Journal of Plankton Research Vol.18 no.10 pp.1767-1779, 1996
P.G.Verity and G.-A.Paffenh6fer
Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA
31411, USA
Abstract. The consumption of photosynthetic and heterotrophic cells by an abundant calanoid copepod species feeding on natural plankton communities was quantified with a state-of-the-art image-analysis system. Late copepodid stages of Eucalanus pilcatus did not ingest bacteria, small photosynthetic and heterotrophic nanoplankton, or the abundant Ceratium spp. in quantifiable amounts. Although diatoms were by far the most abundant cells (in terms of POC H), the copepods ingested a higher percentage of ciliates in relation to their abundance than of diatoms and small heterotrophic dinoflagellates in the first experiment, and ingested a higher percentage of dinoflagellates and ciliates compared to diatoms in the second experiment Heterotrophic cells sufficiently large to be captured were repeatedly preferred by E.pileatus over autotrophs of similar or larger size. Moreover, among the cells which could be individually perceived by this calanoid, larger ones were not preferred over smaller cells, implying that some aspect of food quality can be as significant as prey size.
These results support the notion (e.g. Kleppel, Mar. EcoL Prog. Ser., 99,183-195,1993) that feeding by copepods will be underestimated if ingestion of heterotrophic food organisms is not quantified. While the proposed microscope-based method is comparatively slow (~1 h per sample), it is the only technique which provides detailed information on both the size and trophic composition of ingested prey.
Introduction
Despite early reports of omnivorous diets among calanoid copepods (Marshall,
1924; Mullin, 1966; Petipa et al., 1970), the ease of using fluorometry to estimate rapidly the consumption of phytoplankton in incubation bottles resulted in considerable focus on herbivory. Appreciation of the role of the microbial food web has emphasized the significance of omnivory, because the sheer magnitude of secondary production by hetero- and mixotrophic ciliates, dinoflagellates and nanoplankton (e.g. Lynn and Montagnes, 1991) requires a substantial sink to prevent massive and rapid accumulation of these micrograzers. The latter is seldom observed, in fact the biomass of nano- and microzooplankton appears relatively constant given their growth potential.
Omnivory is characteristic of juvenile as well as adult stages (Stoecker and
Egloff, 1987); estuarine (Gifford and Dagg, 1988), neritic (Turner, 1987a) and oceanic (Mullin, 1966) taxa; and temperate to tropical species (Landry, 1978;
Petipa, 1978). Recognition of this gustatory diversity is salient from several perspectives. Without including dietary contributions from heterotrophic as well as autotrophic prey, the total ration, biomass and egg production of metazooplankton cannot be predicted or causally related to measures of food availability. Moreover, the role of meso- and macrozooplankton in the structure and function of pelagic food webs, particularly contributions by key taxa, cannot be elucidated without fundamental knowledge about their food acquisition potentials, life histories and survival strategies (e.g. Verity and Smetacek, 19%).
The question of contemporary importance, however, is how to quantify omnivory? Examination by brightfield microscopy of the composition of prey communities before and after their modification by meso- and macroconsumers
© Oxford University Press 1767

P.G. Verity and G.-A.Paffenhdfer
Table L Feeding experiments with adult male (M), female (F), and copepodid (C) stages of Eucalanus pileatus
Exp 27-28 June 1994 Exp. 30 June-1 July 1994
Collection Site
Isobath in situ
Temperatures
Exp. Temperature
36°OO'N, 75°25
21 m
W
22.5 to 24.5 °C
21.7
in upper 18 m
36°10'N,75°16' W
30m
22 °C in upper
21.5
14m
Replicate Bottle A B C B
Estimated Number and stages of copepods at start
At end of experiment
18CIII/IV 24 CIII/IV 12 CIII/IV
A i cm
15 CIV
7 CIII
7 CIV
1 M, 1 F,
1CIII,4CIV,
11CV
2M,2CIII,
4 CIV, 16 CV
2 M, 1 CI,
3CIV,7CV
1 M, 1 F,
15 CIV, 8 CV
2 CIII,
8CIV.4CV
has long been the standard method. However, this laborious and somewhat subjective approach (in terms of biomass estimation as well as separating auto- from heterotrophic prey) is at odds with the current interest in measuring biological variables at time and space scales commensurate with the driving physical forces.
