Journal of Plankton Research Vol.19 no.8 pp.1167-1174, 1997
Effects of temperature and salinity on the growth of the red tide
flagellates Heterocapsa circularisquama (Dinophyceae) and
Chattonella verruculosa (Raphidophyceae)
Mineo Yamaguchi, Shigeru Itakura, Keizo Nagasaki, Yukihiko Matsuyama,
Takuji Uchida and Ichiro Imai1
Nansei National Fisheries Research Institute, Ohno, Saeki, Hiroshima 739-04 and
'Faculty of Agriculture, Kyoto University, Sakyo, Kyoto 606-01, Japan
Abstract Growth responses of the red tide flagellates, Heterocapsa circularisquama (Dinophyceae)
and Chattonella verruculosa (Raphidophyceae), were examined with 36 different combinations of
temperature (5-3O°C) and salinity (10-35 PSU). Heterocapsa circularisquama did not grow at or
below a temperature of 10°C. The maximum growth rate of H.circularisquama (13 divisions day 1 )
was obtained with a combination of 30°C and 30 PSU. In contrast, C. verruculosa did not grow at 10
PSU and at temperatures of 25°C or more. The maximum growth rate of C. verruculosa (1.74 divisions
day 1 ) was obtained with a combination of 15°C and 25 PSU. A significant temperature-salinity interaction on growth was found by factorial analysis. Based on the physiological characteristics obtained
in the present study, these novel flagellates have a potential for future outbreaks of red tides in previously unaffected waters.
Introduction
Harmful red tides caused by organisms which have not been recorded previously,
'novel red tides', have recently occurred in coastal waters of western Japan. One
of the most harmful organisms is Heterocapsa circularisquama Horiguchi, Dinophyceae (Horiguchi, 1995), which has caused severe damage to both natural and
cultured bivalves such as pearl oyster, short-necked clam and oyster (Matsuyama
et aL, 1995, 1996; Nagai et aL, 1996). In addition, the organism suppresses the
growth of other phytoplankton by cell contact (Uchida et aL, 1995). The other
organism is Chattonella verruculosa Y. Hara et Chihara, Raphidophyceae (Hara
et aL, 1994), blooms of which have been associated with the mortality of fish such
as yellowtail, amberjack, red and black sea breams in early summer and winter
(Yamamoto and Tanaka, 1990; Baba etaL, 1995). These novel red tide organisms
have been identified since the late 1980s. Since then they have taken the place of
red tide species such as Chattonella antiqua, C.marina and Heterosigma akashiwo,
and in recent years have become the predominant causative organism of harmful
red tides. Therefore, it is important to clarify the mechanisms by which such novel
organisms initiate red tides. Increasing knowledge on the autoecology of these
organisms is urgently needed to establish the biological background for the prediction of blooms and establishing preventative measures.
In this present study, we examined the effects of temperature and salinity on
the growth of cultures of these organisms to evaluate the relative importance of
these factors in the dynamics of natural populations. Because the growth of red
tide organisms under natural conditions is predominantly determined by the
interaction of physicochemical factors (Yamaguchi and Honjo, 1989; Nielsen and
© Oxford University Press
1167
M.Yamaguchl et aL
T0nseth, 1991; Yamaguchi et aL, 1991), a factorial experiment was carried out
over a broad range of both temperature (5-30°C) and salinity (10-35 PSU).
Method
Organisms and culture conditions
Heterocapsa circularisquama (strain HA-2) and C.verruculosa (strain M) used in
this study were isolated from Ago Bay in 1992 and Hiroshima Bay in 1993. The
former strain is axenic and the latter one is unialgal but not axenic. For comparison with the novel species, an axenic clonal culture of Heterocapsa triquetra, a
congeneric species with H.circularisquama, isolated from Hiroshima Bay was also
used in the growth experiment. The strains were maintained in 50 ml flasks containing 25 ml of modified SWM-3 medium (Itoh and Imai, 1987) at 22°C for the
two Heterocapsa species and at 15°C for C.verruculosa under 150 umol photons
m~2 s"1 of cool-white fluorescent illuminations on a 14:10 h light:dark cycle. As
C.verruculosa requires selenium for growth (Imai et aL, 1996), selenite (Na2SeO3)
was added to the medium at a concentration of 2 nM.
