Journal of
Plankton Research
plankt.oxfordjournals.org
J. Plankton Res. (2015) 37(2): 388– 398. First published online February 12, 2015 doi:10.1093/plankt/fbv004
Consequences of a cyanobacteria bloom
for copepod reproduction, mortality and
sex ratio
JONNA ENGSTRÖM-ÖST1,2*, ANDREAS BRUTEMARK1,2, ANU VEHMAA2, NISHA H. MOTWANI3 AND TARJA KATAJISTO2,4
1
ARONIA COASTAL ZONE RESEARCH TEAM, NOVIA UNIVERSITY OF APPLIED SCIENCES AND ÅBO AKADEMI UNIVERSITY, FI-10600 EKENÄS, FINLAND,
3
TVÄRMINNE ZOOLOGICAL STATION, UNIVERSITY OF HELSINKI, FI-10900 HANKO, FINLAND, DEPARTMENT OF ECOLOGY, ENVIRONMENT AND PLANT SCIENCES,
4
STOCKHOLM UNIVERSITY, SE-10691 STOCKHOLM, SWEDEN AND FINNISH ENVIRONMENT INSTITUTE, MARINE RESEARCH CENTRE, FI-00251 HELSINKI, FINLAND
2
*CORRESPONDING AUTHOR: [email protected]
Received August 26, 2014; accepted January 24, 2015
Corresponding editor: Marja Koski
The aim of the study was to measure copepod reproduction, mortality and sex ratio in the field before, during and
after a cyanobacteria bloom during the summer in the western Gulf of Finland. Environment and zooplankton
samples were collected every fortnight, and the copepod Acartia spp. was incubated in the laboratory for reproductive
output, i.e. egg production and egg hatching success. Other responses monitored were female:male ratio, mortality
and body condition. In addition, molecular analyses of the nodularin-producing cyanobacterium Nodularia in Acartia
gut contents (GCs) were assessed. Egg production and body condition decreased with increasing Nodularia GCs.
During the bloom, hatching decreased as a response to Nodularia in the copepod gut. Although not related to cyanobacteria variables, male mortality was higher than female mortality, resulting in a female-biased sex ratio over most of
the summer. The study demonstrates that Acartia reproductive output is constrained by cyanobacteria blooms in the
Baltic Sea, and more generally that copepod population dynamics may be negatively affected by such blooms. This is
especially significant considering that toxin-producing blooms are predicted to increase due to warming.
KEYWORDS: body condition; cyanobacteria; copepods; fitness; gut contents; survival
I N T RO D U C T I O N
Copepods are the most important secondary producers
in the oceans and they represent the interface between
primary producers, microzooplankton and planktivores
(Mauchline, 1998). Fish larvae and other plankton
feeders, such as mysid shrimps, herring and sprat, are
highly dependent on the food resources produced during
the warm summer months in the northern hemisphere
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J. ENGSTRÖM-ÖST ET AL.
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CYANOBACTERIA AND COPEPODS
(Rudstam et al., 1992). Calanoid copepods are the main
prey for these taxa, and Acartia, the focus of the present
study, is a dominant genus in global coastal waters
(Paffenhöfer and Stearns, 1988). Calanoid copepods
respond to increasing food availability and temperature
by rapid reproduction (Viitasalo et al., 1995). However,
growth and reproduction are also dependent on food
quality, such as dietary nitrogen (Kiørboe, 1989), fatty
acids, sterols and amino acids (Ahlgren et al., 2005; Klein
Breteler et al., 2005, Rossoll et al., 2012), as well as availability of heterotrophic prey (Dutz and Peters, 2008) or
presence of cyanobacteria (Ger et al., 2014).
In eutrophic areas, cyanobacteria blooms can be
common (O’Neil et al., 2012) and nutritional quality of
the available phytoplankton community can be low for
zooplankton (Ikawa, 2004). The study area at the entrance to the Gulf of Finland is known for its heavy latesummer cyanobacteria blooms. The main bloom-formers
are diazotrophs, such as the non-MC (microcystin) producer Aphanizomenon flos-aquae, and hepatotoxic Nodularia
spumigena and Dolichospermum spp. ( previously Anabaena).
The blooms are increasing both locally (Suikkanen et al.,
2013) and globally (Paerl and Otten, 2013), and since
they are predicted to benefit from climate change in multiple ways (Paerl and Huisman, 2009), they are likely to
continue increasing in both marine, estuarine and lake
water bodies in the future (reviewed by O’Neil et al.,
2012).
