingly, the key point for clarifying the mechanisms underlying the extensive occurrence of P. donghaiense red tides is to understand the growth features of the species. In our irradiance experiments, the compensation irradiance (I0) and half-saturating irradiance (Ks) of P. donghaiense were low, while the competition coefficient (a, equal to mm/Ks) was high, revealing that it could be adapt to low light conditions (Table 2). a
N. Xu et al. / Harmful Algae 9 (2010) 13–17
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Table 2 Summary of I0 (mmol m2 s1), Is (mmol m2 s1) and a reported for 10 HAB species. HAB species Karenia mikimotoi
I0
Is
0.7
110
Chattonella antiqua Chattonella marina Alexandrium tamarence
10.3 10.5 76
110 110 90
Cochlodinium polykrikoides Gyrodinium instriatum Akashiwo sanguinea Prorocentrum donghaiense Phaeocystis globosa Pseudo-nitzschia pungens
10.38 10.61 14.4 0.1 0.5 0.1
90 70 114 30 60 90
a
References
0.01 0.01 0.01 0.26 0.10 0.35
Yamaguchi and Honjo (1989) Yamaguchi et al. (1991) Yamaguchi et al. (1991) Yamamoto and Tarutani (1997) Kim et al. (2004) Nagasoe et al. (2006) Matsubara et al. (2007) This study Author, unpublished Author, unpublished
is a important indicator used to compare the competitive ability of phytoplankton under low irradiance. On the other hand, photoinhibition was not observed even at the highest irradiance used in this study (230 mmol m2 s1). It has been reported that the maximum irradiance in the surface layer in the field can be 2000 mmol m2 s1 (Kirk, 1983). Field data from 2004 to 2006 showed that the average irradiance in the surface layer was 500–600 mmol m2 s1 during the bloom (Sun et al., 2008). However, it has been reported that P. donghaiense gestated before the blooms and concentrated during the bloom within the subsurface layer (Zhou and Zhu, 2006). In situ shipbased experiments also suggested that the optimal light intensity of P. donghaiense was about 170 mmol m2 s1, which was located in the subsurface layer with water depth of 3–15 m (Sun et al., 2008). Moreover, irradiance above 300 mmol m2 s1 inhibited the growth of the organism (Sun et al., 2008). To clarify the photoinhibition in this species, further study is necessary under higher irradiances than used in the present study. It has been well documented that the requirements for irradiance are clearly different among phytoplankton (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Yamamoto et al., 2002; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). And the adaptability to differing irradiances can not only determine the outcome of interspecific competition among phytoplankton, but can also influence the absorption of nutrients (Fisher et al., 1982). As such, we have compiled a list of compensation irradiances (I0) and competition coefficients (a) for 10 harmful algae which bloom in Asian coastal waters (Table 2). Our findings showed that P. donghaiense, P. pungens and P. globosa had low compensation irradiance (I0) but high competition coefficient (a), suggesting that they are much more tolerant of low light intensity. On the contrary, high I0 and low a could be found in Cochlodinium polykrikoides, Gyrodinium instriatum and Akashiwo sanguinea, which suggested that these species preferred high irradiance and lacks of competitive ability in light limiting conditions. In fact, P. donghaiense appear to bloom in turbid water in spring, while C. polykrikoides bloom during summer when waters are presumably clearer (Kim et al., 2004). Although the suitable temperature range for P. donghaiense was 20–27 8C, it maintained moderate growth rates at 10–20 8C, displaying an ability to persist at lower temperatures. When temperature rose from 15 to 20 8C, the specific growth rate increased twice in the optimal salinity range of 25–35 psu, amounting to 0.60 d1, which was about 80% of the maximum specific growth rate. In addition, P. donghaiense possessed a relatively wide salinity tolerance of 15–40 psu, indicating that it has the ability to adapt to broad fluctuations in salinity. In fact, the ocean currents around Changjiang River estuary and Zhoushan archipelago where P. donghaiense blooms are very complicated and the salinity can vary dramatically (Zhou et al., 2003).
