Microb Ecol (1994) 28:223-235
Controls of the Microbial Loop: Biotic Factors
MICROBIAL ECOLOGYInc. © 1994Springer-Vertag New York
Bacterivory and Herbivory: Key Roles of Phagotrophic Protists in Pelagic Food Webs E.B. Sherr, B.F. Sherr College of Oceanic and Atmospheric Science, Oregon State University, Corvallis OR 97331-5503, USA
Abstract. Research on "microbial loop" organisms, heterotrophic bacteria and phagotrophic protists, has been stimulated in large measure by Pomeroy's seminal paper published in BioScience in 1974. We now know that a significant fate of bacterioplankton production is grazing by < 20-p~m-sized flagellates. By selectively grazing larger, more rapidly growing and dividing cells in the bacterioplankton assemblage, bacterivores may be directly cropping bacterial production rather than simply the standing stock of bacterial cells. Protistan herbivory, however, is likely to be a more significant pathway of carbon flow in pelagic food webs than is bacterivory. Herbivores include both < 20-p~m flagellates as well as > 20-p,m ciliates and heterotrophic dinoflagellates in the microzooplankton. Protists can grow as fast as, or faster than their phytoplankton prey. Phototrophic cells grazed by protists range from bacterial-sized prochlorophytes to large diatom chains (which are preyed upon by extracellularly-feeding dinoflagellates). Recent estimates of microzooplankton herbivory in various parts of the sea suggest that protists routinely consume from 25 to 100% of daily phytoplankton production, even in diatom-dominated upwelling blooms. Phagotrophic protists should be viewed as a dominant biotic control of both bacteria and of phytoplankton in the sea.
Introduction Since Pomeroy's landmark paper in BioScience [68], a broad theme in the conceptualization of microbial food webs has been that of the trophic roles of phagotrophic protists. These organisms graze other microbial cells, remineralize the nitrogen and phosphorus bound up in those prey, and serve as a food resource for higher trophic levels. Excellent reviews of phagotrophic protists as regenerators of inorganic nutrients [16] and as food for metazoans [29, 86] are available elsewhere. Here we would like to focus on our own area of expertise, protistan grazing as a control of biomass and productivity of both heterotrophic and autotrophic microbes [78]. The "microbial loop" envisaged by Azam et al. [3], which drew upon ideas presented by Pomeroy [68], Sieburth et al. [83], and Williams [98], as well as others, solidified the notion that heterotrophic microbes were basically a shunt of carbon and energy from the main phytoplankton-based food web [see 21]. During
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this initial development of microbial food web models, one of the main points of Pomeroy [68], namely that nanophytoplankton were significant primary producers in the sea, was more-or-less overlooked. Although Pomeroy was chiefly concerned with small-sized phytoplankton as contributors to overall microbial respiration, an obvious implication was that smaller grazers, rather than net-caught copepods, might be the main consumers of nanophytoplankton and thus could be significant herbivores in marine systems. This hypothesis was not immediately seized upon in the aftermath of Pomeroy [68], but subsequent discoveries have made it inescapable. Now, twenty years later, the ideas that the "microbial loop" is only one component of a larger microbial food web [78], and that the role of phagotrophic profists as herbivores may be significant (in terms of total carbon-energy flow) are finally taking hold [5, 55, 57]. The categorization of phagotrophic protists as either bacterivores or herbivores has been made more difficult by the discovery of abundant phototrophic prokaryotes, coccoid cyanobacteria, and prochlorophytes in the sea [19, 44, 96]. Thus the process of "bacterivory" includes the consumption of both heterotrophic and autotrophic cells. Below we first summarize recent findings regarding protistan grazing on bacteria, and then examine the case for protists as herbivores in pelagic food webs.
P r o t i s t s as B a c t e r i v o r e s
Following the publication of Pomeroy's paper in BioScience in 1974 [68], increased attention was given to the role of bacteria in pelagic ecosystems. Methods for in situ enumeration of bacterioplankton via epifluorescence microscopy [40] and for determination of in situ bacterial biomass production via radiolabeled thymidine uptake [24] began to be widely used. It was soon apparent that bacteria were an active component of marine food webs. The question of the fate of bacterioplankton production was resolved with the discovery that the abundant nonpigmented flagellated protists observed in seawater could not exist saprophytically [37], but rather were voracious consumers of bacteria [22, 23, 37]. Since these reports, a number of investigators have quantified the rate of consumption of bacteria in the marine pelagial, using a variety of methods. It appears that the flagellate assemblage may graze on the order of 25 to > 100% of the measured daily production of bacterioplankton [15, 56, 64, 73, 76]. Several explanations have been offered for the frequently observed mismatch between rates of bacterial cell production and rates of grazing mortality due to heterotrophic flagellates (i.e., less grazing than production): (1) In some systems, other types of grazers besides nonpigmented flagellates, e.g., mixotrophic phytoflagellates [72] or pelagic ciliates [76], may be important bacterivores. (2) Bacterivores may selectively graze the larger, growing and dividing cells within the bacterioplankton assemblage and thus are directly cropping bacterial production rather than simply the standing stock of bacterial cells [34]. If this were the case, then there need not be a direct balance of cells produced to cells grazed in order for protists to control the in situ abundance of bacterioplankton. In support of this idea, Sherr et al. [77] found that both the average cell size and frequency of dividing cells was lower in grazed compared to nongrazed bacterioplankton assemblages in
Phagotrophic Protists and Pelagic Food Webs
225
Table 1. Acid lysozyme activity (pmole MUF m1-1 sample h -a) in several size fractions (whole water, 20 Ixm screened, 5 Ixm screened, and 0.8 Ixm filtered) of freshly collected coastal seawater. Enzyme assays were done according to the protocol of Gonzalez et al. [35]; enzyme preparations were incubated in the dark at in situ water temperature (9-1 I°C) Sampling date
Whole water
< 20 Ixm
< 5 txm
< 0.8 ~ m
22 June 93 30 July 93 31 July 93 3 September 93
0.235 0.287 0.229 0.043
0.256 0.093 0.097 0.065
0.259 0.127 0.076 0.043
0.096 0.053 0.055 0.003
estuarine water. (3) Currently used methods may either overestimate bacterioplankton production and/or underestimate protistan bacterivory [56, 64]. For example, grazing determinations based on uptake of heat-killed, fluorescently-labeled bacteria (FLB) may underestimate true rates of bacterivory by as much as 50% because bacterivorous flagellates have significantly higher ingestion rates of motile compared to nonmotile bacteria [36, 58], and motile bacteria may be common in marine bacterioplankton assemblages [2]. (4) Finally, protistan grazing may not be the sole loss process for bacterial cells; viral infection has been proposed as (but not yet shown to be) a routine source of mortality for bacterioplankton [69]. We still know little about the bacterial-protist trophic link in the sea. Most work on this link has been conducted in shallow, eutrophic to mesotrophic coastal systems. Present in vivo methods for determination of in situ rates of bacterivory are not very satisfactory for use in oligotrophic systems because of the low abundance and activity of heterotrophic microbes in such systems. A new approach to estimation of bacterivory in situ has recently been developed: quantification of the activity of lysozyme, a digestive enzyme which degrades bacterial cell walls at pH 4.5 (characteristic of food vacuoles) in sonicated aliquots of freshly collected seawater samples [35]. Acid lysozyme activity is calibrated with an independent method of estimating in situ bacterivory; Gonzalez et al. [35] used FLB uptake as the calibration technique. The acid lysozyme method involves measurement of accumulation of a fluorochrome, methylumbelliferone (MUF), which is enzymatically cleaved from a substrate analogue of peptidoglycan, a major structural component of eubacterial cell walls. The acid lysozyme approach is technically simple, does not require manipulation or incubation of living cells, and can greatly improve the resolution of sampling (both in time and space) for community-level rates of bacterivory. In an initial application, we have found between-sample differences in distribution of acid lysozyme activity in size-fractioned coastal seawater (Table 1). In two samples (22 June 1993 and 3 September 1993) most bacterivory, as indicated by acid lysozyme activity, was due to organisms in the < 5 ~m fraction. In two other samples (30 July 1993 and 31 July 1993) more than half of total bacterivory appeared to be associated with the > 20 p~m fraction. Epifluorescence microscopic inspection showed that in the June and September samples, most bacterivores were < 5-1xm free-swimming flagellates, while the July samples had abundant 15-20-1xm-long choanoflagellates and flagellates associated with the detritus of a decaying phytoplankton bloom; the flagellates in the latter samples were largely retained on the 20-1xm screen.
