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Impact of grazing and bioturbation of marine benthic deposit feeders on dinoflagellate cysts
Harmful Algae 2 (2003) 43–50
Impact of grazing and bioturbation of marine benthic deposit feeders on dinoflagellate cysts Agneta Persson a,∗ , Rutger Rosenberg b a
b
Department of Marine Ecology, Göteborg University, P.O. Box 461, SE 40530 Göteborg, Sweden Department of Marine Ecology, Göteborg University, Kristineberg Marine Research Station, SE 45034 Fiskebäckskil, Sweden Received 13 December 2001; received in revised form 31 May 2002; accepted 18 December 2002
Abstract The impact of benthic deposit feeders on marine dinoflagellate cysts was studied by adding a concentrated natural Swedish cyst assemblage to sediment with different deposit feeders in replicate 4-l aquaria. The deposit feeders used were the bivalve Abra nitida, the echinoderm Amphiura filiformis, and the polychaetes Melinna cristata and Nereis diversicolor. These species occur naturally near the Swedish west coast and were selected to represent different ways of feeding. The results showed a significant relative decrease of unfossilizable cyst species; whereas, the common fossilizable species Lingulodinium polyedrum significantly increased in the cyst assemblage after grazing. This work suggests that differences in dinoflagellate cyst compositions can in part be caused by different animal grazing behaviors. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Deposit feeder; Dinoflagellate; Cyst; Sediment; Grazing
1. Introduction Dinoflagellate cysts have been known as microfossils for a long time and are used extensively in oil exploration (biostratigraphy). In the early 1960s, Wall (1965) and Wall and Dale (1966) discovered living equivalents and showed that these were resting stages of dinoflagellates. Since then, surveys of dinoflagellate cysts have been conducted in many countries, especially on toxic species (Bolch and Hallegraeff, 1990; Turgeon et al., 1990; Nehring, 1997; Sonnemann and Hill, 1997; Anderson, 1998; Persson et al., 2000), and the physiology of encystment and excystment pro∗ Corresponding author. Present address: NOAA/NMFS Milford Laboratory, 212 Rogers Avenue, Milford, CT 06460, USA. E-mail addresses: [email protected] (A. Persson), [email protected] (R. Rosenberg).
cesses have been studied (Dale, 1983 and references therein). The cysts constitute a seed bank, and the presence of cysts from toxic species can provide vegetative stages for yearly outbreaks of red tides (Laroque and Cembella, 1990; Anderson, 1997). The outer walls of sporopollenin-walled cyst species can be preserved for millions of years (Bolch and Hallegraeff, 1990). However, not all dinoflagellate cysts are fossilizable (Wall and Dale, 1968; Dodge, 1982; Dale, 1983; Anderson et al., 1985). Viable dinoflagellate cysts are frequently found in copepod pellets (Reid and Boalch, 1987) and feces from shellfish (Bravo et al., 1998). For a discussion on the possibilities of cyst predation and references on the topic, see Persson (2000). Benthic deposit feeders have not previously been studied from the aspect of their influence on the benthic resting stages of protists. The food quality for deposit feeders is variable over the year. In seasons
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with sedimenting phytoplankton blooms, food is ubiquitous, but in the winter months freshly deposited food is sparse. The benthic fauna consists of many species with different feeding modes. Deposit feeders rework the sediment surface more or less continuously and are likely to have a significant impact on the sediment geochemistry, including the distribution of dinoflagellate cysts and pellets (Taghon et al., 1984). Deposit feeders typically process at least the equivalent of one body weight of sediment daily (Lopez and Levinton, 1987). Viable resting cysts of dinoflagellates contain a nutrient supply composed of oil and starch (Dale, 1983). This would be a valuable, high-energy food provided the deposit feeders could extract it in some quantity. In this experiment we studied the impact deposit feeders have on the species composition of a dinoflagellate cyst assemblage, concentrated from natural Swedish sediments.
2. Materials and methods The experiment was conducted at Kristineberg Marine Biological Research Station, Sweden, in August and September 2000. 2.1. Sampling The bivalve Abra nitida (Müller), the brittle star Amphiura filiformis (Müller) and the tube-dwelling polychaete Melinna cristata (Sars) were collected in the Gullmarsfjord area (58◦ 15.80 N, 11◦ 25.97 E to 58◦ 15.90 N, 11◦ 25.97 E) at 36–50 m depth. The sediment was sieved to remove all organisms larger than 0.5 mm. The sieved sediment, to be used as substrate in the experiment, was kept in a large container and thoroughly mixed. Subsamples were taken for determination of the cyst content (stored in darkness at 4 ◦ C until end of experiment). Animals to be used in the experiment were placed in buckets or aquaria with 34‰ running water or aeration. The free-living polychaete Nereis diversicolor (Müller) was collected in shallow waters outside the research station. For obtaining a dinoflagellate cyst assembly with enough cysts, a large amount of sediment had to be sieved. The cysts were concentrated by sieving sediment in 34‰ filtered seawater, and the 25–100 m fraction was used in the experiment. The origin of this
“dinoflagellate cyst fraction” was a mixture of 90% anoxic surface sediment (58◦ 15.10 N, 11◦ 29.85 E, 16–17 m depth) and 10% oxic surface sediment (58◦ 15.59 N, 11◦ 26.08 E, 75 m depth). Subsamples of the thoroughly mixed cyst fraction for determination of start values were taken and stored in darkness, 4 ◦ C until end of experiment. 2.2. Experiment Twenty-four 4-l aquaria (bottom area 195 mm × 100 mm, hight 200 mm) were used in the experiment. The amount of sieved sediment used was 1 l for A. nitida, A. filiformis, N. diversicolor and Control, and 2 l for M. cristata since their tubes were about 15 cm long. Filtered seawater was added to make the total volume 3 l. Each aquarium was aerated. We used five aquaria each for A. nitida, A. filiformis, N. diversicolor and Control, and three for M. cristata (plus one 2-l sediment Control). The animals were counted, weighed and added to the aquaria to acclimatize 5 days before the start of experiment. The aquaria were placed in a constant temperature room at 8.5 ◦ C in darkness. At the start of the experiment, the aeration was turned off and 69 ml of the 25–100 m “dinoflagellate cyst fraction” was added to each aquarium. The dinoflagellate cyst fraction spread evenly and sedimented as an approximately 4 mm thick black layer on top of the other sediment. When the cyst fraction had sedimented, the aeration was turned on again. The deposit feeders were allowed to feed from the cyst fraction for 37 days. The aquaria were observed daily. At the end of the experiment, in all aquaria with A. nitida, M. cristata and N. diversicolor fecal pellets were carefully picked up with a Pasteur pipette. Within each aquarium the pellet samples were pooled. In aquaria with A. filiformis subsamples (five per aquaria) were taken with cut-off 5 ml syringes and divided into fractions from 0 to 1, 1 to 2, 2 to 3 and 3 to 4 cm depth in the sediment. All five samples from the same depths were mixed together resulting in one pooled sample for each depth in each aquarium. In the Control aquaria, surface sediment samples were taken with Pasteur pipettes for comparison with the animals that made pellets (A. nitida, M. cristata and N. diversicolor), and samples were taken with cut-off 5 ml syringes for comparison with A. filiformis. The volume
