Aquatic Toxicology 163 (2015) 102108

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Water contaminated with Didymosphenia geminata generates changes

in Salmo salar spermatozoa activation times
Pamela Olivares a , Paola Orellana a , Guillermo Guerra a , Matas Peredo-Parada b,c ,
Viviana Chavez d , Alfredo Ramirez e , Jorge Parodi a,
a
Laboratorio Fisiologa de la Reproduccin, Escuela de Medicina Veterinaria, Ncleo de Investigacin en Produccin Alimentaria, Facultad de Recursos
Naturales, Universidad Catlica de Temuco, Chile
b
Departamento de Ingeniera en Obras Civiles, Universidad de Santiago de Chile, Chile
c
Plataforma de Investigacin en Ecohidrologa y Ecohidrulica, EcoHyd Ltda, Chile
d
Laboratorio de Investigacin y Educacin, Tonalli Ltda, Chile
e
Laboratorio de Criobiologa y Anlisis de Funcionalidad Espermtica. Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral
de Chile, Valdivia, Chile

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

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

and the effects of molecules within their function, as motility and

viability are related (Parodi, 2014) and computer assisted semen
analysis (CASA) can be used for evaluated toxic effect on spermatozoa (Kime et al., 1996). Microalgae belonging to the family of brown
diatom algae are rich in some molecules, such as antioxidants and
polyphenols such as diadinoxhantina (Lohr and Wilhelm, 1999).
Interestingly, several researches describe the benets of antioxidants and polyphenols in cellular models. However, its toxic effects
on cancer cells (Korkina et al., 2009) are also described, indicating
that a certain dose may be lethal to cell groups. It should be remembered that the dosage perfectly denes the positive or negative
effect of a molecule in a cell model.
We evaluated the effect of D. geminata in the activation of
Atlantic salmon sperm. We used water that was contaminated with
microalgae to evaluate motility time, kinetics and cell viability.
The same tests were carried out with uncontaminated river water
and with Powermilt, a commercial activator. Polyphenols extracted
from the didymo were used to evaluate a possible mechanism of
the effects observed from D. geminata on the spermatozoa functions
studied.
2. Methods
2.1. Collection of D. geminata samples
D. geminata was collected in the Futaleufu and Biobo rivers
in winter and spring of 2014. The samples were transported to
the laboratory in plastic, closed dark boxes, at 10 C. River water
and substrate (river rocks) colonized by the microalgae were also
collected.
2.2. Protocol for maintenance of the D. geminata samples
Collected samples were kept in aquariums, closed recirculating
water systems called articial rivers, for observation in the laboratory. The rocks contaminated with D. geminata were arranged in
the articial rivers, adding 50% of the original river water, plus 50%
distilled water, (total volume of 14 l) making sure to leave a column of water of about 15 cm over the rocks. Articial rivers were
maintained with an expanded polystyrene insulate cover and the
temperature was reduced to an average of 12 C through a cooling gel system. The water was kept in constant ux, using a model
71009 Plaset-Italy brand motor with 30 W potency. This device captures water from the nal part of the articial river, and is connected
to a 1 diameter PVC pipe which returns to the origin of the river
for water discharge, achieving constant ow and aeration. Microscopic and macroscopic changes in the aquarium with D. geminata
were recorded daily.
2.3. Mortality studies of D. geminata
The mortality of D. geminata was observed by visual inspection
with clear eld microscopy, using an inverted Meijie (VT series,
Techno Co., Ltd., Japan) microscope, observing the presence or
absence of granules within the cytoplasm, denominating them
as granular forms (Root and OReilly, 2013). The numbers of D.
Geminata cells, through a 40 lens, were counted, recording the
percentage of those containing granules within the cytoplasm as a
viable form of indication. In order to improve the documentation in
images, a Nomarsky microscope at an objective of 40 (Olympus)
was used for observation of intracellular structures and comparison with the viability obtained using modied neutral red dye.
For this, the samples were left in a 0.01% neutral red dye solution
for 10 min, which has been reported as a means of assessing the
viability of the D. geminata. When a granular red coloration is