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P r o t i s t s as H e r b i v o r e s
An important development in the past few years has been a growing appreciation of phagotrophic protists as consumers of phytoplankton production in the sea (80). Several lines of evidence point to protists as being routinely dominant herbivores in pelagic food webs, particularly in oligotrophic seawater: 1. Except for periodic blooms of large diatoms and dinoflagellates, most primary production in the sea is accomplished by phototrophs in the size range of l to 10 txm. It is increasingly apparent that much of the carbon production in the marine pelagial is accomplished by cells too small to be efficiently grazed by copepods [79]. These primary producers include; eukaryotic cells < 5 ~m, of various taxonomic groups including prasinophytes, prymnesiophytes, and diatoms [45, 74, 90]; coccoid cyanobacteria 0.8-1.5 txm in size [44, 96, 97]; and 0.5-1.0 txm prochlorophytes, phototrophic bacteria containing divinyl chlorophyll a [19, 30]. The photosynthetic prokaryotes may be particularly significant contributors to primary production in oligotrophic regions of the sea [14, 54, 67, 92, 97]. Methods for determining the standing stocks of pelagic prochlorophytes, including counting cells with a flow cytometer [ 14, 62, 92] and assaying the concentration of divinyl chlorophyll a [30, 92], have only recently been developed. Campbell and Vaulot [ 14] found that at Station ALOHA off Hawaii, the prochlorophyte Prochlorococcus sp. was the most abundant component of the phytoplankton assemblage, on the order of 105 cells ml-1, and contributed, on average, 45% of phytoplankton carbon biomass in the mixed layer. Furthermore, Campbell and Vaulot [14] reported that about 30% of the bacterioplankton enumerated via acridine orange staining in surface waters of the central North Pacific were actually prochlorophytes rather than heterotrophic cells. Prochlorophytes are also abundant in the Sargasso Sea [54, 62] and in the Mediterranean Sea [91]. Li et al. [54] found that in the Sargasso during September, prochlorophyte carbon biomass was twice that of cyanobacteria, 50% that of photosynthetic eukaryotes, and 25% that of heterotrophic bacterioplankton. With the possible exception of a few species of gelatinous zooplankton, the only organisms capable of efficiently grazing bacterial-sized primary producers are phagotrophic protists. Protists are also the organisms with the greatest capacity to consume ultraphytoplankton cells in the range of 2-8 Ixm [79]. 2. Herbivorous protists are ubiquitous in the sea, and are diverse in terms of size, taxonomy, and feeding behavior. Ciliates, including tintinnids and aloricate choreotrichs, are currently thought of as "typical" phytoplankton grazers in the microzooplankton, heterotrophic organisms 20-200 txm in size [4, 70]. Certainly ciliates have been shown to be significant grazers in marine systems [15, 70, 93]. However, there are two other major groups of protistan herbivores: < 20 p~m flagellates and heterotrophic dinoflagellates. Nanoflagellates, 2-20 Ixm in size, are commonly relegated to the role of "bacterivore" in models of marine food webs [2, 21]. It now appears that most consumers of < 1-1xm-sized cells are actually flagellates less than 5 txm in size, and that 5-20-1xm flagellates select larger prey, in the general range of 1-10 Ixm [75]. This is the size range of a large part of the biomass of autotrophic cells in the open ocean, as discussed above. There is accumulating evidence for the
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227
significance of nanoflagellate herbivory in situ. Campbell and Carpenter [13] and Caron et al. [17] reported that nanoplanktonic grazers were the main consumers of coccoid cyanobacteria in marine food webs. Caron et al. [17] concluded that in coastal water of the northeastern USA, nanoplankton consumers could remove as much as 54% of the cyanobacterial standing stock per day, and that at times as much as 30% of the total prokaryotic biomass consumed was as cyanobacteria. Sherr et al. [81] found that in a Georgia estuary, the assemblage of 5-15-p~m flagellates cleared 2-6 txm phytoplankton cells at about the same rate as did the ciliate assemblage. Kuosa [47] reported that nanoflagellate grazing consumed about 32-42% of primary production in the summer and fall in the Baltic Sea. An important component of the assemblage of < 20-1xm-sized flagellates appears to be nonarmored, gymnodinoid dinoflagellates. Bjornsen and Kuparinen [6] found that 12-14-txm-sized Gymnodinium sp. were major herbivores in the Southern Ocean, consuming algal cells in the size range of 8-10 p~m. Strom [89] isolated a 10-12-p~m-sized Gymnodinium species from surface waters in the subarctic Pacific. This dinoflagellate resembled a common, similarly-sized herbivorous protist observed in the Georgia estuary [81 ]. Results from the North Atlantic Bloom Experiment in 1989 and 1990 indicate that in the open North Atlantic, the heterotrophic dinoflagellate assemblage was dominated, in terms of both numbers and biomass, by cells < 20 p~m in size; in this case most of the dinoflagellates were 4-8 p~m spindle-shaped species, similar to Katodinium sp. [95]. The abundance and biomass of heterotrophic dinoflagellates 8-100 txm in size often equals or exceeds that of ciliates in the plankton [10, 38, 50, 52, 53, 84]. Dinoflagellates appear to be a particularly important component of the microzooplankton in polar seas. In the Canadian Arctic, Bursa [12] found that heterotrophic dinoflagellates comprised 73% of microzooplankton numbers on an annual basis, and Lessard and Rivkin [51] reported that in McMurdo Sound, Antarctica, up to 97% of the biomass of microzooplankton was due to phagotrophic dinoflagellates. Dinoflagellates may also be major herbivores in Antarctic sea-ice communities [8]. Fecal pellets and empty frustules produced by dinoflagellates feeding on diatoms can comprise a significant fraction of sinking particles in the Southern Ocean [61]. Phagotrophic dinoflagellates are capable of consuming diverse types of prey over three orders of magnitude in size, from < 1-txm prokaryotes to > 200-1xm diatom chains. As Lessard [50] observed, phagotrophic dinoflagellates are "eclectic feeders" having a variety of mechanisms for consuming their prey. Non-armored dinoflagellates can engulf whole prey into food vacuoles, often feeding on organisms nearly as large as themselves. Some species utilize a specialized appendage to pierce the cell wall of prey cells and "suck out" the cytoplasm in a manner analogous to the feeding behavior of spiders [25]. Armored dinoflagellates may also extrude a large, pseudopodial feeding veil which envelopes their prey and permits extracellular digestion [25, 26, 42]. By the latter mechanism, armored species are able to feed on diatom chains much larger than the dinoflagellate cell itself. Thus, the range of prey sizes grazed by phagotrophic dinoflagellates exceeds that of pelagic ciliates, which normally ingest prey < 20 txm in size [46, 70, 93].
