Journal of Plankton Research Vol.18 no.lOpp.1837-1849, 19%
Influences of temperature, salinity and irradiance on growth of
Prorocentrum minimum (Dinophyceae) from the Mediterranean
Sea
D.Grzebyk and B.Berland
Centre d'Odanologie de Marseille, Uniti CNRS 'Diversity biologique et
fonctionnement des icosystimes matins', Station Marine d'Endoume, Rue de la
Batterie-des-Lions, F-13007 Marseille, France
Abstract. A Mediterranean clone of the red-tide forming dinoflagellate Prorocentrum minimum was
studied in vitro for its capacities to adapt to salinity, temperature and light. This clone is euryhaline and
shows optimal growth between 15 and 35%o. After adaptation, slow growth was observed at salinities
as low as 5%o. An apparatus generating crossed gradients of temperature and light allowed 100 combined experimental conditions to be studied. Variations in lighting between 30 and 500 (Amol photons
m~2 s"1 had little effect on growth, and no photoinhibition occurred. The clone can grow between 8
and 31°C, but is thermophilic with an optimal growth between 18 and 26.5°C. As a result of large variations in temperature from 18°C down to 10°C and maintained at 10°C, small spherical structures (8-10
ujn) were observed; they are described as temporary cysts. These results were compared to those
obtained by different authors, in vitro and in situ, notably in the Mediterranean region.
Introduction
The small lenticular dinoflagellate Prorocentrum minimum has a wide geographical distribution. In the Northern Hemisphere, large blooms have been almost
exclusively observed in temperate or subtropical waters bordering the North
Pacific (Russia, China, Japan, Canada), the east and south coasts (including South
Florida) of the USA, the NE Atlantic, the English Channel, the North Sea, the
Baltic Sea, the Mediterranean Sea and the Black Sea. Reports of P.minimum in
temperate waters of the Southern Hemisphere are recent, along the coasts of
Uruguay and New Zealand (Mendez, 1993; Chang, 1995). Prorocentrum
minimum reports in tropical waters are also rare: on the Pakistan coast (Rabbani
et aL, 1990), in the Equator, attested to by one clone kept in the CCMP collection
(Andersen, personal communication), and Exuviaella baltica on the Angolan
coast (Silva, 1953) was perhaps P.minimum. It appears that P.minimum blooms
generally occur in zones affected by freshwater inputs (large deltas, estuaries,
fjords, lagoons) and/or anthropogenic inputs. The increasingly frequent appearance of exceptional blooms of this species has been documented by Smayda
(1990) and Moncheva et aL (1995).
On the French Mediterranean coast, a bloom of Exuviaella sp., but probably
P.minimum, was first noticed in 1970 in the Gulf of Fos which is influenced by the
Rh6ne River (Blanc and Leveau, 1973). Other P.minimum blooms have since been
reported at the mouth of the Rhdne and in other coastal zones and lagoons,
especially by the French Phytoplankton Monitoring Network (REPHY; Belin et aL,
1995).
Prorocentrum minimum is easy to grow in culture and many in vitro
© Oxford University Press
1837
D.Grzebyk and B.Berland
studies have dealt with this species (Berland and Grzebyk, 1991). To date, no
Mediterranean clone of P.minimum has been systematically studied. We have
therefore undertaken several studies to confirm the existence of toxic clones
(D.Grzebyk et al., submitted) and the ecophysiological requirements of a
Mediterranean clone of P.minimum. Such knowledge is necessary to evaluate the
competitive ability of this species and to predict the occurrence of red tides. In this
paper, we give an account of the results obtained in salinity, temperature and light
experiments, and new observations on the formation of temporary cysts.
Method
The clonal strain of P.minimum PmMrs used in this study belongs to the variety
minimum (Hulburt, 1965) (Figure 1A). It was isolated in the Gulf of Marseille
(northern part of the Occidental Mediterranean) and made axenic with antibiotics.
