Daily Photocycle Research Articles

Occurrence, diel patterns, and the influence of melatonin on the photosynthetic performance of cultured Symbiodinium

Dinoflagellata is the earliest phylum in which true circadian regulation of melatonin rhythms has been convincingly demonstrated. Here, diel profiling of melatonin in a cultured member of this phylum belonging to the genus Symbiodinium indicated that melatonin levels oscillate with significant nocturnal peaks. However, unlike in other previously studied dinoflagellate species, the diel rhythmicity of melatonin in Symbiodinium did not persist under constant dark conditions. Thus, the oscillating pattern of melatonin in Symbiodinium is presumed not to be driven by endogenous circadian control of melatonin production, but rather by changes in the daily photocycle, most likely through a mechanism involving the enhanced photo-consumption of melatonin by free radicals. Although direct interactions of melatonin with detrimental radicals have been previously studied in several basal species, including dinoflagellates, none of these investigations addressed the effects that this molecule may have on photosynthesis, a major source of radical species in unicellular algae. In the present work, real-time monitoring of oxygen evolution in Symbiodinium cultures indicated a significant decrease in photosynthesis rates upon treatment with various doses of melatonin. Analyses of chlorophyll a fluorescence and xanthophyll cycle activity confirmed this effect and further revealed that this slowdown may occur through an enhanced engagement of photoprotective mechanisms in melatonin-treated cells. These findings are of great importance as they demonstrate that in certain photoautotroph species, the interactions of elevated melatonin levels with photosynthesis may extend beyond the general purpose of antioxidant protection.

Temporal organization of activity in the brown bear (Ursus arctos): roles of circadian rhythms, light, and food entrainment

Seasonal cycles of reproduction, migration, and hibernation are often synchronized to changes in daylength (photoperiod). Ecological and evolutionary pressures have resulted in physiological specializations enabling animals to occupy a particular temporal niche within the diel cycle leading to characteristic activity patterns. In this study, we characterized the annual locomotor activity of captive brown bears (Ursus arctos). Locomotor activity was observed in 18 bears of varying ages and sexes during the active (Mar-Oct) and hibernating (Nov-Feb) seasons. All bears exhibited either crepuscular or diurnal activity patterns. Estimates of activity duration (α) and synchronization to the daily light:dark cycle (phase angles) indirectly measured photoresponsiveness. α increased as daylength increased but diverged near the autumnal equinox. Phase angles varied widely between active and hibernating seasons and exhibited a clear annual rhythm. To directly test the role of photoperiod, bears were exposed to controlled photoperiod alterations. Bears failed to alter their daily activity patterns (entrain) to experimental photoperiods during the active season. In contrast, photic entrainment was evident during hibernation when the daily photocycle was shifted and when bears were exposed to a skeleton (11:1:11:1) photoperiod. To test whether entrainment to nonphotic cues superseded photic entrainment during the active season, bears were exposed to a reversed feeding regimen (dark-fed) under a natural photocycle. Activity shifted entirely to a nocturnal pattern. Thus daily activity in brown bears is highly modifiable by photoperiod and food availability in a stereotypic seasonal fashion.

Temporal and histological evaluation of melatonin patterns in a ‘basal’ metazoan

While recent advances suggest functional pleiotropy of melatonin in higher organisms, an understanding of the biological significance of this ancient molecule in early evolutionary groups is lacking. Here, endogenous melatonin production was identified for the first time in the sea anemone Actinia equina, a nonsymbiotic hexacorallian cnidarian. Day/night activity profiles of melatonin in this anemone indicated that melatonin levels oscillate with significant nocturnal peaks. However, dynamic changes in melatonin concentration did not persist under constant dark conditions and therefore were not circadian in nature. Thus, the oscillating pattern of melatonin in A.equina is presumed to be the result of alternative, simpler melatonin control mechanism that likely involves direct regulation by the daily photocycle. As nocturnal melatonin signals still potentially provide 'time-of-day' information and can illustrate the seasonally changing length of the biological night, we hypothesize that melatonin may be relevant to temporal coordination of timed processes also in anthozoans. Spatial patterns of melatonin distribution found in this study indicate abundant melatonin distribution in the endodermal filaments wrapped around gametes. This finding supports the possibility that one of the melatonin-responsive processes in this basal metazoan species may involve reproductive functions.

