Bull. Fac. Fish. Nagasaki Univ., No. 71 (1992) 163
Effects of salinity, food level and temperature on the population growth of Noctiluca scintillans (Macartney).
Jung Keun LEE* and Kazutsugu HIRAYAMA
The population growth of Noctiluca scintillans was investigated, using Tetraselmis tetrathelle as food, at salinities ranging from 8.5-34‰, food levels from 1 x 10³-8 × 105 cells/ml and temperatues from 5-32°C. Under every experimental condition, ten to twenty Noctiluca were grown individually in 1 or 3m1 of food suspensions for three to four days. The specific growth rates were obtained from the linear regression analysis of the population growth.
Salinity : The specific growth rate was maximal at approximately 22‰, and decreased differentially with increasing or decreasing salinities. The lowest salinity for the minimal and continual growth was 14%o, but the sudden drop from 34‰ to 14‰ was lethal.
Food Level : There was little growth at the food levels lower than 3 × 104 cells/
ml. At food levels between 3x 104 and 3x 105 cells/ml, the specific growth rate increased proportionally with increasing food levels. With further increases in food level, the rate increased asymptotically. The values of the specific growth rates were 0.03 (S.
D ; 0.16) at 3 × 104 cells/ml, 0.74 (S. D ; 0.16) at 3 x 105 cells/ml and 0.81 (S. D ; 0.12) at 8 × 105 cells/ml.
Temperature : The specific growth rate was maximal around 23°C, and decreased differentially with increasing or decreasing temperatures. All Noctiluca died at 32
°C within a day. It grew at 5°C normally but slowly.
Key words : specific growth rate ; Noctiluca scintillans; Tetraselmis tetrathelle;
A marine protist Noctiluca scintillans is widely distributed from the tropics' to the arctic.' It is one of the dominant plankton in the temperate and tropical seas, and the red tides caused by its high density have been frequently reported from many parts of the world. In Japan, the Noctiluca red tide occurs almost annually between spring and autumn depending on the year and site." In spite of its frequency of occurrance, the relationship between its growth and the basic environmental factors are not well known.
In vitro experiments are necessary to elucidate this information, and a stable culture of Noctiluca is a prerequsite. Noctiluca cultures
have been conducted both in an organically enriched medium' and in the phytoflagellate food suspension.' 6) We found in the prelimi- nary experiments that Tetraselmis tetrathelle has a high food value for Noctiluca, and could establish a stable Noctiluca culture using this food organism.
In this study, we grew Noctiluca individual- ly in 1 or 3m1 of food suspensions under various experimental conditions and clarified the re- lationship between its population growth and the basic environmental factors.
* Graduate School of Marine Science and Engineering.
Materials and Methods
Al∂ctilzaca was collected with a plankton net
from Nomozaki Bay in Nagasaki Prefecture,
southern Japan. It was maintained in culture on a diet of T. tetrathe〃e in about 100rnl of GF/C filtered seaWater in petri−dishes. The
む ロ
culture was carrled out at 22−23 C ln fluorescent
,
light((2−4)×1031ux)on 14L:10D light cycle.
To deter面ne the s.pecific growth rate under each experimental condition, ten to twenty
〈ioctiluca were grown individually in l or 3ml of conditioned food suspensions for three to four days. This corresponded to ten to twenty cases. The experimental vessels used were Falcon multiplates(#3046 for 3ml experiment,
#3047for lml experiment), which were com−
pletely sealed with vinyl tape to prevent evaporation. Linear regression analysis was performed for each case using daily sensus counts, from which the following equation was deduced:
1n Nt=kt十a
where Nt is the number of 1>Octiluca at time t,
kis the specific growth rate, t is the time passed in days and a is constant. When gametocytes7)
were formed(0−3 cases out of ten), the cases were excluded from the analysis. As many specific growth rates as the valid cases were obtained and the mean k with standard
,
deviation, represented the specific growth rate under each experirnental condition.