Other newer techniques, such as high-pressure liquid chromatography (HPLC), can provide chemotaxonomic information about phytoplankton which are potential prey (Strom, 1993; Waterhouse and Welschmeyer, 1995), but not specific data on various non-plastidic prey. With the development of fluorescence microscopy adapted specifically for relatively rapid, precise, and accurate determination of the abundance, size distribution, and biomass of auto- and heterotrophic plankton
(Sieracki etal., 1989a,b; Verity and Sieracki, 1993; Verity etai, 1993), it has become possible to relate in detail the effects of grazers on potential prey fields. Here, we report initial observations of the feeding of late copepodid stages of the subtropical calanoid Eucalanus pileatus on natural particle assemblages. The data are limited to two experiments, the results of which are part of a larger study being conducted through 1996. The purpose here is to describe in detail the method for analysis of the prey community, and offer insights into the power of such a comprehensive tool. The importance of Eucalanus in the zooplankton food web will be described elsewhere; general plankton relations in this region during spring are presented in Verity et al. (1996a).
Method
Eucalanus pileatus is among the abundant calanoid copepods in subtropical neritic waters (Bowman, 1971; Binet, 1977; Valentin, 1984; Madhupratap and
Haridas, 1986; Turner, 1987b). During the latter part of June 1994, E.pileatus occurred abundantly between 35°30' and 36° 30' on the continental shelf north of
Cape Hatteras. Copepods were collected in tows lasting 3-4 min with a net of 202 jun mesh and a 4-1 codend, while the ship was drifting. Microscope observations confirmed that the E. pileatus were undamaged and each had a visible oil globule.
Within 60 min, batches of 12-24 animals (see Table I) were transferred into three
1768

Assessment of prey ingestion by copepods
1900 ml jars (two jars in the second experiment) containing a natural suspension of particles from the depth of maximum occurrence of E.pileatus. Control jars contained no copepods, and all jars were amended with 3 jimol NO31"
1
. The jars were rotated on a ferris wheel at ~0.2 r.p.m. in a water bath maintained close to collection temperatures. Experiments were run over 24 h in a natural light-dark cycle (15:9 h).
At the beginning and end of each experiment, samples were drawn from each control and experimental jar for composition, enumeration and biomass measurements of plankton using a color-imaging cytometry system. The methods used to enumerate and determine the biovolume of plankton cells via image-analysis techniques, and subsequent conversion to cell carbon and nitrogen, have already been developed (Sieracki et al., 1989a,b; Verity et al., 1992; Verity and Sieracki,
1993). They have been used to quantify plankton communities in diverse environments such as the Atlantic Ocean (Verity et al., 1993), Pacific Ocean (Verity et al.,
1996c) and Norwegian fjords (Hansen et al., 1994). Briefly, triplicate subsamples from each bottle were initially fixed with very low concentrations of glutaraldehyde (0.3% final concentration), stained with DAPI (10 (ig ml"
1
final concentration) for 4 min, then momentarily stained with proflavine (1.4 p-g mh
1
final concentration), and finally collected on black 0.4 jxm Nuclepore filters. Filters were covered with a small drop of low-fluorescence immersion oil and a coverslip, and slides were stored frozen. Proflavine and DAPI are ideal stains for imageanalysis studies due to their bright fluorescence.
The samples were analyzed with a color-imaging system consisting of a desktop computer (Pentium 100 mHz with 32 MB RAM and 1 GB hard drive) housing several integrated software packages which operated microscope-mounted hardware and additionally performed image-processing functions. An Olympus BX-
60 fluorescence microscope, which was also capable of brightfield, darkfield, phase contrast and differential interference contrast illumination, provided high-quality initial views of the filtered samples using a wide range of objectives (10-lOOx).
