Journal of Plankton Research Vol.19 no.10 pp.1517-1536, 1997
Effects of UV radiation on grazing by two marine heterotrophic
nanoflagellates on autotrophic picoplankton
Clifford A.Ochs
Department of Biology, University of Mississippi, University, MS 38677, USA
Abstract. Phytoplankton <2-3 \im in diameter, or autotrophic picoplankton, can constitute the
majority of the biomass and productivity of photosynthetic organisms in marine and freshwater
systems. Indirect evidence has indicated that mortality of autotrophic picoplankton occurs principally
at night in the open ocean, but continuously in coastal water, l i e predominant view of the fate of
autotrophic picoplankton production in the ocean is that they are consumed by heterotrophic nanoflagellates. A possible mechanism to explain these observations is that grazing of heterotrophic
nanoflagellates on autotrophic picoplankton is inhibited by ultraviolet radiation (UV), at least in clear
open-ocean environments. A series of laboratory experiments was conducted to examine the effects
of UV radiation on the grazing impact of two heterotrophic nanoflagellates on Synechococcus spp.,
a commonly occurring genus of autotrophic picoplankton. The two nanoflagellates used were Paraphysomonas bandaiensis and Paraphysomonas imperforata. For both nanoflagellates, there was an
inverse relationship between the grazing mortality of Synechococcus and UV irradiance. The grazing
mortality of Synechococcus was reduced less with P.imperforata than with P.bandaiensis. In some
experiments, the effect of UV on the grazing impact of the nanoflagellate populations was caused in
part by UV-related reductions in nanoflagellate survival. However, UV reduced the grazing impact
of nanoflagellates primarily by reducing the rates of consumption of Synechococcus by individual
nanoflagellates, to a degree directly related to UV irradiance. The results suggest that UV radiation
may be an important selection factor in clear open-ocean water, and that in order to predict the effect
of increasing UV radiation on marine microbial plankton communities, we must consider interactions
between trophic levels as well as effects on single trophic levels.
Introduction
In the ocean, and many lakes, a significant fraction of algal biomass and productivity is contributed by photosynthetic cells <2-3 u,m in diameter, called
autotrophic picoplankton (APP) (Stockner and Antia, 1986; Weisse, 1993). At
certain locations and times of the year, autotrophic picoplankton may occur in
densities of 1 X 106 cells ml"1 or more, and contribute up to 50-60% of total
primary productivity (Stockner and Antia, 1986). Unicellular chroococcoid
cyanobacteria in the genus Synechococcus, of which there are a number of strains,
are a well-known and common component of the APP community in marine and
freshwater ecosystems. Recent reviews discussing the ecology of Synechococcus
spp. can be found in Weisse (1993), Stockner (1988), Stockner and Antia (1986)
and Waterbury et al (1986).
Waterbury et al (1986) conducted a detailed study of the population growth of
Synechococcus at two locations: a clear-water site in the northern Sargasso Sea and
a more turbid coastal water site near Woods Hole, Massachusetts. At both
locations, the net growth rate of Synechococcus was calculated from the rate of
change in Synechococcus density during the day. Mortality rates, assumed to be
due to grazing by nanoflagellates, were calculated by the rate of decline in
Synechococcus density at night. Assuming that the night-time mortality rate is
equal to the mortality rate during the day, the gross growth rate of Synechococcus
can be calculated as the difference between the observed daytime growth rate and
© Oxford University Press
1517
GA-Ochs
the night-time mortality rate. By this method, Waterbury et al (1986) calculated
that the diel (24 h) gross growth rate of Synechococcus was 4.4 doublings day 1 , a
rate they considered unrealistically high. To explain the discrepancy, Waterbury et
al (1986) proposed that predation of Synechococcus at the Sargasso Sea site may
be discontinuous, occurring principally at night. If this were so, the actual diel gross
growth rate would be 2.9 doublings day 1 , a value consistent with estimates made
by 14CO2 fixation (Waterbury et al, 1986). At the coastal site, the calculated diel
gross growth rate for Synechococcus was lower (1.5 doublings day 1 ), suggesting to
Waterbury et al (1986) that in coastal waters Synechococcus mortality occurs continuously and perhaps by a wider variety of predators than in the open-ocean site.
A possible mechanism explaining the observations of Waterbury et al. (1986)
is based on the difference in the penetration of UV in coastal versus open-ocean
water. UV is readily absorbed by dissolved organic matter (DOM) and particles
suspended in the water column (Smith and Baker, 1979; Worrest, 1986). Thus, in
a coastal zone exposed to strong tidal mixing, UV is absorbed quickly and would
not be expected to have a significant direct effect on organisms. Conversely, in
clear-water marine systems, where DOM and particles are in low concentration,
even ambient levels of UV can significantly reduce the growth rates of phytoplankton and bacteria down to at least 10 m (Karentz and Lutze, 1990; Gleason
and Wellington, 1993; Herndl et al, 1993). Thus, a diel pattern in the direct effect
of UV on organisms would be more likely to occur in the open ocean than in
coastal waters.
Could parasites or predators of Synechococcus be inhibited by UV radiation?
In the ocean, mortality of Synechococcus results largely from either viral infection (Suttle and Chan, 1994; Fuhrman and Noble, 1995) or grazing by nonpigmented heterotrophic nanoflagellates (reviewed by Caron et al, 1991; Weisse,
1993). The infectivity of marine bacteriophages is reduced by ambient UV compared to samples incubated in the dark (Suttle and Chen, 1992). Recently, Sommaruga et al. (1996) reported that grazing on bacterioplankton in freshwater by
the heterotrophic nanoflagellate Bodo saltans is inhibited by both ambient UVB
and UVA. No previous published studies have examined the effect of UV on the
grazing rates of marine heterotrophic nanoflagellates on cyanobacteria.