The culture experiment was carried out at six temperatures (5, 10, 15, 20, 25
and 30°C) in combination with six salinities (10,15, 20, 25, 30 and 35 PSU) using
a temperate gradient growth chamber (TG-100-AD, Nippon Medical & Chemical Instrument Co. Ltd). Salinities of «30 PSU were obtained by diluting aged
seawater from Hiroshima Bay with ultradistilled water. A salinity of 35 PSU was
obtained by concentrating the natural seawater in a drying oven at 50°C. Enrichment with modified SWM-3 medium was carried out after the salinity adjustments.
Growth experiment
Pre-conditioning to the experimental conditions through stepwise transfer of
stock cultures to each temperature and salinity regime was carried out. If transferred cells grew at the experimental regime, then the culture was conditioned at
that regime for at least 1 month; if not, the growth experiment was not carried
out and the growth rate at that temperature and salinity regime was regarded as
zero. Acclimated stock cultures were inoculated into triplicate PP-capped test
tubes (13 X 150 mm) for each experimental regime. Inoculum size was adjusted
giving -1/100 v/v of pre-cultures.
Growth rates were determined by measuring the in vivo chlorophyll a fluorescence using a Turner Designs Model 10-100 R fluorometer (Brand et aL, 1981).
Growth rates (u; divisions day 1 ) were calculated using data from the exponential portion of the growth curve by least squares regressions of the natural
logarithm of fluorescence on day number (Yamaguchi and Honjo, 1989). The
mean growth rate was calculated using the three independent estimates of u. The
fluorescence decline from the initial value at extreme temperatures and salinities
was not quantified, and was considered to represent a zero growth rate. Analysis
of variance (ANOVA) was used to test the effects of temperature and salinity on
1168
Growth responses of red tide flagellates
the growth rates. On the basis of the ANOVA results, cubic polynomial equations
for the growth rate were constructed.
Results and discussion
Response surface contours of the growth rate of H.circularisquama for the temperature and salinity combinations are given in Figure 1. The organism did not
grow at ^10°C at all salinities. At temperatures of £20°C, H.circularisquama grew
faster at the higher temperatures and salinities. The maximum growth rate of 1.3
divisions day 1 was obtained with the combination of 30°C and 35 PSU, respectively. This result indicates that H.circularisquama prefers high temperatures and
salinities. These results correspond to field observations of the red tides being
associated with high temperatures and salinities. For example, in Fukuoka Bay,
a H.circularisquama red tide occurred at temperatures ranging from 27.7 to
28.0°C and salinities from 32.6 to 32.83 PSU (Yamamoto and Tanaka, 1990). In
Ago Bay, H.circularisquama red tides occurred at 16.9-26.9°C and 30.5-33.4 PSU
in 1992 (Matsuyama etal, 1995), and at 28.1-31.6°C and 33.1-34.1 PSU in 1994
(Matsuyama et ai, 19%). Accordingly, it may be concluded that temperature is
one of the important factors in the initiation of these H.circularisquama red tides,
because the optimum temperature for the growth of H.circularisquama is higher
than the prevailing summer temperatures in these waters.
Iwasaki (1979) summarized the optimum temperature for the growth of red
tide flagellates. Only a few species have an optimum temperature of >30°C. The
Heterocapsa clrcularlsquama
35r
•
30
*
W
20
15
10
5
10
15
20
25
30
Temperature (°C)
Fig. L Response surface contours of the growth rate of H.circularisquama (divisions day-1) as a function of temperature and salinity.
1169
M.Yamagudii el al
present study demonstrated that the maximum growth rate of H.circularisquama
was obtained at 30°C, the highest temperature used in the experiment. Thus,
there is a possibility that the optimum temperature of this organism is higher than
30°C. This physiological characteristic presumably allows them to grow at high
temperatures in summer and predominate over other species. In addition, it can
be inferred that this organism may have originated from habitats of high temperature and salinity, such as subtropical or tropical waters.