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This is not the end of the dinoflagellate story, as it now appears that many bloom-forming autotrophic dinoflagellates are capable of ingesting prey as well. Incidental reports of mixotrophic dinoflagellates are common in the literature [25]. Recently, Bockstahler and Coats [7] reported ingestion of prey, including diatoms and < 20-t~m ciliates, by three common red-tide dinoflagellate species in Chesapeake Bay. Jacobson [41] has also found food vacuoles in several other species of autotrophic dinoflagellates. Thus, we can no longer conceptualize microzooplankton herbivores as being mostly ciliates; phagotrophic dinoflagellates and nanoflagellates are also significant consumers of phototrophic cells. In addition, phagotrophic protists do not simply consume phytoplankton cells that are small enough to escape metazooplankton grazers. Protists are capable of feeding on the entire size range of phytoplankton and may, at times, be in direct competition with herbivorous metazooplankton. 3. Heterotrophic protists are capable of growing as fast as, or faster than, phytoplankton, and thus are able to curtail development of blooms. As Goldman [31] has observed, eukaryotic microbes with a heterotrophic mode of existence, i.e., phagotrophic protists, are able to grow at a faster rate than can autotrophic microbes. Under optimum culture conditions, phytoplankton typically have population growth rates in the range of 0.5 to 2.0 day -1 [18, 71], while optimum growth rates of phagotrophic protists are equal to, or greater than, these rates (Fig. 1). In culture, the growth rate of heterotrophic protists is inversely proportional to cell size and directly related to temperature (Table 2). It is interesting that heterotrophic dinoflagellates have lower growth rates than do ciliates of comparable size (Fig. 1, Table 2); Banse [4] previously showed that autotrophic dinoflagellates have lower maximum growth rates than do diatoms. However, even larger-sized protists growing at cool temperatures can double in numbers within 1-2 days, as long as food is not limiting (Table 1). It has been speculated that the capacity for rapid growth exhibited by phagotrophic protists is one factor that prevents phytoplankton blooms in areas of the ocean where surface nutrients are high but chlorophyll a concentrations remain low, e.g., the subarctic Pacific [57] and the central equatorial Pacific [49; H. Ducklow 1989, The Oceanography Society Meeting, Monterey CA, Abstract 19]. Information on the actual growth rates of herbivorous protists in situ is almost nonexistent at present; this is a research area in need of attention. 4. Measurements of grazing impact of 'microzooplankton' (which also ineludes < 20-p~m herbivores), indicate that this component of the grazing community is responsible for a large share of phytoplankton mortality in all areas of the ocean so far examined. During the past decade, data on microzooplankton grazing impact in situ have steadily accumulated (Table 3). Various habitats in the ocean have been examined: open ocean and coastal waters; polar, temperate, and tropical regions; oligotrophic and eutrophic systems. In all of these regions, microzooplankton grazing was generally equal to half or more of daily phytoplankton production (Table 3). Microzooplankton grazing may account for 100% of daily production, particularly in systems dominated by < 5-p~m-sized phytoplankton cells. Even during diatom blooms in an upwelling system off the Oregon coast, protists, predominately extracellularly-feeding dinoflagellates,
Phagotrophic Protists and Pelagic Food Webs
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Body Size, cubic microns Fig. 1.
Relation of growth rate, (Ix day-~), to body size (Ix3) for phytoplankton and phagotrophic
protists growing under nonlimitingconditionsin culture at 20°C. Data for phytoplanktonare from Table 4 of Raven [71]. Valuesfor protists are from Table 2; in some cases where culturetemperatures were near 20°C, growthrate at 20°Cwas calculatedusinga Q~oof 2.0. p, phytoplankton;f, nanoflagellates; d, dinoflagellates;c, ciliates. consumed 26-50% of daily production [S. Neuer and T. Cowles, manuscript accepted for publication in Mar. Ecol. Prog. Ser.]. As a final note on the subject of the role of phagotrophic protists as grazers of 1-10-1~m-sized cells, in the open ocean up to half of the eukaryotic organisms in this size class are not phototrophic cells, but rather heterotrophic flagellates [82]. The idea that microzooplanktonic grazers, specifically ciliates, are a major source of mortality for bacterivorous flagellates is an integral part of the concept of the microbial loop [3, 21]. Only recently, however, has there been direct evidence for this trophic link. Both Ohman and Snyder [63] and Verity [94] have cultured pelagic ciliates using nonpigmented flagellates as a food source. The fact that both ciliates and phagotrophic dinoflagellates in open ocean plankton assimilate carbon from radiolabeled bacterial cells may also indicate a trophic link mediated by bacterivorous flagellates [52]. Again, more work is needed on this topic.
Conclusions Recent findings compel us to rethink the initial concept of the role of heterotrophic microbes, bacteria, and phagotrophic protists, as simply a side loop "add-on" to the classic phytoplankton-copepod-fish food chain:
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Table 2. Maximum growth rates (ix, day-l), and minimum generation times (T, h), at various temperatures for diverse phagotrophic protists in culture Protist (size, ixm3)
Nanoflagellates Actinomonas mirabilis (75) Monosiga sp. (20) Ochromonas sp. (200) Paraphysomonas vestita (190) Pleuromonas jaculans (50) Pseudobodo tremulans (90) Paraphysomonas imperforata (200-500) Diphanoeca grandis Paraphysomonas imperforata (200-300) Dinoflagellates Oxyrrhis marina (6300) Gymnodinium sp. (1000-1500) Gymnodinium sp. (600-1200) Gyrodinium dominans (20,000-30,000) Protoperidinium hirobis (6400) Protoperidinium spiniriferum (48,000)
Temp (°C)
ixday -1
T (h)
Reference
20 20 20 20 20 20 20-24 15 -1.5 6.0 15
6.0 4.1 4.6 5.5 3.8 3.6 2.4 3.6 0.5-0.8 0.9-1.5 2.3-2.7
2.8 4.1 3.6 3.0 4.3 4.6 6.9 4.6 22-32 11-19 6-7
22 22 22 22 22 22 32 1
0.8-1.3 0.3 0.7 0.3 0.8 1.2 0.23
13-21 55 24 55 21 15 72
33
20
1 12 20 25-27 20 20
20
6 88 60 43 43
Ciliates
Tintinnopsis cf. acuminata (11,000) Eutintinnus pectinis (18,000) Favella sp. Balanion sp. (19,000) Tintinnopsis acuminata (12,000) Tintinnopsis vasculum (120,000) Strombidium reticulatum (40,000) Lohmaniella spiralis (150,000) Strombidium sp. (17,000-33,000) Strobilidium sp. (18,000-22,000) Urotricha furcata (1800)
Plagostrombidium flallax (50,000)
Strombilidium lacustris (113,000)
18 18 7 15 20 7-10 15 20 15 20 25 5 10 15 12 12 15 20 20 5.5 12 15.5 21.5 5.5 12 15.5 21.5 5.5 12 15.5 21.5
1.4 1.4 0.4 1.0 1.4 1.5 2.4 2.9 1.2 1.4 1.9 0.5 1.0 1.0 0.9 1.0 1.4 2.7 1.4 0.46 0.66 0.76 1.72 0.03 0.42 O.57 0.90 0.43 0.70 0.99 1.42
12 12 43 17 12 11 7 5.8 14 11.5 8.7 35 17 17 18.5 17 11.5 6.2 11.5 36 25 22 9.7 555 40 29 18.5 39 24 16.8 11.7
39 39 87
87
93
93 46 46 63 94 59
59
59
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Table 3. Summaryof recent estimates of microzooplanktongrazing impact on phytoplankton in various marine systems, as percent of primary productiongrazed day-1 by organisms < 200 Ixm in size in the water column of shallow systems, or in the mixed layer of the open ocean. The dilution method of Landry and Hassett [48] was used in these studies to estimate grazing rates Location
% Primpro grazed day-1
Reference
Washington, USA coastal waters Celtic Sea Jones Sound, Arctic Baffin Bay, Arctic Halifax Harbour, Nova Scotia Rhode River Estuary, MD, USA Grand Bank, Newfoundland Subarctic Pacific Central equatorial Pacific Northeastern Atlantic Indian Ocean Oregon, USA coastal upwelling blooms
17-52% 13-65% 40-114% 37-88% 47%, 100% 45-104% 50%, 70% 40-60% 44%, 90% 39-115% 70-100% 26-50%
48 9 65 66 28 27 66 89 49 10 11 Neuer & Cowles, ms accepted by MEPS
1. Picoplanktonic primary producers are a significant component of marine food webs. Prochlorophytes can be a substantial fraction of the numbers and biomass of the "heterotrophic" bacterioplankton enumerated via acridine orange direct counts. Other, slightly larger picoautotrophs, coccoid cyanobacteria and picoeukaryotes, can also represent a large part of total phytoplankton biomass and primary production, particularly in the open ocean. This implies that bacterivorous protists must also be consuming bacterial-sized primary producers. Thus phagotrophic protists in the nanoplankton, for the most part flagellates but also including lO-20-p,m ciliates, are likely to have a major role as consumers of picoplanktonic primary production as well as of the secondary production of heterotrophic bacteria.