Were either viral-induced cell lysis or nanoflagellate grazing sensitive to UV,
the effect would be expected to be of most importance in non-turbid open-ocean
sites. Since almost three-quarters of the surface of the earth is covered with ocean,
which, except at coastal regions, has high transparency, UV may be a significant
factor influencing ecological interactions between aquatic organisms over a vast
area of the globe. The purpose of the present study was to examine the effect of
UV approximating ambient irradiances on the grazing activity of two marine
heterotrophic nanoflagellates on Synechococcus.
Method
Synechococcus strains, nanoflagellates and media
The Synechococcus strains used in this study were WH8012 and WH7803 (Waterbury et al, 1986). WH8012 is coccoid with a diameter of -1.5 urn. WH7803 is
1518
Effect of UV on protozoan grazing
ovoid with a lengthwise diameter of ~2 u.m. Both strains were maintained in SN
medium (Waterbury et al, 1986) prepared with artificial seawater. The composition of the artificial seawater was 18.7 kg I"1 Cl", 10.4 kg H Na+, 2.6 kg H SO42-,
1.3 kg I-1 Mg2+, 0.4 kg H Ca2+, 0.4 kg H K+, 0.2 kg h1 HCO3-, 0.006 kg H B and
0.008 kg I"1 Sr2+. The nanoflagellates used in this study, Paraphysomonas
bandaiensis and Paraphysomonas imperforata, are commonly isolated from
marine plankton samples (D.Caron, personal communication). Both species
consume Synechococcus readily in culture (personal observations). Nanoflagellate cultures were maintained on a mixed bacterial assemblage growing in a
0.01 % yeast extract made with artificial seawater and amended with rice grains.
All organisms were cultured at 20°C under a light-dark cycle (12 h light:12 h
dark) at an intensity of photosynthetically active radiation (PAR) of -30 u,mol
UV exposure
The UV source was a Psoralite 2400 light system fitted with F24T12BL/HO UVA
fluorescent lamps situated behind an acrylic shield. The UV spectrum at 0.5 m
produced with this unit (Figure 1) was measured with an Optronics spectroradiometer (Model 742) calibrated with an Optronics OL-200H calibration standard. Using this UV source, the maximum irradiance of wavelength-specific
radiation to which the organisms were exposed in experiments was 0.41 W irr2 at
354 nm, an irradiance at this wavelength that has been measured at between 5.5
0.5
0.4 -
UV-A; 17 Wm" 2
UV-B: 0.35 W m"2
%UV-B/UV-A = 2.1
0.0
280
300
320
340
360
420
Wavelength
Fig. 1. Irradiance of UV source at 0.5 m. Units are W nr 2 nnr 1 .
1519
CA-Octa
Table L Irradiance of UV and PAR of UV source at 05 m. UV irradiance units are W nr 2 . PAR units
are umol s~' nr 2 . The number of neutral density filters (NDF) used is indicated
Filter used
UVA
(320-400 nm)
UVB
(290-320 nm)
%
(UVB/UVA)
PAR
(400-700 nm)
1NDF
2 NDF
3 NDF
Plexiglass UF-3
9.36
5.65
3.40
0.01
0.19
0.12
0.07
0.00
2.03
2.12
2.06
0.00
8.0
5.9
3.7
7.2
and 8.5 m in clear ocean water (Baker and Smith, 1982). The irradiance at 300
nm, 0.008 W m~2, has been measured within the same depth interval (Baker and
Smith, 1982).
Irradiances of UV to which treatments were exposed, and the percentage of
UVB (290-320 nm) to UVA (320-400 nm), are described in Table I. Neutraldensity screening was used to provide a range of UV irradiance. UV was removed
by a cardboard screen or a shield of clear acrylic. The irradiance of broad-band
UVA and UVB to which organisms behind the acrylic shield were exposed was
<0.015 W m"2 and 4 X 10"6 W nr 2 , respectively. The range of broad-band UV
irradiances used and the ratio of UVB to UVA, integrated between 290-320 and
320-400 nm, correspond to measurements of UV made in the Caribbean between
10 and 20 m. For comparison, Gleason and Wellington (1993) measured an integrated UV irradiance of 15.70 (UVA) and 0.32 (UVB) W nr 2 at 10 m in the
Caribbean. At shallower depths, the ratio of UVB to UVA would be higher since
UVB is absorbed more readily by water than is UVA.
Although the broad-band UV irradiance at the treatments corresponds well
with published field measurements of broad-band solar irradiance in water, the
spectral distribution of the UV source differs from the spectral profile of solar
UV. Solar irradiance at the surface of the Earth tends to increase with wavelength
through the UV and up to between 450 and 500 nm in the visible portion of the
solar spectrum (Kirk, 1994). Compared to the solar spectrum, irradiance to which
organisms were exposed in these experiments was relatively depleted in UVA and
visible wavelengths >360 nm.
The UV source produced some PAR, which was measured with a Licor
radiometer (Model Li-1000) equipped with a 4TT cosine-corrected sensor (Table
I). In three of the eight experiments that were conducted, additional PAR of -40
u-mol s"1 m~2 was provided by a bank of fluorescent lights (tube #RB15T8) positioned on the opposite site of the treatments from the UV source (Table II). The
fluorescent lights providing PAR were shielded by a polycarbonate lens to
remove any UV that might be produced.
