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Factors affecting growth and viability of natural diatom populations in the

meso-eutrophic Rimov Reservoir (Czech Republic)

Article  in  Hydrobiologia · July 2015

DOI: 10.1007/s10750-015-2417-8

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Hydrobiologia
DOI 10.1007/s10750-015-2417-8
PRIMARY RESEARCH PAPER
Factors affecting growth and viability of natural diatom
populations in the meso-eutrophic Řı́mov Reservoir
(Czech Republic)
Petr Znachor . Pavel Rychtecký .
Jiřı́ Nedoma . Veronika Visocká
Received: 5 June 2015 / Revised: 22 July 2015 / Accepted: 22 July 2015
Ó Springer International Publishing Switzerland 2015
Abstract Algal population size may vary substan-
tially over the season in response to varying growth
and loss rates strongly affected by environmental
drivers. In our study, we measured growth and
viability of Fragilaria crotonensis, a dominant diatom
of the summer phytoplankton in the small, meso-
eutrophic, dimictic freshwater Řı́mov Reservoir
(Czech Republic). Over two consecutive seasons,
Fragilaria growth and viability were assessed with
PDMPO, a specific Si-deposition tracer in diatoms,
and SYTOX Green, a membrane permeability probe,
respectively. Using multiple linear regression with
stepwise forward selection of environmental vari-
ables, diatom growth and viability were found to be
driven by different factors. Fragilaria growth was
positively affected by daily light exposure during the
24-h incubation with PDMPO and by mean river
discharge into the reservoir over the 10 days before
our measurements. Fragilaria viability, in contrast,
Handling editor: Luigi Naselli-Flores
P. Znachor (&)  P. Rychtecký  J. Nedoma  V. Visocká
Institute of Hydrobiology, Biology Centre of Czech
Academy of Sciences, v.v.i., Na Sádkách 7,
České Budějovice 37005, Czech Republic
e-mail: znachy@gmail.com
P. Znachor  P. Rychtecký  V. Visocká
Faculty of Science, University of South Bohemia,
Branišovská 31, České Budějovice 37005, Czech
Republic
declined markedly with decreased ambient silica
concentration. This indicates that while diatom growth
is tightly related to physical properties of the envi-
ronment, cell viability reflects the availability of silica,
which is essential for generating and maintaining
diatom siliceous frustules. Importantly, there was a
significant negative correlation between Fragilaria
viability and growth, suggesting that seasonal peaks of
diatom growth are coupled with low cell viability.
Keywords Diatoms  PDMPO  SYTOX Green 
Growth rate  Viability  Reservoir
Introduction
Phytoplankton represent the base of aquatic food webs
and constitute almost half of global primary produc-
tion (Geider et al., 2001). Among the phytoplankton,
diatoms are one of the most ubiquitous groups playing
a fundamental role in primary production and biogeo-
chemical cycles (Smetacek, 1999; Armbrust, 2009). In
freshwater lakes and reservoirs, diatoms tend to
dominate in spring, forming a vernal peak of biomass
(Sommer et al., 1986), and in summer or autumn (Horn
& Uhlmann, 1995; Znachor et al., 2008). Their
temporal occurrence patterns are generally attributed
to water column mixing and nutrient availability
(Reynolds, 2006). Diatom cells are encased in a finely
ornamented siliceous frustule composed of two
123