The basic culture medium used was that of Antia and Cheng (1970) as modified
by Antia et al. (1975) with the omission of silicate. All the experiments were
carried out under continuous illumination.
The salinity of the medium was varied by diluting seawater with distilled
water and the supplying solutions of the basic medium, to obtain a series from 5
to 38%o. Two adaptation series were successively carried out at 22°C and at 250
u,mol photons m~2 s"1. The first series was started with inocula from a basic
medium culture (33%o). The second one was started with inocula from the first
series and was, therefore, already adapted; for 5 and 8%o, the inocula came from
the test carried out at 15%o. A third series of experiments, started with inocula
from the second series, was carried out under two irradiances: 250 and 60 n.mol
m-2s-'.
An experiment was carried out using an apparatus which generates crossed
gradients of light and temperature, allowing 100 combinations. It consists of an
aluminium plate, pierced with holes that hold the culture vials. The temperature
gradient (10-31°C) was obtained by circulating hot and cold water at opposite
edges of the plate. The light gradient (30-500 u.mol photons m"2 s~') was
produced using two compact fluorescent tubes placed end to end at one edge of
the aluminium plate. Light was distributed through a plexiglas plate below the
glass vials TTie weakest irradiances were obtained by sticking neutral filters to
the bottom of the vials. The irradiances were measured with a Licor LI-192SB
meter. The experiment was started with a culture that was exponentially growing
at 18°C in the basic medium, with a cell density of 104 ml"1. Aliquots (10 ml) of
this culture were sterilely distributed in all vials previously conditioned in the
apparatus In this way, the selected temperatures were quickly reached in the
vials
Growth was measured by counting cells in a Neubauer haemocytometer. Each
sample was counted at least four times so that, whenever possible, at least 400 algal
cells were counted per sample, giving an accuracy of ±10% (Lund et al., 1958). In
each culture, growth was studied by regular sampling in order to catch the exponential phase and usually until reaching stationary phase. Maximum growth
1838
2
a
o
3
£
00
Fig. 1. (A) Vegetative cell of P.minimum, axenic clone from the Gulf of Marseille, under the scanning electron microscope. (B-G) Cell transformation after an
abrupt change of temperature in culture from 18 to ICT'C. (B) and (C) Temporary cyst under the light microscope, with a pellicular envelope (arrowheads)
between the cyst and the theca. (D) and (E) Pellicular envelopes, with an apical winged spike (arrowhead on Figure IE). (F) (G) Freed spherical temporary
cysts showing an invagination (black arrows). On Figure IF the wlute arrowhead points out an aperture in a pellicular envelope after the release of cyst. On
Figure 1G the cyst accidentally fell inside a theca during sample preparation.
D.Grzebyk and B.Bertand
rates, &,„„ [divisions (div.) day"1], were calculated according to Guillard's formula
(1973), with samples taken during exponential growth.
For scanning electron microscopy, samples were fixed in 2% glutaraldehyde
and gently filtered, rinsed in water, then dehydrated in successive aqueous ethanol
baths (30,50,70%) and finally 100% acetone. Acetone was replaced by liquid CO2
and filters were then dried at the critical point, stuck to a stud and gold plated
before being observed.
Results
Evidence of temporary cysts
For the light-temperature experiments, all cells that were transferred abruptly
from 18 to 10°C became immobile within 24 h and sedimented to the bottom of
the vials. Light microscopy revealed a cytoplasmic contraction into a small spherical structure (cyst) inside the theca (Figure IB and C). When the encysted cells
were returned to conditions near the optimal temperature (20°C), they recovered
into a normal and mobile state within 24 h. The same changes occurred when the
cells were cooled again to 10°C (encystment) and then re-warmed to 20°C (return
to mobile vegetative form). Some of the cysts formed in this way were kept in the
dark at ambient temperature for 3 months. They were revitalized by putting them
back into the light.