1.Salinity
a.Specific growth rates
Noctiluca cultures were grown at four salinities(17,22,27 and 34%・)for more than a week. T. tetrathelle were grown in six salinities
(10,14,17,22,27,and 34%・), each in 100ml of Erd−Schreiber medium in a 200ml flask. The salinity of‡iltered sea water was measured with a digital salinometer(Tsurumi seiki, E−202), and the desired salinities were obtained by diluting filtered sea water with distilled water. The
experiments were run at 23 C on 14L:10D light
cycle. Sixty Noctiluca from each acclimated culture were tested in six salinities. ten Noctiluca
in each salinity. Only those with active tentacle movement were used. They were placed indi−
vidually in 3ml of food suspensions of various salinities, and their growth was monitored for three days. The food suspension was pre−
pared by adding lml of food culture (about 3×
105 cells/ml) to 2ml of filtered sea water of the same salinity, and its resultant concentration was about 1×105 cells/ml.
b. lnfluence of salinity on the stoutness of Noctiluca
Noctiluca were separatey cultured at 23, 28 and 320/oo for three weeks with sufficient food
and those with food vacuoles filled with algal food were used in the experiment. Thirty IVoctiluca from each cultute were transferred into the wells of Falcon # rr!ultiplate, five in each well containing 3ml of filtered sea water.
The multiplates were placed on an electric shaker which reciprocated a distance of 1.5cm at a rate of 155 times/min. Shaking was continued for periods of O, 2, 4, 8, 14, and 24 hours, and surviving Noctiluca were counted 24 hours after the start of shaking.
2. Food level
The algal food used for the experiments,
Ti tetrathelle, was grown in diluted filtered sea water (220/oo) enriched with Erd−Schreiber medium
(1 : 1, volumetrically) to a concentration of 3×
105 cells/ml. Various levels of food suspension were obtained by diluting the food culture with the diluted filtered sea water. Plankton cell countings were done on the food culture using a haemacytometer and the concentration was determined as the average of ten cell counts.
Noctiluca were cultured with sufficient food. and
those were used whose food−vacuoles were filled up with algal food. This experiment was run at 230C, 220/oo and in dim light. The specific growth rate at each food level was obtained by growing ten IVoctiluca individually in lml of food suspensions for four days.
Bull. Fac. Fish. Nagasaki Univ., No. 71 (1992) 165
3。Temperature
Noctiluca were first acclimated at six tem−
peratures for one month, ranging from 5 to 280C, in a multi−chamber incubator accurate
to±0.5 C. Food was given sufficiently. The algal food was also grown at the same tern−
peratures in diluted filtered sea water(22%・)
enriched with Erd−Schreiber medium (1:1,
volumetrically)・一Those〈⑩漉1%6αwere chosen..
whose food vacuoles were filled up with algal food, and tested at each acclimated temperature and.at 32。C. Those tested at 32℃had been
りacclimated at 28 C..The initial food levels were arranged at about 3×105 cells/ml. This ex−
periment was conducted at 22%・on 12L:12D light cycle. The specific growth rate at each temperature was obtained by growing twenty
〈roctiluca individually for three days in lml of food suspensions.
Results
but growth curve diverged by food level from the second day onwards. Thus, the data from the second day on were used in the analysis.
2 0
0 嗣℃︵z鑑︐召︶ ︒2 0 一
︵工︶田トくにエトタOαO 4 2 0 00一比一り国∩﹇の
(17 Oloo)
㏄ α o o2
o
(22 o/.)
/1太
O・6
O・4
O・2
o
一〇.2
一〇4
(27 Ol.)
ヨ
逓一 /
04
O・2
o
(34 01.)
︐
/i*i
Fig. 1 shows the specific growth rates of Noctiluca at various salinities. Regardless of the acclimated salinities. Nocliluca demonstrat一
ed the highest specific growth rates at 220/oo,
and the rates decreased differentially with increasing or decreasing salinities. The lowest salinity for the continual population growth was ユ4%・,but the sudden drop from 34%・to 14%・was lethal. ln lower than 100/oo S, Noctiluca was not viable.
