Consumption of Scrippsiella lachrymosa resting cysts by the eastern oyster (Crassostrea virginica).

ABSTRACT Scrippsiella spp. resting cysts, unlike many other dinoflagellate cysts, possess an outer layer of calcite beneath which is a thin sporopollenin wall. This feature may affect cyst survival through the digestive tract of benthic organisms, when they consume the cysts. The extent of digestibility is related to the degree to which grazing by benthic organisms could influence a benthic cyst population. To test consumption and digestion of a representative Scrippsiella cyst by one benthic grazer, the eastern oyster (Crassostrea virginica) was fed culture-produced resting cysts of the dinoflagellate Scrippsiella lachrymosa under controlled conditions. Cyst recovery from no-oyster, control containers was 97%; therefore, digestive destruction of cysts could be quantified as the difference between cysts added to experimental containers containing oysters and the number of intact cysts recovered after a period of oyster feeding. In each treatment, 18 % of the cysts were destroyed after being ingested at a cell density of 43.4 cysts/mL and 11% were digested at a higher cell density (263.2 cysts/mL). Cysts were observed to become rounded and turn yellow after first losing the outer, calcareous wall as a first step in digestion. In fecal-pellet samples, contents from broken cysts could be found as well as intact cysts and rounded yellow cysts. Viability of ingested cysts was not evaluated, but it seems that Scrippsiella cysts are relatively resistant to digestion by oysters.

KEY WORDS: Scrippsiella lachrymose, Crassostrea virginica, dinoflagellate, cyst, oyster, grazing

INTRODUCTION

As many other dinoflagellates, Scrippsiella species produce resting cysts as part of the life cycle. The formation of resting cysts in dinoflagellates is part of sexual reproduction and occurs at the end of a bloom (Dale 1983). Gametes fuse and form zygotes, which, after some period of time (often 1-2 wk, Lewis 2001), develop into resting cysts and sink to the sea floor. The cysts are dormant for a species-specific period of time a couple of weeks to several months (Dale 1983, Anderson 1998). After dormancy, the cysts remain quiescent until conditions become advantageous for germination (i.e., in the new growing season). Dinoflagellate cysts in the sediment constitute a "seed bank," or source for future blooms (Anderson et al. 1983). Cysts are rich in lipids and starch for survival during the resting period (Xiaoping et al. 1989, Binder & Anderson 1990) that can last for several years. Keafer et al. (1992) reported half-lives of cysts to be in the range of 2-10 y for buried cysts. Living cysts can be as old as 40 y (Lingulodinium polyedrum, Lewis 1988). Several harmful dinoflagellate species (e.g., species of the genus Alexandrium and Gymnodinium catenatum) have resting cysts; therefore, there is a large interest in surveying coastal areas to map the presence of dinoflagellate cysts. Scrippsiella spp. cysts are ubiquitous and cosmopolitan, widespread in the neritic zone but also common in open oceans (Wall et al. 1970, Bolch & Hallegraeff 1990, Nehring 1996, Sonneman & Hill 1997, Persson et al. 2000).

Scrippsiella spp. resting cysts are different from many other dinoflagellate cysts in that they possess an outer layer of calcite crystals (Xiaoping et al. 1989, Lewis 1991), beneath which, is a thin sporopollenin wall. The inner wall is rather fragile and does not persist well in prolonged storage of samples in a closed container (Lewis et al. 1999). In contrast, other cysts with a thick sporopollenin wall are known to be preserved (the empty wall, not alive) for millions of years as "microfossils." The calcitic cysts are not included in most palaentological studies because the traditional method for preparation of dinoflagellate cysts includes removal of organic material by strong acids (Dale 1979), which also dissolves calcium carbonate. Interest in calcitic cysts is, however, increasing, and several studies of recent (Montresor et al. 1994 and Montresor et al. 1998) and fossil cysts (Zonneveld et al. 2000) have been done in the Mediterranean Sea.

Several recent studies have highlighted the possible importance of benthic grazing on the persistence and survival of resting cysts. One study evaluating digestion of Scrippsiella cysts by benthic polychaetes found the cysts to be quite resistant to digestion (Kremp et al. 2003); whereas, Alexandrium fundyense cysts were found to be relatively susceptible to digestion by oysters (Persson et al. 2006). To explore the possible importance of the calcite cyst-wall inclusions in Scrippsiella to grazing resistance, we conducted a series of experiments in which culture-produced resting cysts of Scrippsiella lachrymosa (Lewis 1991) were fed to the eastern oyster (Crassostrea virginica Gmelin, 1791). The same experimental containers and methods were used as in previous studies showing digestion of cultured, toxic Alexandrium fundyense resting cysts by oysters (Persson et al. 2006) to facilitate direct comparison of results.

