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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
a r t i c l e
i n f o
Article history:
Received 4 February 2015
Received in revised form 26 March 2015
Accepted 28 March 2015
Available online 8 April 2015
Keywords:
Didymo
Sperm cells
Toxity
a b s t r a c t
Didimosphenia geminata (didymo), has become a powerful and devastating river plague in Chile. A
system was developed in D. geminata channels with the purpose evaluating the effects of water polluted
with didymo on the activation of Atlantic salmon (Salmo salar) spermatozoa. Results indicate that semen,
when activated with uncontaminated river water had an average time of 60 21 s. When using Powermilt,
(a commercial activator), times of 240 21 s are achieved, while rivers contaminated with D. geminata
achieve a motility time of 30 12 s. Interestingly enough, the kinetic parameters of VSL, VCL and VAP
showed no signicant changes under all of the conditions. Furthermore, the presence of D. geminata
reduces activation time of the samples as the cells age, indicating increased effects in spermatozoa that
are conserved for more than 5 days. D. geminata has antioxidant content, represented by polyphenols;
200 ppm of polyphenol were obtained in this study per 10 g of microalgae. Spermatozoa exposed to these
extracts showed a reduction in mobility time in a dose dependent manner, showing an IC50 of 15 ppm. The
results suggest an effect on spermatozoa activation, possibly due to the release of polyphenols present in
contaminated rivers, facilitating the alteration of sperm motility times, without affecting the viability or
kinetics of the cells. These ndings have important implications for current policy regarding the control
of the algae. Current control measures focus on the number of visible species, and not on the compounds
that they release, which this study shows, also have a problematic effect on salmon production.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Didymosphenia geminata (D. geminata) is a unicellular benthic
diatom. This microalgae known as didymo in Chile, has been
found in the river waters of southern Chile (Riveraet al., 2013), and
has been considered a plague in freshwater sources by the Government of Chile and can affect sh population (Reid et al., 2012).
International studies indicate that D. geminata alters the microenvironment, reduces the sh population (Clearwater et al., 2010)
and perturbs aquatic macro-vertebrate communities and drinking
water system lters (Bergey et al., 2010; Gillis and Chalifour, 2010;
Corresponding author at: Av. Las Mariposas S/N, Campus Dr. Rivas, Universidad
Catlica de Temuco, Temuco, Chile. Tel.: +56 45205564.
E-mail address: jparodi@uct.cl (J. Parodi).
http://dx.doi.org/10.1016/j.aquatox.2015.03.022
0166-445X/ 2015 Elsevier B.V. All rights reserved.
Kilroy et al., 2009). Recently, a toxic effect of microalgae on communities in contaminated rivers was described (Larned and Kilroy,
2014). The removal of the microalgae, also introduces damages
to rivers (Larned and Kilroy, 2014). In Chile, there are no studies that conrm this hypothesis and we must control these effects
with laboratory research to support eld observations (Rivera et al.,
2013). Fish spermatozoa are immobile in the ejaculate, and it is
only after suffering osmotic shock in water that they are activated
and begin to move or swim (Alavi et al., 2009; Takei et al., 2012;
Vladic and Jarvi, 2001). Spermatozoa are labile cells and are affect
by aquatic contamination (Hatef et al., 2013). In the laboratory,
one can replicate this effect using water from rivers or commercial solutions (Figueroa et al., 2013). Different kinetic parameters
can be observed, including mobility time of the masses, either manually (Ubilla and Valdebenito, 2011) or by computerized systems
(Hu et al., 2013). This is a way of measuring their cellular function
103
104
Fig. 1. Laboratory condition for didymo maintaining. A shows image of two articial river used for maintaining dydimo in the lab upper panels control water lower panel
dydimo water, the number in the circles, show point of sample collection for observation of didymo. B shows a time course of the number of didymo live in the different
point of the sample collection.
used for separation of means with p < 0.05. Levels of probability (p)
less than 0.05 were considered statistically signicant. All data was
analysed with the Prism 4.0 statistical program.
3. Results
3.1. Maintenance model of D. geminata in the laboratory
A disadvantage of D. geminata, not have easy accessibility in the
laboratory. Although the D. geminata in conservation model can
be transported, but there is no evidence of its cultured in the exvivo model. An articial river model was developed as shown in
Fig. 1A, to have circulating river water and water contaminated
with D. geminata. River rocks with D. geminata were maintained in
these articial rivers under conditions that were similar between
the two channels, as summarized in Table 1 and Fig. 1B, which show
no signicant physicochemical changes between the channels. In
order to register whether D. geminata are present or not, the articial rivers were monitored over three observation points. Fig. 1A
showed example of articial river used for maintaining the D.
Table 1
Condition of articial rivers. Show a table of the physical condition in control and
didymo articial rivers system.
Parameters measured
Control
Didymo
Total volume
Useful volume
Slope
Channel height
Substratum major, volume
Substratum minor, volume
pH
T
O2
28 (lt)
16 (lt)
1.50%
11.6 (cm)
766 (ml)
9.6 (ml)
7.3
15
150
28 (lt)
16 (lt)
1.50%
766 (ml)
9.6 (ml)
7.2
16
143
geminate. Upper panel, shows a control river and lower panel articial river with D. geminata, the number indicate point of sample
recovery (observation point) for D. geminata viability evaluation.