unequally sized valves, the epivalve fitting onto the
hypovalve like a lid onto its box (Round et al., 1990).
They predominantly divide vegetatively through
mitosis and cytokinesis, during which each daughter
cell inherits one maternal valve as the epivalve, while
the hypovalve is newly synthesized within the mother
cell, which results in one of the two daughter cells
decreasing in size (Round et al., 1990). Once they have
reached a critical size threshold, sexual reproduction is
required to restore maximal cell size (Chepurnov et al.,
2004; Bowler et al., 2010a, b).
Changes in phytoplankton physiological status may
have wide-ranging ecological consequences, and
therefore, knowledge of factors controlling both
growth and decline of algal populations are of crucial
importance. While the factors controlling growth and
cell division have been well-known and intensively
studied, there is little understanding of factors that
affect cell mortality (Franklin et al., 2006; Peperzak &
Brussaard, 2011). Reduction of population size has
traditionally been considered a result of grazing or
sinking (Kirchman, 1999). Recently, changes in cell
viability leading to cell death have become recognized
as an additional and very important loss process that
affects community dynamics, species succession, and
flux of energy to other trophic levels (Bidle &
Falkowski, 2004; Franklin et al., 2006). There is
increasing evidence that the whole process of cell
mortality in the phytoplankton is genetically con-
trolled in a process analogous to programmed cell
death (PCD) in metazoan organisms (Berges &
Falkowski, 1998; Bidle & Falkowski, 2004; Franklin
et al., 2006). The rate of cell death may be accelerated
by environmental stresses, such as nutrient depletion
(Berman-Frank et al., 2004), high irradiance (Berges
& Falkowski, 1998), oxidative stress (Vardi et al.,
1999), excessive salt concentrations, or cell age (Bidle
& Falkowski, 2004). Additionally, infection by viral,
bacterial or fungal pathogens (Brussaard & Riegman,
1998; Bidle & Falkowski, 2004), and allelopathic
interactions (for a review see Legrand et al., 2003;
Leflaive & Ten-Hage, 2007) are factors inducing cell
death in phytoplankton.
The physiological state of microbial cells is not
easy to determine, mainly due to the lack of clear
definition of viable and dead cells. In classical
microbiology, the concept of viable cells is associated
with the ability to divide and give rise to colonies.
Recently, various fluorescence probes coupled with
123
Hydrobiologia
epifluorescence microscopy or flow cytometry have
been successfully applied to viability assessment of
natural phytoplankton populations (Veldhuis et al.,
2001; Garvey et al., 2007; Peperzak & Brussaard,
2011; Gorokhova et al., 2012). The existing
approaches are largely based on evaluation of
metabolic activity and membrane integrity (Peperzak
& Brussaard, 2011; Gorokhova et al., 2012). Cell
membrane-permeable fluorescein diacetate (FDA) and
its derivatives, which are cleaved by intracellular
esterases into a green fluorescent product, have been
commonly applied in several viability assays that
assume that cells lacking esterase activity are dead
(Jochem, 1999; Garvey et al., 2007). Applications of
membrane-impermeant nucleic acid stains (e.g.,
SYTOX Green) are based on the capacity of viable
cells to exclude the fluorescence probe. Positively
stained cells with a compromised cell membrane
(Veldhuis et al., 2001) cannot maintain an electro-
chemical gradient and are classified as dead cells
(Vives-Rego et al., 2000). Conversely, unstained
viable cells are capable of cell division and maintain
population growth.
Traditionally, growth rates of natural phytoplank-
ton populations have been deduced from changes in
cell counts over time (Reynolds, 2006). Due to their
obligate silicon requirement, various Si-tracers have
been recently applied (Shimizu et al., 2001; Descles
et al., 2008; Annenkov et al., 2010) to serve as
proxies for diatom growth (Leblanc & Hutchins,
2005; Znachor et al., 2013). The most commonly
used Si-tracer is 2-(4-pyridyl)-5{[4-(2-dimethy-
laminoethyl-aminocarbamoyl)-methoxy] phenyl}ox-
azole (PDMPO, LysoSensorTM Yellow/Blue DND-
160 (Leblanc & Hutchins, 2005). PDMPO quickly
enters diatom cells and is co-precipitated with silica
within silica deposition vesicles, where new siliceous
frustules are formed, yielding bright green fluores-
cence after UV excitation (Shimizu et al., 2001).
Recently, PDMPO has also been employed to exam-
ine how iron availability in the northeastern subarctic
Pacific Ocean affects silicon cycling by diatom
communities (Durkin et al., 2012) and to confirm
that diatoms are the primary drivers of winter
productivity in Lake Erie (Saxton et al., 2012).
Application of the PDMPO technique allowed for
measurement of seasonal changes in Si deposition of
Fragilaria crotonensis Kitton dominating the sum-
mer diatom assemblage in the meso-eutrophic Řı́mov
Hydrobiologia
Reservoir (Czech Republic; Znachor & Nedoma,
2008; Znachor et al., 2008).
The undisputed importance of both cell growth and
death for phytoplankton population dynamics raises an
intriguing question of how both processes are related
to each other. Proportions of dead cells in algal
populations may vary considerably among various
systems and different phytoplankton communities
(Veldhuis et al., 2001; Agusti et al., 2006) in response
to environmental stressors and biotic interactions.
However, field studies addressing factors affecting
both population growth and viability are virtually
absent. Under laboratory conditions, exponentially
growing cultures have shown high cell viability, while
that viability decreased once the culture reached the
stationary phase (Lee & Rhee, 1997; Agusti &
Sanchez, 2002). However, cell viability may be
independent of the growth rate, as was demonstrated
for the diatom Ditylum brightwellii (T.West) Grunow
(Brussaard et al., 1997a).
In our field study, we applied two fluorescence
methods to estimate growth (PDMPO) and viability
(SYTOX Green) of natural populations of the domi-
nant colonial diatom F. crotonensis in the meso-
eutrophic Řı́mov Reservoir. Part of the whole dataset
(season 2011) has already been analyzed and pub-
lished (Znachor et al., 2013; Rychtecký et al., 2014);
however, our present study has a quite distinct focus.
Rychtecký et al. (2014) examined spatio-temporal
differences in cell viability of three dominant species
(cyanobacterium Aphanizomenon flos-aquae and dia-
toms Asterionella formosa, F. crotonensis). Nutrients
were revealed as a factor affecting Fragilaria viabil-
ity, while light availability was more important for
Aphanizomenon. The authors concluded that cell
viability may vary at both spatial and temporal scales,
which may have broad consequences for the function-
ing of the whole reservoir ecosystem. Using the same
experimental design, Znachor et al. (2013) employed
the PDMPO technique to estimate overall silification
rates of the whole phytoplankton assemblage and also
those of several dominant diatoms. Notably, they
showed that the intensity of PDMPO fluorescence per
cell was tightly related to growth rates of three diatom
species (Asterionella formosa Hassall, Aulacoseira
italica (Ehrenberg) Simonsen, and F. crotonensis)
calculated from changes in cell counts during the
incubation. Diatom growth was driven by daily light
exposure (DLE), pointing out the importance of both
seasonal fluctuations of day length and weather
conditions. In addition, the authors underlined the
fact that growth and loss processes in phytoplankton
may run at different rates along the longitudinal profile
of a canyon-shaped reservoir. In this study, we
compiled a robust dataset that encompassed two
consecutive seasons. Our aims were (i) to discover
whether high diatom growth is coupled with high cell
viability, (ii) to determine the most influential factors
driving seasonal changes in Fragilaria growth and
viability, and iii) to explain the effect of these
variables on diatoms’ physiological state.
Methods
Study site description and sampling
Our study was conducted in the small canyon-shaped
meso-eutrophic Řı́mov Reservoir (Czech Republic;
48°500 5600 N, 14°290 2600 E; 470 m a.s.l.; area 2.06 km2;
volume 34.5 9 106 m3; length 13.5 km; max. depth
43 m; mean depth 16.5 m; mean retention time
100 days; dimictic). It was built in 1974–1978 as a
drinking water reservoir by impounding the River
Malše, the main reservoir tributary, which accounts
for 90% of the water inflow. In 2011 and 2012 (July–
October), we took surface samples (0.5 m depth) at
1–2 week intervals from two sites characterizing lacus-
trine and transition zones of the reservoir according to
(Thornton et al., 1990; Fig. 1). Samples were taken
using a Friedinger sampler. To obtain general trends,
pooled data from both sites and seasons were used.
Field measurement, chemical, and phytoplankton
analyses
Concentration of dissolved oxygen was measured
in situ (WTW 330i Oximeter, WTW, Weilheim,
Germany). Samples for dissolved silica (DSi) and
soluble reactive phosphorus (SRP) assessments were
filtered through filters of 0.4 lm porosity (Macherey
Nagel GF-5, Macherey Nagel, Düren, Germany) in the
laboratory. DSi and SRP were determined spectropho-
tometrically according to Mackereth et al. (1989) and
Murphy & Riley (1962), respectively. Total phospho-
rus (TP) was analyzed according to Kopáček &
Hejzlar (1993). The concentration of NO3-–N was
determined following the procedure of Procházková
123