MATERIALS AND METHODS

Three grazing experiments were conducted with the eastern oyster ( Crassostrea virginica) fed culture-produced Scrippsiella lachrymosa resting cysts. Experimental procedures were modified from Persson et al. 2006.

Cyst Production

Scrippsiella lachrymosa, strain B-10, was grown in f/10 medium with 5% marine sediment extract added (sediment was autoclaved in distilled water and then centrifuged according to the description for making soil extract; modified from Pringsheim 1946). The addition of soil or sediment extract was necessary to make the culture grow well in the Milford, CT (Long Island Sound, USA) seawater, although the culture grows in f/2-enriched Woods Hole seawater (Olli & Anderson 2002, Kremp et al. 2003). Encystment, however, was successfully accomplished in f/2 without nitrogen, as described for this species (Olli & Anderson 2002, Kremp et al. 2003). See Smith and Persson (2004) for further details on the encystment method.

Oysters

Oysters (Crassostra virginica, 3-4 cm shell height), provided by the Noank Aquaculture Cooperative, Noank, CT, USA (January 22, 2003), were kept in running, unfiltered seawater until a few days before each experiment. The temperature of the seawater was increased gradually (2 4[degrees]/day) up to 18[degrees]C, where it was maintained thereafter. Three-to-four days before the start of each experiment, the oysters to be used were placed in a container with aerated, 0.1[micro]m-filtered seawater, which was changed daily, to empty the digestive systems of previously-eaten particles. The first experiment was started February 3, the second on February 11, and the third experiment on March 31. Oysters were healthy and growing rapidly in the unfiltered seawater in which the main food items were planktonic diatoms.

Experiments

Experiment 1: Oyster Clearance of Cysts at a Low Cyst Density

Cysts were collected from the encystment vessels by sieving onto a 20-[micro]m sieve, rinsed with filtered seawater, collected in volumetric flasks, and counted (triplicate) in a Sedgewick-Rafter chamber with a light microscope. The experiment was conducted at 18[degrees]C in a constant-temperature room (24 h light 54 [micro][Em.sup.-2][s.sup.-1]). Dark boxes were placed over the beakers at night to maintain the dark-light cycle in which the cysts were produced.

The experimental containers and associated apparatus were the same as described in Persson et al. (2006). Briefly, two oysters were suspended on stiff plastic screen in 1-L beakers. Magnetic stir-bars were used to keep the cysts suspended and accessible to the oysters. Three sets of triplicate beakers contained feeding oysters, controls with empty oyster shells, and unfed oysters to assure that no previously eaten Scrippsiella cysts were present from the natural food consumed previously.

Initial cyst densities were established at 43.4 cysts/mL in a 1-L volume, and oysters were allowed to feed for 24-h. Then the water in each beaker was poured onto a 20-[micro]m nylon mesh sieve, and the beaker, oysters, net, and stir bar were spray-rinsed repeatedly with 0.l-[micro]m filtered seawater that subsequently was poured onto the sieve. The material on the sieve was transferred (with repeated rinsing) to a 100-mL volumetric flask. The flasks were kept in a refrigerator until counting (within two days).

After rinsing, oysters were put in clean beakers with 1-L of filtered seawater. Oysters were placed on the bottoms of the beakers and there was no mixing or aeration (to avoid destruction of fecal pellets formed), and, after one day, a drop of yellow plastic bead suspension (fluorescent microspheres; Fluoresbrite YG Carboxylate Microspheres 2.00-[micro]m, Polysciences, Inc.) was added to monitor oyster digestive progress and ascertain the excretion of any remaining cysts. After a total time of 48-h after feeding stopped, biodeposits were collected in the same way as described above. They were rinsed onto a 20-[micro]m sieve, rinsed thoroughly, and transferred to 100-mL volumetric flasks. The flasks were kept refrigerated until cysts were counted (within four days). To count cysts recovered, the volumetric flask was shaken thoroughly, and 200-[micro]L aliquots were counted in a Sedgwick-Rafter chamber using a light microscope.

Experiment 2: Clearance Over Time

Two oysters were placed in one beaker with 0.5 L of filtered seawater containing 457 Scrippsiella lachrymosa resting cysts/ mL. The same experimental conditions as in experiment 1 were used. Samples (0.2-mL) were taken every hour for 10 h and then after 23 h 15 min. The remaining cysts were collected on a 20-[micro]m screen by rinsing all components with filtered seawater. Oysters were rinsed, moved to a plastic beaker with filtered seawater, and a drop of yellow microbeads was added. The day after, oysters had defecated yellow pellets, which were collected and rinsed as above. Cysts were counted as described earlier.