Heterocapsa circularisquama red tide was first observed in Uranouchi Bay,
Kochi Prefecture, in 1988 (Yamamoto and Tanaka, 1990). Since then, the organism has spread to the western part of Japan within several years. In the case of
Alexandrium spp., the causative dinoflagellates of paralytic shellfish poisoning,
several explanations have been suggested for the increase in their geographical
range, including the increased abundance of previously unnoticed endemic
species, natural dispersal mechanisms, human-assisted dispersal, or a combination of all the above (Scholin et al., 1995). Considering the above-mentioned
growth characteristics, it is possible that they can survive during the transportation of fishery products such as shellfish, allowing them to seed uncontaminated areas. To prevent their dispersal, freshwater treatment may be a
feasible method because H.circularisquama appears to be intolerant of low
salinities (Figure 1).
For comparison with H.circularisquama, the response of H.triquetra to temperature and salinity is shown in Figure 2. This species grew at temperatures from
5 to 25°C, but not at 30°C. The contour profiles show a gradual reduction of the
Heterocapsa triquetra
' /
30
25
0.8
0-8
0.6
3
20
• H
15
10110
15
20
25
30
Temperature (°C)
Fig. 2. Response surface contours of the growth rate of H.triquetra (divisions day-') as a function of
temperature and salinity.
1170
Growth responses of red tide flagellates
growth rate as the temperature decreases from the optimum, but a more abrupt
decline with temperatures higher than the optimum (Eppley, 1972). In addition,
the tolerable salinity range for growth was very broad over the entire temperature range. The highest growth rate of 0.95 divisions day"1 was observed with the
combination of 15°C and 15 PSU. These findings indicate that H.triquetra is well
adapted to euryhaline conditions. Clearly, the optimum temperature for
H.triquetra is considerably lower than that of H.circularisquama, suggesting that
the physiological characteristics of these congeneric species are quite different.
Figure 3 shows the contour plot of the growth rate of C. verruculosa at the temperature and salinity combinations. This organism did not grow at 10 PSU or at
temperatures of £25°C. The highest growth rate of 1.74 divisions day 1 was
obtained with the combination of 15°C and 25 PSU. Red tides of this species have
occurred at temperatures of 12.3-12.7°C and salinities of 32.56-32.8 in Fukuoka
Bay (Yamamoto and Tanaka, 1990). In Uchinomi Bay, Kagawa Prefecture, a red
tide of Cverruculosa occurred in January 1989 (S.Yoshimatsu, personal communication). Baba et al. (1995) reported that a red tide of Cverruculosa occurred
at a temperature range of 18.8-21.5°C and a salinity range of 32.0-33.09, causing
mortalities of cultured yellowtail in Tokuyama Bay. Therefore, the present results
are in accordance with field observations.
The effects of temperature and salinity on the growth of Cantiqua and
C.marina have previously been examined (Yamaguchi et al., 1991). Comparing
the present results with those obtained for Cantiqua and C.marina, the growth
rate of Cverruculosa is much higher than that of the other two species of
Chattonella verruculosa
35
r
30 - •
25
1
20
15
10L
10
15
20
25
30
Temperature (°C)
Fig. 3. Response surface contours of the growth rate of Cverruculosa (divisions day 1 ) as a function
of temperature and salinity.
1171
M.Yamaguchl et aL
Chattonella, although the optimum temperature of C.verruculosa is comparatively low. In addition, C.antiqua and C.marina did not grow at 10°C, whereas
C.verruculosa showed considerable growth. It is well known that C.antiqua and
C.marina form cysts in their life cycles and overwinter via this form in the bottom
sediments. Those overwintered cysts play an important role in initiating summer
blooms of C.antiqua and C.marina (Imai and Itoh, 1986,1987; Imai et aL, 1991).
However, C.verruculosa presumably does not need to form cysts for overwintering because this species can grow at low temperatures. Conversely, C.verruculosa
may use cysts to endure the high temperatures in summer. Thus, the life cycle
strategy of C.verruculosa might differ from that of C.antiqua and C.marina. It is
of interest to compare the life cycles of species among the genus Chattonella. This
study is now in progress in our laboratory.
Previously, no harmful red tides occurred during winter in Japan except for
Gymnodinium mikimotoi (Terada et al, 1987; Hanai et aL, 1992). However, the
present study demonstrates the physiological capability of C.verruculosa to
bloom in the cold period of the year.
A two-factor ANOVA of the growth rates of H.circularisquama, H.triquetra
and C.verruculosa on temperature and salinity shows significant effects of temperature, salinity and temperature-salinity interaction on their growth (Table I).