Fig. 1 A map of the canyon-shaped Řı́mov Reservoir showing
location of the sampling stations
(1959). The euphotic depth (depth of 1% surface
irradiance) was deduced from vertical profiles of the
photosynthetically active radiation (PAR) obtained by
LI-COR LI-1400 datalogger with spherical quantum
underwater sensor LI 193 SA (LI-COR, Lincoln,
USA). A submersible multiparametric probe (GRYF
XBQ4, Gryf Ltd., Havlı́čkův Brod, Czech Republic)
with temperature sensor was deployed to measure
detailed vertical temperature profiles. The mixing
depth (Zmix) was estimated, according to individual
temperature profiles, as the depth of the thermocline
defined as an onset of largest temperature decrease
123
Hydrobiologia
towards hypolimnion. The Zeu/Zmix ratio was used as a
light availability index. Incident PAR was integrated
using the Minikin QT smart sensor (Environmental
Measuring Systems Company, Brno, Czech Republic)
during the in situ experiments. Phytoplankton samples
were preserved with Lugol solution and stored in the
dark. Species were enumerated employing the Uter-
möhl method and an inverted microscope (Olympus
IX 71; Lund et al., 1958). The mean algal cell
dimensions were obtained for biovolume calculation
using the approximation of cell morphology to regular
geometric shapes (Hillebrand et al., 1999). Data on
inflow rate measured on a daily basis were obtained
from a gauging station upstream of the Řı́mov
Reservoir.
Diatom growth measurement using the PDMPO
labeling technique
To assess Fragilaria growth rates (FGRs), we used a
24-h incubation with PDMPO 2-(4-pyridyl)-5-((4-(2-
dimethylaminoethyl-aminocarbamoyl)methoxy)phenyl)
oxazole, a specific tracer for biogenic Si deposition in
diatoms (Fig. 2; Shimizu et al., 2001). PDMPO
quickly enters diatom cells and is co-deposited with
silica into the newly synthesized frustule, yielding a
bright green fluorescence (Shimizu et al., 2001).
PDMPO fluorescence has been shown to be propor-
tional to the amount of Si deposited (Leblanc &
Hutchins, 2005) as well as to the growth rates of
natural diatom populations under a wide range of
varying environmental constraints (Znachor et al.,
2013). PDMPO fluorescence intensity expressed as
relative fluorescence units (FU) was quantified by
image analysis, as described by Znachor & Nedoma
(2008). Samples from both sampling sites were
incubated in 50 ml glass bottles with PDMPO in situ
near the dam at the depth of 0.5 m, and the PDMPO
(final concentration of 0.125 lmol l-1; Molecular
Probes, Europe BV, Leiden, Netherlands) was added
at the beginning of the incubation. After incubating for
24 h, samples were transported to the laboratory
(within *30 min), and filtered through 10 lm poly-
carbonate filters (Osmonics, Poretics Corp., Liver-
more, CA, USA). Filters were rinsed with distilled
water to eliminate any unbound PDMPO, then placed
upon a drop of immersion oil on a glass slide and
sealed under a glass cover with another drop of oil
above the filter and immediately inspected by
Hydrobiologia