Control and experimental treatments
Approximately 24 h prior to an experiment, a portion of the nanoflagellate
culture, in stationary phase, was filtered first through a Whatman GF/Cfilter,then
through a 1.0 u-m Nuclepore filter. This procedure removed all nanoflagellates,
1520
Effect of UV on protozoan grazing
Table D. Organisms used and light conditions in the experiments. 'Light' refers to whether or not the
treatments were exposed to additional PAR of 40 umol sr[ nr2
Experiment
Synechococcus
strain
Nanoflagellate
species
Initial number of
nanoflagellates mh1
Light
A
B
C
D
E
F
G
H
WH8012
WH7803
WH8012
WH8012
WH8012
WH8012
WH8012
WH8012
P.bandaicnsis
P.bandaiensis
P.bandaiensis
P.bandaiensis
P.imperforata
P.imperforata
P.imperforata
P.imperforata
40
31
17
21
30
30
48
19
No
No
Yes
No
No
Yes
No
Yes
000
000
000
000
000
000
000
000
but enough heterotrophic bacteria passed through the filters to repopulate the
media. The filtered media were used for control treatments in which predation
on Synechococcus was eliminated.
Experiments were initiated by the introduction into sterile artificial seawater
of either Synechococcus strain WH8012 to a final concentration of 10 X 106 ml"1,
or strain WH7803 to a final concentration of 5 X 106 ml"1 (Table II). To treatments containing nanoflagellates, either P.bandaiensis or P.imperforata were
introduced to a final concentration of between 17 and 50 000 cells ml"1, depending on the experiment. High densities of Synechococcus and nanoflagellates were
used in order to reduce the effect that changes in encounter frequency between
Synechococcus and nanoflagellates as a function of changing cell densities could
have on the results. Control treatments (nanoflagellate-free) were prepared for
each UV irradiance. For each treatment, there were four replicates of 25 ml. To
control treatments an aliquot of nanoflagellate-free filtrate, equal in volume to
the additions containing nanoflagellates, was added. All treatments were incubated in UV-transparent Whirlpak bags (Karentz and Lutze, 1990) at 25°C.
Experimental manipulations and sampling
The treatments were suspended 0.5 m from the UV lamps and exposed to UV
radiation as described in Table I. Every 2 h, for 10 h, 2 ml aliquots were withdrawn from each incubation culture. At each sampling, in vivo fluorescence of
the unpreserved aliquots was measured. Fluorescence was measured in a 1-cmwide cuvette by a Sequoia-Turner fluorometer using a 540 nm shortwave pass
filter for excitation and a 585 nm longwave pass filter for emission.
In most experiments, samples for counts of nanoflagellates were collected after
10 h of incubation. In some experiments, nanoflagellates were also collected after
6 h. Heterotrophic bacteria were also enumerated in some experiments. All
samples were preserved with glutaraldehyde (final concentration of 2%).
Enumeration of nanoflagellates and heterotrophic bacteria was by epifluorescence microscopy after staining with the fluorochrome DAPI or acridine
orange. The average densities of organisms in a time interval were calculated by
the equations of Heinbokel (1978). Where nanoflagellate density was determined
for more than one time interval, an average density for the experiment was
1521
CA.O«*s
calculated as the weighted sum of average densities in each of the smaller time
intervals.
To reduce the potential of oxygen decline during the experiments due to photooxidation of DOM, the incubation bags were left open at the top and the treatments gently mixed periodically. Oxygen measured in all treatments at the
conclusion of several experiments was never less than 8.0 mg H.
Calculations of rates of grazing mortality of Synechococcus
Gross growth rates (b) of Synechococcus in each control treatment were estimated as:
b = [1//, - r0]ln(/lc//oc)
(1)
where / 1C and /QC are the relative fluorescence intensities in treatments without
nanoflagellates at times tx and tQ, respectively. The net rate of growth (a) in the
experimental treatments was estimated similarly as:
a = [1/r, - fo]ln(/iE//oE)
(2)
where / 1E and I0E are the relative fluorescence intensities in treatments containing nanoflagellates. Assuming that the gross growth rates were equal in the
control and experimental treatments, the estimated rate of mortality of Synechococcus by grazing, GM (time"1), is:
GM = b - a
(3)
Nanoflagellate-specific grazing rates were calculated as:
GR = GMW(1_,o
(4)
where GR is the nanoflagellate-specific grazing rate (nanoliters cleared of prey
per nanoflagellate per hour) and A',] . ^ is the average density of nanoflagellates
in the time interval between ^ and tx over which GM was calculated.
In these experiments, we used changes in the in vivo fluorescence of the treatment cultures as a means of estimating GM rather than making direct counts of
Synechococcus. Treatment fluorescence is not a reliable indicator for comparison
of the cell density of pigmented organisms exposed to different irradiances or
qualities of light because of variation between treatments that can occur in cellspecific pigment concentrations or fluorescence (e.g. Takano et al., 1995). The aim
of this study, however, was not to estimate changes in Synechococcus density, but
to quantify the effect of UV on grazing activity of heterotrophic nanoflagellates
consuming Synechococcus. Treatment fluorescence can be used to measure GM
accurately if two conditions are met. First, regressions between cell number and
treatment fluorescence in all treatments receiving an identical UV exposure must
have a _y-intercept at the origin. Second, the slopes of regressions between cell
1522
Effect of UV on protozoan grazing
number and treatment fluorescence at a particular UV irradiance and incubation
time must be the same in all treatments, whether or not nanoflagellates are
present. Only if these two conditions are met will the ratio of cell number and
treatment fluorescence be constant across a range of cell numbers, and changes
in treatment fluorescence during an incubation period represent proportionately
equivalent changes in cell number in all treatments receiving the same UV irradiance. Conversely, it is not necessary that the slope of the regression between cell
number and treatment fluorescence be the same in treatments incubated at different UV irradiances, since the influence due to slope cancels in the grazing calculations.
