IOC Science and Communication Centre on Harmful Algae University of Copenhagen

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Module III Section 6, Dinoflagellate Cysts (4 hours)
Contents: 6.1. Suggested reading 6.2. Some basic features 6.3. Taxonomy: classification and identification of harmful species 6.4. Notes on HAB cysts types 6.5. Methods 6.6. References and identification literature 6.7. Glossary

References in bold are available as pdf files on the course CD.

6.1. Suggested reading
Present document, textbook, and the following articles: Anderson D.M., Fukuyo Y. & Matsuoka K. 2004. Cyst methodologies. In: Manual on harmful marine microalgae.(Ed. by G.M. Hallegraeff, D.M. Anderson & A.D. Cembella), pp. 165-189. Monographs on oceanographic methodology 11. UNESCO. Dale B. 1983. Dinoflagellate resting cysts: ”benthic plankton”. In: Survival strategies of the algae (Ed. by G.A. Fryxell), pp. 69-136. Cambridge University Press. Dale, B. & Dale, A.L. 2002. Environmental applications of dinoflagellate cysts and acritarchs. In: Quaternary environmental micropalaeontology, S. Haslett (ed.), Edward Arnold (Publishers), London, 207-240. Suggested web site with cyst illustrations and with text also in Spanish: http://dinos.anesc.u-tokyo.ac.jp/

6.2. Some basic features
Free-living dinoflagellates spend most of their life cycle as unicellular flagellates reproducing asexually by binary fission. However, under certain conditions, sexuality may be induced. During this process gametes are produced, which through fusion produce a swimming zygote (planozygote). The planozygote of some species undergoes meiosis in the plankton, while those of other dinoflagellates, after remaining in the plankton for several days, loose their flagella, build a new resistant cell wall becoming morphologically different from their motile counterpart, and accumulate in sediments as a dormant, non-motile hypnozygote (hypno – sleep) or resting cyst (Fig.1).

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Figure 1. Life-cycle diagram of a cyst producing dinoflagellate Alexandrium tamarense (from Anderson et al. 2004) Of the known species of dinoflagellates, only around 20% are known to form cysts, among which several HAB species are included (eg. several species within the genus Alexandrium,
Pyrodinium bahamense, catenatum). Lingulodinium polyedrum, Protoceratium reticulatum, Gymnodinium

In nature, little is known about the environmental control of sexuality in dinoflagellates. However, it is clear that it is not an automatic response to adverse environmental conditions (Pfiester & Anderson 1987). It most certainly involves complex interactions between physical and chemical parameters and the metabolism of the organisms. For several species, cyst production has been shown to occur in association with a peak in the abundance of the motile stage and towards the end or after a period of exponential growth (Wall & Dale 1968, Ishikawa & Taniguchi 1996, Montresor et al. 1998). Wall and Dale (1968) also noted, for different species, a different ratio of motile cells to encysted forms, rendering difficult to infer from cyst abundances the equivalent phytoplankton concentrations.

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Under laboratory conditions, sexuality in autotrophic species is usually induced by nutrient starvation, particularly nitrogen depletion (Pfiester & Anderson 1987). Recent work has shown that temperature and day-length may also play a role in regulating encystment in several dinoflagellates (Sgrosso et al. 2001). Once formed, dinoflagellate cysts display a dormancy period that may vary from a few days to several months, during which germination apparently cannot occur (Dale 1983, Pfiester & Anderson 1987). This mandatory dormancy period is species specific and is most probably related to endogenous factors inhibiting germination. It should be distinguished from quiescence, which corresponds to the suspension of germination in cysts which are competent to do so but which are inhibited by environmental conditions. The conditions necessary for germination in cysts that have completed their dormancy period is not well defined. Most commonly, temperature has been considered the primary controlling factor, and several authors refer to the existence of a permissive temperature window within which quiescent cysts will germinate, but outside of which they will remain in a resting state (Dale 1983). Other environmental factors affecting dormancy and excystment are less studied, but results with several species indicate that other parameters like the need for oxygen during germination, or exposure to light, either brief pulses or prolonged periods, may be essential (Ishikawa & Taniguchi 1996, Rengefors & Anderson 1998). Cysts are able to withstand harsh environmental conditions, and in general remain viable in sediments for many years. Among the roles that dinoflagellate cysts have been suggested to play are survival through adverse environmental conditions, initiation and termination of blooms through respectively excystment and encystment, species dispersal and genetic recombination (Dale 1983). Cyst production may also be a succesful life-cycle strategy that anchors a species to the most favourable environment for the motile stage, since rapid mass sinking in coastal environments will minimize advective dispersal to unfavorable environments. Cyst studies are, thus, a crucial tool for the understanding of bloom dynamics and the geographical spread of cyst producing HAB species, both natural and human mediated (e.g. ship ballast water).