A. Persson, R. Rosenberg / Harmful Algae 2 (2003) 43–50
of each pellet sample was measured by marking the surface of the sample with a pencil on the test-tube (after sedimenting over night in the constant temperature room in darkness). After sampling with the syringes, the aquaria were emptied in a sieve, and animals were counted and weighed. Microscope slides for cyst counting were made quantitatively according to Persson et al. (2000). A Leitz diaplan microscope was used. Microscope slides were prepared with pellet material from each aquarium of A. nitida and M. cristata. Cysts from every aquarium with animals, and from the surface sediment in the Controls were counted. For A. filiformis and the samples taken with syringes in the Control, “depths” slides were made from samples in one aquarium for each treatment. After counting two slides from each depth, we concluded that the uppermost layer was most affected, and further counting was done on that (0–1 cm) layer only. For N. diversicolor, slides from the pellet sample were counted. Replicate slides from initial samples for the added cyst fraction and from the sieved sediment were also counted. At least 100 cysts were counted per slide. We recorded if cysts had living looking contents, were with contents, whole empty, collapsed, broken or germinated (with archaeopyle), but all were counted—as they would have been in a dinoflagellate cyst survey. Statistics (one-way analysis of √ variance) were made on transformed (x = arcsin x) values according to Underwood (1997), since values were proportions.
3. Results 3.1. The deposit feeders 3.1.1. Visual observations The animals showed no adverse reaction when adding the cyst-sediment suspension; rather, they started to feed. The added cyst-sediment suspension was black at the start but was slowly oxidized. After 1 day, approximately half of this layer had turned to a brown color in all aquaria. At the end of the experiment the surface was oxidized down to approximately 4 mm, while parts of the cyst fraction if present below that depth, were still black. There were no differences (visual or otherwise) between the replicates within
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Table 1 The weight of animals used in the experiment Species
Weight before (g per aquarium)
Weight after (g per aquarium)
Abra nitida Amphiura filiformis Nereis diversicolor
1.32 ± 0.15 1.66 ± 0.30 1.56 ± 0.27
1.44 ± 0.17 1.46 ± 0.11 1.46 ± 0.15
The tube-dwelling Melinna cristata was not weighed. Standard deviations are shown.
each treatment. No significant changes in the weights of animals were seen (Table 1). A. nitida: Animals fed on the sediment surface, picked food with the siphon and moved around in the sediment. The entire sediment surface seemed to be pelletized at the end of experiment. The most clearly differentiated pellets were sampled; these pellets were like small hard oval balls and could be rinsed with seawater without being dissolved. M. cristata: Animals clearly fed on the surface sediment with their tentacles. The surface layer (cyst fraction) was consumed to a radius of approximately 3 cm around each animal tube. Pellets were loose but very distinct and could be picked without touching the underlying sediment. Nereis diversicolor: The animals made burrows throughout the sediment, but there were no indications of the surface being more touched than the underlying sediment. When adding the cyst fraction at the start of the experiment, some of that material fell down into the burrows already made. Pellets were found only in one aquarium. Amphiura filiformis: Animals clearly fed on the surface sediment, rapidly transporting it down along the arms to the disc at 3–4 cm below the surface. Material was also transported up to the sediment surface and mounds were made. Thus, holes and mounds were frequent in the used areas of the aquaria. Parts of the aquaria seemed totally untouched and looked like the Control aquaria. Samples were taken only from the areas with animal activity. 3.1.2. Microscopic inspections of pellets and Control sediment A microscopic examination of the sampled sediment material was made prior to the preparation of slides. There was a great visual difference between the pellet material of A. nitida and M. cristata versus
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the surface sediment collected in the Control aquaria at the end of the experiment. The microscopic examination of the pellets revealed a “sterile” environment with very little life whereas the surface sediment of the Control showed a multitude of various living organisms: several ciliate species, several diatom species and small flagellated protists. No vegetative stages of dinoflagellates were observed. Unidentifiable detritus material and copepod pellets of the size 25–100 m were still present in the Control. The pellets of A. nitida and M. cristata were composed predominantly of particles of very small size (<1 m) although the added food items were all in the size 25–100 m. Both empty and viable dinoflagellate cysts were seen in both pellet and Control samples. In the Control aquaria the surface sediment was untouched but oxidized (based on the color) down to 4 mm depth.
3.2. Changes in cyst composition There was a significant change in the species composition of dinoflagellate cysts (Figs. 1 and 2). The proportion of uncolored, smooth-walled cysts decreased significantly; whereas, the proportion of cysts that are well known from fossil records increased significantly. Lingulodinium polyedrum (Stein) Dodge (paleontological name Lingulodinium machaerophorum): There was no significant difference between initial values and Controls in relative occurrence of L. polyedrum. A significant (P < 0.02) increase in percentage of L. polyedrum cysts was found for A. nitida (Fig. 1). Grazing by M. cristata resulted in a non-significant increase (P = 0.13) (Fig. 1). Grazing by A. filiformis significantly (P < 0.02) increased the percentage of L. polyedrum in the surface sediment (Fig. 2).