An Optronics DEI-470 integrating 2/3" CCD captured analog RGB color images from the filter surface at variable frame rates from 1/10000 s to 2 min. An onboard frame grabber continuously displayed full-frame images, with integration times up to 2 min producing a minimum sensitivity of 0.0025 lux. A Windowsbased imaging software, Image Pro Plus (IPP) for Windows (Media Cybernetics,
Inc.), controlled image capture, enhancement, measurement, analysis and output.
IPP directed an electronic shutter mounted in-line in the microscope light path so that the sample was exposed to excitation for only as long as the camera shutter was open, thus minimizing photobleaching. Images were digitized via an Ima-
Graph ImaScan/Chroma PCI video capture board and displayed on a Nokia 17"
RGB monitor.
Two analytical approaches were used depending on the density on the slide of the particular cell population being analyzed. For cells which were numerous per field and relatively uniform in brightness in a given sample (e.g. phototrophic and heterotrophic nanoplankton), randomly chosen whole fields were analyzed. In this case, all the cells in a field were segmented (identified in the image) and measured automatically. This approach required that a single threshold be used
1769

RGVerity and G.-A.Paffenhdfer
for all cells in the image, but was faster than automatically finding individual cell thresholds. Analysis of an image with 40 cells took ~4 s. The microscope was interfaced with a motorized stage and associated modular automation controller, which permitted the operator to scan transects of variable length across the plankton slides while the computer recorded the fraction of the surface area of the slide which has been examined. For abundant populations, the computer randomly selected individual locations on the slide. The entire process (moving to a given location, focusing, opening an electronic shutter, grabbing an image, closing the shutter and moving to a new location) was automated and computer controlled.
For more rare cells (e.g. ciliates and heterotrophic dinoflagellates), transects of the slide were scanned, and individual cells were isolated and identified by the operator interactively. In this way, densities of rare cells were calculated per volume of sample. Sub-images containing the individual cells were temporarily stored and measured automatically overnight using automatic threshold determination according to the second derivative method, segmentation and cell measurement (Sieracki et al., 1989a).
Dinoflagellates were distinguished from other flagellates based on cell morphology and structure of the nucleus, especially the unique condensed chromosomes visible by DAPI staining. Heterotrophic and autotrophic nanoplankton and dinoflagellates were discriminated by the absence and presence of autofluorescent chloroplasts, respectively. Ciliates were identified by their oral ciliarure and other diagnostic features, e.g. the large plastids in Mesodinium. Diatoms and ceratia were distinguished by their unique external cell walls. Typically, 100 cells of each type were measured in each sample. The average coefficient of variation of triplicate counts of nanoplankton, dinoflagellates, diatoms, ceratia and ciliates was 7, 9, 8, 13 and 10%, respectively. The cell volumes of nanoplankton, dinoflagellates and ciliates were estimated from linear dimensions using the robust assumption that cell depth and cell width are equal dimensions in these small cells
(Sieracki et al., 1989b; see also Verity et al., 1992). The linear dimensions of diatoms and ceratia were measured, and applied to the appropriate stereometric formulae to estimate cell volumes (Edler, 1979). Heterotrophic dinoflagellate and nanoplankton biovolumes were converted to cell carbon using factors of 0.24 pg
C jim~
3
[derived from Lessard (1991) and Verity era/. (1992)] and 0.22 pg C urn"
3
(Borsheim and Bratbak, 1987) respectively. Ciliate carbon was estimated from biovolume using empirically derived conversion factors (Putt and Stoecker, 1989).
The carbon contents of diatoms and ceratia were estimated from cell volumes according to Edler (1979).
Copepod nauplii, which also can serve as prey, were not enumerated because they occurred in insufficient quantities to be adequately accounted for by our method. Since they can perceive, avoid or escape from predators, their contribution as a food organism is expected to be small except for strongly carnivorous copepods like Centropages velificatus (Paffenhdfer and Knowles, 1980).