The interactions between cyanobacteria and zooplankton are complex (Ger et al., 2014). Many studies have used
Daphnia as a model grazer (Wilson et al., 2006; Tillmanns
et al., 2008), as Daphnia grazes on large particles and therefore has the potential to control a bloom (Elser et al., 2000;
Sarnelle et al., 2010). Zooplankton are able to coexist with
cyanobacteria mainly via two key traits: physiological tolerance (i.e. detoxification pathways) and selective ingestion
(Ger et al., 2010 and references therein). In general, species
that avoid ingestion of cyanobacteria by selective feeding
are considered less exposed to, and therefore less tolerant
to cyanobacterial toxins (Ger et al., 2009). On the other
hand, it has been shown that copepods graze more on
cyanobacteria when alternative food is scarce (Gorokhova
and Engström-Öst, 2009; Engström-Öst et al., 2011a).
Zooplankton tend to coexist with, rather than eliminate,
cyanobacteria (Bouvy et al., 2001; Hu et al., 2006).
Although selective grazing is thought to be the dominant
mechanism enabling copepods to thrive under the toxic
conditions typical for cyanobacteria blooms, few studies
have linked food availability to copepod ingestion rates
and fitness-related traits (i.e. reproductive success, mortality, sex ratio and body condition).
On an annual time scale, Acartia spp. (dominated by
A. bifilosa) are the most abundant species of the pelagic
mesozooplankton community in the northern Baltic Sea.
The populations build up in spring and 3 peaks can be
observed before population decline in late autumn
(Viitasalo et al., 1995). Assuming that global warming will
continue according to a business-as-usual CO2 emission
scenario, the dominant zooplankton herbivore populations will be exposed to increasing frequency and duration of cyanobacteria during summer months (Ger et al.,
2014), which constitutes the most important copepod
growth period.
A recent paper shows that Acartia populations have
declined in the Gulf of Finland since the 1970s (Suikkanen
et al., 2013). Thus, possible links between zooplankton and
cyanobacteria deserve further investigation. We measured
reproductive output, in situ mortality as well as condition
factor and sex ratio. The study was conducted during one
summer, i.e. before, during and after a cyanobacteria
bloom (referred to as pre-bloom, bloom and post-bloom).
In addition, DNA of the main toxin-producing cyanobacterium in the Baltic Sea, Nodularia, was measured from
copepod gut contents (GCs). We hypothesize that copepod
survival, fitness, sex-ratio and condition are negatively
correlated with cyanobacterial dominance. The second
hypothesis is that copepod fitness and condition will be
inversely related to the amount of Nodularia DNA in their
guts.
METHOD
Sampling
Five water samples were collected during the morning
every second week, in total seven times between 6 June
and 29 August 2011 at a monitoring station (34 m deep)
in an open area Storfjärden (598500 N, 238150 E), close to
Tvärminne Zoological Station, Baltic Sea. The cyanobacteria bloom occurred at the beginning of August
(.75% of the phytoplankton consisted of filamentous
cyanobacteria; Fig. 1); June and July are considered as
pre-bloom conditions, and mid- and late August as postbloom conditions. The water samples were collected with
a 3-L Limnos sampler from 5 m depth (chlorophyll a
maximum at 5 m; Almén et al., 2014) to measure temperature, pH, salinity, chlorophyll a (hereafter Chl a),
phytoplankton biovolume and cyanobacterial toxin concentrations (intra- and extracellular, measured as microcystin equivalents). Temperature was recorded from the
thermometer mounted in the sampler. For egg production
and hatching measurements, zooplankton were collected
by vertical hauls from 25 m to the surface by 3–4 net tows
with a 200-mm net (Ø 48.5 cm) with 1-L cod-end, in
order to obtain enough animals for incubations. The
copepods perform diel vertical migration during the
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Fig. 1. Chlorophyll a (mg L21) shown as (a) box– whisker plots over
the study period. Central horizontal line of the boxes indicates the
median and the ends of the boxes represent the upper and lower
quartiles of the data. Whiskers mark maximum and minimum values.
(b) Average (n ¼ 5) phytoplankton community composition as
biovolume ( 108 mm3 L21).
summer and were therefore sampled from the whole water
column (Almén et al., 2014). All samples were added to
one 50-L container with water from 10 m depth (below
the thermocline). Five replicate net tows were collected
taken for mortality and sex ratio, respectively (in total 35
during the summer). For GC analysis, zooplankton
samples collected from 25 to 0 m were preserved randomly
in bulk using RNAlater (Ambion) and stored at 2208C
until further analysis (Gorokhova, 2005).
