Reproductive Ecology of the Dominant Dinoflagellate, Ceratium Fusus, in Coastal Area of Sagami Bay, Japan

Journal of Oceanography, Vol. 63, pp. 35 to 45, 2007

Reproductive Ecology of the Dominant Dinoflagellate, Ceratium fusus, in Coastal Area of Sagami Bay, Japan

SEUNG HO BAEK*, SHINJI SHIMODE and TOMOHIKO KIKUCHI

Graduate School of Environmental and Information Sciences, Yokohama National University, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan

(Received 12 March 2006; in revised form 1 September 2006; accepted 1 September 2006)

The seasonal abundance of the dominant dinoflagellate, Ceratium fusus, was investi- Keywords: gated from January 2000 to December 2003 in a coastal region of Sagami Bay, Japan. ⋅ Dinoflagellate The growth of this species was also examined under laboratory conditions. In Sagami Ceratium fusus, ⋅ Bay, C. fusus increased significantly from April to September, and decreased from reproductive November to February, though it was found at all times through out the observation strategy, ⋅ bloom, period. C. fusus increased markedly in September 2001 and August 2003 after heavy ⋅ growth rates, rainfalls that produced pycnoclines. Rapid growth was observed over a salinity range ⋅ Sagami Bay, Japan. of 24 to 30, with the highest specific rate of 0.59 dÐ1 measured under the following conditions: salinity 27, temperature 24°C, photon irradiance 600 µmol mÐ2sÐ1. The growth rate of C. fusus increased with increasing irradiance from 58 to 216 µmol mÐ2sÐ1, plateauing between 216 and 796 µmol mÐ2sÐ1 under all temperature and salinity treatments (except at a temperature of 12°C). Both field and laboratory experiments indicated that C. fusus has the ability to grow under wide ranges of water temperatures (14Ð28°C), salinities (20Ð34), and photon irradiance (50Ð800 µmol mÐ2sÐ1); it is also able to grow at low nutrient concentrations. This physiological flexibility ensures that populations persist when bloom conditions come to an end.

1. Introduction 2003). In addition, red tides occurred from March to July The dinoflagellate genus, Ceratium, is an important in 1997 along the Pacific coast of central Japan from component of marine phytoplankton communities. It has Wakayama to Ibaraki Prefecture (Machida et al., 1999). an extraordinary biogeographical range through all of the Furthermore, C. fusus has a seasonality similar to that of world’s oceans, from the warmest waters of the tropics to C. furca. The cell density of C. fusus at the peak of pro- the cold polar seas (Graham, 1941). Within the North liferation exceeds an average value for red tides (Mulford, Atlantic Ocean and adjacent seas, distribution depends 1963; Dodge and Marshall, 1994). There have been field significantly on water temperature (Dodge and Marshall, and laboratory studies of factors that control seasonal 1994). Some species of the genus Ceratium frequently changes in C. furca, and optimal environmental condi- dominate coastal phytoplankton communities, where they tions for bloom outbreaks have been determined (Baek et contribute substantially to annual primary production al., 2006). There is no similar information for C. fusus, (Nielsen, 1991; Dodge and Marshall, 1994). though a few authors have reported on cell tolerance to Ceratium fusus and Ceratium furca have recently changes in temperature and salinity. For example, C. fusus been recognized as dominant red tide species in eastern is able to survive at temperatures ranging from 1.7 to 27°C Asian areas, such as Chinese coastal water, Hong Kong, and salinities between 14.4 and 34.8 in water from Vir- the Philippine Sea and the Gulf of Thailand etc (Lu, 2003; ginia, USA (Mulford, 1963). Yin, 2003; Lirdwitayaprasit, 2003). Although both spe- Due to difficulties in isolating C. fusus from natural cies are frequently observed in coastal areas of Korea and seawater and subsequent laboratory cultivation, there have Japan, red tides of C. furca have been especially frequent been few ecological or physiological studies of the spe- on the southern coast of Korea since 1995 (Suh et al., cies, and there is a shortage of information obtained under controlled laboratory experiments on the life cycle (including cyst populations), and on nutrient require- * Corresponding author. E-mail: [email protected] ments, light intensities, salinities and water temperatures Copyright©The Oceanographic Society of Japan/TERRAPUB/Springer for optimal growth.

