Download Short-term changes of protozoan control on autotrophic

Journal of Plankton Research Vol.18 no.3 pp.443-462,1996
Short-term changes of protozoan control on autotrophic
picoplankton in an oligo-mesotrophic lake
Jakob Pernthaler, Karel Simek1, Birgit Sattler, Angela Schwarzenbacher,
Jitka Bobkova1 and Roland Psenner
Institute of Zoology and Limnology, University of Innsbruck, Technikerstrafie
25, A-6020 Innsbruck, Austria and' Hydrobiological Institute of the Czech
Academy of Sciences, Na sddkdch 7, Ceski BudijoVice 37005, Czech Republic
Abstract. In May 1994, we investigated the short-term development of the planktonic community in the
epi- and metalimnion of an oligo-mesotrophic lake (Piburger See, Tyrol), focusing on trophic links
between protists and picoplankton, but also including phyto- and zooplankton. Uptake experiments
withfluorescentlylabelled bacteria (FLB) and picocyanobacteria (FIX) were performed in order to
compare the importance of both prey types as carbon sources for bacterivorous protists. Heterotrophic
nanoflagellates (HNF) were responsible for —90% of total protozoan picoplanktivory (FLB + FIX);
ciliates accounted for —10%. A selectivity index related to prey density showed that both HNF and
ciliates clearly preferred FIX over FLB. The mean cell size of autotrophic (prokaryotic) picoplankton
(APP) was nearly three times larger (0.323 jim3) and much less variable than mean bacterial cell volume
(0.122 (inV). Although APP biomass was on average only 8.6% of total picoplankton biomass, picocyanobactena accounted for a mean 15.9% of total HNF carbon uptake. We calculated that total HNF
grazing could match potential APP maximum growth rates at the beginning of the study period. A
strong decrease in HNF individual clearance rate (CR) on APP was clearly related to a fall in the
percentage of choanofiagellates (from 75 to —10% of the HNF community). A simultaneous decrease
in HNF biomass and CR was followed by a steep increase in APP abundance; APP abundance and HNF
biomass were highly negatively correlated both in the epi- and the metalimnion (/-, „ = -0.879, r, META =
-0.907; P < 0.001). Total rotifer abundance increased by a factor of 50 within 2 weeks and was also
negatively correlated with HNF biomass (r, EPI = -0.852, P < 0.001; r, MlrTA = -0.659, P < 0.05). HNF
grazing was found to exert a strong short-term control on picocyanobacteria and this link was probably
broken by an increase in metazooplankton, especially due to rotifer predation on HNF.
Introduction
The role of autotrophic prokaryotic picoplankton (APP) as a component of pelagic marine and freshwater microbial food webs has been reviewed recently
(Stockner and Antia, 1986; Stockner, 1988, 1991; Weisse, 1993). Unlike heterotrophic pelagic bacteria, autotrophic picocyanobacteria have received little attention in terms of their quantitative importance as a possible carbon source for
protozoans (Stockner, 1991; Weisse, 1993, and references therein). Picocyanobacterial cells are mostly considerably larger than the average cell volume of pelagic
bacteria (Weisse and Kenter, 1991) and are readily ingested by bacterivorous
protozoans (Nagata, 1988; Christoffersen, 1994; Simek etal., 1995), although their
nutritional value for protists has been questioned (Caron et al., 1991). The differences between high gross growth rates in laboratory studies and the modest apparent in situ growth rate of picocyanobacteria [Weisse (1993) and references therein]
may indicate nutrient limitation (Stockner, 1991), but also that a high percentage
of APP production is channelled to other plankton organisms. However, it has
been difficult to separate the effects of protozoan grazing clearly from other
© Oxford University Press
443
J.Pernthaler el al.
biological loss processes, like viral lysis of cells (Proctor and Fuhrmann, 1990) or
metazooplankton grazing (Voros et al., 1991; Wehr, 1991).
The quantitative importance of individual components of the plankton community varies considerably throughout the seasons (e.g. Nagata, 1988; Sanders et
al., 1989; Marasse" el al., 1992) and frequently transition periods, especially within
aquatic microbial communities, lie within the range of days (Weisse et al., 1990;
Psenner and Sommaruga, 1992). In afirststep, it is important to follow the seasonal
range of variability for APP and its potential predators (e.g. Weisse, 1988; Burns
and Stockner, 1991; Voros et al., 1991; Sommaruga and Psenner, 1995). As the
generation times of pico- to microzooplankton organisms are usually much shorter
than sampling intervals of seasonal investigations, short-term studies are required
to reconstruct functional (e.g. trophic) interactions between populations and
communities.
In contrast to studies on the 'classic' plankton organisms (e.g. Miracle, 1977),
aquatic microbial ecology has rather neglected spatial, in particular vertical, distribution patterns. However, there are indications that microbial communities can
also differ significantly in the epi- and metalimnion of stratified lakes (Nagata,
1988; Weisse and Kenter, 1991).
The objective of this study was to depict the development of the plankton community during the decline of the phytoplankton spring peak in the epi- and metalimnion of a stratified lake. In particular, we investigated the role of bacteria and
autotrophic prokaryotic picocyanobacteria as prey for protists, changes in bacterial and APP abundance and biomass, as well as bacterial secondary production.
We also attempted a simple evaluation of heterotrophic nanoflagellate (HNF)
community structure and followed the biomass changes of HNF, ciliates, phytoand zooplankton over a period of 5 weeks.
