AQUATIC Vol. 9: 183-189,1995 Published August 31 Aquat microb Ecol 1

Observations on possible life cycle stages of the cf. acuminata, Dinophysis acuta and Dinophysis pa villardi

Brigitte R. ~erland'r*,Serge Y. Maestrini2, Daniel ~rzebyk'

'Centre d'Oceanologie de Marseille (CNRS URA 41), Station marine drEndoume, Chemin de la Batterie des Lions, F-13007Marseille, France 2Centre de Recherche en Ecologie Marine et Aquaculture de L'Houmeau (CNRS-IFREMER), BP 5, F-17137L'Houmeau. France

ABSTRACT: Some aspects of the life-cycle have been investigated in Dinophysis cf. acuminata, the dominant species of the genus along the French Atlantic coast, as well as in D. acuta, a few observa- tions have also been made on the Mediterranean species D. pavillardj. Djnophysls cells occur in 2 clearly distinguished sizes. Small cells typically had a theca thinner than large cells, and cingular and sulcal lists were less developed. Both small and large cells were seen dividing, producing 1 to 4 round intracellular bodies. Some of these round bodies in turn contained many small flagellated cells which escaped through a pore and swam rapidly. Their behaviour after release, and how they might give rise to vegetative cells, has not been observed thus far; we do not believe they are fungal parasites. We pro- pose the following hypothesis to explain our observations: round-shaped bodies, formed inside the veg- etative cells, produce small, motile zoids. These zoids grow and are transformed into apparently vege- tative forms, which later act as gametes. Soon after conjugation, the zygote encysts, sometimes after the first or the second division. This working hypothesis, however, requires further elucidation and confir- mation using different approaches.

KEY WORDS: Dinophysis sp. . Life cycle . Cyst

INTRODUCTION newly developed methods. Identification of different taxa as well as different stages within species is an Although the genus Dinophysis has important aspect of dinoflagellate ecology, since either been known since 1840, when Ehrenberg first de- spatial or temporal species succession may be partly scribed several species, its and ecophysiology triggered by mechanisms maintaining the species' are still poorly understood. capacity to adapt to environmental changes and thus The harm caused by diarrheic toxins which Dinoph- also affect their biological diversity. ysis spp. produce appears to have recently shown a That no Dinophysis species have yet been cultured in considerable increase both in space and time (Ander- the full sense, coupled with their sporadic occurrence in son 1989, Smayda 1990, Belin 1993, Hallegraeff 1993). the sea, has so far seriously limited current knowledge. Furthermore, densities of >105 cells I-', 2 orders of Nonetheless, Reguera et al. (1990) described cells of magnitude higher than those mentioned in the old lit- 2 distinct sizes that Mackenzie (1992) later referred to erature, have recently been reported from different different stages of a sexual life cycle, while Bardouil regions of the world (Freudenthal & Jacobs 1991, Belin et al. (1991) observed formation of cysts which they 1993, Lassus et al. 1993, Subba Rao et al. 1993). Conse- believed to represent a temporary resistant stage. quently, increasing attention is now being focused on Here we report some new forms observed in natural the life cycles, reproductive strategies, nutrition, toxin populations of Dinophysis cf. acuminata and D. acuta, production and taxonomy of Dinophysis spp. using 2 dominant species along the French Atlantic coast, and D, pavillardi, a species occurring in the Mediter- 'E-mail: [email protected] ranean Sea.

