23
Aquatic Ecology 37: 23–35, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Effects of unicellular and colonial forms of toxic Microcystis aeruginosa
from laboratory cultures and natural populations on tropical cladocerans
Aloysio da Silva Ferrão-Filho* and Sandra Maria F. O. Azevedo
Instituto de Biofísica Carlos Chagas Filho, CCS, Bloco G, Cidade Universitária, Ilha do Fundão, 21949-900,
RJ, Brazil; *Author for correspondence (e-mail: [email protected])
Received 30 October 2001; accepted in revised form 27 June 2002
Key words: Feeding inhibition, Growth bioassay, Life-table, Microcystins, Toxic cyanobacteria, Tropical zooplankton
Abstract
Three life-table experiments, two growth experiments and one feeding inhibition experiment, were performed to
study the effects of the toxic cyanobacterium Microcystis aeruginosa on the cladocerans of a tropical lagoon
(Jacarepaguá Lagoon, Rio de Janeiro, Brazil). Different experimental designs were used to estimate toxic effects
of both field samples and laboratory cultures of Microcystis aeruginosa on cladoceran life history parameters and
juvenile growth rates. Effects of nutritional deficiency could be distinguished from toxic effects in experiments
where green algae in high carbon concentration were mixed with Microcystis. Our results show that natural assemblages of Microcystis caused much less pronounced toxic effects than laboratory cultures and that unicellular
forms were more toxic than colonial forms, even though both contained high concentrations of toxins. One possible explanation is that colonies were too large to be ingested by the small Moina micrura and Ceriodaphnia
cornuta. Feeding inhibition by single cells and small colonies seems to be another mechanism that contributes to
the harmful effects of Microcystis on cladocerans, both in the laboratory and in the field. Thus, caution is needed
in extrapolating results from the laboratory to the field. We did find, however, that toxic algae in natural seston
can inhibit growth and reproduction of native cladocerans populations.
Introduction
Many laboratory studies have examined cyanobacteria-zooplankton interactions in the last two decades,
mostly focusing on the effects of cultured strains of
these prokaryotes on cladocerans, especially from the
temperate zone (Lampert 1981, 1982; Nizan et al.
1986; DeMott et al. 1991; Smith and Gilbert 1995).
However, only a few studies have dealt with the deleterious effects of cyanobacteria on tropical zooplankton, even though cyanobacterial blooms are often long lasting in tropical lakes (Ferrão-Filho et al.
2000; Nandini 2000).
Several studies have shown that the toxicity of cyanobacteria is the main factor that decreases the survival and reproduction rates of zooplankton (Fulton
and Paerl 1987a; DeMott and Moxter 1991; DeMott
et al. 1991; Kirk and Gilbert 1992; Jungmann and
Benndorf 1994), although nutritional deficiency may
also cause similar responses (DeMott and Müler-Navarra 1997). Several complicating factors make the
study of cyanobacteria-zooplankton interaction challenging. First, toxic effects are difficult to separate
from effects of nutritional deficiency (DeMott and
Dhawale 1995). Characteristics of cyanobacteria
other than toxicity can affect zooplankton. For example, colonies and filaments and some deterrent compounds released by cyanobacteria can interfere with
feeding rates, leading to low ingestion of carbon and
starvation (Fulton and Paerl 1987b; Haney et al.
1995). The inherent low nutritive value of many cyanobacteria is another matter of controversy, since
previous studies have shown that some non-toxic species are poor food (DeBernardi and Giussani 1990)
while others can be a complementary resource in
mixed diets (DeMott 1998).
24
In addition, different toxic compounds can be involved and exert synergistic effects on zooplankton
(Jungmann and Benndorf 1994). The toxic peptides
known as microcystins are considered the main cyanobacterial toxins involved in poisoning of animals
and man (Carmichael 1992; Jochimsen et al. 1998)
although other toxins have been recently isolated
which are more toxic to Daphnia than microcystins
(Jungmann 1995). Also, feeding inhibition can be
caused either by toxic and non-toxic strains while
acute toxic effects are primarily caused by toxic
strains (Rohrlack et al. 1999). DeMott (1999) has also
tried to separate effects of feeding inhibition (i.e. reduced ingestion) from more direct poisoning effects
of Microcystis and showed that both effects were important for some daphnid species.