Method
Study site and sampling scheme
Piburger See (Tyrol, Austria, 915 m a.s.l.) is a small (13.4 ha) dimictic softwater
lake located in the crystalline part of the Eastern Alps (47°11'N, 10°53'E). It has a
mean depth of 13.7 m and a mean annual total phosphorus concentration of ~8 |ig
I"1. A detailed description of the study site is given in Pechlaner (1979).
Between 4 May and 1 June 1994, samples were collected with a 5 1 SchindlerPatalas sampler from two depths three times a week in order to match roughly the
generation times of the studied microorganisms. Samples were taken above the
deepest point of the lake (24.6 m), between 08:00 and 09:00 h Central European
Time. A composite sample of 151 was drawn from the epilimnion at 0.5,1 and 1.5 m
depth. To localize the thermocline, temperature and oxygen were profiled for each
sampling date with an oxymeter (WTW OXY™ 196; FRG). From the metalimnion, a composite sample of 151 was taken from the depth of maximal temperature
change and from 0.5 m above and below this depth. The metalimnion was usually
situated between 4 and 5 m. Water transparency was measured with a Secchi disk.
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Protistan control of autotrophic picoplankton
Two litres of the mixed samples were required for further phytoplankton and
ciliate analysis. The remaining 13 1 were screened through a 50 (im net and the
metazooplankton collected for later determination. The filtered water was transported to the laboratory within 45 min in plastic tanks at in situ temperature.
Phytoplankton
One litre of water wasfilteredthrough a Whatman GF/C glass fibrefilter.Pigments
were extracted with acetone and chlorophyll a (Chi a) concentration measured
spectrophotometrically after Lorenzen (1967). Phytoplankton samples were fixed
with Lugol's solution and quantified in Utermohl sedimentation chambers on an
inverted microscope. A computer program designed by J.Komarkova" (unpublished) was used to calculate species cell volume via geometric approximations
from size measurements and for conversion into algal fresh mass. Phytoplankton
biomass was transformed into carbon using a conversion factor of 0.11 (Rocha and
Duncan, 1985).
Bacterial and picocyanobacterial abundance and biomass
A 100 ml volume of filtered lake water wasfixedwith 0.2 u,m pre-filtered formalin
(final concentration 2% v/v). Five millilitres of subsample were stained with the
fluorochrome 4',6-diamidino-2-phenylindole (DAPI, 0.2% w/v final concentration) according to Porter and Feig (1980) and filtered onto black membrane
filters (0.2 u,m, Poretics™). Between 400 and 500 bacteria were counted at 1600x
magnification by epifluorescence microscopy (Zeiss Axioplan™) with a BP 365, FT
395, and LP 397 filter set. Size measurement on 400-600 DAPI-stained bacteria
per sample was carried out with a semi-automated image-analysis system
(Psenner, 1993). Mean bacterial volumes were calculated according to Psenner
(1993). For biomass estimation, the mean carbon content per cell was calculated
for each sampling date and layer using the allometric formula of Norland (1993).
The quantification and size measurement of picocyanobacteria was performed in a
similar way as for bacteria, but using the autofluorescence of phycoerythrin for cell
detection (green excitation 510-560/FT580/LP 590 filter set) instead of DAPI
staining (Burns and Stockner, 1991). A conversion factor of 200 fg carbon |j.nr3 was
assumed (Weisse and Renter, 1991).
Bacterial secondary production
Bacterial in situ growth rates were measured via [3H]thymidine incorporation
(Riemann and S0ndergaard, 1986). Triplicate 10 ml subsamples were incubated
for 1 h with [methyl-3H]thymidine (Amersham'"; 5 nM final concentration) at in
situ temperature. Incorporation was stopped by the addition of 0.2 n,m-pre-filtered
buffered formalin (2%finalconcentration). In parallel, pre-fixed blanks were processed for each set of samples to correct for tracer adsorption on other particles.
Samples were filtered on cellulose nitrate filters (Sartorius™; 0.2 u,m pore size)
which had been pre-soaked twice with 5 ml of ice-cold trichloroacetic acid (TCA;
5% concentration). Cell disruption was performed for 5 min with ice-cold TCA
(5% concentration); afterwards, filters were again rinsed three times with TCA
and put into 20 ml polyethylene vials, where 5-10 ml of liquid scintillation
445
J.Pernthaler et al.
cocktail (BeckmanT") were added. After 5-6 h, the samples were measured twice
for 10 min for each sample in a liquid scintillation counter (Beckman'" LS 6000
IC). The number of newly produced bacterial cells was estimated from thymidine
uptake using a conversion factor of 2 x 10'" cells mol"' thymidine (Bell, 1990).
Protozoan abundance, biomass and grazing experiments
To quantify HNF, 20 ml of formalin-fixed sample (2% final concentration) were
stained with DAPI, filtered through 1 u-m pore size black membrane filters (Poretics) and at least 50 individuals were counted with an epifluorescence microscope
(lOOOx magnification; 365/395/397 UV filter set). Samples were processed within
24 h after fixation, and only individuals without plastids (checked for Chi a fluorescence with the 490/510/520filterset) were included in the counts. The length and
width of HNF were measured weekly in the epi- and metalimnetic samples on
at least 40 individuals per sample, and geometrical approximations (prolate
spheroids) were used to estimate HNF mean cell volume. For biomass estimation,
we assumed a conversion factor of 220 fg C u.nr3 (Borsheim and Bratbak, 1987). A
rough evaluation of HNF community structure on a group level was attempted in
the DAPI-stained samples on 40-90 individuals per sampling date according to
their shape, nuclear morphology, and the number and position of flagellae.