Since the amount of food removed by a late copepodid stage of E.pileatus could only be approximated, not knowing in advance the composition and abundance of potential food particles, the concentrations of copepods were deliberately varied
1770

Assessment of prey tngestion by copepods from 12 to 24 CIII/IV per 1900 ml jar on 27 June, and 1 CIII/15 CIV and 7 CIII/7
CFV on 30 June 1994 (Table I). Their stage was estimated from size without a dissecting microscope, based on experience (G.-A.Paffenhofer). Since the age of collected adult female copepods cannot be accurately determined, and copepodids far outnumbered females, we decided to utilize the former for experiments. At the end of each experiment, the surviving copepods were removed from each jar, and their cephalothorax length (to the nearest 0.02 mm) and stage were determined.
Animals which molted to males were not included in the feeding rate calculations.
All others were immediately dried at 60°C and upon return to the laboratory the combined copepod weight from each jar was determined to the nearest 0.1 jig with a Cahn-Balance. Ash-free dry weight was multiplied by a factor of 0.45 to obtain their organic carbon content (G.-A.Paffenh5fer, personal observation).
Growth and grazing coefficients and ingestion rates of each potential prey category were calculated from the end-point sample measurements according to the equations of Frost (1972). Weight-specific ingestion was calculated by normalizing the disappearance of prey per experimental bottle (relative to controls) to the number of copepods and their estimated carbon biomass. In the first experiment, where three bottles containing added Eucalanus were incubated, measurement errors in clearance and ingestion were calculated from mean estimates for each bottle. Here, the estimated errors reflect the variance among the three experimental bottles. In the second experiment, where only two bottles contained added
Eucalanus (a third bottle deliberately contained another copepod genus), the three subsamples from each bottle were treated as separate replicates and errors were estimated from the six combined subsamples. Here, the estimated errors include both intra- and inter-bottle variability.
Results
During each 24 h study, most copepodids molted to the next stage (Table I). Mortality occurred once (Experiment 30 June, jar A) which we assume was due to losing one copepod when closing or opening the respective jar. In one case, we had one more copepod at the end than at the start (Experiment 27 June, jar C) when a late nauplius molted to CI. Copepodid stages of all calanoid genera and nauplii of E.pileatus were removed from each control and experimental jar prior to the addition of experimental animals.
We quantified removal by E.pileatus of seven different groups of potential food organisms (Tables II—III); data on bacteria are not shown because they are well below the minimum prey size of Eucalanus. Cell volumes represent means of triplicate measurements of £100 cells from the two experiments. The phytoplankton community included diatoms (Rhizosolenia alata, R. stolterfothii, Chaetoceros spp.,