For H.circularisquama, all main effects and the two-factor (temperature and
salinity) interaction are significant at the 0.1% level. Fifty-seven and 19% of total
sum of squares are accounted for by the sum of squares for temperature and salinity, respectively. This indicates that temperature is more important than salinity
in contributing to the observed variation in growth rates. For H.triquetra, all main
effects and the two-factor interaction are significant at the 0.1% level, as in
H.circularisquama. Eighty-eight and 2% of total sum of squares are accounted
for by the sum of squares for temperature and salinity, respectively. Thus, temperature is the most important factor contributing to the observed variation in
growth rates. For C.verruculosa, all main effects and the two-factor interaction
Table L Analysis of variance (ANOVA) for the effects of temperature and salinity on the growth rate
of H.circularisquama, H.triquetra and Cverruculosa. Significance level: *•*/> < 0.001
F
Species
Source of variation
d.f.
Sum of squares
Mean square
Heterocapsa
circularisquama
Temperature
Salinity
Interaction
Error
Total
Temperature
Salinity
Interaction
Error
Total
Temperature
Salinity
Interaction
Error
Total
5
5
25
72
107
5
5
25
72
107
5
5
25
72
107
11.319
3.802
4.688
0.176
19.985
9.197
0.159
1.029
0.059
10.445
19.450
13.930
9.901
0.619
43.900
2.264
0.760
0.188
0.002
926.41***
311.23*"
76.74***
1.840
0.032
0.041
0.001
2227.86***
38.58*"
49.83***
3.890
2.786
0396
0.009
452.73***
324.25***
46.09*"
Heterocapsa triquetra
Chattonella
verruculosa
1172
Growth responses of red tide flageDates
are significant at the 0.1% level, and the contribution of main factors is almost
equal (44 and 31 % of total sum of squares are accounted for by the sum of squares
of temperature and salinity, respectively).
On the basis of the ANOVA results, cubic equations of the form:
M = boo + bx0T + bQ\S + bn-TS + b^TS2 + b2vT2S + fr^T3 + feo^3
where u is the growth rate, T is temperature, 5 is salinity and b^ are regression
coefficients, were fitted by the stepwise forward regression method. The multiple
regressions of the growth rate of each species on temperature and salinity
obtained were as follows:
H.circularisquama:
u = -0.25767 + 0.00145- TS - 0.00005- TS2 + 0.00009- T2S - 0.00003- T3
H.triquetra:
u =0.02836 + 0.04218- T + 0.00246- T2 - 0.00013-T3
C.verruculosa:
u = 0.84457 - 0.53171- T + 0.01241-7-5 + 0.03729- T2 - 0.00044-7^-5 - 0.00078-T3
+ 0.05669-5 - 0.00005-53
The regression model fits the data well with adjusted R2 values of 0.88, 0.83 and
0.82 for H.circularisquama, H.triquetra and C.verruculosa, respectively. Based on
these formulae, it is now possible to estimate in situ growth rates of the organisms using temperature and salinity data from field observations (Yamaguchi and
Honjo, 1989; Yamaguchi et al, 1991; Toda et al, 1994).
In conclusion, the present study demonstrated that the novel red tide flagellate
H.circularisquama can tolerate high temperatures and salinities, and C.verruculosa low temperatures. These physiological features are considerably different
from those of the previously prevailing red tide organisms. Based on the physiological characteristics obtained in the present study, there is a possibility for
future outbreaks of those novel red tide organisms in previously unaffected
waters. Consequently, careful monitoring and countermeasures are necessary to
prevent the dispersal of red tide organisms by human activities such as transportation of fishery products and ballast waters. If not, novel red tide organisms
presumably will become 'common' red tide flagellates in temperate waters. In
addition, further study is necessary to examine the effect of other factors (light,
nutrient uptake, competition with other phytoplankton, etc.) which may affect
the success of these organisms.
Acknowledgements
We are grateful to Dr T.Kamiyama who kindly provided us with a unialgal culture
of H.triquetra. Financial support from the Environmental Agency of Japan is
greatly appreciated.
1173
M.YamaguchJ el at
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Received on December 17,1996; accepted on April 15,1997
1174