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J. Great Lakes Res. 25(1):61–77 Internat. Assoc. Great Lakes Res., 1999 The Impact of Zooplankton Grazing on Phytoplankton Species Composition and Biomass in Lake Champlain (USA-Canada) Suzanne N. Levine1,*, Mark A. Borchardt2, Moshe Braner1, and Angela d. Shambaugh1 1School of Natural Resources Aiken Center University of Vermont Burlington, Vermont 05405 2Marshfield Medical Research Foundation Marshfield, Wisconsin 54449 ABSTRACT. Rates of grazing on phytoplankton by macrozooplankton (cladocerans and copepods > 220 µm in length) and microzooplankton (animals < 220 µm, mostly rotifers and nauplii) were determined for Lake Champlain on three occasions using a modified version of the Lehman-Sandgren method. Gradients in grazer density were created in fertilized cubitainers incubated in situ, and clearance rates on specific phytoplankton taxa determined from regressions of algal growth rates on herbivore biomass. Grazers consumed 3 to 26% of the total phytobiomass present and 22 to 139% of net primary productivity daily. Macrozooplankton fed most heavily on algae 5 to 25 µm in size and generally selected dinoflagellates and green algae (6 to 26% of biomass removed per day) over cryptophytes (1 to 8%/day), diatoms (0 to 10%/day) and blue green algae (0 to 6%/day). However, variability in grazing vulnerability among the species within divisions was high. Microzooplankton had greater weight-specific clearance rates than macrozooplankton when consuming diatoms, blue-green algae, and cryptophytes, but were less efficient at harvesting green algae. An experiment in which nutrients and zooplankton were manipulated in a 2 × 3 factorial design indicated that both variables have a net positive impact on phytoplankton growth rates in Lake Champlain, the zooplankton because they excrete required nutrients. Indirect effects of the nutrients vs. grazers experiment included rotifer growth in response to increased algal productivity and harvesting of rotifers and Cladocera by cyclopoid copepods. It was concluded that both nutrients and grazing influenced the structure of Lake Champlain’s phytoplankton community, but that nutrients were generally more important. INDEX WORDS: Lake Champlain, zooplankton, phytoplankton, grazing, nutrients, primary productivity, mortality, edibility. INTRODUCTION terest in biomanipulation as a tool for reducing algal biomass when P reductions are difficult (Reynolds 1994). The potential for grazing to influence algal biomass has long been recognized. The “clear-water phase” that is a feature of many lakes in early summer has been attributed to grazing rates that outpace reproduction, the latter being constrained by nutrient depletion (Sommer et al. 1986). That grazing may account for significant mortality over longer periods of time is apparent in recent re-evaluations of the chlorophyll a- phosphorus relationship in lakes (Hansson 1992, Persson et al. 1992, Mazumder 1994). These show chlorophyll a concentrations increasing about four times more Successful manipulation of forage fish communities through addition or removal of piscivorous fish and of zooplankton communities through change in planktivorous fish densities (Reynolds 1994) has fueled the development of trophic cascade theory (Carpenter et al. 1985), and raised questions about the relative roles of nutrients and grazers in controlling algal biomass and species composition in lakes. While eutrophication management continues to focus on phosphorus control, there is growing in- *Corresponding author. E-mail: [email protected] 61 62 Levine et al. rapidly with rising total P concentration in lakes with impoverished zooplankton communities than in lakes with dense populations of large daphnids. Biomanipulation experiments have been highly successful in reducing algal biomass when one of their outcomes has been increased densities of Daphnia (Liebold 1989, Reynolds 1994). By contrast, manipulations yielding zooplankton communities dominated by copepods or small-bodied cladocerans rarely have affected phytoplankton. When zooplankton manipulations affect phytobiomass, phytoplankton species composition is often altered as well (Porter 1973, Berquist and Carpenter 1986, Proulx et al. 1996, Svensson and Stenson 1991). Discrepancies in the relative proportions of phytoplankton species found in the guts of zooplankton with the proportions present in lake communities suggest that zooplankton frequently feed selectively (Porter 1973, Infante 1978, Berquist and Carpenter 1986), with prey discrimination on the basis of size, shape, toxicity, or nutrient content (Reynolds 1984). Given the complexity of the pelagic foodweb and the many factors that affect phytoplankton growth and mortality, progress in understanding phytoplankton community structure and succession, and an enhanced ability to predict the outcome of biomanipulation or nutrient management, may require the use of numeric models. Modeling of phytoplankton dynamics requires quantitative data on reproductive and mortality rates, however, and these are rarely obtained at the species level. While zooplankton grazing on the total phytoplankton standing stock has been quantified in a large number of lakes, there is only one substantial data set on the mortality of individual phytoplankton species during zooplankton grazing, that of Elser and colleagues for three California lakes (Tahoe, Castle, and Clear; Elser and Goldman 1991, Elser 1992, Elser and Frees 1995). The intense labor investment needed to track individual phytoplankton species has discouraged species-level analysis, as has slow methods development. The most reliable method for grazing assessment is that of Lehman and Sandgren (1985), in which clearance rates on individual phytoplankton taxa are estimated by manipulating grazer densities in experimental systems to multiple levels, measuring the resulting growth rates, and regressing algal growth rates on grazer density. The slope of the regression line is the clearance rate. However, the approach provides meaningful rates only if phytoplankton reproduction is “constant” across experimental sys- tems. Because zooplankton are major recyclers of N and P (Lehman 1980), and these nutrients frequently limit phytoplankton growth rate, manipulation of zooplankton density almost certainly alters reproduction rates under ambient nutrient conditions. To eliminate the effects of zooplankton nutrient regeneration, nutrients may be added to experimental systems. These saturate nutrient uptake and raise phytoplankton growth rate to a maximum (and uniform) velocity (Elser 1992). Lehman and Sandgren’s original method did not include nutrient additions, so that its use was restricted to consideration of the general direction (positive, negative, neutral) of net taxon response to grazer density (Lehman and Sandgren 1985, Berquist and Carpenter 1986). Elser and Goldman (1991) and Elser (1992) used experimental systems with and without zooplankton present and with nutrient saturation to obtain the first estimates of species-specific grazing mortality in lakes. Cyr and Pace (1992) and Cyr (1998) then combined the nutrient additions of Elser with the Lehman-Sandgren method to estimate zooplankton grazing on total phytobiomass (chlorophyll a) in 16 northeastern U.S. lakes, and