Enumeration of ciliates was performed in a similar manner, from the subsamples used for the fluorescently labelled bacteria (FLB) uptake experiments,
but counting at least 50 individuals per sample. Ciliate cell length and width were
measured from each individual inspected for FLB/fluorescently labelled picocyanobacteria (FLC) uptake. Biomass calculation was performed using geometrical volume approximations (prolate sphaeroids) and a conversion factor of 140 fg
C li-m-3 (Putt and Stoecker, 1989).
Protozoan grazing was measured in short-term direct-uptake experiments of
fluorescently labelled prey. Two types of stained prey were prepared: FLB and
(monodispersed) FLC. Two days before the start of the sampling, bacterioplankton was collected from the lake, pre-filtered (1 u,m) and concentrated from 20 1
with an Amicon™ hollow-fibre filtration apparatus. A strain of rod-shaped Synechococcus sp. with the size and shape typical for Piburger See picocyanobacteria
(from the Culture Collection of the Botanical Institute of Trebon, Czech Republic) was cultured and harvested by centrifugation (mean cell volume ± SD: 0.32
± 0.13 |xm3). Both prey types were heat killed and stained with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescin (DTAF) according to Sherr and Sherr (1993).
Grazing experiments were performed on each sampling date from 500 ml of
sample in 1 1 acid-washed glass bottles. Two sets of experiments were carried out
independently for FLB and FLC uptake in both the epi- and metalimnion. After
the sample had undergone a 15 min adaptation period at in situ temperature,
stained cells were added at a concentration of 20-25 % of the natural abundance of
bacteria (for FLB) and 35-45% of APP (for FLC). Subsamples (60 ml) were taken
after 6,10,20 and 30 min,fixedwith alkaline Lugol's solution, followed by boratebuffered, 0.2 nm-pre-filtered formalin (final concentration 2%), and decolourized
with sodium thiosulphate (Sherr and Sherr, 1993). Flagellate and ciliate uptake
446
Protistan control of autotrophic picoplankton
rates were both measured from the same treatment. Samples were processed
within 24 h after fixation. As uptake was generally slow for both HNF and ciliates,
the 20 or 30 min subsamples were used to count ingested FLB and FLC. Subsamples of 20 and 40 ml for HNF and ciliates, respectively, were stained with DAPI,
filtered through a 1 n-m pore size black filter (Poretics) and inspected via epifluorescence microscopy (Zeiss Axioplan, UV light filter set: see above; blue-light filter
set: 450-490/510/520 nm; 1250x magnification). At least 20 ciliates and 50 HNF
were checked for tracer uptake in each sample. Hourly uptake rates were estimated from ingested tracer cells and feeding period assuming a linear ingestion
rate. Samples were fixed 5,10,20 and 30 min after tracer addition during the first
uptake experiments. As the total number of ingested tracer particles was found to
increase almost linearly over this period, only the samples from the 20 min tracer
uptake period were inspected later. On average, 50-80% of inspected HNF were
found with ingested FLB after 20 min and 20-50% of HNF ingested at least one
FLC during this period. Tracer uptake per individual was high for some ciliate
species (e.g. Pelagohalteria viridis, Vorticella aquadulcis-comp\ex), whereas other
species, e.g. Urotricha sp., were not found to take up fluorescent particles at all
(Simek el al., 1996). Total protozoan uptake of tracer particles was calculated as
the product of flagellate plus ciliate in situ abundance and their respective average
uptake rates. Protistan grazing on bacteria and picocyanobacteria was estimated
from the percentage of tracer addition and the in situ abundances of picoplankton.
As a measure of selectivity between FLB and FLC, we chose the 'relative difference of mortality rate' of two prey types, D, calculated according to Jacobs (1974):
r - irp + p
where p is the fraction of FLC of the total number of added tracer particles (FLB +
FLC) and r is the fraction of FLC of the total number of ingested tracer particles
(FLB + FLC).
D takes into account the relative abundance of different food types in the
environment and is thus immune to changes in food composition. This is important, because the concentration of FLB and FLC was modified according to bacterial and APP abundance between successive sampling dates. D varies from -1 to
0 for negative selection and from 0 to +1 for positive selection; at D = 0, there is no
preference for either food type.
Zooplankton
Metazooplankton >50 u.m were preserved in formalin (~4% final concentration).
Subsamples (for rotifers) or the total concentrate (for crustaceans) were counted
on an inverted microscope (Leitz Labovert) using UtermOhl sedimentation chambers. Biomass was estimated from length and width measurements of 25-50 individuals of each species (or stage of development for copepods) from the epi- and
the metalimnion, using geometric volume approximations from Ruttner-Kolisko
(1977) for rotifers and length-weight regressions (Bottrell et al., 1976) for
447
J.Pemthaler el al.
crustaceans. A mean carbon content of 48% of dry matter was assumed for crustaceans (Andersen and Hessen, 1991) and 50% for rotifers (Latja and Salonen,
1978).
Statistical analysis
Data were analysed using the statistical software SIGMASTAT™ (Jandel Corp.).
Prior to other analyses, variables were tested for normality by the KolmogorovSmirnov test. Differences between the epi- and metalimnion were analysed using a
non-parametric alternative to a paired Student's Mest (Wilcoxon matched pairs
test). If no significant difference between the two layers could be detected, the epiand metalimnetic samples were pooled for the calculation of correlations (n = 26),
otherwise they were treated separately (nen = nMETA = 13). Correlations between
pairs of variables were calculated using the non-parametric Spearman rank sum
test (r,).