6.3. Taxonomy: classification and identification of harmful species
(see also IOC/HAB Manual, Chapter 20, Matsuoka and Fukuyo 2004) Morphologically cysts differ from their motile counterparts. Most known cysts are organicwalled, many of which have a highly resistant organic compound forming, or partly forming, the cyst wall. It is usually referred to as dinosporin and is considered to be responsible for the fossilisation of organic-walled cysts and for their variable resistance to both natural decay and laboratory treatments (Dale 1983, Fensome et al. 1993). There are also dinoflagellates capable of producing calcareous cysts. These are a closely related group of phototrophic dinoflagellates (subfamily Calciodinelloideae) that are abundant in marine neritic and oceanic waters and form wide fossil deposits (Dale 1992, D’Onofrio et al. 1999). Dissolution of the outer calcareous wall may leave a thin organic acid-resistant inner wall (Dale 1983). Cyst morphology is distinctive and constant within a species, although variations in process length and robustness may occur (e.g. Ellegaard et al. 2002). Cyst body shape may vary

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considerably between species. It may be obviously related to the shape of the motile stage, or it may be basically spherical. The cyst wall may be smooth and essentially featureless or it may be ornamented with distinctive outgrowths from the wall, like ridges and processes (Fig. 2) (Dale 1983, Fensome et al. 1993).

Figure 2. Cyst of Lingulodinium polyedrum: spherical body cyst with numerous processes, and combination precingular archeopyle (from Fensome et al. 1993) Features found in cyst walls may reflect characteristics of the motile stage, like the sulcus, the cingulum or the plate pattern. However, these features are independent structures from the wall of the vegetative stage. In descriptive cyst terminology it is common to use the prefix “para” to describe these reflected features (e.g. parasulcus, paracingulum, etc.) (Fig.3). This is particularly convenient when both motile stages and cysts are being discussed (Dale 1983).

Figure 3. Comparison of cyst and motile stage descriptive terminology (from Dale 1983)

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One of the main features of dinoflagellate cysts is the excystment aperture in the wall, known as archeopyle. The original definition of archeopyle was based on fossil dinoflagellate cysts and involved the loss of one or more paraplates, i.e. the excystment opening was related to paratabulation (e.g. Fig. 4) (Fensome et al. 1993). More recently, in living cysts other types of excystment openings have been identified through life cycle studies. Matsuoka (1985) proposed the terms chasmic and tremic archeopyle to describe, respectively, a linear to slightly curved opening and a more or less circular opening with no signs of paratabulation. In fossil cyst studies, however, if there is no sign of paratabulation, or the specimen is not obviously dinoflagellate shaped, it cannot be confirmed as a dinoflagellate, and these structures are then designated as acritarchs (Dale 1983, Fensome et al. 1993).