Under a high-magnification light microscope, the cysts appeared as granular
spheres with thin walls. A thin pellicular envelope between the theca and the cyst
itself could be distinguished (Figure 1C).
With the scanning electron microscope, two structures different from normal
cells (measuring 16-18 n,m in length;Figure 1A) were observed. The ovoid pellicular structure (13-15 jim) (Figure ID and E) has a smooth surface covered with
small spines, occasional pores and, at one end, a sort of winged spike. This structure appears to be beneath the theca and to contain the cyst itself, the smaller
spherical structure (8—12 \xm) (Figure IE and G) with a rough surface and an
invagination.
Influences of abiotic factors on growth
Salinity. The first series of experiments ( • , Figure 2A ), carried out at 250 u,mol
m~2 s"1 and 20°C, primarily considered the tolerance of the clone to variation in
salinity (adaptative series). At salinities <10%o (i.e. after an abrupt decrease of
salinity by >22%o), growth did not occur. At both 10 and 12%o, slow growth began
after a lag phase of 5 days. The growth rate and final cell yield were -15-20% of
that obtained under optimum conditions. Between 15 and 38%o, rapid growth
started immediately. The maximum growth rate (1.0 div. day"1) occurred at 33%o
(i.e. the salinity of the basic culture medium).
In the second series, carried out at the same light and temperature conditions,
optimum growth occurred between 15 and 35%o. After the pre-adaptation carried
1840
Factors affecting growth of P.minlmum
200
• adapts tive series
D 25 0 umol nv 2 s~1
•
60 umol rrr 2 s -1
o.o
0
10
20
Salinity
%o
Fig. 2. Influence of salinity on (A) the final cell concentration and (B) the maximum growth rate k^
(during exponential phase) of P.mimmum. The series ( • ) was carried out prior to adaptation to salinity conditions under irradiance of 250 umol photons nr 2 s~'. The other series were carried out after
adaptation to salinity conditions, under irradiances of 60 (•) and 250 umol nr 2 s~' (D).
out during thefirstseries, growth at 10 and 12%o was slightly better, and at 8%o slow
growth also occurred after a lag phase of 2 days.
Salinity-light. In the third series of experiments, different salinity-dependent
growth responses were observed at the two light intensities. At 60 u,mol m~2 s"1
(•, Figure 2), growth occurred between 5 and 38%o, increasing with salinity
between 5 and 17%o, and then remaining stable between 17 and 38%o. At 250 u.mol
m"2 s"1 (•, Figure 2), no growth occurred at 5%o. At other salinities, growth was
higher than in the 60 (xmol m"2 s"1 series. Optimum salinities ranged between 15
and 35%o, with little variation in growth rate
and final cell yield. The highest
fcma* was 1.15 div. day"1 at 17%o.
1841
D.Grzebyk and B.Berland
Light-temperature. Temperature had a predominant effect, affecting both the
final cell yield (Figures 3A and 4A) and the growth rate (Figures 3B and 4B).
Growth occurred between 13 and 31°C at all irradiances, except at 31°C under the
weakest light (46 ji,mol m~2 s"1). The optimum temperature range was wide
(18-26.5°C) in which kmax was 1.13 div. day"1 (26.5°C,475 jimol m~2 s"1)- Above
26.5°C, growth was greatly and rapidly disrupted. In contrast, growth increased
quickly between 13 and 18°C,and was already high in comparison to the optimum.
At 10°C, whatever irradiance, cells became encysted. Nevertheless, this temperature did not prevent growth since other cultures grew slowly after the temperature was gradually lowered to 8°C over 24 h.
Relative to the temperature, the effect of changing irradiance was rather weak.
Indeed, in Figure 3, at each temperature, points representing different irradiances
(30-500 u.mol m~2 s"1) are relatively clustered. No photoinhibition was observed
at the highest irradiances (500 p,mol m~2 s"1) provided by our experimental
apparatus.