Studies on cyanobacteria-zooplankton interactions
have failed to demonstrate toxic effects in the field
(Haney 1987). The high complexity of the natural environment may be one of the reasons. For example,
patchy distribution can provide refuges for zooplankton from the deleterious effects of cyanobacterial
blooms (Haney 1987). Also, complex assemblages of
algae can support good rates of growth and reproduction for zooplankton, even in environments dominated by cyanobacteria (Fulton and Jones 1991).
Some selective filter feeders, like copepods, can discriminate between toxic and non-toxic algae and are
therefore less susceptible to toxic cyanobacteria (DeMott and Moxter 1991). Also, zooplankton communities dominated by small cladocerans may benefit
from the fact that they are not able to ingest large
colonies or filaments and may coexist with toxic cyanobacteria (Fulton and Paerl 1987a; DeMott 1989;
Kirk and Gilbert 1992).
In this study we compare the effects of unicellular
and colonial forms of Microcystis aeruginosa from
both laboratory cultures and natural assemblages on
growth and reproduction of two small tropical cladocerans. Our results support the generalization based
on the temperate cladoceran species and also help explain the difficulties in extrapolating the laboratory
experiments to field conditions.
Methods
Study site, seston samplings and analyses
Jacarepaguá Lagoon is located in the South coast of
Rio de Janeiro State, in the metropolitan zone of Rio
de Janeiro City (Figure 1). It is an oligohaline, shallow (maximum depth: about 1.5 m), hypereutrophic
lagoon with recurrent cyanobacterial blooms, usually
dominated by Microcystis aeruginosa Kützing. The
only cladoceran species that occur in this lagoon are
Ceriodaphnia cornuta Sars and Moina micrura Kurs
(Ferrão-Filho et al. 2002).
During the experiments, seston samples were
taken from the lagoon for phytoplankton counting and
analyses of particulate organic carbon (POC) and microcystins. For phytoplankton analysis, subsurface
water was sampled with a 200 ml glass bottle and
fixed with acetic-lugol solution. For POC analysis,
100–250 ml of water was filtered onto pre-ignited
glass-fiber filters (Sartorius AG 37070, Goettingen,
Germany) and carbon determined according to Strickland and Parsons (1972). Other phytoplankton samples were taken from the lagoon using a 25 ␮m-mesh
net in horizontal hauls and passing the concentrate
through a 200 ␮m sieve to remove large zooplankton. Microcystins were analyzed from seston samples
(2 l) filtered onto glass-fiber filters and from freezedried cells of Microcystis from laboratory cultures
and natural populations, following an extraction
method adapted from Krishnamurthy et al. (1986) and
quantification by High Performance Liquid
Chromatography (HPLC)/diode array technique (see
Ferrão-Filho et al. (2000) for details). Microcystin
content in each sample was expressed in mg g −1 DW
and nominal toxin concentrations in each experimental treatment were calculated based on the carbon
concentration used in the experiments, considering a
carbon/dry weight ratio of 0.5 and expressed in ␮g
microcystins l −1.
Cultures of experimental organisms
All algae and cyanobacteria strains were cultured in
500 ml aerated batch cultures in a temperature-controlled chamber at 23 ± 1 °C, with a light intensity of
40 ␮E m −2 s −1 and a 12:12 h light:dark cycle. The
chlorophytes Ankistrodesmus falcatus (strain
NPIN-1) and Chlamydomonas reinhardtii (strain
UTEX90) were used as food for cladocerans and were
cultured in MBL medium (Stemberger 1981). The Microcystis strains NPLJ-2 and NPLJ-42 used in toxicity experiments were cultured in ASM-1 medium
(Gorham et al. 1964). Strain NPLJ-2 is unicellular,
with cell size of about 5 ␮m, whereas strain NPLJ-42
is colonial, with colonies ranging from 40 to 190 ␮m
(102.5 ± 33.8 ␮m; n = 40) in Greatest Axial Linear
25
Figure 1. Location of Jacarepaguá Lagoon in the State of Rio de Janeiro (Brazil).
Dimension (GALD). Both strains were isolated from
Jacarepaguá Lagoon. These cultures were used between the first and second week after inoculation, during the log phase of growth.
The two cladocerans species, C. cornuta and M.
micrura, both isolated from the study site, were cul-
tured in filtered-autoclaved lagoon water for several
generations prior to the experiments. C. cornuta was
fed Chlamydomonas and M. micrura was fed Ankistrodesmus, both at a concentration of 1.0 mg C l −1.
Cladocerans were kept at the same temperature as algal cultures, although under dim light.