Fig. 2 Microscopic images

of a colonial diatom
Fragilaria crotonensis.
A Nomarski differential
contrast, B composite image
of chlorophyll
autofluorescence (red) and
specific Si-tracer in
diatoms—PDMPO (green),
C composite image of
chlorophyll
autofluorescence and
nucleic acid stain SYTOX
Green (green) in a
formaldehyde-preserved
sample. Scale bar 10 lm

epifluorescence microscopy. PDMPO fluorescence
was measured 30 min after the filter preparation using
image cytometry; *50 diatom colonies were mea-
sured per sample (magnification 2009, Znachor &
Nedoma, 2008). Using the data from 2011, it was
shown that the intensity of PDMPO fluorescence per
Fragilaria cell was tightly related to growth rates
calculated from changes in cell counts during the
incubation (Znachor et al., 2013). In our study,
PDMPO fluorescence per cell was finally converted
to FGRs according to the equation of PDMPO-growth
rate linear regression (Znachor et al., 2013).
SYTOX Green viability assessment
We used SYTOX Green dye (S-77020; Molecular
Probes, Leiden, The Netherlands) to examine cell
viability. The dye is a membrane-impermeable
nuclear stain that penetrates cells with compromised
plasma membranes (non-viable cells) but leaves living
cells unstained (Fig. 2; Roth et al., 1997). Immediately
after transportation to the laboratory, fresh samples
were divided into two subsamples (volume 15 ml);
one subsample remained unamended (live) while the
other was treated with formaldehyde at a final
concentration of 4% for 30 min. The formaldehyde-
treated sample was used as a positive control, since
formaldehyde is known to permeabilize cell mem-
branes (Shapiro, 2003; Peperzak & Brussaard, 2011).
All subsamples were incubated for 15 min in the dark
at a final SYTOX concentration of 1 lM. Both
incubation time and dye concentration were optimized
in preliminary experiments (data not shown). After the
incubation, samples were filtered through polycarbon-
ate filters with 10 lm porosity (Osmonics, Poretics
Corp.), then rinsed with 2 9 5 ml of distilled water
and subsequently placed upon a drop of immersion oil
on a slide and sealed under a glass cover with another
drop of oil above the filter. Emitted fluorescence
(excitation/emission: 448/520–540 nm) was mea-
sured by image cytometry (Znachor & Nedoma,
2008). To assess cell viability, we used the membrane
integrity index (IMI) modified from Peperzak &
Brussaard (2011) and Machado & Soares (2012) and
calculated according to Rychtecký et al. (2014) as