Fig. 1. Species composition of cysts in aquaria with Abra nitida, Melinna cristata, Control and start (initial values). Values are averages from five slides for each of A. nitida, M. cristata and Control, and six slides from start.
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Fig. 2. Species composition of cysts in the upper 1 cm sediment in aquaria with Amphiura filiformis and Control. Values are averages from six slides per treatment.
P. dalei Indelicato et Loeblich III: There was a significant (P < 0.02) increase after grazing by A. nitida and the same tendency (ns, P = 0.13) with M. cristata (Fig. 1). There was no significant difference between A. filiformis and its Control (Fig. 2). P. reticulatum (Claparède et Lachmann) Bütschli (paleontological name Operculodinium centrocarpum): There was significantly (P < 0.01) more P. reticulatum cysts in A. filiformis compared to its Control (Fig. 2). No significant differences were found between start and Control or for A. nitida and M. cristata versus Control (Fig. 1). Spiniferites spp.: These were present in the cyst assemblage, but in too low a proportion to provide reliable statistics. There was, however, a tendency towards increasing percentage after grazing (Fig. 1). Grazing by A. filiformis did not change the percentage Spiniferites spp. (Fig. 2). Scrippsiella spp.: No significant changes were noted for initial versus Control or A. nitida and M.
cristata versus Control (there was a tendency towards reduced levels of Scrippsiella spp. by grazing, but the proportion was too low to provide reliable statistics). A. filiformis had a significantly lower (P = 0.01) percentage of Scrippsiella spp. than its Control (Fig. 2). Brown cysts (Protoperidinium and Diplopsalis-like genera, heterotrophic dinoflagellates): There were no significant differences between initial and Control values. There was a significant (P < 0.01) decrease in brown cysts after grazing by A. nitida and M. cristata, as compared to the Control (Fig. 1 and Table 1). Grazing by A. filiformis caused a near significant decrease in percentage of brown cysts (P = 0.06, Fig. 2). Smooth colorless cysts (a large part were unidentified cysts, but also included were Gonyaulax verior Sournia, Fragilidium spp. and Alexandrium spp.): These were the only cysts for which there was a significant difference between initial values and Control (Fig. 1). The percentage of uncolored cysts decreased from 18 to 10% (P = 0.02) in the Control.
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The decreased percentage of smooth colorless cysts was significant for both A. nitida and M. cristata (Fig. 1) compared to the Control, whereas there was no difference between A. filiformis and its Control. 3.2.1. Changes in cyst concentration During cyst counting in the microscope, we observed that a considerable amount of unidentifiable detritus was present in initial samples whereas the pellet material was deprived of unidentifiable detritus material and consisted mainly of mineral particles, pollen grains and different kinds of resting stages.
4. Discussion Microscopic examination of pellets and Control surface sediment indicated a strong “top–down” Control of deposit feeders on protists in marine sediments. In slides from Control and initial cyst fraction, there was more unidentifiable material (“detritus”) than in the pellets, and at the start there were very few cysts compared to the treatments at the end. The unidentifiable detritus material seemed to be easily degradable (labile) material, compared to the cysts, and can be assumed to have been the main food of the animals during the experiment. We had no possibilities in this experiment to determine if the cysts were avoided or selected as food items. We have to assume that the “food” we added was eaten as it was. In the aquaria at the end of the experiment, the entire surface sediment in A. nitida treatment seemed pelletized and in the aquaria with M. cristata the surface sediment was excavated to a radius of ca. 3 cm around each animal tube. A. filiformis was active only in parts of the aquaria, and here sections of the used material were analyzed. Even if the cysts were not “regarded as” food by the benthic fauna, the cysts were clearly affected by their activities. 4.1. Changes in cyst composition L. polyedrum: This is a highly resistant species well known as a microfossil, and is well studied in many aspects. It is known to be preserved for long times in the sediment. Lewis et al. (1999) germinated L. polyedrum from cysts in sediment stored for 9 years in a refrigerator. The L. polyedrum cysts used in our experiment
are likely to have been at least 1-year old because sediments were taken in early August, and according to Kuylenstierna (personal communication), a L. polyedrum bloom, followed by cyst sedimentation, occurs in September each year. L. polyedrum dominated the cyst assemblage in this experiment and has been shown previously, to be dominant among dinoflagellate cysts at the Swedish west coast (Persson et al., 2000). Cysts of P. dalei, P. reticulatum and Spiniferites spp. are common microfossils. These cysts showed a significant relative increase for some of the deposit feeders used, and in no instances any decrease (Figs. 1 and 2). Scrippsiella spp. cysts have a calcareous outer wall and should thus theoretically be sensitive to acids produced in the fermentation process in anoxic sediments. Previous observations have shown (Persson, personal observation) that the outer wall of living Scrippsiella cysts was dissolved in anoxic sediment. In a survey of calcareous cysts in the Gulf of Naples calcareous cysts were frequently observed in sediments of sand, silt and clay, but seldom in black mud (Montresor et al., 1994). Scrippsiella spp. cysts in this experiment were not affected by the digestion process in A. nitida or M. cristata, but were affected by A. filiformis. This possibly reflects the differences in digestion processes of the animals. The “brown cysts” were predominantly Protoperidinium and Diplopsalis-like species. In this experiment, brown cysts were susceptible to degradation by the activity of deposit feeding animals. Zonneveld et al. (1997) found remarkable differences in the preservation of brown cyst species in different oxygen regimes. Of those found in anoxic sediments only 0.3% occurred in oxidized ones although the origin of the sediments was thought to be the same. Smooth, uncolored cysts showed a significant reduction, both from Control compared to initial and for the treatments with animals compared to Control, indicating that they are either very easily germinated, and the wall then disintegrated, or that they are destroyed by microbial action and by animal activity. Small, naked dinoflagellates often have small and inconspicuous cysts (which were not included in this experiment if they were smaller than 25 m), but such cysts are common in nature (Persson, personal observation). Cysts of the potentially toxic species of the genus Alexandrium were far too few in this experiment