26
Table 1. Summary of all experiments carried out in this study. NPLJ-2 and NPLJ-42 are the Microcystis strains cultured at the laboratory.
Number
Experiment Sample/Strain
Size of cells or colonies
Food concentration
(mg C l −1)
Microcystis/Seston
conc. (mg C l −1)
1
Life-table
Natural
2
3
Life-table
Life-table
0.0 – 1.0
9.5
1.0
0.0 – 1.0
0.0 – 1.0
1.2
0.0 – 1.5
0.0 – 1.0
4
5
6
Growth
Growth
Feeding
Natural
NPLJ-2
NPLJ-42
Natural
Natural
Natural
Colonies: 20–130 ␮m
Whole lagoon water
Single cells and small colonies: 5–20 ␮m
Unicellular: 5 ␮m
Colonial: 40–190 ␮m
Colonies: 20–130 ␮m
Single cells and small colonies: 5–20 ␮m
Single cells and small colonies: 5–20 ␮m
0.0 – 1.0
1.0
1.0
0.0 – 1.0
0.0 – 1.5
0.0 – 0.5
Life-table experiments
Life-table experiments were performed to test the effects of both cultured and naturally occurring Microcystis on the reproduction of cladocerans (Table 1).
In these experiments, 10–20 newborns (<24 h old) of
each cladoceran species were placed individually in
flat-bottom glass tubes containing 30 ml of each treatment. The surviving animals were transferred daily to
new food suspensions and checked for the appearance
of eggs and newborns. The experiments lasted until
animals in the control group reached its fourth clutch.
We estimated the age at first reproduction (primiparous), mean clutch size and total offspring produced
during the experiment. The survivorship and fecundity data were used also to estimate the population
growth rate (life-table r) using a bootstrap technique
(Taberner et al. 1993), with 500 replicates per run and
bias adjusted correction for small cohorts (Meyer et
al. 1986).
Statistical analysis of life-table parameters was
conducted by one-way ANOVA and significant differences among treatments were tested by multiple comparison Tukey’s test. Differences between the little r
values in all treatments were statistically tested using
two-sample t-tests.
Life-table experiments were performed with a variety of natural samples and cultured Microcystis
strains (Table 1). In the first experiment, we used
treatments with Microcystis colonies concentrated
with 25 ␮m plankton net in the lagoon. In the laboratory, these samples were centrifuged at 4000 g × 10
min., and the buoyant colonies staying in suspension
(supernatant) were separated from other sestonic material. When analyzed under a microscope, the supernatant was comprised almost only of large Microcystis colonies, varying from 20 to 130 ␮m (56.2 ± 29.3
␮m; n = 30) in GALD and containing a lot of gas
vacuoles. These colonies were mixed in different concentrations (0.0 to 1.0 mg C l −1) with the green alga
used in the control (A. falcatus), making up a final
concentration of 1.0 mg C l −1. This design was aimed
to test for the effects of an increasing proportion (0–
100%) of toxic cells in the diet, while the total
amount of food remained constant. During this experiment, we also used whole water samples from the
lagoon, passed through a 200 ␮m sieve to remove
large zooplankton. In this case, the seston was used
in its original concentration or diluted with filtered
lagoon water to 25 and 50%, and no extra food was
added to these treatments. The treatments with whole
lagoon water were used to estimate the population
growth rates with natural food (seston). However,
these treatments were not included in the statistical
analysis since they cannot be compared with controls
with green alga.
The second life-table experiment was performed
also with a natural sample collected with the plankton net (25 ␮m). The sample was centrifuged at 4000
g × 10 min., the supernatant with large colonies was
disregarded, and the settled material was used in the
experiments. This sestonic material comprised unicellular, small colonies of Microcystis (5–20 ␮m) and a
small proportion of algae and detritus. Different concentrations (0.0 to 1.5 mg C l −1) of this fraction were
mixed with a fixed amount (1.0 mg C l −1) of the same
green alga (A. falcatus) used as food in the controls.
Although total food concentration increased from 1.0
to 2.5 mg C l −1, this design showed to be more appropriate to separate confounding effects of nutritional deficiency and toxicity than the previous one,
since the increase in the gradient of toxic cells was
accompanied by a constant concentration of highly
nutritional food (see discussion).
