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Melatonin is a molecule found in both prokaryotic and eukaryotic organisms. Its presence has been demonstrated in all examined species of bacteria, plants, invertebrates and vertebrates. In the pineal gland and blood plasma of vertebrates, melatonin reaches its highest levels during the nighttime. However, this rhythm of melatonin production does not seem to occur in all invertebrates. Forexample, in three separate decapod species, the highest level of melatonin synthesis was observed either at night or day (Procambarus clarkii), during the day (Macrobrachium rosenbergii), or the level remained constant (Carcinus maenas). In the last decade, Daphnia has become a model organism for freshwater studies. It is an aquatic equivalent of the fruit fly, Drosophila, in which melatonin was discovered nearly 15 yr ago. Surprisingly, melatonin has never been studied in Daphnia; therefore, we sought to determine whether melatonin is present in this small crustacean.
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Journal of Pineal Research (2009) 46: 242–244
LETTER TO THE EDITOR
Presence of melatonin in Daphnia magna
Magdalena Markowska, Piotr Bentkowski, Malgorzata Kloc, Joanna Pijanowska
DOI: 10.1111/j.1600-079X.2008.00642.x
Melatonin is a molecule found in both prokaryotic
and eukaryotic organisms. Its presence has been
demonstrated in all examined species of bacteria,
plants, invertebrates and vertebrates. In the pineal
gland and blood plasma of vertebrates, melatonin
reaches its highest levels during the nighttime.
However, this rhythm of melatonin production does
not seem to occur in all invertebrates [1]. For
example, in three separate decapod species, the
highest level of melatonin synthesis was observed
either at night or day (Procambarus clarkii) [2, 3],
during the day (Macrobrachium rosenbergii) [4], or
the level remained constant (Carcinus maenas) [5]. In
the last decade, Daphnia has become a model
organism for freshwater studies [6]. It is an aquatic
equivalent of the fruit fly, Drosophila, in which
melatonin was discovered nearly 15 yr ago [7].
Surprisingly, melatonin has never been studied in
Daphnia; therefore, we sought to determine whether
melatonin is present in this small crustacean.
Daphnia magna (clone P3) used in our
experiments originated from the Binnensee, a shallow
brackish lake in North Germany. Experimental
animals were obtained from the second clutch of a
synchronized population of mothers originating from
a single female. The experimental populations were
kept at 22 ± 2°C in filtered lake water and a long-day
summer photoperiod (16:8 L:D – white light : dim red
light) was applied. Daphnia were kept in glass
containers (60 individuals/container) and fed with
Scenedesmus at a concentration of 1.0 mg org. C/L.
For melatonin analysis, harvesting of Daphnia (100 ±
4 hr-old) started 24 hr after any remaining
Scenedesmus were removed. Every 2 hr during the
day and every hour at night, all 60 animals from a
single container were harvested and frozen at – 85°C.
Melatonin is also present in algae [8]; therefore, to be
sure that our measurements reflect genuine levels of
D. magna melatonin, we also examined melatonin
concentrations in Scenedesmus. An experimental
culture of Scenedesmus was propagated under the
same conditions used for D. magna, and according to
the same schedule, 50 mL samples were collected and
immediately frozen at – 85°C.
The melatonin concentration in D. magna
and Scenedesmus obliquus sonicated samples was
measured using an enzyme immunoassay (ELISA)
(RE54021, IBL-Hamburg, Germany) according to the
manufacturer instruction. Melatonin levels were
normalized against total protein (deter- mined using
the Bradford method) in Daphnia or organic carbon
(spectrophotometrically determined at 800 nm
wavelength) in Scenedesmus samples. The
Fig. 1. Melatonin concentration in samples of Daphnia
magna (A) and S. obliquus (B) kept in L:D 16:8. D. magna
melatonin levels are presented as means with SE values for
three independent experiments. a – P= 0.05 ZT 5 versus all
other time points. The black bar denotes the dark phase.
experiment was repeated three times. Statistical
differences between the melatonin concentrations at
different time points were assessed by one-way
ANOVA followed by the Tukey test. We also
examined the localization of melatonin in D. magna
Fig. 2. Immunostaining of melatonin in the tissues of Daphnia magna. (A–C) Area of the head region including the cyclopic
eye (short arrows) and adjacent nerve tissues showing the presence of melatonin in nerve fibers (long arrow), the optic
ganglion (filled circle) and supraesophageal ganglion (asterisk * ). The dark staining within the cyclopic eye is visual
pigment. (D) Area of a longitudinal section through the body showing the presence of melatonin in thorecopods (arrows).