2. Phagotrophic flagellates, particularly dinoflagellates, are a ubiquitous and significant component of the herbivorous microzooplankton. Recent studies in several regions of the world ocean, including the subarctic Pacific, the North Atlantic at the time of the spring bloom, the Indian Ocean, and an upwelling system off the coast of Oregon, USA, have demonstrated that microzooplanktonic grazers are often the major consumers of phytoplankton. In all of these systems, heterotrophic dinoflagellates in the nanoplankton and microplankton size ranges were implicated as major herbivores. Dinoflagellates have a variety of species-specific feeding strategies and can graze cells over a wide size range, from picoplankton to large diatom chains. Based on the latest findings about the structure and functioning of marine pelagic food webs, we should now view phagotrophic protists, ciliates, and flagellates as a biotic control of both bacteria and phytoplankton in the world ocean. Phagotrophic protists are also responsible for much of the regenerative flux of nitrogen and phosphorus in the sea [16], and can be a vital food resource for metazoans [29, 86]. It is clear that phagotrophic protists should be routinely considered as having major trophic roles over a broad range of marine ecosystems, including regions characterized by spring blooms and upwelling. Future
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models of pelagic food webs should formally include protistan as well as metazoo 31ankton consumers of primary production.
References 1. Andersen (t989) Functional biology of the choanoflagellate Diaphanoeca grandis Ellis. Mar Microb Food Webs 3:35-50 2. Azam F, Ammerman JW (1984) The cycling of organic matter by bacterio-plankton in pelagic marine ecosystems: microenvironmental considerations. In: Fasham MJR (ed) Flows of energy and materials in marine ecosystems: theory and practice. Plenum Press, New York, pp 345-360 3. Azam F, Fenchel T, Field JG, Gray JS, Meyer-Reil LA, Thingstad F (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Set 10:257-263 4. Banse K (1982) Cell volumes, maximal growth rates of unicellular algae and ciliates, and the role of ciliates in the marine pelagial. Limnol Oceanogr 27:1059-1071 5. Banse K (1992) Grazing, temporal changes of phytoplankton concentrations, and the microbial loop in the open sea. In: Falkowski PG, Woodhead AD (eds) Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, pp 409-440 6. Bjomsen PK, Kupadnen J (1991) Growth and herbivory by heterotrophic dinoflagellates in the South Ocean, studied by microcosm experiments. Mar Biol 109:397-405 7. Bockstahler KR, Coats WD (1993) Spatial and temporal aspects of mixotrophy in Chesapeake Bay dinoflagellates. J Protozoo140:49--60 8. Buck K, Bolt PA, Garrison DL (1990) Phagotrophy and fecal pellet production by an athecate dinoflagellate in Antarctic sea ice. Mar Ecol Prog Set 60:75-84 9. Burkill PH, Mantoura RFC, Llewellyn CA, Owens NJP (1987) Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Mar Bio193:581-590 10. Burkill PH, Edwards ES, John AWG, Sleigh MA (1993) Microzooplanktonand their herbivorous activity in the northeastern Atlantic Ocean. Deep-Sea Res 40:479-494 11. Burkill PH, Leakey RJG, Owens NJP, Mantoura RFC. ('1993)Synnechococcusand its importance to the microbial food-web of the northwest Indian Ocean. Deep-Sea Res 40:773-782 12. Bursa AS (1961) The annual cycle at iglootik in the Canadian Arctic. II. The phytoplankton. J Fish Res Bd Can 18:563-615 13. Campbell L, Carpenter EJ (1986) Estimating the grazing pressure of heterotrophic nanoplankton on Synechococcus spp. using the sea water dilution and selective inhibitor techniques. Mar Ecol Prog Ser 33:121-129 14. Campbell L, Vaulot D (1993) Photosynthetic picoplankton community structure in the subtropical North Pacific near Hawaii (station ALOHA). Deep-Sea Res 40:2043-2060 15. Capriulo GM, Sherr EB, Sherr BF (1991) Trophic behaviour and related community feeding activities of heterotrophic marine protists. In: Reid PC, Turley CM, Burkill PH (eds) Protozoa and their role in marine processes. (NATO ASI Series G, vo125) Springer-Verlag, Berlin, pp 219-265 16. Caron DA, Goldman JC (1990) Protozoan nutrient regeneration. In: Capriulo GM (ed) Ecology of marine protozoa. Oxford Univ Press, New York, pp 283-306 17. Caron DA, Lira EL, Miceli G, Waterbury JB, Valois FW (1992) Grazing and utilization of chroococcoid cyanobacteria and heterotrophic bacteria by protozoa in laboratory cultures and a coastal plankton community. Mar Ecol Prog Ser 76:205-217 18~ Chisholm SW (1992) Phytoplankton size. In: Falkowski PG, Woodhead AD (eds) Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, pp 213-237 19. Chisholm SW, Olson RJ, Zettler ER, Goericke R, Waterbury JB, Welchmeyer NA (1988) A novel free-living prochlorophyte abundant in the ocean ic euphotic zone. Nature 334:340-343 20. Choi JW, Peters J (1992) Effects of temperature on two psychrophilic ecotypes of a heterotrophic nanoflagellate, Paraphysomonasimperforata. Appl Environ Microbiol 58:593-599 21. Ducklow H (1983) Production and fate of bacteria in the oceans. BioScience 33:494-501 22. Fenchel T (1982) Ecology of heterotrophic microflagellates. II. Bioenergetics and growth. Mar Ecol Prog Ser 8:225-231
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75. Sherr BF, Sherr EB (1991) Proportional distribution of total numbers, biovolume, and bacterivory among size classes of 2-20-p~m nonpigmented marine flagellates. Mar Microb Food Webs 5:227237 76. Sherr BF, Sherr EB, Pedros-Alio C (1989) Simultaneous measurement of bacterioplankton production and protozoan bacterivory in estuarine water. Mar Ecol Prog Ser 54:209-219 77. Sherr BF, Sherr EB, McDaniel J (1992) Effect of protistan grazing on the frequency of dividing cells in bacterioplankton assemblages. Appl Environ Microbiol 58:2381-2385 78. Sherr EB, Sherr BF (1988) Role of microbes in pelagic food webs: a revised concept. Limnol Oceanogr 33:1225-1227 79. Sherr EB, Sherr BF (1991) Planktonic microbes: tiny cells at the base of the ocean's food web. Trends in Ecol & Evol 6:50-54 80. Sherr EB, Sherr BF (1992) Trophic roles of pelagic protists: phagotrophic flagellates as herbivores. Arch Hydrobiol Beih Ergebn Limnol 37:165-172 81. Sherr EB, Sherr BF, McDaniel J (1991) Clearance rates of < 6-p~m fluorescently-labeled algae (FLA) by estuarine protozoa: potential grazing impact of flagellates and ciliates. Mar Ecol Prog Ser 69:81-92 82. Sieburth J.McN. (1984) Protozoan bacterivory in pelagic marine waters. In: Hobbie JE, Williams PJleB (eds) Heterotrophic activity in the sea. Plenum Press, New York, pp 405-444 83. Sieburth J.McN., Smetacek