Results
Differences between the epi- and metalimnion
Water temperature ranged between 13.5 and 16.5°C in the epilimnion, and
between 9.6 and 14°C in the metalimnion, increasing gradually during the study
period (data not shown). The thermocline was located at a depth of 4-5 m; Secchi
disc transparency was between 6 and 7 m. The oxygen concentration was generally
higher in the thermocline (13-14.3 mg 1-') than in 1 m depth (9.1-11.2 mg I"1).
Although temperature was on average 3.2°C higher in the epilimnion than in the
metalimnion, only a few parameters were found to differ significantly in the two
layers. Bacterial and APP mean cell volume as well as APP biomass were higher in
the metalimnion. Ciliate community ingestion and clearance rate of both tracers
also differed between the epi- and metalimnion. Differences between the two
layers could be established for the abundance of all common rotifer species but
Conochilus, for Acanthodiaptomus, total crustacean and rotifer abundance and
biomass, chlorophyll a and diatom biomass.
Phytoplankton
Chlorophyll a (minimum 1.7, maximum 4.1, mean 2.6 u.g I"1) was higher in the metalimnion and tended to decrease in both layers during the study period (Figure 1).
Phytoplankton biomass in the epilimnion fell sharply after thefirstsampling date,
then built up to another maximum around 18 May and finally decreased again
towards the end of the investigation. In the metalimnion, total phytoplankton biomass fell to about one-third of its initial value after the second sampling date and
remained fairly constant thereafter. In the epilimnion, dinoflagellates (Gymnodiniwn spp., Peridinium spp.), chrysophytes (Dichrysis sp., Stichogloea sp.) and
chlorophytes were the main contributers to phytoplankton biomass. In the metalimnion, dinoflagellates, diatoms (Fragillaria crotonensis) and chlorophytes were
the most abundant taxa. Diatoms decreased to almost zero after initially high
abundances in the metalimnion, which coincided with a distinct increase in
448
Protistan control of autotrophic picoplankton
400
5
Epflimnion
„ 300
"L
u
Bin EUGLENO
Q CHLORO
S DINOPH
EZ3 CRYPTO
E 2 BAC1L
B CHRYSO
• CYANO
pn
16
20
25
30 May
Fig. 1. Biomass in carbon units of the important phytoplankton taxa and chlorophyll a concentration in
the epilimnion and metalimnion of Piburger See between 4 May and 1 June 1994. C Y A N O , cyanobacteria; CHRYSO, Chrysophyceae; BACIL, Bacillariophyceae; CRYPTO, Cryptophyceae; D I N O P H ,
Dinophyceae; C H L O R O , Chlorophyceae; E U G L E N O , Euglenophyceae.
cladoceran biomass. Between 16 and 23 May, a peak of Microcystis incerta could be
observed in the epilimnion (Figure 1).
Bacterioplankton and APP
Bacterial abundance varied between 1.73 and 3.54 x 106 ml*1; the mean cell volume
ranged from 0.62 to 0.28 u-m3, which corresponded to 14.3-33.4 fg carbon per cell
according to Norland (1993). No significant differences in bacterial biomass could
be detected between samples from the epi- and metalimnion (Figure 2a and b).
Bacterial abundance and biomass decreased to a minimum between 13 and 20
May, and tended to increase afterwards. An opposite trend was observed for bacterial volume in both layers (Figure 2a and b), but a significant negative correlation
between bacterial abundance and average cell volume was found in the epilimnion
only (/-, gp, = -0.797, P < 0.005). Bacterial abundance and biomass were not significantly correlated with temperature or Chi a.
[3H]Thymidine uptake was extremely variable, indicating rapid changes within
the metabolically active fraction of the bacterioplankton (Figure 3). Bacterial secondary production showed a weak but significant negative correlation with total
bacterial biomass (expressed as carbon) in the epilimnion (r, EPI = -0.593, P < 0.05)
and in the pooled sample (r, = -0.494, P < 0.05).
449
J.Pernthaler el al.
1—
i
'
!
a
Epilimnion
-J 80
_1
0,5
0,4
- 60
2 -
- 40
1
l.llll.
6
II.II il.
II
16
25
|
5
.E
20 - -
lFunr
20
0,3
0
30 May
0,2
1
0,0
0,5
- 80
_
0,4
a.
0,3
0,2
0,1
0,0
6
II
16 20 25 30 May
Abundance —v— Biomass i
i Cell volume
0,5
0,4
0,3
16
20
25
0
30 May
"o
>
0,1
1
0,0
12
10
0,2
I
0,5
Metalimnion
*f\
8
6
6
M
4
4
2 g.
I
6
11
Abundance
16
20
25
0
30 May
—v— Biomass '
0,4
u
|
0,2
S
0,1
a.
0,0
' Cell volume
Fig. 2. Abundance, biomass (in units carbon) and mean cell volume of bacteria (a, b) and picocyanobacteria (APP, c, d) in the epi- and metalimnion between 4 May and 1 June 1994. Note that only mean cell
volumes of APP and bacteria are depicted at the same scale.
450
Protistan control of antotrophic pfcoptankton
EPILIMNION
IINF t r u i n g
Ciliatc grazine
Bact. production
nnnNn
6
11
16
20
25
30
May
16
20
25
30
May
Fifr 3. Bacterial production (lines) and total protozoan grazing on bacteria (stacked bars) in the epiand metahmnion (upper and lower panel) between 4 May and 1 June 1994.