grazing on Cryptomonas in two lakes. In this work the Lehman-Sandgren technique is used with nutrient addition to assess taxon specific phytoplankton loss rates in Lake Champlain. Both macrozooplankton (animals > 220 µm in length; Cladocera and copepodite and adult copepods) and microzooplankton (< 220 µm; mostly rotifers and nauplii) grazing were assessed on three occasions (spring, summer, and fall), and compared with phytoplankton division rates (or primary productivity). A 2 × 3 factorial experiment was also conducted to compare the importance of nutrients (N + P + C) and grazers as controls on phytoplankton biomass in the lake. Lake Champlain is just the fourth lake worldwide, and the first lake outside of California, for which rates of macrozooplankton grazing on specific taxa have been obtained. It is the second lake for which microzooplankton grazing has been assessed (the first was Castle Lake, CA; Elser and Frees 1995). As Lake Champlain shares many physicochemical and biological characteristics with the Laurentian Great Lakes, the data presented here may contribute to an understanding of the phytoplankton dynamics in these important lakes as well as in Lake Champlain. This work was conducted in concert with studies of bacterivory and zooplankton-protozoan interactions in Lake Champlain, and was part of a larger effort to describe and model the lake’s food web (Levine et al. 1998, 1999). Zooplankton Grazing of Phytoplankton in Lake Champlain SITE DESCRIPTION Lake Champlain is a large (170 km long, surface area 1,130 km 2 ), deep (maximum depth 122 m; mean depth 23 m), but narrow (maximum width, 20 km) lake straddling the border between Vermont and New York and extending a short distance into Quebec. Numerous islands, sills, peninsulas, and causeways divide the lake into partially isolated sub-basins. This study took place in the largest of these, known as the “Main Lake” (at 44°27′ N, 73°17′ W, about 1 km northwest of Juniper Island, at the broadest expanse of the lake). The Main Lake is marginally dimictic; it stratifies in summer, establishing an epilimnion with a depth of 10 to 12 m, and, in winter, sometimes freezes for as long as 2 months (February to April). For several months between October and June, however, it mixes to the bottom. The Main Lake is oligotrophic-mesotrophic, with a mean total phosphorus concentration of 0.4 µM (VT Dept. Environ. Consev., unpub. data) and a mean chlorophyll a concentration of 5 µg/L (NY Dept. Environ. Consev., unpub. data). Its phytoplankton community is dominated by diatoms and cryptophytes during much of the year, although blue-green algal dominance is common in late summer (McIntosh et al. 1993). Seasonal phytoplankton biomass distribution is bimodal, with a major peak in spring and a smaller peak in fall. The zooplankton community is a mixture of cycloploid copepods, cladocerans (principally Daphnia galeata mendota and Daphnia retrocurva), and rotifers (McIntosh et al. 1993). Calanoid copepods are comparatively scarce. In a typical year, copepods and rotifers are most abundant in spring, while cladocerans peak in summer. Over 80 species of fish are present in Lake Champlain; Osmerus mordax dentex (rainbow smelt) is the dominant planktivore (Myer and Gruendling 1979). METHODS Grazing Experiments The Lehman-Sandgren method with nutrient addition was used to estimate zooplankton grazing rates. Three levels of grazer density were created in duplicate in transparent 10-L cubitainers for both macrozooplankton (animals retained by a 220 µm mesh; adult and copepodite copepods, and cladocerans) and microzooplankton (< 220 µm; rotifers and nauplii) and the cubitainers incubated in situ for 2 to 3 days. Phytoplankton growth rates in cu- 63 bitainers were obtained by measuring phytoplankton biomass four or more times during an incubation, and calculating the slope of the regression of ln (phytobiomass) against time. These rates then were regressed against grazer biomass to yield estimates of grazer-biomass specific clearance rates (from the slope). The fraction of phytoplankton biomass removed per day was calculated as the product of clearance rate and ambient zooplankton biomass and carbon flow from the product of this value and the C content of the phytoplankton (about 0.5 µg C/ µg dwt; Reynolds 1984). Grazing losses were estimated for several abundant species, for the phytoplankton divisions, and for the phytoplankton community as a whole. Separate estimates for micro- and macrozooplankton grazing were possible because the slope of the relationship between algal growth rate and grazer biomass was not affected by an across-the-board subtraction of grazer biomass from all data points (provided that the biomass subtracted is of the same type, which it was). The sieve used to manipulate macrozooplankton densities allowed microzooplankton to pass freely and thus attain similar densities in all macrozooplankton treatments, while the microzooplankton treatments used water pre-sieved to remove macrozooplankton (some contamination occurred; see below). The experiment was repeated three times, in mid-summer (25 to 27 July) and fall (27 to 29 September) 1994, and in spring (8 to 11 May) 1995. Water and plankton for the experiment were obtained with a clear vertically-oriented 8-L van Dorn bottle which was deployed at 1-m intervals over the depth of the epilimnion (10 m in summer, 18 m in fall) and 2 m into the metalimnion. In spring, when the lake mixed to the bottom, water collection stopped at 20 m. The collected water was pooled in a 240-L tank, and mixed with a paddle, prior to dispersal to cubitainers or sieving. Zooplankton for the “high macrograzer” treatment were obtained with a 202 µm-mesh Wisconsin net (collar off; mouth diameter 0.5 m; a mason jar attached) towed vertically over the same depth interval used for water collection. The captured animals were added to 30 L of lake water (under the water surface to minimize air trapping under cladoceran carapaces) to create a macrozooplankton “concentrate.” 64 Levine et al. The procedure for preparing the experimental treatments was as follows. The “ambient macrograzer” (AMA) cubitainers received unaltered lake water from the reservoir, while the “high macrograzer” (HMA) treatment was created by filling cubitainers with lake water and adding sufficient zooplankton concentrate to increase animal densities about 4 fold (300 to 500 mL). The water remaining in the reservoir then was passed through either a 202 µm plankton net (July) or a 220 µm sieve (September and May) to remove macrozooplankton. The “low macrograzer” (LMA) treatment (which also served as the “ambient micrograzer” (AMI) treatment) consisted of cubitainers filled with this coarsely-sieved water. To create the remaining treatments, some of the 220 µm (or 202 µm) “sievate” was passed through a second 20 µm sieve. The finer “sievate,” which had been largely cleared of micrograzers, was poured into cubitainers to become the “low micrograzer” (LMI) treatment, while animals collected on the sieve were washed back into 20 L of the 220/202 µm “sievate” to produce the “high micrograzer” (HMI) treatment. Once all the cubitainers were filled, KH 2PO 4, NH4Cl, and dextrose (C6H12O6) were added to each to increase P, N, and C concentrations by 3, 5, and 34 µM, respectively. Air was squeezed out