500
400 -
s=
300 200 -
IE
TJ
10
15
20
25
30
Temperature
Fig. 3. Influence of temperature (independently of light) on (A) the final cell concentration (at stationary phase) and (B) the maximuni growth rate kmMX (during exponential phase) of P.minimum, in
the 100 combined conditions of irradiance and temperature.
1842
Factors affecting growth of P.minimum
0.5
35
Fig. 4. Influence of light and temperature on (A) thefinalcell concentration (at stationary phase) and
(B) the maximum growth rate k^ (during exponential phase) of P.minimum, in the 100 combined
conditions of irradiance and temperature.
Discussion
Temporary cysts
To date, no cyst of P.minimum giving a flagellate cell had ever been observed.
Extensive incubations of sediments from the Chesapeake Bay (USA), where
regular blooms of P.minimum occur, did not produce vegetative forms (Tyler and
Seliger, 1981). Yamochi and Joh (1986) induced the formation of mobile forms of
phytoflagellates from sediments from Osaka Bay in Japan, but did not detect
P.minimum, Pmicans and P.triestinum. Overall, the formation and germination of
sexual cysts in the genus Prorocentrum is only known in a few species (Faust, 1990,
1993).
1843
D.Gnebyk and B-Bertand
However, Moncheva (1992) noticed the presence of dark brown, flagellaless
spheres of 12 u,m in diameter during a bloom and in old cultures of P.minimum.
She believed these cysts to be planozygotes from sexual reproduction because
they were formed in eutrophic conditions, when the high cell density was
favourable for a nuclear reorganization and restoration of genetic material. Comparison with the structures we observed is difficult because of the weak magnification of the photographs provided by Moncheva. On the other hand, the shape
of the cysts described here is similar to that described for Peridinium hangoei
Schiller during the decline of a spring bloom in the Baltic Sea (Heiskanen, 1993).
These cysts were assumed to be immature resting cysts rather than temporary
cysts.
In our case, the P.minimum cysts were temporary (or pellicular) cysts; the cyst
structure (thin walled), the conditions of encystment (clonal culture, cold stress,
old cultures) and the return to a mobile vegetative state are similar to those of
temporary cysts in other dinoflagellate species (Prakash, 1967; Dale, 1977; Anderson and Wall, 1978). We also found the two types of structures described above in
old cultures of two other clones of P.minimum: one isolated from the Berre
Lagoon (near Marseille) and the other one from the collection at UTEX (University of Texas, Austin, TX). Moreover, in a declining bloom of P.minimum in a
lagoon in the Sete region (French Mediterranean coast), we found pellicular
forms of this species (similar to those in Figure ID and E) and of P.micans (pellicular forms, but without spines).
When conditions allow it, these cysts would be able to re-sow the water column
with vegetative cells. Since they have never been found in sediments, the cysts must
have a limited lifespan, although some remained viable for 3 months in the dark.
Ecophysiological characteristics
The Mediterranean clone of P.minimum studied here is euryhaline, along with
other well-studied clones. However, differences can be observed between clones.
Maximum growth rates in P.mariae-lebouriae (Tyler and Seliger, 1981) and
P.minimum (Trick el al, 1984; Kondo et al, 1990) were in a wide optimum salinity
range (15—30,15—30 and 12-25, respectively), whereas that of the mariae-lebouriae
strain (Trick et al, 1984) was at 15-20%o, the growth at 27%o being >50% slower.
Our Mediterranean minimum clone and the minimum strain (Trick et al., 1984)
appear similar with respect to salinity, but are different in their response to light.
In the two NE Pacific clones (Trick et al., 1984), the maximum growth rate was at
S90 (imol m~2 s"1, whereas that of the Mediterranean clone increased with
irradiance up to 500 junol m~2 s"1 and did not show photoinhibition at this high
irradiance. For the range of irradiances considered here, the growth did not
change a lot, indicating an efficient photoadaptation.