Fig. 2 shows the survived number after various lengths of shaking of three groups of
・Noctiluca which were acclimated at 22. 28 and
350/oo S, respectively. As the duration of shaking increased, the number of surviving Noctiluca decreased in all the groups and there were no tendencies to indicate any differences among the groups in tolerance to the shaking shock.
Fig. 3 is an example of the time course of the growth of Noctiluca, which were fully fed and exposed to various food levels. The high growths were obtained in the first day of the experiment regardless of the initial food levels,
de wh.Lu−一LLLLLL.L 10 20 30 10 20 30
SAUNITY (e/eo)
Fig. 1. The specific growth rates of N scintillans grown at various salinities. ln the paren−
theses are shown the salinities they were acclimated at before being transferred into various salinities. Vertical lines indicate standard deviations. The open circle on the x−axis signifies that all Noctiluca died at that salinity.(N;the number of八り6−
tiluca:t; time in days: k; the specific growth rate)
︵0仁コσ£oも
」ミ醐い0一﹂Φζσ αUロロΣ⊃Z ゴ﹈り ω﹂N︶
60
40
20
o
Fig. 2.
Acclimated satinity 一 : 22 ont 皿コ : 28脇
〔==コ:35eA・ 2 二2 vl .g di = Dい E 一,一 〇
一一一一一一一一一一一一一一一一一一一一一一一一一一一 黶j
c
幽凶
」_一騨旧_; 」r_剛r騨」 一 」___■」 一
〇 2 4 8 14 DURATION OF SHAKING ( hour)
一
24
一﹂σ緕ゴOQ
The survival number of N. scintillans after various Iengths of shaking.
200
100
0 5
に国口Σ⊃Z﹂﹂Uり
20
10
23e c
︵
22『陶S ●(30ゆ32)
e/
e/ ./e(lo.11)
/e
//e
;≦§メ…≡i羅7)
0 α8
︵Z潔11
Q6@ 鱗
論︶・︵5山・く匡
2 0
エト≧6配OQ一﹂﹇O国巳の
0
SaEinity:22 Oke
so+Z9
i27・su
l一振
娼 ↓ 30十 う 30一
乏
(丁)
Fig. 5.
o
Fig. 3.
LO
︵Zメー− 00 60 0論︶杢︶ω↑くエ↑≧O¢OO一﹂一〇国住の O・4
O・2
o
一〇一2
十
1 2 3 4
DAYS AFTER INOCULAT!ON
The growth curves of N. scintillans which
were exposed to various food levels immediately after being fully fed. The numbers in the parentheses indicate the
food levels (cells ×10 /ml) at the start and end of the experiment. For the food levels lower than 1×10 cells/ml, only the initial food levels are given.
︵23ec
22 9ke S
十 エー ー ー
十1 10 100
FOOD LEVEL (cettsxlo4mt−t)
The specific growth rates of IV. scintillans grown at various food levels. The ex−
planation of marks is given in Fig. 1.
5 10 15 20 25 so
TEMPERATURE (eC)
The specific growth rates of N scintillans grown at various temperatures. The num−
bers next to the vertical lines represent the food levels (cells ×10 /ml) at the start and
end of the experiment. The explanation of marks is given in Fig. 1.
104and 3×105 cells/m1, the specific growth rate increased gradually with increasing food levels,
but it increased asymptotically with further increase in food level. The specific growth rateS
(k)were O.03(S. D;0.16)at 3×104 cells/ml,0.74
(S.D;0.16)at 3×105 cells/ml and O.81(S. D;0.12)
at 8×105 cells/ml.
Fig.5shows the specific growth rates of
〈loctil勿ca at various temperatures. The rate was
む
highest around 23 C, and decreased differential−
ly with increasing or decreasing temperatures.