Experiment 3: Clearance at a High Cell Density of Cysts

This experiment was conducted with a cyst density of 263.2 cysts/mL. The cysts were newly produced and dormant. The experiment was performed almost exactly as the first experiment except the Dark boxes were not used to simulate night, the yellow plastic beads were given directly after transfer to clean beakers, and biodeposits were collected after 24-h as in experiment 2.

Study of Digestive Processes

Microscopic observations were made of cysts in biodeposits from oysters (Crassostrea virginica) fed the pure Scrippsiella lachrymosa cysts. To investigate the effect of acid on the cysts, a small amount of 37% hydrochloric acid, HC1, was added to a microscope slide with resting cysts in a moist pellet sample and the cysts were observed in the microscope.

RESULTS

The results show a highly significant decrease in cysts recovered in experimental treatments with grazing oysters (P < 0.02). At a low cyst density, 18% of the cysts (corresponding to 3,806 cysts/oyster) were digested by the oysters, and at a high cell density, 11% of the cysts were digested (corresponding to 14,610 cysts/oyster). There was a high recovery of cysts from the control (measured in exp. 1; over 97%). The unreplicated experiment, experiment 2, showed a rapid clearance of cysts; after 4 h only 50% of the cysts were left in suspension. Only a small number of cysts could be found in the biodeposits collected after the experiments. Biodeposits were formed within two hours of the beginning of the experiment; therefore, the contents of these could have become recirculated within the experiment and then rinsed and counted together with remaining cysts from the water at the end of the feeding period.

In the detailed study of the digestion process of Scrippsiella cysts, the disintegration of cysts could be followed. As can be seen in Figure 1, red spots typical of Scrippsiella cysts, calcite crystals, and starch grains can be seen embedded within the biodeposits. Some seemingly-unharmed cysts can be seen (Fig. 2), but also common were rounded cells with yellow contents and a spot more orange than red in color (Fig. 3). In healthy cysts, the spot is red. To determine if the small crystals within the pellets were the calcite crystals from cysts, acid was added to some pellet samples. The crystals disappeared, and cysts lost the outer calcite layer and became round in shape; the cell wall became thin, the contents turned yellow, and the red spot became orange. This confirmed that the rounded, yellow cells observed in the fecal pellets were, indeed, partly digested Scrippsiella cysts.

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DISCUSSION

Digestion of 18% and 11% of provided Scrippsiella lachrymosa cysts by oysters in the experiments reported here demonstrates clearly that grazing has the potential to cause a reduction in cyst numbers. Comparing the results from the experiment on S. lachrymosa reported here with another experiment done using the same experimental conditions and the same group of oysters (Persson et al. 2006), Alexandrium fundyense resting cysts were shown to be more susceptible than S. lachrymosa cysts to destruction by Crassostera virginica feeding and digestion; 59% of ingested A. fundyense cysts were digested, and toxins from cysts were accumulated in the oyster soft tissues (Persson et al. 2006). Several previous studies of how resting cysts of different Scrippsiella species responded to grazing by different animals have been reported. The Eastern mudsnail, Ilyanassa obsoleta, actively forages for, consumes, and digests S. lachrymosa resting cysts (Persson et al. 2008). Some reports show a reduction in cyst numbers or viability of cysts; whereas, others show that the animal does not affect the cyst numbers or that the resting cysts may even take advantage of being eaten. Persson and Rosenberg (2003) showed that the common deposit feeder Amphiura filiformis significantly reduced the proportion of S. trochoidea cysts in Swedish sediment. Montresor et al. (2003) investigated the viability of S. trochoidea and S. ramonii resting cysts after grazing by four different copepod species. The majority of cysts were recovered intact in fecal pellets, but the germination capability of grazed cysts was low (although different between copepod species). Chemical damage attributable to enzymes in digestive fluids was suggested as the cause of reduced cyst viability. It is, thus, likely that some intact, viable-looking cysts found in sediment samples cannot germinate because they have been damaged by passage through, for example, a copepod gut.

Kremp et al. (2003) found that resting cystsof S. lachryrnosa (the same strain as used in these experiments) were remarkably resistant to digestion by three different deposit-feeding polychaetes used in feeding experiments. Cysts were not adversely affected, and pelletization even enhanced the germination capability of cysts. It appears as though Scrippsiella spp. cysts may, under some circumstances, even benefit from being ingested. Rengefors et al. (1996) showed uptake of phosphorous in Scrippsiella resting cysts. Fecal pellets could serve as a protective cover and provide a nutrient-rich environment simultaneously. If the grazer has not damaged the cyst mechanically or chemically by the action of acids and/or enzymes in the digestive fluids, being ingested (by the right animal) could be advantageous for cyst survival. Reid and Boalch (1987) suggested that a major route for resting cysts to the bottom may be via sedimentation of zooplankton fecal pellets. Vegetative cells of other Dinoflagellate species exhibited varying degrees of survival through the digestive processes of Oysters (Persson et al. 2006) and several other Bivalve species (Hegaret et al. 2008), which have also exhibited different ingestion behaviors (Hegaret et al. 2007).