35 Fig. 1. The sampling stations.

Ceratium fusus is a dominant red tide species in layers only) and with 6 L Niskin bottles. The sampling Sagami Bay; high cell numbers are observed frequently depths at the two stations were 0 m, 5 m, 20 m and 35 m from April to September, after the spring diatom bloom. at St. 40, and 0 m, 5 m, 20 m, 40 m and 65 m at St. 70. The species sometimes occurs as a red tide under rela- Debris and large-sized plankters in the collected waters tively low nutrient conditions. Population densities in the were removed by filtering through 330 µm mesh on board water column decrease from November to February. This ship immediately after measurement of water tempera- seasonal pattern of occurrence has not been as rigorously ture with a mercury thermometer. Each filtered water sam- assessed as those of other dinoflagellates. In this study, ple was kept in a dark bottle and taken to the laboratory the natural population of C. fusus was monitored to pro- for determination of salinity, inorganic nutrients, chloro- vide information on the relationship between cell abun- phyll a (Chl.a) concentrations, and phytoplankton assem- dance and environmental factors (water temperature, sa- blages. Salinity was measured using an inductive salinom- linity, nutrients) in the coastal waters of Sagami Bay. eter (Model 601 MK-IV, Watanabe Keiki MFG. Co. Ltd.). Laboratory cultures were used to examine optimum physi- Subsamples for the estimation of phytoplankton abun- ological requirements for growth. The experimental re- dances were fixed immediately with 2.5% (final concen- sults were compared to observations of the natural popu- tration) glutal aldehyde solution after filtration through lation to better understand the reproductive ecology of TTTP type 2.0 µm membranes, and stored at 4°C in the the species in Sagami Bay. dark until cells were counted (using a Sedgwick-Rafter chamber). 2. Materials and Methods Duplicate subsamples of >100 ml were filtered onto Whatman GF/F glass fiber filters for analysis of Chl.a. 2.1 Field investigation Each filter was extracted in the dark at 4°C for 24 h in a Sampling was conducted monthly from 2000 to 2003 10 ml brown vial containing 10 ml N,N- at two coastal stations, designated St. 40 and 70 (ca. 40 Dimethylformamide (DMF) (Suzuki and Ishimaru, 1990). m and 70 m depth, respectively) in the north-western part Chl.a concentration was determined fluorometrically on of Sagami Bay, Central Japan (Fig. 1). Sagami Bay faces a Turner Design fluorometer according to the method of the Pacific Ocean and its hydrography is related prima- Holm-Hansen et al. (1965). Water samples for determi- rily to fluctuations of the Kuroshio Current axis. It is also nation of dissolved inorganic nutrients were filtered influenced by the fresh water discharged from the Sagami through Millipore Milex filters (pore size: 0.45 µm). The and Sakawa Rivers as well as the water from Tokyo Bay filtered water was transferred into plastic tubes and kept (Hogetsu and Taga, 1977). Interaction between the cur- in a freezer (Ð20°C) for later measurement of nutrients. rent and river discharges results in stratified waters in The water samples were thawed to room temperature, and Ð Ð Sagami Bay. The surface layer consists of a mixture of nitrite + nitrate-N (NO2 + NO3 ), and phosphate-P 3Ð waters from the Kuroshio Current and fresh waters (Iwata, (PO4 ) concentrations were analyzed using an auto- 1985). analyzer (Bran Luebbe, AACS-II), following analytical Water samples were collected with a bucket (surface methods based on Parsons et al. (1984).

36 S. H. Baek et al. Fig. 2. Seasonal changes in vertical profiles of water temperature and salinity at St. 70, Sagami Bay, Japan (2000Ð2003). Black dots indicate sampling depths.

Rainfall was measured every day during the sam- one month at 22°C under a photon fluence rate of 180 pling period with rain gauges located on the roof of the µmol mÐ2sÐ1 with a 12 L: 12 D cycle (using cool white ° ′ ″ ° ′ ″ Manazuru City Hall (35 09 15 N, 139 08 26 E) and the fluorescent lamps). Enriched seawater (modified T5 me- Odawara office of the Japan Meteorological Business dium, i.e., T1 medium concentrated five-fold) with soil Support Center (35°15′01″ N, 139°09′03″ E). extract was used in the acclimation period. Preliminary µ experiments showed that T5 medium (N: 5 M, P: 0.5 µ 2.2 Isolation and culture of Ceratium fusus M) promoted better cell population growth than T1 me- Individual cells of C. fusus were isolated from natu- dium. The following experiments were conducted after ral assemblages in waters of Sagami Bay during July 2003, T5 medium-acclimation. when water temperature and salinity were approximately 22°C and 32.5, respectively. Each of the isolated C. fusus 2.3 Specific growth rate experiments cells was washed by serial transfer through three drop- We tested the effects of varying light, temperature lets of T1 medium containing H2SeO3 (Ogata et al., 1987; and salinity on the growth rate of C. fusus populations Baek et al., 2006), and then pipetted into one 5 ml well maintained in 100 ml flasks in T5 medium with soil ex- of a 12-well tissue culture plate. For acclimation to labo- tract. Stock cultures containing 3000 cells mlÐ1 of C. fusus ratory conditions, the cells were cultured for a period of were concentrated by gentle reverse filtration through a

