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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
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
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.
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,
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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). ...
... A. SCHWARZENBERGER AND A. WACKER j MELATONIN SYNTHESIS FOLLOWS A DAILY CYCLE IN DAPHNIA 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). ...
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.
... In many other organisms however, when nights get longer in autumn, also the melatonin signal is longer. Melatonin is a molecule which can be found in all kinds of organisms from prokaryotes to eukaryotes and has also been detected in the freshwater micro-crustacean Daphnia (Markowska et al. 2009). In most animals, melatonin is produced at night, which is also the case for D. pulex (Schwarzenberger and Wacker 2015). ...
... In mammals, it is synthesized in the pineal gland in the animals' brains as a result of rhythmic transcription of genes of the circadian clock (Foulkes et al. 1997). Also in Daphnia the highest concentration of melatonin was detected in the nervous system (Markowska et al. 2009), which is in line with findings for several other invertebrates (Vivien-Roels and Pévet 1993). Until today, more and more functions of melatonin have been found in diverse organisms (reviewed in Poeggeler 1993): These functions include adjustment of the circadian clock to the environmental light regime and anti-stress effects. ...
Changes in day-length entrain the endogenous clock of organisms leading to complex responses to photoperiod. In long-lived organisms experiencing several seasons this response of the clock to photoperiod is phenotypically plastic. However, short-lived organisms often experience a single season without pronounced changes in day-length. For those, a plastic response of the clock to different seasons would not necessarily be adaptive. In aquatic ecosystems, zooplankton species like Daphnia live only for some weeks, i.e. one week up to ca. two months. However, they often show a succession of clones that are seasonally adapted to environmental changes. Here, we found that 16 Daphnia clones per each of three seasons ( = 48 clones) from the same pond and year differed in clock gene expression with a homogenous gene expression pattern in ephippia-hatched spring clones and a bimodal expression pattern in summer and autumn populations indicating an ongoing adaptation process. We clearly demonstrate that spring clones were adapted to a short, and summer clones to a long photoperiod. Furthermore, we found that gene expression of the melatonin-synthesis enzyme AANAT was always lowest in summer clones. In the Anthropocene, Daphnia’s clock might be disturbed by light-pollution and global warming. Since Daphnia is a key-organism in trophic carbon transfer, a disruption of its clock rhythm would be devastating for the stability of freshwater ecosystems. Our results are an important step in understanding the adaptation of Daphnia’s clock to environmental changes.
... 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. ...
... The values are expressed as median, quantiles, maximum, and minimum concentration. Procambarus clarkii (Agapito et al., 1995), Uca pugilator (Tilden et al., 2001) and Daphnia magna (Markowska et al., 2009), or in both the photophase and the scotophase as in N. granulata (Maciel et al., 2008). In C. sapidus, circulating melatonin did not exhibit a temporal oscillation in intermolt or premolt crabs, and no concentration difference was seen between stages. ...
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Environmental cues synchronize endogenous rhythms of many physiological processes such as hormone synthesis and secretion. Little is known about the diurnal pattern of hormones and gene expression of the Callinectes sapidus molt cycle. We aimed to investigate in the eyestalk and hepatopancreas of premolt and intermolt C. sapidus the following parameters: 1) the diurnal expression of the ecdysteroid receptor CasEcR isoforms, and the molt inhibiting hormone CasMIH; 2) the diurnal hemolymph ecdysteroid and melatonin levels; and 3) melatonin effects on the transcripts of the above-mentioned genes in intermolt C. sapidus. Ecdysteroid levels were higher in the premolt than the intermolt animals at all time points evaluated (ZTs). Premolt crabs displayed a variation of ecdysteroid concentration between time points, with a reduction at ZT17. No difference in the melatonin level was seen in either molt stage or between stages. In the eyestalk of intermolt animals, CasEcR expression oscillated, with a peak at ZT9, and premolt crabs have a reduction at ZT9; CasMIH transcripts did not vary along 24h in either molt stage. Moreover, the evaluated eyestalk genes were more expressed at ZT9 in the intermolt than the premolt crabs. In the hepatopancreas, CasEcR expression showed a peak at ZT9 in premolt crabs. Exogenous melatonin (10−7 mol/animal) reduced the expression of both genes in the eyestalk at ZT17. In the hepatopancreas, melatonin markedly increased the expression of the CasEcR gene at ZT9. Taken altogether, our results are pioneer in demonstrating the daily oscillation of gene expression associated to molt cycle stages, as well as the daily ecdysteroid and melatonin levels and the remarkable influence of melatonin on the molt cycle of C. sapidus.
... Melatonin has very recently been detected in the crustacean Daphnia [12], which is an important model organism in biological and especially in ecological studies [13]. Moreover, in Daphnia the highest concentration of melatonin was detected in the nervous system [12]. ...
... Melatonin has very recently been detected in the crustacean Daphnia [12], which is an important model organism in biological and especially in ecological studies [13]. Moreover, in Daphnia the highest concentration of melatonin was detected in the nervous system [12]. This situation is comparable to that of mammals, in which melatonin is synthesized in the pineal gland in the brain. ...