Long-term preservation of calcareous cysts in anoxic areas is not likely; whereas, cysts with a sporopollenin-type wall (e.g., Lingulodinium polyedrum, Spiniferites spp. Protoperidinium spp. etc.), and cysts of the genus Alexandrium, are often well preserved in anoxic environments (Dale 1983, Lewis 1988, Keafer et al. 1992) until they are reintroduced to the water column by upwelling or a storm. Immersion in a slightly acidic environment eventually dissolves the outer calcite layer of Scrippsiella cysts (Anderson et al. 1985), and Scrippsiella cysts collected from anoxic sediment in Sweden were without the calcite layer (Persson, personal observations). The anoxic decomposition of organic material produces various organic acids, which helps explain dissolution of the calcite layer in Scrippsiella cysts in anoxic sediments. The wall of Scrippsiella cysts is destroyed in palynological processing (Lewis 1991). Even prolonged storage in a closed container reduces Scrippsiella cyst numbers considerably; Lewis et al. (1999) kept sediment samples in a refrigerator for nine years, during which the count of Scrippsiella crystallina specimens with normal contents decreased to less than 4% of the original number. In a study by Nehring (1996), Scrippsiella trochoidea was the dominant cyst species in the water column and very common as living cysts in sediment, but this species was relatively underrepresented as empty cysts in sediment, attributable to the fragility of these cysts compared with cysts of sporopollenin. Cysts in oyster fecal pellets showed signs of acid degradation as compared with cysts exposed to HCl. The digestive gut of the Crassostrea virginica has been reported to have a pH as low as 5.2 (Galtstoff 1964). This does not take into account additional digestive enzymes, which may be present, but can explain the effects observed.

According to Montresor et al. (1998) the high alkalinity in the Mediterranean Sea favors biomineralization and maintenance of a great diversity of specimens with calcareous covering in the sediments. In their study, calcareous cysts constituted the major part of the entire dinoflagellate cyst assemblage at stations closer to the coast where the sediment was described as silt and sand-silt; whereas, the relative importance of calcareous cysts was lower at deeper stations where the sediment was described as silt-clay, and still lower at the station with sediment described as black mud.

In a population-dynamics study on Scrippsiella spp. by Ishikawa and Taniguchi (1996) it was shown that 9.5 times more cysts were deposited on the sediment than germinated from it on a yearly basis. If this ratio is consistent, and the numbers of cysts in sediment were to remain constant over time, one would expect that 9 of 10 cysts disappear (or become unable to germinate) in different ways, for example because of grazing. Thus, our study demonstrating limited digestive destruction of S. lachrymosa cysts by feeding oysters is consistent with ecological observations indicating a higher loss rate of calcareous cysts in anoxic sediments by chemical processes than in oxygenated sediments by biological processes.

Conclusions

It can be concluded that Scrippsiella lachrymosa cysts are digested by oysters to a certain extent that is measurable with statistical significance, but the decrease is not as high as for other dinoflagellate cyst species; these calcareous cysts can thus be said to be comparatively more resistant to grazing, at least by oysters and by polychaetes as shown previously. Some animals digest Scrippsiella spp. resting cysts; whereas, others do not. It is important to remember that grazing in nature occurs constantly, not only for one day as in our experiments. Repeated grazing by different animals may, over time cause large reductions in cyst numbers, even in comparatively resistant cyst species.

It is possible that chemical processes in anoxic areas cause an even larger decrease in cyst numbers of Scrippsiella cysts than exposure to digestion by animals in oxygenated areas, as the calcareous cysts are common in oxygenated sediments but rare in anoxic sediments. Cysts in fecal pellets showed signs of acid degradation (i.e., loss of calcite, yellowing).

ACKNOWLEDGMENT

The authors thank Anke Kremp, at the Woods Hole Oceanographic Institution for providing the cultures of Scrippsiella lachrymosa along with culturing advice; Jennifer Alix, for assistance with culturing; and Dr. Gary H. Wikfors for guidance and advice. This research was performed while Dr. Agneta Persson held a National Research Council Research Associate-ship Award at the National Oceanic and Atmospheric Administration, National Marine Fisheries Service laboratory in Milford, Connecticut. Mention of trade names does not imply endorsement.

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AGNETA PERSSON (1,2) AND BARRY C. SMITH (1) *

(1) National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center, Milford Laboratory, 212 Rogers Ave., Milford, Connecticut 06460;

(2) Department of Marine Ecology, Goteborg University, Box 461, SE-405 30 Goteborg, Sweden

* Corresponding author. E-mail: [e-mail]