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Previous studies confirmed the presence of melatonin in Daphnia magna and demonstrated diurnal fluctuations in its concentration. It is also known that in several invertebrate species, melatonin affects locomotor activity. We tested the hypothesis that this hormone is involved in the regulation of Daphnia diel vertical migration (DVM) behaviour that is well recognized as the adaptive response to predation threat. Using ‘plankton organs’, we studied the effect of three concentrations of exogenous melatonin (10−5, 10−7, 10−9M) on DVM of both female and male D. magna in the presence or absence of chemical cue (kairomone) of planktivorous fish. Depth distribution was measured six times a day, using infrared-sensitive closed circuit television cameras. Our results showed a significant effect of melatonin on the mean depth of experimental populations, both males and females, but only when melatonin was combined with fish kairomone. Females stayed, on average, closer to the surface than males, both responding to the presence of kairomone by descending to deeper strata. In the presence of exogenous melatonin and with the threat of predation, Daphnia stayed closer to the surface and their distribution was more variable than that of individuals, which were exposed to the kairomone alone. Approaching the surface in the presence of predation threat seems to be maladaptive. We postulate the role of melatonin as a stress signal inhibitor in molecular pathways of response to predation threat in Cladocera. Keywords Daphnia -Depth distribution-Melatonin-Predation
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BIOLOGY OF CLADOCERA
Role of melatonin in the control of depth distribution
of Daphnia magna
Piotr Bentkowski
Magdalena Markowska
Joanna Pijanowska
Published online: 7 March 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Previous studies confirmed the presence
of melatonin in Daphnia magna and demonstrated
diurnal fluctuations in its concentration. It is also
known that in several invertebrate species, melatonin
affects locomotor activity. We tested the hypothesis
that this hormone is involved in the regulation of
Daphnia diel vertical migration (DVM) behaviour
that is well recognized as the adaptive response to
predation threat. Using ‘plankton organs’, we studied
the effect of three concentrations of exogenous
melatonin (10
-5
,10
-7
,10
-9
M) on DVM of both
female and male D. magna in the presence or absence
of chemical cue (kairomone) of planktivorous fish.
Depth distribution was measured six times a day,
using infrared-sensitive closed circuit television cam-
eras. Our results showed a significant effect of
melatonin on the mean depth of experimental popu-
lations, both males and females, but only when
melatonin was combined with fish kairomone.
Females stayed, on average, closer to the surface than
males, both responding to the presence of kairomone
by descending to deeper strata. In the presence of
exogenous melatonin and with the threat of predation,
Daphnia stayed closer to the surface and their
distribution was more variable than that of individu-
als, which were exposed to the kairomone alone.
Approaching the surface in the presence of predation
threat seems to be maladaptive. We postulate the role
of melatonin as a stress signal inhibitor in molecular
pathways of response to predation threat in Cladocera.
Keywords Daphnia Depth distribution
Melatonin Predation
Introduction
The presence of melatonin has been reported in many
organisms, including bacteria (Tilden et al., 1997a),
unicellular algae (Balzer & Hardeland, 1991), higher
plants (Reiter & Tan, 2002), invertebrates (Vivien-
Roels & Pe
´
vet, 1993), and vertebrates (Gern et al.,
Guest editors: M. Silva-Briano & S. S. S. Sarma / Biology of
Cladocera (Crustacea): Proceedings of the VIII International
Cladocera Symposium
P. Bentkowski J. Pijanowska
Faculty of Biology, Department of Hydrobiology,
University of Warsaw, Banacha 2,
02-097 Warsaw, Poland
J. Pijanowska
e-mail: j.e.pijanowska@uw.edu.pl
M. Markowska
Faculty of Biology, Department of Animal Physiology,
University of Warsaw, Miecznikowa 1,
02-096 Warsaw, Poland
e-mail: markosia@biol.uw.edu.pl
P. Bentkowski (&)
School of Environmental Sciences,
University of East Anglia, Norwich NR4 7TJ, UK
e-mail: P.Bentkowski@uea.ac.uk
123
Hydrobiologia (2010) 643:43–50
DOI 10.1007/s10750-010-0134-x
1986). It functions in free radical scavenging, the
regulation of body mass and energetic metabolism,
and is a key component of the biological clock
responsible for modulating physiological rhythms
(for reviews see Hardeland & Poeggeler, 2003;
Pandi-Perumal et al., 2006). With regard to the
effects of melatonin in invertebrates, it has been
shown that application of this hormone can alter
locomotory activity in the cricket Acheta domesticus
(Yamano et al., 2001) and the crayfish Procambarus
clarkii (Tilden et al., 2003); it can influence regen-
eration processes in the planarian Dugesia japonica
(Yoshizawa et al., 1991), the nemertean Lineus
lacteus (Arnoult & Vernet, 1995) and the crab Uca
pugilator (Tilden et al., 1997b); and it can alter life
history and reproduction in the aphid Acyrthosiphon
pisum (Gao & Hardie, 1997) and the dinoflagellate
Gonyaulax polyedra (Balzer & Hardeland, 1991).