O Inter-Research 1995

Berland et al.: Life cycle stages of Dinophysis spp. 185

MATERIAL AND METHODS RESULTS

The port of Antifer, near Le Havre, France, provides Cells of 2 different sizes were observed in Dinoph- exceptionally good conditions for collecting Dinoph- ysis cf. acuminata (Figs. 1 & 2); the smaller cells resem- ysis spp. cells, since high densities, up to 160000 cells bled D, skagii Paulsen. After 1 to 2 wk in the labora- I-',have occurred in summer nearly every year slnce tory, small cells had increased in number; both large 1987 (Lassus et al. 1993). The dominant species is and small cells were seen dividing. Usually small cells similar to D. acuminata (Fig. 12), though it is likely to had a theca thinner than large cells (Fig. ll), and less be a different, undescribed species (Lassus & Bardouil developed cingular and sulcal lists (Figs. 1, 2 & 3). Both 1991). In July and August 1990 and 1992, we harvested large and small cells were also observed in D. pavil- a large number of cells using a protocol for concentra- lardi (Fig. 14). In D. acuta, we also observed a contrac- tion by size fractionation and reverse sedimentation tion of the cytoplasm inside the theca, resulting in for- (Maestrini et al. 1995 -this issue). In 1992, cells of D. mation of a small cell inside a large cell (Fig. 15). pavillardi were collected in the Gulf of Fos, in the One to two wk after sampling, most of the cells, both Mediterranean Sea. In 1993, cells of D. acuta were har- large and small, were seen to have produced 1 or sev- vested in the harbor of Douarnenez, southern Brittany, eral internal spherical bodies of varying sizes (Figs. 2, where a bloom of this species occurred in May. All 4, 5 & 13), up to 4 in Dinophysis acuta. Only a fraction Dinophysis-enriched assemblages were kept in origi- of the cytoplasm contributed to the formation of these nal seawater (10 1 polycarbonate bottles) in subdued bodies; hence, when cells disrupted and the round daylight, without any shaking or turbulence, at 18OC; bodies were released, a part of the cell material was some Dinophysis populations survived up to 3 wk. released into the water. The outer wall of the round Light microscopy. Living and fixed (2 % glutaralde- bodies was smooth. hyde) cells were observed with a Leitz optical micro- Some round bodies were filled with numerous small scope. For scanning electron microscopy, fixed speci- granula (Figs. 6 & 7) showing a colored area. These mens were gently filtered and rinsed with distilled granula appeared to move and escape from the round water and dehydrated through ethyl alcohol (30, 50, 70 and 100%); then the filters were mounted on scan- ning stubs, freeze dried and coated with a gold palladium alloy. Transmission electron micro- scopy. Cells were first fixed in a mixture of glutaraldehyde and caco- dylate (seawater, 5 vols.; glutaralde- hyde 25%, 1 vol.; cacodylate buffer 0.4 M, 4 vols.) at 4OC, for 2 h; then cells were rinsed in a mixture of 0.2 M cacodylate and 0.4 M NaCl for 2 h, followed by 2 rinses with sea- water; cells were finally postfixed with 2% osrnic acid for 1 h, and rinsed twice with seawater and sev- eral times with distilled water. Fixed samples were dehydrated through a graded ethanol series, embedded in 'Durcupan' resin and sectioned; before observation, thin sections were stained with uranyl acetate.

Fig. 11. Dinophysis cf. acruninata.Trans- versal section of a small-size, thin-theca cell, at the flagellar pore level, showing the tongue-like organelle (indicated by a large arrow), and a fraction of 1 large cell 186 Aquat microb Ecol9: 183-189, 1995

Figs 12 to 16. Dinophysis spp. Fig. 12. Vegetative cell of D. cf. acuminata. Fig. 13. Cell of D. cf. acuminata, with 1 cyst inside. Fig. 14. Conjugation of 1 small and 1 large cell of D. pavillardi, strongly attached by lists (vigorous shaking with a micropipette was not able to separate the cells). Fig. 15. Small cells of D. acuta forming in large cells. Fig. 16. Coupled formation of 2 cells of D. acuta, with a bridge shown by the arrow

ULcJ Llll pore (Fig. 6). Then. they were seen as one small cell (Fig. 14). Two cells first come together, gellatec 3pidly swimming, pyriformic cells, 2 pm and used their lists and flagella to wrap around each wide and m long (Figs. 8 & 9). The flagella seemed other (Fig. 14). Later the cells appear to be strongly to be inserted in the anterior part of the cell; they were attached near the sulcal area (Figs. 10 & 16). They not clearly seen, however. Unfortunately, most of these always move together very fast; hence, to enable pic- cells were immediately captured and ingested by cili- tures to be taken, they had to be pipetted and trans- ates, and no further observation has yet been possible. ferred into a few drops of seawater and fixed with glu- With all 3 species, we have observed images which taraldehyde; this treatment did not separate the 2 cells. resembled steps in conjugation, either between cells of We were not able to observe the whole process in a sin- the same size (Figs. 10 & 16) or between one large and gle pair of cells, however. Berland et al.. Life cycle stages of Dinophysis spp. 187

