- Email: [email protected]
Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan
Available online at www.sciencedirect.com
Harmful Algae 7 (2008) 459–463 www.elsevier.com/locate/hal
Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan Mineo Yamaguchi a,*, Haruo Yamaguchi b, Goh Nishitani a, Setsuko Sakamoto a, Shigeru Itakura a a
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 Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan Received 21 February 2007; received in revised form 12 September 2007; accepted 4 October 2007
Abstract To elucidate the mechanism of bloom outbreaks of Chattonella ovata (Raphidophyceae), we investigated the cysts of C. ovata and succeeded in finding them from the bottom sediments of Hiroshima Bay. The morphology of the cysts was mostly hemispherical in shape, with a diameter of ca. 30 mm and height of ca. 20 mm. The cysts were usually adhering to solid materials, such as diatom frustules, yellow-greenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation. Because the morphological characteristic of the cysts was in general agreement with those of Chattonella antiqua and Chattonella marina, it was difficult to differentiate the cysts of these three species. Germination of the cysts of C. ovata was observed at temperatures from 17.5 to 30 8C, but not at 15 8C or below. The number of the germinated cysts increased with increasing temperature and the optimum temperature for germination was 30 8C. Although cysts of C. antiqua and C. marina germinated at temperatures from 15 to 30 8C, optimum temperature of germination was 22.5 8C. The lower limit and optimum temperatures for germination of C. ovata cysts was higher than for C. antiqua and C. marina. The role of cysts in the population dynamics of C. ovata is discussed. # 2007 Elsevier B.V. All rights reserved. Keywords: Chattonella ovata; Cyst; Germination; Morphology; Raphidophyceae
1. Introduction Species of the genus Chattonella (Raphidophyceae) are harmful flagellates which cause mass mortality of cultured fishes in coastal waters (Imai et al., 1998). Although six species have been reported in the genus Chattonella (Hallegraeff and Hara, 1995), so far in Japanese waters the most representative and harmful species 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 from the 1970s through the mid 1980s (Imai et al., 1998). During the 1990s, however, the frequency of harmful blooms caused by C. antiqua and C. marina have decreased in the Seto Inland Sea (Imai et al., 2006). In summer of 2004, however, a massive bloom of Chattonella ovata. Hara et Chihara (Hara
* Corresponding author. Tel.: +81 829 55 0666; fax: +81 829 54 1216. E-mail address: [email protected] (M. Yamaguchi). 1568-9883/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2007.10.002
et al., 1994) occurred over almost the entire area of the Seto Inland Sea and caused damage to cultured fish (Hiroishi et al., 2005). This is the first record of a bloom and damage to fisheries caused by C. ovata in Japan (Yamaguchi et al., 2008), although vegetative cells have been observed since the mid 1980s (Yoshimatsu and Ono, 1986). Cysts of harmful algae play an important role in species dispersal, bloom initiation and termination, and survival under unfavorable conditions (Anderson and Wall, 1978; Anderson, 1998). Therefore, information on the distribution and abundance in natural sediments as well as the germination characteristics of cysts is essential for understanding the ecology and bloom dynamics of harmful algae. The morphology and physiological ecology of the cysts of C. antiqua and C. marina have already been thoroughly examined (Imai and Itoh, 1987, 1988; Imai, 1989, 1990; Imai et al., 1989; Yamaguchi and Imai, 1994). Although the existence of cysts of C. ovata in the bottom sediment was expected (Imai, 1990), its morphology and germination characteristics are still unknown.