In an earlier study, we found that melatonin is
present in the cladoceran Daphnia magna, being
located mainly in the optic nerves and ganglia as well
as in thoracopodes. It exhibits a diurnal rhythm, with
the peak abundance occurring during the light hours
(Markowska et al., 2009). Kashian & Dodson (2004)
detected no effect of melatonin application on either
the growth rate or reproduction of Daphnia.
Zooplankton diel vertical migration (DVM) as an
anti-predator strategy has been observed in a number of
prey–predator interactions (e.g. Bollens & Frost, 1989;
Dawidowicz et al., 1990; Neill, 1990; Dawidowicz,
1993; Loose, 1993). In Daphnia, migratory behaviour
is induced by the chemical cues (kairomones) released
to water by planktivorous fish (Dawidowicz & Loose,
1992a). The most typical pattern of DVM is searching
the dark refuge against visual predation in the deep
strata during the day and approaching the surface at
night, to exploit the epilimnetic resources. Reduced
growth and reproduction rates in low temperatures of
deeper water are well-documented costs of avoiding
the risk of being eaten (Dawidowicz & Loose, 1992a).
Though ecological significance of DVM has been well
recognized (e.g. Ringelberg, 1999), the understanding
of its physiological and biochemical mechanisms is
still poor.
As melatonin is known to alter locomotory activity
depending on light conditions, we tested the hypoth-
esis that it is involved in the regulation of the
phenomenon of DVM in Daphnia, the onset and
offset of which are triggered by light.
Materials and methods
Daphnia magna
clone P3 originating from Binnen-
see, a shallow brackish lake in North Germany
inhabited by fish (Lampert, 1991), was used in the
experiment. Animals were obtained from the third
or later clutches of a synchronized population of
mothers, originating from a single female. Mothers
were kept at a temperature of 22 ± 2°C, under a long
day photoperiod (16L:8D), at a density of 10 ind. l
-1
in water from a shallow, eutrophic lake near Warsaw,
that had been stored and aerated for several weeks
and then filtrated through a glass fibre filter (GF/C,
Sartorius) prior to use. They were fed the algae
Scenedesmus obliquus at a concentration of
1mgC
org
l
-1
. Algae were grown in xenic (contain-
ing bacteria) batch cultures on Z/4 medium, under
continuous illumination from white fluorescent
lamps, at 21 ± 1°C.
The experiment was conducted in a ‘plankton
organ’: an apparatus described by Dawidowicz &
Loose (1992a). Glass flow-through tubes (60 cm
long, 1.5 cm in diameter) were arranged vertically in
an aquarium (60 cm 9 100 cm 9 15 cm) located
in an air-conditioned darkroom. Medium (same as in
mothers’ culture) was delivered individually to each
tube using a peristaltic pump (Ismatec). Thermal
stratification in the tubes (21 ± 1°C at the surface
and 10 ± 0.5°C at the bottom) was achieved by
heating the surface waters in the aquarium and
cooling the bottom. Experimental animals (neonates
born within the previous 8 h) were transferred to
eight tubes, at three individuals per tube. Altogether,
eight treatments were applied by making additions to
the media in the separate tubes:
1 One control (i.e. medium without additions)
2–4 Three different melatonin concentrations
(10
-5
,10
-7
,10
-9
M)
5 Fish kairomone
6–8 Fish kairomone plus three different melatonin
concentrations (10
-5
,10
-7
,10
-9
M).
The fish kairomone used was an extract of the
faeces of Leucaspius delineatus (Cyprynidae) fed
with live D. magna, prepared according to the
procedure of Slusarczyk & Rygielska (2004) and
stored frozen (-20°C) before use. The concentration
of kairomone was equivalent to the presence of one
fish in 10 l of water. Melatonin (Sigma) was
44 Hydrobiologia (2010) 643:43–50
123
dissolved in acetone (0.1% solution) and stored in a
refrigerator (4°C). The same acetone concentration
was in treatment 1 and 5. Earlier studies have shown
that low concentrations of acetone do not influence
Daphnia and can be used as a melatonin solvent
(Kashian & Dodson, 2004). The media in the tubes
were changed twice a day: after the laboratory sunrise
and before the laboratory sunset.
During daytime, the system was illuminated from
above by four halogen bulbs (20 W, 12 V, Phillips)
shining through a frosted plexiglass screen to provide
homogeneous diffused light. Apart from the front
panel, the sides of the aquarium were covered with
black paper. Two monochromatic closed circuit
television (CCTV) cameras were installed in front
of the aquarium and focused on the upper and lower
parts of the tubes, respectively. The cameras were
connected to a PC, which recorded 1-min long videos
at 4, 8 and 12 h after light-on (day), and 2, 4 and 6 h
after light-off (night). At night, the visible light was
turned off and the system was illuminated by 100
infra red light emitting diodes (TSAL-6400, peak
wavelength 940 nm), only when video recordings
were made. The diodes were placed inside the
aquarium in two rows (50 in each) in two plexiglass
tubes, positioned behind the flow-through tubes
containing Daphnia (Fig. 1). The first video was
recorded in the morning on the first day of the
experiment and recording was continued over the
next 5 days. After the completion of the experiment,
the position of individual Daphnia in each tube was
recorded at each time point (with 0.5 cm accuracy)
while viewing the video. Depth of each animal was
determined in the very first moment when it appeared
on the screen. For analysis, video tapes from different
treatments were randomly selected.