Environmental variables
pH was measured with a Jenway 3510 pH meter, calibrated daily according to NIST (www.nist.gov) with three
buffers ( pH 4, 7 and 10). Salinity was measured with a
salinometer in the laboratory.
Chl a concentration was measured by filtering a
known volume of water on GF/C filters (Whatman),
which were frozen until analysis. Ten millilitres of
ethanol were added, the sample was extracted for 24 h
and the absorbance measured with a spectrophotometer
(Shimadzu UV-2501 PC) (Arvola, 1981).
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Samples (50 mL) for determining phytoplankton and
protist cell density and biovolume were transferred into
plastic centrifuge tubes and preserved with acid Lugol’s
solution and stored in the cold (88C) and dark until
analysis. In order to estimate the biovolume of the dominant taxa, cells were counted in sedimentation chambers
according to Utermöhl (1958), where 10 mL samples
were allowed to sediment for at least 24 h. From each
replicate, the first 10 encountered individuals of each
species/taxa were measured for biovolume using an
ocular micrometer and calculated following approximate
stereometrical formulas provided by Olenina et al. (2006).
The samples were counted and measured using an
inverted Leitz Labovert microscope equipped with a 40
optical lens (ocular magnification 12.5). Quantification
of the picocyanobacterium Synechococcus in the field was
made according to detailed instructions in Motwani and
Gorokhova (Motwani and Gorokhova, 2013).
The intra- and extracellular microcystin concentrations
were analysed with ELISA (detection limit 0.16 mg L21),
using a microcystin plate kit (EnviroLogix, Portland, ME,
USA), according to the kit instructions. Microcystin concentrations were analysed from 100 to 300 mL samples collected on Whatman GF/C filters (intracellular toxin) using
low-vacuum pressure (,0.2 bar 20 kPa21 0.2 atm21), and
from the filtrates (extracellular toxin). Filters were put into
scintillation vials and frozen at 2208C until determination
of toxins as in Engström-Öst et al. (Engström-Öst et al.,
2011b). The frozen dissolved toxin samples (GF/C filtered
seawater) were completely thawed and properly stirred,
before analysis. A negative control and a standard curve
were measured accordingly. The toxin concentration was
determined by dual reading from the absorbance at
450 nm and as reference 655 nm with a photometer
(Tecan infinite M200), using Magellan software.
Copepod egg production and hatching, sex
ratio, mortality and condition factor
After transporting the samples to the laboratory, 150
female Acartia spp. were picked at in situ temperature
under a dissecting microscope and put into five 120-mm
false bottom chambers in 250 mL of 0.2-mm filtered
seawater (Sartorius Sartobran). Females were unfed to
obtain egg numbers produced from past resources (background). The chambers were constructed to prevent egg
cannibalism; only eggs could pass through the mesh. The
females were incubated in in situ temperature (12– 228C)
under a 16:8 h light:dark regime in climate chambers for
24 h and the eggs from each replicate were counted
under a microscope (Leica 25). Half of the eggs from
each replicate were put into petri dishes in filtered seawater to estimate egg hatching (EH) success (%), and the
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other half were dyed and frozen for egg viability measurements (data not shown). The hatching period was terminated after 2 – 4 days by adding acid Lugol’s solution
to the samples (Katajisto, 2003). Termination of hatching
varied as hatching is dependent on temperature, i.e.
slower in cold water (Katajisto, 2003).
The copepod sex ratio (females:males) samples were
emptied into dark 1-L glass bottles, preserved with 10 mL
acid Lugol’s solution (1%), and stored at 88C. The sample
was poured through a 200-mm sieve and washed with tap
water into a measuring cylinder, which was filled to
50 mL. The sample was moved to a beaker and the measuring cylinder was carefully rinsed with a known volume
of water, which was poured into the beaker. The beaker
was stirred and a 2–5 mL subsample collected. More aliquots were added if ,100 animals (Acartia spp. females
and males) were counted from the first aliquot. Sparse
samples were counted in full. Samples were counted in a
counting chamber with a stereomicroscope. Prior to
counting, a few drops of sodium thiosulphate were added
to bleach the animals darkened by the Lugol’s solution.
The Acartia abundances were corrected for subsample size,
as well as sampling depth and net diameter. Sex ratio estimates were based on microscopy counts (on average, 109
females versus 68 males counted sample21).