Single cells of Synechococcus-type cyanobacteria increased in abundance by
about a factor of four (from 2.31 to 11.5 x 10* ml-'), with no statistical difference
between the epi- and metalimnion (Figure 2c and d). The mean cell volume of APP
ranged from 0.237 to 0.446 u.m3 (47.4-89.2 fg C cell-').
Protozoa
Heterotrophic nanoflagellate numbers ranged from 1.05 to 3.71 x 103 ml-'. Their
biomass tended to decrease continuously throughout the period of investigation
after an initial peak on the second sampling date (Figure 4a). The epilimnion and
metalimnion did not differ significantly in either abundance or biomass of HNF,
which both showed significant negative correlations with bacterial abundance if
samples from both layers were pooled, but not with bacterial biomass (Table I).
HNF biomass was also negatively correlated with total rotifer abundance and biomass (data not shown) in both the epi- and metalimnion (Table I, Figure 6). At the
beginning of the investigation, the HNF community consisted of -75 % choanoflagellates, 20% chrysomonads and 5% bodonids. The relative importance of these
groups changed substantially during the sampling period: choanoflagellates
decreased to almost 10%, chrysomonads nearly doubled in relative abundance
(35%), bodonids and Katablepharis-likeflagellatesmade up another 20% of all
HNF. Between 10 and 25 % of the nanoflageUates per sample could not be assigned
to any of the above groups. This change in community composition was reflected in
451
J.Pernthaler el al.
Table I. Spearman rank correlations of abundance, biomass and particle uptake of heterotrophic nanoflagellates with selected parameters for the time series from 4 May to 1 June 1994 (n = 13 for the epi- and
metalimnion; n - 26 for the pooled sample).
B
BBM
APP
CILI
ROTIF
CRUST
0.143
0.049
0.072
HNF
number
EPI
META
ALL
-0.654*
-0.225
-0.410*
-0.478
0.319
-0.035
-0.571*
-0.626*
-0.559**
0.330
0.451
0.452*
-0.484
-0.368
-0.444*
HNF
biomass
EPI
META
ALL
-0.478
-0.451
-0.404*
-0.451
0.308
-0.020
-0.879***
-0.907***
-0.829***
0.676*
0.324
0.551**
-0.852***
-0.659**
-0 701***
0.044
-0.071
0.030
TGR
(FLB)
EPI
META
ALL
-0.143
-0.132
-0.122
-0.033
0.484
0.263
—
—
—
—
TGR
(FLC)
EPI
META
ALL
-
-
-0.709**
-0.538
-0.592**
-
-
EPI, epilimnion; META, metalimnion, ALL, pooled data from both layers; B, bacterial abundance;
B BM, bacterial biomass (units carbon); APP, picocyanobacterial abundance; CILI, biomass of nanociliates < 50 (j.m; ROTIF, rotifer abundance; CRUST, crustacean abundance; TGR (FLB), total HNF
grazing rate on FLB; TGR (FLC), total HNF grazing rate on FLC.
*P < 0.05; **P< 0.01 ;*•*/>< 0.001. -, not determined.
a marked decrease in HNF clearance rate on FLC (Figure 5c). Significant negative
correlations were found between APP abundance and biomass and HNF biomass,
as well as between APP abundance and HNF grazing on FLC (Table I). FLB
uptake by HNF was highly correlated with FLC uptake (r, = 0.802, P < 0.001);
however, a clear positive selectivity for FLC could be observed (Table II; with a
mean DPLC^PLB of 0.33).
Ciliate abundance and biomass ranged from 4.8 to 32.7 individuals ml"1, and
decreased drastically after the third sampling date (Figure 4b). Inversely, the mean
individual feeding rate on bacteria increased substantially (minimum 2.2, maximum 587, average 176 bacteria ciliate1 h~'), reflecting a shift in community structure (data not shown). Uptake of APP also increased towards the end of the
investigation (minimum 0.8, maximum 35.3, mean 14.3 APP ciliate1 Ir1) and also
ciliates selected positively for FLC (mean D^ „_ ^ = 0.40). A more detailed
report on ciliate bacterivory in Piburger See is given elsewhere (Simek etal., 1996).
Although individual uptake rates were high, ciliate community grazing on bacteria
and APP was on average only 10.2 and 11.2% of total protozoan uptake (Figure 3).
Ciliate and HNF total biomass development showed a similar trend during the
study period (Table I).
Protozoa consumed on average 29.8% of bacterial standing stock per day (epilimnion 7.68-66.24%; metalimnion 12.96-62.16%) or 69.4% of mean bacterial
production (i.e. of newly produced cells calculated from labelled thymidine
uptake). Protozoan total grazing on bacteria was maximal on the third sampling
date and decreased to a minimum on May 23 and 20 in the epilimnion and meta452
Protistan control of autotrophic picoplankton
METALIMNION
EPILIMNION
a
100
- Rotifers fV
- 600
p \ ~
450
75
u
a
;
/
^
:
25
125
' Crustaceans
100
75
u
150 ]
0
0
100
300"
75 "
50
50 •
25
25 '
1
0
6
11 16 20 25 30 May
Biomass
6
11 16 20 25 30 M»y
Abundance
Fig. 4. Abundances and biomass of (s) aplastidicflagellates(HNF), (b) ciliates <50 jim, (c) rotifers and
(d) crustaceans in the epi- and metalimnion (left and right panels, respectively) between 4 May and 1
June 1994.
limnion, respectively. Bacterial production was higher than protozoan grazing in
nine out of 13 cases in the epilimnion and in six cases in the metalimnion (Figure 3).
