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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
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 . 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) , 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.
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 ; 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 .
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.