Figure 4. Examples of intercalary archeopyles within the order Peridiniales, family Congruentidiaceae (from Fensome et al. 1993). Species producing cysts with processes show, in any given population of cysts, some variability in length, namely some of the cysts have shorter processes (see Fig. 7). In general these represent a small fraction of the cyst population. However, in low salinity environments there is a proportional increase in specimens with markedly reduced processes, and high proportions of the latter group may indicate brackish conditions (Dale 1996, Ellegaard et al. 2002).

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6.4. Notes on HAB cyst types
6.4.1. Alexandrium
This group is very difficult to identify because of the lack of clear morphological characteristics. In general the cyst wall is colourless, without any surface ornaments, and a pigment body is present in the cytoplasm. A shapeless gelatinous layer sometimes covered by detritus material covers many cysts. The archeopyle and operculum are generally difficult to observe, and in most cases are of unknown characteristics. Cyst shape may vary from round to elliptical, depending on species, but the same cyst may show different shape depending on cyst orientation. These cysts maybe easily be confused with other non-HAB groups, for instance within the genus Scrippsiella (when the calcareous plates are not present) or Gonyaulax verior, and only excystment experiments may clarify the identity.

6.4.2. Pyrodinium bahamense (Fig. 5)
Cysts are spherical covered with numerous long and generally strong processes. Processes are cylindrical to tubiform (hollow) with capitate distal endings. In the same specimen processes vary. Most are simple, but others tend to bifurcate at the tip (arrow head) or even almost down to the base (arrow). Fully joined processes may also occur. Cyst wall is colourless generally with a granular surface, and with no paratabulation. The archeopyle is epicystal. In certain cyst assemblages where Pyrodinium bahamense coexists with Lingulodinium polyedrum and Protoceratium reticulatum, the morphological variations of the cyst processes within each cyst group may render difficult the correct identification of these 3 species. In some specimens only if the archeopyle is clearly visible one can be sure of the correct cyst identification.

Figure 5 . Pyrodinium bahamense. Figures illustrate processes and the hemicyst resulting from the typical epicystical archeopyle. Hemicyst 56 µm x 40 µm.

6.4.3. Lingulodinium polyedrum (Figs. 2 and 6)
Cysts are spherical, with numerous spines that are variable in length and form. Spines have enlarged bases and then taper. In one cyst, some of the tips maybe found to have small spinules that are easily visible with interference contrast (DIC) and are a good diagnostic

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character (Fig. 2). Specimens with very reduced processes maybe recorded, sometimes reduced to small nodules on cyst surface. Archeopyle is formed by the loss of 3 to 4 precingular paraplates (Figs. 2 and 6. Cyst wall is colourless and granular. No paratabulation is visible.

Figure 6. Lingulodinium polyedrum showing typical combination precingular archeopyle.

6.4.4. Protoceratium reticulatum (Fig. 7)
Cysts are spherical with numerous processes with capitate tips (Fig. Fb). Processes are long and simple, usually slender. Specimens with very reduced processes may be recorded, sometimes reduced to small nodules on cyst surface (Fig. 7a). The archeopyle is simple formed by the loss of the third precingular paraplate. Cyst wall is colourless. A. B.

Figure 7. Protoceratium reticulatum A. Specimen with very short processes, archeopyle visible (diameter 42 µm); B. Specimen with typical long capitate processes in optical section (diameter 38 µm, processes 8-10 µm).

6.4.5. Gymnodinium catenatum (Fig. 8)
Spherical brown cyst with microreticulate ornamentation which probably reflects the amphiesmal vesicles on motile stage. The paracingulum, parasulcus and acrobase are evidenced by the alignment of the paravesicles (Fig. 8). The cyst of G. catenatum may be confused with cysts of other non-toxic species namely G. microreticulatum and G. nolleri. Cysts of the latter two species are smaller, but particularly the upper limit of G. nolleri may overlap with the lower limit of G. catenatum. Other differences at the paracingulum level may also exist.

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A.

B.

Figure 8. Gymnodinium catenatum. A. Cyst under the light microscope. Paracingulum is evidenced by two parallel rows of paravesicles (diameter 39 µm). B. SEM, paracingulum and para-apical groove visible.