For mortality, mesozooplankton samples were concentrated from the cod-end on a 250-mm sieve and carefully
flushed into 100 mL dark glass bottles and immediately
stained with 0.05 mL non-toxic aniline blue stain (45 M
stock solution; 0.34 g mL21 Milli-Q water) mL21 seawater (Bickel et al., 2009). Sampling was performed as
carefully as possible to minimize mortality due to sampling. Aniline blue (Methyl blue for microscopy, Fluka)
stains dead individuals, whereas the live appear unstained, and it is commonly used in fresh- and brackish
water studies (Bickel et al., 2009). The samples were incubated in a closed cool-box in ambient temperature water
for 15 – 25 min and immediately processed after arrival
in the laboratory. Samples were concentrated on a
120-mm mesh disc, rinsed with filtered seawater to
remove excess stain and frozen at 2208C. At least 100
individuals of the most abundant group, usually live
females, were counted. Samples were counted within 2
weeks. The Acartia female (in situ) mortality percentage
was calculated as follows: the number of dead females/
total number of Acartia adults 100 (%).
To estimate copepod nitrogen content, individual live
Acartia females were collected and measured for prosome
length (Inverted Leica DM IRB, 125 magnification),
and rinsed twice in Milli-Q water and transferred to tin
cups with forceps, sterilized with ethanol following rapid
combustion. Nine or 10 individuals were added to each
of five replicate samples, and dried in 608C for at least
24 h. Nitrogen content was measured with a spectrometer (Europe Scientific, ANCA-MS 20 –20), as described
in Grasshoff (Grasshoff, 1976). Copepod condition factor
was determined as a ratio of copepod body weight as nitrogen (mg N) to the cube of prosome length (mm3)
(Durbin and Durbin, 1978).
DNA isolation and qPCR assay
Female Acartia spp. were picked from bulk zooplankton
sample under a dissecting stereo microscope with
forceps, rinsed in artificial seawater and transferred in
groups (10 –16 ind. sample21) into 1.5 mL microcentrifuge tubes containing 40 mL of 10% Instagene Chelex
(Bio-Rad). For DNA extraction, these samples were incubated for 30 min at 1058C and the resulting lysate was
centrifuged at 12 000g for 5 min. The supernatant was
used to quantify N. spumigena in Acartia spp.
The SYBR Green approach was applied to amplify a
200 bp fragment of N. spumigena 16S rDNA with a
StepOne real-time cycler (Applied Biosystems) using the
toxic Nodularia-specific primer (Moffitt et al., 2001) and
universal reverse primer 1494R (Neilan et al., 1997). GCs
of Nodularia are reported as Nodularia, similar to the phytoplankton counts. The primers used in this study have been
tested successfully to evaluate grazing on N. spumigena by
mysids (Gorokhova, 2009) and copepods (Gorokhova
and Engström-Öst, 2009; Engström-Öst et al., 2011a).
DNA from exponentially growing N. spumigena cultures
was extracted to obtain standards for the target gene
(Becker et al., 2002). The standard curves were constructed using N. spumigena DNA in a series of 10-fold
dilutions (8.4 to 8.4 1024 ng). In order to avoid false
negatives or positives, we analysed the reference samples
(i.e. contamination control or positive control) which
were prepared using freshly hatched Artemia spp. nauplii
(San Francisco Bay Brand; 10 ind. sample21) to ensure
that zooplankton DNA does not produce positive amplification, and thereafter treated similarly as the zooplankton
samples. All qPCR reactions were performed in triplicate
using the KiCqStart SYBR Green qPCR Ready Mix
(Sigma), and sterile water was employed as a negative
control for each set of qPCR reactions. We found positive
amplifications in neither the false-positive control nor in
the false-negative control. All copepod field samples were
spiked with an intermediate standard concentration
(0.084 ng) to ensure that the sample concentration could
be detected in the range of the standard curve; the calculated sample concentrations were then adjusted with the
spiked concentration (Engström-Öst et al., 2011a). The
lower detection limit of the qPCR assay is 8.4 1024 ng.
The linearity of the standard curve was R 2 . 0.99, with
amplification efficiency in the range of 95 –100%.