460
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
In the present paper, we report the occurrence of cysts of C. ovata in the bottom sediments of Hiroshima Bay, examine the effect of temperature on the germination of the cysts, and discuss the role of cysts in the population dynamics of C. ovata. 2. Materials and methods 2.1. Sediment sampling Sediment samples were collected with a gravity coresampler (inner diameter 4 cm) from 12 stations in Hiroshima Bay (Fig. 1) in June 2005. The top 1 cm of each sediment sample from the replicate samples was sliced off, mixed, placed in a plastic container and stored in the dark at 10 8C. 2.2. Detection of cysts Five gram (wet weight) aliquots of the sediment samples were suspended in filtered-autoclaved seawater, sonicated and sieved through plankton netting to obtain the size fraction between 20 and 150 mm. The material remaining on the 20 mm netting was finally resuspended in 10 ml filtered-autoclaved seawater. The sediment suspension was used for microscopic observations using an inverted epifluorescence microscope under blue light excitation. For observing the cysts, 0.1–0.2 ml of the sediment suspension was placed in a Sedgwick-Rafter chamber with 1 ml filtered-autoclaved seawater. Cyst-like cells were isolated with a capillary tube under the inverted epifluorescence microscope referring to the morphological features of previously known species of Raphidophytes such as C. antiqua, C. marina and Fibrocapsa japonica (Imai and Itoh, 1988; Yoshimatsu, 1992). Each isolated cyst-like cell was incubated in a well of a 48-well tissue culture plate (Costar) with 0.5 ml filtered-autoclaved seawater at 22 8C, under an illumination of 50 mmol photons m 2 s 1 with a 12hL:12hD photo-cycle. After an incubation period of 14 days, each vegetative cell that germinated from cyst-like cells was
identified on the basis of the morphology of cultured strains of C. ovata, C. antiqua and C. marina (Hara et al., 1994). 2.3. Enumeration of cysts For enumerating cysts, sediment suspensions were prepared as mentioned above and examined using an inverted epifluorescence microscope under blue light excitation. For counting cysts, 0.1 ml of sediment suspension was placed in a Sedgwick-Rafter chamber with 1 ml filtered-autoclaved seawater and the enumeration made on the basis of characteristics of autofluorescence and morphology of the cysts (Imai and Itoh, 1988). The number of cysts in the sediment suspension was determined at 100 magnification, and counts were made in triplicate. The cyst count was expressed as the number of the cyst per gram wet sediment. 2.4. Effect of temperature on the germination of the cysts The effect of temperature on the germination of the cysts was examined by an extinction dilution method (Imai et al., 1984) using the sediment sample collected from St. 12 in 2005 (Fig. 1). Five to 15 g of the sediment sample was suspended in filtered-autoclaved seawater, sonicated and sieved through plankton netting to obtain the size fraction between 20 and 150 mm. This fraction was suspended in 50 ml of filteredautoclaved seawater. After serial 10-fold dilutions (10 1 to 10 2) with filtered-autoclaved seawater, 1 ml aliquots of the diluted suspension were inoculated into five replicate test tubes containing 1 ml of filtered-autoclaved seawater. To avoid the growth of diatoms, GeO2 was added to the seawater at a final concentration of 1 mg l 1. These test tubes were incubated at 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 8C under an illumination of 50 mmol photons m 2 s 1 with a 12hL:12hD photo-cycle. Appearance of the vegetative cells of Chattonella spp. (C. antiqua, C. marina and C. ovata) in each tube was examined after an incubation period of 7–14 days using an inverted epifluorescence microscope. The tubes in which vegetative cells of Chattonella spp. were observed were scored as positive. The most probable number (MPN) of the cysts of Chattonella spp. was then calculated referring to the MPN tables (Itoh and Imai, 1987). 3. Results and discussion
Fig. 1. Sampling stations in Hiroshima Bay, the Seto Inland Sea.
A total 329 cyst-like cells were isolated from the bottom sediment samples. After incubation, 14 cells were successfully germinated. Ten of them were identified as vegetative cells of C. ovata (Figs. 2 and 3) and the remaining four were identified as those of C. antiqua or C. marina (Fig. 4). The cysts of C. ovata were mostly hemispherical in shape, with a diameter of ca. 30 mm and height of ca. 20 mm. These cysts were usually adhering to solid materials such as diatom frustules, yellowgreenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation (Fig. 2). The morphological characteristics of the cysts generally agreed with those of C. antiqua/C. marina a (Fig. 4; Imai and Itoh, 1988).
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
461
Fig. 2. Cultured vegetative cells of Chattonella ovata (A) and the cyst (B) from which the vegetative cell germinated (arrow).