Skeletonema costatum, Cerataulina sp., Leptocylindrus danicus), dinoflagellates
(Ceratium tripos, Clineatum, Cfurca, Prorocentrum spp.), and small photosynthetic nanoplankton (cryptomonads, prasinophytes, coccolithophorids). This community is similar to that previously reported for these waters (Marshall and
Ranasinghe, 1989). Heterotrophic (= aplastidic) nanoplankton included species of
Paraphysomonas, Bodo, Leucocryptos, and choanoflagellates. Heterotrophic
1771

|~5 Table n. Ingestion and clearance rates of E.piieatus on 27-28 June 1994. See Method for details
Food taxon Cell volume
(u,m
3
)
Mean cell concentration
(ugCI-
1
)
Ingestion rates
(ng C copepod-
1
Bottles h"
1
)
Phototrophic nanoplankton
Heterotrophic nanoplankton
Heterotrophic dinoflagellates
Ciliates
Ceratia
Diatoms
Total
Weight-speciGc consumption
(u.g Cu.gr
1
C of copepod per 24 h)
1.1 X 10
2
1.6 X 10
2
4.6 X 10
2
1.3 X 10
3
-
1.6 x 10
4
1.8 x 10
4
32
28
7
14
12
78
171
A
0
0
19.0
84.1
0
304.0
407.1
0.55
B
0
0
15.8
63.3
0
246.2
325.3
0.61
C
0
0
15.5
72.5
0
279.8
367.8
0.59
x (SD)
0(0)
0(0)
16.8(1.9)
73.3 (10.4)
0(0)
276.7 (29.0)
366.7 (40.9)
0.58 (0.03) -
Clearance rates
(ml copepod-
1
h"
1
)
Bottles
A
0
0
2.7
6.0
0
3.9
3.7
B
0
0
2.3
4.5
0
3.2
2.9
C
0
0
2.2
5.6
0
3.6
3.3
x (SD)
0(0)
0(0)
2.4 (0.3)
5.4 (0.8)
0(0)
3.6 (0.4)
3.3 (0.4)
-
Table HI. Ingestion and clearance rates of E.piieatus on 30 June-1 July 1994. See Method for details
Food taxon Cell volume
(ujrr
1
)
Mean cell concentration
("•g C I"
1
)
Ingestion irates
(ng C copepod"
1
h~')
Bottles
x (SD)
Phototrophic nanoplankton
Heterotrophic nanoplankton
Heterotrophic dinoflagellates
Ciliates
Ceratia
Diatoms
Total
Weight-specific consumption
(u.g C u,g-' C of copepod per 24 h)
1.6 X 10
U X 10
2
6.5 x 10
2
2.4 x 10
3
1.4 X 10
4
-
13 x 10
4
2
40
24
4
19
8
70
165
A
0
0
25.7
109.9
0
226.6
361.2
0.67
B
0
0
20.4
88.2
0
203.6
312.2
0.82
0(0)
0(0)
23.9 (3.7)
99.0(13.3)
0(0)
215.1(15.2)
336.5 (32.0)
0.74 (0.10) -
A
0
0
6.8
6.1
0
3.3
3.6
Clearance rates
(ml copepod"' h~')
Bottles
B
0
0
5.1
4.6
0
2.9
3.1
-
.r (SD)
0(0)
0(0)
6.0 (0.9)
5.3 (0.8)
0(0)
3.1(0.3)
3.4 (0.3)
yj ft u
3.
CL
P
>
5
<?
g-
O :

Assessment of prey Digestion by copepods
Table IV. Mean clearance (± SD) of several dinoflagellate taxa by Eucalanus in the experiment during
27-28 June
Dinoflagellate
Unidentified heterotroph sp.
Katodinium rotundatum
Prorocentrum minimum
Heterocapsa sp.
Prorocentrum micans
Cell size
(p.m)
5-7
8-10
12-14
17-19
22-25
Cell volume
(tun
3
)
118 (37)
384(60)
1215 (221)
2162 (344)
3620 (617)
Clearance
(ml copepocH h~')
0.2 (0.2)
1.9 (03)
3.4 (03)
4.1 (0.4)
4.0 (0.5)
Cell sizes are approximate dimensions of cell length and width. Cell volumes of the two smallest taxa were calculated assuming a circular cross-section. Cell volumes of P.minimum, Heterocapsa, and
P.micans were calculated assuming dorsoventral flattening of 10, 20, and 50%, respectively (Edler,
1979; Verity era/., 1992).
(= aplastidic) dinoflagellates were represented by both thecate and athecate taxa, especially spindle-shaped morphotypes of the latter. Ciliates included both plastidic and aplastidic taxa, with the common genera being Strombidium, Lohman-
niella and Strobilidium.