Ochs and Eddy (1997), using data from several experiments in which grazing
rates were measured by both changes in treatment fluorescence and Synechococcus density, found no significant difference in results. In this study, an
additional test was conducted specifically to evaluate the two assumptions
described above. For this test, Synechococcus WH8012 and WH7803 were incubated in Whirlpak bags at the two extremes of UV irradiance used in the grazing
experiments: 0 and 9.36 W nr 2 . For each strain of Synechococcus, and each UV
irradiance, there were three 25 ml replicates with nanoflagellates and three replicates in which nanoflagellates were excluded, prepared as described above.
Samples were removed from each treatment after 10 h of UV exposure. Each
sample was diluted with particle-free artificial seawater to a final concentration
of 50, 25 and 12.5% of the cell density in undiluted samples. Treatment fluorescence was measured in each dilution. Synechococcus density in the undiluted
samples was determined by epifluorescence microscopy; for diluted samples,
density was calculated using the density in the undiluted sample and the dilution
factor. The slope and intercept of plots comparing Synechococcus density and
treatment fluorescence in treatments with and without nanoflagellates, but
exposed to the same irradiance, were compared for significant differences.
Measurements of GM, calculated by changes in density and treatment fluorescence, were also compared.
Toxicity test
Whirlpak bags are commonly used for studies of growth and photosynthesis of
marine phytoplankton, but have not been tested for their effect on nanoflagellate
grazing when exposed to UV. A preliminary test was conducted to examine the
possibility that UV-irradiated bags or culture media might inhibit nanoflagellate
grazing activity. Twenty-five milliliters of sterile nanoflagellate culture media
were added to Whirlpak bags (n = 16) and to 50 ml glass serum bottles (n = 8),
and allowed to incubate for 24 h. During this incubation period, half of the bags
were exposed to UV for 12 h at an irradiance of 17.3 W nr 2 (2% UVB). The other
eight bags and all eight bottles were kept in the dark during this period. After
24 h, all bags and bottles were inoculated with 5 X 106 cell ml"1 of Synechococcus
WH8012. Half of each group of bags and half the bottles were also inoculated
with P.bandaiensis to a final concentration of -40 000 nanoflagellates ml"1; the
other half of the bags and bottles were inoculated with an equal volume of
1523
CA.Ochs
nanoflagellate-free filtrate as described above. Four replicates of each treatment
were placed in the dark and treatment fluorescence measured after 5,10 and 20 h.
Thus, the bags and culture media were exposed to UV, but the nanoflagellates
and Synechococcus were not. Short-lived molecules that may be produced by
photochemical reactions (Mopper and Zhou, 1990) were not evaluated for their
effect on nanoflagellate grazing; previous studies have found no effect of these
compounds on nanoflagellate motility (Hader, 1993).
Statistical analysis
Differences between treatments in the rates of grazing mortality (GM) and cellspecific flagellate grazing rates (GR), and in changes in flagellate density, were
examined by one-way ANOVA. Where the data were not normally distributed,
the Kruskal-Wallis one-way analysis of variance on ranks was used. For isolation
of treatment differences, the Student-Newman—Keuls test was used. Statistical
significance was set at P < 0.05.
Results
Evaluation of treatment culturefluorescenceas a measurement of grazing
mortality
For Synechococcus WH8012, both assumptions necessary for an evaluation of
GM based onfluorescencechanges were validated (Figure 2). In both the absence
of UV and at the highest irradiance of UV used in these experiments, the slopes
of the relationships of Synechococcus density and treatment fluorescence after
10 h of incubation, with and without nanoflagellates present, were not significantly different. Also, the y-intercept for each plot is not significantly different
from zero, indicating a constant relationship of cell number and fluorescence.
Consequently, measurements of GM, calculated by cell number and by fluorescence, were not significantly different for WH8012 (data not shown).
For Synechococcus WH7803, the y-intercept of all plots was at the origin, but
the slopes of the plots for treatments in which there were nanoflagellates were
steeper than those for treatments in which nanoflagellates were absent. The
reason for the difference in slopes is unknown, but were this difference to be consistent GM would be underestimated by changes in treatment fluorescence. The
steeper the slope of the relationship in the treatments with flagellates compared
to the slope in the absence of flagellates, the greater is the underestimation of
GM. In this test, GM calculated by changes in fluorescence underestimated GM
calculated by changes in cell number by between 20 and 40%, depending on the
dilution factor (data not shown). As the underestimations of GM in treatments
exposed to the two extremes in UV irradiance were not significantly different
(P > 0.05), and because Synechococcus WH7803 was used in only one of the eight
experiments, no corrections were made to account for these underestimations of
grazing. Any errors in the calculation of GM, because they would influence
treatments at both extremes of UV irradiance equally, do not affect conclusions
regarding the relative effect of UV on the grazing activity of nanoflagellates.