Therefore, incubation of cysts and observation of the germinated vegetative cells are necessary to discriminate the cysts of these species. Fig. 5 shows the horizontal distribution of the cysts of Chattonella spp. in Hiroshima Bay determined by the direct count method using epifluorescence microscope. As mentioned above, the number of cysts given in the figure include those for all
three species of Chattonella. Chattonella cysts were observed at all the stations except for St. 9 (Fig. 1). Cyst concentration ranged from 0 (i.e., not detected) to 227 cysts/g wet sediment. Major cyst deposition areas were observed in the central part of the bay (west of Etajima Island). In this bay, a cyst survey was conducted in 1987. The results showed that the number of the cysts was at most 2 cysts/g wet sediment (Imai, 1990). The present study indicates
Fig. 3. Cysts from which the vegetative cell of C. ovata germinated (arrows).
462
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
Fig. 4. Cysts from which the vegetative cell of Chattonella antiqua or Chattonella marina germinated (arrows).
that the cyst abundance has since increased markedly. The high abundance of cysts is probably caused by the deposition of newly formed cysts during the preceding bloom of C. ovata that occurred in the summer of 2004. Thus the majority of the cysts were expected to be those of C. ovata. Therefore it is important that Hiroshima Bay be monitored for further, potential outbreaks of blooms of C. ovata. The effect of temperature on the germination of the cysts was investigated by the extinction dilution method using natural sediment samples (Fig. 6). Although just germinated
cells of C. ovata could be distinguished from those of C. antiqua and C. marina from the morphological characteristics of the chloroplasts (Hara et al., 1994), it was difficult to discriminate the latter two species. Therefore, calculation of MPN was done combining C. antiqua with C. marina. Germination of C. ovata cysts was observed at temperatures from 17.5 to 30 8C, but not at 15 8C or below. In addition the number of the germinated cysts increased with increasing temperature, with the optimum temperature for the germination 30 8C (Fig. 6). Although the germination of the cysts of C. antiqua and C. marina occurred from 15 to 30 8C, the optimum germination temperature was 22.5 8C (Fig. 6). The present
Fig. 5. Horizontal distribution of the cysts of Chattonella spp. (C. antiqua, C. marina and C. ovata) in Hiroshima Bay in July 2005. Nd = not detected.
Fig. 6. Effect of temperature on germination of the cysts of Chattonella spp. (C. antiqua, C. marina and C. ovata) collected in Hiroshima Bay in July 2005.
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
results for C. antiqua and C. marina are in accordance with those reported by Imai (1990). Thus, the lower limit and temperatures optimum for the germination of C. ovata cysts are 2.5 and 7.5 8C higher, respectively, than that of C. antiqua/C. marina. Furthermore, our study showed that the optimum temperature for the growth of the vegetative cells of C. ovata was 30 8C (Yamaguchi et al., unpublished data), which is 5 8C higher that those of C. antiqua and C. marina (Yamaguchi et al., 1991). These physiological features of the cysts and vegetative cells suggest that C. ovata is well-adapted to warm water environments. The present study demonstrated that C. ovata has a dormant cyst stage, indicating further studies are necessary to determine the whole life history of the organism, such as the dormancy characteristics and the process of the cyst formation. 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´s-Altamirano et al., 2006). It has been suggested that human activities such as ship’s ballast water displacements and the transportation of fishery product are the major causes of the geographical expansion of harmful algae (Hallegraeff, 1993). Cysts or resting stage cells play an important role in this species dispersal because they have higher tolerance to changes in environmental conditions (Anderson and Wall, 1978; Anderson, 1998). Since there is a possibility for future outbreaks of C. ovata in previously unaffected waters, careful monitoring of this species is necessary, especially in subtropical and tropical areas. References Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transition. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Berlin, pp. 29–48. Anderson, D.M., Wall, D., 1978. Potential importance of benthic cysts of Gonyaulax tamarensis and G. excavata in initiating toxic dinoflagellate blooms. J. Phycol. 14, 224–234. Barraza-Guardado, R., Corte´s-Altamirano, R., Sierra-Beltra´n, A., 2004. Marine die-offs from Chattonella marina and Ch. cf. ovata in Kun Kaak Bay Sonora in the Gulf of California. In: Wyatt, T. (Ed.), Harmful Algae News., vol. 25. IOC of UNESCO, pp. 7–8. Corte´s-Altamirano, R., Alonso-Rodriguez, R., Sierra-Beltra´n, A., 2006. Fish mortality associated with Chattonella marina and C. cf ovata (Raphidophyceae) blooms in Sinaloa (Mexico). In: Wyatt, T. (Ed.), Harmful Algae News, vol. 31. IOC of UNESCO, pp. 7–8. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79–99. Hallegraeff, G.M., Hara, Y., 1995. Taxonomy of harmful marine raphidophytes. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual
463
on Harmful Marine Microalgae. UNESCO Publishing, Paris, pp. 365– 371. Hara, Y., Doi, K., Chihara, M., 1994. Four new species of Chattonella (Raphidophyceae, Chromophyta) from Japan. Jpn. J. Phycol. 42, 407–420. Hiroishi, S., Okada, H., Imai, I., Yoshida, T., 2005. High toxicity of the novel bloom-forming species of Chattonella ovata (Raphidophyceae) to cultured fish. Harmful Algae 4, 783–787. Imai, I., 1989. Cyst formation of the noxious red tide flagellate Chattonella marina (Raphidophyceae) in culture. Mar. Biol. 103, 235–239. Imai, I., 1990. Physiology, morphology, and ecology of cysts of Chattonella (Raphidophyceae), causative flagellates of noxious red tides in the Inland Sea of Japan. Bull. Nansei Natl. Fish. Res. Inst. 23, 63–166 (in Japanese with English abstract). Imai, I., Itoh, K., 1987. Annual life cycle of Chattonella spp., causative flagellates of noxious red tides in the Inland Sea of Japan. Mar. Biol. 94, 287–292. Imai, I., Itoh, K., 1988. Cysts of Chattonella antiqua and C. marina (Raphidophyceae) in sediments of the Inland Sea of Japan. Bull. Plankton Soc. Jpn. 35, 35–44. Imai, I., Itoh, K., Anraku, M., 1984. Extinction dilution method for enumeration of dormant cells of red tide organisms in marine sediment. Bull. Plankton Soc. Jpn. 31, 123–124. Imai, I., Itoh, K., Anraku, M., 1989. Dormancy and maturation in the cysts of Chattonella spp. (Raphidophyceae), red tide flagellates in the Inland Sea of Japan. In: Okaichi, T, Anderson, D.M., Nemoto, T. (Eds.), Red tides: Biology, Environmental Science, and Toxicology. Elsevier, New York, pp. 289–292. Imai, I., Yamaguchi, M., Watanabe, M., 1998. Ecophysiology, life cycle, and bloom dynamics of Chattonella in the Seto Inland Sea Japan. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Berlin, pp. 95–112. Imai, I., Yamaguchi, M., Hori, Y., 2006. Eutrophication and occurrences of harmful algal blooms in the Seto Inland Sea. Jpn. Plankton Benthos Res. 1, 71–84. Itoh, K., Imai, I., 1987. Raphidophyceae. In: A Guide for Studies of Red Tide Organisms, Japan Fisheries Resources Conservation Association, Shuwa, Tokyo, pp. 122–130 (in Japanese). Lu, S., Hodgkiss, I.J., 2001. More raphidophyte blooms in South China waters. In: Wyatt, T. (Ed.), Harmful Algae News, vol. 22. IOC of UNESCO, pp. 1–2. Yamaguchi, M., Imai, I., 1994. A microfluorometric analysis of nuclear DNA at different stages in the life history of Chattonella antiqua and Chattonella marina (Raphidophyceae). Phycologia 33, 163–170. Yamaguchi, M., Imai, I., Honjo, T., 1991. Effects of temperature, salinity and irradiance on the growth rates of the noxious red tide flagellates Chattonella antiqua and C. marina (Raphidophyceae). Nippon Suisan Gakk. 57, 1277– 1284. Yamaguchi, H., Sakamoto, S., Yamaguchi, M., 2008. Nutrition and growth kinetics in nitrogen- and phosphorus-limited cultures of the novel red tide flagellate Chattonella ovata (Raphidophyceae). Harmful Algae 7, 26–32. Yoshimatsu, S., 1992. Life history studies on two species of Alexandrium (Dinophyceae) and three species of Raphidophyceae in Seto Inalnd Sea. Bull. Akashiwo Res. Inst. Kagawa Pref. 4, 1–90. Yoshimatsu, S., Ono, C., 1986. The seasonal appearance of the red tide organisms and flagellates in the southern Harima-Nada, Inland Sea of Seto. Bull. Akashiwo Res. Inst. Kagawa Pref. 2, 1–42.