The resolution of the CCTV images limited the
number of tubes that could be examined simulta-
neously to 8 (i.e. each of the separate treatments
without replicates). The whole experiment was per-
formed three times with female Daphnia, but only
once with males.
For analysis of the depth distribution of female
Daphnia, five-way independent ANOVA was applied
(SPSS Statistics 16.0), with day, hour, the presence or
absence of kairomone, melatonin concentration and
replicate/experiment as independent variables. As the
experiment was run only once for males, four-way
independent ANOVA was used to statistically analyze
their depth distribution.
Results
Daphnia exposed to the chemical trace of fish
presence stayed deeper in the water than those kept
free of kairomone. Furthermore, the former animals
performed DVMs, approaching the surface during the
night and descending towards the bottom during the
day, although the difference between control and
kairomone treatment was less pronounced in males
than in females (Fig. 2). However, the differences in
behaviour due to the threat of predation became
clearly visible after 48 h of exposure, with the mean
depth of Daphnia females differing significantly
between the first 2 days and the remaining part of
the experiment, so that two homogeneous groups
could be differentiated with a Tukey HSD post hoc
test. These results are consistent with earlier research
showing that smaller (i.e. younger) Daphnia are less
vulnerable to fish predation and prefer surface waters,
also during the daytime (Hansson & Hylander, 2009).
Fig. 1 Experimental setup
of the ‘plankton organ’ used
to examine the effects of
melatonin and threat of
predation (fish kairomone)
on the depth distribution of
Daphnia. The position of
the infra red light emitting
diodes (IR LED) used for
illumination during
nocturnal video recording is
shown. Aquarium
dimensions are
60 cm 9 100 cm 9 15 cm
Hydrobiologia (2010) 643:43–50 45
123
In males, three separate homogeneous groups were
differentiated: on day 1, day 3 and days 2, 4, 5
(Tukey HSD post hoc test). Therefore, data collected
after the first 48 h of the experiment were selected for
further analysis. Since then, the mean day depth of
females in the presence of kairomone averaged
32.2 ± 2.5 vs. 3.3 ± 0.5 cm in the control treatment,
whereas in males it was 38.7 ± 1.7 vs. 14.9 ±
3.2 cm (mean ± SE).
In females, the presence of kairomone, melatonin
concentration, light conditions, day and hour of the
experiment had significant effects on Daphnia depth
selection, and all interactions of these main effects
were also significant (Table 1). In the absence of fish
kairomone, all animals stayed close to the water’s
surface in all treatments, regardless of the melatonin
concentration (Fig. 3A). However, a clear effect of
melatonin on Daphnia depth distribution was
observed in the presence of fish kairomone: Daphnia
exposed to melatonin tended to stay on average
12 cm closer to the surface (their mean day depth was
20 ± 1.4 vs. 32.2 ± 2.5 cm in the presence of
kairomone alone) and their distribution was highly
variable (Fig. 3B). A highly significant interaction
between the concentration of melatonin and the
presence of kairomone was identified (Table 1).
A significant difference was only observed between
the highest concentration of melatonin and no
exogenous melatonin in the medium (Table 2).
We also observed significant effects of all main
factors on the mean depth of Daphnia males (Table 3).
All interactions were significant with the exception of
that between melatonin concentration and hour
(Table 3). Males stayed close to the surface (albeit
deeper than females) in the absence of kairomone,
regardless of the melatonin concentration (Fig. 4A). In
the presence of the chemical trace of fish, melatonin
had a significant effect on male depth selection
(Fig. 4B), their mean day depth averaging 24.2 ±
1.6 vs. 38.7 ± 1.7 cm in the presence of kairomone
alone and the strength of its impact depending on the
concentration of the hormone. The lowest melatonin
concentration caused the strongest effect, forcing
males to approach the surface (Table 4).
Discussion
Our results clearly indicate that depth distribution of
D. magna can be influenced by melatonin. To our
knowledge, this is the first evidence that exogenous
melatonin can affect Daphnia migratory behaviour.
In the presence of fish kairomone, melatonin
appeared to ‘push’ animals closer to the surface.
This influence was observed only when the applied
melatonin was accompanied by the threat of preda-
tion, suggesting the involvement of this hormone in
the response to stress. This function of melatonin has
also been proposed in vertebrates. In several studies,
it has been observed that melatonin counteracts the
effects of stress (Reiter et al., 2000, 2008; Bob &
Fedor-Freybergh, 2008
).
Constant residing in surface layers brings fitness
advantages, since high food availability and high
temperature enable faster growth and reproduction as
Fig. 2 Depth distribution (mean ± SEM) of Daphnia females
(A) and males (B) in the presence and absence of fish
kairomone. Grey bars indicate night hours
46 Hydrobiologia (2010) 643:43–50
123
compared with deeper layers of water column, less
profitable in terms of temperature and food conditions
(Guisande et al., 1991; Dawidowicz & Loose, 1992a).