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for us to have the possibility to draw any conclusions about their susceptibility to benthic animal activity. It is difficult to believe that dinoflagellate cysts could constitute a valuable food source for “bottom fauna”. Dinoflagellate cysts are generally a small part of the total sedimenting material (<1‰), although periodically (at the end of a dinoflagellate bloom) they are more common (Nehring, 1996; Montresor et al., 1998). Resting cells of many kinds are present in the sediment and can theoretically give nourishment to benthic animals during periods of starvation or during short periods of massive sedimentation of resting stages. Some of these resting stages are very resistant whereas some are fairly easily disintegrated. In nature, a repeated consumption on the same sediment particles occurs, by different species, one feeding on the fecal pellets of others (Taghon et al., 1984). The cysts may be eaten many times, also the empty walls. With time, fossilizable cysts are concentrated and sensitive cysts presumably are consumed. The deposit feeders used in this study (A. nitida, M. cristata, A. filiformis and N. diversicolor) have different modes of feeding and affect the cyst assemblages differently. Thus differences in the “cyst bank” might depend both on spatial differences in the composition and abundance of the fauna as well as on differences in cyst production and accumulation. This leads to difficulties in interpreting the differences in cyst assemblages between places or countries. It is possible that some cysts are relatively resistant to the digestion processes of many animals, but are not preserved for a long time in an anoxic environment (as can be hypothesized for cysts with an outer wall of calcareous material, since acids are produced by anaerobic digestion, and acids are known to dissolve calcium). Other species that survive for several years in anoxic sediments may be rapidly consumed/digested when present in oxic environments (brown cysts). In nature, most cysts are deposited in oxygenated environments (i.e. with fauna) since most marine environments are oxic at the surface (Chester, 1990). There is a need for more grazing experiments on dinoflagellate cysts and especially on living cysts of harmful species. 5. Conclusions Benthic animal activity resulted in a changed species composition of dinoflagellate cysts with a
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significant relative increase of fossilizable species while the percentage of sensitive species decreased significantly. The different benthic animals affected the dinoflagellate species composition slightly differently, indicating that differences in cyst seed banks in part can be attributable to the species composition of deposit feeding animals.
Acknowledgements We want to thank Dr. Barrie Dale for valuable comments on an earlier version of the manuscript. We are grateful to Berne Petersson and Sylve Robertsson on R/V Oscar von Sydow for being helpful at sampling and to Gunnar Persson for assistance in the time-consuming work of concentrating cysts. Financial support was provided by Kapten C. Stenholms foundation and the Hierta-Retzius foundation. References Anderson, D.M., 1997. Diversity of harmful algal blooms in coastal waters. Limnol. Oceanogr. 42, 1009–1022. Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transitions. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Heidelberg, pp. 29–48. Anderson, D.M., Lively, J.J., Reardon, E.M., Price, C.A., 1985. Sinking characteristics of dinoflagellate cysts. Limnol. Oceanogr. 30, 1000–1009. Bolch, C.J.S., Hallegraeff, G.M., 1990. Dinoflagellate cysts in recent marine sediments from Tasmania, Australia. Bot. Mar. 33, 173–192, and references therein. Bravo, I., Franco, J.M., Reyero, M.I., 1998. PSP toxin composition of three life cycle stages of Gymnodinium catenatum. In: Reguera, B., Blanco, J., Fernández, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and IOC of UNESCO, Grafisant, Santiago de Compostela, pp. 356–358, and references therein. Chester, R., 1990. Marine Geochemistry. Allen & Unwin, London. Dale, B., 1983. Dinoflagellate resting cysts: benthic plankton. In: Fryxell, G.A. (Ed.), Survival Strategies of the Algae. Cambridge University Press, Cambridge, pp. 69–136, and references therein. Dodge, J.D., 1982. Marine Dinoflagellates of the British Isles. Her Majesty’s Stationery Office, London. Laroque, R., Cembella, A.D., 1990. Ecological parameters associated with the seasonal occurrence of Alexandrium spp. and consequent shellfish toxicity in the lower St. Lawrence estuary (eastern Canada). In: Graneli, E., Sundström, L.,
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Edler, L., Anderson, D.M. (Eds.), Toxic Marine Phytoplankton. Elsevier, New York, pp. 368–373. Lewis, J., Harris, A.S.D., Jones, K.J., Edmonds, R.L., 1999. Long-term survival of marine planktonic diatoms and dinoflagellates in stored sediment samples. J. Plankton Res. 21, 343–354. Lopez, G.R., Levinton, J.S., 1987. Ecology of deposit-feeding animals in marine sediment. Q. Rev. Biol. 62, 235– 260. Montresor, M., Montesarchio, E., Marino, D., Zingone, A., 1994. Calcareous dinoflagellate cysts in marine sediments of the Gulf of Naples (Mediterranean Sea). Rev. Palaeobot. Palynol. 84, 45–56. Montresor, M., Zingone, A., Sarno, D., 1998. Dinoflagellate cyst production at a coastal Mediterranean site. J. Plankton Res. 20, 2291–2312. Nehring, S., 1996. Recruitment of planktonic dinoflagellates: importance of benthic resting stages and resuspension events. Int. Revue Ges. Hydrobiol. 81, 513–527. Nehring, S., 1997. Dinoflagellate resting cysts from recent German coastal sediment. Bot. Mar. 40, 307–324. Persson, A., 2000. Possible predation of cysts—a gap in the knowledge of dinoflagellate ecology? J. Plankton Res. 22, 803– 809. Persson, A., Godhe, A., Karlson, B., 2000. Dinoflagellate cysts in recent sediments from the west coast of Sweden. Bot. Mar. 43, 69–79.