of the cubitainers to prevent animal trapping in an air space, and the cubitainers were suspended from anchored floating frames in Burlington Harbor at a depth of 1.5 m. They were incubated 44, 48, and 64 h in summer, fall, and spring, respectively. Light intensities in the cubitainers were generally below the threshold for photoinhibition for most phytoplankton groups (< 1,000 µmol/m 2 /s), but still sufficiently high during mid-day (> 300 µmol/m2/s) to saturate photosynthesis. Sixty-milliliter phytoplankton samples were collected in triplicate from the cubitainers immediately before incubation, on 3 or 4 occasions during the first 24 h of incubation, and daily thereafter. One percent acid Lugol’s solution was used as a phytoplankton preservative. Zooplankton were collected at the beginning of each experiment by passing 10 L of the water from the mixing tank through either a 64 µm (July and September 1994) or 20 µm-mesh sieve (May 1995), and washing the retained animals into sample bottles. These samples were used to estimate lake standing stocks. To estimate zoobiomass in the cubitainers, the entire contents of each cubitainer were sieved (same mesh sizes as the initials) at the end of each experiment. The animals collected on sieves were anesthesized in carbonated water prior to preservation in 5% buffered sucrose formalin (Haney and Hall 1973) with rose bengal stain. Algal species composition and cell densities were determined by direct microscopic counts of settled samples using an Olympus inverted light microscope (150 random fields, or > 300 cells of the dominant species). The biovolume of each phytoplankton species was estimated from its cell dimensions and geometry. For common species, cell dimensions were measured during every incubation and in all treatments; scarce species were measured during at least one incubation. Algal dry weight was estimated from biovolume using the conversion factor 0.47 pg dwt/µm3 (Reynolds 1984). Zooplankton were enumerated and measured with the same compound microscope. The entire sample was counted for macrozooplankton, while subsamples were used to estimate microzooplankton numbers. Dead and moribund animals and nonfeeding stages (eggs and embryos) were counted but not included in biomass estimates. The biomass of rotifers was estimated from shape dimensions and geometry (Ruttner-Kolisko 1977), and that of copepods and cladocerans from biomass-length relationships (McCauley 1984, Culver et al. 1985). To the extent possible, the relationships used were for animals preserved as in our study. Preservation can cause shrinkage of animals, although it is generally minimal (5 to 10%) in 4 to 5% sucrose formalin (Dumont et al. 1975, Culver et al. 1985). The first two copepodite instars of cyclopoid copepods were included in the estimates of herbivore biomass as these animals consume substantial amounts of phytoplankton. Later stages (especially adults, which contributed > 90% of copepod biomass in the experiment) are predominantly carnivorous, and thus were not included. Primary Productivity The y-intercepts of the Lehman-Sandgren regression lines provided estimates of algal division rates in cubitainers (growth in the absence of grazers). Multiplying these rates by phytoplankton biomass and C content yielded estimates of net primary productivity (NPP) under the conditions of the experiment (1.5 m incubation depth, and nutrient enrichment). To assess primary productivity within the lake, the 14C technique in a laboratory incubator was used (Fee et al. 1992). Water was collected as in the grazing experiments on the second day of the Zooplankton Grazing of Phytoplankton in Lake Champlain last two grazing studies (in September and May), and incubated with 14C-labelled bicarbonate at five light intensities and at ambient temperature. A LiCor light meter and submersible probe were used to estimate light extinction rates at the sampling site, while a second light meter with an aerial probe monitored solar irradiance over the course of the grazing experiments. The numerical model of Fee (1990) was employed to estimate daily primary production from the data on solar irradiance, light extinction, and productivity-light relationships. Dissolved inorganic carbon was estimated from sample alkalinity and pH. The Nutrients vs. Grazers Experiment To evaluate the relative importance of nutrients and grazers in determining Lake Champlain’s phytoplankton standing stocks, a 2 × 3 factorial experiment was performed (31 July to 4 August 1995). Water and zooplankton were collected as in the grazing experiments (depth interval, 0 to 15 m) and the same “high,” “ambient,” and “low” macrograzer treatments were created, except that each treatment was repeated in six, rather than two, cubitainers. Half of the cubitainers at each grazer level then were enriched with C, N, and P (as in the grazing experiments), while the other half were left unfertilized. Duplicate chlorophyll a samples (750 mL) were collected at the beginning of the experiment from the tanks used to fill cubitainers, and from the cubitainers after 23 and 92 hr of incubation at 1.5 m depth. Pigments collected on GFF filters (nominal pore size 0.7 µm) were extracted in hot ethanol (Sartory and Grobbelaar 1984), and their absorbance read on a Shimadzu UV-VIS 160U spectrophotometer, using the monochromatic procedure (665 nm) with phaeophytin correction (Lorenzen 1967). Phytoplankton growth rates were calculated from the rate of change in chlorophyll a concentration over the incubation. Triplicate phytoplankton samples (for microscope counts) also were taken from cubitainers at the beginning and end of the 4day experiment and those in the fertilized systems were used to obtain a fourth set of grazing and NPP rate estimates. Zooplankton were collected at the experiment’s end using the procedures described earlier. Two-way analysis of variance (ANOVA) allowed assessment of the relative contributions of nutrients and zooplankton in controlling phytoplankton growth rates. 65 RESULTS Grazing Studies Experimental Conditions The three grazing studies were conducted at the beginning, middle, and end of the growth season to maximize the number of phytoplankton species examined and to provide a sense of grazing rate variance. Physicochemical and biological conditions were markedly different during the three experiments (Table 1). In May, the lake was isothermal, with a water temperature of 5°C, and nutrient concentrations relatively high. The lake was fully stratified in July, with a mixed layer depth of 10 m and an epilmnion temperature of 22°C, whereas in September, thermocline erosion was underway (epilimnion temperature, 17°C; mixed-layer depth, 18 m). Phytoplankton biomass was far greater in May than during the other experiments (Fig. 1) and strongly dominated by diatoms (96%), in particular Aulacoseira sp. (67%). The July phytoplankton community was dominated by green algae (Mougeotia sp., Oocystis spp., and Eudorina elegans) and cryptophytes (Chroomonas sp. and Cryptomonas sp.), and the September community, by diatoms and cryptophytes, although blue-green and green algae were also common. During two of the three grazing experiments, Cladocera (Daphnia galeata mendotae, Daphnia retrocurva, and Eubosmina coregoni) were the principal herbivores present (Fig. 2). The