123
 Hydrobiologia

IMI ¼ 1  ðFa =Fmax Þ
where Fa is the mean fluorescence intensity of cells
untreated with formaldehyde and Fmax is the mean
fluorescence intensity of formaldehyde-killed cells
(permeabilized membranes, positive control, Peperzak
& Brussaard, 2011). A higher value of this index
indicates higher viability of Fragilaria cells. At least
fifty colonies were counted to obtain mean values of
fluorescence intensity.
Statistical analysis
All samples were measured in duplicates. A multiple
linear regression with stepwise forward selection
(SigmaPlot, Systat Software, Inc., Chicago, IL, USA)
was made to relate Fragilaria growth and viability to
the environmental variables measured (for the list, see
Table 1), accepted as significant when P \ 0.05. A
simple linear correlation was employed to relate diatom
growth and viability and to test the explanatory power
of river discharge on Fragilaria growth (Prism 5,
GraphPad Software Inc., La Jolla, USA).
ð1Þ both years the sampling periods were characterized by
low precipitation, resulting in inflow rates very similar
to the long-term average of 2.9 m3 s-1 (Table 1).
Thermal stratification was fully developed over both
sampling periods. High photosynthetic rates of the
phytoplankton in summer resulted in high pH values
(average: 9.8, range: 6.3–12.1) and oxygen oversatu-
ration (up to 170%). Dissolved nutrient concentrations
(Table 1) varied markedly over the study and corre-
sponded to the meso-eutrophic state of the reservoir, as
well as to the TP (average: 21.6 lg l-1, range:
10.4–94.4 lg l-1) and chlorophyll a (average:
28.3 lg l-1, range: 0.41–140 lg l-1) concentrations.
Phytoplankton biovolume (average: 15.4 mm3 l-1,
range: 0.11–84.2 mm3 l-1) peaked in early September
and decreased with the progress of autumn. The
colonial diatom F. crotonensis was the dominant
component of the phytoplankton, contributing an
average of *30% (range: 0.1–76%) to the overall
biomass. A large set of 42 measurements of environ-
mental variables, listed in Table 1, was used for
statistical analysis of the factors affecting the growth
and viability of the diatom.

Results Factors affecting growth and viability

of Fragilaria crotonensis
Season characteristics
There was a significant negative correlation between
For two consecutive seasons (2011 and 2012, July– Fragilaria viability and growth (r2 = 0.41,
October) we measured the growth and viability of a P \ 0.001; Fig. 3), suggesting that seasonal peaks of
common freshwater diatom Fragilaria crotonensis. In diatom growth are coupled with low cell viability.
Using multiple linear regression with stepwise for-
ward selection of environmental variables, diatom
Table 1 Mean values with ranges of the parameters used as growth and viability were found to be driven by
explanatory variables for diatom growth and viability in mul-
tiple linear regression (pooled data from 2011–2012) different factors. For Fragilaria viability, the concen-
tration of dissolved silica (DSi; F = 67, P \ 0.001)
Mean Range
and the concentration of nitrate nitrogen N (NO3-N;
Temperature (°C) 19.9 6.8–24.6 F = 5.96, P \ 0.05) were selected as the significant
Discharge Q(1–10 days) (m3 s-1)a 2.9 1.5–6.5 predictors. The resulting regression model was
Daily light exposure (mol m-2 day-1) 9.2 1.6–15.7 Viability ¼ 0:151  DSi  0:293  NO3 -N
Dissolved oxygen (mg l-1) 9.9 6.6–14.7
þ 0:0744 ðr 2 ¼ 0:66Þ:
Mixing depth Zmix (m) 4.5 2.0–15
Light availability index Zeu/Zmix 0.9 0.3–1.8 For FGR, the average inflow rate measured 10 days
Soluble reactive phosphorus SRP (lg l-1) 6.0 2.7–15.0 before the sampling date (Q(1–10 days); F = 20.6,
Dissolved silica DSi (mg l-1) 3.24 0.23–5.83 P \ 0.001) and the DLE during the sampling date
Nitrate nitrogen NO3--N (mg l-1) 0.47 0.06–1.01 (i.e., during the incubation with PDMPO; F = 16.9,
a
Average discharge in the river inflow 10 days before P \ 0.001) were selected by the model. The best-fit
sampling and measurement regression model was