6.4. Methods
Once dinoflagellate cysts are formed, they behave as fine silt particles and accumulate in bottom sediments (Dale 1996). At any given time cyst assemblages reflect an integrated balance between cyst production, transportation, sedimentation and preservation. Most dinoflagellates producing resting cysts are coastal/neritic and in these environments cyst and sediment transport are limited (Dale 1983, Montresor et al. 1998). Thus, mapping cysts in coastal surface sediments, in well selected sedimentary basins, provides an integrated information, both in time and space, on local cyst production and distribution (Dale 1983, 1996, Montresor et al. 1998). These studies have also proven very useful in giving early warning for the presence of species not previously detected in the plankton. By contrast, in deep sea sediments, results with sediment traps indicate that apart from a few cysts representing local production (oceanic species), most cysts recovered in bottom sediments represent cysts transported by currents or older material resuspended from adjacent shelves (Dale & Dale 1992). Several methods have been proposed for mapping cysts in sediments (Anderson et al. 2004). One that is particularly easy to use, non-expensive and that has proven good results is the one described in Dale and Dale (2002) (Fig. 9). Sediment samples are collected from shore or from a small boat, using a hand-made simple sucking device, made from a propipette bulb and a funnel with a lead weight, attached to opposite ends of a plastic tubbing ca. 20 m long. This device allows retrieval of the upper 1-2 cm of sediment, including the top flocculent layer.

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Figure 9. Sediment sucker (from Dale & Dale 2002). Samples, when possible, are prepared the same day of collection, or the following day, to prevent premature cyst germination. Otherwise sediments must be kept cool and in the dark, and temperature changes must be avoided. For cyst identification and enumeration using permanent slides, 10 to 20 cm3 of wet sediment is placed in a 100 cm3 Erlenmeyer and covered with de-mineralised water (sea water for incubation experiments). The sediment suspension is then manually agitated and sonicated for 1 min (e.g. Bransonic 1210 ultrasonic bath). After sonication, samples are wet sieved through a 100 m sieve onto a 25 m sieve. The fraction remaining on the 25 m sieve is washed into a 15 ml centrifuge tube and centrifuged for 5 min at 2500 rpm. To further concentrate dinoflagellate cysts and eliminate most of the mineral particles, the supernatant is discarded and a high density solution of sodium polytungstate (SPT) (2.016 g cm-3) (Johnson Matthey GmbH Alfa) maybe added to the pellet (modified from Munsterman & Kerstholt 1996, Bolch 1997). The pellet is then homogenized with the aid of a Vortex and centrifuged for 15 min at 1000 rpm. The lighter organic fraction floated off by the heavy liquid is then collected with a Pasteur pipette and rinsed with de-mineralised water through a 25 m sieve. When samples are very poor, the pellet maybe re-suspended and centrifuged once again to increase cyst recovery. The fraction retained on the 25 m sieve is collected in a 15 ml centrifuge tube, followed by centrifugation for 5 min at 2500 rpm. The supernatant is pipetted off leaving a final volume between 1 ml and 2 ml, depending on sample concentration. Aliquots are mounted on microscope slides with glycerine jelly and sealed with wax.

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If cysts are to be isolated for cyst/motile stage studies, aliquots of the sample are observed under the light microscope in a Sedgwick Rafter chamber. Single cyst are isolated by micropipette and washed in filtered sterilized seawater from the sampling site, before being transferred to culturing cell wells with the same water, or diluted culturing media (e.g. f/50) ( Diagram illustrating sampling processing

Palynological treatment – information on

Raw sediment

Incuba

Mixed cultures – information on viable

Cleaning – ultrasound b h

Sieving: 100μm onto

High density solution- Sodium polytungstate (2.016 g cm-3)