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Statistical analyses
Differences in copepod size-corrected egg production
rate (EPR divided by prosome length), EH success, mortality ( percentage of dead individuals; female, male,
total), adult copepod sex ratio and female condition
factor between the samplings were tested using one-way
analysis of variance (ANOVA) and pairwise comparisons
with Tukey’s honest significant difference (HSD) test. The
proportion data (EH and mortality) were arcsinetransformed, the EPR data were Box–Cox-transformed
and the sex ratio data log-transformed in order to fullfil
the assumptions of ANOVA, which were checked using
the Shapiro – Wilk normality test and the Fligner–
Killeen test of homogeneity of variances. Model fit
validations were made using residual diagnostics. The
difference in mortality between sexes was analysed using
a paired t-test. To test if bloom conditions explain the
variation in EPR, EH, mortality, sex ratio and condition
factor, they were plotted against the explanatory variables
Chl a, cyanobacteria biomass and Nodularia GC. If the
plots showed relationships between the variables, linear
or polynomial regression analyses were made, depending
on which one gave the better fit. Instead of regression
analysis, a generalized linear model (GLM) with binomial error structure and probit link function was used for
the EH proportion data. Residual diagnostics were conducted for validation of the model fit. In the GLM for
EH, residuals revealed two highly influencing values
(behind Cook’s distance contour 1). Therefore, EH was
also tested without these two values. Transformations
were made for the response variable to gain better fit for
the data and linear response. However, despite the transformations, a second-degree polynomial function gave
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the best fit for EH and condition factor data. The effect
of bloom conditions (Chl a, GC Nodularia, Anabaena þ
Aphanizomenon þ Nodularia biomass, Anabaena biomass,
Aphanizomenon biomass, Nodularia biomass) on EPR was
tested with and without the last sampling (29 August),
because the low egg production prevented an appropriate
model fit (only significant results reported; Table I). We
report the F-statistics or x 2 statistics for regression models
and GLMs, respectively, of the explanatory variable that
gave the best fit (R 2 or AIC, Akaike information criterion)
for the data. The statistical analyses were conducted
using R 3.0.2 software (R Core Team, 2013).
R E S U LT S
Conditions in the study area
The temperature increased steadily from 11.4 (6 June) to
22.28C (1 August), whereas salinity decreased from 6.3
(6 June) to 5.6 (29 August) (Table I). Chl a varied during
the summer from 1.5 (6 June, Fig. 1a) to 17.9 mg L21
(1 August), and peaked when A. flos-aquae was the dominating cyanobacterium (Fig. 1). Aphanizomenon flos-aquae
was also abundant at the beginning of July (Fig. 1b).
Although not dominant, Nodularia was present in samples
collected in July and August. The picocyanobacterium
Synechococcus sp. did not vary in abundance over the
season. Dissolved microcystin concentrations were under
detection level throughout the season, but one peak was
detected during the bloom. Intracellular microcystin
was detected during the whole season (except at the
beginning of June) with one peak at the beginning of
July and another at the beginning of August. The highest
Table I: Cyanobacteria variables (Chlorophyll a, GC) of Nodularia, Anabaena þ
Aphanizomenon þ Nodularia biovolume combined and separately) explaining egg production rates
EPR, EH success, mortality, sex ratio and condition factor
Chl a
GC Nodu
EPR (linear regression)
EH success (GLM)
Whole data: polynomial response,
binomial errors, probit link function
AIC ¼ 439.91
2
x(2,n ¼ 35) ¼ 36.19, P , 0.001
Tot. mortality
Male mortality
Female mortality
Sex ratio
Condition factor
(polynomial regression)
No relationships with bloom variables
No relationships with bloom variables
No relationships with bloom variables
No relationships with bloom variables
Whole data: Box– Cox-transformed adjusted R 2 ¼ 0.622
F1,33 ¼ 56.82, P , 0.001
Without 29 August: log-transformed adjusted R 2 ¼ 0.880
F1,28 ¼ 213.8, P , 0.001
Without two highly influencing values (one replicate from
samplings 5 and 6): polynomial response, binomial
errors, probit link function
AIC ¼ 193.87
x 2(2,n ¼ 33) ¼ 81.47, P , 0.001
Polynomial response
R 2 ¼ 0.539
F2,32 ¼ 20.91, P , 0.001
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concentration was measured during the bloom (0.28 mg
g21 C). The abundance of ciliates and phytoplankton
(Fig. 1b) followed the Chl a development quite closely,
showing two abundance peaks in June and August. The
abundance of adult Acartia varied on average between 21
and 3620 ind. m23 during the summer.