calanoid copepods Diaptomus sicilis and Diaptomus minutus shared dominance with Cladocera during the May experiment, but otherwise were scarce. The rotifer community was consistently dominated by Keratella spp, but Polyarthra spp., Noltholca sp. (May 1995), and Kellicottia longispina were present as well. The predaceous rotifer Asplanchna sp. was common in July and large enough to be included in the macrozooplankton. Cyclopoid copepods were the most significant components of the carnivorous zooplankton community. These animals were almost as abundant as Cladocera in May (dominants Diacyclops thomasi, Acanthocyclops robustus, and Tropocyclops prasinus prasinus), scarce in July, and the major contributors to zoobiomass in September (A. robustus, Mesocyclops edax, and D. thomasi). Extent of Zooplankton and Phytoplankton Manipulation Through Sieving and Addbacks Through sievings and additions of netted animals, 6 to 60 fold gradients in herbivore biomass 66 Levine et al. TABLE 1. Conditions at the sampling site during the 4 experiments. The chemistry data are from the Vermont Department of Environmental Conservation, and pertain to samples taken within a week of the experiments. All values pertain to the mixed layer, and are for daytime. Physicochemical Parameters Mixed layer depth (m) Secchi depth (m) Temperature (°C) TP (µM) TDP (µM) TN (µM) DIN (µM) TN:TP DIN:TP DSi (µM) July 1994 Grazing Studies Sept. 1994 May 1995 Nutrients vs. Grazers July 1995 10 5 22 0.32 0.13 29 12 89 36 2.9 18 6.5 17 0.29 0.13 38 10 130 33 25 70 — 5 0.35 0.19 49 — 139 — — 12 6 22 0.29 0.23 23 — 79 — 8.2 Biological Parameters Chlorophyll a (µg/L) 1.5 2.1 6.3 1.8 Phytobiomass (µg dwt/L) 146 274 1,264 20 Zooplankton Biomass (µg dwt/L) 79 1,282 695 1,144 Herbivore Biomass (µg dwt/L)1 79 371 485 648 Primary Productivity in fertilized cubitainers (mg C/m3/d) 15 13 119 5 in the lake, areal (mg/m2/d)2 — 349 1,660 — Limiting Nutrient3 N+P N+P None 1Excludes biomass associated with late-stage cyclopoid copepods. 2Cloudless conditions are assumed for the sake of comparability. The estimates include phytoplankton exudates and thus are approximately gross primary productivity. The values for the cubitainers are net productivity, without respiration and exudates). 3Results of enrichment experiments conducted within a week of the grazing studies at the same sampling site (Levine et al. 1997). were created within the macrograzer portion of our experiments (0.06 to 3.4, 0.003 to 0.25, and 0.09 to 0.58 mg.dwt zoop./L, in May, July, and September, respectively). The biomass of micrograzers present in the macrograzer treatments was both fairly uniform, and a minor portion of total zoobiomass (Fig. 2). Thus the estimates of macrograzing can be considered to be relatively free of a “micrograzer influence.” Duplication of macrozooplankton biomass within treatments was sometimes poor, but this was not an issue, as data from each cubitainer were entered separately into the regression analysis. In the microzooplankton portion of the study, the greatest herbivore biomass present in cubitainers exceeded the lowest by 200 to 1500 fold. Some of the LMI cubitainers were almost devoid of animals (0.0001 to 0.0005 mg dwt/ L), while zoobiomass in the HMI cubitainers reached 0.61, 0.019, and 0.24 mg .dwt/ L in May, July, and September, respectively. Sieving of lake water through a 220 µm sieve or a 202 µm net prior to micrograzer manipulation succeeded in removing > 95% of macrozooplankton biomass. However, it was not uncommon for one or two macrograzers to slip through or around the sieve. Because these animals had biomasses three orders of magnitude greater than those of the individual microzooplankton, and microzooplankton were relatively scarce, the estimates of total herbivore biomass in the micrograzer treatments were affected by macrozooplankton contamination; macrograzers made up from 13 to 57% of the biomass in the AMI treatments, and 36 to 44% of that in the HMI treatments. In estimating microzooplankton clearance rates, the influence of macrozooplankton grazing was accounted for as follows: macrozooplankton clearance rates for the taxa of in- Zooplankton Grazing of Phytoplankton in Lake Champlain FIG. 1. The biomass of different phytoplankton groups in cubitainers at the beginning (I) and ending (F) of incubations with macrozooplankton at three different densities (low (LMA), ambient (AMA), and high (HMA). The two sets of columns indicate duplicate treatments. Incubation times were 44, 48, and 64 h in July, October, and May, respectively. terest (from the macrozooplankton experiment) were multiplied by the biomass of macrograzers in cubitainers to yield estimates of the fraction of phytobiomass removed per day. These were added to the measured growth rates, yielding estimates of growth rates in the absence of macrograzer contamination. Finally, the corrected growth rates were regressed on micrograzer biomass to obtain the clearance rates reported. Phytoplankton communities were not altered by macrozooplankton sievings and addbacks during the July and September experiments (Fig. 1). In 67 FIG. 2. The biomass of major zooplankton groups in experimental cubitainers at the end of the three grazing studies. HMA, AMA, and LMA refer to high, ambient, and low macrograzer densities; HMI, AMI, and LMI, to high, ambient, and low micrograzer densities. The two sets of columns indicate duplicate treatments. The “lake” samples were taken at the initiation of the experiments from the 240 L tank used to fill cubitainers. Lack of rotifers and nauplii in the “lake” in July is due to these two samples accidentally being concentrated with a 90 µm rather than a 64 or 20 µm sieve prior to counting; the sieve probably allowed rotifers to pass. May, however, colonial diatoms (Aulacoseira sp., Tabellaria spp., and Fragilaria crotonensis) dominated the phytoplankton. These colonies were sufficiently large to be partially retained by the zooplankton net and added to the HMA treatment along with zooplankton. Thus the HMA treatment 68 Levine et al. FIG. 3. The biomass of different phytoplankton groups in cubitainers at the beginning (I) and ending (F) of incubations with microzooplankton at three different densities (low (LMI), ambient (AMI), and high (HMI). The two sets of columns indicate duplicate treatments. Three levels of microzooplankton density were used. Incubation times were 44, 48, and 64 h in July, October, and May, respectively. had a phytoplankton biomass 2 to 3 times greater than the AMA and LMA treatments. Manipulation of micrograzers with a 20 µm sieve did not influence cryptophyte abundance in cubitainers, but did affect the densities of larger algae, especially those forming colonies (Fig. 3). The HMI cubitainers therefore contained about three times as much phytobiomass, and the LMI cubitainers only one half as much, as was present in the lake (AMI). The expected effects of these manipulations on grazing rate estimates are considered below. Primary Productivity The y-intercepts of the Lehman-Sandgren curves provided estimates of phytoplankton division rates in the cubitainers, albeit under the nutrient- and light-enhanced conditions of the incubations. These rates were 0.20, 0.09, and 0.18 /d in July, September, and May, respectively. Multiplying these values by phytobiomass and C content