The animals not exposed to fish kairomone stayed,
indeed, close to the surface. When planktivorous
visually hunting fish are present, the risk of being
eaten during the day surpass potential benefits, unless
Daphnia are small enough not to become vulnerable
(Hansson & Hylander, 2009). Since metabolic costs
of up and down swimming are negligible in Daphnia
(Dawidowicz & Loose, 1992b), the optimal strategy
to overcome this trade-off is to descend deeper during
the day, to seek for a dark refuge against visually
hunting fish and to exploit the resources of surface
water during the night time. Approaching closer to
the surface under predation threat by melatonin-
treated animals seems to be maladaptive since it
exposes Daphnia to an increased risk from the fish. It
has recently been shown that melatonin is able to
reduce the expression of Hsp 40, Hsp 70 and Hsp 90
genes elevated after stress treatment (adriamycin,
lipopolysaccharide or arsenite) in several cell culture
systems (Catala
´
et al., 2007; Esposito et al., 2008;
Lin et al., 2008). Changes in the expression of heat
shock proteins (Hsp 60, Hsp 70, Hsp 40) are a
potential marker of kairomone-induced predator
Table 1 Effects of
different factors on depth
distribution of Daphnia
females (main effect and
interactions, five-way
independent ANOVA)
Factor df FP
Day 4 52.488 \0.0005
Hour 5 14.504 \0.0005
Kairomone 1 2149.0 \0.0005
Melatonin 3 4.972 0.002
Replicate 2 458.209 \0.0005
Day 9 hour 20 5.939 \0.0005
Day 9 kairomone 4 85.711 \0.0005
Day 9 melatonin 12 5.423 \0.0005
Day 9 replicate 8 24.487 \0.0005
Hour 9 kairomone 5 3.854 0.002
Hour 9 melatonin 15 4.278 \0.0005
Hour 9 replicate 10 18.815 \0.0005
Kairomone 9 melatonin 3 18.558 \0.0005
Kairomone 9 replicate 2 137.921 \0.0005
Melatonin 9 replicate 6 19.575 \0.0005
Day 9 hour 9 kairomone 20 5.335 \0.0005
Day 9 hour 9 melatonin 60 1.692 0.001
Day 9 hour 9 replicate 39 6.667 \0.0005
Day 9 kairomone 9 melatonin 12 2.789 0.001
Day 9 kairomone 9 replicate 8 25.750 \0.0005
Day 9 melatonin 9 replicate 24 3.201 \0.0005
Hour 9 kairomone 9 melatonin 15 3.065 \0.0005
Hour 9 kairomone 9 replicate 10 10.528 \0.0005
Hour 9 melatonin 9 replicate 30 2.552 \0.0005
Kairomone 9 melatonin 9 replicate 6 16.187 \0.0005
Day 9 hour 9 kairomone 9 melatonin 60 1.663 0.001
Day 9 hour 9 kairomone 9 replicate 39 5.909 \0.0005
Day 9 hour 9 melatonin 9 replicate 117 1.864 \0.0005
Day 9 kairomone 9 melatonin 9 replicate 24 5.594 \0.0005
Hour 9 kairomone 9 melatonin 9 replicate 30 2.942 \0.0005
Day 9 hour 9 kairomone 9 melatonin 9 replicate 111 1.959 \0.0005
Hydrobiologia (2010) 643:43–50 47
123
stress (Pijanowska & Kloc, 2004; Pauwels et al.,
2005). It may be, therefore, postulated that one of the
mechanisms responsible for the effect of exogenous
melatonin on Daphnia depth selection involves
reduction of kairomone-induced Hsp expression and
thus, reversal of the physiological effect of kairo-
mone action.
The effect of melatonin on Daphnia appeared
more pronounced during the day than at night. In
female Daphnia there was no linear correlation
between the exogenous melatonin concentration and
the strength of the response to its presence. In males,
however, the lowest melatonin concentration pro-
duced the most visible effect, with animals in this
treatment group migrating closest to the surface.
Previously, we demonstrated that melatonin levels in
Daphnia are highest in the middle of the light phase
(Markowska et al., 2009). Therefore, it is possible
that sensitivity to melatonin is also higher during the
day. As Daphnia produces melatonin in a rhythmical
manner and, simultaneously, its diurnal behaviour is
influenced by exogenous melatonin, it may be
speculated that this hormone plays a role in the
regulation of a light-triggered response to predation
in cladocerans.
As we do not know how much of the hormone is
absorbed by these animals from the medium or the
resultant ratio between endo- and exogenous levels of
melatonin, any attempt to explain this phenomenon
remains highly speculative at this stage. Further
Fig. 3 Effect of different melatonin concentrations on depth
distribution (mean ± SEM) of Daphnia females in the absence
(A) and presence of fish kairomone (B). Grey bars indicate
night hours
Table 2 Comparison of the impact of different melatonin
concentrations on depth distribution of Daphnia females
(Tukey HSD post hoc test, P = 0.05)
Melatonin
concentration
N Mean depth (cm)
Subset 1 Subset 2
0 M 471 17.293
10
-9
M 514 16.011 16.011
10
-7
M 499 16.665 16.665
10
-5
M 504 15.860
P 0.438 0.073
Table 3 Effects of different factors on depth distribution of
Daphnia males (main effect and interactions, four-way independent
ANOVA)
Factor df FP
Day 4 55.739 \0.0005
Hour 5 21.230 \0.0005
Kairomone 1 383.835 \0.0005
Melatonin 3 49.439 \0.0005
Day 9 hour 20 4.539 \0.0005
Day 9 kairomone 4 17.387 \0.0005
Day 9 melatonin 12 6.542 \0.0005
Hour 9 kairomone 4 5.989 \0.0005
Hour 9 melatonin 15 2.468 0.002
Kairomone 9 melatonin 3 5.916 0.001
Day 9 hour 9 kairomone 20 2.507 \0.0005
Day 9 hour 9 melatonin 60 1.911 \0.0005
Day 9 kairomone 9 melatonin 12 10.262 \0.0005
Hour 9 kairomone 9 melatonin 15 2.251 0.005
Day 9 hour 9 kairomone 9 melatonin 42 2.142 \0.0005
48 Hydrobiologia (2010) 643:43–50
123
investigations are necessary to identify the direct
pathways (including molecular) by which melatonin
affects Daphnia behavioural responses to biotic stress.