The combination of low salinity and low light reduced growth, in a similar way
to that observed with a combination of low salinity and low temperature (Tyler
and Seliger, 1981). However, the growth occurring at the low salinity limit (5%o)
and low light (60 tunol m~2 s~') (Figure 2) showed the positive interaction in this
combination for increasing the clonal tolerance to a strong salinity decrease.
1844
Factors affecting growth of P.mlnimum
Our Mediterranean clone of P.minimum is also eurythermic even if temperature directly affected its growth rate in a significant manner. With optimum
growth at 18-26°C, this clone is clearly thermophilic, along with P.mariaelebouriae (Tyler and Seliger, 1981) and P.minimum (Kondo et al, 1990), with
respect to the P.minimum studied by Trick et al. (1984) with a maximum growth
rate at 13-23°C.
Maximum growth rate of P.minimum
During our in vitro experiments at various salinities, temperatures and irradiances, the Mediterranean P.minimum var. minimum showed a k^^^ of 1.15 div.
day"1. Other P.minimum (or P.mariae-lebouriae) growth rates found in the literature are globally comparable with those in our clone (Table I). However, one may
wonder whether strains identified as P.minimum may or may not have higher
growth rates than those called P.mariae-lebouriae, such as in Trick etal. (1984). It
would, therefore, be interesting to study whether the physiological differences are
related to morphological differences in the minimum and mariae-lebouriae
varieties. However, it was never indicated whether species names used in these
studies could be referred to the varieties defined by Hulburt (1965).
Table L Summary of P.minimum growth rates in various in vitro and in situ studies
Growth rate
Identification
In vitro
k^ = 1.15 div. day 1 P.minimum
1.36 div. day~'
0.8 div. day 1
039 div. day 1
(in two clones)
0.8 div. day-'
P.minimum
P.minimum
P.minimum
0.6 div. d a y '
P.mariaelebouriae
P.mariaelebouriae"
P.mariaelebouriae'
P.mariaelebouriae"
0.8 div. day"1
0.46 div. day 1
0.36 div. day 1
In situ
0.85 div. day-'
1
0.7 div. day-
2.0 div. day 1
0.72 div. day
1
1.2-2.2 div. day-'
P.minimum
P.mariaelebouriae
P.mariaelebouriae
P.minimum
P.minimum
ExuviaeUa
cordatab
Conditions of work
Reference
Continuous light £250 (imol m~2 s~',
2:20^, 17%o
Continuous light, 23°C, 18%o
LD 14:10,30 (imol rrr2 sr1,18°C,29%o
Continuous light (500 (imol nr 2 s"1),
15°C,33%o
Continuous light >90 (imol rrr2 s"1,
18°C 15-30%o
Continuous light >90 (imol nr 2 s~',
24°C, 15-20%o
LD 12:12 (300 (imol nr 2 s"1),
16-26°C,6-30%o
LD 12:12 (0-300 junol m"2 s-1),
15°C, 15%o
LD 12:12 (258 junolm^s- 1 ),
15°C, 15%«
This work
Diffusion chambers, Cheasapeake
Bay (USA)
Phytoplankton cages, Cheasapeake
Bay (USA)
Diffusion chambers, Narragansett
Bay (USA)
Net rate of increase, 17-19%o,
Baltic Sea
Net rate of increase, >20°C 11-15V
Black Sea
From Kondo et al., 1990
Sakshaug etal., 1984
Johnsen and Sakshaug,
1993
Trick etal., 1984
Trick etal., 1984
Tyler and Seliger, 1981
Harding et al., 1983
Coats and Harding,
1988
Owens et al., 1977
Tyler and Seliger, 1981
Furnas, 1982a,b
Olsson and Edler, 1991
Sukhanova et al., 1988
a Same clone.
b
Probably P.minimum according to Marasovic et al. (1990).