Bacteria, photosynthetic and heterotrophic nanoplankton, and Ceratium spp.
were not eaten; the former three groups probably because the cells were too small to be collected efficiently, the latter perhaps because the Ceratium were morphologically complex. Heterotrophic dinoflagellates, ciliates and diatoms were ingested in all bottles containing Eucalanus. In the first experiment (Table II), ciliates were cleared at significantly higher rates than the larger diatoms, which were cleared at significantly higher rates than the smaller dinoflagellates. Ingestion of diatom carbon was significantly greater than that of ciliates and dinoflagellates due to the
6-fold higher biomass of diatoms. In the second experiment (Table III), clearance of ciliates and dinoflagellates was not significantly different, but both were significantly greater than clearance of the larger diatoms. Again, more diatom carbon was ingested due to the greater abundance of diatoms. Thus, in both experiments, the smaller heterotrophs were cleared at higher rates than the larger phototrophic diatoms The average percent reduction in prey abundance in bottles containing
Eucalanus was 17,20 and 24% (Experiment 1) and 19,24 and 21% (Experiment 2) for diatoms, ciliates and dinoflagellates, respectively (data not shown).
There was also a size-based component to feeding by Eucalanus. In the June
27-28 experiment, several taxa of photosynthetic dinoflagellates of varying size were present in sufficient abundance to enumerate (Table IV). While they did not contribute substantially to the daily ration of the copepods, their disappearance during the incubation was proportional to cell size. The smallest heterotrophic dinoflagellates of 5-7 u,m were not significantly collected by Eucalanus. The larger Katodinium (8-10 \LVO) and Prorocentrum minimum (12-14 \\.m) were cleared at 1.9 and 3.4 ml copepod"
1
h"
1
, respectively, while cells of Heterocapsa sp.
and Prorocentrum micans, ranging from 17 u,m to 25 jim, were removed at 4 ml copepoch
1
h"
1
. The three highest clearance rates were significantly greater than that of Katodinium, which was significantly greater than the smallest cells.
On 27-28 June, the copepods ingested daily between 55 and 61% of their body weight (Table II), and on 30 June-1 July ingested 67 and 82% (Table III). When
1773

P.G.Verity and C-A.PalTeiihofer
Table V. Mean prey concentration (per cent of total prey carbon in ingested categories) and ingestion rates (per cent of total prey carbon which was ingested)
Food type Experiment 27-28 June
1994
Experiment 30 June-1
1994
July
Mean cell concentration
A B C Mean cell concentration
A B
Heterotrophic dinoflagellates
Ciliates
Ceratia
Diatoms
Total
6%
13%
11%
70%
100%
5%
2 1 %
0%
74%
100%
5%
19%
0%
76%
100%
4%
20%
0%
76%
100%
4%
19%
8%
69%
100%
7%
30%
0%
63%
100%
7%
28%
0%
65%
100% comparing prey availability to prey ingestion, ciliates and heterotrophic dinoflagellates amounted to 24-26% of ingested carbon in the first study, although they represented only 19% of the standing stock of food items which were clearly large enough to be eaten (Table V). In the second study, 35-37% of the ingested food was ciliates and dinoflagellates, although they represented only 23% of available food carbon (Table V). In each experiment, the copepods ingested a higher percentage of heterotrophic than autotrophic food particles in relation to those available.
Discussion
Hundreds of papers have been published on zooplankton feeding using various methodologies for more than 60 years, with the general goal of determining the amount and type of food consumed by respective stages, species or groups
(Paffenhofer, 1988). In any improved protocol, all potential food particles should be considered and quantified, and the behavior of the grazers must be known when interpreting the data. Various studies have shown that copepods make decisions about what and how much they eat (e.g. Price, 1988; Vanderploeg et ai,
1988). The present results illustrate that late copepodid stages of E.pileatus ingest more heterotrophs than autotrophs in relation to their abundance, and that cell size and prey composition are equally important variables influencing ingesticn.