103

observed, it is indicated as a viable form of D. geminata (Jellyman

et al., 2010).
2.4. Extraction of polyphenols and confocal images
D. geminata samples were frozen with liquid nitrogen, subsequently ground and maintained at 4 C. Soon after, 20 mL of
deionized water was added and rupture was performed by ultrasound with a Misonix XL2000 Series sonicator, 10 pulses of 30 s
in 1 min intervals until decomposition of the complex for all of
the samples. They were then incubated for 20 min at 30 C, being
agitated and then ltered by gravity. Samples were then passed
through a double gauze lter and a No. 2 (125 mm) Wartman as
described by Fernndez et al. (2013). The nal extract was subjected to a measurement of polyphenols. Folin & Ciocalteu reagent
was used; following the protocol described by Lowry at 1951 Optical density was measured at 517 nm of the reaction. The samples
were frozen and passed through HPLC to identify their prole.
The protocol described by Lohr and Wilhelm (Lohr and Wilhelm,
1999) was modied, using the extracts for measurement with a
C18 (Macherey-Nagel, Duren, Germany) column, measuring retention at 440 nm. Then, the presence of diadinoxantina was identied
in the yellow fraction. The fresh samples were also observed in a
spectral confocal Olympus microscope (Olympus Fluoview 1000,
USA), the samples were observed from an excitation of 280 nm to
680 nm, obtaining images of the stimulated structures. The images
of the different channels and the DIC images were combined using
the ImageJ program.
2.5. Collection and handling of semen samples
A selection was made of adult male Atlantic salmon, (Salmo
salar) coming from sh farms associated with the Catholic University of Temuco. Prior to the extraction of semen, sh were
anesthetized with BZ-20 at 0.015%. Then, through an abdominal
massage or stripping, the semen samples were extracted, which
were maintained with oxygen, placed in hermetically sealed plastic
containers and refrigerated. In the laboratory, semen was diluted
in a proportion of 1:2 (semen: sperm diluent), composed of 18.8 g
NaCl/L CaCl2, 3H2O 2 g/L KCl 72 g/L, NaH2PO4, H2O 4.1 g/L, NaHCO3
1 g/L, MgSO4, 7H2O 2.3 g/L and glucose 1 g/L. All procedures were
performed at 4 C. The samples were agitated and aerated daily
by injecting compressed oxygen. For observation, samples were
mounted on a slide, adding 10 L of Atlantic salmon (S. salar) semen
and quickly mounted on the NIKON Labophot 2 optical microscope.
Once the samples were focused, 20 L of well water, river water,
contaminated river water, and water with the Powermilt activator
which was diluted with distilled water (1), was applied. Protocol
were taken to examine the time from the beginning of sperm movement, until all cells in the visual eld stopped moving, their kinetics
were analyzed through a Computer Assisted Semen Analysis (CASA)
system in the ImageJ program, curvilinear velocity (VCL), straight
line velocity (VSL) and straight line velocity (VAP) are described.
Through this image analysis, the kinetic values were quantied
under various conditions (Parodi et al., 2015). A video was made
under a 10 zoom lens, as to generate a greater contrast with the
microscope in a lter phase, for the Nikon 10 lens and in the shortest possible time after the activation of sperm, in order to capture
the maximum amount of information. All solutions were kept at
4 C.
2.6. Statistical analysis
The results are presented as the average standard error mean
(SEM). An ANOVA variance analysis was performed, comparing all
observations. A post-test was applied and the Bonferroni test was

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P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

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.

3.2. Effects of D. geminata on spermatozoa viability and function

The microalgae, D. geminata, has not been identied as a toxic
agent, although it has been suggested that it alters the ecology of
rivers through mechanisms that are unclear or not described in the
literature. Studies at the cellular level have not yet begun, and studies do not suggest cytotoxicity. Fish spermatozoa in general are very
labile cells with high sensitivity to environmental changes (Cabrita
et al., 2011). The effect on their viability, as shown in Fig. 2A, indicate
no increase in the number of dead cells when exposed to water from
rivers contaminated with D. geminata, nor to water from articial
rivers, maintained in the laboratory. Fig. 2B shows a quantication
of the microphotographs and no signicant changes in any of the
conditions. These ndings suggest that didymo does not induce
cell death in this model. S. salar spermatozoa undergo an activation
process when subjected to osmotic shock and present movement
at that moment which can last a few minutes. The effects of the
presence of D. geminata on spermatozoa were explored, recording
activation time through the ImageJ-CASA program. The time that
the samples spend moving was recorded. We observed spermatozoa in water with D. geminata to have 50% reduction in activity

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

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.

when compared to uncontaminated river water. To complete this

study, evaluations were made of the effects of the presence of D.
geminata on activation during the retention period of the samples.
Fig. 3B shows a progression curve for the effect of time on viability
in control cells, which are activated with river water with Powermilt and water contaminated with D. geminata. The gure shows
evidence that the samples are viable after being conserved for 20
days. Interestingly, when samples that were conserved in the laboratory were activated with water contaminated with D. geminata,
they showed a signicant decrease in activation times after 5 days
of conservation. This suggests that after days of conservation of the
sperm samples, they become more sensitive to the presence of D.
geminata, and their viability can be affected by undened mechanisms. Powermilt, a commercial solution (Figueroa et al., 2013) and
osmotic shock activates S. salar spermatozoa motility which lasts
several minutes. In the samples, this was diluted from its stock 10
to 1 with deionized water. We constructed a V/V concentration
curve for Powermilt diluted with water from D. Geminata contaminated rivers, noting that there is a type of dose-response effect and
that the dilution of Powermilt with water reduces this effect with
IC50 of 15 0.7 for D. geminata activation time as shown in Fig. 3C.
This evidence suggests that water contaminated with D. geminata
alters the activation time of spermatozoa samples, even inhibiting
the potentiating effect of commercial solutions such as Powermilt.
3.3. Observation of spermatozoa kinetics in the presence of D.
geminata contaminated waters.
Using low-cost protocols implemented in the laboratory (Parodi
et al., 2015) kinetic changes were observed, particularly of the VCL,
VSL and VAP values. The previous data indicated a change in the