Reid, C.R., Boalch, G.T., 1987. A new method for the identification of dinoflagellate cysts. J. Plankton Res. 9, 249–253. Sonnemann, J.A., Hill, D.R.A., 1997. A taxonomic survey of cyst-producing dinoflagellates from recent sediments of Victorian coastal waters, Australia. Bot. Mar. 40, 147–177. Taghon, G., Nowell, A.R.M., Jumars, P.A., 1984. Transport and breakdown of fecal pellets: biological and sedimentological consequences. Limnol. Oceanogr. 29, 64–72. Turgeon, J., Cembella, A.D., Therriault, J.C., Beland, P., 1990. Spatial distribution of resting cysts of Alexandrium spp. in sediments of the lower St. Lawrence estuary and the Gaspé coast (eastern Canada). In: Graneli, E., Sundström, L., Edler, L., Anderson, D.M. (Eds.), Toxic Marine Phytoplankton. Elsevier, New York, pp. 238–243. Underwood, A.J., 1997. Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance. Cambridge University Press, Cambridge, 528 pp. Wall, D., 1965. Modern hystrichospheres and dinoflagellate cysts from the woods hole region. Grana Palynol. 6, 297–314. Wall, D., Dale, B., 1966. ‘Living fossils’ in western Atlantic plankton. Nature 211, 1025–1026. Wall, D., Dale, B., 1968. Modern dinoflagellate cysts and evolution of the peridinales. Micropaleontology 14, 265–304. Zonneveld, K.A.F., Versteegh, G.J.M., de Lange, G.J., 1997. Preservation of organic-walled dinoflagellate cysts in different oxygen regimes: a 10,000 year natural experiment. Mar. Micropaleontol. 29, 393–405.
Impact of grazing and bioturbation of marine benthic deposit feeders on dinoflagellate cysts Agneta Persson a,∗ , Rutger Rosenberg b a
b
Department of Marine Ecology, Göteborg University, P.O. Box 461, SE 40530 Göteborg, Sweden Department of Marine Ecology, Göteborg University, Kristineberg Marine Research Station, SE 45034 Fiskebäckskil, Sweden Received 13 December 2001; received in revised form 31 May 2002; accepted 18 December 2002
Abstract The impact of benthic deposit feeders on marine dinoflagellate cysts was studied by adding a concentrated natural Swedish cyst assemblage to sediment with different deposit feeders in replicate 4-l aquaria. The deposit feeders used were the bivalve Abra nitida, the echinoderm Amphiura filiformis, and the polychaetes Melinna cristata and Nereis diversicolor. These species occur naturally near the Swedish west coast and were selected to represent different ways of feeding. The results showed a significant relative decrease of unfossilizable cyst species; whereas, the common fossilizable species Lingulodinium polyedrum significantly increased in the cyst assemblage after grazing. This work suggests that differences in dinoflagellate cyst compositions can in part be caused by different animal grazing behaviors. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Deposit feeder; Dinoflagellate; Cyst; Sediment; Grazing
1. Introduction Dinoflagellate cysts have been known as microfossils for a long time and are used extensively in oil exploration (biostratigraphy). In the early 1960s, Wall (1965) and Wall and Dale (1966) discovered living equivalents and showed that these were resting stages of dinoflagellates. Since then, surveys of dinoflagellate cysts have been conducted in many countries, especially on toxic species (Bolch and Hallegraeff, 1990; Turgeon et al., 1990; Nehring, 1997; Sonnemann and Hill, 1997; Anderson, 1998; Persson et al., 2000), and the physiology of encystment and excystment pro∗ Corresponding author. Present address: NOAA/NMFS Milford Laboratory, 212 Rogers Avenue, Milford, CT 06460, USA. E-mail addresses: [email protected] (A. Persson), [email protected] (R. Rosenberg).
cesses have been studied (Dale, 1983 and references therein). The cysts constitute a seed bank, and the presence of cysts from toxic species can provide vegetative stages for yearly outbreaks of red tides (Laroque and Cembella, 1990; Anderson, 1997). The outer walls of sporopollenin-walled cyst species can be preserved for millions of years (Bolch and Hallegraeff, 1990). However, not all dinoflagellate cysts are fossilizable (Wall and Dale, 1968; Dodge, 1982; Dale, 1983; Anderson et al., 1985). Viable dinoflagellate cysts are frequently found in copepod pellets (Reid and Boalch, 1987) and feces from shellfish (Bravo et al., 1998). For a discussion on the possibilities of cyst predation and references on the topic, see Persson (2000). Benthic deposit feeders have not previously been studied from the aspect of their influence on the benthic resting stages of protists. The food quality for deposit feeders is variable over the year. In seasons
1568-9883/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1568-9883(03)00003-9
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with sedimenting phytoplankton blooms, food is ubiquitous, but in the winter months freshly deposited food is sparse. The benthic fauna consists of many species with different feeding modes. Deposit feeders rework the sediment surface more or less continuously and are likely to have a significant impact on the sediment geochemistry, including the distribution of dinoflagellate cysts and pellets (Taghon et al., 1984). Deposit feeders typically process at least the equivalent of one body weight of sediment daily (Lopez and Levinton, 1987). Viable resting cysts of dinoflagellates contain a nutrient supply composed of oil and starch (Dale, 1983). This would be a valuable, high-energy food provided the deposit feeders could extract it in some quantity. In this experiment we studied the impact deposit feeders have on the species composition of a dinoflagellate cyst assemblage, concentrated from natural Swedish sediments.