Reproductive Ecology of Ceratium fusus 37 Fig. 3. Variation in rainfall in the north-western part of Sagami Bay, Japan. White and black bars indicate average rainfall in month and total rainfall over five days preceding sampling date, respectively.

20 µm Nitex mesh. Elevated concentrations were needed the lowest in March. The water column was well mixed because densities did not increased above 1500 cells vertically from November to March, and gradually strati- mlÐ1, even under optimal conditions. One ml of the cell fied thereafter. Salinity varied from 23.0 to 34.7 during concentrate was transferred into each 100 ml conical flask. the sampling period, and low salinities were frequently Thus, the initial cell density was 30 mlÐ1. The densities recorded in summer due to rainfall (Figs. 2 and 3). In were determined as means of three replicate cell counts particular, salinity decreased drastically from 34.5 to 24.5 using a 1 ml cell counts using a 1 ml Sedgwick-Rafter in September 2001, and from 34.5 to 23 in August 2003, counting chamber under a microscope. The experiments probably due to heavy rain two to four days prior to sam- were run three times. pling in both months. In contrast, we did not find salinities Five temperature (12, 16, 20, 24 and 28°C), six sa- of less than 30 after relatively high rainfall (>100 mm) in linity (17, 20, 24, 27, 30 and 34) and six photon irradi- June of 2000 and 2002. Large rainfall events were re- ance regimens (0, 58, 180, 230, 600 and 800 corded from May to October during all 4 years (Fig. 3). µmol mÐ2sÐ1) were established. Cells were incubated at The largest annual rainfall during the study period oc- 20, 24 and 28°C under all six salinity conditions. They curred in 2003. Average rainfalls five days prior to each were also incubated at 12 and 16°C at a salinity of 34. sampling day were in excess of 100 mm on four occa- Experiments ran four days under a 12L: 12D cycle. The sions during summer (June to September) in each sam- specific growth rate (µ) was estimated from: pling year. Concentrations of nitrate + nitrite-N ranged from µ µ µ = ln(Nt/N0)/t, >0.02 M on July 2003 to 20.49 M on August 2003, and the mean values during four sampling years were ± µ µ where N0 and Nt represent the initial and final cell densi- 3.61 2.86 M (Fig. 4). The highest (20.49 M) nitrate ties and t represents incubation time (day). + nitrite-N value was recorded in surface water on Au- gust 2003 after heavy rainfall. The second highest con- 2.4 Data analysis centration was observed on July 2001, although there was We examined relationships among cell densities and no rain in the day (July 7 to 13) preceding the sampling environmental parameters (Temperature, Salinity, Chl.a, date. Phosphate-P concentrations ranged from >0.02 µM Ð Ð 3Ð µ NO2 + NO3 and PO4 ) in the field using Pearson’s on May 2001 to 1.62 M on July 2001, with a mean value correlation analysis. A significance level of P < 0.05 was of 0.30 ± 0.23 µM over the four sampling years (Fig. 3). used in all statistical analyses. 3.2 Chl.a concentrations and phytoplankton assemblages 3. Results Chl.a concentrations at 5 m depth in the two station are shown in Fig. 5. The Chl.a concentrations ranged from 3.1 Abiotic factors 0.02 mg mÐ3 on November 2002 to 9.66 mg mÐ3 on March Seasonal changes in environmental factors were al- 2001 at St. 40. The concentrations remained at low val- most identical at the two stations. Therefore, we show ues through September to January in each of four years, only the results obtained at St. 70. and tended to increase from February to July. Eight peaks Water temperature varied from 12 to 29°C during the (>5 mg mÐ3) of Chl.a concentration were observed at both sampling period (Fig. 2). In each sampling year, the high- stations. Seasonal changes in Chl.a concentrations were est temperature was recorded in August or September and almost identical at the two stations. Spring blooms, rec-