Long-term field data (Wang and Huang, 2003; Zhou and Zhu, 2006) supports the hypothesis that temperature plays a principal role in the succession of dominant phytoplankton species in the vicinity of the Yangtze River estuary. According to the field data from 2002 to 2004, initiation of P. donghaiense blooms in early to mid-April within the subsurface layer leads to large blooms across the East China Sea in early May. These massive blooms of P. donghaiense frequently occur at temperature of 18–20 8C. When temperatures increase to 23 8C, both P. donghaiense and S. costatum become co-dominant species and when temperatures exceed 23 8C, S. costatum typically dominates (Wang and Huang, 2003). Monitoring data from the spring of 2005 confirmed that temperature is a key factor responsible for the succession of dominant species in the East China Sea. In the spring of 2005 when water temperatures were cooler, the occurrence of P. donghaiense red tide was delayed from early May to late May, following massive bloom caused by diatoms (Zhou and Zhu, 2006). Nevertheless, the average water temperature in early to mid-April of 2005 was 2– 3 8C lower than that in April of 2004 (15 8C), suggesting that the growth of P. donghaiense growth could have been restricted and the lower temperature may have favored of the growth of diatoms other than S. costatum. Moreover, nutrient input, particularly silicon and nitrogen, was evidently increased by the excessive runoff of Changjiang River from November 2004 to March 2005, and this may have contributed to the outbreak of diatom blooms. With the rapid increase in temperature and drawdown of nutrients, P. donghaiense gradually become dominant and formed bloom in late May. Therefore, short-term climate variability seems to have a significant impact on the outbreak and succession of large-scale HABs along the east Chinese coast through alterations of environmental conditions such as temperature and the delivery of nutrients (Zhou and Yu, 2007). Changjiang River estuary and its adjacent area are directly influenced by the Changjiang River and the Taiwan warm current, which converge in the region and form the vital plume front and convergent bell (Zhou et al., 2003). The water turbidity in the region is generally high and there are typically excessive nutrients in water (Zhou et al., 2003). When the temperature, salinity and irradiance conditions are ideal for maximal growth, individual red tide species may rapidly proliferate to form blooms in the convergence of fronts in the area. Therefore, it can be concluded that the superior adaptability of P. donghaiense to low and moderate irradiance (2 to >230 mmol m2 s1), moderate temperatures (20 8C), and a wide range of salinity (20–40 psu) should be the primary reasons responsible for the outbreak of large-scale P. donghaiense red tides along the east Chinese coast. Acknowledgements We wish to thank anonymous reviewers who kindly provided helpful insights on the manuscript. We are pleased to acknowledge Dr. Christopher J. Gobler of School of Marine and Atmospheric Sciences, Stony Brook University for kind assistance in modification. This work was supported by National Natural Science Foundation of China (NSFC) (Grant No. 40776078, 40876074, U0733006); National Basic Research Program of China (Grant No. 2001CB409700); and National High Technology Research and Development Program of China (Grant No. 2006AA09Z178).[SS] References Ellegaard, M., Christensen, N.F., Moestrup, 1., 1993. Temperature and salinity effects on growth of a non-chain-forming strain of Gymnodinium catenatum (Dinophyceae) established from a cyst from recent sediments in the Sound (1resund), Denmark. J. Phycol. 29, 418–426. Fisher, T.R., Carlson, P.R., Barber, R.T., 1982. Carbon and nitrogen primary productivity in three North Carolina estuaries. Estuar. Coast. Shelf. S. 15, 621–644.