Eucalanus pileatus cannot represent all calanoids, but has food acquisition behavior similar to other abundant calanoid genera, e.g. Paracalanus and Temora
(G.-A.Paffenhofer, personal observation). Larger protozoa (>10 p,m length or width) are ingested at high rates by various stages and species of calanoid copepods otherwise known to be herbivorous (e.g. Petipa, 1978; Stoecker and Egloff,
1987; Gifford, 1993; Fessenden and Cowles, 1994; Ohman and Runge, 1994). For example, at similar abundances of larger phytoplankton and microzooplankton, the percentage of non-plant material (protozoa and metazoa) in gut contents of
Calannspacificus ranged from 23 to 99%, and of Clausocalanus sp. from 55 to 98%
(Kleppel et al., 1988). These observations were based on pigment identification; the advantage of the approach used here is that information on sizes and specific taxonomic groups is simultaneously available.
1774

Assessment of prey Digestion by copepods
Others have used inverted microscope observations to quantify ingestion of protists. For example, C.pacificus females did not ingest measurable amounts of phytoplankton when cell sizes were small (<20 urn, 170 u,g C H)>
D u t
gTazed heavily on ciliates (>10 ujn, 11 u,g C H; Fessenden and Cowles, 1994). In contrast,
C.pacificus copepodid stage V ingested phytoplankton (mostly diatoms) when it occurred at high concentrations and was of large size (>20 u,m, 2230 u,g C I"
1
) and did not ingest measurable amounts of ciliates (15 jig C H). Thus, sizes and abundances seem to have affected grazing rates on phytoplankton and ciliates. Adult female Cfinmarchicus from two different environments in the Gulf of St Lawrence
(Ohman and Runge, 1994) always cleared ciliates, which were more voluminous than diatoms and dinoflagellates, at higher rates than other food organisms, even when the diatoms amounted to 1460 u.g C I"
1
and the ciliates to only 3.1 u.g C I"
1
.
Ciliates were represented by Lohmanniella and Strombidium, the latter ranging from -10000 to 30000 u,m
3
. Dinoflagellates were cleared at rates similar to those of diatoms. Both of these studies (Fessenden and Cowles, 1994; Ohman and Runge,
1994) show that the genus Calanus has the highest clearance rates on ciliates when these are larger than the accompanying food organisms.
In the present study, late copepodids of E.pileatus cleared ciliates, which were
5-10 times less voluminous than diatoms, at a far higher rate than the latter
(Tables II and III). In the second experiment (Table III), clearance rates on heterotrophic dinoflagellates surpassed those on diatoms, although the average cell volume of dinoflagellates was only 5% of that of the diatoms. Differences in encounter rates between copepods and motile heterotrophs versus non-motile diatoms (e.g. Gerritsen and Strickler, 1977) are insufficient to explain the large differences in clearance. It is apparent from the absolute values of clearance rates that all three types of ingested food were actively eaten by E.pileatus. Why might large, easily perceivable and ingestible food particles not be preferably ingested?
Calanoids which create a feeding current use it to perceive food particles at a distance. Here the first decision is made whether a particle is worth ingesting ('coarse or preliminary evaluation'). After gathering the particle and displacing it to the mouth, the sensors there aid the copepod in deciding whether to ingest or reject this particle ('close or fine-tuned examination'). Thus, when food is scarce, longdistance perception may be the dominant process concerning food ingestion (Paffenhofer and Lewis, 1990), i.e. food quantity appears to be more important than quality. When food is abundant, as in the present experiments, tasting at the mouth may be more important, i.e. food quality may be the deciding factor (e.g. Vanderploeg et al., 1988; PaffenhCfer et al., 1995). However, this idea remains conjecture until it becomes possible to assess differences in food quality or nutritional status in natural plankton communities.