Fig. 1B shows a plot graph of time course of viability of D. geminata
in our laboratory. At 0 days, when the experiment beginning 90% of
viability of the D. geminata are observed. After 50 days of maintenance in the laboratory 50% of the microalgae survived for 40 last
days of monitoring we observed a reduction of the live form of D.
geminate. This suggests that it is possible to maintain viable values
of 50% of D. geminata for months, which can be used as a source of
D. geminata for laboratory studies.
105
activation time, without affecting the viability of the samples. Evaluations were made on whether the kinetics, another function of
spermatozoa, was affected by the presence of D. geminata. In Fig. 4A,
a record of the traces of sperm movement is observed in the different activation conditions using the CASA ImageJ program. Fig. 4B
shows the observation of the VCL in the various conditions, not
showing signicant changes in samples. Fig. 4C and D show graphs
for VSL and VAP where no signicant changes were observed in
the kinetic parameters either, but when assessing the progressive
mobility, a reduction of 50% was observed in the presence of D.
geminata, as summarized in Fig. 4E. These experiments suggest that
there is no effect on the kinetics of spermatozoa activation when D.
geminata contaminated water was used. Although activation time
decreased, it did not seem to affect motility. This evidence indicates
that didymo alters correct spermatozoa function.
3.4. Polyphenols, presence and possible D. geminata effector
molecule
Fig. 2. Sperm cells viability exposed to didymo. In A image of the sperm cells in the
absence (river) or presence of didymo (river/didymo) In B a graph bar of quantication in the gure A from cell viability. The microphotographes are representative of
9 different experiment in different condition and the bar show means SEM.
106
Fig. 3. The didymo alter function of sperm cells. A shows bar graph of activation time of sperm cells, exposed to river water, powermilt, distillated water or water contaminated
with didymo. In B, graph time activation, in samples conserved for several days, exposed to river water, powermilt or water contaminated with didymo. In C shows a curve
doses response, the time activation of sample exposed to powermilt plus increased proportion of water contaminated with didymo, with IC50 of 16 07 didymo V/V. The bar
and the dot show means SEM from 12 independent experiments. *p < 0.05 (ANOVA).
2014). Our ndings indicate that the viability of S. salar spermatozoa is not affected when exposed to water contaminated with D.
geminata (Fig. 2B). However, the reaction time is affected, as well
as the mass of motile spermatozoa (Fig. 3A). A decrease in activation time of 50% was shown (Fig. 3A), altering the functions of
Fig. 4. Kinetic of sperm cells exposed to didymo. A shows a traces record of the sperm cells, exposed to river water, powermilt or water contaminated with didymo. In B,
bar graph of VCL in absences or presences of water contaminated with didymo, in C bar graph of VAP in absences or presences of water contaminated with didymo, in D bar
graph of VSL in absences or presences of water contaminated with didymo, in E bar graph of motility of sample in absences or presences of water contaminated with didymo.
The bar show means SEM from 12 independent experiment. *p < 0.05 (ANOVA).
107
Fig. 5. Polyphenols present in didymo extract and the effect over sperm cells. A shows confocal image of fresh didymo exposed to 280688 nm of excitation. In B, bar graph
of concentration of polyphenols obtained from fresh sample of didymo, dry didymo from river, didymo with extend protocol of extraction and didymo dry in the laboratory,
in C doses response curve of polyphenols effect over time of activation with IC50 of 10 0,7 ppm of polyphenol. The bar and dot show means SEM from 9 independent
experiment.
spermatozoa. The Powermilt compound has been used as a spermatozoa activator, as described by Figueroa et al. (2013). This
compound produced an excellent response in our samples with an
average of 4 min of activation (Fig. 3C). In order to understand the
active mechanism of D. geminata, observations were made whether
it was able to inhibit the effects of Powermilt on the activation
times. We observed that upon dilution with water contaminated
with D. geminata, of the Powermilt stock (10) to a working solution of (1) with distilled water plus contaminated D. geminata
water, in increasing proportions (Fig. 3D) a decreased effect of Powermilt on activation was observed, inhibiting it fully when 100% of
D. geminata was used. This suggests that the release of molecules
into river water, with D. geminata would be responsible for the
reduction of the activation time of the S. salar spermatozoa without affecting the kinetics of these spermatozoa (Fig. 4). Another
study with metals in water, showed effects on kinetic parameters
(Dietrich et al., 2010), but our results showed the contaminated
water altered the activation processes, except for the already activated spermatozoa. This suggests a complex mechanism which
reduces the number of cells being able to be activated, but not the
quality of this activation, a different mechanism of classic contamination, such as metal in the waters.
The organic content of D. geminata has proved to be rich in
antioxidant polyphenols common to brown algae such as diadinoxantina. This type of molecule has cellular effects, ranging from
benecial to toxic, depending on concentration. In the samples
maintained or recovered from rivers, it was possible to show that
there is a ratio of about 200 ppm of polyphenols per 10 g of D.
geminata, and that it is possible to be extracted (Fig. 5B). In order
to explore whether it is possible to associate this organic content to the functional effects of D. geminata, the spermatozoa were
108
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