2. Materials and methods The experiment was conducted at Kristineberg Marine Biological Research Station, Sweden, in August and September 2000. 2.1. Sampling The bivalve Abra nitida (Müller), the brittle star Amphiura filiformis (Müller) and the tube-dwelling polychaete Melinna cristata (Sars) were collected in the Gullmarsfjord area (58◦ 15.80 N, 11◦ 25.97 E to 58◦ 15.90 N, 11◦ 25.97 E) at 36–50 m depth. The sediment was sieved to remove all organisms larger than 0.5 mm. The sieved sediment, to be used as substrate in the experiment, was kept in a large container and thoroughly mixed. Subsamples were taken for determination of the cyst content (stored in darkness at 4 ◦ C until end of experiment). Animals to be used in the experiment were placed in buckets or aquaria with 34‰ running water or aeration. The free-living polychaete Nereis diversicolor (Müller) was collected in shallow waters outside the research station. For obtaining a dinoflagellate cyst assembly with enough cysts, a large amount of sediment had to be sieved. The cysts were concentrated by sieving sediment in 34‰ filtered seawater, and the 25–100 m fraction was used in the experiment. The origin of this
“dinoflagellate cyst fraction” was a mixture of 90% anoxic surface sediment (58◦ 15.10 N, 11◦ 29.85 E, 16–17 m depth) and 10% oxic surface sediment (58◦ 15.59 N, 11◦ 26.08 E, 75 m depth). Subsamples of the thoroughly mixed cyst fraction for determination of start values were taken and stored in darkness, 4 ◦ C until end of experiment. 2.2. Experiment Twenty-four 4-l aquaria (bottom area 195 mm × 100 mm, hight 200 mm) were used in the experiment. The amount of sieved sediment used was 1 l for A. nitida, A. filiformis, N. diversicolor and Control, and 2 l for M. cristata since their tubes were about 15 cm long. Filtered seawater was added to make the total volume 3 l. Each aquarium was aerated. We used five aquaria each for A. nitida, A. filiformis, N. diversicolor and Control, and three for M. cristata (plus one 2-l sediment Control). The animals were counted, weighed and added to the aquaria to acclimatize 5 days before the start of experiment. The aquaria were placed in a constant temperature room at 8.5 ◦ C in darkness. At the start of the experiment, the aeration was turned off and 69 ml of the 25–100 m “dinoflagellate cyst fraction” was added to each aquarium. The dinoflagellate cyst fraction spread evenly and sedimented as an approximately 4 mm thick black layer on top of the other sediment. When the cyst fraction had sedimented, the aeration was turned on again. The deposit feeders were allowed to feed from the cyst fraction for 37 days. The aquaria were observed daily. At the end of the experiment, in all aquaria with A. nitida, M. cristata and N. diversicolor fecal pellets were carefully picked up with a Pasteur pipette. Within each aquarium the pellet samples were pooled. In aquaria with A. filiformis subsamples (five per aquaria) were taken with cut-off 5 ml syringes and divided into fractions from 0 to 1, 1 to 2, 2 to 3 and 3 to 4 cm depth in the sediment. All five samples from the same depths were mixed together resulting in one pooled sample for each depth in each aquarium. In the Control aquaria, surface sediment samples were taken with Pasteur pipettes for comparison with the animals that made pellets (A. nitida, M. cristata and N. diversicolor), and samples were taken with cut-off 5 ml syringes for comparison with A. filiformis. The volume
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of each pellet sample was measured by marking the surface of the sample with a pencil on the test-tube (after sedimenting over night in the constant temperature room in darkness). After sampling with the syringes, the aquaria were emptied in a sieve, and animals were counted and weighed. Microscope slides for cyst counting were made quantitatively according to Persson et al. (2000). A Leitz diaplan microscope was used. Microscope slides were prepared with pellet material from each aquarium of A. nitida and M. cristata. Cysts from every aquarium with animals, and from the surface sediment in the Controls were counted. For A. filiformis and the samples taken with syringes in the Control, “depths” slides were made from samples in one aquarium for each treatment. After counting two slides from each depth, we concluded that the uppermost layer was most affected, and further counting was done on that (0–1 cm) layer only. For N. diversicolor, slides from the pellet sample were counted. Replicate slides from initial samples for the added cyst fraction and from the sieved sediment were also counted. At least 100 cysts were counted per slide. We recorded if cysts had living looking contents, were with contents, whole empty, collapsed, broken or germinated (with archaeopyle), but all were counted—as they would have been in a dinoflagellate cyst survey. Statistics (one-way analysis of √ variance) were made on transformed (x = arcsin x) values according to Underwood (1997), since values were proportions.
3. Results 3.1. The deposit feeders 3.1.1. Visual observations The animals showed no adverse reaction when adding the cyst-sediment suspension; rather, they started to feed. The added cyst-sediment suspension was black at the start but was slowly oxidized. After 1 day, approximately half of this layer had turned to a brown color in all aquaria. At the end of the experiment the surface was oxidized down to approximately 4 mm, while parts of the cyst fraction if present below that depth, were still black. There were no differences (visual or otherwise) between the replicates within
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Table 1 The weight of animals used in the experiment Species
Weight before (g per aquarium)
Weight after (g per aquarium)
Abra nitida Amphiura filiformis Nereis diversicolor
1.32 ± 0.15 1.66 ± 0.30 1.56 ± 0.27
1.44 ± 0.17 1.46 ± 0.11 1.46 ± 0.15
The tube-dwelling Melinna cristata was not weighed. Standard deviations are shown.