N. Xu et al. / Harmful Algae 9 (2010) 13–17 Guillard, R.R.L., 1973. Division rates. In: Stein, J.R. (Ed.), Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press, Cambridge, pp. 289–311. Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Cultures of Marine Invertebrate Animals. Plenum Press, New York, pp. 29–60. Harrison, P.J., Waters, R.E., Taylor, F.J.R., 1980. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J. Phycol. 16, 28–35. Kim, D., Matsuyama, Y., Nagasoe, S., Yamaguchi, M., Yoon, Y.H., Oshima, Y., Imada, N., Honjo, T., 2004. Effect of temperature, salinity and irradiance on the growth of the harmful red tide dinoflagellate Cochlodinium polykridoides Margalef (Dinophyceae). J. Plankton. Res. 26, 61–66. Kirk, J.T.O., 1983. Light and Photosynthesis in Aquatic Ecosystems. Cambridge University Press, Cambridge, pp. 219–253. Lederman, T.C., Tett, P., 1981. Problems in modeling the photosynthesis-light relationship for phytoplankton. Bot. Mar. 24, 125–134. Lu, D., Goebel, J., 2001. Five red tide species in genus Prorocentrum including the description of Prorocentrum donghaiense Lu sp. Nov. from the East China Sea. Chin. J. Oceanogr. Limnol. 19 (4), 337–344. Lu, D, Goebel, J., Qi, Y, Zou, J., Gao, Y., 2002. Prorocentrum donghaiense—a high biomass bloom-forming species in the East China Sea. Harmful Algae News, An IOC Newsletter on Toxic Algae and Algal Blooms. 23, 1–5. Lu, D., Goebel, J., Qi, Y., Zou, J., Han, X., Gao, Y., Li, Y., 2005. Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species. Harmful Algae 4 (3), 493–505. Lu, D., Qi, Y., Goebel, J., Zou, J., Gao, Y., 2003. Redescription of Prorocentrum donghaiense Lu and comparison with relevant Prorocentrum species. Chin. J. Appl. Ecol. 14 (7), 1060–1064 (in Chinese, with English abstract). Matsubara, T., Nagasoe, S., Yamasaki, Y., Shikata, T., Shimasaki, Y., Oshima, Y., Honjo, T., 2007. Effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea. J. Exp. Mar. Biol. Ecol. 342, 226–230. Nagasoe, S., Kim, D., Shimasaki, Y., Oshima, Y., Yamaguchi, M., Honjo, T., 2006. Effects of temperature, salinity and irradiance on the growth of the red tide dinoflagellate Gyrodinium instriatum Freudenthal et Lee. Harmful Algae 5, 20– 25.
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Sun, B., Wang, X., Li, Y., Wang, C., Wang, A., Liang, S., Zhang, C., 2008. Effects of irradiance on blooms of the dinoflagellate Prorocentrum donghaiense Lu in the coastal area in East China Sea. Environ. Sci. 29 (2), 362–367 (in Chinese, with English abstract). Sunda, W.G., Graneli, E., Gobler, C.J., 2006. Positive feedback and the development and persistence of ecosystem disruptive algal blooms. J. Phycol. 42, 963–974. Wang, J., Huang, X., 2003. Ecological characteristics of Prorocentrum dentatum and the cause of harmful algal bloom formation in China Sea. Chin. J. Appl. Ecol. 14 (7), 1065–1069 (in Chinese, with English abstract). Yamaguchi, M., Honjo, T., 1989. Effect of temperature, salinity and irradiance on the growth of the noxious red tide flagellate Gymnodinium nagasakiense (Dinophyceae). Nippon Suisan Gakkaishi 55, 2029–2036 (in Japanese, with English abstract). Yamaguchi, M., Imai, I., Honjo, T., 1991. Effect of temperature, salinity and irradiance on the growth of the noxious red tide flagellate Chattonella antique and C. marina (Rhaphidophyceae). Nippon Suisan Gakkaishi 57, 1277–1284 (in Japanese, with English abstract). Yamaguchi, M., Itakura, S., Nagasaki, K., Matsuyama, Y., Uchida, T., Imai, I., 1997. Effect of temperature and salinity on the growth of the red tide flagellates Heterocapsa circularisquama (Dinophyceae) and Chattonella verruculosa (Rhaphidophyceae). J. Plankton. Res. 19, 1167–1174. Yamamoto, T., Oh, S.J., Kataoka, Y., 2002. Effect of temperature, salinity and irradiance on the growth of the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae) isolated from Hiroshima Bay. Jpn. Fish. Sci. 68, 356–363. Yamamoto, T., Tarutani, K., 1997. Effect of temperature, salinity and irradiance on the growth of toxic dinoflagellate Alexandrium tamarense isolated from Hiroshima Bay, Japan. Jpn. J. Phycol. (Sorui) 45, 95–101 (in Japanese, with English abstract). Zhou, M., Yan, T., Zou, J., 2003. Preliminary analysis of the characteristics of red tide areas in Changjiang River estuary and its adjacent sea. Chin. J. Appl. Ecol. 14 (7), 1031–1038 (in Chinese, with English abstract). Zhou, M., Yu, R., 2007. Mechanisms and impacts of harmful algal blooms and the countmeasures. Chin. J. Nat. 29 (2), 72–77 (in Chinese, with English abstract). Zhou, M., Zhu, M., 2006. Progress of the Project ‘‘Ecology and Oceanography of Harmful Algal Blooms in China’’. Adv. Earth Sci. 21 (7), 673–679 (in Chinese, with English abstract).