(E) High magnification of a melatonin-positive thorecopod. (F) Longitudinal section through a whole Daphnia with positive
staining for melatonin, and (G) negative control. A, B, D–G = ZT 8, C = ZT 20. The scale bar is equal to 60 μm in A, 25 μm
in B, 40 μm in C, 100 μm in D, 11 μm in E and 200 μm in F and G.
by immunostaining. Daphnia collected at ZT 8 and
ZT 20 were fixed in Bouin fixative (Sigma-Aldrich,
San Louis, MO, USA) for several hours. Paraplast
(Fisher Scientific, IL, USA) embedded samples were
cut to 10 μm sections using a microtome. After
blocking with caseine blocking buffer (Bio-Rad
Laboratories, Hercules, CA, USA), the sections were
incubated with primary rabbit polyclonal anti-
melatonin antibody (1:200 dilution, Abcam,
Cambridge, MA, USA) for 1 hr and thereafter with
secondary anti-rabbit antibody conjugated to alkaline
phosphatase (1:200 dilution Calbiochem, San Diego,
CA, USA), stained using NBT/BCIP (Roche,
Indianapolis, IN, USA), postfixed overnight in 2%
formalin in PBS, then dehydrated, cleared, mounted
in Permount and photographed under a Nikon
microscope.
Melatonin was present in samples of D.
magna collected at all time points, with the maximum
concentration occurring during the light phase. At
time point ZT 5, levels of melatonin were
significantly higher than those found during the dark
phase (Fig. 1A). Concentrations of melatonin in S.
obliquus were below the assay detection limit (Fig.
1B). We were also able to confirm the presence of
melatonin in D. magna tissues by immunostaining
with anti-melatonin antibody. Melatonin was present
in individuals harvested at all stages of the light cycle
(i.e. both day and night), with highest levels detected
in the cyclopic eye nerve fibers, optic and
supraesophageal ganglions and the thorecopods (Fig.
2). To our knowledge, these results represent the first
evidence for the presence of melatonin in Daphnia
magna. In animals kept under a L:D 16:8 light
regime, melatonin exhibited a circadian rhythm with
the maximum concentration occurring during the light
phase. Melatonin was localized in structures
connected with the optical system and surprisingly it
was also detected in the thorecopods. Further studies
are required to determine whether the observed
rhythm of melatonin is generated endogenously or
induced by external factors such as light and food.
For instance, in rats kept on a restricted diet, serum
melatonin and pineal NAT levels were found to be
altered compared with those in rats fed ad libitum [9].
Moreover, melatonin synthesis in vertebrates is
controlled by light conditions in the environment. It is
also important to identify the physiological functions
of melatonin in D. magna. In vertebrates, melatonin is
known to modulate circadian locomotor activity; so
an analogous role in Daphnia migratory behavior,
well recognized as an adaptive response to predation,
and in other rhythmical behaviors, is quite plausible.
Acknowledgements
This study was supported by Polish Ministry of
Science and Higher Education, grant no. 6 P04F 036
26. This research was performed at the faculty of
Biology, University of Warsaw, Poland and at the
Methodist Hospital Research Institute, Houston,
USA.
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Cite:
Markowska M, Bentkowski P, Kloc M, Pijanowska J. (2009). Presence of melatonin in Daphnia
magna. J Pineal Res 46: 242–244.
... Due to rhythmic expression of genes from the melatonin synthesis pathway, melatonin is produced in the brain's pineal gland of vertebrates, which is in response to signals originating from the endogenous clock (Foulkes et al., 1997). Similarly, in Daphnia the highest concentration of melatonin has been detected in the head region [and in the thoracopods; (Markowska et al., 2009)]. Also, it has been shown that melatonin is present in Daphnia day and night, which suggests a cyclical change in melatonin concentration over the light -dark cycle. ...
... The information of day length is transmitted by the duration of the signal by the hormone melatonin (Lincoln, 2006). Melatonin is a molecule which can be found in all kinds of organisms from prokaryotes to eukaryotes and has very recently been detected in Daphnia (Markowska et al., 2009). In vertebrates, the rate-limiting enzyme of melatonin synthesis is the AANAT (Klein, 2007). ...
