(Dinophyceae) from the Mediterranean Sea

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

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

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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.

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

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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


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

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 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


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.

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. Downloaded from plankt.oxfordjournals.org by guest on July 17, 2011

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.

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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

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


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.

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

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).

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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).

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

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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

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

References Anderson,D.M. and Wall,D. (1978) Potential importance of benthic cysts of Conyaulax lamarensis and G.excavata in initiating toxic dinoflagellates blooms. / PhycoL, 14,224-234. Antia,NJ. and ChengJ.Y. (1970) The survival of axenic cultures of marine planktonic algae from prolonged exposure to darkness at 20 °C. Phycologia, 9,179-184. Antia,N.J., Berland.RR., BoninJ3J. and Maestrini,S.Y. (1975) Comparative evolution of certain inorganic sources of nitrogen for phototrophic growth of marine microalgae. / Mar. Biol. Assoc. UK, 55, 515-539. Beker3- (1986) Communautes phytoplanctoniques en milieu cotier a salinity variable (Etang de Berre, golfe de Fos). Th. Dip. Res. Univ., Univ. Aix-Marseille II, 112 pp. Belin.C, Beliaeff3-, Raffing, Rabia>l. and IbanezJ'. (1995) Phytoplankton time-series data of the French phytoplankton monitoring network: toxic and dominant species. In Lassus,P, Arzul.G., Erard-Le Denn,E., Gentien,P. and Marcaillou-Le Baut,C (eds), Harmful Marine Algal Blooms. Lavoisier, Paris, pp. 771-776. Berland3- and GrzebykJ). (1991) Prorocenlrum minimum. In Soumia^A., Belin.C, Berland,B., Erard-Le Denn,E.,Gentien,P.,Grzebyk,D., Marcaillou-Le Baut,C, Lassus,P. and Partensky,F. (eds), Le phytoplancton nuisible des cdtcs de France. De la biologie d la prtvention. Programme National 'Efflorescences algales marines', CNRS-IFREMER, pp. 101-113. Blanc,F. and Leveau,M (1973) Plancton et eutrophie: aire d'dpandage rhodanienne et golfe de Fos (traitement mathematique des donnees). Th. Dr Etat, Univ. Aix-Marseille II, 681 pp. Brand,L.E., Murphy.L-S., Guillard,R.R.L. and Lee,H.T. (1981) Genetic variability and differentiation in the temperature niche component of the diatom Thalassiosira pseudonana. Mar. Biol,62,103-110. Cervetto,G, Gaudy,R., Pagano,M., Saint-Jean,L., Verriopoulos,G., Arfi,R. and Leveaujtf. (1993) Diel variations in Acartia tonsa feeding, respiration and egg production in a Mediterranean coastal lagoon. J. Plankton Res., IS, 1207-1228. ChangJI.F. (1995) The first records of Gymnodinium sp. nov. (c£ breve) (Dinophyceae) and other harmful phytoplankton species in the early 1993 blooms in New Zealand. In Lassus^P, Arzul.G., Erard-Le Denn.E., Gentien.P. and Marcaillou-Le Baut,C (eds), Harmful Marine Algal Blooms. Lavoisier, Paris, pp. 27-32. CoatsJ5.W. and Harding,L.W. Jr (1988) Effect of light history on the ultrastructure and physiology of Prorocentrum mariae-lebouriae (Dinophyceae). / PhycoL, 24,67-77. Dale,B. (1977) Cysts of the toxic red-tide dinoflagellate Gonyaulax excavate (Braarud) Balech from Oslofjorden, Norway. Sarsia, 63,29-34. Faust>i.A. (1990) Cysts of Prorocentrum marinum (Dinophyceae) in floating detritus at Twin Cays, Belize mangrove habitats. In Graneli,E-, Sundstr0m3-, EdlerJ- and Anderson,D.M. (eds), Toxic Marine Phytoplankton. Elsevier, New York, pp. 138-143.

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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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Faust.M.A. (1993) Sexuality in a toxic dinoflagellate, Prorocentrum lima. In Smayda,TJ. and Shimizu.Y. (eds), Toxic Phyloplanklon Blooms in the Sea. Elsevier, Amsterdam, pp. 121-126. Folack J. (1986) Variations mensuelles de la biomasse et de la production du phytoplancton d'une zone cotiere d'int^rfit aquicole: Anse de Carteau, Golfe de Fos. Th. 3eme Cycle, University Aix-Marseille II, 167 pp. Furnas,MJ. (1982a) An evaluation of two diffusion culture techniques for estimating phytoplankton growth rates in situ. Mar. Biol.,70,63-72. Furnas,MJ. (1982b) Growth rates of summer nannoplankton (