Harmful Algae 7 (2008) 459–463 www.elsevier.com/locate/hal
Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan Mineo Yamaguchi a,*, Haruo Yamaguchi b, Goh Nishitani a, Setsuko Sakamoto a, Shigeru Itakura a a
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 Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan Received 21 February 2007; received in revised form 12 September 2007; accepted 4 October 2007
Abstract To elucidate the mechanism of bloom outbreaks of Chattonella ovata (Raphidophyceae), we investigated the cysts of C. ovata and succeeded in finding them from the bottom sediments of Hiroshima Bay. The morphology of the cysts was mostly hemispherical in shape, with a diameter of ca. 30 mm and height of ca. 20 mm. The cysts were usually adhering to solid materials, such as diatom frustules, yellow-greenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation. Because the morphological characteristic of the cysts was in general agreement with those of Chattonella antiqua and Chattonella marina, it was difficult to differentiate the cysts of these three species. Germination of the cysts of C. ovata was observed at temperatures from 17.5 to 30 8C, but not at 15 8C or below. The number of the germinated cysts increased with increasing temperature and the optimum temperature for germination was 30 8C. Although cysts of C. antiqua and C. marina germinated at temperatures from 15 to 30 8C, optimum temperature of germination was 22.5 8C. The lower limit and optimum temperatures for germination of C. ovata cysts was higher than for C. antiqua and C. marina. The role of cysts in the population dynamics of C. ovata is discussed. # 2007 Elsevier B.V. All rights reserved. Keywords: Chattonella ovata; Cyst; Germination; Morphology; Raphidophyceae
1. Introduction Species of the genus Chattonella (Raphidophyceae) are harmful flagellates which cause mass mortality of cultured fishes in coastal waters (Imai et al., 1998). Although six species have been reported in the genus Chattonella (Hallegraeff and Hara, 1995), so far in Japanese waters the most representative and harmful species 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 from the 1970s through the mid 1980s (Imai et al., 1998). During the 1990s, however, the frequency of harmful blooms caused by C. antiqua and C. marina have decreased in the Seto Inland Sea (Imai et al., 2006). In summer of 2004, however, a massive bloom of Chattonella ovata. Hara et Chihara (Hara
* Corresponding author. Tel.: +81 829 55 0666; fax: +81 829 54 1216. E-mail address: [email protected] (M. Yamaguchi). 1568-9883/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2007.10.002
et al., 1994) occurred over almost the entire area of the Seto Inland Sea and caused damage to cultured fish (Hiroishi et al., 2005). This is the first record of a bloom and damage to fisheries caused by C. ovata in Japan (Yamaguchi et al., 2008), although vegetative cells have been observed since the mid 1980s (Yoshimatsu and Ono, 1986). Cysts of harmful algae play an important role in species dispersal, bloom initiation and termination, and survival under unfavorable conditions (Anderson and Wall, 1978; Anderson, 1998). Therefore, information on the distribution and abundance in natural sediments as well as the germination characteristics of cysts is essential for understanding the ecology and bloom dynamics of harmful algae. The morphology and physiological ecology of the cysts of C. antiqua and C. marina have already been thoroughly examined (Imai and Itoh, 1987, 1988; Imai, 1989, 1990; Imai et al., 1989; Yamaguchi and Imai, 1994). Although the existence of cysts of C. ovata in the bottom sediment was expected (Imai, 1990), its morphology and germination characteristics are still unknown.