... The first time point (9:30 pm) served as calibrator for relative gene expression and was always set as 1. increased gene expression followed by a peak in melatonin production is conspicuous. The clear one peak/one nadir production of melatonin in D. pulex was different from earlier findings for another Daphnia species: In D. magna, the highest peak of melatonin production ( 100 pg melatonin per mg protein) was measured during the light phase (Markowska et al., 2009). Unfortunately, a direct comparison between Daphnia and other arthropods is not really feasible, since most studies focused on melatonin concentrations of brains, eyestalks or heads and not of whole organisms (Vivien-Roels and Pévet, 1993). ...
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... In Crustaceans, on the other hand, there is no consensus as the time of the day at which melatonin peaks in the various species as well as if this hormone displays an oscillatory pattern (Agapito et al., 1995;Tilden et al., 1997;Maciel et al., 2008;Markowska et al., 2009;Sainath et al., 2013;Han et al., 2018). As to melatonin actions in crustaceans, it has already been demonstrated that the indoleamine plays a role in limb regeneration (Tilden et al., 1997), ecdysteroid production (Sainath S. B. and Reddy P. S., 2010;Girish et al., 2015), antioxidant defense Geihs et al., 2016;She et al., 2019), color change (Nery et al., 1999), locomotor activity , and hyperglycemia (Sainath SB. and Reddy PS., 2010;Maciel et al., 2014;Yang et al., 2018), among others. ...
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In the past 30 years, Daphnia has become a model organism in aquatic ecology. I review the changing concepts and paradigms in plankton ecology as reflected in the work on Daphnia. The availability of radiotracers favoured a new physiological approach that resulted in better energetic models and more reliable estimates of filtering rates. This led to deeper insights into the role of herbivore grazing on phytoplankton and microbial communities, and nutrient recycling. It provided a conceptual basis for general hypotheses on predictable seasonal successions (e.g. the PEG model). On the other hand, increasing knowledge about selective predation on zooplankton triggered population dynamic models and gave explanations for changing community structures. The Size-Efficiency-Hypothesis generated a framework for studies on trade-offs between competitive ability and susceptibility to predation. Daphnia was now in the centre of interaction-based concepts, being predator and prey at the same time. It was the backbone of practical applications of the theory in food-web manipulations. When ultimate factors came into the focus, Daphnia played an important role in explaining striking phenomena like diel vertical migration and cyclomorphosis. Its central position in food-webs, the unique propagation mode, easy cultivation and accessibility by molecular genetic methods made it a favourite object for studies in evolutionary ecology, concerning local adaptation, evolution of defences and life histories, induced phenotypic change, and genetic diversity. The large advantage of Daphnia over other biological model organisms is that its importance in pelagic freshwater systems is undoubtedly known. Hence there is a direct way of applying the results to ecological systems.
Article
In crustaceans, melatonin has been detected in the central nervous system and some other organs. The aim of this study was to analyze the melatonin content in the visual system of Procambarus clarkii, by means of radioimmunoassay, at different day-night phases. We have also studied the action of exogenous melatonin on the main properties of the electroretinogram (ERG) circadian rhythm. Experiments were conducted with 25 specimens maintained under controlled conditions of 16°C and 12 h of light alternating with 12 h of darkness. Eyes where cut in dim red light and shock frozen with liquid nitrogen and pulverized in a mortar until a homogenous powder was obtained. Melatonin was extracted with acetone, followed by centrifugation, diluted with an equal volume of equa bidest to ensure freezing at −80°C for at least 90 min and lyophilization at the same temperature. Lyophilizates, after having been dissolved in RIA buffer, were used for determinations of melatonin. Long-term recordings of electrical responses to light (ERG) were obtained for 10 or more consecutive days. At the 5th day, a single dose of melatonin was injected and its effects on amplitude and period of the ERG circadian rhythm were measured. Melatonin concentrations differed considerably depending on the circadian time and attained a maximum during dark phase. Among the crustaceans, Procambarus clarkii represents the first case in which melatonin peaks during the night following the typical pattern known in the majority of organisms. After melatonin injection, period and amplitude of the ERG circadian rhythm were increased. This effect suggests the involvement of melatonin in the oscillators underlying the generation and expression of circadian rhythms in crayfish.