27
In the third life-table experiment, two toxic Microcystis strains cultured in the laboratory were used:
NPLJ-2 (unicellular), and NPLJ-42 (colonial) (see
strains characteristics above). Both strains were used
in mixtures with the green alga A. falcatus in different proportions (0–50%), making up a total concentration of 1.0 mg C l −1. Toxicity of both strains was
confirmed in preliminary acute tests using Daphnia
similis and controls with starved animals, following
Lampert (1981). Except in the treatment with whole
lagoon water, all experiments were run in filtered and
autoclaved lagoon water.
Juvenile growth rates
Simultaneous with the first two life-table experiments, juvenile growth rates of M. micrura were estimated in two growth bioassays, using the same natural samples and treatments as in the life-table
experiments. Initially, we placed 50 newborns (< 24
h) in each of the three replicate 500 ml bottles with
either whole lagoon water or the different food mixtures. At time 0 and after 2 days and 4 days, a group
of 5 to 10 animals were taken from each of the replicate bottles and placed in pre-tarred aluminum foil
containers and dried overnight at 60 °C. These containers were then weighed in a microbalance (Mettler
Toledo UMT-2) to the nearest 0.1 ␮g. Growth rates
were calculated using the equation:
g ⫽ 关ln共M t兲 ⫺ ln共M o兲兴/t,
where M o and M t are mean individual mass initially
and after t days. Since Moina is a small, fast-growing
species, and it reaches maturity in about 2 or 4 days,
we estimated the exponential growth rates during the
juvenile stage: from 0–2 days in the first bioassays
and from 0–4 days for the second bioassay.
Statistical analysis was conducted by one-way
ANOVA and significant differences among treatments
were tested by multiple comparison Tukey’s tests.
Feeding inhibition experiment
The inhibitiory effect of Microcystis on the feeding
rates of cladocerans was tested using an experimental
design similar to that of the second life-table experiment (Table 1), with the seston fraction (5–20 ␮m).
Polystyrene microspheres (6.4 ± 1.4 ␮m; Seragen Diagnostics, USA) were used as tracers for estimating
the filtering rates of cladocerans (DeMott 1986). Food
mixtures comprised 1.0 mg C l −1 of the green alga
and additions of 0.25 and 0.50 mg C l −1 of Microcystis. Controls consisted of animals fed only 1.0
mg C l −1 of the green alga (A. falcatus). The polyestyrene beads were added to the food mixtures and
controls in a concentration of about 3,000 particles
ml −1. After one hour of acclimation to food mixtures,
beads were added and animals were exposed for
about 7 min and then anaesthetized with carbonated
water and placed in vials with 4% formalin. For
counting the beads, a tissue solubilizer (TS-2 Research International Corp., USA) was added to each
animal placed on a glass slide and covered with a
cover slip. After about 1–2 h, the beads inside the gut
became visible and were counted under a microscope
(100X). The filtering rate for each animal in each
treatment was calculated with the formula:
FR ⫽ 共N/3,000兲 ⫻ 共60/t兲,
where FR is the filtering rate in ␮l animal −1 h −1; N is
the number of beads counted in the animal’s gut and
t is the feeding time.
Statistical analysis was conducted by one-way
ANOVA and significant differences among treatments
were tested by multiple comparison Tukey’s tests.
Table 1 summarizes all the experimental designs
used in this study.
Results
Life-table experiments
The Microcystis colonies from the lagoon used in this
experiment (supernatant) did not cause severe harmful effects in the reproduction of cladocerans (Figure 2). Only in C. cornuta reproduction was significantly decreased with the Microcystis colonies. This
effect was, however, not proportional to the Microcystis concentration. For this species, only total offspring produced (F 3,32 = 5.5, p = 0.004) and r (t-test,
p < 0.05) had significantly lower values in the Microcystis treatment than in the controls. For M. micrura,
only the age at first reproduction was significantly
lower in these treatments (F 3,58 = 12.8, p < 0.001).
The average microcystin content in Microcystis from
the supernatant sample was 3.9 mg g −1 DW, and microcystin concentrations in the Microcystis treatments
ranged from 2.0 to 8.0 ␮g l −1. Although microcystins
were present in relatively high concentration in whole
28
Figure 2. First life-table experiment performed with the sample collected on April 03, 1997, from the natural population with 25 ␮m-mesh
size net (see also Table 1). Controls consisted of 1.0 mg C l −1 of green alga (0.0 mg C l −1 of toxic cells), and treatments with Microcystis
consisted of mixtures of colonies in the supernatant fraction and green alga in different proportions, in a total concentration of 1.0 mg C l −1.