Acknowledgements This research was supported by
Ministry of Science and Higher Education (Poland) grants 2
P04F 036 26 and N304 094135.
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... Endogenous melatonin is produced over a day-night cycle, with a peak at midnight following the gene expression of the clock and aanat (Schwarzenberger and Wacker, 2015). It has been shown that exogenous melatonin influences Daphnia: It decreases the heart rate of D. magna (Kaas et al., 2009) and attenuates stress responses due to crowding (Schwarzenberger et al., 2014) and kairomones (Bentkowski et al., 2010;Schwarzenberger et al., 2014). Whether Daphnia's endogenous melatonin concentration is also a signal for (seasonal) change in photoperiod is unclear. ...
... A continuous increase in gene expression might be adaptive for a preparation of subsequent behavioural and physiological responses before night time falls: e.g. the observed diel vertical migration (DVM) to deeper water levels already at the end of the day (e.g. Harris, 1963;Bentkowski et al., 2010), and a timely onset of melatonin synthesis. This is well imaginable because melatonin synthesis genes (which are probably controlled by the circadian clock) also show a continuous increase in gene expression at the end of the day. ...
Article
Nearly all organisms show daily and seasonal physiological and behavioural responses that are necessary for their survival. Often these responses are controlled by the rhythmic activity of an endogenous clock that perceives day length. Day length differs not only between seasons but also along latitudes, with different seasonal day lengths between the north and the south. Both seasonal and latitudinal differences in day length are discussed to be perceived/processed by the endogenous clock. Some species are distributed over a wide range of latitudes; it should be highly adaptive for these species to be able to time physiological responses (e.g. migration behaviour and diapause) according to the organisms’ respective photoperiod, i.e. their respective seasonal and latitudinal day length. The mediator of day length is the indoleamine hormone melatonin which is synthesized by melatonin-producing enzymes (AANAT and HIOMT). These enzymes are in turn controlled by an endogenous clock. The ubiquitous aquatic keystone organism Daphnia possess clock and melatonin synthesis genes that are rhythmically expressed over 24 hours. We were able to show that the 24-hour rhythm of D. magna’s clock persists in constant darkness and is thus truly circadian. In one particular photoperiod, all D. magna clones produced a similar melatonin concentration due to a fixed AANAT activity. However, we have demonstrated that clones originating from different latitudes are adapted to their respective photoperiod by showing a geographic cline in clock and downstream melatonin synthesis gene expression. These findings hint at the problem locally adapted organisms face when they are forced to leave their respective photoperiod, e.g. because of climate change-driven range-expansion. If such a species is incapable of adjusting its endogenous clock to an unknown photoperiod, it will likely become extinct.
... Therefore, the role of endogenous melatonin has not been investigated, yet. Until now, only very few studies have tested the effects of exogenous melatonin on Daphnia: Addition of melatonin significantly decreased the heart rate of D. magna (Kaas et al., 2009), and attenuated or changed stress responses due to crowding (Schwarzenberger et al., 2014) and kairomone exposure (Bentkowski et al., 2010;Schwarzenberger et al., 2014). ...
... This is supported by the finding that exogenous melatonin leads to the alteration of locomotory activity in the cricket Acheta domesticus (Yamano et al., 2001), which is normally controlled by the circadian clock [e.g. in Drosophila (Allada and Chung, 2010) and in crustaceans (Arechiga et al., 1993)]. Similarly, in Daphnia, a treatment with exogenous melatonin has been shown to change the response to predators by disturbing diel vertical migration ( Bentkowski et al., 2010), which has also been suggested to be under circadian control . ...
Article
In freshwater systems, Daphnia has been demonstrated to show adaptive responses following the light–dark cycle. The adjustment of these responses to the change of day and night is probably transmitted via the hormone melatonin. The rate-limiting enzyme in melatonin synthesis is the arylalkylamine N-transferase (AANAT). We identified three genes coding for insect-like AANATs in Daphnia, of which we measured the gene expression in an ecologically relevant light–dark cycle. We demonstrated that Daphnia's insect-like AANAT gene expression oscillated in a daily manner, and that the highest peak of expression after the onset of darkness was followed by a peak of melatonin production at midnight. Moreover, we could show an oscillation of endogenous melatonin synthesis in Daphnia. In most organisms, melatonin synthesis is due to rhythmic expression of genes of the circadian clock, since transcription of aanats is directly linked to a circadian transcription factor. We could demonstrate that putative clock genes and insect-like AANAT genes of Daphnia were equally expressed. Therefore, we propose that melatonin synthesis is coupled to the expression of Daphnia clock genes, and that insect-like AANATs of crustaceans have a similar function as AANATs of vertebrates: The initiation of melatonin synthesis. In future studies with Daphnia, it will be necessary to take the time of day into account since melatonin concentrations might influence stress responses.