DISCUSSION (Figs. 8 & 9) is reported for the first time. We saw this ephemeral phenomenom 3 times in Dinophysis cf. Several Dinophysis species have been reported to acuminata, always during the same period of the year, show cells of 2 distinct sizes, each of which was previ- late August. These round-shaped bodies resembled ously described as a separate species. Accordingly, the cysts described by Faust (1990, 1993) for different following pairs of former species are now associated: species of Prorocentrum. Similar bodies have been also D. sacculus and D. skagii (Bardouil et al. 1991). D. seen in other , including the toxic acuta and D. dens (Reguera et al. 1990, MacKenzie Nitzscllia pungens (Subba Rao et al. 1991). 1992, Hansen 1993), D. caudata and D. diegensis It has been suggested that zoid-like bodies seen in (Moita & Sampayo 1993), D. fortii and D. lapidistrigill- dinoflagellates could be fungal parasites, such as forrnis (in Bardouil et al. 1991), D. norvegica f. debilor chytrids (Rosowski et al. 1992), and freshwater dinofla- and D. f.crassior (Hansen 1993). gellates of the genera Ceratium and Peridinium have Mackenzie (1992) argued that each size type repre- indeed been reported to be infected by chytridiales sents a different gamete in a sexual life cycle, consis- (Canter 1961, 1968). In contrast, in none of the Dinoph- tent with the anisogamous gamete phenomena ob- ysis cf. acurninata and D. acuta we observed did we served in some dinoflagellates (Pfiester 1984). The see any sporangia or on the thecae (in the conjugation couplets we observed and the existence zoosporic fungi, sporangia are epibiotic rather than of a planozygote with 2 trailing flagella, the result of endobiotic) or any thread-like rhizoidal system inside fusion by 2 cells, seen once by McLachlan (1993) the cell typical of chytrids (Canter & Jaworski 1978). in Dinophysis acuminata, also support the idea that The ultrastructure shown in the micrographs of D. cf. sexual occurs. In contrast, Partensky & acurninata sampled 1 wk before the formation of round Vaulot (1989) argued that the 2 size forms of Gyrnno- bodies did not indicate infestation by fungi or any dinium cf. nagasakiense are not gametes, but reflect a other organisms. growth strategy; the smaller ones, which use photosyn- Many dinoflagellates are known to parasitize marine thetic active radiation best, divide faster when envi- and metazoans (Cachon 1964, Cachon & ronmental conditions are favourable, while the larger Cachon 1967). Fritz & Nass (1992) showed the pres- ones are more resistant under adverse conditions. ence in Dinophysis norvegica of the endoparasite Historically, this 'depauperating' process was first dinoflagellate Amaebophr-ya ceratii, which was previ- described by Silva (1971) in several dinoflagellates; ously known to parasitize Gonyaulax catenella (Taylor without any physiological interpretation, however. 1968). However, all the sections we have observed in Our findings indicate that both the large and the D. cf. acuminata and D. acuta differed markedly from small cells are capable both of vegetative division and the trophont given by A. ceratiiin the host cell (Figs. 13 of forming round bodies; we suggest that these round & 14 in Fritz & Nass 1992).Moreover, the cyst-contain- bodies result from . In other words, ing Dinophysis cells never differed from the vegetative small cells should not be solely considered as gametes; cells, while trophont-hosting A. ceratii cells become they could act both as vegetative and sexual cells, giant (Taylor 1968). In contrast, Dinophysis cf. acumi- according to the life cycle stage. This process would nata zoids somewhat resembled motile zoids of the reflect a high flexibility of the life cycle and ability to non-cyst-forming dinoflagellate Noctiluca miliaris cope with changes in environmental conditions. which were assumed by Zingmarck (1970) to be Cysts formed by Dinophysis spp. were first observed gametes capable of conjugating and of giving a zygote. by Bardouil et al. (1991) who believed they were a tem- To tentatively summarize: due to the lack of serial porary, resistant stage. Then, Moita & Sampayo (1993) observation of the whole process, several hypotheses argued that small cells in D. acuta (D. dens) and D. tri- can be proposed to explain the images we have pos or D. caudata (D. diegensis) were gametes, and observed. Although at present the parasite hypothesis that the resulting zygote was identical to the 'resting cannot be discarded, we prefer the following one. cyst stage'. In their fixed samples of natural popula- Round bodies form inside the vegetative cells, and pro- tion~,the different forms intermediate between a typi- duce small, motile zoids; these zoids could be gametes, cal vegetative cell and a cyst were separated only by except that conjugation of zoids was not observed gradual differences, perhaps suggesting an encyst- while conjugation of vegetative-shaped cells was ment. According to these authors, the encystment clearly seen (Figs. 10, 14 & 16). Hence, it is likely that process seemed to begin with the disappearance of the zoids grow and give apparently normal vegetative cingular and sulcal lists followed by the production of Dinophysis cf. acuminata and D, acuta forms, which an outer membrane. The cysts were oval. later act as gametes. Soon after conjugation, the zygote The formation of intracellular round bodies which encysts, sometimes after the first or the second divi- themselves can produce many small motile cells sion. Aquat microb Ecol9: 183-189, 1995