Responses by copepods to environmental
conditions
Most of the copepod response variables monitored
between June and August showed significant differences
over time (Fig. 2 and Table II). The size-corrected EPR
was highest in pre-bloom conditions at the beginning of
June and lowest in post-bloom conditions at the end
of August, whereas second lowest during the bloom (beginning of August; Fig. 2a). EH did not change significantly (Fig. 2b). Copepod condition factor (mg N mm23)
remained fairly constant in pre-bloom conditions, but
decreased during the bloom, as well as during postbloom conditions at the end of August (Fig. 2c). Male
mortality changed over time, whereas the female mortality did not (Fig. 2d). Male and female mortality differed
Fig. 2. Box–whisker plots for (a) size-corrected ( prosome) egg production rates (EPR female21 24 h21 mm21), (b) hatching success (%), (c)
condition factor (mg N mm23), (d) female and male mortality (%) and (e) sex ratio (females:males). Central horizontal line of the boxes indicates the
median and the ends of the boxes represent the upper and lower quartiles of the data. Whiskers mark maximum and minimum values. Letters
above the boxes indicate Tukey’s HSD post hoc results, e.g. AB above a box indicates that the column differs significantly from the first (A) and the
second (B) box.
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Table II: Average (min– max) of measurements
from the seven samplings performed during the
study period
Average (min –max)
EC microcystin (mg L21)
IC microcystin (mg g C21)
pH
Salinity
Temperature (8C)
Acartia spp. (ind. m23)
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Table III: Results from ANOVA analyses of
copepod variables presented in Fig. 2
SS
df
MS
F
P-value
20.866
0.172
121.3
,2e216***
Box – Cox transf
0.03 (0.00 –0.18)
0.076 (4.1 1024 –2.75 1021)
8.42 (8.18 –9.00)
5.8 (5.6 –6.3)
17.8 (11.4 –22.2)
747 (21 – 3620)
Presented averages and ranges are based on averages for five replicates
per sampling.
EC, extracellular; IC, intracellular.
Size-corrected EPR
Sampling
125.20
6
Residuals
4.82
28
Hatching successarcsin transf
Sampling
0.6572
6
Residuals
1.5374
28
Total mortalityarcsin transf
Sampling
0.08059
6
Residuals
0.19492 28
arcsin transf
Male mortality
Sampling
0.149
6
Residuals
0.284
28
Female mortalityarcsin transf
Sampling
0.0559
6
Residuals
0.2055
28
Sex ratio (females:males)log transf
Sampling
17.786
6
Residuals
2.788
28
Condition factor
Sampling
1.1883
6
Residuals
0.7022
28
0.10953
0.05491
1.995
0.1
0.013432
0.006961
1.929
0.111
0.02483
0.1014
2.448
0.0498*
0.009316
0.007340
1.269
0.303
2.9643
0.0996
0.19804
0.02508
29.77
7.897
6.44e211***
4.88e205***
produced with higher amounts of ingested cyanobacterium (Fig. 3a and Table III). When the last sampling occasion was left out (mean EPR 0.15 eggs female 24 h21),
Nodularia GC explained even more the variation in EPR.
Copepod EH showed a negative polynomial response
with Chl a concentration and Nodularia DNA GC
(Table III). The response indicates that hatching increases
slightly with increasing Chl a or GC, until a threshold
whereby further increase in Chl a or GC levels results in
lower EH. The condition factor showed a significant
negative polynomial response with Nodularia GC. It
decreases with increasing GC of Nodularia, but the decrease stabilized as a response to high GCs (Fig. 3b).
Mortality (male, female or total) and sex ratio did
not show any relationship with cyanobacteria or Chl a
variables (Table I).
DISCUSSION
GCs and reproductive output
Fig. 3. Scatterplot of (a) size-corrected egg productions rates (EPR,
eggs female21 24 h21 mm21) and (b) condition factor (mg N mm23) as
a function of GC of Nodularia (ng DW ind.21).
significantly ( paired t-test, t34 ¼ 2.87, P ¼ 0.0071) and
female/male sex ratio also showed large changes over
time, varying between 0.6 and 6.4 (Fig. 2e).
GC of Nodularia DNA explained several responses by
copepods in the field. EPR showed a strong negative
relationship with Nodularia GC, indicating fewer eggs
EPR showed a strong negative relationship with Chl a,
indicating support for our hypothesis that EPR are decreasing during bloom conditions. The current results
contrast with those from the Southern Baltic Sea where
reproductive rates were positively correlated with Chl
a concentrations (Diekmann et al., 2012). Our results
suggest that the available phytoplankton during bloom
conditions in the study area is of low quality for copepods
(cf. Suikkanen et al., 2013). In fact, the Chl a peaks were
dominated by filamentous cyanobacteria A. flos-aquae,
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suggesting that the cyanobacterium has a negative
impact on copepod reproduction. Few studies have investigated how Aphanizomenon affects copepods, but Sellner
et al. (Sellner et al., 1994) reported low grazing and poor
condition of Baltic Sea Acartia in A. flos aquae blooms.