1524
Effect of UV on protoxoan grazing
80
70
60
50
40
30
20
10
0
i
i
i
i
i
i
i
i
r
0 2 4 6 6 101214161820
\
i
i
i
i
i
i
i
0 2 4 6 8 101214161820
Synechococcus abundance (X 10° ml)
Fig. 2. Regressions of Synechococcus abundance and relative fluorescence at 10 h in experiments to
test the assumptions of using treatment fluorescence to determine grazing mortality, (a) and (b) for
Synechococcus WH8012 at 0.0 and 9.5 W nr 2 , respectively, (c) and (d) WH7803 at 0.0 and 9.5 W nr 2 .
Nanoflagellates present (•); nanoflagellates absent (#).
Effects of UV on grazing activity of Paraphysomonas
Effects of UV exposure on GM for two time periods in all eight experiments are
summarized in Table III. Calculated over 6 h of exposure, in each experiment
using P.bandaiensis there was a significant decline in GM corresponding to an
increase in UV irradiance. In three of these four experiments, there was a decline
in GM even at the lowest irradiance. Generally, differences between treatments
in the effect of UV on GM were larger when calculated over the 10 h exposure
period compared to the 6 h exposure period, indicating that the impact of nanoflagellate grazing in reducing Synechococcus became increasingly depressed as
the time of UV exposure increased. In each of the four experiments using
P.bandaiensis, there was a significant linear relationship between UV irradiance
and the degree of reduction of GM after both 6 and 10 h (Table III).
Effects of UV exposure on the grazing mortality of Synechococcus with the
nanoflagellate P.imperforata were less pronounced than with P.bandaiensis
(Table III). Calculated over 6 h of exposure, in only one of the four experiments
using P.imperforata (experiment G) was there a significant difference between
treatments in GM; this difference was only evident at the highest UV irradiance.
Over a 10 h period in two of these four experiments (experiments E and G), there
were significant differences in GM between the two treatments receiving the least
1525
CA.Ochs
Table in. Grazing mortality (GM) in treatments exposed to different levels of UV as a percentage of
the treatment shielded from UV (the 0.0 W m~2 column). Measurements are for two time periods of
exposure: 0-6 and 0-10 h. Each value is the mean of four replicates. In an experiment, values having
the same letter are not significantly different from each other at P < 0.05. P values are for linear
regressions of UV irradiance and the GM percentages (n = 16)
Experiment
Nanoflagellate
A
P.bandaiensis
B
P.bandaiensis
C
P.bandaiensis
D
P.bandaiensis
E
P.imperforata
F
P.imperforata
G
P.imperforata
H
P.imperforata
Interval (h)
0-6
0-10
0-6
0-10
0-6
0-10
0-6
0-10
0-6
0-10
0-6
0-10
0-6
0-10
0-6
0-10
UV irradiance (W m" :! )
P
0.0
3.5
5.7
9.5
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
100a
91b
66b
102a
81b
86b
82b
82b
65b
106a
92a
100a
92a,b
91a
81b
93a
85a,b
74c
58c
89a
66b
81b
67c
69c
43c
97a
77b
85a
87b
88a
66c
78a
73b
59d
32d
25b
22c
60c
43d
31d
22c
94a
60c
91a
83b
70c
61c
80a
82a,b
<0.0001
<0.0001
0.0011
<0.0001
0.0014
<0.0001
<0.0001
•cO.0001
0.2379
<0.0001
03171
0.0002
0.0009
<0.0001
0.0282
0.0397
UV irradiance and the two treatments receiving the highest UV irradiance.
Significant regressions of UV irradiance and GM in two of the four experiments
using P.imperforata after 6 h, and in all experiments following 10 h, suggest a weak
UV-related effect (Table III).
Differences between the two nanoflagellate species in the degree of response
to UV irradiation are also indicated by regressions of GM and UV dose (irradiance X exposure time) (Figure 3). In each of the experiments with P.bandaiensis, there was a significant inverse relationship of GM and UV dose. Variance in
the regression lines in three of these four experiments (A, C and D) was small (r2
> 0.87). In contrast, there tended to be a poorer relationship of GM and UV dose
when P.imperforata was used. In only two of the four experiments (experiments
G and H) was there a statistically significant relationship of GM and UV dose,
and in all experiments using P.imperforata there was substantial variance in the
regressions (r2 < 0.55).
As examples of patterns in the time courses of treatment fluorescence, the
results of two experiments are shown in Figure 4. In treatments in which nanoflagellates were not present (control treatments), fluorescence increased, an indication of either reproduction and/or cell-specific changes in fluorescence of
Synechococcus during the experiment. The rate of decline in fluorescence in
experimental treatments, in which nanoflagellates were present, tended to be
highest at lower irradiances of UV (Figure 4a and c). The effect of UV on the
rate of removal of Synechococcus in these experiments, corrected for fluorescence changes in control treatments, is illustrated in Figure 4b and d. As indicated by the values of GM recorded in Table III, the higher the UV irradiance,
the less the rate of removal of Synechococcus.
1526
Effect of UV OD protozoan grazing
140 -
a
120 -
•
100 80 60 40 C
A
B
D
20 0 -
1
I
I
I
I
I
I
250
300
350
400
e>
F
E
H
50
100
150
200
250
300
350
400
2
UV Irradiation (J m" , unweighted)
Fig. 3. Regressions of UV irradiation and the percentage of GM compared to control treatments.
Regression lines are for individual experiments as indicated (Table II). For each experiment, all data
from samples collected at 2 h intervals are included, (a) Experiments using P.bandaiensis: (•) experiment A, (•) B, (A) C, (•) D; (b) experiments using P.imperforata: (•) experiment E, ( • ) F, (A) G,
(•)H.