Concentrated cyst

Cyst assemblage

Cyst – motile stage

Permanent slides mounted on

Fresh mounts, counting in

Single cyst isolation and incubation in culturing cell wells;

6.6. References and identification literature
Anderson D.M., Fukuyo Y. & Matsuoka K. 2004. Cyst methodologies. - In: Manual on harmful marine microalgae.(Ed. by G.M. Hallegraeff, D.M. Anderson & A.D. Cembella), Monographs on oceanographic methodology 11. UNESCO, pp. 165-189. Bolch C.J. 1997. The use of sodium polytungstate for the separation and concentration of living dinoflagellate cysts from marine sediments. - Phycologia 36: 472-478. Dale A.L. & Dale B. 1992. Dinoflagellate contributions to the sediment flux of the Nordic Seas. - In: Dinoflagellate contributions to the deep sea. (Ed. by S. Honjo) pp. 45-73. Ocean Biocoenosis Series No. 5. Woods Hole Oceanographic Institution, Woods Hole, MA.

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Dale B. 1983. Dinoflagellate resting cysts: ”benthic plankton”. - In: Survival strategies of the algae (Ed. by G.A. Fryxell), pp. 69-136. Cambridge University Press. Dale B. 1992. Dinoflagellate contributions to the open ocean sediment flux. (Ed. by S. Honjo) pp. 1-23. Ocean Biocoenosis Series No. 5. Woods Hole Oceanographic Institution, Woods Hole, MA. Dale B. 1996. Dinoflagellate cyst ecology: modeling and geological applications. - In: Palynology: principles and applications (Ed. by J. Jansonius & D.C. McGregor) pp. 1249-1275. Vol. 3, American Association of Stratigraphic Palynologists Foundation. Dale, B. & Dale, A.L. 2002. Environmental applications of dinoflagellate cysts and acritarchs. - In: Quaternary environmental micropalaeontology, S. Haslett (ed.), Edward Arnold (Publishers), London, 207-240. D’Onofrio G., Marino D., Bianco L., Busico E. & Montresor M. 1999. Toward an assessment on the taxonomy of dinoflagellates that produce calcareous cysts (Calciodinelloideae, Dinophyceae): a morphological and molecular approach. - Journal of Phycology 35: 1063-1078. Ellegaard, M., Lewis, J. & Harding, I. 2002. Cyst-theca relationship, life cycle, and effects of temperature and salinity on the cyst morphology of Gonyaulax baltica sp. nov. (Dinophyceae) from the Baltic Sea area. - Journal of Phycology 38: 775-789. Fensome R.A., Taylor F.J.R., Norris G., Sarjeant W.A.S., Wharton D.I. & Williams G.L. 1993. A classification of living and fossil dinoflagellates. Micropaleontology Special Publication Number 7. Sheridan Press, Hanover, Pennsylvania. 351 pp. Ishikawa A. & Taniguchi A. 1996. Contribution of benthic cysts to the population dynamics of Scrippsiella spp. (Dinophyceae) in Onagawa Bay, northeast Japan. Marine Ecology Progress Series 140: 169-178. Matsuoka, K. 1985. Archeotype structure in modern gymnodinialean dinoflagellate cycsts. Rev. Paleobot. Palynol. 44: 217-31 Matsuoka, K. & Fukuyo, Y. 2004. Taxonomy of cysts. In: Manual on harmful marine microalgae.(Ed. by G.M. Hallegraeff, D.M. Anderson & A.D. Cembella), pp. 563-592. Monographs on oceanographic methodology 11. UNESCO. Montresor M., Zingone A. & Sarno D. 1998. Dinoflagellate cyst production at a coastal Mediterranean site. Journal of Plankton Research 20: 2291-2312. Munsterman D. & Kerstholt S. 1996. Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Paeobotany and Palynology 91: 417422. Pfiester L.A. & Anderson D.M. 1987. Dinoflagellate reproduction. In: The biology of dinoflagellates. (Ed. by F.J.R. Taylor), pp. 611-648. Botanical Monographs vol. 21. Blackwell Scientific Publications, London. Rengefors K. & Anderson D.M. 1998. Environmental and endogenous regulation of cyst germination in two freshwater dinoflagellates. Journal of Phycology 34: 568-577. Sgrosso S., Esposito F. & Montresor M. 2001. Temperature and daylength regulate encystment in calcareous cyst-forming dinoflagellates. Marine Ecology Progress Series 211: 77-87 Wall D.& Dale B. 1968. Modern dinoflagellate cysts and evolution of the Peridiniales. Micropaleontology 14: 265-304.