The copepod size-corrected EPR between June and
August was strongly explained by the Nodularia GC in
female copepods, indicating that consumption of toxinproducer Nodularia had negative consequences for the
numbers of eggs produced. Even though our results
clearly show a negative effect of cyanobacteria on copepods, it is not clear if it is due to low food quality of
Nodularia or toxin, produced by N. spumigena. Nodularia spumigena is the main toxin producer in the area, since the
Baltic A. flos-aquae is a non-MC (microcystin) producer
(reviewed by Stal et al., 2003; Vahtera et al., 2010) and
Dolichospermum ( previously Anabaena) has neither been
found to produce MC in the current study area (Halinen
et al., 2007). During post-bloom conditions, when daily
EPR was between 0 and 2 eggs, seemingly also other
non-cyanobacterial late season factors affected egg production (such as food, cooling, mixing, predation;
Viitasalo et al., 1995). Further, at the end of the season, it
is possible that the females are older and thereby produce
fewer offspring (Rodrı́guez-Graña et al., 2010).
EH success showed a polynomial response with Chl a
and Nodularia GC, indicating that EH increased slightly
with low concentrations but decreased with higher concentrations of Chl a or Nodularia GC (Table I). EH was
lowest during the actual cyanobacteria bloom at the beginning of August (Chl a 18 mg L21; GC 0.74 ng DW
Nodularia ind.21). Our hypothesis suggesting that copepods would produce fewer but higher quality offspring
during bloom conditions is therefore not supported by
these results. The females’ strategy to balance offspring
number and quality when food quality is decreasing is
commonly shown in terrestrial animals (Mappes and
Koskela, 2004: bank vole; Wilson et al., 2009: Soay
sheep), but more seldom in the marine environment
(Barrett, 2014). Vehmaa et al. (Vehmaa et al., 2013) report
fewer eggs of Acartia, but increased egg viability in cyanobacteria treatments (10% biomass of toxin-producer
N. spumigena and 90% chlorophyte Brachiomonas). It is therefore possible that when the female feeds more on toxinproducing Nodularia, EH is constrained, probably due to
low digestibility of ingested food, the toxin nodularin
(Karjalainen et al., 2007) or other bioactives (Suikkanen
et al., 2006). Our result has to be interpreted with caution:
although Chl a and GC explained the variation in hatching success significantly, hatching did not show particularly
large variation over the season. Controlled laboratory
experiments are necessary to test for the detailed effects of
copepod’s consumption of cyanobacteria on EH.
Previous studies show that copepods tend to feed
on filamentous cyanobacteria, including hepatotoxic
strains, such as N. spumigena when other food is scarce
(Gorokhova and Engström-Öst, 2009; Engström-Öst
et al., 2011a). Copepods fed on Nodularia during the whole
study period, less when the biovolume was low and more
when the biovolume was high (cf. Isari et al., 2013), which
suggests that they were not able to efficiently select
against N. spumigena (Gorokhova and Engström-Öst,
2009; Engström-Öst et al., 2011a). Nodularia spumigena may
also have presented the copepods with a valuable nutrient or compound, otherwise not available (Hogfors et al.,
2013; Vehmaa et al., 2013), although note that ingestion
of high amounts of N. spumigena negatively influenced
EPR (Fig. 2b). Regardless of the argument presented
above, our field study shows that the cyanobacteria
Nodularia is consumed at moderate rates by populations of
the copepod Acartia spp. in the Baltic Sea (cf. Wannicke
et al., 2013).
Sex ratio, mortality and body condition
Acartia is the most common copepod in the Baltic Sea
and its population peaks occur in May, July and
September/October. In autumn, the population declines
as a consequence of cooling, mixing and increased predation by herring (Viitasalo et al., 1995). Katajisto et al.