The densities of nanoflagellates after 10 h in all four experiments using
P.bandaiensis and two experiments using P.imperforata are shown in Figure 5. In
four of these experiments, the highest density of nanoflagellates at 10 h occurred
in either the UV-shielded treatment or the treatment exposed to the lowest UV
irradiance. In experiments B and G, there were, for unknown reasons, reductions
in the densities of nanoflagellates in the UV-shielded treatments as well as in the
UV-exposed treatments, with little difference between any treatments in the
density of nanoflagellates at 10 h. In experiment H, after an initial decrease of
nanoflagellate densities in all treatments within the first 6 h, there was an increase
in densities in the two treatments exposed to the lowest UV irradiances (data not
1527
CA-Ocbs
130 -i
lO.O-
oo3.55.79.5-
120 110 -
~1
O
2
4
6
Time (hours)
8
10 12
6
I
8
I
I
10 12
Time (hours)
Fig. 4. Results of two experiments to test the effect of UV radiation on nanoflagellate grazing, (a)
and (c) Percentages of initial treatment culture fluorescence through time for experiment C. The
numbers indicate UV exposure (W nr2) in treatments with (+) or without (-) nanoflagellates. (b) and
(d) Trends in Synechococcus abundance in treatments containing nanoflagellates corrected for
Synechococcus growth. Data are from experiment E. Trends were produced by adjusting initial fluorescence values in samples with nanoflagellates by rates of grazing mortality of Synechococcus [GM in
equation (3)] calculated every 2 h. Results are expressed as a percentage of the initial culture fluorescence. In all plots, each point is the mean of four replicates.
shown). Consequently, despite clear differences in nanoflagellate densities at
10 h, the average densities of nanoflagellates over the course of experiment H
were not significantly different between treatments (P > 0.05).
Nanoflagellate-specific GR were calculated for each of the experiments for
which measurements of nanoflagellate densities were made (Table IV). Over the
10 h incubation period, GR was inversely related to UV irradiance in all experiments using P.bandaiensis, and in experiment G in which P.imperforata was used.
In contrast, in experiment H the relationship of UV irradiance and GR was not
significant.
Microscopic observations of living cells of P.bandaiensis after several experiments revealed obvious effects of UV on swimming behavior. Nanoflagellates
exposed to 9.5 W m~2 of UV moved slowly in small circles compared to
1528
Effect of UV on protozoan grazing
50000
0 0 3.5 5.7 s 5
0 hrs
0 0 3.5 5.7 9.5
10hre—
Ohre
— 1 0 hrs—
-2,
Time and UV Irradiance (W m )
Fig. 5. Densities of nanoflagellates at time 0 and 10 h after the start of incubation at different UV
irradiances. Letters in each plot indicate the experiment (Table n). Error bars are 1 SD. Notice that
the range of the y-aris is not the same in each plot.
nanoflagellates exposed to a lesser UV irradiance which moved more rapidly and
in varied directions. These observations, although evident to several observers,
were not quantified. In experiments using P.imperforata, a noticeable effect of
UV on swimming behavior was not evident.
In all experiments, the inoculum of nanoflagellates into experimental treatments included heterotrophic bacteria present in the nanoflagellate cultures. For
TaMe IV. Per capita grazing rates of nanoflagellates (GR) in treatments exposed to different levels of
UV as percentages of the treatment shielded from UV. All measurements are for a 10 h period of
exposure. Each value is the mean of four replicates. Values having the same letter are not significantly
different from each other at P < 0.05. P values are for the linear regressions of UV irradiance and the
GR percentages (/1 = 16)
Experiment
A
B
C
D
G
H
Nanoflagellate
P.bandaiensis
P.bandaiensis
P.bandaiensis
P.bandaiensis
P.imperforata
P.imperforata
UV irradiance (W n r 2 )
P
0.0
3.5
5.7
9.5
100a
100a
100a
100a
100a
100a
70b
77b
76b
62b
73b
106a
63c
65b
68c
47c
56c
80a
38d
21c
49d
24c
60c
86a
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0658
1529
CA-Octo
Table V. Ratio of Synechococcus to all bacteria (including Synechococcus)at 10 h in treatments
without nanoflagellates. Ratios were compared by a r-test
Experiment
1
2
UV irradiation
(W m-2)
0.0
9.5
0.0
9.5
Ratio (SD) at time after start of
experiment
0
10
0.72 (0.04)
0.72 (0.04)
0.73 (0.09)
0.73 (0.09)
0.55 (0.07)
0.66 (0.08)
0.75 (0.03)
0.76 (0.03)
P value of r-test
0.08
0.83
this reason, heterotrophic bacteria were also introduced into the nanoflagellatefree control treatments. In the experimental treatments, these bacteria are an
alternative food source for the nanoflagellates, and were the proportion of bacteria to Synechococcus to differ significantly between treatments over the time
course of the experiments, estimates of grazing of Synechococcus would represent variable proportions offlagellategrazing activity on all available food items.
To account for this possibility, in two experiments the ratio of Synechococcus to
all bacteria (including Synechococcus) was compared in control treatments
exposed to the extremes of UV exposure. In both experiments, the differences
were not significant (Table V). In other words, Synechococcus were not 'diluted'
to a greater extent in treatments in which predation on Synechococcus was least.