yielded net primary production (NPP) estimates of 15, 13, and 119 mg C/m3/d. These rates can be compared with 14C estimates of gross primary productivity (GPP) at the incubation depth during September and May, 56 and 184 mg C/m3/d respectively. The latter were greater, despite the fact that they were for unfertilized conditions, because they include C exuded from algae or respired during the 2 day incubations used to estimate NPP. For the lake as a whole, areal primary production (GPP) estimates of 236 and 1,093 mg C/m2/d were obtained in September and May, respectively. These rates (the first ever reported for Lake Champlain) reflect partially cloudy weather. Under cloudless conditions, the areal rates would be 349 and 1,660 mg C/m2/d. The mixed layer was much deeper in May than in September (70 vs. 18 m), and the phytoplankton, although more abundant, were more light limited (Levine et al. 1999). The mean volumetric GPP rates for the mixed layer during September and May were 13 and 16 mg C/m 3 /d, respectively. Under cloudless conditions, they would be 19 and 24 mg C/m3/d. Grazing Rates Rates of phytoplankton loss from Lake Champlain as a result of macrograzing were very low in July and September 1994, < 1% of total phytobiomass per day, and only modestly higher in May (10% per day). Losses to microzooplankton were slightly greater and followed the same seasonal trends, 4, 3, and 15% per day, in July, September, and May, respectively. Microzooplankton made up a relatively small proportion of total zoobiomass, but had weight-specific clearance rates four or more times those observed among macrozooplankton (Tables 2 vs. 3), hence the similarity in grazing pressure. Expressed as a percentage of the NPP in cubitainers, macrozooplankton and microzooplankton removal rates for total phytobiomass were 2, 0, and 56% and 20, 33, and 83% in July, September, and May, respectively. Thus in May, grazers removed more phytobiomass per day than was produced, Zooplankton Grazing of Phytoplankton in Lake Champlain 69 TABLE 2. Clearance rates (CR; mL/day / µg dwt zoopl.) for macrozooplankton feeding on phytoplankton and estimates of percentage algal biomass lost per day during the four studies in Lake Champlain. Rates are given for the total phytoplankton community, for algal divisions containing > 10% of phytoplankton biomass, and for a selection of individual species. Clearance rates significantly different from zero at P > 0.05 are indicated with an asterisk. GALD = greatest axial linear dimension. Taxon Total GALD — July 1994 C R % Lost 0.06 0.4 Sept. 1994 C R % Lost –0.02 0 May 1995 C R % Lost 0.26 10 July 1995 C R % Lost 0.17 11 — 0.18* –0.01 0.15 — — — 0.19 — 1.69* 0.41* 0.13* 0.08 0.07 — By Division: Greens Cryptophytes Diatoms Blue greens Dinoflagellates — — — — — By Species: Mougeotia sp. Coelastrum scabrum Cryptomonas sp. Chroomonas sp. Aphanizomenon flos-aquae Chroococcales Fragilaria crotonensis Tabellaria sp. Aulacoseira sp. Centrales1 1An unidentified, small species, 18 1.8 13 — — — — — — 11 — — 1.8* 58 — — — — 7 1.79 12 0.28 9 — — 0.46 28 4 –0.59 0 0.06 2 — — 0.09 6 32 — — –0.05 0 — — — — 2 — — — — 0.14 9 27 — — 0.00 0 — — — — 17 — — –0.01 0 — — — — 13 — — — — 0.16 8 — — 2 –5.63 0 — — — — 0.05 3 not colonial. This estimate does not include grazing on other Centrales, like Aulacoseira. 0.92 0.12 –0.68 –0.39 2.11 6 1 0 0 15 — 7 0 6 — — — 10 — 89 26 8 5 4 — TABLE 3. Clearance rates (CR; mL/day / µg dwt zoopl.) for microzooplankton feeding on phytoplankton and estimates of percentage phytobiomass lost per day, during the three grazing studies in Lake Champlain. Rates are given for the total phytoplankton community and for algal divisions containing > 10% of phytoplankton biomass. Clearance rates significantly different from zero at P > 0.05 are indicated with an asterisk. Taxon Total CR 2.28 By division: Greens Cryptophytes Diatoms Blue-greens 0.81 4.40 — — July 1994 % Lost 4 1 7 — — contributing to the collapse of the spring bloom. For the September and May experiments, grazing can also be expressed as a percentage of the mean GPP rate in the 18 and 70 m-deep mixed layers; the values are 22 and 658%. Sept. 1994 CR % Lost * 1.07 3 May 1995 CR % Lost * 0.83 15 — 2.15* 0.33 2.64* — — 1.12* — — 6 1 7 — — 20 — Discrepancies in taxon vulnerability to macrograzers were apparent both at the Division level and among species within a Division (Table 2). During July, dinoflagellates lost 15% of their standing stock to grazing daily, while green algae lost 6%, 70 Levine et al. and cryptophytes, 1%. Blue-green algae had negative clearance rates (grew more rapidly in the presence of grazers), and thus probably were largely invulnerable to grazing. In September, when dinoflagellates and green algae were scarce, cryptophytes and blue-green algae were subject to greater grazing mortality, 7 and 6% per day, respectively. Diatoms also were present at this time, but apparently not utilized by grazers (loss rate, 0% per day). In May, diatoms, the biomass dominant, lost an average of 10% of their biomass per day to macrograzers, which in this case included copepods as well as Cladocera. Micrograzers consumed cryptophytes, blue-green algae, and diatoms at higher rates than did macrograzers (Tables 2 vs. 3). During the spring bloom, they removed 20% of diatom biomass daily. Micrograzers were somewhat less efficient than macrograzers at green algal consumption, perhaps because the green algae present were relatively large. Table 2 includes rates of macrozooplankton grazing on some of the more abundant phytoplankton species. Grazing by microzooplankton at the species-level was not measured because of the low densities of the species involved. Among the cryptophytes, Cryptomonas sp. was more vulnerable to macrozooplankton predation (11 to 12% loss per day) than Chroomonas sp. (loss rate, 0 to 2% per day). Among the blue-green algae, the Chroococcales were more heavily grazed than Aphanizomenon flos-aquae, and, among diatoms, Aulacoseira sp. suffered greater grazing losses than Fragilaria crotonensis and Tabellaria sp. Many of the clearance rates given in Tables 2 and 3 are not significantly different from zero at a P level of 0.05. This is partly because clearance rates were, in fact, very low, but also because of scatter related to the patchy distribution of large (especially colonial) and rare phytoplankton species and the strong influence of individual animal length on zoobiomass estimates (all animals were assumed to have a mean length, when length actually varied). Although the regressions would have been greatly improved by the omission of outliers, this was avoided, given that there was no knowledge of what the correct grazing rates were. The reporting of large grazing rate estimates not significantly different from zero should be viewed as provisional. Nutrients Versus Grazers The nutrients vs. grazers experiment took place at the same time of year as the July grazing experi- ment, but a year later. Epilimnion depth, temperature, and nutrient concentrations were nearly identical during the two studies. Phytoplankton species composition was similar in the 2 years (Figs. 1 vs. 4), but phytobiomass was 7 times lower and herbivore biomass, 8 times greater in 1995. In addition, cyclopoid copepods (mostly Mesocyclops edax) were abundant in 1995, but scarce in 