460
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
In the present paper, we report the occurrence of cysts of C. ovata in the bottom sediments of Hiroshima Bay, examine the effect of temperature on the germination of the cysts, and discuss the role of cysts in the population dynamics of C. ovata. 2. Materials and methods 2.1. Sediment sampling Sediment samples were collected with a gravity coresampler (inner diameter 4 cm) from 12 stations in Hiroshima Bay (Fig. 1) in June 2005. The top 1 cm of each sediment sample from the replicate samples was sliced off, mixed, placed in a plastic container and stored in the dark at 10 8C. 2.2. Detection of cysts Five gram (wet weight) aliquots of the sediment samples were suspended in filtered-autoclaved seawater, sonicated and sieved through plankton netting to obtain the size fraction between 20 and 150 mm. The material remaining on the 20 mm netting was finally resuspended in 10 ml filtered-autoclaved seawater. The sediment suspension was used for microscopic observations using an inverted epifluorescence microscope under blue light excitation. For observing the cysts, 0.1–0.2 ml of the sediment suspension was placed in a Sedgwick-Rafter chamber with 1 ml filtered-autoclaved seawater. Cyst-like cells were isolated with a capillary tube under the inverted epifluorescence microscope referring to the morphological features of previously known species of Raphidophytes such as C. antiqua, C. marina and Fibrocapsa japonica (Imai and Itoh, 1988; Yoshimatsu, 1992). Each isolated cyst-like cell was incubated in a well of a 48-well tissue culture plate (Costar) with 0.5 ml filtered-autoclaved seawater at 22 8C, under an illumination of 50 mmol photons m 2 s 1 with a 12hL:12hD photo-cycle. After an incubation period of 14 days, each vegetative cell that germinated from cyst-like cells was
identified on the basis of the morphology of cultured strains of C. ovata, C. antiqua and C. marina (Hara et al., 1994). 2.3. Enumeration of cysts For enumerating cysts, sediment suspensions were prepared as mentioned above and examined using an inverted epifluorescence microscope under blue light excitation. For counting cysts, 0.1 ml of sediment suspension was placed in a Sedgwick-Rafter chamber with 1 ml filtered-autoclaved seawater and the enumeration made on the basis of characteristics of autofluorescence and morphology of the cysts (Imai and Itoh, 1988). The number of cysts in the sediment suspension was determined at 100 magnification, and counts were made in triplicate. The cyst count was expressed as the number of the cyst per gram wet sediment. 2.4. Effect of temperature on the germination of the cysts The effect of temperature on the germination of the cysts was examined by an extinction dilution method (Imai et al., 1984) using the sediment sample collected from St. 12 in 2005 (Fig. 1). Five to 15 g of the sediment sample was suspended in filtered-autoclaved seawater, sonicated and sieved through plankton netting to obtain the size fraction between 20 and 150 mm. This fraction was suspended in 50 ml of filteredautoclaved seawater. After serial 10-fold dilutions (10 1 to 10 2) with filtered-autoclaved seawater, 1 ml aliquots of the diluted suspension were inoculated into five replicate test tubes containing 1 ml of filtered-autoclaved seawater. To avoid the growth of diatoms, GeO2 was added to the seawater at a final concentration of 1 mg l 1. These test tubes were incubated at 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 8C under an illumination of 50 mmol photons m 2 s 1 with a 12hL:12hD photo-cycle. Appearance of the vegetative cells of Chattonella spp. (C. antiqua, C. marina and C. ovata) in each tube was examined after an incubation period of 7–14 days using an inverted epifluorescence microscope. The tubes in which vegetative cells of Chattonella spp. were observed were scored as positive. The most probable number (MPN) of the cysts of Chattonella spp. was then calculated referring to the MPN tables (Itoh and Imai, 1987). 3. Results and discussion
Fig. 1. Sampling stations in Hiroshima Bay, the Seto Inland Sea.
A total 329 cyst-like cells were isolated from the bottom sediment samples. After incubation, 14 cells were successfully germinated. Ten of them were identified as vegetative cells of C. ovata (Figs. 2 and 3) and the remaining four were identified as those of C. antiqua or C. marina (Fig. 4). The cysts of C. ovata were mostly hemispherical in shape, with a diameter of ca. 30 mm and height of ca. 20 mm. These cysts were usually adhering to solid materials such as diatom frustules, yellowgreenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation (Fig. 2). The morphological characteristics of the cysts generally agreed with those of C. antiqua/C. marina a (Fig. 4; Imai and Itoh, 1988).
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
461
Fig. 2. Cultured vegetative cells of Chattonella ovata (A) and the cyst (B) from which the vegetative cell germinated (arrow).
Therefore, incubation of cysts and observation of the germinated vegetative cells are necessary to discriminate the cysts of these species. Fig. 5 shows the horizontal distribution of the cysts of Chattonella spp. in Hiroshima Bay determined by the direct count method using epifluorescence microscope. As mentioned above, the number of cysts given in the figure include those for all
three species of Chattonella. Chattonella cysts were observed at all the stations except for St. 9 (Fig. 1). Cyst concentration ranged from 0 (i.e., not detected) to 227 cysts/g wet sediment. Major cyst deposition areas were observed in the central part of the bay (west of Etajima Island). In this bay, a cyst survey was conducted in 1987. The results showed that the number of the cysts was at most 2 cysts/g wet sediment (Imai, 1990). The present study indicates
Fig. 3. Cysts from which the vegetative cell of C. ovata germinated (arrows).