each treatment. No significant changes in the weights of animals were seen (Table 1). A. nitida: Animals fed on the sediment surface, picked food with the siphon and moved around in the sediment. The entire sediment surface seemed to be pelletized at the end of experiment. The most clearly differentiated pellets were sampled; these pellets were like small hard oval balls and could be rinsed with seawater without being dissolved. M. cristata: Animals clearly fed on the surface sediment with their tentacles. The surface layer (cyst fraction) was consumed to a radius of approximately 3 cm around each animal tube. Pellets were loose but very distinct and could be picked without touching the underlying sediment. Nereis diversicolor: The animals made burrows throughout the sediment, but there were no indications of the surface being more touched than the underlying sediment. When adding the cyst fraction at the start of the experiment, some of that material fell down into the burrows already made. Pellets were found only in one aquarium. Amphiura filiformis: Animals clearly fed on the surface sediment, rapidly transporting it down along the arms to the disc at 3–4 cm below the surface. Material was also transported up to the sediment surface and mounds were made. Thus, holes and mounds were frequent in the used areas of the aquaria. Parts of the aquaria seemed totally untouched and looked like the Control aquaria. Samples were taken only from the areas with animal activity. 3.1.2. Microscopic inspections of pellets and Control sediment A microscopic examination of the sampled sediment material was made prior to the preparation of slides. There was a great visual difference between the pellet material of A. nitida and M. cristata versus
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the surface sediment collected in the Control aquaria at the end of the experiment. The microscopic examination of the pellets revealed a “sterile” environment with very little life whereas the surface sediment of the Control showed a multitude of various living organisms: several ciliate species, several diatom species and small flagellated protists. No vegetative stages of dinoflagellates were observed. Unidentifiable detritus material and copepod pellets of the size 25–100 m were still present in the Control. The pellets of A. nitida and M. cristata were composed predominantly of particles of very small size (<1 m) although the added food items were all in the size 25–100 m. Both empty and viable dinoflagellate cysts were seen in both pellet and Control samples. In the Control aquaria the surface sediment was untouched but oxidized (based on the color) down to 4 mm depth.
3.2. Changes in cyst composition There was a significant change in the species composition of dinoflagellate cysts (Figs. 1 and 2). The proportion of uncolored, smooth-walled cysts decreased significantly; whereas, the proportion of cysts that are well known from fossil records increased significantly. Lingulodinium polyedrum (Stein) Dodge (paleontological name Lingulodinium machaerophorum): There was no significant difference between initial values and Controls in relative occurrence of L. polyedrum. A significant (P < 0.02) increase in percentage of L. polyedrum cysts was found for A. nitida (Fig. 1). Grazing by M. cristata resulted in a non-significant increase (P = 0.13) (Fig. 1). Grazing by A. filiformis significantly (P < 0.02) increased the percentage of L. polyedrum in the surface sediment (Fig. 2).
Fig. 1. Species composition of cysts in aquaria with Abra nitida, Melinna cristata, Control and start (initial values). Values are averages from five slides for each of A. nitida, M. cristata and Control, and six slides from start.
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Fig. 2. Species composition of cysts in the upper 1 cm sediment in aquaria with Amphiura filiformis and Control. Values are averages from six slides per treatment.
P. dalei Indelicato et Loeblich III: There was a significant (P < 0.02) increase after grazing by A. nitida and the same tendency (ns, P = 0.13) with M. cristata (Fig. 1). There was no significant difference between A. filiformis and its Control (Fig. 2). P. reticulatum (Claparède et Lachmann) Bütschli (paleontological name Operculodinium centrocarpum): There was significantly (P < 0.01) more P. reticulatum cysts in A. filiformis compared to its Control (Fig. 2). No significant differences were found between start and Control or for A. nitida and M. cristata versus Control (Fig. 1). Spiniferites spp.: These were present in the cyst assemblage, but in too low a proportion to provide reliable statistics. There was, however, a tendency towards increasing percentage after grazing (Fig. 1). Grazing by A. filiformis did not change the percentage Spiniferites spp. (Fig. 2). Scrippsiella spp.: No significant changes were noted for initial versus Control or A. nitida and M.
cristata versus Control (there was a tendency towards reduced levels of Scrippsiella spp. by grazing, but the proportion was too low to provide reliable statistics). A. filiformis had a significantly lower (P = 0.01) percentage of Scrippsiella spp. than its Control (Fig. 2). Brown cysts (Protoperidinium and Diplopsalis-like genera, heterotrophic dinoflagellates): There were no significant differences between initial and Control values. There was a significant (P < 0.01) decrease in brown cysts after grazing by A. nitida and M. cristata, as compared to the Control (Fig. 1 and Table 1). Grazing by A. filiformis caused a near significant decrease in percentage of brown cysts (P = 0.06, Fig. 2). Smooth colorless cysts (a large part were unidentified cysts, but also included were Gonyaulax verior Sournia, Fragilidium spp. and Alexandrium spp.): These were the only cysts for which there was a significant difference between initial values and Control (Fig. 1). The percentage of uncolored cysts decreased from 18 to 10% (P = 0.02) in the Control.
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The decreased percentage of smooth colorless cysts was significant for both A. nitida and M. cristata (Fig. 1) compared to the Control, whereas there was no difference between A. filiformis and its Control. 3.2.1. Changes in cyst concentration During cyst counting in the microscope, we observed that a considerable amount of unidentifiable detritus was present in initial samples whereas the pellet material was deprived of unidentifiable detritus material and consisted mainly of mineral particles, pollen grains and different kinds of resting stages.