CONCLUSION trum hma. In: Smayda TJ, Shimizu Y (eds) Toxic phyto- blooms in the sea. Proc 5th Int Conference on Dinophysis cf. acuminataand Dinophysis acutamay Toxic Marine , 28 Oct-l Nov 1991, New- port, USA. Elsevier SCIPub, New York, p 121-126 have a sexual reproduction and a life cycle different Freudental AR, Jacobs J (1991). Observations on Dinophysis from those already known for dinoflagellates. The acuminata and Dinophysis norvegica in Long Island presence of 1 or several zoid producing bodies, if later waters: toxicity, occurence following diatom-discolored confirmed, is new for these organisms. Lack of avail- water and CO-occurence with Ceratium. In: Smayda TJ (ed) Abstract 5th Int. Conference on Toxic Marine Phyto- able culture has hitherto prevented observation of suc- plankton, Oct 28-Nov 1 1991, p. 45 cessive stages and timing of the gametogenesis; hence Fritz L, Nass M (1992) Development of the endoparasitic further experiments are needed, including DNA mea- dinoflagellate Amaebophrya ceratii within host dinofla- surement and transmission electron microscopy. Full gellate species. J. Phycol 28(3):312-320 reconstruction of their life cycle, as well as a better Hallegraeff GM (1993) A review of harmful algal blooms and their apparent global increase. Phycologia 32(2):79-99 knowledge of their nutritional regime, could perhaps Hansen G (1993) Dimorphc indviduals of Dinophysis acuta be useful in developing culture methods. and D. norvegica (Dinophyceae) from Danish waters. Phy- cologia 32(1):73-75 Lassus P, Bardouil M (1991) Le complexe 'Dinophysis acumi- Acknowledgements. This study was supported by the 'Pro- nata': identification des especes le long des cbtes fran- gramme National Efflorescences Algales Toxiques'. We qaises. Algol 12(1):1-9 warmly thank Captain Cirot, M. Ferme and their teams for Lassus P, Proniewski F. Maggi P, Truquet P, Bardouil M (1993) help during sampling at Antifer, and Dr lan Jenkinson Wind-induced toxic bloms of Dinophysis cf. acuminata in (ACRO, La Roche Canillac) for improving the English version. the Antifer area (France). In. Smayda TJ, Shirnizu Y (eds) Toxic phytoplankton blooms in the sea. Proc 5th Int Con- ference on Toxic Marine Phytoplankton, 28 Oct-l Nov LITERATURE CITED 1991, Newport, USA. Elsevier Scl Pub, New York, p 519-523 Anderson DM (1989) Toxic algal blooms and red tides: a Mackenzie L (1992) Does Dinophysis have a sexual life cycle? global perspective. In: Okalchi T, Anderson DM, Nemoto J Phyc0128:399-406 T (eds) Red tides: biology, environmental science, and tox- Maestrini SY, Berland BR, Grzebyk D, Spano AM (1995) icology. Elsevier Sci Pub, New York, p 11-16 Dinophysis spp. cells concentrated from nature for experi- Bardouil M, Berland B, Grzebyk D, Lassus P (1991) L'exis- mental purposes, uslng size fractionation and reverse tence de kystes chez les Dinophysales. C r Acad Sci Paris, migration. Aquat microb Ecol9:177-182 ser 111 312:663-669 McLachlan JL (1993) Evidence for sexuality in a species of Belin C (1993) Dlstributlon of Dinophysis spp. and Alexan- Dinophysis. In: Smayda TJ, Shimizu Y (eds) Toxic phyto- driurn rninutwn along French coasts since 1984 and their plankton blooms in the sea. Proc 5th Int Conference on DSP and PSP toxicity levels. In: Smayda TJ, Shirnizu Y Toxic Marine Phytoplankton. 