462
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
Fig. 4. Cysts from which the vegetative cell of Chattonella antiqua or Chattonella marina germinated (arrows).
that the cyst abundance has since increased markedly. The high abundance of cysts is probably caused by the deposition of newly formed cysts during the preceding bloom of C. ovata that occurred in the summer of 2004. Thus the majority of the cysts were expected to be those of C. ovata. Therefore it is important that Hiroshima Bay be monitored for further, potential outbreaks of blooms of C. ovata. The effect of temperature on the germination of the cysts was investigated by the extinction dilution method using natural sediment samples (Fig. 6). Although just germinated
cells of C. ovata could be distinguished from those of C. antiqua and C. marina from the morphological characteristics of the chloroplasts (Hara et al., 1994), it was difficult to discriminate the latter two species. Therefore, calculation of MPN was done combining C. antiqua with C. marina. Germination of C. ovata cysts was observed at temperatures from 17.5 to 30 8C, but not at 15 8C or below. In addition the number of the germinated cysts increased with increasing temperature, with the optimum temperature for the germination 30 8C (Fig. 6). Although the germination of the cysts of C. antiqua and C. marina occurred from 15 to 30 8C, the optimum germination temperature was 22.5 8C (Fig. 6). The present
Fig. 5. Horizontal distribution of the cysts of Chattonella spp. (C. antiqua, C. marina and C. ovata) in Hiroshima Bay in July 2005. Nd = not detected.
Fig. 6. Effect of temperature on germination of the cysts of Chattonella spp. (C. antiqua, C. marina and C. ovata) collected in Hiroshima Bay in July 2005.
M. Yamaguchi et al. / Harmful Algae 7 (2008) 459–463
results for C. antiqua and C. marina are in accordance with those reported by Imai (1990). Thus, the lower limit and temperatures optimum for the germination of C. ovata cysts are 2.5 and 7.5 8C higher, respectively, than that of C. antiqua/C. marina. Furthermore, our study showed that the optimum temperature for the growth of the vegetative cells of C. ovata was 30 8C (Yamaguchi et al., unpublished data), which is 5 8C higher that those of C. antiqua and C. marina (Yamaguchi et al., 1991). These physiological features of the cysts and vegetative cells suggest that C. ovata is well-adapted to warm water environments. The present study demonstrated that C. ovata has a dormant cyst stage, indicating further studies are necessary to determine the whole life history of the organism, such as the dormancy characteristics and the process of the cyst formation. 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´s-Altamirano et al., 2006). It has been suggested that human activities such as ship’s ballast water displacements and the transportation of fishery product are the major causes of the geographical expansion of harmful algae (Hallegraeff, 1993). Cysts or resting stage cells play an important role in this species dispersal because they have higher tolerance to changes in environmental conditions (Anderson and Wall, 1978; Anderson, 1998). Since there is a possibility for future outbreaks of C. ovata in previously unaffected waters, careful monitoring of this species is necessary, especially in subtropical and tropical areas. References Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transition. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Berlin, pp. 29–48. Anderson, D.M., Wall, D., 1978. Potential importance of benthic cysts of Gonyaulax tamarensis and G. excavata in initiating toxic dinoflagellate blooms. J. Phycol. 14, 224–234. Barraza-Guardado, R., Corte´s-Altamirano, R., Sierra-Beltra´n, A., 2004. Marine die-offs from Chattonella marina and Ch. cf. ovata in Kun Kaak Bay Sonora in the Gulf of California. In: Wyatt, T. (Ed.), Harmful Algae News., vol. 25. IOC of UNESCO, pp. 7–8. Corte´s-Altamirano, R., Alonso-Rodriguez, R., Sierra-Beltra´n, A., 2006. Fish mortality associated with Chattonella marina and C. cf ovata (Raphidophyceae) blooms in Sinaloa (Mexico). In: Wyatt, T. (Ed.), Harmful Algae News, vol. 31. IOC of UNESCO, pp. 7–8. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79–99. Hallegraeff, G.M., Hara, Y., 1995. Taxonomy of harmful marine raphidophytes. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual
463
on Harmful Marine Microalgae. UNESCO Publishing, Paris, pp. 365– 371. Hara, Y., Doi, K., Chihara, M., 1994. Four new species of Chattonella (Raphidophyceae, Chromophyta) from Japan. Jpn. J. Phycol. 42, 407–420. Hiroishi, S., Okada, H., Imai, I., Yoshida, T., 2005. High toxicity of the novel bloom-forming species of Chattonella ovata (Raphidophyceae) to cultured fish. Harmful Algae 4, 783–787. Imai, I., 1989. Cyst formation of the noxious red tide flagellate Chattonella marina (Raphidophyceae) in culture. Mar. Biol. 103, 235–239. Imai, I., 1990. Physiology, morphology, and ecology of cysts of Chattonella (Raphidophyceae), causative flagellates of noxious red tides in the Inland Sea of Japan. Bull. Nansei Natl. Fish. Res. Inst. 23, 63–166 (in Japanese with English abstract). Imai, I., Itoh, K., 1987. Annual life cycle of Chattonella spp., causative flagellates of noxious red tides in the Inland Sea of Japan. Mar. Biol. 94, 287–292. Imai, I., Itoh, K., 1988. Cysts of Chattonella antiqua and C. marina (Raphidophyceae) in sediments of the Inland Sea of Japan. Bull. Plankton Soc. Jpn. 35, 35–44. Imai, I., Itoh, K., Anraku, M., 1984. Extinction dilution method for enumeration of dormant cells of red tide organisms in marine sediment. Bull. Plankton Soc. Jpn. 31, 123–124. Imai, I., Itoh, K., Anraku, M., 1989. Dormancy and maturation in the cysts of Chattonella spp. (Raphidophyceae), red tide flagellates in the Inland Sea of Japan. In: Okaichi, T, Anderson, D.M., Nemoto, T. (Eds.), Red tides: Biology, Environmental Science, and Toxicology. Elsevier, New York, pp. 289–292. Imai, I., Yamaguchi, M., Watanabe, M., 1998. Ecophysiology, life cycle, and bloom dynamics of Chattonella in the Seto Inland Sea Japan. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Berlin, pp. 95–112. Imai, I., Yamaguchi, M., Hori, Y., 2006. Eutrophication and occurrences of harmful algal blooms in the Seto Inland Sea. Jpn. Plankton Benthos Res. 1, 71–84. Itoh, K., Imai, I., 1987. Raphidophyceae. In: A Guide for Studies of Red Tide Organisms, Japan Fisheries Resources Conservation Association, Shuwa, Tokyo, pp. 122–130 (in Japanese). Lu, S., Hodgkiss, I.J., 2001. More raphidophyte blooms in South China waters. In: Wyatt, T. (Ed.), Harmful Algae News, vol. 22. IOC of UNESCO, pp. 1–2. Yamaguchi, M., Imai, I., 1994. A microfluorometric analysis of nuclear DNA at different stages in the life history of Chattonella antiqua and Chattonella marina (Raphidophyceae). Phycologia 33, 163–170. Yamaguchi, M., Imai, I., Honjo, T., 1991. Effects of temperature, salinity and irradiance on the growth rates of the noxious red tide flagellates Chattonella antiqua and C. marina (Raphidophyceae). Nippon Suisan Gakk. 57, 1277– 1284. Yamaguchi, H., Sakamoto, S., Yamaguchi, M., 2008. Nutrition and growth kinetics in nitrogen- and phosphorus-limited cultures of the novel red tide flagellate Chattonella ovata (Raphidophyceae). Harmful Algae 7, 26–32. Yoshimatsu, S., 1992. Life history studies on two species of Alexandrium (Dinophyceae) and three species of Raphidophyceae in Seto Inalnd Sea. Bull. Akashiwo Res. Inst. Kagawa Pref. 4, 1–90. Yoshimatsu, S., Ono, C., 1986. The seasonal appearance of the red tide organisms and flagellates in the southern Harima-Nada, Inland Sea of Seto. Bull. Akashiwo Res. Inst. Kagawa Pref. 2, 1–42.