Light exposure mediates circadian rhythms of rhizosphere microbial communities

Microbial community circadian rhythms have a broad influence on host health and even though light-induced environmental fluctuations could regulate microbial communities, the contribution of light to the circadian rhythms of rhizosphere microbial communities has received little attention. To address this gap, we monitored diel changes in the microbial communities in rice (Oryza sativa L.) rhizosphere soil under light–dark and constant dark regimes, identifying microbes with circadian rhythms caused by light exposure and microbial circadian clocks, respectively. While rhizosphere microbial communities displayed circadian rhythms under light–dark and constant dark regimes, taxa possessing circadian rhythms under the two conditions were dissimilar. Light exposure concealed microbial circadian clocks as a regulatory driver, leading to fewer ecological niches in light versus dark communities. These findings disentangle regulation mechanisms for circadian rhythms in the rice rhizosphere microbial communities and highlight the role of light-induced regulation of rhizosphere microbial communities.Subject terms: Microbial ecology, Community ecology     

Eyeless Mexican Cavefish Save Energy by Eliminating the Circadian Rhythm in Metabolism

The eyed surface form and eyeless cave form of the Mexican tetra Astyanax mexicanus experience stark differences in the daily periodicities of light, food and predation, factors which are likely to have a profound influence on metabolism. We measured the metabolic rate of Pachón cave and surface fish at a fixed swimming speed under light/dark and constant dark photoperiods. In constant darkness surface forms exhibited a circadian rhythm in metabolism with an increase in oxygen demand during the subjective daytime, whereas cave forms did not. The lack of circadian rhythm in metabolism leads to a 27% energy savings for Pachón cave fish compared to surface fish when comparing both forms in their natural photoperiods. When surface forms were tested under constant dark conditions they expended 38% more energy than cave forms under equivalent conditions. Elimination of the circadian rhythm in metabolism may be a general feature of animals that live in perpetually dark food-limited environments such as caves or the deep sea.     

Reactive oxygen species can modulate circadian phase and period in Neurospora crassa

Reactive oxygen species (ROS) may serve as signals coupling metabolism to other cell functions. In addition to being by-products of normal metabolism, they are generated at elevated levels under environmental stress situations. We analyzed how reactive oxygen species affect the circadian clock in the model organism Neurospora crassa. In light/dark cycles, an increase in the levels of reactive oxygen species advanced the phase of both the conidiation rhythm and the expression of the clock gene frequency. Our results indicate a dominant role of the superoxide anion in the control of the phase. Elevation of superoxide production resulted in the activation of protein phosphatase 2A, a regulator of the positive element of the circadian clock. Our data indicate that even under nonstress conditions, reactive oxygen species affect circadian timekeeping. Reduction of their basal levels results in a delay of the phase in light/dark cycles and a longer period under constant conditions. We show that under entrained conditions the phase depends on the temperature and reactive oxygen species contribute to this effect. Our results suggest that the superoxide anion is an important factor controlling the circadian oscillator and is able to reset the clock most probably by activating protein phosphatase 2A, thereby modulating the activity of the White Collar complex.     

Early Chronotype and Tissue-Specific Alterations of Circadian Clock Function in Spontaneously Hypertensive Rats

Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.     

Photoperiodic time measurement in the aphid Megoura viciae

Megoura produces parthenogenetic virginoparae in long day conditions, gamic oviparae in short days. The nature of this photoperiodic response has been analysed by rearing parent apterae in a wide range of circadian and non-circadian light cycles. By varying the light and dark components independently in a two-component cycle it has been established that the time measuring function is associated primarily with the dark period. There is no evidence that an endogenous circadian oscillation is implicated: thus (a) the ‘short day’ response is abolished by ‘night interruptions’ positioned in the early or late night. But this bimodal response pattern remains unchanged when the duration of the ‘main’ photoperiod is varied from ca. 6 hr to at least 25·5 hr. The stability of the maxima within the scotophase is inconsistent with the ‘coincidence’ models of photoperiodic timing that have been proposed. It is suggested that the essential timing process operates on the hour-glass principle, beginning anew with the onset of each period of darkness; (b) night interruption experiments employing very long (up to 72 hr) scanned dark periods yielded response maxima explicable in terms of the hour-glass hypothesis but did not reveal any circadian relationship between the maxima.The ‘dark reaction’ comprises a sequence of four stages, definable by the effects of light. Stage 1, extending from dark hr 0 to ca. 2·5, is fully photoreversible: at the next dark period the entire timing sequence is repeated up to the 9·5 hr critical night length. Towards the end of stage 1 reversibility is gradually lost and after a light interruption the reaction is resumed from a later time equivalent than dark hr 0; the subsequent critical night length is therefore reduced. The extent of the photoreversal is related to light duration. The period of maximum light insensitivity (stage 2) is attained at the end of the fourth hour. From ca. dark hr 5 to just short of the critical night length light exerts an increasingly promotive action which favours the production of virginoparae. This dark process is not photoreversible. Stage 4, which begins at hr 9·5, marks the end of the timing sequence. Light will not then annul the non-promotive action of the previous long night.Light has three effects which are determined by its duration and position within the cycle. The two terminal effects, mentioned above, are associated with the interception of dark stages 1 and 3 by either short (1 hr) or longer photoperiods. Light also prepares or primes the dark period timer. Thus the critical length is increased, and timing accuracy lost, if the preceding photoperiod is less than ca. 6 hr. Light during stage 4 has a priming action but no terminal function. Repeated cycles are ‘read’ in various ways, depending on the cycle structure. For example, if light intercepts stage 3, a two-component cycle is interpreted as the overlapping sequence light/dark/light. One and the same photoperiod then acts terminally in respect of the preceding dark period and as a primer for the next dark period.There is also a mechanism for summing the promotive effects produced by repeated interruption of dark stage 3. With complex (four-component) cycles both halves of the same cycle may contribute. ‘Product accumulation’ falls below threshold if the frequency of presentation of a given promotive cycle is too low. This occurs if there are very long, relatively non-promotive dark components. Such cycles are accepted as ‘continuous darkness’.     