28 Oct-l Nov 1991, New- (eds) Toxic phytoplankton blooms in the sea. Proc 5th Int port, USA. Elsevier Sci Pub, New York, p 143-146 Conference on Toxic Marine Phytoplankton, 28 Oct- Moita MT. Sampayo MA de M (1993) Are there cysts in the 1 Nov 1991, Newport, USA. Elsevier Sci Pub, New York, genus Dinophysis? In: Srnayda TJ, Shimizu Y (eds) Toxic p 469-474 phytoplankton blooms in the sea. Proc 5th Int Conference Cachon J (1964) Contribution a l'etude des Peridiniens para- on Toxic Marine Phytoplankton, 28 Oct- l Nov 1991. New- sites. Cytologie, cycles evolutifs. Ann Sci nat Zoo1 12eme port, USA. Elsevier Sci Pub, New York, p 153-157 ser V1:l-158 Partensky F, Vaulot D (1989) Cell differentiation of the bloom- Cachon J, Cachon M (1987) Parasitic dinoflagellates. In: Tay- forming dinoflagellate Gymnodinium cf. nagasakiense. lor FJR (ed)The biology of dinoflagellates. Blackwell Sci J Phycol 24(3).408-415 Pub, Oxford, p 571-610 Pflester LA (1984) Sexual reproduction. In: Spector DL (ed) Canter HM (1961) Studies on British chytrids. XVIII. Further Dinoflagellates. Acad Press, London. p. 181-199 observations on species invahng planktonic algae. Nova Reguera B, Bravo I,Fraga S (1990) Distribution of Dinophysis Hedwigia 3:73-78 acuta at the time of a DSP outbreak In the nas of Ponteve- Canter HM (1968) Studies on British chytrids. XXVIII. Rhizo- dra and Vigo (Galicia, NW Spain). Comm Meet Int Coun phyd~ummobile sp. nov. parasitic on the resting of Explor Sea CM-ICESlL.14, 12 p Ceratium hirundeua D.F. Mull. from the plankton. Proc Rosowski JR, Johnson LM, Mann DG (1992) On the report of Limn Soc Lond 179:197-201 gametogenesis, oogamy, and uniflagellated sperm in the Canter HM, Jaworsh GHM (1978) The isolation, mainte- pennate diatom Nitzschia pungens (1991; J Phycol 27: nance and host range studies of a Chytrid Rhizophydiurn 21-26). J Phycol 28(4):570-574 planktonicum Canter emend., parasitic on AstedoneUa Silva ES (1971) The 'small' form in the life cycle of dinoflagel- formosa Hassall. Ann Bot 42:967-979 lates and its cytological interpretation. In: Farinacc1 A (ed) Faust MA (1990) Cysts of Prorocentrum marinum (Dino- Proc 2nd Plankton Conference, Roma, 1970, Techni- phyceae) in floating detritus at Twin Cays, Belize man- scienza, p 1157-1 167 grove habitats. In: Graneh E, Sundstrom B, Edler L, Smayda TJ (1990) Novel and nuisance phytoplankton blooms Anderson DM (ed) Toxic marine phytoplankton. Proc in the sea: evidence for a global epidemic. In: Graneli E, Fourth Int Conterence Toxic Marine Phytoplankton, Sundstrom B, Edler L, Anderson DM (ed) Toxic marine 26-30 June 1989, Lund, Sweden. Elsevier Pub, New York, phytoplankton. Proc 4th Int. Conference Toxic Marine p 138-143 Phytoplankton. 26-30 June 1989, Lund, Sweden. Elsevier Faust MA (1993) Sexuality in a toxic dinoflagellate, Prorocen- Pub, New York, p 29-40 Berland et al.. L~fecycle stages of Dinophys~sspp. 189

Subba Rao DV. Partensky F. Wohlgeschaffen G, Li WKW Basin, eastern Canada. Mar Ecol Prog Ser 9?:117-126 (1991) Flow cytometry and microscopy of gametogenesis Taylor FJR (1968) of the toxin-producing dinofla- in Nitzschia pungens, a toxic-forming, marine diatom. J gellate Gonyaulax catenella by the endoparasitic dinofla- Phycol 2?(1):21-26 gellate Amaebophyra ceratii. J Fish Res Bd Can 25(10): Subba Rao DV, Pan Y, Zitko V, Bugden G, Mackeigan K 2241-2245 (1993) Diarrhetic shellfish poisoning (DSP)associated with Zingmark RG (1970) Sexual reproduction in the dinoflagellate a subsurface bloom of D~nophysisnorvegica in Bedford Noctiluca rnll~arlsSuriray. J Phycol 6:122-126