Contents lists available at ScienceDirect
Harmful Algae journal homepage: www.elsevier.com/locate/hal
Effects of temperature, salinity and irradiance on the growth of the harmful dinoflagellate Prorocentrum donghaiense Lu Ning Xu *, Shunshan Duan, Aifen Li, Chengwu Zhang, Zhuoping Cai, Zhangxi Hu Institute of Hydrobiology, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 September 2008 Received in revised form 19 June 2009 Accepted 19 June 2009
The effects of temperature, salinity and irradiance on the growth of the causative organism of large-scale red tide along the east Chinese coast Prorocentrum donghaiense were examined in the laboratory. The optimum irradiance for growth was >30 mmol m2 s1. A moderate specific growth rate of 0.33 d1 was observed at 2 mmol m2 s1, the minimum irradiance in the experiments, and photoinhibition did not occur even at 230 mmol m2 s1, the maximum irradiance in the experiments. From 42 different combinations of temperature (10–31 8C) and salinity (10–40 psu) under saturated irradiance, P. donghaiense exhibited its maximum specific growth rate of 0.77 d1 at a combination of 27 8C and salinity of 30 psu. Optimum growth rates of >0.60 d1 were observed at temperatures ranging from 20 to 27 8C and at salinities from 25 to 35 psu. The organism was able to grow at temperatures ranging from 10 to 27 8C and at salinities from 20 to 40 psu, but it could not grow at temperature 31 8C or at salinities 10 psu. Temperature had the greatest influence on the growth rate, followed by salinity and the interaction between temperature and salinity. It is noteworthy that P. donghaiense adapts to low irradiance, low temperature ranging from 10 to 20 8C and a broad range of salinities, demonstrating its ability to dominate in estuaries, and bloom in early spring. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Growth rate Irradiance Prorocentrum donghaiense Salinity Temperature
1. Introduction The East China Sea is an important fishery area; meanwhile it contains a number of natural reserves, and hence plays an important role in the Chinese coastal ecosystems. Changjiang River, which is the largest river in China and the third largest in the world, is the primary source of freshwater to the East China Sea. Historical data showed that during the past 20 years, the Changjiang River estuary and its adjacent sea has changed into an area where red tides frequently occur, since about 1/4 of red tide in China occurred in this area (Zhou et al., 2003). In the 1980s and early 1990s the red tide-causative species were mainly Skeletonema costatum, Pseudo-nitzschia pungens and Noctiluca scientillans, which bloomed after June every year. However, this species have been displaced by extensive Prorocentrum blooms along the east Chinese coast, near Changjiang River estuary and Zhejiang coast have been found in early summer in recent years (2000–2006). Such blooms extended to 10,000 km, lasted for one month, and had severe impacts on the aquaculture and the marine ecosystem. Although there are some lingering controversies regarding its taxonomy, the causative organism has been affirmed as a new
species Prorocentrum donghaiense Lu (Lu and Goebel, 2001; Lu et al., 2002, 2003, 2005). It has been reported that blooms of the same species have also occurred in Japan, South Korea and Turkey. The outbreaks of red tides are associated with some complex ecological and oceanographic processes, and can be affected by a variety of environmental factors (Sunda et al., 2006). Among them, water temperature, salinity and light are believed to be the most basic factors for the survival and reproduction of red tide organisms. Multiple laboratory studies have confirmed that environmental factors such as water temperature, salinity and light can significantly influence the growth rate of harmful algal species, and thus play a crucial role in the formation and demise of blooms (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Yamamoto et al., 2002; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). Since the physiology of P. donghaiense Lu is yet to be investigated, in the present study, we examined the effects of water temperature, salinity and irradiance on the growth of this described affirmed species under nutrient-replete laboratory conditions. 2. Methods 2.1. Organism and culture conditions