... Many Daphnia clones are migrating to deeper water layers during the day in order to escape UV light and fish predation (Loose and Dawidowicz, 1994;Rhode et al., 2001;Ekvall et al., 2015); thus, they are confronted with diurnal changes in food quality and quantity. In Daphnia, the involvement of the clock in diel vertical migration (DVM) is likely (Cellier-Michel et al., 2003), especially because DVM is inhibited by the application of exogenous melatonin (Bentkowski et al., 2010). Since food quantity often is high at the surface of freshwater bodies (where Daphnia are situated during night) and low in deeper water levels (to which Daphnia sink to during day) a circadian expression and activity of digestive enzymes is expected. ...
Article
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Cryptochromes are evolutionary ancient blue-light photoreceptors that are part of the circadian clock in the nervous system of many organisms. Cryptochromes transfer information of the predominant light regime to the clock which results in the fast adjustment to photoperiod. Therefore, the clock is sensitive to light changes and can be affected by anthropogenic Artificial Light At Night (ALAN). This in turn has consequences for clock associated behavioral processes, e.g., diel vertical migration (DVM) of zooplankton. In freshwater ecosystems, the zooplankton genus Daphnia performs DVM in order to escape optically hunting predators and to avoid UV light. Concomitantly, Daphnia experience circadian changes in food-supply during DVM. Daphnia play the keystone role in the carbon-transfer to the next trophic level. Therefore, the whole ecosystem is affected during the occurrence of cyanobacteria blooms as cyanobacteria reduce food quality due to their production of digestive inhibitors (e.g., protease inhibitors). In other organisms, digestion is linked to the circadian clock. If this is also the case for Daphnia, the expression of protease genes should show a rhythmic expression following circadian expression of clock genes (e.g., cryptochrome 2). We tested this hypothesis and demonstrated that gene expression of the clock and of proteases was affected by ALAN. Contrary to our expectations, the activity of one type of proteases (chymotrypsins) was increased by ALAN. This indicates that higher protease activity might improve the diet utilization. Therefore, we treated D. magna with a chymotrypsin-inhibitor producing cyanobacterium and found that ALAN actually led to an increase in Daphnia’s growth rate in comparison to growth on the same cyanobacterium in control light conditions. We conclude that this increased tolerance to protease inhibitors putatively enables Daphnia populations to better control cyanobacterial blooms that produce chymotrypsin inhibitors in the Anthropocene, which is defined by light pollution and by an increase of cyanobacterial blooms due to eutrophication.
... Simple predator-induced movements constitute the basis of complex migratory phenomena such as DVM or diel horizontal migration (DHM) (Lass & Spaak, 2003). Most research on predatorinduced behavior in Daphnia has focused on the DVM behavior as a strategy to lessen predation risk (Bentkowski, Markowska, & Pijanowska, 2010). The highly significant linear relationship between life history and behavior in the present study may indicate a cost tradeoff between them. ...
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Cladoceran species are important model organisms for studying aquatic ecology and evolution and are textbook examples of inducible defense against predators. To test the defense traits of Moina macrocopa in response to different fish species that co-occur or do not co-occur with the prey species in their native habitat, we conducted experiments with the three fish species Rhodeus ocellatus (wild species), golden fish Carassius auratus (artificial breeding species), and Danio rerio (model organism used in laboratories). We measured the size at maturity, time to reproduction, size of brood, and the moving rate of M. macrocopa. This crustacean species exhibited earlier reproduction time and increase in offspring number when exposed to R. ocellatus kairomone. Such changes in the life history of M. macrocopa in response to R. ocellatus were more sensitive than those to artificially raised fish (C. auratus and D. rerio). In addition, the moving rate of Moina exposed to R. ocellatus kairomone was significantly lower than to other tested fish species after the third instar stage. We found a coupling of life history and behavior-related responses evoked by R. ocellatus kairomone. These results provide evidence supporting energy re-allocation between life history and behavior in inducible defenses of M. macrocopa.
... In invertebrates, the relationship is less established and shows inconsistencies across taxa (see Jones et al. 2015 for a recent overview). Notwithstanding species-specific differences in peak concentrations, variation in melatonin concentrations are linked to shifts in behaviour in a number of invertebrates (Thakurdas et al. 2009, Yamano et al. 2001, Tosches et al. 2014, perhaps the best known of which is the diel vertical migration in Daphnia (Bentkowski et al. 2010). Of considerable interest is that the observed differences in the cycle of melatonin are often unrelated to organismal activity periods; thus, both nocturnal and diurnal species have their melatonin peak during periods of darkness. ...