123
Hydrobiologia
Fig. 3 Relationship between growth and index of membrane
integrity (IMI) of Fragilaria crotonensis. Increasing values of
the membrane integrity index indicate increasing cell viability
FGR ¼ 1:26  Qð110 daysÞ
þ 0:311  DLE  1:52 ðr 2 ¼ 0:52Þ:
The simple linear relationships between viability
and ambient silica (r2 = 0.60, P \ 0.001) and nitro-
gen (r2 = 0.02, n.s.) are shown in Fig. 4. Fragilaria
growth was positively affected by DLE during the
24-h incubation with PDMPO (r2 = 0.52, P \ 0.001;
Fig. 5A) and mean daily inflow rate preceding our
measurements by 10 days (r2 = 0.34, P \ 0.001;
Fig. 5B). Since the gauging station upstream of the
reservoir provides daily data on the river inflow, we
tested the explanatory power of the average inflow rate
from 1 to 30 days preceding each measurement. No
significant correlation was found with the inflow rate
on the day when PDMPO experiments were con-
ducted, but when preceding average inflow rates were
used, the strength of the correlation increased to reach
a maximum at 10 days prior to diatom growth
measurement (Fig. 6).
Discussion
Due to the high year-to-year variations in measured
parameters, we used pooled data from two seasons to
obtain a robust dataset allowing identification of the
most influential factors affecting viability and growth
of the diatom species recurrently dominating the
phytoplankton in the reservoir. We did not address
differences in measured parameters between sampling
sites since spatio-temporal heterogeneity along the
reservoir has been analyzed elsewhere (Znachor et al.,
2013, Rychtecký et al., 2014). The most interesting yet
controversial finding in this study is the close and
statistically significant inverse relationship between
growth rate and viability of F. crotonensis. We
consider it appropriate to discuss a possibility of
potential methodological limits in either PDMPO or
the SYTOX Green technique.
Applicability of fluorescence techniques in field
studies
PDMPO fluorescence has been shown to be propor-
tional to the amount of Si deposited (Leblanc &
Hutchins, 2005) and thus provides a useful indirect
measure of the rate of recruitment of new cells.
Previous measurements of Fragilaria PDMPO fluo-
rescence in the vertically separated populations
implied that diatom growth rates attained at the
euphotic depth are markedly lower than those at the
surface (Znachor & Nedoma, 2008). Additionally,
significantly higher numbers of Fragilaria cells per
colony and cell width at surface also provide an
indirect indication of higher growth rates maintained
in a light-saturated environment (Znachor &
Nedoma, 2008). Using the data from the surface
and at euphotic depth, Znachor et al. (2013) per-
formed a simple linear regression model on Fragi-
laria that explained 75% of the variability in the
relationship of PDMPO per cell fluorescence versus
growth rates. Both PDMPO fluorescence and growth
rates were always lowest at euphotic depth but higher
variation was observed at the surface (Znachor et al.,
2013), suggesting that factors other than growth rates
may affect silification rates. Nevertheless, consider-
ing a wide range of diatom growth rates under varying
environmental constraints, PDMPO technique can be
used as a proxy for their growth (Znachor et al.,
2013).
Numerous studies have shown that SYTOX Green
is an appropriate tool for studying phytoplankton
viability (Veldhuis et al., 1997; Peperzak & Brussaard,
2011). However, it has been pointed out that in natural
phytoplankton populations, there might be a contin-
uum from brightly stained to unstained cells, preclud-
ing a trustworthy classification of a cell as either
SYTOX positive (dead) or SYTOX negative (viable)
123
 Hydrobiologia

Fig. 4 Relationship
between index of membrane
integrity (IMI) of Fragilaria
crotonensis and
concentration dissolved Si
and NO3-N. Increasing
values of the membrane
integrity index indicate
increasing cell viability

Fig. 5 Relationship
between growth rate of
Fragilaria crotonensis and
daily light exposure (DLE)
(A) and mean daily flow rate
into the reservoir in the
10 days before our
measurements (B)

(Veldhuis et al., 2001; Agusti & Sanchez, 2002). To
overcome these problems, we introduced the mem-
brane integrity index, which includes a measurement
of the fluorescence intensity in a formaldehyde-killed
positive control. To validate the relationship between
the index and viability, cell suspensions of intact- and
formaldehyde-killed cells were combined in different
proportions and stained with SYTOX green (see
Rychtecký et al., 2014). For the cultivated Fragilaria
strain, the membrane integrity index was closely
correlated to the proportion of dead cells added to a
suspension of live cells (r2 = 0.60, P \ 0.001; for
details see Rychtecký et al., 2014). Therefore, we
assume that the index can be used as a measure of cell
viability, as suggested by Peperzak & Brussaard
(2011) and Gorokhova et al. (2012).
Diatom growth and viability
It is difficult to admit that high growth rates might be
coupled with low cell viability. Nevertheless, there are
studies reporting similar phenomena and suggesting
their ecological implications. Using a culture of a
marine diatom Chaetoceros calcitrans Paulsen, (Veld-
huis et al., 2001) observed a high percentage of cells
with damaged membranes coinciding with active cell
division in the same population, which might possibly
act as the starting population under favorable condi-
tions. While the importance of the simultaneous
presence of actively growing and non-viable cells in
populations of certain species for fitness of its own
population is obscure, loss of cell membrane integrity
accelerates the efflux of small metabolites into water,