38 S. H. Baek et al. Ð Ð 3Ð Fig. 4. Seasonal changes of vertical profiles of nutrients (NO2 + NO3 -N and PO4 -P) at St. 70, Sagami Bay, Japan (2000Ð 2003). Black dots indicate sampling depths.

ognizable by high Chl.a concentrations (>5 mg mÐ3) following mixing of the water column in winter, were mostly dominated by diatoms such as Eucampia spp., Rhizosolenia spp., and Chaetoceros spp. The phytoplankton assemblages during the summer periods consisted mainly of dinoflagellates, such as Ceratium furca and C. fusus. When the two species bloomed at both stations in September 2001 and May 2002, C. fusus made up >90% of total phytoplankton cell density. In addition to Ceratium species, other dinoflagellates, such as Prorocentrum spp. and smaller diatoms, such as Nitzschia spp., increased in abundance after heavy rainfall during summer. Fig. 5. Variation in Chl.a concentrations at 5 m depth during the study period. White and black bars indicate St. 40 and 3.3 Temporal variation of C. fusus abundance St. 70, respectively. Arrows indicate point in time when C. fusus accounted for more than ca. 80% of total Population densities of C. fusus were measured at phytoplankton abundance.

Reproductive Ecology of Ceratium fusus 39 Fig. 6. Seasonal changes in cell density (cells lÐ1) of Ceratium fusus by depth at St. 40 (a) and St. 70 (b) in Sagami Bay, Japan (2000Ð2003). Black dots indicate sampling depths.

Table 1. Pearson correlation coefficients (r) indicating relationships between environmental factors and cell density of Ceratium fusus between 2000 and 2003.

Ð Ð 3Ð Depth Temperature Salinity Chl.a NO2 +NO3 PO4 Temperature Ð0.152 Salinity 0.455* Ð0.492* Chl.a Ð0.478* Ð0.024 Ð0.304 Ð Ð NO2 + NO3 0.249 Ð0.567* 0.137 Ð0.229* 3Ð PO4 0.325* Ð0.634* 0.342* Ð0.316* 0.776* C. fusus Ð0.276* 0.144 Ð0.160 0.222* Ð0.299* Ð0.244

*Significant correlations (P < 0.01).

both stations in each sampling year (Fig. 6). Total abun- Marked seasonal blooms of the species were observed dances at St. 70 were slightly lower than those at St. 40. during the periods from April to August in 2000Ð2002. In Populations remained at low densities through October 2003, abundance was exceptionally high in both summer to January, and increased from April to September. and winter in comparison with the previous three years.

40 S. H. Baek et al. Fig. 7. Changes in growth rates of Ceratium fusus with increasing photon irradiance at different salinity (a: 34, b: 30, c: 27, d: 24, e: 20, f: 17) and temperature conditions. Error bars are SD.

During the study period, individual blooms persisted for m depth was always higher at St. 40 than at St. 70, except no more than one month at both stations. in 2000 when the average value was higher at St. 70. Maximum densities of C. fusus occurred between the The relationship between environmental factors and surface and 20 m depth. Subsurface maxima were fre- abundances of C. fusus during the 4 years are shown in quently observed at 5 m depth, with sharply decreasing Table 1. The abundance of the species was not signifi- abundances with increasing depth at each station during cantly correlated with water temperature and salinity. In the summer period. In contrast, during winter periods of contrast, the abundance was significantly negatively cor- 2002 and 2003 at both stations, cells were observed related with water depth (r = Ð0.276, p < 0.01), nitrate + throughout the water column (as a result of vertical mix- nitrite-N concentrations (r = Ð0.299, p < 0.01), and phos- ing). The annual average density calculated from 0 to 5 phate concentration (r = Ð0.244, p < 0.01).

Reproductive Ecology of Ceratium fusus 41 µmol photons mÐ2sÐ1. However, growth rates at salinities of 34, 20 and 17 were lower than those in the range from 24 to 30. At a salinity of 17, morphologically abnormal cells without apical horns were observed during incubation. At salinity <14 (data not shown), we also observed cells damaged by phenomena such as cytolysis (Fig. 8).