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Light represents one of the most reliable environmental cues in the biological world. In this review we focus on the evolutionary consequences to changes in organismal photic environments, with a specific focus on the class Insecta. Particular emphasis is placed on transitional forms that can be used to track the evolution from (1) diurnal to nocturnal (dim-light) or (2) surface to subterranean (aphotic) environments, as well as (3) the ecological encroachment of anthropomorphic light on nocturnal habitats (artificial light at night). We explore the influence of the light environment in an integrated manner, highlighting the connections between phenotypic adaptations (behaviour, morphology, neurology and endocrinology), molecular genetics and their combined influence on organismal fitness. We begin by outlining the current knowledge of insect photic niches and the organismal adaptations and molecular modifications that have evolved for life in those environments. We then outline concepts and guidelines for future research in the fields of natural history, ethology, neurology, morphology and particularly the advantages that high throughput sequencing provides to these aspects of investigation. Finally, we highlight that the power of such integrative science lies in its ability to make phylogenetically robust comparative assessments of evolution, ones that are grounded by empirical evidence derived from a concrete understanding of organismal natural history.
... The use of fish kairomones for inducing zooplankton DVM would allow easier and more detailed experiments. Unfortunately, the chemical structure of the kairomones is not yet known in detail, although there are some first results suggesting some molecules (Akkas et al. 2010, Bentkowski et al. 2010 ...
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Circadian rhythms enable organisms to mediate their molecular and physiological processes with changes in their environment. Although feeding behavior directly affects within-organism processes, there are few examples of a circadian rhythm in this key behavior. Here, we show that Daphnia have a nocturnal circadian rhythm in feeding behavior that corresponds with their diel vertical migration (DVM), an important life history strategy for predator and UV avoidance. In addition, this feeding rhythm appears to be temperature compensated, which suggests that feeding behavior is robust to seasonal changes in water temperature. A circadian rhythm in feeding behavior can impact energetically demanding processes like metabolism and immunity, which may have drastic effects on susceptibility to disease, starvation risk, and ultimately, fitness.
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Melatonin is a ubiquitous and conservative biogenic amine implicated in a variety of bio-physiological processes. Although the daily variations of melatonin concentrations have been determined in many crustacean species, results have been conflicting. The present study was undertaken to evaluate the effect of different photoperiods on the circadian rhythms of melatonin in Eriocheir sinensis by using high performance liquid chromatography. The circadian rhythms of melatonin in the eyestalk of E. sinensis were influenced by external light stimulation. When under consistent light (24L:0D) and consistent dark (0L:24D), the melatonin rhythm in eyestalks and hemolymph was disturbed with respect to that of the control group (12L:12D). However, disturbance owing to different photoperiod treatments decreased with increasing treatment duration. Our results underscore the exquisite sensitivity of melatonin content to different photoperiods. These findings may have real-world implications in terms of the disruption of circadian rhythms induced by exposure to different photoperiods.
Article
This study is the first to examine the circadian rhythms of melatonin in Eriocheir sinensis and Palaemonetes sinensis, two economically important crustaceans. We collected haemolymph and optic lobes from both species every 4 h over a whole day cycle. Melatonin content was measured with high-performance liquid chromatography. E. sinensis haemolymph exhibited significant (p < 0.05) peaks in melatonin at 16:00 (0.180 ± 0.020 μg·mL⁻¹) and 24:00 (0.244 ± 0.055 μg·mL⁻¹), while eyestalks had significant peaks at 16:00 (72.377 ± 18.100 μg·eyestalk⁻¹) and 24:00 (98.756 ± 30.271 μg·eyestalk⁻¹). In P. sinensis, melatonin peaked significantly only at 16:00 in optic lobes (12.493 ± 1.475 μg·eyestalk⁻¹) (p < 0.05); no significant peaks were present in haemolymph. Thus, both E. sinensis and P. sinensis exhibit species-specific melatonin rhythms. Time of day should therefore be considered when examining the physiological status of both crustaceans, given the potential influence of fluctuating daily melatonin concentrations.
Article
Pea aphids, Acyrthosiphon pisum, were fed on artificial diet containing various concentrations of melatonin. Under long-day conditions (16h light:8h dark) their progeny included males and virginoparous/oviparous (asexual/sexual) intermediate females, which normally occur only in short days or around critical night-length. Endogenous melatonin in pea aphids was measured by radioimmunoassay and verified by parallelism with a melatonin standard curve and by thin layer chromatography. However, melatonin titres showed large variations and although they tended to be higher during the scotophase than during the photophase they were not significantly different. The possibility of melatonin being involved in photoperiodism is discussed.
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In a laboratory batch culture experiment, females of Daphnia Magna were exposed to five different experimental media containing either: (1) water from an aquarium with fish, (2) extract of fish faeces, (3) mixture of both media, (4) extract of homogenised conspecific Daphnia, or (5) control water without the addition of extra cues. The experiment was planned to test potential pathways of excretion of the chemical cues that induce resting‐egg formation in D. magna and to find an effective way of collecting these chemical cues. The results indicate that fish faeces are the prevailing source of the chemical cues that induce resting‐egg production in D. magna. The ease of collection and the possibility of storing it in a frozen state make it a convenient cue for inducing diapause response in Daphnia. The results of the experiment imply that in natural conditions Daphnia may face high concentration of the inductive signals once migrating to the bottom zone where fish faeces commonly accumulate.