123
Hydrobiologia
Fig. 6 Testing of the explanatory power of flow rates supplying
the Řı́mov Reservoir on growth rates of Fragilaria crotonensis.
Linear correlation was used on flow-rate-averaged periods of
1–30 days preceding the measurement of diatom growth. The
highest correlation coefficient was found for an average value of
10 days used in a regression model
and thus, may have significant ecological implications
for the functioning of the microbial loop (Brussaard
et al., 1995; Orellana et al., 2013). It has been reported
that bacterial carbon demand was supplied by cell
lysis, accounting for termination of a Phaeocystis
bloom in the North Sea (Brussaard et al., 1995). A
more complex interrelationship between a green alga
Dunaliella salina (Dunal) Teodoresco and bacterium
Halobacterium salinarum Elazari-Volcani has been
recently described (Orellana et al., 2013). Similarly to
our study, high growth rates of Dunaliella were
observed, even though a significant portion of the
Dunaliella population showed low viability and
underwent PCD at night following daytime growth.
PCD in Dunaliella resulted in release of cell con-
stituents, such as glycerol, which could have been used
not only by other still-living Dunaliella but also by
concurrent bacteria. In addition, Halobacterium re-
mineralized the dissolved material, releasing nutrients
and promoting algal growth (Orellana et al., 2013). In
the Řı́mov Reservoir, Fragilaria viability was driven
mainly by concentrations of dissolved Si. Nonetheless,
it is rather unlikely that the possible mechanism
affecting changes in Fragilaria viability would be
potential acquisition of Si via its dissolution from
frustules of non-viable diatom cells, as observed in
marine systems (for a review see Ragueneau et al.,
2000). Moreover, it is also difficult to see how
nutritional benefit could be restricted to conspecifics
in a diverse planktonic environment.
The inverse relationship between Fragilaria
growth and viability raises intriguing questions about
possible mechanisms that may explain that apparent
paradox. In our opinion, there are two principle ways
to explain why both processes were tightly correlated:
(1) Fragilaria growth is inherently associated with
temporary loss of membrane integrity, thus mimicking
decrease in viability measured using the membrane
permeability probe SYTOX Green; and (2) Fragilaria
growth and loss of viability are independent processes
but are controlled by similar or correlated environ-
mental factors. As for the first possibility, since
diatoms appear to undergo cell division in a specific
way, differing from that of plant or animal cells (De
Martino et al., 2009; Bowler et al., 2010a; Huysman
et al., 2014), we may speculate that in certain phases of
the cell cycle membrane integrity may be temporarily
lowered, which allows SYTOX Green to enter the cell.
If true, application of the SYTOX Green technique in
diatoms must have been interpreted cautiously. How-
ever, no solid evidence supporting or disproving this
speculation could be found in the literature; only two
clues have been found. Testing various techniques for
evaluation of cell viability in the ballast water, (Reavie
et al., 2010) occasionally observed motile (viable)
pennate diatoms with SYTOX Green-stained nuclei.
Another indication that positive staining with mem-
brane-impermeable stains does not necessarily mean
loss of cell viability is available from a study of yeasts.
It has been shown for Saccharomyces cerevisiae
Hansen that, despite stress-induced loss of membrane
permeability (tested with propidium iodide), some
cells were able to restore their membranes and remain
viable (Davey & Hexley, 2011). Irrespective of stress
conditions, a small portion of cells exhibited tempo-
rary membrane permeability, which may indicate cell-
cycle dependence, as is also possible for Fragilaria.
The extent to which phytoplankton populations may
recover from partial loss of membrane permeability is
unclear. Field measurement did not allow for testing
the above hypothesis, but we highlight it here as a
possibility for future investigation using, for example,
synchronized diatom cultures under well-defined
laboratory conditions.
The fact that Fragilaria growth and viability were
correlated in our dataset with different environmental
variables is consistent with the other aforementioned
possibility: that growth and loss of viability may be
two independent processes. Cell viability of F.
123