1845
D.Grzefoyk and RBertand
In situ, very high growth rates (2.0 and 2.2 div. day"1) have sometimes been
observed (Furnas, 1982a,b; Sukhanova et al., 1988). Finally, P.minimum shows
rather high growth rates among all dinoflagellates, as can be seen in Tang (1995).
However, these growth rates are clearly lower than those of coastal diatoms such
as Skeletonema costatum (1.5-5.9 div. day"1) and Leptocylindrus danicus (1.2-3.3
div. day"1) (Furnas, 1982a), two ubiquitous species whose blooms often precede
those of P.minimum.
Ecophysiological characteristics and environmental conditions of P.minimum
blooms
All data obtained in vitro show that P.minimum does not have strict requirements
for salinity, light or temperature. This physiological plasticity is supported in situ
by the wide spatiotemporal distribution of this species.
Moreover, it appears that blooms of P.minimum are quite common when
growth conditions are suboptimum. For example, due to its ability to grow at a
non-negligible rate of 0.2-0.3 div. day~' at low salinities, low temperatures and low
light, P.minimum may possess a competitive advantage in cold turbid environments that receive inputs from the land and rivers. In many cases, the temperature
may not be the major factor limiting the development of P.minimum. In Lake
Nakanoumi in Japan, P.minimum red tides generally occur in winter when the cold
desalinated waters (3-12°C, 9-15%o) are not optimum for a clone isolated in these
waters (Table I; Kondo et al, 1990).
Temperature could be implied in succession, but only through interactions with
other environmental factors (Karentz and Smayda, 1984). Among others, a salinity decrease generally resulting in increasing nutrient concentrations could be a
determining factor in the activation of P.minimum blooms. In many cases, blooms
are concomitant with salinity decreases (Olsson and Edler, 1991; Mendez, 1993),
and occur after heavy rainfall such as in Obidos Lagoon in January-February 1983
(13-22%o, 9-15-C) (Silva, 1985).
In Mediterranean waters or areas with a Mediterranean climate as well, temperature does not appear to be the main factor controlling P.minimum blooms,
except during winter when water temperatures can reach 4-5°C in the shallow
coastal zones. Blooms usually occur between April and June, and between
October and November (Silva, 1985; Folack, 1986) when it is often rainy. In the
extremely eutrophic Berre Lagoon, P.minimum is prominent all year round when
salinities vary from 5 to 27%o and temperatures from 4 to 27°C, except in August
and September when the concentration of nitrates is particularly low (Beker,
1986; Cervetto et al., 1993) or because of a very great desalination (only 2-3%o)
during the particularly rainy years 1977-1978 (Kim, 1981). On the other hand, in
the Northern Adriatic Sea, P.minimum blooms only occurred in summer in conditions of warm desalinated stratified waters (Marasovic, 1986).
Finally, peculiar hydrological conditions (sometimes regional specificities) may
prevail in the occurrences of P.minimum blooms. In addition, the diversity in
clonal ecophysiological characteristics shown by in vitro studies may partially
explain the wide spatiotemporal distribution of this dinoflagellate. It would be
1846
Factors affecting growth of P.minimum
interesting to study whether this diversity may be genetically determined, as it has
been shown with ubiquitous diatoms (Gallagher, 1980,1982; Brand et al, 1981;
Gallagher et al, 1984).
In conclusion, this Mediterranean P.minimum exhibits a broad range of tolerance in relation to salinity, light and temperature. The existence of temporary cysts
shown here might be an extra advantage for this species when the environmental
conditions become unfavourable. Since cysts appear to be absent in sediments,
their lifespan might be limited, or the ecophysiological plasticity of this opportunist dinoflagellate might allow it rapidly to re-form the vegetative cells without
the environmental conditions needing to be optimum.
Acknowledgements
This study was funded in France by the 'Programme National Efflorescences
Algales Toxiques'. The authors thank Mrs C.Bezac for the preparations for scanning electron microscopy and the printing of photographs. They also thank the
reviewers for their useful comments.
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Received on May 26,1995; accepted on May 3,1996
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