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Melatonin is a ubiquitous molecule and widely distributed in nature, with functional activity occurring in unicellular organisms, plants, fungi and animals. In most vertebrates, including humans, melatonin is synthesized primarily in the pineal gland and is regulated by the environmental light ⁄ dark cycle via the suprachiasmatic nucleus. Pinealocytes function as ‘neuroendocrine transducers’ to secrete melatonin during the dark phase of the light ⁄ dark cycle and, consequently, melatonin is often called the ‘hormone of darkness’. Melatonin is principally secreted at night and is centrally involved in sleep regulation, as well as in a number of other cyclical bodily activities. Melatonin is exclusively involved in signaling the ‘time of day’ and ‘time of year’ (hence considered to help both clock and calendar functions) to all tissues and is thus considered to be the body’s chronological pacemaker or ‘Zeitgeber’. Synthesis of melatonin also occurs in other areas of the body, including the retina, the gastrointestinal tract, skin, bone marrow and in lymphocytes, from which it may influence other physiological functions through paracrine signaling. Melatonin has also been extracted from the seeds and leaves of a number of plants and its concentration in some of this material is several orders of magnitude higher than its night-time plasma value in humans. Melatonin participates in diverse physiological functions. In addition to its timekeeping functions, melatonin is an effective antioxidant which scavenges free radicals and up-regulates several antioxidant enzymes. It also has a strong antiapoptotic signaling function, an effect which it exerts even during ischemia. Melatonin’s cytoprotective properties have practical implications in the treatment of neurodegenerative diseases. Melatonin also has immuneenhancing and oncostatic properties. Its ‘chronobiotic’ properties have been shown to have value in treating various circadian rhythm sleep disorders, such as jet lag or shift-work sleep disorder. Melatonin acting as an ‘internal sleep facilitator’ promotes sleep, and melatonin’s sleep-facilitating properties have been found to be useful for treating insomnia symptoms in elderly and depressive patients. A recently introduced melatonin analog, agomelatine, is also efficient for the treatment of major depressive disorder and bipolar affective disorder. Melatonin’s role as a ‘photoperiodic molecule’ in seasonal reproduction has been established in photoperiodic species, although its regulatory influence in humans remains under investigation. Taken together, this evidence implicates melatonin in a broad range of effects with a significant regulatory influence over many of the body’s physiological functions.
Article
For Daphnia hyalina the amplitudes of vertical migrations were 3 and 60 m d-1; food conditions were 0.1 and 1.0 mg C liter-1. None of the investigated parameters (fecundity, individual growth rate, age of maturation) was significantly different between long- and short-distance migrating populations at high levels of food, whereas at low levels of food only the individual growth rate was higher in the short-distance migrating Daphnia. Results suggest that the energy spent to travel the vertical distance does not account for the metabolic costs of diurnal vertical migration. -from Authors
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SYNOPSIS. Melatonin has many actions in vertebrates, with some considered hormonal. But are some melatonin actions more ancient than others? A survey of the tissues which synthesizemelatonin demonstrates that some are more recent vertebrate characters compared to others, indicating that melatonin action in these tissues also is more recent. The lateral eyes and pineal organs appear to be very ancient sources of melatonin and any action this molecule has within these tissues should be considered primordial. We hypothesize that melatonin's first actions (functions) were paracrine, that is, operating within these photoreceptive structures to facilitate the process of photoreception. Such actions have been documented. It is hypothesized that melatonin synthesis occurred at nightwithin the pineal organs and retinas of ancient vertebrates, as is the case among extant vertebrates. Accompanying the nightly synthesis of melatonin for paracrine function, secretion of melatonin either incidental or for detoxification by the liver occurred, providing a faithful template of the onset and/or duration of the scotophase. This nightly pulse of melatonin could provide important timing information to distant tissues capable of receiving the signal. The number of physiological systems within vertebrates "using" thenightly circulating melatonin pulse, and the apparent increased importance of circulating melatonin in timing physiological events in mammals, like reproduction, is the result of recent cooptation.
Article
Predator evasion is the most commonly hypothesized reason for diel vertical migrations undertaken by a wide variety of planktonic organisms in lakes and seas, yet direct evidence remains elusive. We tested the predation hypothesis by exposing enclosed populations of a marine copepod Acartia hudsonica to caged or free-ranging individuals of their natural predator, the planktivorous fish Gasterosteus aculeatus. After little more than a week, adult copepods changed their vertical distribution and migration behavior depending on the presence or absence of predation. Only free-ranging fish induced vertical migration in the copepod population. Caged fish had no effect, indicating that vertical migration was not a simple chemically mediated response of copepods to the predator. Rather, copepods seemed to react to the presence of predators by other means, perhaps visual or mechanical stimuli, and to exhibit a downward escape response which, because encounters with visually orienting fish occur chiefly in the daytime, effectively limited the copepods' occurrence in the upper water column to the night-time hours. Alternatively, because fish imposed heavy mortality on copepods, it is possible that selective predation altered the proportions of individuals with fixed, genetically determined migration behaviors. We suggest experiments to distinguish these alternatives.