X-axis indicates the carbon concentration of Microcystis only. There were 10 replicate animals for C. cornuta and 16 for M. micrura. Data
are means ± SE for each treatment.
water samples (16.1 ␮g l −1) containing natural populations of Microcystis (data not shown), no evidence
of toxic effects was found. In general, cladocerans
showed a better performance with the seston diet than
with the green alga. Both cladocerans reproduced earlier in whole water (1 or 2 days). For C. cornuta, the
mean clutch size and total number of offsprings were
higher in the seston diet. The population growth rate
(r) was near to the maximum growth rate (0.41–0.43
d −1) and was similar for both species in whole lagoon
water. The Microcystis concentration was relatively
high (1.2 mg C l −1) in lagoon water, although most
colonies were > 20 ␮m.
In the second life-table experiment, there was
clearly a negative effect of Microcystis cells in the
seston fraction on reproduction of both cladocerans
(Figure 3). The age at first reproduction was significantly prolonged for both species in higher concen-
29
trations of toxic cells (F 3,117 = 51.2, p < 0.001). There
was a significant decrease in mean clutch size (F 3,117
= 118, p < 0.001), total offspring (F 3,117=56.8, p <
0.001) and r value (t-test, p < 0.05) for both species
as the seston concentration increased. However, there
was a significant interaction between cladoceran species and concentration of toxic cells in the age at first
reproduction (F 3,117 = 5.1, p = 0.002), fecundity
(F 3,117 = 36.9, p < 0.001) and total offspring (F 3,117 =
13.3, p < 0.001), showing that M. micrura was more
affected than C. cornuta. This cladoceran had negative to nearly zero population growth rates with
seston concentrations of 1.0 mg C l −1. Because in the
seston concentration of 1.5 mg C l −1 only one individual of M. micrura reproduced, demographic data
were not calculated. Although C. cornuta was also
affected by Microcystis, it had positive r values in
each concentration of toxic cells. The microcystin
content in seston was 3.1 mg g −1 DW, and microcystin concentrations in the seston treatments ranged
from 1.6 to 9.4 ␮g l −1.
In the third experiment, in which only laboratory
Microcystis strains were used, the effects of the two
Microcystis strains on cladocerans differed (Figure 4).
The age of first reproduction of both species was significantly affected (F 1,128 = 117, p < 0.001), having a
delay of 1 or 2 days in the higher concentrations of
the unicellular strain treatment. The mean fecundity
of C. cornuta was significantly decreased by both
strains (unicellular: F 3,68 = 34.6; colonial: F 2,51 =
27.3, both p < 0.001), although this reduction was
about the same in both treatments and concentrations
and, therefore, Microcystis concentration had only a
minor effect on r value. There was, however, a clear
effect of the unicellular strain NPLJ-2 concentration
on the total offspring produced by C. cornuta (F 3,68 =
70.3, p < 0.001). For M. micrura, there was a significant and strong negative effect of the unicellular
strain on fecundity (F 3,60 = 147, p < 0.001), on total
offspring (F 3,60 = 36.7, p < 0.001) and on r value (ttest, p < 0.05). The reproduction of this cladoceran,
however, was little or not affected at all by the colonial strain NPLJ-42. There were only minor effects on
mean clutch size (F 2,43 = 19.7, p < 0.001) and on total offspring produced (F 2,43 = 3.7, p = 0.033). These
reductions in the reproductive output of M. micrura
were, however, not reflected in r values for the animals exposed to Microcystis colonies, because these
values did not significantly differ from the controls
with green alga (t-test, p > 0.05). We also performed
an ANOVA using strain as a blocking factor to test
for the effect of strain and interactions between cladoceran species and strains. Interestingly, there were
significant interactions for the two main population
parameters that account for the r value, fecundity
(F 1,166 = 130, p < 0.001) and total offspring (F 1,166 =
89.3, p < 0.001). In the treatment with the unicellular
strain, C. cornuta performed better than M. micrura,
whereas the opposite was observed in the treatment
with the colonial strain. The microcystins content was
similar in both strains, varying from 4.5 to 5.8
mg g −1 DW. Microcystin concentrations in the treatment with the unicellular strain ranged from 1.0 to 4.5
␮g l −1 and in the treatments with the colonial strain
from the 2.9 to 5.8 ␮g l −1.