1994 (Fig. 2 vs. 5). This was essentially a “clear water” phase. Samples taken 23 h into the experiment indicated little change in chlorophyll a concentrations relative to initial values (Fig. 6), suggesting that phytoplankton may need a day or more to “gear up” for nutrient assimilation and division (Reynolds 1984), and also that grazing was not particularly intense. FIG. 4. The biomass of major phytoplankton groups in experimental cubitainers at the beginning (I) and end (F) of the nutrients vs. grazers experiment, July 1995. The three sets of columns indicate triplicate treatments. FIG. 5. The biomass of major zooplankton groups in experimental cubitainers at the end of the nutrients vs. grazers experiment, July 1995. The three sets of columns indicate triplicate treatments. Zooplankton Grazing of Phytoplankton in Lake Champlain 71 FIG. 6. Chlorophyll a concentrations in experimental cubitainers 0, 23 and 92 h into the nutrients vs. grazers experiment, July 1995. Initial concentrations were measured for the water used to fill cubitainers of similar treatment. The three sets of columns indicate triplicate treatments. FIG. 7. Phytoplankton growth rates vs. (a) total zooplankton biomass and (b) herbivore biomass (late stage cyclopoids excluded) of July 1995. Trend lines are linear regressions. The one shown in Panel b is for fertilized cubitainers. After 92 h, however, most nutrient-enriched cubitainers had chlorophyll a concentrations substantially above initial values, as did cubitainers with elevated zooplankton densities. By contrast, those cubitainers that were left unfertilized and with zooplankton densities at or below ambient levels showed no increase in chlorophyll a. Two-way ANOVA indicated that both nutrient and zooplankton levels had a statistically significant (p < 0.05), and positive, impact on phytoplankton growth rates during the 3 days of the experiment when chlorophyll a levels increased (23 to 92 h). The nutrientzooplankton interaction term was trivial. A plot of phytoplankton growth rate in cubitainers versus total zooplankton biomass (herbivores plus carnivores) revealed only a weak relationship between the two variables under nutrient enrichment, but a strong positive relationship when no nutrients were added (Fig. 7). Growth rates declined sharply with herbivore biomass in the fertilized sys- tems, but, in the unfertilized systems, they first increased and then decreased as herbivore biomass rose above 0.5 mg/L. The slope of the relationship between growth rates and herbivore biomass for fertilized and unfertilized systems was similar above the 0.5 mg/L threshold, suggesting that in both treatments, phytoplankton divided at maximal rates when zooplankton were dense enough to keep the nutrient supply high. Thus the slopes obtained reflected grazing impacts alone. Analysis of zooplankton communities at the end of the experiment revealed some unexpected treatment side effects. As intended, total zooplankton and also cyclopoid biomass in cubitainers increased along the gradient LMA to AMA to HMA, while nauplii densities were uniform (micrograzers were not manipulated). Cladoceran biomass, however, proved to be greater in the AMA than in the HMA cubitainers, and rotifer biomass peaked at the lowest macrozooplankton level (Fig. 5). Furthermore, 72 Levine et al. rotifer biomass (mostly Polyarthra and Keratella) was significantly greater in fertilized than in unfertilized cubitainers. These results suggest that the 4day incubation was too long for maintenance of the zooplankton community structure imposed by the treatments. Rotifers, which can reproduce rapidly due to small size and parthenogenesis, were able to exploit the greater productivity of the nutrient-enriched systems and increase in numbers, while cyclopoid copepods managed to exert a grazing impact on rotifer densities. Cyclopoid predation may have also been responsible for the reduced cladoceran densities in the HMA treatments. Macrograzer densities created in fertilized cubitainers permitted an additional set of grazing rate estimates for mid-summer (Table 2). The findings were similar to those obtained during the year before: green algae were more heavily grazed than diatoms, cryptophytes, and blue-green algae (26% vs. 4 to 8% of biomass/d), and among the cryptophytes, Chroomonas sp. was grazed less than Cryptomonas sp. (6% vs. 28% loss/d). The clearance rate for the phytoplankton community as a whole was 0.17 mL per g of zooplankton/d, or 11% of phytobiomass daily. Using change in chlorophyll a concentration in the fertilized cubitainers to calculate community grazing mortality yielded a nearly identical result (0.18 mL/g zooplankton/d or 11% of biomass/d). All grazing estimates derived from the nutrients versus grazers experiment may be slightly below real values because of the growth of rotifers in the fertilized LMA treatment. These animals likely contributed to a lowering of the algal growth rate in the cubitainers involved, with the result that the growth rate vs. zoobiomass curve was less steep than it otherwise would be. Net primary productivity was especially low during this experiment (5 mg C/m3/d). DISCUSSION Macrozooplankton Grazing on Phytoplankton This study provided much needed data on taxonspecific rates of phytoplankton mortality due to zooplankton grazing. Modeling of phytoplankton community dynamics requires such information, but only one study prior to this has yielded rate estimates for taxonomic levels finer than the entire community. With this study, the list of analyzed species is expanded from warm temperate species to those found in boreal regions and the Great Lakes. Like Elser and Goldman (1991) and Elser (1992), substantial variability in the vulnerability of phytoplankton taxa to macrozooplankton consumption was found. Some species lost as much as 58% of their biomass per day to grazers, while others were unaffected. A few had negative grazing rates, indicating that they grew more quickly when zooplankton were abundant than when they were rare. These species are likely immune to grazing and benefit from the suppression of more vulnerable phytoplankton species, either because this suppression releases resources for their use or because the suppressed species are active in allelochemical production. Much of this assessment was done at the Division level. Most conclusions were in agreement with previous findings and with widely-accepted concepts about the role that grazers play in influencing algal community structure; a few were not. Because Cladocera dominated during all but the May experiment (when calanoid copepods were also important), these observations pertain primarily to this group of herbivores. One important paradigm of phytoplankton ecology is the existence of low grazing pressure on blue-green algae, which presumably are toxic (Arnold 1971), of low nutrient content (Lampert 1981), or too large and filamentous for easy consumption (Lampert 1981, Infante and Abella 1985). Blue-green grazing mortality was consistently minimal (< 1% per day) during the experiments. Another group frequently described as relatively inedible is the dinoflagellates (Porter 1977, Sommer et al. 1986). However, higher grazing rates were measured on this group than on any other, and these results are not unique. Elser (1992) reported grazing