Brown microalgae, including didymo, contain antioxidants in

the form of polyphenols (Lohr and Wilhelm 1999). A presence of
diadinoxantina is described in this type of microalgae, with diverse
biological effects. Polyphenols have recognized benecial effects
on cell physiology, but toxic effects have also been reported. As
described by Paracelsus, a classic physician and researcher in pharmacology, the benecial or toxicological effects of a compound or
molecule depend on the concentration used, as quoted in this manual of pharmacology and toxicology from Dr. Eichlotz (Eichholtz,
1948). Polyphenols were extracted from D. geminata samples, using
protocols described in (Fernndez et al., 2013), which were modied for microalgae. Fig. 5A displays a confocal image, which shows
organic content of D. geminata in red and green. Fig. 5B shows
content achieved in ppm from various sources of D. geminata.
These extracts were used in experiments where we observed the
activation time of spermatozoa when exposed to increasing concentrations of the extract, nding that an IC50 dose of activation
time is 10 0.7 ppm, as shown in Fig. 5C. Our ndings suggest
that D. geminata can reduce the activation time because of the
release of polyphenols into rivers and that there is a doseresponse
effect when this type of extract is used in the S. Salar spermatozoa
functions. This suggests that there are mechanisms by which D.
geminata affects the correct operation of closed systems such as
rivers.
4. Discussion
Evidence suggests an effect of water contaminated with D. geminata on the motility time of S. salar spermatozoa. A decrease is
observed of the activation time and percentage of motile cells,
without affecting the viability of the spermatozoa nor motile spermatozoa kinetics. These effects could be mediated by polyphenol
content present in D. geminata and can be released into rivers contaminated with this microalga. D. geminata has become a pollution
problem in the rivers of southern Chile. Its detection started in
rivers in the Los Lagos Region, but since then, there have been at
least three more regions reported that contain this plague and projections are that it will continue to spread if barriers are not raised.
No evidence exists that it can be maintained in a cultivated system,
despite rapid growth (Sundareshwar et al., 2011). On the contrary, it
is possible to nd research indicating that under various conditions,
it is not feasible to keep D. geminata in closed systems (Czarnecki,
1988). A model of articial rivers was developed (Fig. 1A) using
river substrates, in the form of rocks with D. geminata. A number
of viable forms of D. geminata of over 50% were able to be maintained for more than two months, as shown in Fig. 1C. This allowed

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P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

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).

for conservation of D. geminata contaminated water for several

weeks and facilitated experiments in the laboratory. In other models, the effects of D. geminata in the micro ora of rivers have been
described, reporting changes mainly in aquatic species, but with no
reports of similar effects in more complex forms (Richardson et al.,

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).

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

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

activated with increasing concentrations of the extracts, as shown

in Fig. 5C, showing a doseresponse effect when inhibiting time
activation of the samples with an IC50 of 15 ppm of polyphenols. We
conclude that the effect observed in waters contaminated with D.
geminata is mediated by the presence of polyphenols, but we cannot
discard other mechanisms of action for altering cellular function.
Recent studies have suggested more toxic effects of D. geminata
in vertebrates (Richardson et al., 2014) and in benthic river communities (Gillis and Chalifour, 2010) or of the treatments used for
removal (Larned and Kilroy, 2014). This information on the effects
of the microalgae is just recently being explored. The data suggests
that didymo can have a particular effect on activation time of S. salar
spermatozoa. This is evidence of alterations in cell physiology, possibly due to the presence of polyphenols, but more importantly, our
ndings indicate that the effects of this plague are more complex
and their effect should be closely watched new study about fertility condition of the sh in contaminated river are required and the
evidence show a hypothesis for explain sh population reduction
in our rivers. D. geminata should be considered as a toxicological
agent that affects cell function.
Acknowledgements
Jorge Parodi receives contributions from MECESUP 0804, this
research program was supported by technical assistance from UCT
278-2472 Didy 2013. We are indebted to Professor Ian Scott for his
translation, revision and editing. Language editing services were
provided by www.journalrevisions.com. We are indebted to Dr.
Valdevenito from BIOACUI-UCT for providing samples of spermatozoa and Powermilt. We thank Dr. Romero for access to the confocal
microscope.

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P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

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