む八「octiluca grew at 5 C, but all died within a day
at、31−32 C. The food levels during the ex−
periment were between about 3×105 cells/ml and about 5×105 cells/ml.
Fig. 4.
The growths of algal food during the experi−
ment under these experimental conditions were less than 200/o of the initial food levels.
Fig. 4 shows the relationship between the specific growth rate of Noctiluca and food level.
There was little ,growth of Noctiluca at the food level lower than 3×104 cells/ml. Between 3×
Discussion
The results in Fig. 1 demonstrate that Noctilzaca is adaptable to wide range of salinities when the salinity change is slow and gradual,
and that the most suitable salinity for growth of IVoctiluca is around 220/oo. The growth of algal food during the salinity experiment is considered not to affect the results obtained because T. tetrathelle is euryhaline and grows at a similar rate in salinities ranging from 10 to 340/oo.8 The salinity at which the highest
Bull. Fac. Fish. Nagasaki Univ., No.71 (1992) 167
growth rate is found is not necessarily the optimal salinity for population increase. The physiological stoutness (i. e., tolerance to the
adverse environment)must be considered as well. The results in Fig.2show that〈loctilu ca grown at 22%・for three weeks is as phys−
iologically stout as those grown at higher salini−
ties. From these results, it can be concluded that approximately 22%・is the optimal salinity for ノ>bctiluCa.
Because the results of the salinity ex−
periment showed that the optimal salinity for ノ>Octiluca is 22%・, the food level experiment was carried out at 22%・, The results in Fig.3show that the growth of algal food during the ex−
periment was suppressed enough under these experirnental conditions not to influence the results obtained. The relationship between the specific growth rate of Noctiluca and food level drew a typical S−shaped growth curve(Fig.4).
The maximal rate shows that, with enough food and a suitable environment 1>Octiluca can divide ,
more than twice a day. However, the fact that 〈loctiluca started to grow around 3×104 cells/で ml indicates that 1>Octiluca in nature may be in serious food deficient state as far as living food is concerned. This food deficient state seem to be rectified by phagotrophy9)or possibly by using dissolved organ!c matter4), but the extent of rectification has yet to be studied.
The results in Fig.5show thatハloctiluca is eurythermal and the optimal temperature is
approximately 23 C. Based on the results of food level experiments, this experiment was done at the high food levels where the growth rate of Noctiluca increases asymptotically in order that the varying food levels during the experiment would not affect the results ob一
tained. Noctiluca grows normally at 5 C to see the fact that they contained as much food in their food vacuoles as those at higher temperatures. In the temperate sea,〈loctiluca suddenly disappears in the summer season.10)
From the fact that all〈loctiluca died at 32。C,
the lethal effect of high temperature seems to
be one of the causes of the disappearance.
However, a tropical strain of green八ioctiluca,
which contains flagellated symbionts, grows well around 300C.i) This indicates that the tolerance to high temperature may be different by strain.
Though八ioctiluca has a potential to grow at a speed of more than one division a day, it is highly improbable that such a high growth rate will be found in nature because the most favorable environments (i. e., 220/oo S, 220C and plenty of food) are hardly met at the same time.
The high salinity will always suppress the full growth of Noctiluca, and it would be a rare case for them to meet even the threshold level of living food, even during the spring time when the phytoplankton flourish. Occasionally, the food organisms appear in high density, but this state doesn t last long. ln this respect, the
bloom of Noctiluca seems to be a visual phenomenon which is not necessarily related to the sudden growth of it.
With respect to the maintenance and increase of the Noctiluca population, the rela−
tive importance of its tolerance to adverse environments and its ability to use non−living organic matter as food source have yet to be studied.
References
1) Sweeney, B. M. (1971):Laboratory studies of a green Noctiuca from New Guinea. J.
phycol., 7, 53−58.
2) Tibbs, J. F. (1967): On some planktonic protozoa taken from the track of drift station ARLIS 1. 1960−1961. Arctic, 20, 247−
254.