The mean loss of APP standing stock due to total protozoan grazing was 79.2%
day 1 . This value was exceeded by far at the beginning of the investigation (epilimnion 230.4%, metalimnion 319.2%) and fell steeply afterwards (Figure 5a and b).
Accordingly, abundance and biomass of picocyanobacteria were highly negatively
correlated with total protozoan biomass (r, = -0.894, -0.814, P < 0.001).
453
J.PernthaJer el al.
100
F.PII.IMNION
TGR on APP
% Carbon
[
80
\\
<
from APP
=
2
40
sa
3
ll.i.iihiiil
20 U
i?
METALIMNION
TGR on APP
3 -
% Carbon
from APP
80 £
<
E
60 £
40 3
TGR
20
0
60
50
40
gj
30
20
2
S5
10
z>
C R ^ p epilimnion
A
CR APP metalimnion
rzzi
V, choanoflagellates
80
60
40
ice r;
&
m
c
A
20
0
16
20
25
30 Miy
Fig. S. Heterotrophic flagellate grazing on picocyanobacteria (APP). (a) and (b) Total grazing rate
(TGR) on APP in the epi- and metalimnion. Solid bars represent the contribution of APP to HNF total
carbon uptake from picoplanktivory (%). (c) HNF clearance rates of APP (C/?Afr) in the epi- and
metalimnion, and the percentage of choanoflagellates of the total HNF community (pooled data from
both layers).
Zooplankton
Total rotifer abundance was 12 individuals I"1 at the first sampling date and
increased to >600 individuals 1"' in both the epi- and metalimnion during the
course of the investigation. The most abundant species were Conochilus unicomis,
Keratella cochlearis and Polyarthra dolichoptera in the epilimnion, and C.unicornis and Kellikottia longispina in the metalimnion. Asplanchna priodonta and
C.unicomis were the most important contributors to rotifer biomass in both
454
Protistan control of autotrophtc picoplankton
100 1,1
r j
00
25
2
10-
B
o
o-
r = 0.52
50
^ — ^ ,
„
o
O O#
o
- ..*.
5
CD
o
•
2,5
10
100
200
300
400
Epilimnion
Metalimnion
1
500
700
600
Rotifer abundance pnd 1"']
Fig. 6. Rotifer abundance versus HNF biomass (log scale) pooled from all samples between 4 May and 1
June 1994. Dotted lines: 95% prediction interval of regression (solid line).
layers. Rotifers constituted between 10 and 30% of total zooplankton biomass in
the epilimnion, and 5-10% in the metalimnion (Figure 4). The most common crustacean species were Daphnia longispina, Bosmina longirostris, Cyclops vicinus
and Acanthodiaptomus denticornis. Crustacean abundance was very variable
between successive sampling dates and only a few parameters could be statistically
linked to either abundance or biomass of cladocerans or copepods. Cyclops vicinus
showed a negative correlation with rotifers in the metalimnion (r, META = -0.626, P
< 0.05), particularly with K.longispina (r, META = -0.763, P < 0.05) and P.dolichoptera(r.META = -0.682, P< 0.05).
Zooplankton total biomass (dry weight) ranged from 34 to 250 \ig I 1 and was on
average twice as high in the metalimnion (Figure 4d), due to higher cladoceran
abundance. In the metalimnion, Chi a and total zooplankton biomass were significantly correlated (r, META = -0.709, P < 0.01); substituting total zooplankton biomass by the sum of Bosmina and Daphnia biomass resulted in an even better
relationship (r, META = -0.751, P < 0.005).
Discussion
Bacterivory and HNF community structure
In this study, HNF were by far more important consumers of (auto- and heterotrophic) picoplankton than ciliates, which agrees with a previous report on bacterivory in Piburger See (Sommaruga and Psenner, 1995). The development of
bacterial abundance or biomass could not be related to any of the other measured
parameters and changes in bacterial numbers were small compared with the fluctuations of bacterial production (Figure 3). We did not observe any correlation of
bacterial abundance or biomass with HNF, as has been suggested by Beminger et
al. (1991). Neither did wefindsignificant correlations with phytoplankton biomass
or metazooplankton. Our grazing experiments showed that protozoan grazing corresponded on average to nearly 70% of bacterial production, but the observed
short-term variability of bacterial productivity (Figure 3) puts serious doubts on
the calculation of 'the percentage of production consumed', as this may vary by
455
J.Pernthaler el a I.
Table II. Average HNF grazing rates on bacteria and picocyanobacteria, and selectivity index D
(Jacobs. 1974). Positive values of D indicate a preference for APP over bacteria
1994
Cyanobacteria
Bacteria
04-05
06-05
09-05
11-05
13-05
16-05
18-05
20-05
23-05
25-05
27-05
30-05
01-06
Selectivity index (D)
Grazing rate (cells HNF- 'h-')
EP1
META
EPI
META
18.6
12.7
24.0
14.0
11.6
8.7
6.8
3.5
3.1
12.9
6.9
15.4
12.6
20.0
13.0
18.2
12.8
14.3
14.8
9.8
4.6
4.4
11.3
9.6
13.0
11.8
1.4
1.2
11
1.1
0.6
0.6
0.5
0.5
0.2
0.9
0.6
0.7
0.8
0.9
0.8
1.0
1.1
0.4
0.7
0.3
1.0
0.4
0.7
0.6
0.8
0.7
EPI
META
0.72
0.53
0.31
0.38
0.12
0.21
0.30
0.56
034
0.31
0.47
0.29
0.39
0.44
0.71
0J5
0.46
-0.21
0.02
-0.22
0.58
0.34
0.26
0.28
0.36
0.23
EPI, epilimnion; META, metalimnion.
more than one order of magnitude within a few days (e.g. in the metalimnion:
minimum 21.6%, maximum 1475%). Averaging bacterial production over weekly
or monthly sampling intervals does not yield information about the dynamic processes within the bacterioplankton and may also be very far from estimates of the
mean proportion. However, even our intense sampling scheme could not reconstruct any continuity of bacterial secondary production, but rather pronounced the
occurrence of extremely rapid changes (Psenner and Sommaruga, 1992).