Juvenile growth rates
In the first growth rate bioassay, the animals showed
rapid growth in all treatments until the second day,
but a decline in growth rates was observed from the
second to the fourth day, after they had reached maturity. Thus, estimation of juvenile growth rates in
this experiment was based on the first two days only.
As in the first life-table experiment, the Microcystis
colonies from the supernatant fraction did not have
significant negative effects on M. micrura growth
(Figure 5). Growth rates were even higher in the mixtures of Microcystis and green alga, although there
were no significant differences between these treatments and the control with green algae (F 3,8 = 1.5, p
= 0.284). Animals in the whole water samples
(seston) had growth rates comparable with green algae and Microcystis treatments (0.64–0.68 d −1).
In the second growth bioassay, the animals grew
exponentially until the fourth day, but the growth
rates declined from the fourth to the sixth day. Thus,
the animals were assumed to reach their adult stage
on the fourth day and juvenile growth rates were estimated based on the first four days of growth. Contrary to the results of the first growth bioassay, Microcystis cells from the seston fraction had a
significantly negative effect on the growth rate of M.
micrura (Figure 5). Growth rates were similar in the
control and from 0.25–0.50 mg C l −1, although
growth rates were significantly lower in the concentrations ranging from 1.0–1.5 mg C l −1 of toxic cells
(F 4,10 = 56.9, p < 0.001).
30
Figure 3. Second life-table experiment with the sample collected on April 17, 1997, from the natural population with 25 ␮m-mesh size net.
Controls as in Experiment 1 (Figure 1), and treatments with Microcystis consisted of mixtures of single cells and small colonies (5–20 ␮m)
in the seston fraction with a fixed amount of green alga (1.0 mg C l −1). X-axis indicates the carbon concentration of seston only. There were
20 replicate animals for both species. Data are means ± SE for each treatment.
Feeding inhibition experiment
The two cladocerans responded differently in the
feeding inhibition experiment (Figure 6). The filtering rate of M. micrura was severely inhibited by both
concentrations of natural samples of Microcystis
(seston), with reductions of 70.8 and 80.9% compared
with the control treatment in 0.25 and 0.50 mg C l −1
of seston, respectively. In contrast, C. cornuta was
little affected, the reduction in feeding being only
small at higher concentration of Microcystis in the
diet.
Discussion
The results of the life-table experiments showed that,
although high concentrations of Microcystis cells and
toxins were present in the natural samples, negative
effects were not always observed. In the first life-table
31
Figure 4. Third life-table experiment performed with laboratory cultures of Microcystis. Treatments are: green alga as the control, unicellular
Microcystis strain (open symbols) and colonial Microcystis strain (black symbols). Controls consisted of 1.0 mg C l −1 of green alga (0.0
mg C l −1 of toxic cells), and treatments of mixtures of Microcystis and green algae in different proportions, in a total concentration of 1.0
mg C l −1. X-axis indicates the carbon concentration of Microcystis only. There were 20 replicate animals for both species. Data are means ±
SE for each treatment.
experiment, when Microcystis colonies from supernatant samples were used in mixtures with green alga,
there was some decrease in the reproduction of C.
cornuta relative to controls with green alga alone.
There was, however, no significant effect of Microcystis colonies on reproduction and growth rate of M.
micrura. Despite the relatively high toxin content of
these colonies (3.9 mg microcystins g −1 DW), the effects on growth and reproduction of cladocerans were
small. These colonies were also large (average size
56.2 ± 29.3 ␮m), which may have prevented cla-
docerans from ingesting them efficiently. The negative effect observed in the reproduction of C. cornuta
may be attributed to the low nutritive value of Microcystis for this cladoceran, because there was no significant effect of colony concentration on the population growth rate. In addition, despite the high biomass
of Microcystis (1.2 mg C l −1) and high concentrations
of microcystins in whole water samples (16.1 ␮g l −1),
the reproduction of both cladocerans and growth rate
of M. micrura seem not to have been negatively affected by seston. Animals showed good growth and
32
Figure 5. Juvenile growth rates of Moina micrura fed with green
alga (control = 1.0 mg C l −1) and natural samples of Microcystis.
Treatments are: colonies of Microcystis (supernatant) mixed in different proportions with the green alga, and seston containing Microcystis as single cells and small colonies (5–20 ␮m) mixed in
different concentrations with a fixed amount (1.0 mg C l −1) of
green alga. X-axis indicates the carbon concentration of Microcystis or seston only. Data are means ± SE of three replicate bottles
per treatment.