3) Takayama, H. (1984): Red tide organisms occurring in coastal waters of Hiroshima prefecture−II: IVoctiluca scintillans (Macart−
ney). Bull. Hiroshima Fish. Res. Lab., 14,
25−29.
4) McGinn, M. P. (1962) : Axenic cultivation of Noctiluca scintillans. J. Protozool., 16
(suppl.), 13.
5) Nawata, T., and T. Sibaoka (1976) : Ionic composition and pH of the vacuolar sap in
marine dinoflagellate Noctiluca. Plant Cell
Physiol. 17, 265-272.
6) Takayama, H. (1977) : Culture of Noctiluca scintillans (Macartney).* Bull. Plankton Soc.
Jap., 24 (2), 159-162.
7) Zingmark, R. G. (1970) : Sexual reproduction
in the dinoflagellate Noctiluca miliaris suriray. J. Phycol. 6, 122-126.
8) Okauchi, M. N. (1988) : The mass culture and food value of Tetraselmis tetrathelle.**
Saibai Giken, 14 (2), 85-110.
9) Kimor, B. (1981) : The role of phagotrophic
dinoflagellates in marine ecosystems. 15.
European marine biology symposium. Kiel (GRG) : lower organisms and their role in the food web. Rheinheimer, G. ; Fluegel, H. Lenz, Z. Zeitschel, B. (eds.). Institut
fuer Meereskunde, Kiel (FRG). 164-173.
10) Uhlig, G. and G. Sahling (1982) : Rhythms and distributional phenomena in Noctiluca
miliaris. Ann. Inst. oceanogr., Paris, 58 (S), 277-284.
夜光虫 (Notiluca scintillans) の 増 殖 と塩 分, 餌 料 密 度,温 度 との 関 係
李 正 根 ・平 山 和 次
Tetraselmistetrathelleを 餌 料 と し て,10‑20尾 の 夜 光 虫 を1尾 ず つ 個 別 に1‑3m1の 餌 料 懸 濁 液 に 入 れ,い ろ い ろ の 飼 育 条 件 で 飼 育 し,そ の 増 殖 を 調 べ た 。3‑4日 間 の 増 殖 経 過 か ら 日 間
比 増 殖 率 を 算 出 し,そ の 平 均 値 を 求 め た 。
(8.5-34‰) : 22‰で 最 大 の比 増 殖 率 が得 られ,そ れ 以 上,そ れ 以 下 の塩 分 で は塩 分 の増 加 あ る い は減 少 と と もに比 増殖 率 は減 少 した 。 以上の塩分 では安定的な増殖がみ られた
が, 34‰ の海水か ら直接14‰ の海 水 に移 す と死 滅 した 。
餌 料 密 度(1×10ウ‑8×105ce11s/ml):餌 料 密 度 が3×104cells/ml以 下 で は,夜 光 虫 は ほ と ん ど 増 殖 し な か っ た 。3×IO4cells/mlか ら3×105cells/mlの 間 で は 餌 料 密 度 の 増 加 と と も に 比 増 殖 率 も増 加 し た が,そ れ 以 上 の 餌 料 密 度 で は 比 増 殖 率 の 増 加 の 割 合 は 低 下 し た 。 比 増
殖率の値 (22‰, 23C) は3×104cells/mlで0.03/日(S.D.:0.16),3×105cells/mlで0.74 (S.D;0.16),8×105cells/mlで0.81/日(S.D.;0.12)で あ っ た 。
温 度(5‑32℃):最 大 比 増 殖 率 は 約23℃ で え られ,そ れ 以 上,ま た は そ れ 以 下 の 温 度 で は 減 少 し た 。32℃ の 高 温 で は す べ て の 夜 光 虫 は1日 以 内 に 死 滅 し た が,5℃ の 低 温 で も正 常 に 増 殖
し た 。
* : in Japanese with English summary
** : in Japanese 塩 分