V, Lenz J (1978) Pelagic ecosystem structure: heterotrophic compartments of plankton and their relationship to plankton size fractions. Limnol Oceanogr 33:12251227 84. Smetacek V (1981) The annual cycle of protozooplankton in Kiel Bight. Mar Biol 63:1-11 85. Steele JH (1974) The structure of marine ecosystems. Harvard University Press, Cambridge, Mass. 86. Stoecker DK, Capuzzo JMcD (1990) Predation on protozoa: its importance to zooplankton. J Plankton Res 12:891-908 87. Stoecker DK, Davis LH, Provan A (1983) Growth ofFavella sp. (Ciliata: Tintinnina) and other microzooplankters in cages incubated in situ and comparison to growth in vitro. Mar Biol 75:293-302 88. Strom SL (1991) Growth and grazing rates of the herbivorous dinoflagellate Gymnodinium sp. from the open subarctic Pacific Ocean. Mar Ecol Prog Ser 78:103-113 89. Strom SL, Welchmeyer NA (1991) Pigment-specific rates of phytoplankton growth and microzooplankton grazing in the open subarctic Pacific Ocean. Limnol Oceanogr 36:50-63 90. Thomsen HA (1986) A survey of the smallest eukaryotic organisms of the marine phytoplankton. In: Platt T, Li WKW (eds) Photosynthetic picoplankton. Can Bull Fish Aquat Sci 214:121-130 91. Valour D, Partensky F (1992) Cell cycle distribution of prochlorophytes in the northwestern Mediterranean Sea. Deep-Sea Res 39:727-742 92. Veldhuis MJW, Kraay GW (1990) Vertical distribution and pigment composition of a picoplanktonic prochlorophyte in the subtrophical North Atlantic: a combined study of HPLC-analysis of pigments and flow cytometry. Mar Ecol Prog Ser 68:121-127 93. Verity PG (1985) Grazing, respiration, excretion, and growth rates of tintinnids. Limnol Oceanogr 30:1268-1281 94. Verity PG (1991) Measurement and simulation of prey uptake by marine planktonic ciliates fed plastidic and aplastidic nanoplankton. Limnol Oceanogr 36:729-750 95. Verity PG, Stoecker DK, Sieracki ME, Burkill PH, Edwards ES, Tronzo CR (1993) Abundance, biomass, and distribution of heterotrophic dinoflagellates during the North Atlantic Spring Bloom. Deep-Sea Res 40:227-244 96. Waterbury JB, Watson SW, Guillard RRL, Brand LE (1979) Widespread occurrence of a unicellular, marine, planktonic cyanobacterium. Nature 227:293-294 97. Waterbury JB, Watson SW, Valois FW, Franks DG (1986) Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. In: Platt T, Li WKW (eds) Photosynthetic picoplankton. Can Bull Fish Aquat Sci 214:71-120 98. Williams PJLeB (1981) Incorporation of microheterotrophic processes into the classical paradigm of the planktonic food web. Kieler Meerresforsch Sonderh 5:1-28
Controls of the Microbial Loop: Biotic Factors
MICROBIAL ECOLOGYInc. © 1994Springer-Vertag New York
Bacterivory and Herbivory: Key Roles of Phagotrophic Protists in Pelagic Food Webs E.B. Sherr, B.F. Sherr College of Oceanic and Atmospheric Science, Oregon State University, Corvallis OR 97331-5503, USA
Abstract. Research on "microbial loop" organisms, heterotrophic bacteria and phagotrophic protists, has been stimulated in large measure by Pomeroy's seminal paper published in BioScience in 1974. We now know that a significant fate of bacterioplankton production is grazing by < 20-p~m-sized flagellates. By selectively grazing larger, more rapidly growing and dividing cells in the bacterioplankton assemblage, bacterivores may be directly cropping bacterial production rather than simply the standing stock of bacterial cells. Protistan herbivory, however, is likely to be a more significant pathway of carbon flow in pelagic food webs than is bacterivory. Herbivores include both < 20-p~m flagellates as well as > 20-p,m ciliates and heterotrophic dinoflagellates in the microzooplankton. Protists can grow as fast as, or faster than their phytoplankton prey. Phototrophic cells grazed by protists range from bacterial-sized prochlorophytes to large diatom chains (which are preyed upon by extracellularly-feeding dinoflagellates). Recent estimates of microzooplankton herbivory in various parts of the sea suggest that protists routinely consume from 25 to 100% of daily phytoplankton production, even in diatom-dominated upwelling blooms. Phagotrophic protists should be viewed as a dominant biotic control of both bacteria and of phytoplankton in the sea.
Introduction Since Pomeroy's landmark paper in BioScience [68], a broad theme in the conceptualization of microbial food webs has been that of the trophic roles of phagotrophic protists. These organisms graze other microbial cells, remineralize the nitrogen and phosphorus bound up in those prey, and serve as a food resource for higher trophic levels. Excellent reviews of phagotrophic protists as regenerators of inorganic nutrients [16] and as food for metazoans [29, 86] are available elsewhere. Here we would like to focus on our own area of expertise, protistan grazing as a control of biomass and productivity of both heterotrophic and autotrophic microbes [78]. The "microbial loop" envisaged by Azam et al. [3], which drew upon ideas presented by Pomeroy [68], Sieburth et al. [83], and Williams [98], as well as others, solidified the notion that heterotrophic microbes were basically a shunt of carbon and energy from the main phytoplankton-based food web [see 21]. During
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this initial development of microbial food web models, one of the main points of Pomeroy [68], namely that nanophytoplankton were significant primary producers in the sea, was more-or-less overlooked. Although Pomeroy was chiefly concerned with small-sized phytoplankton as contributors to overall microbial respiration, an obvious implication was that smaller grazers, rather than net-caught copepods, might be the main consumers of nanophytoplankton and thus could be significant herbivores in marine systems. This hypothesis was not immediately seized upon in the aftermath of Pomeroy [68], but subsequent discoveries have made it inescapable. Now, twenty years later, the ideas that the "microbial loop" is only one component of a larger microbial food web [78], and that the role of phagotrophic profists as herbivores may be significant (in terms of total carbon-energy flow) are finally taking hold [5, 55, 57]. The categorization of phagotrophic protists as either bacterivores or herbivores has been made more difficult by the discovery of abundant phototrophic prokaryotes, coccoid cyanobacteria, and prochlorophytes in the sea [19, 44, 96]. Thus the process of "bacterivory" includes the consumption of both heterotrophic and autotrophic cells. Below we first summarize recent findings regarding protistan grazing on bacteria, and then examine the case for protists as herbivores in pelagic food webs.