Article
N-acetyltransferase (NAT) and melatonin were determined in the optic lobe of the giant freshwater prawn Macrobrachium rosembergii de Man. The prawns were divided into three groups: fast-growing “jumper” males; slow-growing “laggard” males; and females. Both NAT and melatonin levels in the jumper and laggard males were comparable, whereas those of the female were significantly lower. The results suggested a sexual dimorphism in the NAT and melatonin in the optic lobe of this species. It was also found that when one optic lobe was isolated, the level of NAT and melatonin in the contralateral optic lobe did not show a compensatory increase in either males or females. On the contrary, melatonin was suppressed in the remaining optic lobes in both sexes.
Article
This study investigated the effect of dietary composition and food access schedule on the rhythmicity of serum melatonin and pineal N-acetyltransferase (NAT) activity. Wistar rats maintained on a 12:12 h light-dark cycle were assigned to two dietary groups: a group fed rat chow and a group fed a choice between a protein-rich and a carbohydrate-rich diet. Each dietary group was further divided based on feeding schedule, with food available between 0800 and 1600 h or ad lib access to food. Regardless of dietary condition, total food and carbohydrate intake of rats having free access to food was higher than under the restricted food access schedule. Protein intake of rats fed the dietary choice was lower with the restricted access than in the free access. In rats fed the dietary choice, melatonin levels and NAT activity were significantly decreased with restricted access compared to free access. Such results were not found in rats offered restricted chow. This study suggests that the rhythms of melatonin secretion and NAT activity can be altered by dietary composition.
Article
Oxidation of melatonin was followed by measuring chemiluminescence emitted during pyrrole ring cleavage, a process leading to the main oxidation product of this indoleamine, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). Radical reactions of melatonin were studied in two variants of a moderately alkaline (pH 8) H2O2 system, one of which contained hemin as a catalyst. In both systems, light emission from melatonin oxidation lasted several hours. Time courses and turnover rates depended on the presence or absence of hemin; the catalyst enhanced light emission many-fold. In the two reaction systems, the presence of hydrogen carbonate (HCO)(3)(-) enhanced chemiluminescence by more than 10-fold, indicating scavenging of carbonate radicals. In the presence of 10% dimethylsulfoxide (DMSO) or 1 m mannitol, HCO(3)(-)-dependent as well as independent light emissions were only partially inhibited. With regard to the stimulatory effect of HCO(3)(-), this implies a formation of carbonate radicals (CO)(3)(-) independent of hydroxyl (OH) radicals, presumably involving superoxide anions abundantly present in the system. Tiron, a scavenger of superoxide anions, strongly and almost instantaneously inhibited chemiluminescence, in accordance to the requirement of this reactive oxygen species for AFMK formation and its involvement in -radical formation. Melatonin's capability of scavenging CO(3)(-) may contribute to its protective potency.
Article
Melatonin has been detected in bacteria, eukaryotic unicells, macroalgae, plants, fungi and various taxa of invertebrates. Although precise determinations are missing in many of these organisms and the roles of melatonin are still unknown, investigations in some species allow more detailed conclusions. Non-vertebrate melatonin is not necessarily circadian, and if so, not always peaking at night, although nocturnal maxima are frequently found. In the cases under study, the major biosynthetic pathway is identical with that of vertebrates. Mimicking of photoperiodic responses and concentration changes upon temperature decreases have been studied in more detail only in dinoflagellates. In plants, an involvement in photoperiodism seems conceivable but requires further support. No stimulation of flowering has been demonstrated to date. A participation in antioxidative protection might be possible in many aerobic non-vertebrates, although evidence for a contribution at physiological levels is mostly missing. Protection from stress by oxidotoxins or/and extensions of lifespan have been shown in very different organisms, such as the dinoflagellate Lingulodinium, the ciliate Paramecium, the rotifer Philodina and Drosophila. Melatonin can be taken up from the food, findings with possible implications in ecophysiology as well as for human nutrition and, with regard to high levels in medicinal plants, also in pharmacology.
Article
A widespread occurrence of melatonin in plant kingdom has been reported. The circadian rhythm in the level of melatonin observed in both unicellular algae and higher plants, suggests a role in regulation of photoperiodic and rhythmic phenomena, i.e. a similar function for melatonin in both plants and animals. Evidence has been obtained for a role of melatonin in plant morphogenesis, but more research is needed to ascertain other suggested physiological roles in higher plants (seed dormancy regulation, radical scavenger activity, interaction with calmodulin) as well the ecological significance of the high melatonin levels recorded in alpine plants. Setting-up more reliable analytical methods for melatonin detection and quantification is a basic requirement to get more insight into melatonin roles in plant physiology and ecology.