UV pre-exposure of the Whirlpak bags and culture media (Figure 6) did not
affect nanoflagellate grazing activity. In all treatments in which nanoflagellates
were present, whether or not they were previously irradiated, the density of
Synechococcus declined rapidly, as crudely estimated by changes in fluorescence.
There was little difference influorescenceamong treatments that did not contain
nanoflagellates and no decline influorescenceover time, indicating that Synechococcus density in the dark was stable in the absence of nanoflagellates.
Discussion
The results of these experiments are consistent with a hypothesis that exposure
to UV, over a range of irradiances that would be encountered in the open ocean,
reduces the grazing impact of marine heterotrophic nanoflagellates on Synechococcus. In each of the eight experiments using two different nanoflagellates,
reductions in GM calculated over 6 and/or 10 h of exposure were correlated with
UV irradiance (Table III). Reductions of GM upon UV exposure may result from
UV-related reductions in survival of nanoflagellates, or UV-related reductions in
nanoflagellate-specific GR. In some, but not all, of the experiments, there were
clear UV-related declines in nanoflagellate density (Figure 5). However, in none
of the six experiments in which both GM and GR were determined did normalization of GM by the average nanoflagellate abundance alter or substantially
weaken the trends observed in GM (Tables III and IV). Even in experiment H,
in which UV-related effects on nanoflagellate densities at the end of the incubation were most apparent, average densities over the course of the experiment
1530
Effect of UV on protozoan grazing
22
20 18 -
16 14 -
12 10 8 -
6 4 2 -
WP+UV(NP)
WP(NP)
Glass (NP)
WP+UV(P+)
WP(P+)
Glas»(P+)
0
5
10
15
20
25
Time (hours)
Fig. 6. Results of experiment to test the effect of UV pre-irradiation (+UV) of culture media and
Whirlpak bags (WP) on the rate of predation of Syntchococcus. Treatments incubated in glass were
not UV irradiated.
were not sufficiently different to explain the patterns observed in GM strictly as
a function of these densities. These results indicate that UV exposure reduces the
grazing impact of nanoflagellates on Synechococcus primarily by reducing the
nanoflagellate-specific GR to a degree that is directly related to UV irradiance,
at least over a 10 h incubation period.
Differences in the sensitivity of microorganisms to UV exposure may be an
important environmental selection factor in the organization of protozoan (Sommaruga et aL, 1996) and phytoplankton (Calkins and Thordardottir, 1980;
Worrest, 1981; Karentz, 1994) communities. Reasons for differences between the
two nanoflagellate species in the effect of UV on GM are unclear (Table III,
Figure 3). As discussed above, for both species, patterns observed in the effects
of UV on GM and GR were similar. Assuming a similar correspondence between
GM and GR in experiments E and F, for which nanoflagellates were not determined, these results, with the possible exception of experiment G, suggest that
per-capita grazing of P.imperforata tends to be less sensitive or perhaps responds
more slowly to UV exposure than grazing of P.bandaiensis. Visual observations
support this conclusion, but these observations were not quantified and are considered tentative.
Grazing of individual nanoflagellates on picoplankton may be inhibited as a
consequence of reduced palatability of UV-stressed food (Hessen et ai, 1995), or
by disruption of the predator's ability to locate food. Studies with pigmented
1531
CA.Odu
nanoflagellates of various kinds have shown that UVB can result in inactivation
of flagella (Hessen et al, 1995). Disruption of flagellar movement could impair
motility of the organism (Hader and HSder, 1989), disrupt flagella-induced
feeding currents (Fenchel, 1986), or both. In these experiments, it was observed
repeatedly that P.bandaiensis were less motile in high-UV treatments compared
to treatments in which UV irradiance was ^3.5 W m~2. A reduction in nanoflagellate motility would reduce encounter frequency between predator and prey,
although this effect was probably minimized in these experiments by using a high
concentration of prey. In nature, UV-induced interference with motility may
reduce the ability of some nanoflagellates to migrate into an area of lesser UV
exposure during the hours of maximum irradiance, as has been observed for dinoflagellate assemblages (Tilzer, 1973).
In addition to increasing the mortality of heterotrophic nanoflagellates, or
reducing the grazing rate of individual nanoflagellates, UV may affect the rate of
grazing of Synechococcus by altering the relative availability of an alternative
food source. Growth rates of heterotrophic bacteria, an alternative food source
for nanoflagellates in these experiments and in the ocean, are inhibited by
ambient UV (Karentz and Lutze, 1990; Herndl et al, 1993). In these experiments,
however, differences in the relative abundance of potential prey items between
treatments do not explain the results (Table V).
For nanoflagellate grazing in the open ocean to occur primarily on a nocturnal
basis (Waterbury et al, 1986), GR must be only temporarily inhibited, or losses
of nanoflagellates during the day must be compensated for by reproduction at
night. Donkor et al. (1993) have measured limited recovery of motility in filamentous cyanobacteria after short periods of UVB exposure, but the extent,
timing and mechanisms of recovery of motility of protozoa from UV exposure
are not known. Are nanoflagellate growth rates sufficiently high to compensate
for possible diel UV-induced mortality? Goldman and Caron (1985) measured
specific growth rates of P.imperforata, grown on a diet of phytoplankton, of
between 2.3 and 2.5 day-1 at 20-24°C. Whether or not this growth rate, which
corresponds to a doubling time of ~7 h, is sufficiently rapid to allow diel recovery
from UV-induced mortality depends on the magnitude of the loss, the effect of
UV on nanoflagellate growth rates, the time required for molecular repair
mechanisms to operate, and the relative importance of other causes of mortality.