P r o t i s t s as B a c t e r i v o r e s
Following the publication of Pomeroy's paper in BioScience in 1974 [68], increased attention was given to the role of bacteria in pelagic ecosystems. Methods for in situ enumeration of bacterioplankton via epifluorescence microscopy [40] and for determination of in situ bacterial biomass production via radiolabeled thymidine uptake [24] began to be widely used. It was soon apparent that bacteria were an active component of marine food webs. The question of the fate of bacterioplankton production was resolved with the discovery that the abundant nonpigmented flagellated protists observed in seawater could not exist saprophytically [37], but rather were voracious consumers of bacteria [22, 23, 37]. Since these reports, a number of investigators have quantified the rate of consumption of bacteria in the marine pelagial, using a variety of methods. It appears that the flagellate assemblage may graze on the order of 25 to > 100% of the measured daily production of bacterioplankton [15, 56, 64, 73, 76]. Several explanations have been offered for the frequently observed mismatch between rates of bacterial cell production and rates of grazing mortality due to heterotrophic flagellates (i.e., less grazing than production): (1) In some systems, other types of grazers besides nonpigmented flagellates, e.g., mixotrophic phytoflagellates [72] or pelagic ciliates [76], may be important bacterivores. (2) Bacterivores may selectively graze the larger, growing and dividing cells within the bacterioplankton assemblage and thus are directly cropping bacterial production rather than simply the standing stock of bacterial cells [34]. If this were the case, then there need not be a direct balance of cells produced to cells grazed in order for protists to control the in situ abundance of bacterioplankton. In support of this idea, Sherr et al. [77] found that both the average cell size and frequency of dividing cells was lower in grazed compared to nongrazed bacterioplankton assemblages in
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Table 1. Acid lysozyme activity (pmole MUF m1-1 sample h -a) in several size fractions (whole water, 20 Ixm screened, 5 Ixm screened, and 0.8 Ixm filtered) of freshly collected coastal seawater. Enzyme assays were done according to the protocol of Gonzalez et al. [35]; enzyme preparations were incubated in the dark at in situ water temperature (9-1 I°C) Sampling date
Whole water
< 20 Ixm
< 5 txm
< 0.8 ~ m
22 June 93 30 July 93 31 July 93 3 September 93
0.235 0.287 0.229 0.043
0.256 0.093 0.097 0.065
0.259 0.127 0.076 0.043
0.096 0.053 0.055 0.003
estuarine water. (3) Currently used methods may either overestimate bacterioplankton production and/or underestimate protistan bacterivory [56, 64]. For example, grazing determinations based on uptake of heat-killed, fluorescently-labeled bacteria (FLB) may underestimate true rates of bacterivory by as much as 50% because bacterivorous flagellates have significantly higher ingestion rates of motile compared to nonmotile bacteria [36, 58], and motile bacteria may be common in marine bacterioplankton assemblages [2]. (4) Finally, protistan grazing may not be the sole loss process for bacterial cells; viral infection has been proposed as (but not yet shown to be) a routine source of mortality for bacterioplankton [69]. We still know little about the bacterial-protist trophic link in the sea. Most work on this link has been conducted in shallow, eutrophic to mesotrophic coastal systems. Present in vivo methods for determination of in situ rates of bacterivory are not very satisfactory for use in oligotrophic systems because of the low abundance and activity of heterotrophic microbes in such systems. A new approach to estimation of bacterivory in situ has recently been developed: quantification of the activity of lysozyme, a digestive enzyme which degrades bacterial cell walls at pH 4.5 (characteristic of food vacuoles) in sonicated aliquots of freshly collected seawater samples [35]. Acid lysozyme activity is calibrated with an independent method of estimating in situ bacterivory; Gonzalez et al. [35] used FLB uptake as the calibration technique. The acid lysozyme method involves measurement of accumulation of a fluorochrome, methylumbelliferone (MUF), which is enzymatically cleaved from a substrate analogue of peptidoglycan, a major structural component of eubacterial cell walls. The acid lysozyme approach is technically simple, does not require manipulation or incubation of living cells, and can greatly improve the resolution of sampling (both in time and space) for community-level rates of bacterivory. In an initial application, we have found between-sample differences in distribution of acid lysozyme activity in size-fractioned coastal seawater (Table 1). In two samples (22 June 1993 and 3 September 1993) most bacterivory, as indicated by acid lysozyme activity, was due to organisms in the < 5 ~m fraction. In two other samples (30 July 1993 and 31 July 1993) more than half of total bacterivory appeared to be associated with the > 20 p~m fraction. Epifluorescence microscopic inspection showed that in the June and September samples, most bacterivores were < 5-1xm free-swimming flagellates, while the July samples had abundant 15-20-1xm-long choanoflagellates and flagellates associated with the detritus of a decaying phytoplankton bloom; the flagellates in the latter samples were largely retained on the 20-1xm screen.