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6.7. Glossary
English Acritarch Archeopyle Benthic resting stage Calcareous cyst Cavate type Chorate type Cingulum Cyst Dinocyst Dinoflagellate cyst Dinospore Dinosporin Dormancy Ecdysis Ecdysal cyst Encystment Excystment Features reflected Germination Holomorph Hypnozygote Hypocyst Hystrichospheres Incubation Life cycle Meiocyte Opercular pieces Operculum Organic-walled cyst Paleontological classification Palynological technique Paracingulum Paraplate Parasulcus Parasuture Paratabulation Español Acritarco Arqueopilo Estadio de reposo bentónico Quiste calcáreo Tipo cavado Tipo corado Cingulum, Cíngulo Quiste Dinoquiste Quiste de dinoflagelado Dinospora Dinosporina Dormancia Ecdisis Quiste ecdísico Enquistamiento Desenquistamiento Caracteres reflejados Germinación Holomorfo Hipnocigota Hipoquiste Histricosfera Incubación Ciclo de vida Meiocito Piezas operculares Operculum, Opérculo Quiste de pared orgánica Clasificación paleontológica Técnica palinológica Paracingulum, Paracíngulo Paraplaca Parasulcus Parasutura Paratabulación Português Acritarca Arqueópilo Quisto/cisto de resistência bentónico/bentônico Quisto/cisto calcáreo Tipo cavado Quisto/cisto de processos longos, com comprimento superior a 1/3 do diâmetro Cingulum, Cíngulo Quisto/cisto Quisto/cisto de dinoflagelado Quisto/cisto de dinoflagelado Esporo de dinoflagelado Dinosporina Dormência Ecdisis/ecdise Quisto/cisto ecdísico Enquistamento/encistamento Desenquistamento/germinação Caracteres reflectidos/refletidos Germinação Holomórfico Hipnozigoto Hipoquisto/hipocisto Histricosfera Incubação Ciclo de vida Meiocito Peças do opérculo Opérculo Quisto/cisto com parede orgânica Classificação paleontologica Técnica palinológica Paracingulum, Paracíngulo Paraplaca Parasulco Parasutura Paratabulação

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Pellicle cyst Planozygote Process Proximate type

Quiste pelicular Planocigota Proceso Tipo próximo (contiguo)

Quisto/cisto pelicular Planozigoto Processo Quisto/cisto morfologicamente semelhante à forma vegetativa; Os processos quando presentes têm um comprimento inferior a 1/10 do diâmetro do quisto/cisto. Quiescente Quisto/cisto de resistência Período de latência/repouso Colector/coletor de sedimentos Bacia sedimentar Crivagem; Lavagem em crivos calibrados Esporopolenina Fases do ciclo de vida Quisto/cisto temporário Camada floculenta Transporte na água de lastro de navios Zigoto

Quiescence Resting cyst Resting period Sediment trap Sedimentary basin Sieving technique Sporopollenin Stages in life cycle Temporary cyst Top flocculent layer Transport via ships´ ballast water Zygote

Quiescencia Quiste de reposo Período de reposo Trampa de sedimentos Cuenca sedimentaria Técnica de tamizado Esporopolenina Estadios en el ciclo de vida Quiste temporario Capa floculenta superior Transporte vía agua de lastre Cigota