4. Discussion Donaghay and Osborn (1997) stated that, ecologi- cally, bloom dynamics seem to be dominated by interac- tions between biological and physical processes that occur over a broad range of temporal and spatial scales. Morse (1947) reported that Ceratium furca reached an especially high density in warm water above the pycnocline in Patuxent River, Maryland, USA. Dense populations of C. fusus have been observed mostly near pycnoclines in stratified water columns, in accordance with the general observation that the pycnocline is a nec- essary precondition for the development of dinoflagellate populations (Donaghay and Osborn, 1997). Pycnoclines also play an important role in the occurrence of subsurface populations and their occurrence has often been in- terpreted as an underlying factor in phytoplankton patchiness (Rasmussen and Richardson, 1989; Horner et al., Fig. 8. Cell morphology of Ceratium fusus. (a) Normal vegetative cell; arrow points to flagella. (b) and (c) show ab- 1997). At our sampling sites, stratification of the water normal forms; white arrow indicates cytolysis in yellow- column developed gradually from spring to summer. Red brown chloroplasts of and the nucleus. tides of C. fusus broke out 5 days after heavy rainfall on 20 August 2003 (Kinoshita, personal communication). Three days later (8 days after the rain), high abundances of C. fusus were observed when surface water was at rela- 3.4 Specific growth rate tively low salinity (24), and strong pycnocline layers ap- Laboratory experiments were conducted to investi- peared, especially near the surface. The results suggest gate effects of temperature, salinity and irradiance on the that the development of the dinoflagellate populations specific growth rate of C. fusus (Fig. 7). Five tempera- probably requires a stabilized water column under the tures between 12 and 28°C were tested at salinity value pycnocline, subsequent to nutrient addition by natural 34 only (Fig. 7a). At 12°C, the specific growth rates of C. rainfall inputs during the rainy season from late spring to fusus decreased gradually with increasing photon irradi- summer. ance between 53 to 183 µmol mÐ2sÐ1, after which there Cell densities of C. fusus decreased gradually from was no growth up to the highest photon irradiance of 796 the late summer to autumn. Factors that may be related to µmol mÐ2sÐ1. Although the growth rates increased with the decline are: (1) breakdown of summer stratification increasing photon irradiance from 58 to 796 µmol mÐ2sÐ1 with decreasing temperature, and (2) continuous high sa- at all temperatures above 16°C, the highest growth oc- linity condition of more than 34 during the period of re- curred at 24 and 28°C. Accordingly, in salinity treatments duced rainfall after October. Elbrächter (1973) reported below 34 salinity, we measured growth at the following that water temperature clearly influenced generation time temperatures: 20, 24 and 28°C. The specific growth rates for Ceratium species. Our results from the field survey of C. fusus increased gradually with increasing photon and laboratory experiments indicate that the growth rates irradiance from 58 to 216 µmol mÐ2sÐ1 in all salinity treat- of C. fusus in the field are probably limited by a gradual ments (except at 12°C and a salinity of 34). Growth rates water temperature decline (to <16°C) and continuous high reached a plateau between 216 and 796 µmol mÐ2sÐ1. salinity (>34) during the fall (though the doubling time Photoinhibition did not occur, even at 796 µmol mÐ2sÐ1, of C. fusus in the field is likely shorter in the laboratory). the maximum photon irradiance used in this study. Our results suggest that, in addition to the low growth In salinities from 24 to 30, high specific growth rates rate caused by falling temperatures in autumn, the reduc- occurred at >24°C and the highest rate was 0.59 dÐ1 in tion of the field populations results from vertical and hori- the following treatment combination: 27, 24°C and 600 zontal diffusion induced by vertical water mixing after