* Corresponding author. Tel.: +86 20 85224366; fax: +86 20 85220239. E-mail address: [email protected] (N. Xu). 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.06.002
A strain of P. donghaiense, provided by Dr. Songhui Lu, Jinan University, Guangzhou, China, was previously isolated from the
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East China Sea, where red tides occurred. An axenic culture was obtained through repeated washing using capillary pipettes. Silicate was eliminated from an f/2 culture medium (Guillard, 1975) and artificial seawater (salinity of 30.5 psu, Harrison et al., 1980) was used as the culture medium. The culture was maintained at constant temperature (24 8C) and irradiance (120 mmol m2 s1) in a 12 h:12 h (light:dark) photoperiod cycle. 2.2. Irradiance experiments The irradiance experiments were carried out in an artificial climate incubator (CC275TL2H, Hangzhou, China) following the method of Kim et al. (2004). The pre-culture lasted for 1 week at 24 8C and 140 mmol m2 s1 irradiance from cool-white fluorescent bulbs with a 12 h light:12 h dark cycle. The cultures were inoculated with 500 cells ml1 in three identical 50 ml capped test tubes (25 mm 150 mm) containing 35 ml of the modified f/2 medium and were gently mixed twice daily. Using this density of inoculum, the complete growth cycle lasted for 12–15 days. Eleven different irradiance levels (2, 4, 7, 15, 30, 60, 90, 110, 140, 170, 230 mmol m2 s1) were established by wrapping the tubes with UV-absorbing vinyl screens. A Li-190SA quantum light meter (LICOR Biosciences) was used to confirm the irradiance levels by wrapping the receiver sensor with UV-absorbing vinyl screens. Every 1–2 days, 100 ml sample was taken from each test tube. A light microscope was used to determine the growth phase by counting the cells using a Sedgwick–Rafter counting chamber. The specific growth rates (m, d1) of samples in the exponential growth phase were calculated according to the method of Guillard (1973) by a least squares fit of a straight line to the data after logarithmic transformation. Eq. (1), modified from Lederman and Tett (1981), was used to describe the relationship between growth rate and irradiance:
m¼
mm ðI I0 Þ ðI þ K s 2I0 Þ
(1)
where m is the specific growth rate (day1), mm is the maximum specific growth rate (day1), I is the irradiance (mmol m2 s1), I0 is the compensation irradiance (mmol m2 s1) and Ks is the irradiance at mm/2 (half-saturation light intensity).
Fig. 1. Specific growth rate (d1) of P. donghaiense as a function of irradiance level.
during the exponential growth phase were calculated as described above. 2.4. Statistical analysis Analysis of variance (ANOVA) was used to determine the effects of temperature and salinity on the growth rate. Cubic polynomial equations were determined based on the ANOVA results (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Ellegaard et al., 1993; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). The computer application SPSS 11.5 for windows (SPSS, Chicago, IL, USA) was used for the statistical analysis. 3. Results 3.1. Effect of irradiance on growth The specific growth rate of P. donghaiense was 0.33 d1 at 2 mmol m2 s1, the minimum irradiance set in this study (Fig. 1). Growth of the organism saturated at 30 mmol m2 s1 and that the optimum irradiance for growth was >30 mmol m2 s1. Photoinhibition did not occur even at the maximum irradiance of 230 mmol m2 s1. The following hyperbolic equation described the exponential growth phase:
2.3. Temperature and salinity experiments Additional growth experiments were conducted using a crossed factorial design with 42 different combinations of six temperatures (10, 15, 20, 24, 27, 31 8C) and seven salinities (10, 15, 20, 25, 30, 35, 40 psu) under the same light irradiance condition (about 120 mmol m2 s1). Salinities <30.5 psu were prepared by diluting artificial seawater with de-ionized water, and salinities >30.5 psu were prepared by evaporating artificial seawater in a drying oven at 70–80 8C. To minimize shock to the inoculum due to rapid changes in temperature and salinity, cultures were pre-acclimated to the desired experimental conditions by stepwise transfer over a period of 1–4 weeks according to the method outlined by Yamaguchi and Honjo (1989) and Kim et al. (2004). If the transferred cells did not grow under the experimental regime, the growth experiment was not carried out, and the growth rate at that particular combination of temperature and salinity was regarded as zero. Acclimated stock cultures were inoculated into three identical capped test tubes (25 mm 150 mm) containing 35 ml of modified f/2 medium (Guillard, 1975) without silicate for each experimental regime. All test tubes were inoculated with 500 cells ml1 and were gently mixed twice daily. Every 1–2 days, 100 ml samples were removed for microscopic examination. Growth rates
m¼
0:67ðI 0:1Þ ðI þ 2:41Þ
(2)
The compensation irradiance (I0) was 0.1 mmol m2 s1. The maximum growth rate (mm) and half-saturating irradiance (Ks) were 0.67 d1 and 2.61 mmol m2 s1, respectively. 3.2. Effects of temperature and salinity on growth Growth of P. donghaiense was observed at 10–15 8C, with the maximum growth rate of 0.2 d1 within this range of temperatures. The specific growth rates increased as temperatures were increased from 15 to 20 8C. Additionally, the growth rate appeared to increase slightly between 20 and 27 8C, attaining the maximum growth rate of 0.77 d1 under the combination of temperature 27 8C and salinity 30 psu. However, P. donghaiense could not grow when the temperature exceeded 31 8C (Fig. 2). The impact of salinity on P. donghaiense growth exhibited as a bell-shaped curve. P. donghaiense displayed optimal growth at the salinities of 25–35 psu, while the growth was prohibited as the salinity was up to 40 psu. Growth limitation was apparent at 20 psu, and the organism could not survive at salinity <15 psu (Fig. 3).
N. Xu et al. / Harmful Algae 9 (2010) 13–17
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Fig. 2. Specific growth rate (d1) of P. donghaiense as a function of temperature at salinity of 30 psu.
Fig. 4. Contour plots for specific growth rate (d1) of P. donghaiense as a function of temperature and salinity.
Table 1 Summary of two-way ANOVA of growth rate of P. donghaiense as a function of temperature, salinity and their interaction. Source of variation Temperature Salinity Temperature salinity Error Total Fig. 3. Specific growth rate (d1) of P. donghaiense as a function of salinity at temperature of 24 8C.
The interaction of temperature and salinity had significant effect on the growth rates of P. donghaiense. Temperature affected the range of salinity for optimal growth of P. donghaiense. The broadest range of salinities for optimal growth (from 15 to 40 psu) was observed at 24 8C, whereas the range of salinities for optimal growth tended to be narrow. The contour plots for the specific growth rate of P. donghaiense as a function of temperature and salinity are shown in Fig. 4. P. donghaiense had a relatively wide range of temperature and salinity for optimum growth. At temperatures of 20–27 8C and salinities of 20–40 psu, growth rates exceeded 0.60 d1, 80% of the maximum value. Therefore, P. donghaiense seems well adapted to conditions found in spring in the East China Sea. Two-way ANOVA indicated highly significant effects of temperature, salinity and the temperature–salinity interaction on the growth rates of P. donghaiense at the 0.1% level (Table 1). Of the total sum of squares, 49%, 31% and 19% were accounted for by the sum of squares for temperature, salinity and the temperature– salinity interaction, respectively. This indicates that the contribution of temperature was the highest. On the basis of the ANOVA results, cubic equations could be developed as the form shown in Eq. (3):
m ¼ b00 þ b10 T þ b01 S þ b11 TS þ . . . þ b30 T 3 þ b03 S3
(3)
where m is the specific growth rate, T the temperature, S the salinity and bnn are the regression coefficients, fitted by means of the stepwise forward regression method. The multiple regressions
*
d.f.