rates on Gymnodinium similar to ours, and Proulx et al. (1996) described the blooming of dinoflagellates in a Quebec lake following the removal of zooplankton through planktivorous fish introduction. Dinoflagellate vulnerability may depend on size, and the species seen during the experiments were relatively small (except for Ceratium hirundinella). Among the phytoplankton generally considered most edible are cryptophytes, chrysophytes, and small non-colonial diatoms (Porter 1977, Sommer et al. 1986). Chrysophytes were too rare in Lake Champlain during this study to permit estimation of their losses to grazers, while the response of cryptophytes to grazing was mixed. Cryptomonas sp. was as strongly grazed as green algae and dinoflagellates, while Chroomonas sp. suffered minimal losses. Many other researchers have reported heavy Zooplankton Grazing of Phytoplankton in Lake Champlain grazing on Cryptomonas (e.g., Porter 1977, Lehman and Sandgren 1985, Knisely and Geller 1986). The Cryptomonas clearance rates measured by Elser and Goldman (1991) and Cyr and Pace (1992) were even greater than those reported here. That Chroomonas sp. is poorly grazed may contribute to its status as the most abundant phytoplankter in Lake Champlain (McIntosh et al. 1993). Minimal grazing was observed on diatoms except for Aulacoseira sp., which was grazed at a rate of 8% per day in May 1995. Because the diatoms present during this study were primarily colonies large in at least one dimension, this was expected. Elser and Goldman (1991) measured similarly low rates of diatom consumption in California lakes. That diatom losses were greater in May than July and September may be related to the greater densities of calanoid copepods present. Calanoids are more efficient than Cladocera at consuming large particles (Peters and Downing 1984). Green algae are a highly diverse group which apparently contains both edible and inedible species (Porter 1977, Sommer et al. 1986). This group was found to be one of the most heavily grazed in Lake Champlain, although some of the algae used as prey items (e.g., Mougeotia sp.) have been previously classified as inedible (Knisely and Geller 1986). In their study of California lakes, Elser and Goldman (1991; Elser 1992) measured low grazing rates on most green algae, but relatively high rates for certain species of Cosmarium, Oocystis, Quadrigula, and Selenastrum. There were no obvious clues as to why some species within phytoplankton divisions were more vulnerable to grazing than others. In some cases, size may have been a factor. Three of the poorly grazed species, Tabellaria sp., Fragilaria crotonensis, and Aphanizomenon flos-aquae, were colonies too large to fit easily into the gap of most cladoceran carapaces or into the mouths of most copepods (Gliwicz 1977). Two colonial species, Aulacoseira sp. and Mougeotia sp., were moderately grazed, but these algae form singular filaments that grazers can attack by holding down one end and pulling (or biting) cells off the other (Vanderploeg and Paffenhöfer 1985). Very small algae may slip though the filtering setae of grazers (Gliwitz 1977) and thus be captured at a lower rate than larger algae. The two smallestsized species that were assessed, Chroomonas sp. (4 µm) and an unidentified centric diatom (2 µm), were very poorly grazed. A third small-celled species, Chroococcus sp., was moderately grazed, 73 but it forms small colonies that increase its effective size. The moderately to heavily grazed species in the experiments were 5 to 25 µm in their greatest axial linear dimensions (GALD), or formed colonies of this size. The literature indicates that the optimal food size for most zooplankton is 10 to 20 µm GALD (Svensson and Stenson 1991). Differential species vulnerability to grazers within phytoplankton divisions presents a challenge to modelers of phytoplankton dynamics. Modeling the dynamics of dozens of phytoplankton species is cumbersome. Therefore, phytoplankton are often compartmentalized by division rather than by species (e.g., Lehman et al. 1975, Scavia et al. 1988). These results, and Elser’s, indicate that mean division loss rates mask important dynamics at the species level. A more fruitful approach may be the modeling of a selection of particularly abundant species. Microzooplankton Grazing on Phytoplankton In a recent study of Castle Lake, CA, Elser and Frees (1995) showed that the traditional emphasis of grazing studies on macrozooplankton may greatly underestimate overall grazing mortality. Phytoplankton removal rates by micrograzers in this lake (5 to 22% of phytobiomass per day) overlapped or exceeded removal rates by macrograzers (5 to 12% per day). This study of micrograzing in Lake Champlain also indicated phytoplankton losses on par with the losses to macrozooplankton (3 to 14% vs 1 to 10% of phytobiomass per day). Micrograzers had significantly higher biomass-specific clearance rates than macrograzers when feeding on most phytoplankton divisions (cryptophytes, blue-green algae, and diatoms). Only green algae appeared to be more easily cleared from suspension by macro- than micrograzers. The green algal species involved (e.g., Mougeotia sp., Oocystis spp., and Coelastrum scabrum) were relatively large or colonial in nature, and thus presumably difficult for a small animal to handle. Phytoplankton species > 20 µm in size were manipulated substantially by the sievings and addbacks preceding the micrograzer experiment. All phytoplankton divisions except the Cryptophyta were affected. Because clearance rates decline with food density (as the inverse of the cube root; Peters and Downing 1984) and growth rates rise with diminished mortality, the impact of unequal phytoplankton densities on the Lehman-Sandgren curves is expected to be a reduction in slope (as the point 74 Levine et al. at high grazer density rises), and thus an underestimation of grazing rates. Thus microzooplankton may be even more important grazers than the values in Table 4 suggest. A recent study by Cyr (1998) suggested that zooplankton communities dominated by rotifers or copepod nauplii generally have higher biomass-specific clearance rates than communities dominated by Cladocera or adult copepods. Champlain is that of a slowly-growing phytoplankton community cropped by zooplankton feeding at a similarly slow pace. Analysis of zooplankton-protozoan-bacterial interactions in the cubitainers indicated that, while phytoplankton were the principal food source for zooplankton in May, bacteria and heterotrophic protozoa were more important in July and September (Levine et al. 1999). Total Grazing Loss Micro- and macrozooplankton together removed from 2 to 21% of the phytoplankton biomass in the mixed layer of Lake Champlain daily. Cyr and Pace (1992) reported exactly the same range of removal rates for 30 phytoplankton communities in 16 northeastern U.S. lakes (they analyzed grazers > 80 µm, and thus included the bulk of micrograzers). The similarity of the two ranges indicates that temporal variability in grazing rates may be of a similar order of magnitude to lake-to-lake variability, and should be considered when phytoplankton grazing is modeled. Comparisons of grazing loss rates with phytoplankton replacement rates have more ecological significance than comparisons with standing