42 S. H. Baek et al. breakdown of stratification in the latter part of year. the Chesapeake Bay, USA. However, we found that spe- Nutrient concentrations are often regarded as impor- cific growth rates of C. fusus decreased at salinity 17. In tant in determining bloom scale and period. Relatively addition, cells of both C. furca and C. fusus were irre- high abundances of Ceratium in oligotrophic conditions versibly damaged at salinity below 14. Baek et al. (2006) are closely related to the occurrence of phagotrophy, found that growth rates of C. furca in culture were simi- which compensates for low nutrient levels (Norris, 1969; lar and relatively high at salinities between 17 to 34. Al- Weiler, 1980). In this study, high abundances of C. fusus though there are some variations between the studies, the were usually observed during months when nutrient con- results indicate that high growth rates of C. fusus are centrations were low. In addition, there were significant stimulated by relatively low salinities (24Ð30), as com- negative correlations between the densities of the spe- monly encountered in coastal waters. cies and nutrient concentrations (nitrate + nitrite-N and Most dinoflagellates dominating coastal waters pro- phosphate-P; Table 1). We successfully isolated the spe- duce two different types of non-motile cells, i.e., a tem- cies from natural assemblages with T1 medium, which porary cyst and/or a resting cyst, in their life cycle. The has a fairly low nutrient level (nitrate ≤1.0 µM and phos- resting cysts in particular have an important ecological phate ≤0.1 µM). The medium concentration was similar role as a seed for recurrent blooms. However, there are to the seawater level during the bloom period of the spe- no reports of resting cyst formations by C. fusus. The cies. According to Dortch and Whitedge (1992) and Justic mechanism by which vegetative cells recruit from the et al. (1995), growth of phytoplankton species may be initial stage before the bloom has not been well under- considered limited when concentrations of dissolved in- stood. We found that C. fusus cells adapt to a wide-range organic nitrogen (DIN; nitrate, nitrite and ammonium) and of variations in water temperature, salinity, irradiance and phosphate are <1.0 and 0.2 µM, respectively. However, reduced nutrient concentrations. Observations on seasonal because Ceratium species are motile, their relatively high variations in abundance suggest that C. fusus is able to division rates might also reflect an ability to change ver- sustain a population in the water column throughout the tical positions to find an optimal depth for nutrient avail- year. Small numbers of cells in the water column, par- ability; this would be an advantage over non-motile forms. ticularly during the winter, may play an important sur- Downward nocturnal migration to nutrient-rich water lay- vival role, allowing the population to sustain adequate ers allows uptake of nitrogen (Cullen and Horrigan, 1981) levels (without cyst formation) to initiate the next bloom. and phosphorus (Watanabe et al., 1988), giving an ad- Ceratium species (C. furca, C. fusus and C. tripos) vantage to photosynthetic cells that subsequently migrate have been considered primarily photosynthetic, but, food upward to the surface layer during the day (Eppley et al., vacuoles were observed by Bockstahler and Coats (1993) 1968; Heaney and Eppley, 1981). We found that nutrient and Li et al. (1996). Smalley and Coats (2002), and concentrations in the deeper water layers in the field were Mouritsen and Richardson (2003) noted that distributions comparatively high, even when values at the surface were of these potentially mixotrophic Ceratium species (C. low (nitrate + nitrite-N: <0.5 µM, phosphate-P: <0.1 µM) furca, C. fusus and C. tripos) are strongly influenced by in early summer. the vertical and horizontal distribution of ciliate prey in a Because of difficulties in isolation and culture from stratified estuary. Smalley et al. (2003) also found that natural seawater, there is limited information on the the feeding of C. furca in the culture occurred when cells growth rate of C. fusus. Our laboratory experiments pro- had been growing under N- or P-depleted conditions, mote a better understanding of the physiology and life while nutrient-replete cells did not ingest prey. There is a strategies of the species. The results of growth measure- need for research into the detail of mixotrophy in C. fusus. ments at various temperature and irradiance combinations In conclusion, C. fusus can survive through indicate an ability to tolerate a wide range of temperature unfavorable environment changes and small surviving (16 to 28°C), with highest growth rates recorded at 24 populations may play an important role in seeding the and 28°C (Fig. 7). There is clearly growth stimulation at next bloom (C. fusus does not have cyst stages). Our re- the temperatures encountered in temperate or tropical sults also indicate that the species probably requires strati- ocean water, and vegetative cells have an obvious ability fication in the water column for remarkable population to overwinter when water temperature is >12°C. growth subsequent to nutrient addition by natural rain- C. fusus in culture was able to tolerate a wide range fall inputs during the rainy season from late spring to sum- of salinity (17 to 34), with higher growth rates at 24, 27 mer. and 30 (Figs. 7b to d). Nordli (1953) reported rapid growth of this species in the field at temperatures over 24°C and Acknowledgements salinities from 20 to 25. In contrast, Smalley and Coats We are grateful to Profs. S. Taguchi and T. Toda, and (2002) reported that C. furca appeared to be restricted to Dr. A. Shibata of Soka University for their invaluable low salinities of >10, and was most abundant at ca. 14 in discussion on this study and permission to use instruments

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