Sum of squares
5 6 30 84
5.010 3.118 1.944 0.098
125
10.169
Mean square
F
1.002 0.520 0.065 0.001
857.804* 444.901* 55.466*
p < 0.001
of the specific growth rate of P. donghaiense on temperature and salinity obtained were as follows:
m ¼ 1:98246 þ 0:121461T þ 0:0854S 0:00513TS 0:00012T 3 þ 0:000226T 2 S 0:000057TS2 0:0000047S3
ðR2 ¼ 0:906Þ
(4)
The regression model fits the observed data well, with the adjusted R2 value of 0.906. Although the in situ growth rate can be influenced by a series of environmental factors such as irradiance, nutrients and grazing pressure etc., this model provides a valuable reference to estimate the in situ growth rates as a function of temperature and salinity under saturating light and nutrients. 4. Discussion Four stages, namely initiating stage, developing stage, proliferating stage and dispersing stage, describing the process of largescale P. donghaiense blooms in the East China Sea in spring have been proposed by Zhou and Zhu (2006), who believed that the developing stage is the most important stage determining the timing and scale of subsequent outbreak of the blooms (Zhou and Zhu, 2006). Correspondingly, the key point for clarifying the mechanisms underlying the extensive occurrence of P. donghaiense red tides is to understand the growth features of the species. In our irradiance experiments, the compensation irradiance (I0) and half-saturating irradiance (Ks) of P. donghaiense were low, while the competition coefficient (a, equal to mm/Ks) was high, revealing that it could be adapt to low light conditions (Table 2). a
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Table 2 Summary of I0 (mmol m2 s1), Is (mmol m2 s1) and a reported for 10 HAB species. HAB species Karenia mikimotoi
I0
Is
0.7
110
Chattonella antiqua Chattonella marina Alexandrium tamarence
10.3 10.5 76
110 110 90
Cochlodinium polykrikoides Gyrodinium instriatum Akashiwo sanguinea Prorocentrum donghaiense Phaeocystis globosa Pseudo-nitzschia pungens
10.38 10.61 14.4 0.1 0.5 0.1
90 70 114 30 60 90
a
References
0.01 0.01 0.01 0.26 0.10 0.35
Yamaguchi and Honjo (1989) Yamaguchi et al. (1991) Yamaguchi et al. (1991) Yamamoto and Tarutani (1997) Kim et al. (2004) Nagasoe et al. (2006) Matsubara et al. (2007) This study Author, unpublished Author, unpublished
is a important indicator used to compare the competitive ability of phytoplankton under low irradiance. On the other hand, photoinhibition was not observed even at the highest irradiance used in this study (230 mmol m2 s1). It has been reported that the maximum irradiance in the surface layer in the field can be 2000 mmol m2 s1 (Kirk, 1983). Field data from 2004 to 2006 showed that the average irradiance in the surface layer was 500–600 mmol m2 s1 during the bloom (Sun et al., 2008). However, it has been reported that P. donghaiense gestated before the blooms and concentrated during the bloom within the subsurface layer (Zhou and Zhu, 2006). In situ shipbased experiments also suggested that the optimal light intensity of P. donghaiense was about 170 mmol m2 s1, which was located in the subsurface layer with water depth of 3–15 m (Sun et al., 2008). Moreover, irradiance above 300 mmol m2 s1 inhibited the growth of the organism (Sun et al., 2008). To clarify the photoinhibition in this species, further study is necessary under higher irradiances than used in the present study. It has been well documented that the requirements for irradiance are clearly different among phytoplankton (Yamaguchi and Honjo, 1989; Yamaguchi et al., 1991, 1997; Yamamoto et al., 2002; Kim et al., 2004; Nagasoe et al., 2006; Matsubara et al., 2007). And the adaptability to differing irradiances can not only determine the outcome of interspecific competition among phytoplankton, but can also influence the absorption of nutrients (Fisher et al., 1982). As such, we have compiled a list of compensation irradiances (I0) and competition coefficients (a) for 10 harmful algae which bloom in Asian coastal waters (Table 2). Our findings showed that P. donghaiense, P. pungens and P. globosa had low compensation irradiance (I0) but high competition coefficient (a), suggesting that they are much more tolerant of low light intensity. On the contrary, high I0 and low a could be found in Cochlodinium polykrikoides, Gyrodinium instriatum and Akashiwo sanguinea, which suggested that these species preferred high irradiance and lacks of competitive ability in light limiting conditions. In fact, P. donghaiense appear to bloom in turbid water in spring, while C. polykrikoides bloom during summer when waters are presumably clearer (Kim et al., 2004). Although the suitable temperature range for P. donghaiense was 20–27 8C, it maintained moderate growth rates at 10–20 8C, displaying an ability to persist at lower temperatures. When temperature rose from 15 to 20 8C, the specific growth rate increased twice in the optimal salinity range of 25–35 psu, amounting to 0.60 d1, which was about 80% of the maximum specific growth rate. In addition, P. donghaiense possessed a relatively wide salinity tolerance of 15–40 psu, indicating that it has the ability to adapt to broad fluctuations in salinity. In fact, the ocean currents around Changjiang River estuary and Zhoushan archipelago where P. donghaiense blooms are very complicated and the salinity can vary dramatically (Zhou et al., 2003).