4. Discussion Microscopic examination of pellets and Control surface sediment indicated a strong “top–down” Control of deposit feeders on protists in marine sediments. In slides from Control and initial cyst fraction, there was more unidentifiable material (“detritus”) than in the pellets, and at the start there were very few cysts compared to the treatments at the end. The unidentifiable detritus material seemed to be easily degradable (labile) material, compared to the cysts, and can be assumed to have been the main food of the animals during the experiment. We had no possibilities in this experiment to determine if the cysts were avoided or selected as food items. We have to assume that the “food” we added was eaten as it was. In the aquaria at the end of the experiment, the entire surface sediment in A. nitida treatment seemed pelletized and in the aquaria with M. cristata the surface sediment was excavated to a radius of ca. 3 cm around each animal tube. A. filiformis was active only in parts of the aquaria, and here sections of the used material were analyzed. Even if the cysts were not “regarded as” food by the benthic fauna, the cysts were clearly affected by their activities. 4.1. Changes in cyst composition L. polyedrum: This is a highly resistant species well known as a microfossil, and is well studied in many aspects. It is known to be preserved for long times in the sediment. Lewis et al. (1999) germinated L. polyedrum from cysts in sediment stored for 9 years in a refrigerator. The L. polyedrum cysts used in our experiment
are likely to have been at least 1-year old because sediments were taken in early August, and according to Kuylenstierna (personal communication), a L. polyedrum bloom, followed by cyst sedimentation, occurs in September each year. L. polyedrum dominated the cyst assemblage in this experiment and has been shown previously, to be dominant among dinoflagellate cysts at the Swedish west coast (Persson et al., 2000). Cysts of P. dalei, P. reticulatum and Spiniferites spp. are common microfossils. These cysts showed a significant relative increase for some of the deposit feeders used, and in no instances any decrease (Figs. 1 and 2). Scrippsiella spp. cysts have a calcareous outer wall and should thus theoretically be sensitive to acids produced in the fermentation process in anoxic sediments. Previous observations have shown (Persson, personal observation) that the outer wall of living Scrippsiella cysts was dissolved in anoxic sediment. In a survey of calcareous cysts in the Gulf of Naples calcareous cysts were frequently observed in sediments of sand, silt and clay, but seldom in black mud (Montresor et al., 1994). Scrippsiella spp. cysts in this experiment were not affected by the digestion process in A. nitida or M. cristata, but were affected by A. filiformis. This possibly reflects the differences in digestion processes of the animals. The “brown cysts” were predominantly Protoperidinium and Diplopsalis-like species. In this experiment, brown cysts were susceptible to degradation by the activity of deposit feeding animals. Zonneveld et al. (1997) found remarkable differences in the preservation of brown cyst species in different oxygen regimes. Of those found in anoxic sediments only 0.3% occurred in oxidized ones although the origin of the sediments was thought to be the same. Smooth, uncolored cysts showed a significant reduction, both from Control compared to initial and for the treatments with animals compared to Control, indicating that they are either very easily germinated, and the wall then disintegrated, or that they are destroyed by microbial action and by animal activity. Small, naked dinoflagellates often have small and inconspicuous cysts (which were not included in this experiment if they were smaller than 25 m), but such cysts are common in nature (Persson, personal observation). Cysts of the potentially toxic species of the genus Alexandrium were far too few in this experiment
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for us to have the possibility to draw any conclusions about their susceptibility to benthic animal activity. It is difficult to believe that dinoflagellate cysts could constitute a valuable food source for “bottom fauna”. Dinoflagellate cysts are generally a small part of the total sedimenting material (<1‰), although periodically (at the end of a dinoflagellate bloom) they are more common (Nehring, 1996; Montresor et al., 1998). Resting cells of many kinds are present in the sediment and can theoretically give nourishment to benthic animals during periods of starvation or during short periods of massive sedimentation of resting stages. Some of these resting stages are very resistant whereas some are fairly easily disintegrated. In nature, a repeated consumption on the same sediment particles occurs, by different species, one feeding on the fecal pellets of others (Taghon et al., 1984). The cysts may be eaten many times, also the empty walls. With time, fossilizable cysts are concentrated and sensitive cysts presumably are consumed. The deposit feeders used in this study (A. nitida, M. cristata, A. filiformis and N. diversicolor) have different modes of feeding and affect the cyst assemblages differently. Thus differences in the “cyst bank” might depend both on spatial differences in the composition and abundance of the fauna as well as on differences in cyst production and accumulation. This leads to difficulties in interpreting the differences in cyst assemblages between places or countries. It is possible that some cysts are relatively resistant to the digestion processes of many animals, but are not preserved for a long time in an anoxic environment (as can be hypothesized for cysts with an outer wall of calcareous material, since acids are produced by anaerobic digestion, and acids are known to dissolve calcium). Other species that survive for several years in anoxic sediments may be rapidly consumed/digested when present in oxic environments (brown cysts). In nature, most cysts are deposited in oxygenated environments (i.e. with fauna) since most marine environments are oxic at the surface (Chester, 1990). There is a need for more grazing experiments on dinoflagellate cysts and especially on living cysts of harmful species. 5. Conclusions Benthic animal activity resulted in a changed species composition of dinoflagellate cysts with a
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significant relative increase of fossilizable species while the percentage of sensitive species decreased significantly. The different benthic animals affected the dinoflagellate species composition slightly differently, indicating that differences in cyst seed banks in part can be attributable to the species composition of deposit feeding animals.
Acknowledgements We want to thank Dr. Barrie Dale for valuable comments on an earlier version of the manuscript. We are grateful to Berne Petersson and Sylve Robertsson on R/V Oscar von Sydow for being helpful at sampling and to Gunnar Persson for assistance in the time-consuming work of concentrating cysts. Financial support was provided by Kapten C. Stenholms foundation and the Hierta-Retzius foundation. References Anderson, D.M., 1997. Diversity of harmful algal blooms in coastal waters. Limnol. Oceanogr. 42, 1009–1022. Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transitions. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Heidelberg, pp. 29–48. Anderson, D.M., Lively, J.J., Reardon, E.M., Price, C.A., 1985. Sinking characteristics of dinoflagellate cysts. Limnol. Oceanogr. 30, 1000–1009. Bolch, C.J.S., Hallegraeff, G.M., 1990. Dinoflagellate cysts in recent marine sediments from Tasmania, Australia. Bot. Mar. 33, 173–192, and references therein. Bravo, I., Franco, J.M., Reyero, M.I., 1998. PSP toxin composition of three life cycle stages of Gymnodinium catenatum. In: Reguera, B., Blanco, J., Fernández, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and IOC of UNESCO, Grafisant, Santiago de Compostela, pp. 356–358, and references therein. Chester, R., 1990. Marine Geochemistry. Allen & Unwin, London. Dale, B., 1983. Dinoflagellate resting cysts: benthic plankton. In: Fryxell, G.A. (Ed.), Survival Strategies of the Algae. Cambridge University Press, Cambridge, pp. 69–136, and references therein. Dodge, J.D., 1982. Marine Dinoflagellates of the British Isles. Her Majesty’s Stationery Office, London. Laroque, R., Cembella, A.D., 1990. Ecological parameters associated with the seasonal occurrence of Alexandrium spp. and consequent shellfish toxicity in the lower St. Lawrence estuary (eastern Canada). In: Graneli, E., Sundström, L.,
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