(Katajisto et al., 1998) showed that the sex ratio of Acartia
is female-biased in the study area during summer. The
mostly female-biased Acartia sex ratio supports our hypothesis and conforms in general to previous results
whereby copepod sex ratios commonly show variation
and tend to depart from the expected 1:1 ratio (Gusmão
and McKinnon, 2009), and are often shown to be
female-biased (reviewed by Hirst and Kiørboe, 2002;
Kiørboe, 2006). According to Kiørboe (Kiørboe, 2006),
this sex skew is largely attributed to the higher mortality
of males. This was also observed in the current study;
male mortality was higher than female mortality during
the summer season (Fig. 2d and Table II). The average
female and male mortality we monitored (5 – 15%) in the
western Gulf of Finland are supported by findings in
other areas. Elliott and Tang (Elliott and Tang, 2011)
reported that on average, 9% of A. tonsa females and 40%
of the A. tonsa males were dead in Chesapeake Bay on the
eastern coast of the USA. Our hypothesis stating that
total mortality increases during bloom conditions was
rejected, as there were no changes over time or in response to cyanobacteria. Even though the measured sex
ratio was not explained by any of the cyanobacteria variables, it differed over time between samplings, and one
reason for higher male mortality could be the low food
quality, in general (see discussion above). Finiguerra et al.
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(Finiguerra et al., 2013) have shown that males are less tolerant to starvation and show lower survivorship during
times of food limitation. Avery et al. (Avery et al., 2008)
showed that A. hudsonica males were fewer during a
harmful dinoflagellate bloom. If males are fewer than
females due to poor feeding conditions, and the females
have low body condition (see below; Sellner et al., 1994),
it may have negative consequences for population dynamics, and cause decreasing Acartia populations, as
detected in long-term monitoring data from the Gulf of
Finland (Suikkanen et al., 2013). Other influencing
factors include predation, disease, pollution, parasites,
hypoxia, warming and turbulence, and indirectly declining body size (Hirst and Kiørboe, 2002; Avery et al., 2008;
Bickel et al., 2009; Elliott et al., 2013). Clearly, the underlying mechanism of copepod mortality during the growth
season needs to be further investigated.
Acartia condition factor (mg N mm23) showed big differences over the season, and was inversely proportional
to Nodularia GC, supporting our hypothesis that condition
factor is decreasing during bloom conditions. Thus, our
results indicate that the lower condition factor may be
caused by the ingestion of Nodularia by Acartia. As
Nodularia is an N2-fixer and rich in nitrogen (Wannicke
et al., 2012), one would assume that copepods are able to
incorporate ingested Nodularia-N into biomass. Why body
N decreases when the copepod feeds on Nodularia can be
due to poor incorporation of ingested nitrogen into
biomass (Loick-Wilde et al., 2012; Wannicke et al., 2013).
Loick-Wilde et al. (Loick-Wilde et al., 2012) showed that
copepods incorporated N. spumigena-nitrogen at rates
seven times lower than nitrogen originating from the
high-quality cryptophyte Rhodomonas salina.
CONCLUSIONS
This study demonstrates that Acartia reproductive output
may be constrained by hepatotoxic Nodularia blooms in
the Baltic Sea, which was indicated by the negative relationship between egg production and the amount of
Nodularia DNA in copepod guts. Nodularia GC also had a
strong negative effect on female condition, possibly due
to nutritional inadequacy or toxic effects. Although our
results clearly show the negative effect of cyanobacteria
on copepods, we do not know how much of this was
driven by low nutrition or due to ingestion of nodularinproducing Nodularia cells. A previous study comparing
toxin-producing and non-toxin-producing strains of
Nodularia found no difference in egg numbers of copepods
provided with the different cyanobacteria strains as food
(Koski et al., 1999). Overall mortality of the Acartia population during the summer varied between 5 and 15%.
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Males showed higher mortality than females, and the sex
ratio was female-biased almost the whole summer.
Combined, our results suggest that cyanobacteria blooms
may affect copepod population dynamics. This is especially significant considering that cyanobacteria blooms
are predicted to increase, as climate change continues
unabated. It would also be important to study how
Aphanizomenon DNA in copepod GCs affects the fitness as
Aphanizomenon is the main-bloom forming cyanobacteria
species in the Baltic Sea.
AC K N OW L E D G E M E N T S
We warmly thank Prof. Elena Gorokhova (Stockholm
University) for guidance and support. We are deeply indebted to Bettina Grönlund for valuable help in the field
and the lab. Tvärminne Zoological Station provided
board, accommodation and POC/PON analyses. The
Finnish Environment Institute (SYKE) provided lab
space and infrastructure for toxin analysis. The reviewers
are gratefully acknowledged for their valuable input on
the manuscript.
FUNDING
Funding from Academy of Finland ( projects 125251,
255566, 276947), Maj and Tor Nessling Foundation,
Norden Havgruppen, Walter and Andrée de Nottbeck
Foundation and Kone Foundation is acknowledged.
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