One premise of the FLB/FLC direct uptake approach is that all feeders (in this
case HNF) ingest particles randomly and at approximately the same rate. The
theoretically expected number of 'empty' HNF cells at a given incubation period
can thus be calculated assuming a Poisson distribution of ingested FLB per protist
(McManus and Okibo, 1991). However, it has been reported (e.g. Simek and
Chrzanowski, 1992) that there are large differences in the uptake efficiencies of
various bacterivorous flagellates isolated from the same natural community under
laboratory conditions. So far, this has been widely ignored infieldstudies on HNF
grazing, mainly because fluorescent microscopy commonly used in direct uptake
studies yields very poor information about protist taxonomy (McManus and
Okibo, 1991). Recently, in situ grazing rates of various freshwater ciliate taxa have
been reported, combining fluorescent and other staining methods (Simek et al.,
1995), but no such studies exist for heterotrophic flagellates, although new in situ
techniques for HNF taxonomy have been described (Lim etal., 1993). Even if our
analysis is very rough, it indicates that shifts in the 'taxonomic' composition of the
HNF community influence the grazing impact on bacteria and APP considerably.
Choanoflagellates, which are specialized at filtering bacteria from suspensions
(Leadbeater, 1991), were the dominant members of the HNF community at the
456
Protisian control of autotrophic picoplankton
beginning of the investigation, but disappeared almost completely towards the
end. In parallel, the individual HNF clearance rate on APP (Figure 5c) and bacteria (data not shown) decreased strongly, thus amplifying the fall in bacterivory
suggested by decreasing total HNF abundance (Figure 4a). The relative share of
more and less active picoplankton consumers within the HNF will therefore
tighten or loosen the trophic coupling of these two plankton components.
HNF grazing on APP
In this study, APP cells were found to be predominantly monodispersed even
though protozoa exerted a considerable grazing pressure; this contradicts the
hypothesis that the appearance of colony-forming APP species is a reaction to
predation, as discussed by Stockner (1991). Although HNF are commonly
assumed to be the most important consumers of picocyanobacteria, quantitative
evidence is scarce (Stockner, 1991; Weisse, 1993). Considering diel fluctuation of
HNF feeding rates on fluorescent particles (Christoffersen, 1994), we expect our
grazing data (from morning hours) to be under- rather than over-estimated. The
combined data from APP and HNF standing stock development (Figures 2c and d,
4a) and from our feeding experiments (Figure 5) suggest that the observed rise in
APP abundance is primarily top-down mediated. We calculated that at the beginning of the investigation, APP gross growth rates would have to be between 1.15
and 3.2 day-1 just to balance out HNF-imposed mortality, which is in the upper
range of growth rates observed for exponentially increasing picocyanobacteria in
laboratory cultures [Weisse (1993) and references therein]. A multiple linear
regression of HNF and ciliate biomass versus cyanobacterial abundance explained
68% of the observed variability (adjusted r2 = 0.679, F = 27.431, P < 0.001; (3HNF =
-0.394, P < 0.005; PCIUATC; = -0.556, P < 0.001). Including temperature did not
increase the predictive power of the correlation. As the grazing pressure from
HNF decreased (Figure 5), we observed a steep rise of APP abundance. It reached
a maximum towards the end of the study, but did not continue to increase linearly
during the whole period (Figure 2c and d). This suggests that another regulatory
mechanism becomes active soon after the tight control of HNF on picocyanobacteria is lost. APP growth may be limited by grazing from metazooplankton
(Weisse, 1988; Burns and Stockner, 1991; Weisse and Kenter, 1991). This does not
explain why we do not observe stronger APP reduction in the metalimnion, where
the biomass of effective APP consumers (Daphnia and Bosminia; Gliwicz, 1977;
Burns and Stockner, 1991; V6r6s et ai, 1991) was significantly higher (Figure 4).
However, as nutrients are considered an important limiting factor for picocyanobacterial growth in oligotrophic systems (Stockner, 1991), it is possible that the
sudden increase of APP to a new level of abundance indicates a switch from topdown (predation) to bottom-up (nutrient-limitation) control (Psenner and Sommaruga, 1992).
The trophic link between HNF and unicellular picocyanobacteria in this study is
tightened by the observed positive selectivity of HNF for APP over bacteria
(Table II), which has also been reported by Christoffersen (1994). This agrees with
the observation by Simek and Chrzanowski (1992) and others that HNF selectively
457
J.Pernthaler et al
ingest larger cell sizes within the bacterioplankton. Our results are in contradiction
to those of Caron et al. (1991), who concluded that protozoa do not select for or
against natural Synechococcus cells. Using mean bacterial and APP cell carbon
contents from each sampling date, we calculated that APP contributed on average
15.9% to the total carbon uptake through bacterivory on FLB and FLC by HNF
(minimum 5.4%, maximum 41.1%) (Figure 5). This is particularly remarkable as
the biomass of picocyanobacteria was on average more than one order of magnitude below bacterial biomass (Figure 2). A similar trend was also found for picoplanktivorous ciliates in Piburger See (Simek et al., 1996). The importance of this
food resource for HNF was nearly twice as large as its contribution to picoplankton
biomass and will thus be underestimated if only APP standing stock development
is considered.