Diel mercury-concentration variations in streams affected by mining and geothermal discharge

Diel variations of concentrations of unfiltered and filtered total Hg and filtered methyl Hg were documented during 24-h sampling episodes in water from Silver Creek, which drains a historical gold-mining district near Helena, Montana, and the Madison River, which drains the geothermal system of Yellowstone National Park. The concentrations of filtered methyl Hg had relatively large diel variations (increases of 68 and 93% from morning minima) in both streams. Unfiltered and filtered (0.1-microm filtration) total Hg in Silver Creek had diel concentration increases of 24% and 7%, respectively. In the Madison River, concentrations of unfiltered and filtered total Hg did not change during the sampling period. The concentration variation of unfiltered total Hg in Silver Creek followed the diel variation in suspended-particle concentration. The concentration variation of filtered total and methyl Hg followed the solar photocycle, with highest concentrations during the early afternoon and evening and lowest concentrations during the morning. None of the diel Hg variations correlated with diel variation in streamflow or major ion concentrations. The diel variation in filtered total Hg could have been produced by adsorption-desorption of Hg2+ or by reduction of Hg(II) to Hg0 and subsequent evasion of Hg0. The diel variation in filtered methyl Hg could have been produced by sunlight- and temperature-dependent methylation. This study is the first to examine diel Hg cycling in streams, and its results reinforce previous conclusions that diel trace-element cycling in streams is widespread but often not recognized and that parts of the biogeochemical Hg cycle respond quickly to the daily photocycle.

Hippocampal Melatonin Receptors Modulate Seizure Threshold

The pineal hormone melatonin has been shown to enhance hippocampal excitability. We therefore investigated whether inactivation of hippocampal melatonin receptors affects behavioral seizures. Intrahippocampal infusions were performed in rats to study the effect of different melatonin receptor antagonists on behavioral activity, EEG, and seizure susceptibility. Experiments were conducted at 2 times of the day that coincided with the peak and trough of the daily melatonin rhythm. Local infusion of the Mel(1b) receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT) into the hippocampus, but not the overlying neocortex, significantly increased seizure latency and in some cases provided complete protection against seizure development. In addition, 4-P-PDOT suppressed open field activity and hippocampal EEG amplitude. The mixed Mel(1a)/Mel(1b) receptor antagonist luzindole also increased seizure latency but to a lesser degree than 4-P-PDOT. The behavioral effects of Mel(1b) receptor inhibition were comparable to those of the gamma-aminobutyric acid (GABA)(A) receptor agonist muscimol and were observed during the dark phase (2400-0200 h) but not the light phase (1200-1400 h) of the daily photocycle. The anticonvulsant effect of intrahippocampal infusion of 4P-P-DOT was blocked by coadministration of the GABA(A) antagonist bicuculline. Our results suggest that nocturnal activation of hippocampal Mel(1b) receptors depresses GABA(A) receptor function in the hippocampus and enhances seizure susceptibility.

Diel dynamics of chlorophylls in high‐nutrient, low‐chlorophyll waters of the equatorial Pacific (180°): Interactions of growth, grazing, physiological responses, and mixing

In situ diel variations of extracted chlorophyllous pigments, beam attenuation by particles (cp), and in vivo chlorophyll fluorescence (Fiv) were investigated during a 5‐day time series in high‐nutrient, low‐chlorophyll waters of the equatorial Pacific (date line = 180°). Samples were taken hourly at 10 depths in the upper 100 m during the first 48 hours, then sampling frequency decreased to 3 hours. In the 30–70 m layer the integrated chlorophyll concentrations, cp, and Fiv increased during the light period, but the minima and, especially, maxima were not fully synchronized. The lowest values of total chlorophyll a (Tchl a = chlorophyll a + divinyl‐chlorophyll a) occurred around 5–6 hours, slightly (0–2 hours) before that of cp and Fiv. Tchl a reached a maximum around 1500 hours ± 1 hour, clearly before cp (1700 hours) and Fiv (1900 hours). In the 0–30 m layer, diel variations of the integrated chlorophyll concentrations, cp, and Fiv were clearly out of phase. They showed a nocturnal increase in Tchl a, starting around midnight and peaking in early morning (0900 hours). In contrast, cp increased only during the light period in the upper 30 m, and variations of Fiv were largely opposite to those of extracted Tchl a. Specific phytoplankton growth (μ0) and grazing loss (g) rates were estimated from diel variations in the 30–70 m layer and compared to independent rate estimates from experimental incubations. These results are discussed in the context of physical processes and physiological responses of the cells to the daily photocycle.