crotonensis reflected the availability of silica, which is
essential for generating and maintaining diatom
siliceous frustules. There is growing evidence that
cell viability in phytoplankton may be markedly
reduced by environmental stresses, such as nutrient
depletion (Brussaard et al., 1997b; Berman-Frank
et al., 2004). Diatom response to decreasing Si
concentration appears to be species-specific. Using
SYTOX Green staining, cell viability was evaluated in
three marine diatom species in response to various
silicate concentrations, and only the viability of
Thalassiosira antarctica Comber decreased with Si,
while no effect was observed for Chaetoceros brevis
Schütt and Ch. calcitrans (Timmermans et al., 2007).
In our study, the lowest ambient Si concentration
dropped to 0.23 mg l-1, which obviously posed
nutrient stress and reduced the viability of Fragilaria
cells. In marine systems, diatom blooms are often
followed by an abrupt decrease in ambient Si concen-
trations (Bowler et al., 2010b), which may in turn lead
to accelerated cell death and bloom termination
(Agusti & Sanchez, 2002; Timmermans et al., 2007).
This may also occur in fresh water, since a negative
correlation between diatom abundance and ambient Si
concentrations has been observed in the Řı́mov
Reservoir in years when overall diatom biomass was
ten-fold higher than in this study (Znachor, unpub-
lished results).
Physical factors affecting diatom growth
FGRs were tightly related to physical properties of the
environment, specifically DLE and inflow rates. These
findings clearly show that changes in actual irradiance
have an immediate effect on diatom growth, which is
in addition also determined by environmental factors
in the period preceding our in situ measurement. The
significant positive correlation of Fragilaria growth
with contemporaneous light conditions indicated that
seasonal dynamics of the diatom reflected both
variations in the day length over the season as well
as actual weather conditions. Under cloudy conditions,
Fragilaria growth was much lower than under clear
skies, similarly as observed in 2011 for two diatom
species (Asterionella formosa, F. crotonensis; Zna-
chor et al., 2013).
River inflows represent one of the major forcings of
ecosystem function in canyon-shaped reservoirs
(Thornton et al., 1990). Phytoplankton seasonal
123
Hydrobiologia
dynamics in a reservoir have a close relationship with
hydrodynamic changes in particular—the inflow
regimes and subsequent mixing processes that dis-
tribute inflow nutrients (Šimek et al., 2011). It has
been demonstrated that hydrological differences are
sufficient to account for interannual variation in spring
diatom crops in Ford Lake (Michigan, USA, Ferris &
Lehman, 2007). Using empirical modeling, Fornarelli
et al. (2013) found that river inflow exerts a strong
control over diatom seasonal dynamics in Fitzroy Falls
Reservoir. High inflow episodes produce turbulences
that allow diatoms to remain entrained in the upper
illuminated surface layers. In addition, river inflow
carrying large nutrient concentrations may fuel phy-
toplankton growth (Rychtecký & Znachor, 2011). In
our study, diatom growth was positively affected by
high inflow rates preceding actual measurements by
10 days. The delay in diatom biological response
corresponds to water transport times, reflecting both
long-term averaged inflow rates and the morphology
of the reservoir (Hejzlar et al., 1993; Vyhnálek et al.,
1994, Hejzlar, unpublished data).
If growth and changes in viability of Fragilaria are
two independent processes controlled by different
environmental drivers, how then to explain their
statistical coupling, and why is the correlation nega-
tive? The reason may lie in mutual correlation of the
environmental variables measured. We suggest that
high DLE, on one hand, stimulates Fragilaria growth
but, on the other hand, increased diatom growth causes
nutrient exhaustion and in turn nutrient stress, leading
to decreasing viability. In the absence of significant
cell removal by sedimentation or grazing, the ultimate
effect on population size depends upon the interplay
between environmental constrains driving the phyto-
plankton physiological state, which may be highly
dynamic, as demonstrated by a discovery of diel
patterns in growth and viability of marine phytoplank-
ton (Llabres et al., 2011).
In summary, using a large set of environmental
variables from two consecutive seasons, we showed
that growth of the dominant diatom in the meso-
eutrophic freshwater Řı́mov Reservoir was tightly
related to physical properties of the environment,
while cell viability reflected the availability of silica. It
also provided evidence that peaks of population
growth may be coupled with low cell viability;
however, a better understanding of that paradox awaits
future work.
Hydrobiologia
Acknowledgements We are grateful to all the people who
participated in field sampling and laboratory analyses, namely J.
Vı́tková, V. Hejzlarová, J. Kroupová, and J. Šindelářová. This
study was supported by the Czech Science Foundation under
research grants P504/11/2177 and P504/11/2182, and minor
support was provided by the Grant Agency of the University of
South Bohemia (Project 142/2010/P). English correction was
made by Anton Baer.
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