Photocontrol of Dark Circadian Rhythms in Stomata of Phaseolus vulgaris L

Stomatal diffusion resistance in primary leaves of Phaseolus vulgaris L. which had been grown in light:dark cycles followed a marked circadian rhythm when the plants were transferred to continuous darkness. Reentrainment of the rhythm required more than one inductive change in photoperiod. The phasing of the rhythm of dark stomatal opening was contolled primarily by the light-on (dawn) signal, whereas the rhythm of dark closure was related to the light-off (dusk) signal. The evidence points to a dual control of the circadian clock in which a product of photosynthesis plays a major role. No evidence for phytochrome involvement in the phasing of the rhythm was found. An influence of phytochrome on the amplitude of the stomatal rhythm was observed in which removal of phytochrome-far-red absorbing form caused rapid damping.     

Lesions of the Intergeniculate Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in Masking Responses to Photic Stimuli in the Nile Grass Rat

Light influences the daily patterning of behavior by entraining circadian rhythms and through its acute effects on activity levels (masking). Mechanisms of entrainment are quite similar across species, but masking can be very different. Specifically, in diurnal species, light generally increases locomotor activity (positive masking), and in nocturnal ones, it generally suppresses it (negative masking). The intergeniculate leaflet (IGL), a subdivision of the lateral geniculate complex, receives direct retinal input and is reciprocally connected with the primary circadian clock, the suprachiasmatic nucleus (SCN). Here, we evaluated the influence of the IGL on masking and the circadian system in a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), by determining the effects of bilateral IGL lesions on general activity under different lighting conditions. To examine masking responses, light or dark pulses were delivered in the dark or light phase, respectively. Light pulses at Zeitgeber time (ZT) 14 increased activity in control animals but decreased it in animals with IGL lesions. Dark pulses had no effect on controls, but significantly increased activity in lesioned animals at ZT0. Lesions also significantly increased activity, primarily during the dark phase of a 12:12 light/dark cycle, and during the subjective night when animals were kept in constant conditions. Taken together, our results suggest that the IGL plays a vital role in the maintenance of both the species-typical masking responses to light, and the circadian contribution to diurnality in grass rats.     

Circadian oxygen consumption rhythm of the flour beetle, Tribolium confusum

Oxygen consumption is circadian (about 24 hr) rhythmic in sterilized Tribolium confusum adults kept on a 12 hr light-12 hr dark regimen. High values for oxygen consumption occur shortly after ‘light-on’ and low ones at the beginning of the dark span. When the light-dark cycle is advanced by 6 hr, the oxygen consumption rhythm is phase-shifted within about 6 days; with a delay of the light-dark schedule (a return to the initial regimen) the oxygen consumption rhythm re-adjusts in 3 days. This directedness or polarity of the phase-shift adjustment constitutes a quantitative circadian system characteristic shared by organisms studied thus far.     

Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver

In a previous study we showed that rats fed ad libitum and maintained on a 12-h light/ 12-h dark cycle demonstrated out-of-phase circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis. As part of an investigation of the factors regulating both the generation of these cycles and their dissimilarity, this paper ompares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/12-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis.     

Characterization of Circadian Behavior in the Starlet Sea Anemone,Nematostella vectensis

Background

Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.

Methodology/Principal Findings

To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.

Conclusions/Significance

These data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.     

Contribution of Drosophila TRPA1-Expressing Neurons to Circadian Locomotor Activity Patterns

In both vertebrates and invertebrates, Transient Receptor Potential (TRP) channels are expressed in sensory neurons and mediate environmental stimuli such as light, sound, temperature, and taste. Some of these channels, however, are expressed only in the brain and their functions remain incompletely understood. Using the GAL4/UAS binary system with a line in which the GAL4 had been knocked into the trpA1 locus in Drosophila, we recently reported new insights into TRPA1 localization and function, including its expression in approximately 15% of all circadian neurons. TRPA1 is expressed in lateral posterior neurons (LPNs), which are known to be highly sensitive to entrainment by temperature cycles. Here, I used the bacterial sodium channel, NaChBac, to examine the effects of altering the electrical properties of trpA1 neurons on circadian rhythms. My results indicate that circadian activity of the flies in the morning, daytime, and evening was affected in a temperature-dependent manner following TRPA1 neuronal activation. Remarkably, TRPA1 neuron activation in flies kept at 18°C impacted the morning peak of circadian activity even though TRPA1 is not expressed in morning cells. Taken together, these results suggest that the activation of TRPA1-expressing neurons may differentially coordinate light/dark circadian entrainment, depending on the temperature.     

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1^−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2^−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 ^−/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.     

Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (¹³C₆-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.