Figure 6. Feeding inhibition experiment with Moina micrura and
Ceriodaphnia cornuta fed with green alga (control = 1.0 mg C l −1)
and natural seston. Same design as second growth experiment (Figure 5), but using only two concentrations of Microcystis.
reproduction with natural seston and performed even
better than with the green alga used as control. Although Microcystis concentration was high in whole
water samples, much of the food was composed of
phytoplankton other than Microcystis, such as chlorophytes and diatoms (0.8 mg C l −1), and also detritus.
Moreover, in these samples, Microcystis was mostly
( ⬃ 65%) in the form of colonies with sizes varying
between 20 and 40 ␮m. One possible explanation for
the better growth and reproduction of cladocerans
with natural seston may be the higher food quality
provided by mixed diets of natural algae assemblages.
Also, the absence of negative effects of seston, even
in the presence of toxic Microcystis, may be also due
to the predominance of colonies that are too large to
be ingested by these small cladocerans.
When Microcystis from the natural seston assemblage was offered as single cells and small colonies
(5–20 ␮m), there was a strong negative effect on reproduction of both cladocerans (Figure 3). There was
a reduction in demographic parameters as the concentration of Microcystis cells increased, showing a doseresponse effect, consistent with a toxic effect. The
same effect was observed in the growth rate experiment with M. micrura. Since in these experiments we
mixed a high concentration of good food (1.0
mg C l −1 of green alga) with Microcystis, we can rule
out nutritional confounding effects of the first experimental design, in which the concentration of green
alga decreased with increasing concentration of toxic
cells. In the seston fraction, most of the Microcystis
colonies were in the edible size range (5–20 ␮m),
which may have caused the pronounced toxic effect.
Nevertheless, the inhibition of feeding rates (Figure 6) and decrease in food intake, may have led to a
decrease in fecundity in both cladocerans, especially
in M. micrura, which starve faster than C. cornuta
(Ferrão-Filho et al. 2000).
Similar effects were observed when we used laboratory cultures of Microcystis (Figure 4). The unicellular strain NPLJ-2 decreased reproduction rates of
both cladocerans while the colonial strain NPLJ-42
decreased slightly only C. cornuta reproduction rates.
These results corroborate the hypothesis that colony
size of Microcystis is an important factor determining
its toxicity for cladocerans (Bendorf and Henning
1989). In nature, Microcystis often blooms in colonial
form. Colony formation in cyanobacteria has been
regarded as a protection mechanism against ingestion
in addition to toxicity against predation by zooplankton (Bendorf and Henning 1989; Fulton and Paerl
1987a). Zooplankton can also induce colony or filament formation by cyanobacteria, both in the laboratory and in the field (Lampert 1987; Haney 1987).
33
Thus, cladocerans would be more susceptible to toxic
Microcystis at the beginning of the bloom formation,
when colonies are small.
Other studies also show that small cladocerans are
more sensitive to single celled than to the colony form
of Microcystis (Fulton and Paerl 1987b; Nandini
2000). Fulton and Paerl (1987b) showed that small
cladocerans such as Diaphanosoma brachyurum are
less inhibited by colonial Microcystis than large ones
such as Daphnia ambigua. In spite of the generalized
idea that large cladocerans are more susceptible to
Microcystis blooms than small cladocerans, this study
and also Ferrão-Filho et al. (2000) showed that there
is no relationship between sensitivity and body size,
since the small cladoceran C. cornuta was affected
both by single cells and colonies.
In situ ingestion rates of small cladocerans such as
Moina micrura and Diaphanosoma excissum on colonial Microcystis were shown to be very low, and
colonies >40 ␮m were not ingested at all (Jarvis et
al. 1987). Also, assimilation efficiency of Microcystis
is low, compared with green algae (Hazanato and Yasuno 1987; Henning et al. 1991). However, Hazanato
and Yasuno (1987) showed that decomposing colonies of Microcystis (<40 ␮m) could be utilized by M.
micrura as a good food source for growth and reproduction.
These findings are in accordance with our observations, and may explain in part why other studies
have failed to show toxic effects of seston during cyanobacterial blooms (Threlkeld 1979; Pace et al.