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P r o t i s t s as H e r b i v o r e s
An important development in the past few years has been a growing appreciation of phagotrophic protists as consumers of phytoplankton production in the sea (80). Several lines of evidence point to protists as being routinely dominant herbivores in pelagic food webs, particularly in oligotrophic seawater: 1. Except for periodic blooms of large diatoms and dinoflagellates, most primary production in the sea is accomplished by phototrophs in the size range of l to 10 txm. It is increasingly apparent that much of the carbon production in the marine pelagial is accomplished by cells too small to be efficiently grazed by copepods [79]. These primary producers include; eukaryotic cells < 5 ~m, of various taxonomic groups including prasinophytes, prymnesiophytes, and diatoms [45, 74, 90]; coccoid cyanobacteria 0.8-1.5 txm in size [44, 96, 97]; and 0.5-1.0 txm prochlorophytes, phototrophic bacteria containing divinyl chlorophyll a [19, 30]. The photosynthetic prokaryotes may be particularly significant contributors to primary production in oligotrophic regions of the sea [14, 54, 67, 92, 97]. Methods for determining the standing stocks of pelagic prochlorophytes, including counting cells with a flow cytometer [ 14, 62, 92] and assaying the concentration of divinyl chlorophyll a [30, 92], have only recently been developed. Campbell and Vaulot [ 14] found that at Station ALOHA off Hawaii, the prochlorophyte Prochlorococcus sp. was the most abundant component of the phytoplankton assemblage, on the order of 105 cells ml-1, and contributed, on average, 45% of phytoplankton carbon biomass in the mixed layer. Furthermore, Campbell and Vaulot [14] reported that about 30% of the bacterioplankton enumerated via acridine orange staining in surface waters of the central North Pacific were actually prochlorophytes rather than heterotrophic cells. Prochlorophytes are also abundant in the Sargasso Sea [54, 62] and in the Mediterranean Sea [91]. Li et al. [54] found that in the Sargasso during September, prochlorophyte carbon biomass was twice that of cyanobacteria, 50% that of photosynthetic eukaryotes, and 25% that of heterotrophic bacterioplankton. With the possible exception of a few species of gelatinous zooplankton, the only organisms capable of efficiently grazing bacterial-sized primary producers are phagotrophic protists. Protists are also the organisms with the greatest capacity to consume ultraphytoplankton cells in the range of 2-8 Ixm [79]. 2. Herbivorous protists are ubiquitous in the sea, and are diverse in terms of size, taxonomy, and feeding behavior. Ciliates, including tintinnids and aloricate choreotrichs, are currently thought of as "typical" phytoplankton grazers in the microzooplankton, heterotrophic organisms 20-200 txm in size [4, 70]. Certainly ciliates have been shown to be significant grazers in marine systems [15, 70, 93]. However, there are two other major groups of protistan herbivores: < 20 p~m flagellates and heterotrophic dinoflagellates. Nanoflagellates, 2-20 Ixm in size, are commonly relegated to the role of "bacterivore" in models of marine food webs [2, 21]. It now appears that most consumers of < 1-1xm-sized cells are actually flagellates less than 5 txm in size, and that 5-20-1xm flagellates select larger prey, in the general range of 1-10 Ixm [75]. This is the size range of a large part of the biomass of autotrophic cells in the open ocean, as discussed above. There is accumulating evidence for the
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significance of nanoflagellate herbivory in situ. Campbell and Carpenter [13] and Caron et al. [17] reported that nanoplanktonic grazers were the main consumers of coccoid cyanobacteria in marine food webs. Caron et al. [17] concluded that in coastal water of the northeastern USA, nanoplankton consumers could remove as much as 54% of the cyanobacterial standing stock per day, and that at times as much as 30% of the total prokaryotic biomass consumed was as cyanobacteria. Sherr et al. [81] found that in a Georgia estuary, the assemblage of 5-15-p~m flagellates cleared 2-6 txm phytoplankton cells at about the same rate as did the ciliate assemblage. Kuosa [47] reported that nanoflagellate grazing consumed about 32-42% of primary production in the summer and fall in the Baltic Sea. An important component of the assemblage of < 20-1xm-sized flagellates appears to be nonarmored, gymnodinoid dinoflagellates. Bjornsen and Kuparinen [6] found that 12-14-txm-sized Gymnodinium sp. were major herbivores in the Southern Ocean, consuming algal cells in the size range of 8-10 p~m. Strom [89] isolated a 10-12-p~m-sized Gymnodinium species from surface waters in the subarctic Pacific. This dinoflagellate resembled a common, similarly-sized herbivorous protist observed in the Georgia estuary [81 ]. Results from the North Atlantic Bloom Experiment in 1989 and 1990 indicate that in the open North Atlantic, the heterotrophic dinoflagellate assemblage was dominated, in terms of both numbers and biomass, by cells < 20 p~m in size; in this case most of the dinoflagellates were 4-8 p~m spindle-shaped species, similar to Katodinium sp. [95]. The abundance and biomass of heterotrophic dinoflagellates 8-100 txm in size often equals or exceeds that of ciliates in the plankton [10, 38, 50, 52, 53, 84]. Dinoflagellates appear to be a particularly important component of the microzooplankton in polar seas. In the Canadian Arctic, Bursa [12] found that heterotrophic dinoflagellates comprised 73% of microzooplankton numbers on an annual basis, and Lessard and Rivkin [51] reported that in McMurdo Sound, Antarctica, up to 97% of the biomass of microzooplankton was due to phagotrophic dinoflagellates. Dinoflagellates may also be major herbivores in Antarctic sea-ice communities [8]. Fecal pellets and empty frustules produced by dinoflagellates feeding on diatoms can comprise a significant fraction of sinking particles in the Southern Ocean [61]. Phagotrophic dinoflagellates are capable of consuming diverse types of prey over three orders of magnitude in size, from < 1-txm prokaryotes to > 200-1xm diatom chains. As Lessard [50] observed, phagotrophic dinoflagellates are "eclectic feeders" having a variety of mechanisms for consuming their prey. Non-armored dinoflagellates can engulf whole prey into food vacuoles, often feeding on organisms nearly as large as themselves. Some species utilize a specialized appendage to pierce the cell wall of prey cells and "suck out" the cytoplasm in a manner analogous to the feeding behavior of spiders [25]. Armored dinoflagellates may also extrude a large, pseudopodial feeding veil which envelopes their prey and permits extracellular digestion [25, 26, 42]. By the latter mechanism, armored species are able to feed on diatom chains much larger than the dinoflagellate cell itself. Thus, the range of prey sizes grazed by phagotrophic dinoflagellates exceeds that of pelagic ciliates, which normally ingest prey < 20 txm in size [46, 70, 93].
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This is not the end of the dinoflagellate story, as it now appears that many bloom-forming autotrophic dinoflagellates are capable of ingesting prey as well. Incidental reports of mixotrophic dinoflagellates are common in the literature [25]. Recently, Bockstahler and Coats [7] reported ingestion of prey, including diatoms and < 20-t~m ciliates, by three common red-tide dinoflagellate species in Chesapeake Bay. Jacobson [41] has also found food vacuoles in several other species of autotrophic dinoflagellates. Thus, we can no longer conceptualize microzooplankton herbivores as being mostly ciliates; phagotrophic dinoflagellates and nanoflagellates are also significant consumers of phototrophic cells. In addition, phagotrophic protists do not simply consume phytoplankton cells that are small enough to escape metazooplankton grazers. Protists are capable of feeding on the entire size range of phytoplankton and may, at times, be in direct competition with herbivorous metazooplankton. 3. Heterotrophic protists are capable of growing as fast as, or faster than, phytoplankton, and thus are able to curtail development of blooms. As Goldman [31] has observed, eukaryotic microbes with a heterotrophic mode of existence, i.e., phagotrophic protists, are able to grow at a faster rate than can autotrophic microbes. Under optimum culture conditions, phytoplankton typically have population growth rates in the range of 0.5 to 2.0 day -1 [18, 71], while optimum growth rates of phagotrophic protists are equal to, or greater than, these rates (Fig. 1). In culture, the growth rate of heterotrophic protists is inversely proportional to cell size and directly related to temperature (Table 2). It is interesting that heterotrophic dinoflagellates have lower growth rates than do ciliates of comparable size (Fig. 1, Table 2); Banse [4] previously showed that autotrophic dinoflagellates have lower maximum growth rates than do diatoms. However, even larger-sized protists growing at cool temperatures can double in numbers within 1-2 days, as long as food is not limiting (Table 1). It has been speculated that the capacity for rapid growth exhibited by phagotrophic protists is one factor that prevents phytoplankton blooms in areas of the ocean where surface nutrients are high but chlorophyll a concentrations remain low, e.g., the subarctic Pacific [57] and the central equatorial Pacific [49; H. Ducklow 1989, The Oceanography Society Meeting, Monterey CA, Abstract 19]. Information on the actual growth rates of herbivorous protists in situ is almost nonexistent at present; this is a research area in need of attention. 4. Measurements of grazing impact of 'microzooplankton' (which also ineludes < 20-p~m herbivores), indicate that this component of the grazing community is responsible for a large share of phytoplankton mortality in all areas of the ocean so far examined. During the past decade, data on microzooplankton grazing impact in situ have steadily accumulated (Table 3). Various habitats in the ocean have been examined: open ocean and coastal waters; polar, temperate, and tropical regions; oligotrophic and eutrophic systems. In all of these regions, microzooplankton grazing was generally equal to half or more of daily phytoplankton production (Table 3). Microzooplankton grazing may account for 100% of daily production, particularly in systems dominated by < 5-p~m-sized phytoplankton cells. Even during diatom blooms in an upwelling system off the Oregon coast, protists, predominately extracellularly-feeding dinoflagellates,
Phagotrophic Protists and Pelagic Food Webs
229
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Body Size, cubic microns Fig. 1.