Food intake inhibition by melatonin in goldfish ( Carassius auratus)

Feeding regulation by monoamines, neuropeptides and certain hormones has been studied in fish, but a possible role of melatonin is unknown. The purpose of the present study was to investigate the effects of melatonin on food intake in goldfish. Fishes were housed in 12L:12D and injected with different doses of either melatonin or 2-iodomelatonin. Two routes of administration, intracerebroventricular and intraperitoneal injections, and two times of the daily photocycle, midday and midnight, were tested. Food intake was measured at 2, 5 and 8 h postinjection. Melatonin and its analog, 2-iodomelatonin intracerebroventricularly injected had no effect on food intake at any time. However, intraperitoneal injections of both indoleamines significantly reduced food intake at different postinjection times. The inhibitory effect of melatonin was blocked by intraperitoneal administration of its antagonist, luzindole. These results demonstrate the in vivo efficiency of luzindole as melatonin antagonist, and thus provide a useful experimental tool to investigate melatonin functions. In conclusion, both melatonin and its agonist 2-iodomelatonin administered peripherally, inhibit food intake in goldfish, and this inhibitory effect appears to be mediated via luzindole-sensitive melatonin receptors. Our results strongly suggest that melatonin is involved in the peripheral satiety mechanisms in goldfish.

Daily changes in thyroid activity in the frog Rana perezi: Variation with season

Plasma triiodothyronine (T 3) and thyroxine (T 4) levels, as well as thyroid free (f) and bound (b) thyroid hormones (TH) content, were determined by radioimmunoassay (RIA) in adult Rana perezi frogs during a 24 h cycle in winter, spring, summer, and autumn. Significant daily changes in plasma T 3 levels were present in all the seasons except for winter, being the lowest values observed during the scotophase. In contrast, plasma T 4 only showed significant changes in spring, following a similar pattern to the one described for T 3. Thyroid fT 3 content did present day/night significant changes only in spring showing high contents at early scotophase. Mean fT 4 content was higher at the beginning of light phase than during the rest of daily photocycle in spring and autumn, but significant differences appeared only in autumn. Regarding the thyroid bound content of TH, bT 3, and bT 4 presented significant daily changes in spring and autumn. However, different profiles were observed in these two seasons. High bound contents were found at early photo-and scotophase with lower values at late dark phase in spring, whereas higher contents were detected at this time in autumn. The present results indicate the existence of seasonally changing daily fluctuations in thyroid activity in Rana perezi and it seems that an interaction between photoperiod and temperature plays a role in the regulation of these daily changes.

Differential characteristics and regulation of arylamine and arylalkylamine N-acetyltransferases in the frog retina ( Rana perezi)

Arylamine N-acetyltransferase activity (A-NAT: E.C.2.3.1.5) from Rana perezi retina was studied using p-phenetidine as specific substrate. Enzyme characteristics and regulation were compared with respect to the arylalkylamine N-acetyltransferase (AA-NAT: E.C.2.3.1.87) from the same tissue. A-NAT activity is distributed in both neural retina and choroid-pigmented epithelium complex, showing a 10-fold higher specific activity in neural retina. In contrast, AA-NAT activity is restricted to neural retina. Subcellular localization in neural retina indicated that both enzymatic activities are in the supernatant fraction (39,000 g , 20 min). p-Phenetidine acetylation was linear as a function of the neural retina amount in the assay ( 1 16 to 1 retina), and it is insensitive to phosphate buffer pH in the range 6.5–8.4. A-NAT kinetic showed a hyperbolic shape for both cosubstrates. Kinetic constants were K M = 11.2 μM, V max = 0.49 nmol/h/mg prot. for p-phenetidine (50 μM acetyl-CoA), and K M = 113.4 μM, V max = 3.1 nmol/h/mg prot. for acetyl-CoA (5 mM p-phenetidine). The additivity test for both enzymatic activities in retina homogenates demonstrated that both acceptor amines do not compete for the catalytic sites. Serotonin addition in the assay modifies differentially the kinetic characteristics of both enzymes. Serotonin acted as a strong mixed inhibitor, mainly competitive in nature (competitive K i = 18.1 μM; non-competitive K i = 1.9 mM) for AANAT. However, it acted as a weak inhibitor with respect to A-NAT, mainly non-competitive, (competitive K i = 5.7 mM; non-competitive K i = 8.7 mM). Attending to the low affinity ( K i) for A-NAT, it can be concluded that serotonin is a poor substrate for this enzyme. The environmental regulation of both NAT activities was clearly different. Low temperature (5°C) significantly increased the retinal AA-NAT activity in cultured eyecups with respect to high temperature (25°C), whereas A-NAT was not affected by temperature. In addition, AA-NAT is regulated by a daily photocycle, showing a marked increase at subjective midnight with respect to midday at both culture temperatures. On the contrary, A-NAT did not show this daily oscillation. In conclusion, frog retina exhibits an A-NAT activity with distinctive characteristics, such as substrate specificity, reaction requirements, and regulation by photoperiod and temperature, which allows it to be discriminated from AA-NAT activity. Retinal A-NAT is unable to acetylate arylalkylamines in vitro, and thus, probably does not contribute to melatonin synthesis in vivo.