1984; Larsson et al. 1985; Matveev and Balseiro
1990; Fulton and Jones 1991). Fulton and Jones
(1991) found no toxic effects on survivorship and fecundity of Daphnia parvula despite the high densities of M. aeruginosa in water samples from Potomac
River (Washington D.C., USA). Although these authors did not measure toxins directly in their study,
they suggested that the lack of toxic effects could be
attributed to the absence of toxic strains of Microcystis and the presence of sufficient alternative food
sources. In our study, there were also sufficient edible
algae in the natural samples, such as chlorophyceans
and diatoms, to support growth in the cladocerans
species used.
Nandini (2000) also tested for toxic effects of natural samples of Microcystis on tropical cladocerans
and rotifers. She separated single cells from the colonial Microcystis by sonication and filtering the sonicated suspension in a 35 ␮m sieve but microcystins
were not measured. We, however, did measure micro-
cystins in the natural samples and showed that they
contained substantial amounts of toxins. Moreover,
the toxin concentrations were similar among the samples used in our experiments, which explains the similarities in the toxic effects observed when we used
concentrated natural samples and cultured Microcystis. This emphasizes the toxicity potential of Microcystis in both laboratory and in field.
Cladocerans differed in their sensitivity to toxic
cells: M. micrura seems to be more sensitive to single
cells, and C. cornuta more to colonies of Microcystis
than M. micrura. Earlier, Ferrão-Filho et al. (2000)
also observed the same pattern of sensitivity between
these two cladocerans, with M. micrura being more
sensitive to unicellular Microcystis strains than C.
cornuta. Nandini (2000) also found similar results to
ours, with Moina macrocopa being more sensitive to
single celled Microcystis than C. cornuta. Although
small cladocerans are less likely to be affected by
large colonies and filaments of cyanobacteria (Gliwicz and Siedlar 1980; Fulton and Paerl (1987a,
1987b)), the high sensitivity of C. cornuta to colonial
Microcystis may be explained by some mechanical
interference with feeding process or by the low nutritive value of this food. The latter hypothesis is less
likely since this cladoceran also had low reproduction
performance in the 1:1 mixtures of colonial Microcystis and green algae. In addition, both unicellular
and colonial strains of Microcystis had relatively high
amounts of toxin, the latter having slightly more microcystins. However, it is not likely that C. cornuta
was more affected by the toxins of the colonial Microcystis, since the size of the colony was too big to
be ingested by this cladoceran. The lack of significant
differences between the responses of C. cornuta to
both strains in the third life-table experiment indicates
that there was no size-dependent toxicity of Microcystis strains for this cladoceran. In the same experiment, there was also a weak effect of strain concentration on C. cornuta. These facts suggest that C.
cornuta was less affected by the toxins of the Microcystis, but more likely by interference of the colonies
on the feeding process.
DeMott (1999) has demonstrated that some cladocerans, like Daphnia magna, exhibited a strong
feeding inhibition after 1-h exposure to toxic Microcystis but an increased filtering rate after 24-h exposure. He suggested that these animals adjust their ingestion of toxic algae so that there is a trade-off
between the effects of poisoning and reduced ingestion and starvation. Ferrão-Filho et al. (2000) also
34
showed that the filtering rate M. micrura was strongly
inhibited after 1-h exposure to the strain NPLJ-2, but
no recovery was observed after 20-h exposure time.
Thus, these results are consistent with the observation
of DeMott (1999) that the absence of recovery in
some cladocerans indicates a direct effect of poisoning. The lower inhibition of C. cornuta after 1-h exposure to toxic cells, therefore, also suggests higher
resistance of this cladoceran to toxins.
In conclusion, the effects of toxic Microcystis
blooms on cladocerans and other zooplankton species
will depend on the size of the colonies present and
on the species of the zooplankton as well. Our results
with different field samples and laboratory cultures,
provided information on the mechanisms involved in
toxicity of Microcystis blooms and different responses
of cladocerans to these toxic resources. Most studies
involving laboratory cultures of Microcystis, usually
in the unicellular form, probably overestimated the
adverse effects of this cyanobacterium on cladocerans. In nature, these effects must be less pronounced
than in the laboratory, and this is likely to be among
the reasons why toxic effects predicted from laboratory studies have not been confirmed in the field
(Haney 1987). Future studies should focus on the
question of how toxic blooms of cyanobacteria can
act as selection mechanism in plankton communities
and also how zooplankton can contribute to the formation of toxic blooms.
Acknowledgements
We thank Conselho Nacional de Pesquisa (CNPq) for
the financial support and Reinaldo Bozelli for the use
of his microbalance. We thank also Bill DeMott and
Alan Tessier for their comments and suggestions on
the manuscript.
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