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Background The widespread occurrence of melatonin in prokaryotes as well as eukaryotes indicates that this indoleamine is considerably old. This high evolutionary age has led to the development of diverse functions of melatonin in different organisms, such as the detoxification of reactive oxygen species and anti-stress effects. In insects, i.e. Drosophila, the addition of melatonin has also been shown to increase the life span of this arthropod, probably by reducing age-related increasing oxidative stress.Although the presence of melatonin was recently found to exist in the ecological and toxicological model organism Daphnia, its function in this cladoceran has thus far not been addressed. Therefore, we challenged Daphnia with three different stressors in order to investigate potential stress-response attenuating effects of melatonin. i) Female and male daphnids were exposed to melatonin in a longevity experiment, ii) Daphnia were confronted with stress signals from the invertebrate predator Chaoborus sp., and iii) Daphnia were grown in high densities, i.e. under crowding-stress conditions.ResultsIn our experiments we were able to show that longevity of daphnids was not affected by melatonin. Therefore, age-related increasing oxidative stress was probably not compensated by added melatonin. However, melatonin significantly attenuated Daphnia¿s response to acute predator stress, i.e. the formation of neckteeth which decrease the ability of the gape-limited predator Chaoborus sp. to handle their prey. In addition, melatonin decreased the extent of crowding-related production of resting eggs of Daphnia. Conclusions Our results confirm the effect of melatonin on inhibition of stress-signal responses of Daphnia. Until now, only a single study demonstrated melatonin effects on behavioral responses due to vertebrate kairomones, whereas we clearly show a more general effect of melatonin: i) on morphological predator defense induced by an invertebrate kairomone and ii) on life history characteristics transmitted by chemical cues from conspecifics. Therefore, we could generally confirm that melatonin plays a role in the attenuation of responses to different stressors in Daphnia.
... In vertebrates, this indeloamine hormone is produced in the brain's pineal gland (Foulkes et al., 1997). Similarly, the highest concentration of melatonin in Daphnia has been detected in the head region (Markowska et al., 2009). In crustaceans, many rhythmically expressed genes have been detected that are probably controlled by the clock, e.g., immune genes in Daphnia (Rund et al., 2016). ...
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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.
... period, cycle, clock, timeless and cryptochrome 2) has been observed one hour after the onset of darkness in a 16:8 h light-dark cycle (Schwarzenberger and Wacker, 2015). Melatonin has also been detected in Daphnia, with the highest concentration in the head region and the thoracopods (Markowska et al., 2009). 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). ...
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.
... MEL is an universal and multifunctional molecule found in both vertebrates and invertebrates (Markowska et al., 2009;Reiter et al., 2010). In vertebrates, this hormone, synthesized in the pineal gland, is known to regulate many physiological processes such as seasonal reproduction (Pang et al., 1998), locomotor activity (Underwood et al., 2001) and immunity (Guerrero & Reiter, 2002;Majewski et al., 2005a, b;Piesiewicz et al., 2012a;Turkowska et al., 2013). ...
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Previously, we have demonstrated the postembryonic development of chicken (Gallus gallus domesticus L.) pineal gland functions expressed as changes in melatonin (MEL) biosynthesis. Pineal concentrations of MEL and its precursor serotonin (5-HT) were shown to increase between the 2nd and 16th day of life. We also found that levels of the mRNAs encoding the enzymes participating in the final two steps of MEL biosynthesis from 5-HT: arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as their enzymatic activities, were raised during postembryonic development. Moreover, the manner of these changes was season-of-hatch dependent, even in animals kept under constant laboratory conditions (L:D 12:12). The most pronounced changes were seen in the concentrations of 5-HT and MEL, as well as in Aanat mRNA level and its enzymatic activity. The high daily variability in 5-HT content suggested that season- and age-dependent changes in the activity of the chicken pineal gland might rely on the availability of 5-HT, i.e. it may be limited by changes in pineal tryptophan (TRP) and/or 5-hydroxytryptophan (5-HTP) levels as well as by the activity of tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC): two enzymes participating in the conversion of TRP to 5-HT. The present study was undertaken with the following objectives: (1) to examine whether the pineal concentration of the 5-HT precursors TRP and 5-HTP exhibit age- and season-related changes; (2) to look for season-related differences in the transcription of the Tph1 and Ddc genes encoding enzymes TPH and AADC; (3) to identify the step(s) in postembryonic development in which these season-related variations in pineal gland function are most pronounced. Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held in L:D 16:8 conditions were kept from the day of hatch in L:D 12:12 conditions. At the age of 2 or 9 days, animals were sacrificed every 2 or 4 h over a 24-h period and their pineal glands were isolated under dim red light and processed for the measurement of (i) the pineal content of TRP, 5-HTP and 5-HT, and (ii) the level of Tph1 and Ddc mRNAs. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The pineal levels of TRP and 5-HT as well as the Tph1 and Ddc transcripts changed during postembryonic development in a season-related way. Whereas, the 5-HTP concentration did not vary between animals from both age groups, regardless of the season. Circadian rhythmicity of all the measured parameters was dependent on both the age and the season of hatch, and was greatest in older animals in the summer. These findings indicated that the efficiency of season-related MEL biosynthesis, reported previously, is limited by 5-HT availability and this limitation depends on the transcription of both the Tph1 and Ddc genes. Moreover, Ddc mRNA level in 9-d-old birds changed rhythmically, even though this gene is generally considered to be arrhythmic.