stocks. However, estimates of species-specific reproductive rates are almost as rare as species-specific grazing mortality estimates. The estimates of reproductive rates reported here were for artificial conditions (more light and nutrients than normally enountered in the lake), but they are useful in forming first order approximations of grazing as a fraction of production. The combined mortality induced by macro- and microzooplankton grazing balanced from 22% to 139% of the NPP measured in fertilized cubitainers. Thus, grazing is a more important phenomenon in Lake Champlain than the slow biomass loss rates suggest. Grazing should be more significant yet when related to the lower NPP rates expected in the absence of nutrients. When contrasted with the mean primary productivity of the lake’s mixed layer (the 14C estimates), grazing mortality was relatively minor in September (22%), but exceeded primary production by 6 fold in May. Cyr and Pace (1993) reported that, on average, zooplankton remove 51% of lacustrine primary production annually. Reproductive rates in the cubitainers were on the low end of values reported for natural phytoplankton communities in the literature, 0.1 to 0.2 versus 0.1 to 0.9 per day (Reynolds 1984). Thus the image of phytoplankton dynamics that emerges for Lake Nutrients Versus Grazers as Phytoplankton Controls The nutrients vs. grazers experiment supported the view that nutrients play a major role in regulating algal biomass in Lake Champlain. Combined additions of N, P, and C to cubitainers increased phytoplankton growth rates, even at the highest grazer level (ANOVA indicated a nutrient level effect at p < 0.03). Other researchers conducting field experiments of a similar design have noted the same effect (Lehman and Sandgren 1985, Berquist and Carpenter 1986, Vanni 1987, Kivi et al. 1993, Spencer and Ellis 1998). At the division level, response to nutrients was more variable. Diatoms and green algae generally increased with fertilization, while cryptophytes and blue-green algae sometimes were negatively affected, or showed no consistent response (Fig. 4; also Levine et al. 1997). Lehman and Sandgren (1985) and Berquist and Carpenter (1986) also found negative responses to fertilization among some species in their experiments. While it is generally expected that grazers should reduce phytobiomass and thus growth rates, ANOVA indicated a significant (p = 0.01) positive relationship between the phytoplankton growth rates in cubitainers and macrozooplankton level. Lehman and Sandgren (1985) and Berquist and Carpenter (1986) also observed phytoplankton species whose growth rates increased on elevation of zooplankton levels. Both Kivi et al. (1993) and Spencer and Ellis (1998) reported a lack of relationship between chlorophyll a concentration and grazer level in nutrient versus grazer experiments, although some positive trends were found in Spencer and Ellis’s study. Kivi et al. (1993) observed a slightly positive relationship between primary productivity and grazer level, while Elser and MacKay (1989) reported a stronger relationship. What is implied by the outcome of this experiment and those of others is that nutrient recycling by zooplankton has a greater impact on phytoplankton dynamics than does grazing mortality. By examining algal growth in fertilized and unfertilized cubitain- Zooplankton Grazing of Phytoplankton in Lake Champlain ers separately, it was confirmed that the positive relationship between growth rates and zooplankton level was due to algal response in the nutrient-poor systems. For the fertilized cubitainers, there was a negative relationship between growth rate and herbivore biomass. The nutrients vs. grazers experiment was perhaps most valuable in revealing the tremendous intertwining of feeding and recycling relationships in the lake, and thus the potential for indirect consequences to resource or trophic level manipulations. Nutrient additions stimulated algal growth, which in turn allowed rotifer growth to increase, providing more food for cyclopoid copepods. This was just one of the “cascades” observed. The rotifer increase also drove down protozoan populations, allowing bacterial densities to rise (Levine et al. 1999). These results argue strongly for a modeling approach to phytoplankton ecology, as models are needed to track multiple permeations of effects. They also suggest that time limits are necessary on the field incubations used to calculate grazing rates. Four days is obviously too long. One to 2 days may be more reasonable, given that rotifer increases were not observed during grazing studies of this length. Brief incubations create problems when grazing rates are low, however. Substantial sample replication and intensive phytoplankton counting are necessary to obtain grazing curves with reasonably high r2 values. Chlorophyll a analysis yielded estimates of total phytoplankton grazing mortality similar to those obtained for phytobiomass, and thus this low-labor method may be used when species-level analyses are not required. Future Research Future research on zooplankton grazing in Lake Champlain should include separate night time and day time analyses. This study did not take into account the fact that macrozooplankton undertake extensive vertical migrations that cause them to concentrate at the surface at night and disperse at depth during the day. The analysis also should be extended to other lake sub-basins, and to wintertime sampling. Assessment of cyclopoid copepod feeding dynamics is desirable, as these largely carnivorous species are common in the lake and may indirectly affect rates of herbivory. They may also engage in omnivory when animal foods are scarce (Adrian and Frost 1993), although they are believed to be inefficient feeders on algae of the small size common to Lake Champlain (Knoechel and Holtby 75 1986). Had cyclopoids been included in the estimates of herbivore biomass, the values obtained for biomass-specific clearance rates would have been reduced by 5 to 98% (estimates of phytoplankton loss rates would not be greatly affected as their determination involves multiplication of the lower clearance rates by greater zoobiomass). Improved assessment of micrograzing is another priority. The dilution method of Landry and Hassett (1982) may allow grazers to be manipulated without affecting phytoplankton. However, when animal and algal populations are already small, dilution further increases variability in density determinations and leads to low r2 values for grazer curves (J. Lehman, Univ. Mich.) A larger goal is extension of species-specific grazing analyses to a diversity of lakes around the world, and comparison of these losses with reproductive rates. Cyr’s (1998) recent comparison of grazing in lakes with different zooplankton community types indicates that calanoid copepods and rotifers can exert as much grazing mortality on phytoplankton as Cladocera under oligotrophic conditions. Thus it is particularly important that studies of species-specific grazing mortality expand beyond the cladoceran-dominated lakes investigated to date. ACKNOWLEDGMENTS We thank H. McKinney for assistance in the field and laboratory, S. 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Submitted: 8 December 1997 Accepted: 18 October 1998 Editorial handling: Marlene S. Evans