Mean clearance rates of ciliates were essentially the same in the two experiments: 5.4-5.9 ml copepod-
1
h"
1
. The same was true of diatoms: 3.1-3.6 ml copepod"
1
h"
1
. However, clearance of heterotrophic dinoflagellates was significantly lower on June 27-28 (2.4 ml copepod-
1
h"
1
) than on June 30-July 1 (5.9 m copepod-
1
h"
1
). It may not be coincidental that the mean cell size of the heterotrophic dinoflagellates was smaller in the experiment with lower clearances. The mean cell volume on June 27 (4.6 X 10
2
jim
3
) was equivalent to a mean cell
1775

P.G.Verity and G.-A.Paffenhdfer
diameter of 9.5 p.m; the corresponding cell size on June 30 was 11 u.m. While a difference of 1.5 u.m is small, the results from Table IV imply that Eucalanus may experience a decrease in particle capture efficiency in this size range. Previous observation of Eucalanus feeding suggested that it is able to capture Thalassiosira
pseudonana cells averaging 6 fj,m with sufficient efficiency to fill the copepod guts, but does not perceive them individually, whereas the larger 12 |xm Thalassiosira
weissflogii was perceived as individual cells (Price etal., 1983). While the different cell sizes of Thalassiosira undoubtably contribute to sensitivity thresholds, this prey item is not a good choice for determining capture efficiencies, because of its extensive production of extracellular p-chitan threads, which cause the actual cell diameter to be 5-10 times larger than the valve diameter (Gifford et al., 1981;
Verity and Villareal, 1986). The data in Table IV suggest that capture efficiency increases dramatically as cell size increases from 5 to 10 u,m. This is further supported by the lack of significant feeding on the small co-occurring photosynthetic and heterotrophic nanoplankton, whose mean cell diameter was <5 \t.m (Tables II and III).
The clearance rates presented here for diatoms (70-78 u.g C I"
1
) are about half those found in laboratory studies using diatoms of similar size and concentration
(Paffenhofer and Knowles, 1978). In natural communities,different potential food types include dead and elongated algae, detritus, fecal pellets and aggregates, many of which would be gathered, but not ingested. Preliminary analysis of nearby stations in these waters during this cruise indicated that the carbon biomass of living plankton and non-living detritus was similar (Verity et al., 1996a,b). Presumably, this would contribute to lower clearance and ingestion rates when compared to studies with a single or few food types because, during handling of an unwieldly, or subsequently unacceptable particles (e.g. Ceratium sp.), ingestions of other (high-quality) particles are difficult to accomplish. For example, Paracalanus juveniles ingested far more when offered one food species (T.weissflogii) in comparison to being offered three (Paffenhofer, 1984). In support of this notion,
E.pileatus CV tastes individual fecal pellets of Paracalanus sp. for >1 s before deciding on ingestion or rejection (G.-A.Paffenhofer, unpublished observation), which should reduce their ingestion of prey which can be analyzed more quickly.
In the future, direct visual observations of the entire feeding process over time (e.g.
Paffenhdfer etal., 1995) are required to examine this hypothesis that an increasing number of different food particles would reduce the overall ingestion rate.
To assess the impact of metazooplankton on prey communities fully, knowledge of the potential and actual food organisms is essential. The method described here is another step in that direction. It is somewhat laborious, but a reliable, replicable and data-intensive method which can be used over endpoint incubations, as done here, or in time-series experiments by collecting samples at regular intervals.
Under the assumption that all cells which disappear are ingested in their entirety, detailed image analysis provides considerably more information than less laborintensive methods. For example, the results of this study suggest that feeding by
Eucalanus is influenced both by the composition and size of potential food particles. Given the diversity and different functional roles of potential prey organisms, this approach, when applied to the abundant stages and species of
1776

Assessment of prey digestion by copepods zooplankton will improve our understanding of the size- and species-dependent nature of interactions between zooplankton and their prey (Vinogradov et al.,
1977). Eventually, equally detailed prey assessments must be accomplished in situ rather than during bottle incubations.
Acknowledgements
The authors thank L.R.Bulluck III, D.Gibson and P.Lane for technical assistance, and the captain and crew of the R/V 'Columbus Iselin'. V.Patrick prepared the manuscript. This research was supported by contract no. DE-F602-92ER61419 from the US Department of Energy and NSF grant OCE-90-22318.
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Received on September 1,1995; accepted on April 12,1996
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