Relation of growth rate, (Ix day-~), to body size (Ix3) for phytoplankton and phagotrophic
protists growing under nonlimitingconditionsin culture at 20°C. Data for phytoplanktonare from Table 4 of Raven [71]. Valuesfor protists are from Table 2; in some cases where culturetemperatures were near 20°C, growthrate at 20°Cwas calculatedusinga Q~oof 2.0. p, phytoplankton;f, nanoflagellates; d, dinoflagellates;c, ciliates. consumed 26-50% of daily production [S. Neuer and T. Cowles, manuscript accepted for publication in Mar. Ecol. Prog. Ser.]. As a final note on the subject of the role of phagotrophic protists as grazers of 1-10-1~m-sized cells, in the open ocean up to half of the eukaryotic organisms in this size class are not phototrophic cells, but rather heterotrophic flagellates [82]. The idea that microzooplanktonic grazers, specifically ciliates, are a major source of mortality for bacterivorous flagellates is an integral part of the concept of the microbial loop [3, 21]. Only recently, however, has there been direct evidence for this trophic link. Both Ohman and Snyder [63] and Verity [94] have cultured pelagic ciliates using nonpigmented flagellates as a food source. The fact that both ciliates and phagotrophic dinoflagellates in open ocean plankton assimilate carbon from radiolabeled bacterial cells may also indicate a trophic link mediated by bacterivorous flagellates [52]. Again, more work is needed on this topic.
Conclusions Recent findings compel us to rethink the initial concept of the role of heterotrophic microbes, bacteria, and phagotrophic protists, as simply a side loop "add-on" to the classic phytoplankton-copepod-fish food chain:
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Table 2. Maximum growth rates (ix, day-l), and minimum generation times (T, h), at various temperatures for diverse phagotrophic protists in culture Protist (size, ixm3)
Nanoflagellates Actinomonas mirabilis (75) Monosiga sp. (20) Ochromonas sp. (200) Paraphysomonas vestita (190) Pleuromonas jaculans (50) Pseudobodo tremulans (90) Paraphysomonas imperforata (200-500) Diphanoeca grandis Paraphysomonas imperforata (200-300) Dinoflagellates Oxyrrhis marina (6300) Gymnodinium sp. (1000-1500) Gymnodinium sp. (600-1200) Gyrodinium dominans (20,000-30,000) Protoperidinium hirobis (6400) Protoperidinium spiniriferum (48,000)
Temp (°C)
ixday -1
T (h)
Reference
20 20 20 20 20 20 20-24 15 -1.5 6.0 15
6.0 4.1 4.6 5.5 3.8 3.6 2.4 3.6 0.5-0.8 0.9-1.5 2.3-2.7
2.8 4.1 3.6 3.0 4.3 4.6 6.9 4.6 22-32 11-19 6-7
22 22 22 22 22 22 32 1
0.8-1.3 0.3 0.7 0.3 0.8 1.2 0.23
13-21 55 24 55 21 15 72
33
20
1 12 20 25-27 20 20
20
6 88 60 43 43
Ciliates
Tintinnopsis cf. acuminata (11,000) Eutintinnus pectinis (18,000) Favella sp. Balanion sp. (19,000) Tintinnopsis acuminata (12,000) Tintinnopsis vasculum (120,000) Strombidium reticulatum (40,000) Lohmaniella spiralis (150,000) Strombidium sp. (17,000-33,000) Strobilidium sp. (18,000-22,000) Urotricha furcata (1800)
Plagostrombidium flallax (50,000)
Strombilidium lacustris (113,000)
18 18 7 15 20 7-10 15 20 15 20 25 5 10 15 12 12 15 20 20 5.5 12 15.5 21.5 5.5 12 15.5 21.5 5.5 12 15.5 21.5
1.4 1.4 0.4 1.0 1.4 1.5 2.4 2.9 1.2 1.4 1.9 0.5 1.0 1.0 0.9 1.0 1.4 2.7 1.4 0.46 0.66 0.76 1.72 0.03 0.42 O.57 0.90 0.43 0.70 0.99 1.42
12 12 43 17 12 11 7 5.8 14 11.5 8.7 35 17 17 18.5 17 11.5 6.2 11.5 36 25 22 9.7 555 40 29 18.5 39 24 16.8 11.7
39 39 87
87
93
93 46 46 63 94 59
59
59
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Table 3. Summaryof recent estimates of microzooplanktongrazing impact on phytoplankton in various marine systems, as percent of primary productiongrazed day-1 by organisms < 200 Ixm in size in the water column of shallow systems, or in the mixed layer of the open ocean. The dilution method of Landry and Hassett [48] was used in these studies to estimate grazing rates Location
% Primpro grazed day-1
Reference
Washington, USA coastal waters Celtic Sea Jones Sound, Arctic Baffin Bay, Arctic Halifax Harbour, Nova Scotia Rhode River Estuary, MD, USA Grand Bank, Newfoundland Subarctic Pacific Central equatorial Pacific Northeastern Atlantic Indian Ocean Oregon, USA coastal upwelling blooms
17-52% 13-65% 40-114% 37-88% 47%, 100% 45-104% 50%, 70% 40-60% 44%, 90% 39-115% 70-100% 26-50%
48 9 65 66 28 27 66 89 49 10 11 Neuer & Cowles, ms accepted by MEPS
1. Picoplanktonic primary producers are a significant component of marine food webs. Prochlorophytes can be a substantial fraction of the numbers and biomass of the "heterotrophic" bacterioplankton enumerated via acridine orange direct counts. Other, slightly larger picoautotrophs, coccoid cyanobacteria and picoeukaryotes, can also represent a large part of total phytoplankton biomass and primary production, particularly in the open ocean. This implies that bacterivorous protists must also be consuming bacterial-sized primary producers. Thus phagotrophic protists in the nanoplankton, for the most part flagellates but also including lO-20-p,m ciliates, are likely to have a major role as consumers of picoplanktonic primary production as well as of the secondary production of heterotrophic bacteria.
2. Phagotrophic flagellates, particularly dinoflagellates, are a ubiquitous and significant component of the herbivorous microzooplankton. Recent studies in several regions of the world ocean, including the subarctic Pacific, the North Atlantic at the time of the spring bloom, the Indian Ocean, and an upwelling system off the coast of Oregon, USA, have demonstrated that microzooplanktonic grazers are often the major consumers of phytoplankton. In all of these systems, heterotrophic dinoflagellates in the nanoplankton and microplankton size ranges were implicated as major herbivores. Dinoflagellates have a variety of species-specific feeding strategies and can graze cells over a wide size range, from picoplankton to large diatom chains. Based on the latest findings about the structure and functioning of marine pelagic food webs, we should now view phagotrophic protists, ciliates, and flagellates as a biotic control of both bacteria and phytoplankton in the world ocean. Phagotrophic protists are also responsible for much of the regenerative flux of nitrogen and phosphorus in the sea [16], and can be a vital food resource for metazoans [29, 86]. It is clear that phagotrophic protists should be routinely considered as having major trophic roles over a broad range of marine ecosystems, including regions characterized by spring blooms and upwelling. Future
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models of pelagic food webs should formally include protistan as well as metazoo 31ankton consumers of primary production.
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