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Melatonin, N-acetyl-5-methoxytryptamine, is an indole mainly synthesized from tryptophan in the pineal gland and secreted exclusively during the night in all the animals reported to date. While the pineal gland is the major source responsible for this night rise, it is not at all the exclusive production site and many other tissues and organs produce melatonin as well. Likewise, melatonin is not restricted to vertebrates, as its presence has been reported in almost all the phyla from protozoa to mammals. Melatonin displays a large set of functions including adaptation to light: dark cycles, free radical scavenging ability, antioxidant enzyme modulation, immunomodulatory actions or differentiation–proliferation regulatory effects, among others. However, in addition to those important functions, this evolutionary ‘ancient’ molecule still hides further tools with important cellular implications. The major goal of the present review is to discuss the data and experiments that have addressed the relationship between the indole and glucose. Classically, the pineal gland and a pinealectomy were associated with glucose homeostasis even before melatonin was chemically isolated. Numerous reports have provided the molecular components underlying the regulatory actions of melatonin on insulin secretion in pancreatic beta-cells, mainly involving membrane receptors MTNR1A/B, which would be partially responsible for the circadian rhythmicity of insulin in the organism. More recently, a new line of evidence has shown that glucose transporters GLUT/SLC2A are linked to melatonin uptake and its cellular internalization. Beside its binding to membrane receptors, melatonin transportation into the cytoplasm, required for its free radical scavenging abilities, still generates a great deal of debate. Thus, GLUT transporters might constitute at least one of the keys to explain the relationship between glucose and melatonin. These and other potential mechanisms responsible for such interaction are also discussed here.
Melatonin is a highly conserved molecule present in nearly all studied species. In mammals and birds, it is well accepted that the circulating levels of this hormone function during daytime, allowing the synchronization of many circadian rhythms. The cellular mechanisms of melatonin action are produced through the activation of specialized membrane and nuclear receptors. However, the mechanism of their actions, from signaling pathways to the integrative level, is far from being understood. Comparative studies provide important contributions toward understanding the action of melatonin. Studies in invertebrates, notably in crustaceans and insects, can greatly help to elucidate various aspects of melatonin action: the common challenge of organisms to face their temporal organization has apparently been achieved by a phylogenetically conserved strategy. In this chapter we will compile information about the following: 1) the presence of melatonin in invertebrates 2) their biological effects, and 3) their mechanisms of action.
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In vertebrates, it is now clearly demonstrated that the pineal gland is implicated in conveying photoperiodic information via the daily pattern of melatonin secretion. Invertebrates, like vertebrates, use photoperiodic changes as a temporal cue to initiate physiological processes such as reproduction or diapause. How this information is integrated in invertebrates remains an unsolved question. Our review will be an attempt to evaluate the possible role of melatonin in conveying photoperiodic information in invertebrates. It is now well demonstrated in both vertebrates and invertebrates that melatonin as well as its precursors or synthesizing enzymes are present in various organs implicated in photoreceptive processes or in circadian pacemaking. Melatonin, serotonin or N-acetyltransferase have been found in the head, the eyes, the optic lobe and the brain of various invertebrate species. In some species it has also been shown that melatonin is produced rhythmically with high concentrations reached during the dark period. Moreover, the physiological effects of melatonin on various periodic processes such as rhythmic contractions in coelenterates, fissioning of asexual planarians or reproductive events in flies have been reported in the literature. All these results support the hypothesis (refs 36, 37) that melatonin is not solely a pineal hormone but that it may be an evolutionary conservative molecule principally involved in the transduction of photoperiodic information in all living organisms.
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Invertebrates have circadian rhythms and exhibit photoperiodism and colour changes. While they lack pineal glands, those that have been investigated contain melatonin. Until now, melatonin has been shown to be present in the photoreceptor organs of this species, but the presence of the rate-limiting enzyme in melatonin synthesis, serotonin-N-acetyltransferase (SNAT, EC has not been investigated. We report here the presence of melatonin and the enzyme SNAT in the eyes (globe plus eyestalk) of the freshwater crayfish Procambarus clarkii. Both melatonin and SNAT activity exhibit circadian variations, with their acrophase during the light phase and their nadir during darkness. These rhythms have the same period, but they are 180° out of phase with respect to those described in vertebrates. SNAT is seemingly different to that reported in vertebrates since EGTA, a calcium chelator, has no protective function as it does in vertebrates.
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.
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.
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.
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.
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.
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.
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.