Long-term field data (Wang and Huang, 2003; Zhou and Zhu, 2006) supports the hypothesis that temperature plays a principal role in the succession of dominant phytoplankton species in the vicinity of the Yangtze River estuary. According to the field data from 2002 to 2004, initiation of P. donghaiense blooms in early to mid-April within the subsurface layer leads to large blooms across the East China Sea in early May. These massive blooms of P. donghaiense frequently occur at temperature of 18–20 8C. When temperatures increase to 23 8C, both P. donghaiense and S. costatum become co-dominant species and when temperatures exceed 23 8C, S. costatum typically dominates (Wang and Huang, 2003). Monitoring data from the spring of 2005 confirmed that temperature is a key factor responsible for the succession of dominant species in the East China Sea. In the spring of 2005 when water temperatures were cooler, the occurrence of P. donghaiense red tide was delayed from early May to late May, following massive bloom caused by diatoms (Zhou and Zhu, 2006). Nevertheless, the average water temperature in early to mid-April of 2005 was 2– 3 8C lower than that in April of 2004 (15 8C), suggesting that the growth of P. donghaiense growth could have been restricted and the lower temperature may have favored of the growth of diatoms other than S. costatum. Moreover, nutrient input, particularly silicon and nitrogen, was evidently increased by the excessive runoff of Changjiang River from November 2004 to March 2005, and this may have contributed to the outbreak of diatom blooms. With the rapid increase in temperature and drawdown of nutrients, P. donghaiense gradually become dominant and formed bloom in late May. Therefore, short-term climate variability seems to have a significant impact on the outbreak and succession of large-scale HABs along the east Chinese coast through alterations of environmental conditions such as temperature and the delivery of nutrients (Zhou and Yu, 2007). Changjiang River estuary and its adjacent area are directly influenced by the Changjiang River and the Taiwan warm current, which converge in the region and form the vital plume front and convergent bell (Zhou et al., 2003). The water turbidity in the region is generally high and there are typically excessive nutrients in water (Zhou et al., 2003). When the temperature, salinity and irradiance conditions are ideal for maximal growth, individual red tide species may rapidly proliferate to form blooms in the convergence of fronts in the area. Therefore, it can be concluded that the superior adaptability of P. donghaiense to low and moderate irradiance (2 to >230 mmol m2 s1), moderate temperatures (20 8C), and a wide range of salinity (20–40 psu) should be the primary reasons responsible for the outbreak of large-scale P. donghaiense red tides along the east Chinese coast. Acknowledgements We wish to thank anonymous reviewers who kindly provided helpful insights on the manuscript. We are pleased to acknowledge Dr. Christopher J. Gobler of School of Marine and Atmospheric Sciences, Stony Brook University for kind assistance in modification. This work was supported by National Natural Science Foundation of China (NSFC) (Grant No. 40776078, 40876074, U0733006); National Basic Research Program of China (Grant No. 2001CB409700); and National High Technology Research and Development Program of China (Grant No. 2006AA09Z178).[SS] References Ellegaard, M., Christensen, N.F., Moestrup, 1., 1993. Temperature and salinity effects on growth of a non-chain-forming strain of Gymnodinium catenatum (Dinophyceae) established from a cyst from recent sediments in the Sound (1resund), Denmark. J. Phycol. 29, 418–426. Fisher, T.R., Carlson, P.R., Barber, R.T., 1982. Carbon and nitrogen primary productivity in three North Carolina estuaries. Estuar. Coast. Shelf. S. 15, 621–644.
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