Effects of metazooplankton on protozoa
The late spring to early summer clear-water phase has been recognized as a period
during which microzooplankton are suppressed or outcompeted by macrozooplankton (Sommer et al., 1986; Giide, 1988; Arndt and Nixdorf, 1990; Simek et al.,
1990). Protozoans are known to be sensitive to direct interference from rotifers
(Arndt, 1993; Gilbert and Jack, 1993) and crustaceans (Sanders and Porter, 1990;
Wickham and Gilbert, 1991). In spite of increasing temperature and a constant or
even increasing supply of picoplankton and small phytoplankton (Figures 1 and 2c
and d), we observed a sharp fall in protozoan biomass at the beginning of the investigation (Figure 4a and b), which can be assigned to metazooplankton predation.
The highly significant negative correlation between rotifers and HNF in our study
(Table I, Figure 6) is at least partly a causal (predator-prey) relationship (cf.
Arndt, 1993; Berninger et al., 1993). Bogdan and Gilbert (1982) found clearance
rates of Keratella and Polyarthra on Chlamydomonas that were slightly below
those of Bosmina longirostris. Daphnia rosea (comparable in size with D.longispina in this investigation) showed only 2-8 times higher clearance rates than
B.longirostris at various concentrations of Chlamydomonas (DeMott, 1982).
Assuming similar clearance rates for aplastidic and plastidicflagellatesof the same
size range, and considering the steep increase in rotifer abundance, they should
have an important impact on HNF mortality in our study (Figure 6).
Assessing the influence of cladocerans and copepods on the studied microbial
food web is difficult because of the numerous maxima and minima of the crustacean biomass (Figure 4d). This extreme variability has been explained by the
horizontally patchy distribution of crustaceans (Hehenwarter, 1980; Verreth,
1990). Piburger See is morphologically meromictic (Pechlaner 1977), so horizontal
patchiness is probably not an effect of wind action, but of internal seiches and
horizontal circular water movement (Hehenwarter, 1980), and can also be a consequence of zooplankton age structure (Siebeck, 1960a). We might also consider
possible changes in the mean population depth of various crustacean species
between sampling dates due to weather conditions (Siebeck 1960b), as we
observed a frequent change of sunny and cloudy days during the study period.
Unfortunately, it is not possible to reconstruct such shifts from the pooled water
458
Protistan control of autotrophic pkoplankton
samples from two layers. It is also difficult to evaluate predation on crustaceans in
Piburger See, as the lake is subject to annual restocking with trouts (Salmo trutta m.
fario and m. lacustris) for hobbyfishing.It is, however, known that roaches (Rutilus
rutilus) in Piburger See feed closer to the surface than the second main crustacean
predator, perch (Percafluviatilis)(R.Hofer, personal communication). From an
earlier study, we know that diel vertical migration of D.longispina does occur in
Piburger See, but probably only during the summer months (Pernthaler et al., in
preparation).
The higher abundances of cladocerans (data not shown) in the metalimnion did
not appear to affect protozoan biomass stronger than in the epilimnion. However,
protozoans, in particular nanociliates, are already reduced efficiently at low abundances of cladocerans. Wickham and Gilbert (1991) could show that susceptible
ciliate communities are suppressed by Daphnia densities <1 individual I"1. Jack
and Gilbert (1993) reported no overall difference in ciliate mortality rates induced
by Daphnia pulex or B.longirostris. In particular, small ciliates (<50 u-m) were
found to be most vulnerable to direct cladoceran interference. In our study, the
threshold level at which ciliates are suppressed and HNF decreased below 2 x 103
individuals I 1 was around 2-6 individuals of Daphnia and 10-30 of Bosmina per
litre.
Conclusions
This study presents a situation where changes in water temperature are small
enough to be ruled out as the driving force behind the observed short-term variability in the biotic parameters of the system. HNF bacterivory was even more
tightly coupled with community structure than with changes in density (Figure 5c),
particularly with the percentage of choanoflagellates. This may be one explanation
for the virtual absence of a link between HNF and bacterial abundance in natural
environments (Gasol and Vaqu6,1993). The selective consumption of picocyanobacteria by HNF is another possible reason for the large scatter in the relationship
between bacteria and HNF proposed by Berninger et al. (1991). Our data show
that the contribution of picocyanobacteria to the diet of HNF is nearly twice as
high as their proportion of total picoplankton biomass, a phenomenon which has
recently been described for ciliates as well (Simek et al., 19%). HNF grazing can
effectively control the abundance of picocyanobacteria under certain circumstances and metazooplankton predation on HNF will break this link. A tight predator-prey relationship between protists and APP has been reported from other
freshwater systems as well, and is probably a common phenomenon across lakes of
different trophic state (Christoffersen, 1994; Simek et al., 1996).
Acknowledgements
We thank Helga Mliller, Ruben Sommaniga and two anonymous referees for
helpful comments on earlier versions of the manuscript, and Jaroslav Vrba and
Mirek Mazek for help during sampling. This study was supported by the Austrian
Ministry of Research (GZ 45.281/3-IV/6a/93).
459
J.PernthaJer et al.
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Received on March 20, 1995; accepted on November 8, 1995
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