Several caveats must be considered before the results of these experiments can
be applied to the natural environment. First, various kinds of colored and colorless compounds have been identified in bacteria and algae that serve as UV sunscreens, and that help to protect against UV damage to nucleic acids or other
sensitive biomolecules (Goodwin, 1980, Garcia-Pichel and Castenholz, 1993).
Organisms cultured in the absence of UV, because they may have reduced concentrations of these molecular sunscreens, are likely to be more susceptible to
UV damage than natural organisms (Paerl et al, 1985). Most heterotrophic
nanoflagellates are nearly transparent, perhaps making them relatively more susceptible than pigmented organisms to the detrimental effects of UV exposure.
Differences in the sensitivity to UV exposure of grazers and pigment-containing
1532
Effect of U V on protozoan grazing
prey such as Synechococcus may result in a separation in time of growth of the
prey and feeding of the grazers (Bothwell et al., 1994).
A second consideration is that the nanoflagellates used in these experiments
may not be representative of other marine protozoa in their sensitivity to UV
irradiation. In fact, P.imperforata has recently been described as a 'weed' which,
although common in occurrence, only becomes abundant under favorable culture
conditions (Lim et al., 1997). Perhaps the density of P.imperforata is low in surface
waters due to poor survival upon prolonged UV irradiation, as suggested by the
results of experiment H (Figure 5).
The dose of UV which an organism receives depends on both the irradiance
and the length of time of exposure. The length of time plankton are exposed to
UV on a sunny day depends on the depth at which the organisms occur, the angle
of the sun, the rate and depth of mixing, and daylength. Daylength and solar incidence are a function of latitude and season. Although the sun may shine almost
all night during summer at high latitudes, the sun remains low on the horizon,
reducing the depth to which UV penetrates the water column. In equatorial
regions, sunlight strikes the ocean surface at a less oblique angle, resulting in UV
penetration to deeper depths for a longer period of time (Dahlback et al, 1989).
The UV irradiances used in this study are similar to broad-band UVA and UVB
irradiances made between 10 and 20 m in the ocean (Gleason and Wellington,
1993), and were sufficient to reduce grazing of P.bandaiensis within 6 h of
exposure. At shallower depths, the effect of UV radiation on protistan grazing
activity may be more severe.
Finally, in nature many organisms, including bacteria, algae and some
protozoa, are capable of enzymatic repair of UVB-induced DNA damage, or
photoreactivation. Photoreactivation repairs dimers formed between adjacent
pyrimidine bases, the primary DNA photoproducts of UVB irradiation (discussed in Karentz, 1994). These dimers, if they are not repaired, interfere with
transcription and can lead to cell death. Photoreactivation requires radiation in
the UVA/shortwave visible range (Mitchell and Karentz, 1993). In these experiments, radiation in this portion of the spectrum relative to radiation in the UVB
was less than under natural solar illumination, even in those experiments in which
additional visible illumination was provided. Assuming that the nanoflagellates
used in these experiments are capable of photoreactivation, it is probable that
this process was inhibited. However, a restricted capacity for photoreactivation
is unlikely to be the only reason for impairment of grazing. In recently conducted
laboratory experiments using UVA alone, at irradiances similar to those used in
these experiments, there were significant reductions of nanoflagellate grazing
rates (Ochs and Eddy, 1997). These effects could not have been caused by pyrimidine dimers because UVA is only slightly absorbed by nucleic acids (Mitchell and
Karentz, 1993). Thus, although inhibition of photoreactivation may have contributed to the reduction of grazing rates observed in the present experiments,
other processes that damage biological macromolecules, such as UVA-induced
production of reactive oxygen species (Fridovich, 1986), are probably of equal or
greater significance. Donkor et al. (1993) have shown that UVA alone at natural
irradiances can impair the motility of marine and freshwater phytoplankton.
1533
CA.Ochs
Thinning of the stratospheric ozone layer has raised concerns that enhanced
levels of UV will disrupt ecological processes in aquatic environments, particularly at low latitudes. Yet, exposure to ambient levels of UV that occur in middle
and low latitudes, relatively unaffected by recent changes in ozone concentration,
also may influence physiological processes and behavior of organisms in freshwater and marine habitats (HSder and Worrest, 1991; Bothwell et al, 1993;
Karentz et al, 1994). Most research on the biological effects of ambient UV has
focused on single species or on broad ecosystem properties such as rates of
primary productivity (Helbling et al, 1992) or biodiversity (Worrest et al, 1978).
The results of this study, and recent studies conducted under natural sunlight,
indicate that UV can also have significant effects on trophic-level interactions
between a variety of micro- and macroorganisms (Bothwell et al, 1994; Sommaruga et al, 1996).
Acknowledgements
Nanoflagellate and Synechococcus cultures were provided by David Caron and
John Waterbury of the Woods Hole Oceanographic Institute. I thank John
R.Beard of Schering-Plough HealthCare Products for excellent technical assistance, and L.Eddy, K.Overstreet, K.Pigott, K.Rhew, D.Washington and C.Webb
for laboratory assistance. The manuscript was greatly improved by the critical
reading of B.Baca, B.Libman, G.Herndl and an anonymous reviewer. This
research was begun while the author was a student in the 1993 Microbial Diversity
Course at the Marine Biological Laboratory in Woods Hole, MA. This paper is
dedicated to the memory of Richard Behmlander.
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Received on May 13, 1996; accepted on June 10, 1997
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