Alpha‐1 Adrenoceptor Involvement in the Control of Melatonin Secretion in the Golden Hamster

The nocturnal peak in pineal and serum melatonin content was reduced following administration of the selective alpha-1 adrenoceptor antagonist prazosin in a dose of 0.25 mg/kg. The effect was pronounced one hour post treatment during the late dark phase of the daily photocycle. These data confirm the reported findings that the hamster pineal sensitivity to adrenergic challenge is confined to the second part of the dark phase and indicate that postsynaptically located alpha-1 adrenoceptors are also involved in the physiological control of melatonin synthesis and/or release in that species.

Factors influencing the affinity and capacity of T3-binding sites in hepatic nuclei of rainbow trout,Oncorhynchus mykiss

The effects of several variables were tested on the in vitro binding affinity (Ka) and maximum binding capacity (MBC) of saturable 3,5,3′-triiodo-L-thyronine (T3)-binding sites (putative thyroid hormone receptors) extracted from hepatic nuclei of immature rainbow trout held on a 12L:12D photocycle at 12°C. Neither Ka nor MBC varied during the daily photocycle, nor were they influenced by the growth rate, food ration, or a 13-fold chronic elevation in plasma T3. However, MBC was inversely correlated with fish size and was depressed significantly by two injections of 17β-estradiol (E2) or starvation for 3 or 12 days. The Ka was uninfluenced in either case. Since both E2 and starvation also depress T3 levels in the blood, the decrease in MBC could be secondary to this change. The drug ipodate, which depresses plasma T3, also lowered the MBC. However, MBC remained low in ipodate-treated trout given exogenous T3, thus suggesting that the effect of ipodate was not due to T3 deprivation. It remains to be shown, therefore, that plasma T3 depression itself can modify MBC. It is concluded that increasing body size and catabolic states, both of which are associated with a decrease in specific growth rate, tend to decrease the abundance of hepatic T3 receptors.

Response to pinealectomy and blinding in vitellogenic female frogs (Rana perezi) subjected to high temperature in autumn

The present experiments were carried out to investigate the effects of pinealectomy and bilateral enucleation on the ovarian activity in Rana perezi frogs maintained in 12-h light--12-h dark photoperiod and 20 +/- 1 degrees C during the vitellogenetic growth in late autumn. These environmental conditions, mainly temperature, induce a gonadal and metabolic response similar to that observed in the natural habitat in summer: a marked ovarian follicular regression, a depletion of the energetic resources from fat bodies and liver, and a minimum in oestradiol circulating levels. This response is partially blocked by pinealectomy and blinding. Protein phosphorus, as an index of vitellogenic proteins, and total ovary lipid content were significantly higher in pinealectomized and blinded frogs with respect to sham-operated animals. Likewise, oestradiol concentrations showed a significant increase during the dark phase of the daily photocycle in pinealectomized and blinded animals. From our results, we can suggest that the arrest of vitellogenesis, the depletion of energetic resources, and the regulation of oestradiol levels induced by the high temperature in Rana perezi frogs can be influenced, at least in part, by the pineal complex and lateral eyes.

Insect photoperiodism: the linden bug,Pyrrhocoris apterus, a species that measures daylength rather than nightlength

Independent variation of the dark and light components of the daily photocycle has shown that the linden bug,Pyrrhocoris apterus, unlike other species, ‘measures’ daylength rather than nightlength. Greatly extended dark periods coupled with a short photophase (a Nanda-Hamner protocol) shows peaks and troughs of diapause at about 16-h intervals, an extremely short period for a circadian clock. If circadian oscillations are involved in photoperiodic time measurement in this species, a photoinducible phase might lie in the early rather than the late subjective night.

Photic relations and experimental dissociation of circadian rhythms in pineal composition and running activity in rats.

Abstract— It has been shown previously that pineal composition and some of its enzyme activities in mammals appear to be responsive to light and darkness in vivo, possibly indirectly. One such possible indirect mechanism would be by whole animal activity and the sympathetic innervation of the pineal. Study of the rhythmic changes in the pineal ethanol‐soluble fraction (mostly lipid) and in whole animal running activity in normal, adult male rats in standard daily photocycle and then in continuous light, shows rapid dissociation of the two rhythms in continuous light, with pineal composition being more light‐dependent.The free‐running circadian rhythm in the male rat's running activity in both standard daily photocycle and continuous light is shown to be similar in normal and in pinealectomized animals. This negative finding is relevant with regard to an hypothesized pineal function in lower tetrapods in regulation of locomotor activity in response to solar radiation.